GENETIC AND SOMATIC
EFFECTSOF IONIZING RADIATION
United Nations Scientific Committee on the Effectsof Atomic Radiation
1986 Report to the General Assembly, with annexes

UNITED NATIONSNew York, 1986

NOTE

The report of the Committee without its annexes appears as Official Records of theGeneral Assembly, Forty-first Session, Supplement No. 16 (A/41/16).
The designations employed and the presentation of material in this publication do
not imply the expression of any opinion whatsoever on the part of the Secretariat of theUnited Nations concerning the legal status of any country, territory, city or area. or of its
authorities, or concerning the delimitation of its frontiers or boundaries.

UNITED NATIONS PUBLICATION
Sales No. E.86.IX.9
ISBN 92-1-142123-3004800P

ANNEX A

Genetic effects of radiation

CONTENTSParagraphs

Paragraphs

INTRODUCTION ...................... .

I.

THEPREVALENCEOFNATURALL Y OCCURRING MENDELi ANAND
CHROMOSOMALDISORDERS
AND
THOSE
OF
COMPLEX AETIOLOGY ........ .
A.

B.
C.

D.
E.
F.
II.

Concepts and definitions ....... .
l\lendelian disorders ........... .
Chromosomal anomalies ....... .
I. Chromosomal anomalies m
spontaneous abortions andstill births .••....••........
2. Chromosomal anomalies infetuses diagnosed pre-natallyin amniocentesis programmes
3. Chromosomal anomalies innew-born children ......... .
4. Chromosomal anomalies inmentally retarded or otherwise handicapped individuals
5. Chromosomal anomalies insubfertile males ........... .
6. Direct chromosome analysesof hum~n spermatozoa andof fertilized eggs: further
results ................... .
7. Fragile sitt:s .............. .
Congenital anomalies .......... .
Other multifactorial disorders (excluding congenital anomalies) ... .
Summary and conclusions ...... .

THE APPLICATION OF RECOMBINANT DNA TECHNOLOGY TO
THE ANALYSIS OF THE HUMAN
GENOME AND GENETIC DISEASE
A.

General principles ..••......•...
I. Production of DNA fragments: the use of restriction
endonucleases ......•..•••.

2.

3.

4.

2-113

2
3-5
6-72

C.

Restriction fragment length polymorphisms (RFLPs) ........... .

133

Recombinant DNA technology inhuman genetics and medicine .... .
I. Direct analysis of genes .... .
2. Indirect analysis of genetic
disease using gene probes todetect linked polymorphisms
3. Indirect analysis of genetic
disease using cloned DNA segments to detect linked DNA
polymorphisms ........... ,4. Other applications of recombinant DNA technology

I 58-165

Summary and conclusions ...... .

166-172

GENES, CHROMOSOMES AND
CANCER •....•..••..............

I 73-227

The oncogenes of retro,·iruses(,·-ones) .•................. · · ·174-175

B.

Proto-oncogenes (proto ones) or
cellular oncogenes (c-oncs) ... , , .

176-178

C.

Cellular gene oncogenesis •......

I 79- I 82

D.

Relationships between cellular
oncogenes detected in transfection
assavs and the transforming genesof a~ute transforming retro,·iruses

183

E.

Other transforming genes ...... .

184

F.

How man}' oncogenes? ......•...

185

22-26
27-30
E.

73-88

A.

89-93
94-113

I 14-172
I 16-131

I 17-118

128-13 I
132

19-21

III.

I 26-127

Construction of libraries ...... , .

14-18

31-34
35-72

I 19-125

B.

D.

7-13

Incorporation of the DNA
fragments into a suitable
vector .............. , .... ·Cloning and the productionof multiple copies of theinserted DNA fragment .....
Selection and characterizationof recombinant clones ..... .

134-165
135-146147-150

151-157

27

Paragraphs

Paragraphs

G.

H.

I.

J.
K.

IV.

!\lechanisms of aclil'ation of proto·
oncogenes ................... .
I. Point mutations in protooncogenes ............... .
2. Attachment of strong promotors/enhanccrs to protooncogenes ............... .
3. Promotor (enhancer) insertion
near proto-oncogenes ...... .
4. Amplification of proto-oncogenes .................... .
5. DNA transposition and rearrangements ......•.......
Human oncogene localizations
and the acth·ation of c-oncogenes
through chromosomal aberrations
Oncogenic transformation of cells
in 1·itro (in transfection experiments)
1·ersus oncogenic acti1·ation andpathogenesis of neoplasms in I ho
Growth factors: mechanism ofaction and relation to oncogenes ..
Summary and conclusions ...... .

MOVABLE GENETIC ELEME!'l:TS
AND SPONTANEOUS MUTATIONRATES ......................... .
A.
B.
C.

D.

E.
F.
G.

Bacteria ..................... .
Yeast ..................•.....
Drosophila .............•......
I. MR-chromosomes ........ .
2. The P-M system of hybrid dysgenesis ...............•...
3. The I-R system ........•...
4. Interrelationships between the
MR, P-M and 1-R systems ...
5. Mobile genetic elements inferred from biochemical studies ..................... .
6. Similarities between Drosophila transposable elements
and those in other systems .. .
\touse ...................... .
Man ..............•..........
Rele1ance in the context of risk
evaluation ................... .
Summary and conclusions ...... .

VI.
186-196

OTHER RELEVANT DATA.......

A.

187-190191-192
193
194
195-196

I 97-206

207-211212-218219-227228-290230-238239-241242-263
243-247248-251252
253-255

256-261262-263264-269
270-271B.

272-274
275-290

V. EXPERIMENTAL DATA ON THE
MECHANISMS INVOLVED JN THE
PRODUCTIONOFCHROMOSOMAL ABERRATIONS IN CELLSIN VITRO ...................... .
A.

B.

C.

D.

28

Nature of the primary DNA lesions
inrnlved in the formation ofchromosomal aberrations •.....•.
The role of DNA repair processes in
the formation of chromosomal
aberrations induced by ionizing
radiation .................... .
I. Inhibitors of DNA synthesis
2. Inhibitors of DNA repair ••.
Prematurely condensed chromosomes: a model system for 1·isualizing effects of DNA damage, repair
and inhibition of repair at the le1elof chromosome structure ....... .
Summary and conclusions

291-335

292-301

302-325304-307308-325c.
326-330331-335

S1udies in experimental mammal~
including primate species ....... .
I. Productionofheritable
tumours 111 mice and rats
following irradiation and/or
chemical treatment ....... .
2. Radiation-induced congenital anomalies in mice ..... .
3. Further data on spontaneousand radiation-induced nondisjunction and chromosome
loss in mice ............. .
4. Further data on the induction of reciprocal translocations 111 mouse spermatogonia ................... .
5. Elimination of x-ray-induced
chromosomal
aberrations
induced in pre-ovulatory
mouse oocytes by early
embryonic death ......... .
6. Aneuploidy and structuralaberrations(spontaneousand radiation-induced) in thegerm cells of rodents ..... .
7. Induction of reciprocal translocations in spermatogonia of
non-human primate species
8. Qualitative
comparison
between spontaneous andradiation-induced mutations
in mice ....•.•...........
9. Molecular analysis of mouse
specific locus mutations ....
10. Genetic and cytogenctic
effects of incorporated radioactive isotopes in mice .....
11. Sensitivity of the mouse,
Chinese hamster and primate
oocytcs to the killing effectsof radiation ............. .
12. Summary and conclusions ..
Studies with in vitro systems .....
I. Spontaneous and radiationinduced thioguanine resistantcells in human lymphocytes
2. Induction of mutations andchromosome aberrations inmammalian (including human)
somatic cells at low doses anddose rates ................ .
3. Molecular approaches to the
studv of mutations and DJSA
repair in mammalian somatic
cells ............•.......•.
4. Use of somatic cell hybrids toidentify repair genes and theirchromosome locations inhuman cells .......•......•
5. Inducible (SOS-like) responses to DNA damage and adaptive responses in mammaliancells .............••.... ,.·6. Summary and conclusions ...
Interspecific comparisons of karyotypes among primate species:
rclernnce for the choice of primate
species close to man for radiation
cytogenetic studies ............ .

336-483336-430

336-342343-346347-363364-370371

372-386387-390391-394395

396-402403-410411-430
431-483431-438439-440

441-450

451-453454-466
467-476477-483

Paragraphs

VII.

RE-EXA~1INATIONOFAND
FURTHER APPROACHES TO RISKASSESSMENTS ................. .

484-515

A.

484-497

B.

C.

Mutational damage ........... .
I. Consistency of the genetic
findings in Hiroshima andNagasaki studies with those
expectedfromradiation
genetic studies with mice ....
2. Risks from the induction ofrecessive mutations ........ .
3. First-generation litter size
reduction following irradiation of spermatogonial stem
cells in male mice: a measureof deleterious effects of
induced mutations (including
deletions) and translocations
Chromosomal damage .. , ...... .
I. An estimate of the percentage
of conceptions with an unbalanced reciprocal translocation that survive birth ...
2. Model radiation studies withhuman
peripheral
blood
lymphocytes and fibroblasts
to estimate the proportion ofbalanced reciprocal translocations, which if induced ingerm cells may generate viable
imbalances after meiosis ... .
Summar} and conclusions ...... .

Paragraphs

VIII.

CURRENT RISK ASSESSMENTS ..

516-526XI.

ESTIMATES OF DETRIMENT FOR
SPONTANEOUSLY ARISING MENDELIAN, CHROMOSOMAL AND
OTHER DISORDERS ............ .

527-547A.

484-488B.

489-490C.

491-497498-510D.

E.

498-502F.

The effects of Mendelian disorders
on human health ......•..•.....
Detriment associated \\ith chromosomal anomalies assessed in termsof psychological disturbances,learning problems, growth andde,·elopment ................. .
Detriment associated with spontaneously arising congenital anomalies in man estimated using
years of life lost or impaired asindicators .................... .
Comparison of the estimates of
detriment for Mcndelian, chromosomal and other disorders ...... .
Frequency and economic burden ofgenetic disease in a paediatrichospital ................ :. : ... .
Summary and conclusions .... ·:· - ..

528-532533-535536-541

542

543
544-547
Page

503-510511-515

Introduction
1. The 1982 UNSCEAR Report [U I] presented acomprehensive review of data on the genetic effects ofionizing radiation in eukaryotes together with quantitative estimates of genetic risks in man. Since then.
some advances have been made in a number of areas
relevant to the work of the Committee. The purposeof this review is to cover these advances. focusing
attention on the following main themes: (a) theprevalence of naturally occurring Mendelian andchromosomal disorders and those of complex aetiology
in humans; (b) the application of recombinant DNA
technology to the analysis of the human genome andgenetic disease; (c) further data on genes. chromosomes and cancer in man; (d) movable genetic
elements and their relevance to the estimation of
spontaneous mutation rates and evaluation of genetic
risks using the doubling dose method: (c) basic studies
on the mechanisms of induction of chromosomal
aberrations in eukaryotic systems including mammalianand human somatic cells; and (f) other data that bear,either directly or indirectly. on the evaluation ofgenetic radiation hazards in humans and on thequantification of genetic detriment.

Tables ............................•.....

110

References ............................. .

133

I.

THE PREY ALENCE OF NATURALLYOCCURRING MENDELIAN AND
CHROMOSOMAL DISORDERS AND THOSEOF COMPLEX AETIOLOGYA.

CONCEPTS AND DEFINITIONS

2. Under the heading Mendelian disorders are included autosomal dominant, X-linked and autosomal
recessive conditions. inherited in a simple Mendelianfashion. Different genotypes are thus due to two or
more alleles at a single locus (so-called unifactorial or
unilocal inheritance). The chromosomal disorders arethose that are associated with structural or numericalchanges of chromosomes. Disorders of complex
aetiology include: (a) congenital anomalies (which arepresent at birth irrespective of whether they aredetected at that time or not): and (b) other disorders,most of which are not manifest at birth but havevariable ages of onset, many manifesting late in life. A
major proportion of disorders of complex aetiology
do not show a simple Mendelian pattern of inheritance.
Twin and family studies suggest that their aetiology ismultifactorial, depending on polygenic genetic pre29

disposition and environmental factors, which may also
be multiple. Among the features common to these
disorders are, firstly. the proportion of first-degree
relatives affected is much larger than in the general
population and, secondly. there is a sharp decrease in
the proportion affected on passing from first- tosecond- to third-degree relatives. The terms irregularly
inherited. polygenic, and multifactorial are often used
to denote these disorders. In this document, the term
multifactorial disorders is used here synonymouslywith disorders of complex aetiology and, for convenience, is considered under two headings: (a) congenital anomalies: and (b) other multifactorial disorders.
B.

MENDELIAN DISORDERS

3. Some new data on the prevalence of certain
Mendelian disorders have become available since thepublication of the 1982 UNSCEAR report [G20-G22,
G6 I-G63. H22. Y6]. These, however, do not require any
revision of the Committee's earlier overall prevalence
estimates to be made for these disorders (1.0%.
autosomal dominant and X-linked, 0.25%, autosomal
recessives). Similarly. the review of Matsunaga [MI]on this subject is based essentially on the samematerial that was extensively reviewed in the 1977
[U2] and 1982 [lJ I] UNSCEAR reports and will not
be gone into here.
4. The findings of Altukhov [A 10] (subsequently
published by Dubinin and Altukhov [D23]) on thefrequencies of rare (non-polymorphic) electrophoretic
variants in normal healthy new-borns (500 children),and in premature infants and babies with multiple
congenital anomalies (227 infants) were briefly alluded
to in the 1982 UNSCEAR report. These data showed
that: (a) the frequencies of these \'ariants (at over
20 genetic loci coding for the synthesis of enzyme
proteins and erythrocyte antigens) were about anorder of magnitude higher in premature infants and inbabies with congenital anomalies than in the healthy
new-born controls; and (b) a sizeable proportion of
the variants in the study group was due to de novo
mutations. suggesting that mutations of this type
could play an important role in the genesis ofcongenital defects and/or prematurity.
5. In order to confirm these results. Neel andMohrenweiser (N40] conducted a study to determine
rare electrophoretic variant frequencies (haemoglobin
and a set of serum proteins and erythrocyte antigens)in blood samples derived from 178 children who were
either born prematurely or had congenital anomalies(31 premature, 27 with chromosomal abnormalities
and physical defects and 120 with congenital anomalies
not known to be associated with chromosomal
anomalies). Similar data (the placental cord blood
series) from new-born infants in Ann Arbor provided
information on control frequencies of these variants(see also [N 17)). A total of 5341 determinations(10,603 locus tests) involving 45 proteins were made in
the study group and 108,963 determinations (215.609
locus tests) in the control. There were 15 rare variantsin the study group (0.00141 per locus tested) and 236
30

in the control (O._oo I 09 per locus tested). Thus, there
was no strong evidence for an excess of rare variantsin the study group; furthermore all these variants
were prese?t in one or the other p~rent, indicating no
new mutational events.

C.

CHROMOSOMAL ANOMALIES

6. New data on chromosomal anomalies in man
have come from cytogcnetic studies involving spontaneous abortions and still births, new-born babies,
amniocentesis programmes, mentally retarded or otherwise handicapped individuals, infertile males, direct
analysis of human spermatozoa and of fertilized eggs
and heritable fragile sites. These data will be discussed in
the following paragraphs.
1.

Chromosomal anomalies in spontaneous abortions
and still births

7. The cytogenetics of pregnancy wastage has recently
been reviewed by Boue et al. (878). Hook [HI]re-examined the available data [H2, H62, SL WI] on
the contribution of chromosomal anomalies to spontaneous abortions and still births from the standpointof gestational age. His analysis, summarized in Table I,
shows that 40% of spontaneous abortions occurring inweeks 5-28 are associated with chromosomal anomalies.
In the weeks 5-7 the rate is lower (17.5%), rising tonearly 50% in weeks 8-15. decreasing to about 30% at
16-19 weeks and dropping to 10% at 20-27 weeks. The
overall rate estimated by Hook of 40% of spontaneousabortions associated with chromosomal anomalies islower than the 50% commonly assumed. The latter
figure, however. allows for dev~lopmental arrests thatprobably occur before the fifth week of gestation(although in these cases, the pregnancy might beunrecognized and such early specimens are seldom
received for examination). In still births (those born
dead at or after 28 weeks), the overall rate is 5.7%
[Kl. SI].
8. Hook used the results of the above analysis toobtain approximate estimates of cytogenetic abnormalities for all embryonic and fetal deaths, including
still births. These estimates. summarized in Table 2,show that about 15.5% of all conceptuses recognizedfrom the fifth week of gestation onwards do notsurvive to live birth. The proportion of all recognizedpregnancies associated with both embryonic or feta!
death and a cytogenetic abnormality is 4.5%. Thus
4.5/ 15.5 or roughly 30% of all embryonic and feta!
deaths recognized after the fourth week of gestation
are associated with a chromosomal abnormality. Since
about 0.6% of new-born babies carry chromosomal
anomalies [Ul]. the proportion of recognizable conceptions with a chromosomal anomaly can be estimatedas 4.5 + (0.6% X 84.5%) = 5.0%.
9. The 1982 UNSCEAR report discussed data on thedelineation of the parental origin of the extra chromosome in individuals with trisomy-21. using cytologicallydetectable heteromorphism of chromosome 21. Those
results, from over 300 informative families, showed that,while there were considerable variations between studies
with respect to the proportion of cases attributable tomaternal or paternal non-disjunction. most trisomy-21
resulted from non-disjunction occurring in maternal
meiosis-I (65-80%); the contribution of errors inspermatogenesis and maternal meiosis-II was small. but
significant (20-35%).
10. Hassold et al. [H23] have extended this approach
to trisomies involving chromosomes 3, 4. 9, 13, 14. 15.
16, 21 and 22 in spontaneous abortions. Chromosome
heteromorphisms of the parents and trisomic fetuses
were compared using either QFQ-banding or Qbanding with dichloromethoxyacridine/spermine for
evaluation oftrisomies-3. -4, -13. -15, -21 and -22 andeither CBG-banding or distamycin/DAPI staining.
Their material consisted of 204 single trisomies mentioned above (66 informative cases), 9 mosaic trisomies(3 informative cases) and 9 double trisomies (5 informative cases). The results demonstrate that nondisjunction at maternal meiosis-I is the most likely
source of the additional chromosome for all trisomies
studied and this was the case at all maternal ages. In 61
of the 66 single trisomies, all 3 mosaic trisomies and 3 ofthe 5 double trisomies. the extra chromosome was ofmaternal origin. The proportion of paternally derived
cases varied considerably among the trisomies: thus, forinstance. the frequency of paternal non-disjunction was
22% (2/9) for both trisomies-I3 and -21, but only 2%( 1/48) for the remaining trisomies.
11. When these data were pooled with similar ones
published in the literature, giving a total of 338( 137 informative) cases of single trisomies, the evidence
for the increased contribution of paternal non-disjunction to trisomy-21 relative to other trisomies
remained strong, i.e.. in spontaneous abortions.
trisomy-21 is more likely than other trisomies to bepaternally derived. The maximum-likelihood estimate
for paternally derived trisomy-21 was about 0.25,
twice that for any other individual trisomy andapproximately three times that for all trisomies otherthan 21. However, the pooled data did not bear out
the high rate of paternal non-disjunction observed forchromosome 13.
12. It is worth pomtmg out that the technique ofusing chromosomal heteromorphism to identify theorigin of extra chromosomes in trisomies is currently
being supplemented by that involving restriction
fragment length polymorphisms (RFLPs). This aspect
is considered later.
13. In another study, Hassold and Chiu [H43]examined the effect of maternal age on the incidenceof chromosomally normal spontaneous abortions andof different categories of chromosome abnormality(sex-chromosomal monosomy, trisomies and polyploids) among all clinically recognized human pregnancies. The data on 2264 spontaneous abortions
karyotyped from 1976 to 1983 were used for thispurpose. The main findings were as follows: (a) the
overall rate of spontaneous abortions was 8.3%,
although this varied significantly with maternal age,
namely, the rate was relatively constant from age 20 to
30 years (6-8%). but from the mid-30-year-old on-

wards there was a marked increase. with more than
25% of all registered pregnancies terminating in
spontaneous abortion by age 40 years: (b) the hospitalbased value of 8.3% is probably an underestimate of thereal level of fetal wastage: (c) the overall rate ofchromosome abnormality was 50.4%, with trisomy
accounting for nearly 30% of the abortions and sexchromosome monosomy and polyploidy for approximately 10% each; (d) the relative proportions of thefour different categories of abortion (chromosomallynormal. sex-chromosome monosomy, trisomy andtriploidy) changed significantly with maternal age.
trisomy accounting for 20% of abortions involving
women under 25 years of age, for approximately 33%of those occurring in the 30-35 year age-group and for
67% in women aged 40 years and older; correspondingly. the relative proportions of the other three
categories of abortion declined with increasing maternal
age; and (e) the authors estimated that. for women
aged 35-39 years. about 10% of all recognizedconceptions were trisomic and for those 40 years andolder. this figure was around 30%; they also foundthat their data are in general agreement with thosepublished by Aymes and Lippman-Hand [AI5] andWarburton et al. [W36].

2.

Ch'romosomal anomalies in fetuses diagnosed
pre-natally in amniocentesis programmes

14. Schreinemachers et al. [S50] analysed the resultsof 19,672 pre-natal cytogenetic diagnoses reported to
the New York State Chromosome Registry and to theUnited States Interregional Chromosome Register
System for the period 1977-1980 and arrived at
estimates of numerical sex-chromosomal and autosomal anomalies. The data pertain to women who
were 35 years or older, for whom there was no known
cytogenetic risk for a chromosomal abnormality except
maternal age. The overall frequencies were the following: 47.XYY: 10 (0.051%); 47.XXY: 25 (0.127%);
47,XXX: 20 (0.102%); 45,X: 5 (0.025%): 47,+21: 179(0.910%): 47.+ 18: 49 (0.249%) and 47.+ 13: 12 (0.06 I%).
Taking maternal age into account, the authors estimated expected rates at amniocentesis from a regression analysis on the observed rates, using a firstdegree exponential model. 0 The expected rates werecompared with estimated rates by maternal age in live
births, which were derived from various studies
carried out in the late 1960s and early I 970s,
summarized earlier by Hook [H24].
15. The main findings are the following: (a) the rates
at amniocentesis for XXX and XXY increase withmaternal age, with those for XXY being almost
identical to those in live births. suggesting no late feta)
mortality for this condition; the rates for XYY show aslight decrease with maternal age, which is also
consistent with a low late feta! mortality; (b) the rates
for 47,+21 at amniocentesis and at live birth areapproximately parallel. with the latter being about
80% of the former; (c) those for 47 ,+ 18 at amnio0y = exp (a + bx) where x is the maternal age at the time of
expected Jive birth and y is the rate of the cytogenetic defect at age x;
x is on the average 0.4 years higher than the age at the time ofamniocentesis.

31

centesis and live birth are also parallel, but the latterare only about 30% of the former. which is consistent
with the high feta! mortalitv associated with thiscondition; and (d) the rates of 47,+ 13 at amniocentesis
show an increase with maternal age. which is not as
marked as that estimated from data on live births.
16. Similar results on structural chromosome abnormalities have been recently published by Hook et al.
[H24] on the basis of data reported to the registries
mentioned above (New York State Chromosome
Registry, for the period 1977-1981 and the US
Interregional Chromosomal Register System, up to
the middle of 1980). The primary indication was,again. advanced maternal age. There were. in theseseries, 27,225 fetuses among which 61 (0.224%) had
one or another kind of structural abnormality. detectedusing banding techniques (non-mosaic and mosaic
cases). The breakdown was the following: balanced
reciprocal translocations. 14 (0.051 %): balanced
Robertsonian translocations, non-Robertsonian translocations and supernumeraries etc., 35 (0.129%): andinversions, 4 (0.015%). Among these were 33 nonmosaic de novo rearrangements (2 inversions,
9 balanced reciprocal translocations, 6 balanced
Robertsonian translocations and 16 unbalanced
rearrangements). Of interest was the finding that thefrequency of supernumerary markers and fragments(0.07%) was about threefold higher than that expectedon the basis of live birth studies (about 0.02%; ref.
[J 10]). For these abnormalities. there was a trend foran association with advanced maternal age. The
frequency of Robertsonian translocations, however,
was only about one-fifth of that in new-horns.
17. Van Dyke et al. [VI] published the results ofchromosome analyses carried out in amniocentesis
programmes at the Henry Ford Hospital in Detroit,
incorporating the data from two similar studies [G23,C28]. These data pertain to balanced autosomal
rearrangements recorded in 8 I 58 amniocenteses forwhich advanced maternal age was the principal
indication. Analysis with banding techniques revealed
that 33 fetuses (0.40%) carried such rearrangements.
Of these. 14 (0.17%) carried balanced reciprocaltranslocations, 9 (0.1 I%) carried balanced Robensoniantranslocations and 10 (0.12%) carried inversions (involving chromosomes 2, 3, 4. 6. 10. II, 16, 18).
Among these were 7 de novo rearrangements (4 reciprocal translocations, 2 Robertsonian translocations
and I inversion). which give a mutation rate estimateof 4.3 10- 4/gamete/generation. This rate is higher than
the figure of 1.88 10- 4 /gamete/generation estimatedby Jacobs (J 10) on the basis of data from surveys of
new-barns involving 59.452 babies. Van Dvke et al.
[VI] suggest that: (a)· while maternal-ag~ amniocentesis series would provide a biased sample withrespect to the frequencies of trisomy-21 or dominant
mutations, this is not likely to be true with regard to
the frequencies of balanced structural rearrangements;and (b) the higher frequencies. as well as the higher
mutation rates, recorded in their work reflect theefficiency of detecting inversions and reciprocal translocations in banded chromosome preparations.
18. It is worth noting that the frequencies ofbalanced structural rearrangements in the work of
32

Van Dyke et al. [VI] are all higher than those in theseries of Hook et al. [H24], in spite of the fact thatbanding methods were used in both. The reasons forthis discrepancy are not clear.
3.

Chromosomal anomalies in new-born children

19. In its 1982 report. UNSCEAR summarized theresults of 10 cytogenetic surveys on the prevalence ofchromosomal anomalies in new-born children. In 7 ofthese surveys (59,465 babies) conventional Giemsa
staining procedures were used, while in the other three(7549 babies) banding methods were used. Among the67,014 babies screened, 424 (0.63%) were chromosomally abnormal. the breakdown being as follows:
158 (0.24%) sex-chromosomal anomalies. 95 {0.14%)
autosomal numerical anomalies. I 34 (0.20%) balanced
autosomal structural rearrangements. and 37 (0.06%)
unbalanced structural rearrangements and supernumerary chromosomes. With the sample sizes
involved, there were no striking differences between
the banding and conventional staining methods in theefficiency of detection of the chromosomal anomalies
except for inversions.
'
20. Recent results from cytogenetic surveys of newborns carried out in Norway [H 13) and Denmark[NI], in addition to results from some pre-natal
genetic studies (e.g .. ref. (VI]) (banding methods wereused in all these). suggest that earlier studies of newborns (without banding methods) have underestimatedthe frequencies of inversions and of reciprocal translocations. In the Norwegian survey [H 13], chromosome analyses were made in blood lymphocytes
derived from umbilical cord blood of 1830 consecutively born infants. Two G-banded cells were
karyotyped from each individual. When one or bothof these cells showed chromosome aberrations. at least
another 20 cells were studied and supplemented with
C-, Q- and R-banding methods when necessary. The
Danish survey of Nielsen et al. [N 1) is essentially acontinuation of their earlier work [NI 1]. Five BrdUtreated cells from each of 3658 children were photographed and analysed: from 20 to 50 cells wereanalysed when one or more of these cells showed achromosome aberration and further cells were examinedwhen mosaicism was suspected. The Q- and/or
C-banding methods were used for structural chromosome aberrations for chromosomal variants where
further analyses with other staining methods wereindicated.
21. Table 3 compares the main results of these twosurveys with the earlier Danish survey (the ethnic
background is fairly similar for these two populations)and with the pooled results of the earlier cytogenetic
surveys in which no banding methods were employed.
It can be seen that the total frequencies in the
Norwegian and the new Danish surveys ( 1.97 and2.02%) are substantially higher. relative to the earlier
Danish survey (0.83%) and to the one based on thepooled results of earlier surveys (0.63%). Particularly
noteworthy is the much higher rate of detection of
inversions (most of these, however, involve chromosome 9 and are currently regarded as variants) and ofreciprocal translocations in the two new surveys.

4.

Chromosomal anomalies in mentally retarded orotherwise handicapped individuals

22. Rasmussen et al. [Rl] presented data'on the prevalence of chromosomal abnormalities among mentallyretarded persons in Arhus. Denmark. In this area
there were 2157 patients, both institutionalized andliving at home. and chromosome analysis was possible
in 1905 cases. Of these. 359 (18.8%) had a chromosomal abnormality: 281 (14.7%) with Down syndrome(260 cases due to trisomy-21. 14 due to translocationsband 7 mosaics), 45 (2.4%) with one or another kind of
autosomal anomaly other than Down syndrome (7 casesof autosomal numerical anomalies. 20 cases of autosomal structural anomalies, including unbalanced translocations. deletions. duplications etc .. 12 cases of inversions predominantly of chromosome 9, and 6 cases ofbalanced reciprocal translocations) and 35 cases (1.8%)of sex-chromosomal anomalies including mosaics. In
comparison with similar data summarized in thereview of Jacobs et al._ [JI], and other data subsequently published [Al, FI, K2]. the Danish results
show that the prevalence frequencies in each of
the different subgroups (Down syndrome. autosomal
anomalies other than Down. and sex-chromosomal),are higher (Table 4 ). The authors attribute thesedifferences to the use of banding methods in theDanish study, which was not the case in the othersurveys.
23. Coco and Penchaszadeh [Cl] published a cytogenetic study of 200 children with mental retardationand three or more major or minor congenital anomalies. These cases were selected from the populationof patients referred to the Genetics Unit of theChildren's Hospital of Buenos Aires for diagnostic
evaluation because of mental retardation and multiple
congenital anomalies. Patients with a clinical diagnosis
of Down, Turner or Klinefelter syndromes were
excluded, but a clinical suspicion of any otherchromosomal syndrome was not considered adequate
reason for exclusion. In all cases. the chromosomes
were studied with conventional staining methods andwith at least one of the banding techniques. The age ofthe patients varied from new-born to 18 years, roughly
one-half being under one year old.

20%, with a mean of l I .0%. These variations in thefrequencies are due to several factors. including thefollowing: (a) the lack of strict comparability of the
populations (some studies sampled institutions for theretarded, others investigated hospitalized patients):(b) differences in age distributions; (c) the degree ofcare taken to exclude known or recognizable syndromes; and (d) the particular cytogcnetic techniquesused.
26. Dykes [D3] presented the results of what isperhaps the most extensive survey of severely handicapped children. Included in this survey are over
10,000 severely handicapped children under the ageof 16 in New South Wales. Australia. It showed that7228 children, representing 71. 7% of the total. werehandicapped by one or more of the following diseases
or disabilities: mental retardation, 6758: Down syndrome, 1162; and cerebral palsy, 1419. The total
number of chromosomal abnormalities in the survey
was 1214, with 1162 of these (95.7%) being in children
with Down syndrome. The anomalies detected in theother 52 (4.3%) children were sex-chromosomal aneuploidy including mosaicism, autosomal trisomies,
balanced and unbalanced translocations. ring chromosomes, partial deletions and duplications of chromosomes. All these children, besides being mentallyretarded (the degree of severity ranging from mild toprofound). had one or more congenital malformations.

5.

Chromosomal anomalies in subfertile males

25. Table 5 compares the results of similar studies inwhich chromosome analyses were made of patientswith mental retardation and multiple congenital anomalies. It can be seen that the frequencies of chromosome anomalies in such patients vary from 2.5% to

27. Dutrillaux et al. [024] compiled the resultsof chromosome studies (in blood lymphocytes) on
958 patients who attended two male infertility clinics
in Paris. Of these, 150 were studied without chromosome banding and the remainder with banding techniques. On the basis of sperm analysis. the patientswere classified into those with azoospermia (absence oflive spermatozoa in semen. 278 cases), predominantly
oligospermia (scarcity of spermatozoa in the semen,
specifically. Jess than 20 J06/ml. 486 cases), predominantly asthenospermia (weakness or loss ofvitality of the spermatozoa) and/or teratospermia(abnormal sperm morphology) and a much lessdefined category, namely unknown and miscellaneous(97 cases). A total of 58 chromosome anomalies weredete~ted (6.1 %), the frequencies being different in the
different groups (azoospermics, 37/278 or 13.3%:
oligospermics. I 5/486 or 3. I%; astheno/teratospermics,
2/97 or 2.1 % and the unknown and miscellaneousgroup. 4/97 or 4. I%). The details of these chromosome anomalies are given in Table 6. Not shown in
the Table is the finding that among the sex-chromosomal anomalies. all but 3 cases (of inversion of the
Y chromosome) were found in the azoospermic males(33/278 or 11.8%); among the autosomal anomalies,
4 were observed in azoospermic males (4/278 or I .4%)
and 12 among oligospermic males (12/486 or 2.5%).
Thus, there is a clear correlation between sex-chromosom-;tl anomalies and azoospermia, mostly, although
not exclusively, due to cases of Klinefelter syndrome.

hNine 14/21, one 15/21, four 21/23 translocations and one
involving chromosome 21 and an unidentified chromosome.

28. The authors compared their results with similar
ones published in the literature. taking into account

24. Of the 200 patients, 42 (21 %) had chromosome
anomalies. The breakdown is as follows: 11 cases(5.5%) of autosomal (chromosome 9. 13. 18. 22)
trisomes; 5 cases (2.5%) of sex-chromosomal aneuploidies (XYY. XXY and mosaics); 19 cases (9.5%) of
de novo structural rearrangements (deletions, rings.
unbalanced rearrangements and extra marker chromosomes). 2 cases ( l %) of unidentifiable supernumerarychromosomes and 5 cases {2.5%) of unbalanced
genomes originating from a balanced structural rearrangement in one of the parents.

33

the fact that there were heterogeneities in patient
selection (in particular, the proportion of azoospermic
males among all infertiles) and differences in methodsof chromosome analysis and of data reporting. In
these studies, the overall frequencies of chromosomal
anomalies ranged from 2.2 to 29%, with the proportion of azoospermics in the range 7-100% (someauthors did not report on the latter).
29. Reliable estimates of Lhe prevalences of chromosomal anomalies in subfertile men are hampered by alack of similar data for an unselected population of
adult males. Dutrillaux et al. [D24] therefore made acomparison with the frequencies recorded in surveysof new-borns and the results are summarized in Table 7.
The general conclusion is that the prevalence ofchromosomal anomalies in subfenile males is about
an order of magnitude higher than in new-borns.
although the frequencies of specific abnormalities
show a great deal of variation.
30. Results similar to those of Dutrillaux et al. [D24]have also been published by Micic et al. [M5 l ]. In thelatter study, lymphocyte chromosomes of 820 infertile
men were examined in banded preparations. The data
are summarized in Table 8. where it can be seen thatthe frequency of infertiles with chromosomal anomalies is 7.3% (60/820), i.e .. similar to that reportedby Dutrillaux et al. Of these males, 77 (9.4%) had
chromosomal variants, the significance of which is notclear, although a similar high frequency of variant D
and G group chromosomes has also been reported byMoreau and Teyssier [M52] ( JO% among infertiles
versus 4% in fertile men). As in the study ofDutrillaux et al., all patients with sex-chromosomal
anomalies were azoospermic. the most frequent among
them being those with an XXY constitution. Other
interesting aspects of the data of Micic et al. include:(a) the relatively high incidence of pericentric inversions in chromosome 9 among infertiles (2.8%) who
were either oligo- or azoospermic (this frequency is
still higher than those recorded by Hansteen et al.
[H 13] and Nielsen et al. [NI] in studies of new-borns
discussed earlier); (b) the finding of 1.1 % of balanced
autosomal translocations. a figure that is in agreementwith that of Dutrillaux et al. ( 1.6% or 15/958): and(c) the absence of structural aberrations of the X or Y
chromosomes (in contrast to the finding of such
aberrations by Dutrillaux et al.) [D24].

6.

Direct chromosome analyses of human spermatozoa
and of fertilized eggs: further results

31. The data of Rudak et al. [R 7]. representing thefirst direct analyses of the chromosome constitution ofhuman spermatozoa after fertilization of hamster(Mesocricetus auratus) eggs, were presented in the1982 UNSCEAR report [U I]. These authors analysed
60 spermatozoa from one normal male and found a
5% frequency of aneuploidy. These studies have now
been extended by Martin et al. [M 11] and Brandriff etal. [822, 838]. In the work of Martin and colleagues,
chromosome analyses of !OOO spermatozoa from33 normal men (21 of proven fertility and 12 of
34

unproven fertility: age range, 22-50 years) were performed. Of the spermatozoa analysed, 8.5% wereabnormal (range. 0-28%), of which 5.2% were aneuploid and 3.3% had structural aberrations. About halfof the aneuploid sperms were hypohaploid; the rest
were hyperhaploid with a very small proportion(0.1 %) having double aneuploidy. All chromosome
groups were represented among the aneuploid complements. but there were none with 24. YY constitucion. This finding is at variance with other studies inwhich fluorescent Y bodies were used as markers (inthose, which were reviewed in the 1982 UNSCEAR
report, the frequency of sperm with 2 Y bodies ranged
from I to 5%). Most structural anomalies werechromosome breaks (22/33). but the anomalies were
not equally distributed among the sperm of the 33
males: 2 males had 11 of the 33 structural anomalies.
32. The paper of Brandriff et al. (822] reports dataon 2468 spermatozoa from I I males (repeat sampling
was carried out for 2 individuals after 14 or 16 months)
and includes data published earlier [838). The males
were between 21 and 49 years of age. The overallfrequency of abnormal spermatozoa was 9.4% ( I. 7%
were aneuploid. with a frequency range of 0.6-3.1 %,and 7. 7% had structural abnormalities, with a rangeof 1.5-15.8%). Again. as in the work of Martin et al.
[M 11], aneuploid chromosome complements involved
all groups, and the majority of structural anomalies.
in order of frequency, were chromosome breaks.
followed by acentric fragments, chromatid exchanges,
chromatid breaks, deletions, dicentrics, translocations
and duplications. The repeat sampling of spermatozoa
from two males did not reveal any significant changes
in the frequencies of abnormal sperm. Parallel studies
with lymphocytes showed that, in six individuals. thepercentages of structurally abnormal cells were significantly higher in sperm than in lymphocytes byfactors of up to 8. but there were no correlations
between lymphocyte and sperm data.
33.

The subsequent work of Manin et al. [M53,
M81] was focused on the analysis of sperm complements of 13 cancer patients before radiotherapy and atregular intervals thereafter. The men were from 19 to
47 years of age and the estimated doses to the testes
ranged from 0.4 to 5 Gy. Before radiotherapy. thefrequency of chromosomal abnormalities in sperm
was zero in all men. After radiotherapy. the majority
were azoospermic for two years. Analysis of chromosomes of 4 men at 12 and 24 months showed highfrequencies of abnormal sperm (12.5% and 12.7%);
after 36 months (during which time sperm production
was recovered in most men). the frequency of abnormalities in 8 men was 20.9%. which was significantly
higher than in control donors (8.5%). For individual
men, the range of abnormalities was from 6 to 67%
and there was a significant correlation between testicular dose and the frequency of abnormalities. Bothnumerical and structural abnormalities were present.
34. Angell et al. [A9] reported on studies dealing
with in vitro fertilization of human oocytes by humansperm. Oocytes were removed from ovaries of7 women(aged between 31 and 39) at Japaroscopy and sperm
was collected from 7 healthy males aged between 27

and 39. all with informed consent. Fertilization was
carried out in small petri dishes under carefully
controlled conditions. Complete chromosomal analysis
was possible in only 3 embryos (8-cell stage). Twoof these were chromosomally abnormal (22,X,-15;
47,XY.+D). The haploid embryo contained only
maternal chromosomes and in the diploid embryo, theextra D chromosome could not be more precisely
identified. Of the further 8 embryos that were analysed
for their DNA content (microfluorimetric methods).
2 appeared to be haploid (excluding the one mentioned
above that was chromosomally analysed). The authorsemphasize that over 100 babies have now been bornafter in vitro fertilization without any apparent
chromosomal abnormality and that the abnormalitiesof the kind found in their study would clearly lead toearly embryonic loss. and probably contribute to thehigh failure rate after embryo transfer.

7.

(a)

Fragile sites

General aspects and classifica1ion

35. In the 1982 UNSCEAR report, some of theproperties of the heritable fragile sites then known inhuman chromosomes and the association between thefragile site on the X chromosome with macroorchidism
and mental retardation were discussed. Since then,considerable progress has been made (e.g .. [B2, C 10.
GI. G25, H3, Hl3. KIO. 09. S2, S24-S27, S55-S57,
T3. T4]). Well over 40 fragile sites have now been
discovered and are currently subdivided into two
major groups. the rare (or relatively so) and thecommon or constitutive. The rare fragile sites havebeen classified, according to the conditions of" tissue
culture under which they are expressed. into folatesensitive, distamycin-A inducible and BrdU requiring.
Nineteen rare fragile sites are currently known.

(i)Rare fragile sites

36. Group I: Folate-sensitive fragile sites. There are
16 of these at 2qll. 2ql3, 6p23. 7pll, 8q22. 9p21,
9q32, 10q23, llql3, llq23, 12ql3, 16pl2. 19pl3,
20pl 1, 22qI3 and Xq27. They are termed folatesensitive because removal of folic acid (and thymidine)from the culture medium was the first factor found tobe essential for their demonstration in lymphocyte
cultures, i.e., they are invisible in complete media but
can be seen in deprived media (such as medium 199)
deficient in folic acid and thymidine. The recently
discovered fragile site at 19pl3 [T30) was found to be
expressed in medium containing low folate and byaddition of FdU. whereas high folate, thymidine andthymidine analogues inhibited its expression.
37. Group 2: Distamycin-A inducible fragile sites arethose at I6q22 and 17pl2. These are not dependent
upon conditions of tissue culture but are enhanced by
the addition of distamycin A to lymphocyte cultures[ClO]. The site at l 7p 12 was not originally accepted asa fragile site but is now included in this group in viewof the finding that it fulfils the definition of a fragilesite[S5S. SS8,S59).

38. Group 3: BrdU-requiring fragile site. The fragilesite at 10q25 was independently discovered by Scheresand Hustinx [S60] and by Sutherland et al. [S61). It is
expressed only if BrdU or related compounds arepresent in the culture medium some hours prior toharvest of cell cultures for chromosome studies.

(ii)

Common fragile sites

39. Paradoxically. less is known about these thanabout the rare ones. The common fragile sites were
initially regarded as hot spots or autosomal lesions
and not as fragile sites, because until recently they
were not known to be inducible. did not appear to be
heritable and were usually seen in only a small
proportion of the metaphases [SSS].
40. Group I: Aphidicolin-inducible fragile sites.
Glover et al. [G25] showed that fragile sites at 2q31,
3p 14, 6q26, 7q32, 16q23 and Xp22 (a total of six) are
weakly induced by the same conditions as those thatinduce folate-sensitive sites. but are more strongly
induced by aphidicolin (a specific inhibitor of DNApolymerase alpha) and most effectively by these
conditions together.
41. Group 2: 5-azacytidine-inducible fragile sites.
Discovered recently by Sutherland et al. [SS?]. thesefragile sites in bands lq42 and 19ql3 are induced by
5-azacytidine. an analogue of cytidine: maximum
induction is observed when the inducing agent is
added 5-8 hours prior to harvest.
42. Group 3: BrdU-inducible fragile sites. Also
discovered by Sutherland et al. [S57], these occur at
6q 13. 9p2 l, I Oq2 I and maximum induction is observed
when BrdU is added 4-6 hours prior to harvest. (Notethat band 9p21 contains a common fragile site and arare folate-sensitive site; a detailed comparison ofthese by high resolution banding has not yet been
performed and hence it is unknown whether they arecoincident.)(iii)

Properties of rare and common fragile sites

43. The cytological appearance of fragile sites isvariable, but there are several essential properties ofrare fragile sites: (a) there is a non-staining gap ofvarying width that usually involves both chromatids;(b) the fragile site is always at exactly the same pointon the chromosomes in all individuals or kindred;(c) the fragile site is inherited in a Mendelian
codominant fashion: and (d) the fragile site exhibits
fragility under conditions of induction, as evidenced
by acentric fragments. deleted chromosomes, triradial
figures, etc. [S53]. The triradial (or multiradial) is themost spectacular cytogenetic manifestation of thefragile site and is essential for distinguishing fragile
sites from other phenomena causing chromosome
breakage.
44. The common fragile sites have the following
properties [D27]: (a) they occur in cells of all
individuals tested with the particular set of conditions35

at the same chromosomal location: (b) they aredetectable in both homologues and are homozygous ina proportion of cells: (c) they usually occur within
G-positive bands (most of the rare ones occur in
G-negative bands); and (d) they do not seem to exhibit
specific triradial formation characteristic of most of
the rare fragile sites. The common fragile sites arepresumably heritable. but family studies have not yet
been performed [S57] because of the low frequency atwhich some of these are expressed and the problem ofidentifying the separate homologues of chromosomes
without C-band or fluorescent variants.
45. The fragile sites mentioned above are the confirmed ones. Other possible rare fragile sites (at lq32,
2p 11, 5q35, 22q, Xq26 and Yq 12) and common fragile
sites (at I p22, I p32, 1p36, 1q25, 2p 13, 2q33, 3p24,
3p27, 5q31, 7p31. 7q22, 8q22, 9q32, l lpl3, I4q24,
22qI2, Xq22, and probably many others) reported in
the literature and discussed by Sutherland and Hecht[S53] need confirmation.

46. There are some suggestions that the expressionof the fragile site at Xq27 is decreased in blood
specimens delayed in transit to the laboratory [J 11,
B26], and Fonatsch [F 18) reported a marked decreasein the frequency of expression of fragile-X in bloodthat had been stored for five days prior to culturecompared to freshly cultured blood. Daniel et al.
[D27] noted that when the blood was stored for 2-4
days at 4°C or at 25°C prior to culture. there was a
decline in the expression of fragile-X relative to
unstored samples and this decline was less pronounced
in the samples stored at 4°C. Storage of the samples
for 1-5 days and at 4°C. 25°C. 37°C and 39°C prior toculture revealed that the per cent expression of thefragile-X was inversely related to the temperature andduration of storage, the decline being more pronounced
at temperatures higher than 4°C and with longer storage
periods.

(b)(i)

The fragile sire on rhe X chromosome andirs associarion wirh menral rerardarion

Localizarion

Xq28 and the fragile site are linked (see also [H26]).
The recent success in cloning and mapping of thehuman coagulation factor IX gene to the region from
Xq26 to Xq28 by several laboratories and the identification of a common Taql restriction fragment length
polymorphism (RFLP) [C57. C58, J 19. 120, K35]
catalyzed the search for linkage between the fragile X
gene and the factor IX Taql RFLP. In the work of
Camerino et al. [C29]. out of 17 informative meioses
in two families. recombination between these genes
was not observed. resulting in an estimate of thegenetic distance of less than 12 centimorgans (cM).
Warren et al. [W37] carried out linkage analyses witha factor IX cDNA probe in a family' with the fragile-Xlinked mental retardation and were able to identify arecombinant individual (among two affected brothers),suggesting that the linkage between these two genes is
not as close as the data of Camerino et al. [C29] mightsuggest. Recombination between these genes has also
been observed in the work of Choo et al. [C59]. (Seealso Davies et al. [D43) and Mulligan et al. [M60].)(ii)

Expression and relation ro age and IQ

49. Herbst et al. [H4] considered that over 1%
expression of fra(X) was necessary to identify the
carrier status with certainty. Age further complicatesthe expression of fra(X) in lymphocytes and there arereports suggesting that fra(X) is more difficult to
detect in older carriers [JI I. C30, 824]. In the absenceof serial studies over time in carriers, it is difficult tobe certain whether an age effect might explain why
there are problems in detecting the fra(X) in carrier
females.
50. Chudley et al. [ C3 I] studied eight Saskatchewan
families (37 affected males and 22 carriers) andshowed a significant inverse relationship between age
and the frequency of fra(X) expression; they also
found that in carrier females the frequency of fra(X)expression and IQ were inversely related and that in
affected ma1es this relationship was less pronounced.
Fishburn et al. [F 16] observed that mildly retardedfra(X) heterozygotes had a significantly higher percentage of fra(X) expressing lymphocytes, comparedwith intellectually normal heterozygotes.

47. It is now firmly established that the X-linked
fragile site (associated with mental retardation) is near
the end of Xq. There has been some uncertainty,
however, regarding its exact location, i.e., whether thesite is in band Xq27, Xq28 or at the Xq27-Xq28
interface. In a scanning electron microscope study.
Harrison et al. [H63] localized the fragile site at
Xq27.3. In their preparations. the fragile site appeared
as an isochromatid gap in the majority of cases.
confirming light microscopy observations. Utilizing
high resolution banding. Brookwell and Turner [B23]
assigned the fragile site to band Xq27, close to the
Xq27-Xq28 interface. A more recent study by Krawczun
et al. [K34], in which high-resolution banding was also
used confirmed the localization to band Xq27.3.

51. In 28 heterozygotes from the same families as
those studied by Chudley et al. [C3I] (one-third ofthese were mentally retarded), Knoll et al. [K 18]
investigated the frequency of fra(X) expression andfra(X) replication pattern. The retarded carriers had ahigher frequency of early replicating fra(X). in addition
to higher overall frequencies of fra(X) expression than
did normal carriers. i.e., mental retardation in females
heterozygous for fra(X) may be largely a function of
the proportion of cells with an early replicating. active
X-chromosome possessing the fragile site. The early
replicating fra(X) accounted for more of the variability
in IQ than the late replicating one. a finding inaccordance with the results of Uchida and Joyce [US]and Uchida et al. [U6]. When the data were adjusted

48. In genetic linkage studies in Sicilian families.
Filippi et al. [F 15] reported that the G6-PD locus at'The family consisted of 2 grandparents, 2 parents, 2 affectedsons (brothers), I affected and I normal daughter and I son
unavailable for study.

36

for age, the nature of the relationships between IQ
and proportion of early replicating fra(X), between IQ
and proportion of late replicating fra(X) and between
IQ and frequency of fra(X) did not change, suggestingthat age had a minimal effect.
52. In the work of Brondum-Nielsen et al. [B24]
involving 63 obligate and potential carriers of fra(X).
a. difference was found between normal and mentallyretarded carriers in the expression of fra(X), in thatthe mean percentage of positive cells was 8% in theformer versus 31 % in the latter. Furthermore, there
was an inverse relationship between the percentage of
positive cells and age in normal carriers, whereas
retarded carriers generally showed high percentages at
all ages (see also Fryns [F29]). Fra(X) carriers ofnormal intelligence with low fra(X) expression had atendency towards higher frequencies of late replicating
fra(X), whereas those with high fra(X} expression had
a tendency towards early replication of fra(X). Thesefindings are at variance with those recorded by Knollet al. [Kl8]: fra(X) carriers of normal intelligenceexpressing fra(X) in relatively high frequency showeda tendency towards an excess of late replicating fra(X)chromosomes as did the normal intelligent carriers
with a low frequency of fra(X).
53. The results of studies on fra(X) replication
patterns and their relationship to IQ do not allow
unambiguous statements to be made on whether Xinactivation plays an important role in determining
the intelligence of carriers of fra(X). Furthermore, as
Sutherland notes (see [09)). the use of BrdU labellingto study fra(X) expression is questionable, since thiscompound is known to inhibit fra(X) expression; latereplicating fra(X) chromosomes are less likely to
express the fragile site than early replicating ones,
leading to an apparent excess of early replicating
fra(X) chromosomes. Additionally, there is the questionof whether X-inactivation as seen in lymphocytes
reflects X-inactivation within the central nervous
system. an aspect more likely to be relevant tointellectual function.
54. The results of cytogenetic analyses of individuals
in 110 pedigrees of fra(X} syndrome presented by
Sherman et al. (S54] show that: (a) mentally retarded
males have a higher frequency of fra(X) positive cells
than mentally impaired females and the latter havehigher frequencies of these cells than normal females:(b) there is no effect of marker expression on IQ in
affected males. but there is a significant inverse
relationship between IQ and the expression of fra(X)in females: and (c) there is a small but significant
effect of age on fra(X) frequency (a decrease in fra(X)positive cells) in both males and females, but infemales it is restricted to those of normal intelligence,
retarded females showing no significant effect.
(iii)

Prevalence and mutation rate estimates

55. Currently, about 25% (58/243) of the X-linked
mutations listed in McKusick's catalogue [M21] leadto, or are associated with, mental retardation [09].
Herbst and Miller [H 14] estimated that the minimal

prevalence of non-specific X-linked mental retardationin British Colombia was 18.3 per 10~ males and thatabout one-half of this (9.2 per 10 4 males) was due tofra(X). The latter estimate was arrived at using
cytogenetic data (on the prevalence of males with
fra(X) chromosome among families with X-linked
mental retardation) published in the literature [JI I,
J 13, H30, T20]. They argued that, if the latter estimate
is valid. fra(X) would be, next to trisomy-21, the mostcommon chromosomal abnormality associated withmental retardation.
56. Again, based on literature data, Turner andJacobs [TI 9] arrived at a lower estimate of 4-5 per 10 4
males for the prevalence of fra(X)-linked mental
retardation. In a subsequent paper on segregation
analysis of data from I 10 families of fra(X) mental
retardation (using the programmes SERGAN and
POINTER developed by Morton and colleagues),
Sherman et al. [S54] found that: (a) there was a deficit
of at least 20% of affected males/ the most plausible
explanation for which was. in their opinion. incomplete
penetrance of the gene in some affected males, suchmales being indistinguishable from normal on both
psychometric and cytogenetic tests (cases of apparently
asymptomatic males transmitting the gene to theirdaughters have in fact been reported in the literature[C29. 026. Fl7. Fl9. JI2. M29. NI8. R29]); (b) the
penetrance of mental retardation in· carrier females
was about 30% and of mental impairment and/or
fra(X) expression. 56%: (c) there was no evidence forthe occurrence of sporadic cases among affected
males, i.e .. all the males had received the fra(X) genefrom their carrier mothers: and (d) assuming normal
segregation, the data were consistent with a prevalenceof fra(X}linked mental retardation in females of 4.1
per 10 4 (which is nearly the same as that for males}
and a mutation rate estimate in male germ cells of
7.2 10- 4 /locus/generation, or. in relation to germ cellsof both sexes. of 2.4 10- 4/locus/generation (theiranalysis provided no evidence for mutations in females).
57. In a further segregation analysis of the fra(X)syndrome in 96 pedigrees, Sherman et al. [S85] found
that: (a) the segregation pattern for any degree ofmental impairment was similar to that in the I 10
pedigrees discussed above: (b} for the combined data,
the best estimate of penetrance of mental impairment
was 79% in males and 35% in females: and (c) there
was little evidence for sporadic cases among affected
males.

(iv) Prevalence of fra(XJ among mentally retarded
individuals
58. Kahkonen et al. [K 10] made a cytogenetic studyof 150 male patients with mental retardation ofdsuch a deficit of affected males was not observed in the study ofFryns [F29]. In his study, 55 carrier females gave birth to 299 children,
171 males and 128 females. Thirteen boys died early in infancy andthe majority of them were said to be mentally subnormal. Of thetotal of 171 males, only 56 were mentally normal. The ratio of
mentally retarded (102) to mentally normal {56) males was 1.82 : I(even with the exclusion of the 13 boys who died).

37

unknown origin in Finland. Six of these patients (4%)
had the fragile-X: one of them had no family historyof mental retardation, four had one or more mentallyretarded first-degree relatives and one had a family
history suggestive of X-linked mental retardation. In
other similar studies, the proportion of those withfragile-X has varied from 2% in Australia [S28] to 6%
in Sweden [82] to 16% in South Africa [V3]. In theirreview of published cases of non-specific X-linked
mental retardation (n = 187 males), Tariverdian andWeck [T3] found that the fragile-X was present in a
majority ( 170) of cases. The range of lymphocytes
showing the fragile site. however, varied widely(3-61 %). The results with obligate female carriers havebeen disappointing. Of 85 such cases discussed by theabove authors, only 45 showed the fragile-X in 0.538% of cells.
59. In an earlier study on the prevalence of fra(X)positive males among an unselected series of severely
mentally retarded boys (IQ< 50) born during theyears 1959-1970 in the northern Swedish county ofVasterbotten, Blomquist et al. [B2] found that 6 out of
96. or 6%, were fra(X) positive (subsequently, one
more case was found). In an extension of this work toless severely mentally retarded children of both sexes,
Blomquist et al. [B25] recorded that 5 out of 110 boys
(4.5%) and none of 61 girls had the fra(X). Thus, thefra(X) was seen in 2.9% of the total series of 171
children. Relating the total number of severely andless severely mentally retarded fra(X) positive children
so far found (12) to the total number of births for theperiod 1959-1970 and alive at one year of age (40.871),
the authors estimated that the prevalence of fra(X)syndrome in that Swedish county was about l per
3000 new-borns.

60. Sutherland [S86] published the results of his
continuing population cytogenetic studies carried out
in Australia on unselected neo-nates, retardates inmental institutions, special schools for the retarded,
sheltered workshops for the mentally handicappedand in hospital referral patients. This paper also
incorporates the data from his earlier [S5] studies.
Among 3558 neo-nates, there were no cases of fra(X)and this was also true of those in sheltered workshops(0/128). Among institutionalized retardates, 1.6% of
the males (7/444) and none of the females (0/80) werefra(X) positive. In hospital referral patients. thefrequencies were 5.3% in males ( 13/2283) and 1.6% infemales (3/ 1890).

61. By far the highest frequency of fra(X) among
institutionalized retardates has been recorded in the
study of Fryns [F29], in which 57 of the 354 males(16.1%) and l of 30 females (3.3%) were fra(X)positive. These 58 patients were found to belong to
37 different families. These and other data bearing on
the prevalence of fra(X) are summarized in Table 9,which shows that the frequencies are generally higher
among males in mental institutions and in thosereferred for cytogenetic examinations than amongfemales.
38(v)

Prevalence offra(X) among autistic children

62. In 1970, Sivasankar [S87] reported an increase in
the rate of chromosomal breakage in leucocyte culturesfrom autistic children! Subsequently, Turner et al.
[T20] described the fragile-X syndrome in an autisticboy and this association was later reported by several
others [840, B41, 844, G37, G38, L21, L22]. In a
recent Swedish multicentre study, 102 cases (83 boys
and 19 girls) of infantile autism (age range, 2-24 years)
were examined for fra(X) [B39]. Thirteen of the
83 autistic boys (15.7%) had the fra(X) expressed in
1-70% of their lymphocytes, but none of the 19 girls
was fra(X) positive. Of the 13 boys who were fra(X)positive, I was classified as having normal intelligence(IQ> 85), 1 was subnormal (IQ= 70-84), 7 had mild
mental retardation (IQ= 50-69) and 4 were severely
mentally retarded (IQ= 0-49).

(c)

Au!Osomal Jo/ate-sensitive fragile sires:
prevalence estimates and effects

63. Sutherland's recent paper [S86] summarizes theresults of screening for folate-sensitive rare autosomal
fragile sites, including those he published earlier [SS].
those of Turner et al. [T3 I] and those of some studies
carried out in France [R48. G39, QI]. His own results
show that in unselected neo-nates. the frequency isquite low (5/3438 or 0.15%) and significantly higher
in all the other groups: referral patients, 0.38%(16/4173); institutionalized retardates. 0.95% (5/524);
those attending special schools, 1.0% (5/502); andthose in sheltered workshops, 1.6% (2/128). Of the
28 autosornal folate-sensitive fragile site ascertainments
in his laboratory. where both parents have beenstudied, the mother was the carrier parent 22 times
and only once was the father the carrier (in theremaining five instances neither parent could beshown to be a carrier). The figure for referral patients
cited above (0.38%) is higher than those reported byRaoul (0.14%, 11/8000) [R48]. Quack et al. (0.14%,
14/1 O,OOO)[Q I]. and Guichaoua et al. (0.21 %. 17/7786)[G39].
64. The results of Sutherland's other study [S6] on
the prevalence of BrdU-requiring fragile site at 10q25
in 1026 unselected neo-nates. 901 patients referred tofor chromosomal studies and 87 institutionalized
retardates showed no differences between these groups.
Apart from one homozygote (found in the group of
neo-nates), all the rest were heterozygotes for the!Oq25 site. The frequencies were as follows: neo-nates,
2.3% (24/ I 026); patients. 2.4% (22/901 ): and retardates.
3.4% (3/87).
65. The distamycin-requiring fragile sites at l 7p 12
and I6q22 are the ones about which least is known
from a population point of view. Although they weredetected in all the groups studied, it is only recently
eAutism is a form of behaviour and thinking observed in young
children, in which the child seems to concentrate upon himself or
herself without regard for reality. It often appears as excessive
shyness, fearfulness or aloofness and later as withdrawal andintrospection. It may be an early manifestation or part of achildhood type of schizophrenia.

that the conditions required for their expression havebeen delineated [S56]. In Sutherland's [S86] study, thefragile site at 17p 12 was seen in I out of 368 neo-nates(0.27%) and the one at 16q22 was not detected in thisgroup; 8 out of 491 patients had the 16q22 (1.6%) and1 in 491 had the 17pl2 (0.02%). Sanfilipo et al. [S87]induced the fragile site at 16q22 with 15 mg/I BrdU
added 7 h prior to harvest and found it in 4/155
institutionalized retardates (2.6%) and 14/ I 444 patients( 1.0%) referred for diagnostic cytogenetics.
66. Apart from the fragile site at Xq27. for which theevidence of association with one form of mental
retardation is strong, the question of whether otherfragile sites may be associated with adverse phenotypic effects is not entirely resolved. Sutherland and
Hinton [S51] recorded the presence of fra(12q13) in akindred involving six members of five generations. but
without clinical abnormalities. Giraud et al. [G24].
however, reported two cases of fra(l2ql3), one in aboy with severe mental retardation and the other inthe mother of a malformed infant with a normal
karyotype (no clinical details were given). The reports
of Donti et al. [025] and of Morie-Petrovic and Laca
[M28] show that the fra(l2q 13) may be associatedwith phenotypic abnormalities (round head. small
chin. low set ears. abnormally long fingers andconsiderable psychomotor retardation). The only difference between these cases is the presence of a
Robertsonian translocation (13q; 14q; familial) in thecase described by Morie-Petrovic and Laca (in addition to the fra(l2ql3}). In neither case was the fragilesite familial. Morie-Petrovic and Laca speculate thatthe phenotypic abnormalities may not be a co11sequence of the presence of the fragile site per se, but
could be due to a loss, in some cells. of a large partof 12q.

(d)Fragile sites and propensity to chromosome
breakage

67. There are indications that certain autosomal
fragile sites may predispose the chromosomes tobreakage [S52, C30]. Hecht and Hecht [H28, H29]
made a systematic study to explore this possibility.
From the data recorded in the New York State
Chromosomal Registry. they extracted information on
the location of break-points leading to constitutional
chromosomal anomalies (deletions, duplications, inversions and non-Robertsonian translocations) found inamniocentesis studies and in those on spontaneousabortions, still births and new-borns.
68. The break-points were individually comparedwith the location of 44 known fragile sites (S53].
Based on a haploid karyotype of 400 bands [16) andthe assumption that each band has the same probabilityof breakage, it would be expected that, by chancealone, 11.0% of the breaks would be coincident withthe fragile sites (44/400). In the rearrangements
recorded in the amniocentesis studies. there was a
total of 278 break-points of which 59 (21%) wereobserved to be in bands containing the fragile sites,
compared to an expectation of 11.0%. In thoserecorded in the abortions, still births and new-barns,

165 ( 18.5%) of the total of 894 break-points were
found in the bands with fragile sites. again a significant excess. compared with the expected 11.0%: inthis work, no break-points in rearrangements were
found in 8 bands containing the fragile sites (2q 11,
3pl4. 6q26. 9q32, 16pl2, 16q23, Xq26 and Xq27).
69. The total data with the separation of the breakpoints according to the origin of the rearrangements(maternal, paternal or unknown) are given in Table 10.
It is clear that these data are in line with the conceptthat certain fragile sites may predispose the chromosome;s to breakage and formation of rearrangements
in meiosis. The authors caution that in view of theheterogeneity of the data base from which thepertinent information was extracted. more evidence isrequired to provide convincing proof that fragile sites
are regions in the chromosomes predisposed to breakage in meiosis.
70. Braekeleer [B42] raised some objections on themethodology used by Hecht and Hecht [H28, H29] intheir analysis, the principal points being that: (a) the
latter authors considered only the total number ofbands; (b) all chromosome bands do not have thesame length; and (c) the chance of having a break in aband is dependent on its length. Therefore, it would
be more accurate to consider the probability of a
relationship between fragile sites and predisposition tochromosome breakage according to the relative lengthsof the bands. On this hypothesis, Braekeleer reanalysed the data of Hecht and Hecht using Monte
Carlo simulation methods. He found that there wasindeed a significant relationship between fragile sites
and rearrangements scored at amniocentesis as well asin spontaneous abortions, still births and new-horns,
but the levels of statistical significance were lower
than those of Hecht and Hecht. Braekeleer stressedthat further studies are needed to assess whether
fragile sites are predisposing to meiotic chromosome
breakage and structural rearrangements per se.
71. At the experimental level, Yunis and Soreng[Y 11] tested whether the expression of the common
fragile sites could be enhanced by specific culturing
regimes and whether at such sites there would be morechromosome breaks. Lymphocytes were grown inmodified Eagle's medium (MEM) Jacking in folic acidand thymidine (FTD medium). In one set of cultures,
FdU was added during the last 24 h in culture: and ina second set. FdU and caffeine were added (the latter
during the last 6 h in culture). Chromosome preparations were made by appropriate procedures to reveal
670-850 bands per haploid set. The results showed
that: (a) 51 common fragile sites could be delineated;!
(b) in the FTD + FdU + caffeine regime. there was a
10-fold enhancement (as a minimum) in the expressionof fragile sites over the FTD + FdU regime; (c) there
were more breaks in the FTD + FdU + caffeine group(519/100 cells) than in the other group (45.6/100
cells), most of the breaks expressed in the presence of
caffeine being represented in the 51 common fragile/The fragile sites were distributed in the different chromosomes asfollows (the number of sites given in parentheses): I (5). 2 (6). 3 (3).
4 (2), 5 (I), 6 (3), 7 (5), 8 (3), 9 (2), 10 (3), 11 (4), 12 (I), 13 (2),
14 (2), 16 (2), 17 (I), 18 (2), 20 (I), 22 (I) and X (2).

39

sites (74-82%) and the rest being scattered among theother bands (frequently in Giemsa-negative ones):(d) the relative distribution of breaks among the
51 fragile sites was similar in the presence or absenceof caffeine, indicating that the expression of thesefragile sites is not elicited but only enhanced bycaffeine; and (e) triradial configurations, typicallyobserved at the rare (heritable) fragile sites were also
observed at sites 3p 14.2, 6q25.3. 8q24. I. 16q22.l.
16q23.2 and Xq22.I.

(e)

Fragile sites and break-poinrs of certain specificchromosomal changes in cancer

72. Evidence is rapidly accumulating [843, LI I, Y4.
Y 11] to suggest that some non-random chromosomalchanges involved in certain cancers have break-points
that coincide with the fragile sites (Table 11). Six otherchromosomes (2, 7, 12, 17 and 20) that are consistently
involved in rearrangements or numerical changes inmalignant cells also contain fragile sites. although
localized to different chromosome bands. In addition,
10 of the 12 amosomes carry both a cellular oncogene
and a fragile site, but they coincide only in the case ofchromosome 8 (fragile site and c-mos at 8q22). The
cellular oncogenes of chromosomes 6. 8. 9 and 11 arein the same regions as the cancer-related break-points
with c-myb on 6q I 5-q24, c-myc on 8q24, c-abl on
9q34 and c-rasHI on 11 p 15. On the basis of all thesefindings, it has been suggested [843, LI I, Y4, YI I]
that fragile sites may act as predisposition factors forcertain chromosomal rearrangements in human neoplasias and that some of them may represent oncogenic
sites.

D.

CONG EN IT AL ANOMALIES

73. In its 1982 report, UNSCEAR used a prevalence
figure of 430/10 4 live births for congenital anomalies(CAs) in man. one that was estimated by Trimble andDoughty [T9] for the population of British Columbia,
Canada. Although the data of Myrianthopoulos andChung [M 12] for the United States of America. andsome of the data of Czeizel [Cll, CI2] for Hungary,were mentioned, no systematic comparisons weremade between these three sets of data. Czeizel andSankaranarayanan [CJ 3] have now published extensive data on the prevalence of CAs in Hungary andhave also made the comparisons mentioned above
with the aims of: (a) examining the extent to which theprevalences of different CAs in Hungary agree withthose in the other two studies: and (b) using the
Hungarian prevalences to estimate detriment due tospontaneously arising CAs. Since such comparisons
are useful both in the context of assessing the load due
to spontaneously arising CAs and of estimating
possible risks due to radiation exposures. the general
problems inherent in such comparisons. the nature of
the three sets of data and the kinds of comparisons
made by these authors are summarized below.
74. The difficulties involved in comparing prevalencesof CAs in different parts of the world have been
discussed in a number of publications (e.g., [C25, C32,

40

L20, L23, N35. S84, W35]: see also section J.C. in
Annex C to the present report). Among the various
factors that are responsible for the differences inprevalences that have been recorded in the different
studies are: the source of data (epidemiological studies,registries). study design (prospective. retrospective).
the efficiency of ascertainment (dictated by the size ofthe material. the available expertise. whether thescreening included still births and live births. whether
autopsy was performed on stillborn babies, the periodof follow-up, if any, in the case of live births),
definition, diagnostic criteria and classification schemes
adopted, geographical, regional, ethnic and other
differences. It is thus obvious that an average figure(from different studies), either for the total prevalenceor for individual conditions. is unlikely to reflect thesituation in a global context and is admittedly crude.
Such a figure will undoubtedly mask the existence ofreal differences-sometimes large-between studies orcountries, for which there may be a genetic basis.
75. The large-scale collaborative study of Stevenson
et al. [S84] is illustrative in this regard. In this study,
which involved a series of consecutive births in
24 centres in 16 countries and the outcomes of 421,781
pregnancies, the summed frequency of all major CAs
was 127/10 4 total births with a range between 31/10 4
and 225/ J04 in the different centres located in various
parts of the world. Among the major conclusions thatemerged from this study are: (a) CAs recognized atbirth are predominantly of types that are suspected tohave complex genotypic as well as environmental
contributions to their aetiology; (b) the frequency of
the CAs present at birth, if all children were followed
up for a few years. would be increased by about 50%,although the numbers missed at birth depend on thetype of CA and vary from zero upwards; and (c) there
are large and sometimes real geographical, regionaland ethnic differences in the estimates of several
specific CAs (e.g .. neural tube defects, cleft lip with orwithout cleft palate, hip dislocation or dysplasia).
These findings lend support to many views based on
previous evidence. have been amply documented in a
number of subsequent studies and need to be taken
into account when international comparisons ofprevalences are made.
76. When comparing the Hungarian data with thosefrom the United States and British Columbia studies.
it is worth pointing out that the authors' choice of
these two sets of data for comparisons were dictatedby the following specific considerations: first. theUnited States data come from a prospective study andrepresent the results of a co-operative endeavour of
12 major medical centres (the so-called Collaborative
Perinatal Project) which was designed from the outset
to ensure ''nearly complete ascertainment and accurate
assessment of the malformations in the new-born"[M 12]: consequently, these data were expected toprovide a perspective of the situation if the ascertainment efficiency were close to 100%. Secondly, theBritish Columbia data with certain modifications now
constitute a major basis for the evaluation of genetic
radiation hazards in man; it is therefore instructive
to inquire whether major differences between theHungarian and British Columbia data exist, particularly if the Hungarian data could be used as a basis
for detriment calculations (see chapter VIII).
77. In the study from the United States, the analyses
were based on data from about 56,000 pregnant
women who had been followed up from the first few
months of pregnancy, and 52,257 deliveries (24. 153
whites, 25,126 blacks and 3.978 other ethnic groups)
with the children having been followed up throughone year of age. Myrianthopoulos and Chung did not
use the scheme recommended in the Manual on
International Classification of Diseases (hereafter tobe referred to as ICD) [15]. The anomalies wereclassified into major and minor and, within them,single and multiple. As Myrianthopoulos and Chungstated. " ... the decision to assign a malformation to
the major or minor group, though it followed certain
guidelines. was in the last analysis. arbitrary and wasbased on expert advice as well as our experience andintuition, rather than any one criterion or set ofcriteria ... Multiple malformations were defined as
those ... found in association with malformation of
the same or any other system or type ... "78. The Hungarian data are more extensive and are
based on a total of 1,992,773 births from 1970 to 1981.
In Hungary, all deliveries take place in hospitals andthe reporting of CAs diagnosed through the first yearof life has been mandatory since 1962. Likewise.
autopsies of all dead infants are also obligatory. The
data derive from two principal sources: populationbased epidemiological surveys for specific CAs andthe Hungarian Congenital Malformation Registry(HCMR). The epidemiological studies were initiatedin the late 1960s and used all possible sources ofascertainment. Data pertaining to about 30% of theconditions (i.e .. ICD entries) to be discussed later(which correspond to about 80% of the case-load)come from this source. The completeness of ascertainment for these is close to 100%.
79. The HCMR has been operational since 1970 andhas multiple sources of ascertainment. For obvious
reasons. the data pertaining to the earlier years(particularly 1970) are likely to be underestimates. The
five years between 1977 and 1981 represent those of
most reliable birth prevalences. a result of increasing
diagnostic expertise and consequently more complete
reporting of cases to the HCMR. The general completeness of ascertainment for these five years is on theorder of 80%, although there are significant differencesin ascertainment efficiency, depending on the kinds ofCAs. The HCMR is the source for about 70% of theconditions (i.e., !CD entries) accounting for about
20% of the cases, for which no epidemiological data
are available. It is worth mentioning that both theepidemiological studies and the maintenance of the
HCMR (and analysis of the data reported to it) are
carried out by the Department of Human Geneticsof the National Institute of Hygiene, Budapest, andthus they complement each other. Furthermore, theepidemiological studies provide a means of controllingthe completeness of the reporting to the HCMR andof introducing appropriate corrections when necessary.

80. In the Hungarian work, all the CAs are registeredusing the ICD code numbers. In the WHO manual,
the CAs are listed in Chapter XIV (entries 740-759),
which includes chromosomal anomalies. but excludes
congenital inguinal and umbilical hernias (!CD numbers
550 and 553) and congenital tumours (ICD numbers
227-228). Many teratologists, however, consider thelatter three entities also as CAs. The CAs have beenclassified into two groups: isolated and multiple.
Isolated CAs are those structural defects that can betraced back to one localized error in morphogenesis.
In contrast, the multiple CAs represent the concurrenceof two or more different (i.e .. differently localized
errors in morphogenesis) major CAs in the same
individual. The Hungarian data presented in the paperof Czeizel and Sankaranarayanan refer mostly to theisolated group. although some multiple ones have also
been included.
81. The British Columbia data derive from twosources: (a) the actual number of cases of congenitally
malformed children born in British Columbia andrecorded in the Registry for Handicapped Children
and Adults: and (b) non-registered cases ascertained
through a province-wide surveillance system of CAs.
For this study period. there were 756,304 live births.
The ICD system was used ro classify the CAs.
82. Table 12 represents a comparison of birthprevalences recorded in the United States, Hungarianand British Columbia studies. The following generalpoints should be made: (a) in the Hungarian andBritish Columbia studies, the prevalence figures are
based on the number of affected individuals, whereas
in the other study these figures refer to the number ofCAs; (b) the prevalence figures for the United States
are those extracted bv Czeizel and Sankaranarayanan
from the large appendix table of Myrianthopoulos andChung, which in their view would correspond to thoseof their isolated CAs (the actual numbers of CAs-andthus not of affected individuals-on which the estimatedprevalences given in column 3 are based. are given inparentheses): and (c) it was considered useful toseparate congenital dislocation of the hip from thegroup of musculoskeletal and skeletal anomalies (!CD754-756) and present totals including and excluding
the above condition, the reason being that thiscondition has a very high birth prevalence in Hungary(257.7/10 4 ). In what follows. unless otherwise stated,
the term prevalence will be used to denote births
affected with CA (and not the number of CAs).
83. Considering first the overall prevalence figures(i.e .. for conditions covered by !CD entries 740-759,
550, 553 and 227-228). it can be seen that theprevalence of 849.2/104 total births for the UnitedStates is higher than that for Hungary (735.9/10 4 totalbirths) by about 13%. Such a comparison between live
birth prevalences in Hungary and British Columbia is
not feasible since Trimble and Doughty's corrected
figure of 428/10 4 live births is only applicable to ICD
entries 740-759.
84. If the comparisons are restricted to ICD entries
740-759 (and thus include the congenital dislocation of
the hip). the estimated prevalences in the United
41

States range from 704.0/10 4 to 846. l/ 10 4 total births,K
which is again higher than that for Hungary (614.9/10 4
total births). It can also be noted that the live birthprevalence of 597.4/10 4 for Hungary is also higher
than that for British Columbia (428/10 4 births).
85. The exclusion of the congenital dislocation of
the hip (again limiting consideration to ICD entries
740-759) makes only a marginal difference to the total
prevalence in the United States (the range is now from632.1/10 4 to 775.0/10 4 total birthsh). However, the
Hungarian figure is substantially reduced to 357.2/10 4
total births. For British Columbia a comparable
calculation cannot be reliably made; however, if thepercentage contribution of this condition in thecorrected estimate of 428/ 10 4 is the same as that basedon the actual observations during the period 1952-1972(the latter is 6.3% or 1063/16, 791 ), then the prevalence
would be marginally reduced from 428/ I0 4 to 401.8/ 10 4live births. For Hungary, the reduction is from 597.4
to 339.7/10 4 live births.
86. A close inspection of columns 3 and 5 ofTable 12 (United States versus Hungary comparisons;taking into account the fact that the United States
figures refer to the number of CAs and those for
Hungary to the number of affected births) shows thatthe prevalences are fairly similar for anencephaly andspina bifida. congenital cardiovascular malformations,anomalies of the urogenital system and for chromosomal anomalies (most of the cases of chromosomal
anomalies in the above two studies, as well as that ofBritish Columbia. were trisomy-21). The agreement isless good for other malformations. such as cleft lip
with or without cleft palate, anomalies of the eye. ear,face and neck, anomalies of the respiratory system
and anomalies of the digestive system, the figuresfrom the United States being generally higher thanthose for Hungary. Particularly noteworthy is thefinding that the prevalence of musculoskeletal andskeletal anomalies in the United States (even with theexclusion of the congenital dislocation of the hip) is
about 6 times, and the prevalence of integumental
anomalies more than 10 times. those in Hungary.
Such a condition-by-condition comparison between
the Hungarian and British Columbia figures is inappropriate, since the British Columbia data summarized in Table 12 for the different conditions refer
to uncorrected estimates.
87. The principal conclusions that emerge from thesecomparisons are the following: (a) the total estimatedn'he estimate of 704.0 is arrived at by assuming that the numberof cases affected with hernias and congenital tumours is the same as
the number of these CAs (= 350) and that 1051 - 350 = 1701 cases
had 2330 CAs or 1.37 CAs per affected case. Thus, 964.5/1.37 = 704.0.
The value of 846.1 is arrived at by assuming that 2051 affectedbinhs had 2330 CAs or roughly 1.14 per affected birth. Thus thenumber of affected births is= 964.5/1.14 = 846.1.
hThe assumptions underlying these calculations are the same as
those explained in footnote g. The estimate of 632.1 is arrived at byassuming that the number of births with congenital dislocation of
the hip, hernias and congenital tumours is the same as the total
number of 1hese CAs (i.e., 177 + 350 = 527) and that a iota! of
2051 - 527 = 1524 births had 2153 CAs or roughly 1.41 CAs/
affec1ed birth. Thus, 891.2/1.41 = 632.1. To arrive at the figure of
775.0, it is assumed that the affected births is 2051 - 177 = 1874.
These had 2153 CAs or 1.15/affected case. Thus, 891.2/1.15 = 775.

42

birth prevalences of CAs (!CD entries 740-759. 550,
553, 227-228) range from 849.2/ 10 4 total births in the
United States to 735.9/10 4 total births in Hungary;with the exclusion of the hernias (ICD 550 and 553)
and congenital tumours (ICD 227-228), the estimatedfigures range from 704.0/ 10 4 to 846.1/ 10 4 total births
in the United States to 614.9/10 4 total births in
Hungary: (b) in live births. the CAs defined by the
ICD entries 740-759 have a prevalence ranging from428/10 4 in British Columbia to 597.4/10 4 in Hungary,but over 40% of this total for Hungary is due tocongenital dislocation of the hip, a condition known
to have a high prevalence in that country: and (c) in
principle. it is possible to have similar or nearly
similar total prevalences in different countries. but therelative contributions of the different CAs to the total
could be different: this fact should be clearlv borne inmind in attempting to arrive at average values
applicable in a global context.
88. In this document, the Committee decided to use
the Hungarian prevalence figure for CAs of 600/ 10 4live births for two principal reasons: (a) this figure per
se does not seem to be an unrealistic average
considering the three sets of data reviewed in thepreceding paragraphs: and (b) only on the basis of the
Hungarian data. it has been possible to arrive atestimates of detriment for CAs and this has been oneof the principal aims of the Committee. The Committee is aware of the fact that congenital dislocationof the hip has a very high birth prevalence in
Hungary; however, as discussed in chapter VIII, thecontribution of this condition to real detriment(expressed as lost or actually impaired life) is so smallthat it does not distort the overall picture of detrimentassociated with CAs as a whole.

E. OTHER MULTIFACTORIAL DISORDERS(EXCLUDING CONGENITAL ANOMALIES)
89. On the basis of their data on the prevalenceof Mendelian, chromosomal and other disorders in
British Columbia, Canada, Trimble and Doughty [T9]estimated that the total prevalence of multifactorial
disorders other than congenital anomalies was on theorder of 4 70/ I 04 live births. As may be recalled, theirestimate was based on a follow-up of individuals frombirth to age 21. Since manv of these multifactorial
disorders are not scored at· birth and have variableages of onset with manifestation late in life, it isobvious that their estimate is applicable only to thosethat manifest themselves before age 21 and thisunderestimates their prevalence in the population as awhole.
90. The preliminary results of Czeizel et al. (unpublished. 1986) obtained from an extensive analysisof data in several epidemiological studies carried out
in Hungary and in other parts of the world strongly
support the thesis that the population prevalence of
these multifactorial disorders is at least about 6000/ 10 4 •Some of the essential aspects of their data and analysis
are briefly summarized below.
91. Since a compilation of an exhaustive list of these
disorders with adequate and reliable information onprevalences, ages of onset, etc., would be a formidable
task for several reasons, they excluded from consideration: (a) those disorders whose population prevalences
are equal to or less than 1/104 ; (b) pre-senile andsenile dementias and other cerebral degenerative
disorders (such as Pick, Creutzfeldt-Jacob and Alzheimer diseases with a high prevalence in the agegroup of 70 and above): (c) refractory errors of the eye(myopia, hypermetropia. astigmatism). strabismus,
and tooth anomalies. although again. many of these
disorders have a significant genetic component in theiraetiology (the decision to exclude them stems from thefact that in spite of their high population prevalences,possibilities for visual or dental corrections exist inalmost all countries and after such corrections, life
impairment is minimal): (d) mental retardation, blindness and deaf-mutism. not only because these conditions are aetiologically or clinically quite heterogeneous. but also because they merit a separate anddetailed analysis; (e) those disorders for which theevidence on the genetic contribution is either inadequate or equivocal (e.g.. pernicious anaemia,
migraine. Parkinson disease. neurosis, alcoholism);(f) those with too many different clinical end-points
such as atherosclerosis. which is associated withmyocardial infarction, senile dementia. cerebral
haemorrhage, hypertension and obesity: (g) infectious
diseases; and (h) all cancers, in spite of evidence
suggesting that there is some genetic component intheir aetiology (this exclusion was made because thesubject is very complex and deserves to be treated in
its own right).
92. The disorders included in their preliminary compilation together with estimates of their prevalences in
Hungary and some of their important epidemiologicalfeatures are summarized in Table 13. It should be
stressed that the disorders included in this selected list
are by no means homogeneous either clinically oraetiologically. Nevertheless. the compilation provides
some idea of the kinds of disorders that fall under the
category being considered in this section. It is worth
mentioning that the figures were standardized for the
study population. Thus. for instance. when only thedata for the adult population (above age 14) wereavailable. the prevalence figure was reduced by 21%
since the population in the 0-14 age range represented
21 % of the total population. The wide range of figures
published in the literature for other countries no
doubt reflects the scope. size and nature of thedifferent studies. the age-groups included in theanalysis and the variations in diagnosis, time. locationand the degree of ascertainment.
93. It is obvious that: (a) the population prevalenceof around 6000/104 (considering the fact that several
disorders have been excluded) is much higher thanthat reported in the British Columbia survey, which
only included, however, those multifactorial disorders(other than congenital anomalies) that become manifest before the age of 21: (b) the Hungarian prevalence
figures for individual conditions are not out of linewith those published for other parts of the world: and(c) the actual frequency of affected individuals will beless than 60% because many of them will suffer from
more than one of these disorders.

F.

SUMMARY AND CONCLUSIONS

94. New data on the prevalence of certain Mendeliandisorders in man that have become available since thepublication of the 1982 UNSCEAR report do not
warrant any revision of the Committee's earlier
assessments in this regard. The earlier findings of
Dubinin and Altukhov that the frequencies of rareelectrophoretic variants (at over 20 genetic loci coding
for the synthesis of enzyme proteins and erythrocyte
antigens) are significantly higher in premature infants
and in those with multiple congenital anomalies, could
not be confirmed in an independent study of a similar
nature carried out by Neel and Mohrenweiser.
95. A re-analysis of data on the contribution ofchromosomal anomalies to spontaneous abortions
and still births shows that about 40% of spontaneousabortions and 5. 7% of still births are associated withchromosomal anomalies. The figure of 40% spontaneous abortions pertains to those that occur inweeks 5-28 of pregnancy (UNSCEAR's figure of 50%
discussed in its earlier reports allows for developmental arrests that may occur before the fifth week ofgestation). The currently available information suggeststhat about 5% of recognized conceptions are associatedwith chromosomal anomalies.
96. Studies on the parental ongm of the extra
chromosome in trisomies in spontaneous abortions
show that. in most cases, the trisomies result from
non-disjunction in maternal meiosis-I.
97. New data have been published on the effect ofmaternal age on chromosomally normal and abnormal
spontaneous abortions. These show that the overall
rate of spontaneous abortions is relatively constantfrom age 20 to 30, but, beginning in the mid-30s. there
is a marked increase and, by 40 years of age, morethan 25% of all registered pregnancies terminate inspontaneous abortions. In chromosomally abnormal
spontaneous abortions, trisomies accounted for 20%
of spontaneous abortions involving women under
25 years of age. but approximately 33% for those in
the 30-35-year age group and no Jess that 67% in
women aged 40 years or more.
98. The rates of numerical chromosomal anomalies
in pre-natal (amniocentesis) cytogenetic studies havebeen determined and compared with maternal-age
adjusted rates in live births. The results show that thefrequencies of XXX and XXY conditions increase
with maternal age while that for XYY shows a slightdecrease with maternal age. The rates of trisomy-21
and for trisomy-18 in Jive births are about 20% and70%. respectively. lower than the corresponding ones
at amniocentesis. Another cytogenetic investigation
showed that the prevalence of balanced structural
rearrangements at amniocentesis is higher than in
new-borns and that the mutation rate estimate (basedon de novo balanced rearrangements) of 4.3 10- 4;
gamete/generation calculated on the basis of thesedata is higher than that of 1.88 10--i;gamete/generation
derived earlier on the basis of data from new-born
surveys.
43

99. Recent results from new-born cytogenetic surveys
using banding methods in Norway and in Denmark,despite the relatively small sample sizes involved.
support the view that in the earlier surveys. which did
not use chromosome banding methods, the frequenciesof reciprocal translocations and inversions had been
underestimated. These new data suggest that the totalbirth prevalence of chromosomal anomalies may be as
high as 2%, but 30-50% of this frequency is made up
of autosomal inversions, primarily of chromosome 9,that are regarded as variants rather than anomalies
per se.
100. The prevalence of chromosomal anomalies inmentally retarded or otherwise handicapped individuals
and in those with mental retardation and one or morecongenital anomalies is higher (8 to 20%) than in newborns. Similarly. in subfertile males the frequency ofchromosomal anomalies is also higher, with XXY
being the major contributor. Furthermore, there is aclear correlation between sex-chromosomal anomalies
and azoospermia. the prevalence of these anomalies
being much higher among azoospermic males than in
new-borns (XXY. JOO-fold higher; XX males. 160-foldhigher).
10 I. The results of further studies on direct analysisof the chromosome constitution of human spermatozoa(after fertilization of hamster eggs in vitro) have been
published. There is a high degree of inter-individual
variation, the frequencies of chromosomal anomalies
ranging from 0-28% in the different males studied.
The anomalies detected include chromatid breaks,
chromosome breaks, chromatid exchanges, acentric
fragments. hypo-ploidy and hyper-ploidy. In cancer
patients who underwent radiotherapy treatments, thesefrequencies were higher than before radiotherapy.
102. Considerable work has been carried out on
fragile sites in human chromosomes during the last 2-3
years. The fragile sites arc currently classified into two
broad groups, the rare and the common or constitutive.
Among the rare fragile sites are 16 folate-sensitive sites(the fragile site on the long arm of the X associatedwith mental retardation and macro-orchidism is included in this group), 2 distamycin-A-inducible sites,
and I BrdU-requiring site. Among the common ones are:
6 aphidicolin-inducible sites, 2 azacytidine-inducible
sites, and 3 BrdU-inducible ones. In addition to thesites that have been confirmed, at least 7 rare fragile
sites and 17 common ones are grouped under the
category of possible fragile sites needing further
confirmation.
103. The fragile site on the X-chromosome that is
associated with macro-orchidism and one form of Xlinked mental retardation has now been localized toband Xq27.3.
104. Most fragile-X carrier females are not mentallyretarded: there is an inverse relationship between thefrequency of fragile-X expression in lymphocytes andIQ (e.g .. mildly retarded females have higher frequencies offragile-X expression than their intellectuallynormal counterparts). An inverse relationship between

44

fragile-X expression and IQ is also found in affected
males, but it is less pronounced than in carrier females
or not demonstrable at all.
105. There is also an inverse relationship between
age and the frequency of fragile-X expression inmentally normal heterozygotcs (older females havelower frequencies of fragile-X expression) and retarded
carriers show higher frequencies at all ages.
106. The mentally retarded carrier females appear tohave higher frequencies of early replicating fragile-X
active X-chromosome than their mentally normal
counterparts. but this relationship needs further study.
107. Analysis of the available data on the prevalenceof non-specific. X-linked mental retardation in males
suggests a rate of about 18.3/10 4 males: about onehalf of this is due to fragile-X, which makes the latterthe second major contributor to mental retardation,
after trisomy-21. A more recent estimate of theprevalence of fragile-X associated mental retardationis 4-5/10 4 males. Genetic analysis of 110 fragile-X
pedigrees allows a mutation rate estimate of about
2.4 10- 4/Jocus/generation (both sexes considered).
108. The results so far available from population
cytogenetic studies show that the prevalence of fragileX is very low in unselected neo-nates (in fact, nofragile-X positive case has been detected in 3558 neonates of both sexes screened) but higher in institutionalized retardates, in those attending special schools forthe retarded and in hospital referral patients; theprevalence in these last groups ranges from 1.6 to
16. l %. obviously depending on the severity of theretarded individuals included in the different studies.
Likewise, the limited data on autosomal folatesensitive rare fragile sites suggest a low prevalence in
unselected neo-nates (0.15%) and significantly higher
ones in hospital referral patients, in children attending
special schools and in institutionalized retardates(range from 0.38 to 1.6%). Data on other rare fragile
sites (i.e .. distamycin-inducible and BrdU-requiring
sites) are Jess extensive.
109. There are reports in support of the working
hypothesis that autism may be associated with thepresence of the fragile-X chromosome. There are noconclusive data demonstrating that fragile sites (otherthan the Xq27) may be associated with adverse
phenotypic effects such as congenital anomalies.
110. Data have been published that support thethesis that certain fragile sites may predispose thechromosomes carrying them to spontaneous breakage(visualized under specific lymphocyte culture conditions) or may be associated with the break-points inconstitutional chromosome rearrangements (seen in
spontaneous abortions. still births and in new-horns).
There is also some evidence suggesting that nonrandom chromosomal changes associated with certain
specific cancers may have break-points that coincide
with, or are in proximity to. the fragile sites and thatsome of the latter may represent oncogenic sites.
However. further data are needed to validate thesefindings.

111. The birth prevalence of isolated and multiple
congenital anomalies in man has been re-assessed bycomparing the estimates made in the British Columbia
study with those of other studies in Hungary and theUnited States. The total estimated birth prevalence of
these anomalies (included in chapter XIV of theInternational Classification of Diseases and conditions
such as inguinal and umbilical hernias and congenital
tumours not included in the above chapter) range
from 849.2/10 4 total births in the United States to735.9/ 10 4 total births in Hungary: with the exclusionof hernias and congenital tumours, the estimatedfigures range from 704.0/ I 0 4 to 846.1/ 10 4 total births
in the United States to 614.9/10 4 rotal births in
Hungary. In live births. the congenital anomalies
included in chapter XIV of the Manual on theInternational Classification of Diseases alone have aprevalence ranging from 428/10 4 in British Columbia
to 597.4/10 4 in the other series. while over 40% of thetotal for Hungary is due to congenital dislocation of
the hip, a condition known to have a high prevalencein that country.
112. Considering the three sets of data. the Committee concluded the Hungarian prevalence figure forcongenital anomalies of about 600/10 4 Jive births is areasonable average.
113. A similar, but preliminary re-assessment of the
population prevalence of other multifactorial disorders in man has been made. In this work, only
conditions that have a population prevalence of atleast 1/104 have been included. Excluded from consideration were those conditions with high prevalencesin age groups of 70 and above. refractory errors of theeve, tooth anomalies, mental retardation, blindness
a~d deaf-mutism, those conditions with a multiplicity
of clinical end-points. disorders for which the evidenceof genetic contribution is either inadequate or equivocal. infectious diseases and cancers. The overall
prevalence (of about 25 entities) is on the order of
6000/10 4 individuals for Hungary. This figure is
within the range reported in the literature for othercountries but is an order of magnitude higher thanthat estimated in British Columbia (470/10 4 ). It
should be stressed, however, that in the latter study,
only those mullifactorial disorders that become manifest before the age of 21 were included.

THE APPLICATION OF RECOMBINANT
DNA TECHNOLOGY TO THE ANALYSIS OF
THE HUMAN GENOME AND GENETICDISEASEII.

114. The latest update [M57) of McKusick's 1983
Compendium [M21] contains details of 1789 conditions, mostly diseases or pathological states determined at distinct gene loci. which are well documented
as being inherited in a Mendelian manner. The
breakdown is as follows: 1059 autosomal dominants,
610 autosomal recessives and 120 X-Iinked ones. In
addition. a further 1886 conditions are included (971
autosomal dominants, 774 autosomal recessives and

14 I X-linked ones) where the evidence of Mendelian
inheritance is not complete. Apart from the fact thatthe total number of loci so far identified is a verysmall proportion of the total number of genes in man(variously estimated to be between 50,000 and 100,000;
see [ M2 I)), only about 450 have been mapped to
specific chromosomes and the molecular basis fordisease is known only in a small proportion of these
conditions [M24).
115. The advent of recombinant DNA technology,
one of molecular biology's most powerful tools, is
now rapidly changing this situation. The applicationof these techniques to the human genome during thelast few years is providing new opportunities forincreasing the speed and precision of gene mapping,for defining the nature of the genetic lesions involved,
for analysis of the action of specific genes in healthand disease. including cancer, and for formulating
new approaches in the management of heritable
disorders. The potential, current achievements andfuture prospects of recombinant DNA technology
have already been reviewed (e.g .. [BJ6-Bl8, DJ5, Ell.
EIS. Jl4. M58. Rl6, WJ7)). The present section is
focused on some of the principal developments thathave resulted from the use of recombinant DNA
applications to human disease. The remarkable insights
into oncogenic transformation that have emerged
through the use of this technology are considered in alater chapter.

A.

GENERAL PRINCIPLES

116. The British Genetic Manipulation Advisory
Group (see [M58]) defined recombinant DNA technology as follows: "the formation of new combinations of heritable material bv the insertion of nucleic
acid molecules. produced by· whatever means outside
the cell, into any virus, bacterial plasmid or othervector system so as to allow their amplification andthen incorporation into a host organism in which they
do not naturallv occur but in which thev are capableof continued pr~pagation". The major t;chniques andtheir biochemical and enzymological aspects havebeen extensively discussed in a number of publications(e.g., [DI5, Ell. R50. S39. W8-WIO, W40]). The
actual recombinant part of the technology. whereby
new combinations of genetic material are generated, isrelatively well defined and can be considered underfour main headings: (a) the generation of DNA
fragments using, for example. restriction endonucleases;(b) the incorporation of these fragments into asuitable vector: (c) the introduction of the vector intoa particular host organism. which is then grown in
culture to produce clones with multiple copies of anincorporated DNA fragment: and (d) the selection andharvesting of clones that contain a specific DNA
fragment.
I.

Production of DNA fragments: the use ofrestriction endonucleases

117. Although DNA can be reduced to fragments bymechanical shearing, the process is random because45there is no way of ensuring that a particular DNA
sequence is isolated. The discovery in 1970, by Smithand Wilcox [S91] and Kelly and Smith [K38], of agroup of enzymes that occur in microorganisms(referred to as class 11 restriction endonucleases) andthat were found to cleave the DNA at sequencespecific sites, therefore, represented a major milestone
in recombinant DNA technology (see also [M59]). In
general, the target sequences are composed of particular tetra-, penta-, hexa-, or hepta-nucleotides. At
present, well over 500 different restriction enzymes,
with at least 100 different recognition specificities, are
known [FIO, K37, Rl 7]. Each enzyme is designatedaccording to the organism from which it is derived(for instance. EcoRI is an enzyme obtained from
Escherichia coli strain R and was the first such
enzyme isolated). Some enzymes may cleave the samesite but are obtained from different organisms (e.g.,
HindIII and Hsu!) or recognize the same sequence butcleave at different sites within the sequence (Xmal andSmal). Table 14 provides some illustrative examples of
the commonly used restriction enzymes and their
specificities.
118. Because of the complementary base-pairing in
the DNA [adenine (A) with thymine (T) and guanine(G) with cytosine (C)]. restriction endonucleases produce double-strand breaks. For instance, the enzyme
EcoRI specifically clea\es the DNA between the bases
G and A in the sequence -GAATTC- producing
staggered ends;
5' - G : A A T T

3' - C

L--------,

C - 3'T T A A : G - 5'

-G
-CTTA

I
AATTCG-The staggered ends with complementary bases arecalled sticky or cohesive ends. Other enzymes, however, produce ''blunt-ended" fragments, e.g., Smal.
5' - CCC
3'- G G G
-CCC
-GGG

G G G- 3'
CCC - 5'GGGCCC-The different types of ends produced by restncuon
enzymes have important implications in the strategy
subsequently used to join two DNA molecules together.
2.

Incorporation of the DNA fragments
into a suitable ~·ector

119. When a restriction enzyme produces sticky
ends, as in the case of EcoRI, and is used on both the
DNA to be incorporated into a vector (foreign DNA)as well as on the DNA of the vector itself, the cohesive
ends will come together and be held by hydrogenbonding between the corriplementary bases. The twomolecules can then be sealed (ligated) and stabilized
by the joining enzyme, DNA ligase. There are several
types of ligases and some (e.g .. T4 DNA ligase) will
actually link together DNA molecules with blunt ends.
The choice of a particular restriction enzyme and46

ON A joining strategy depends on a number of factors.
In general. preference is given to an enzyme that has asingle site on both the foreign and vector DNAs andthat is readily available and relatively inexpensive.
120. The vectors used for cloning are of different
types. Plasmids, bacteriophage lambda derivatives.
bacteriophage M 13 and cosmids are the most commonly used ones to clone fragments of foreign DNA
and propagate them in E. coli. Despite differences insize and structure, these four types of vectors share thefollowing properties: (a) they can replicate autonomously in E. coli, even when joined covalently to aforeign DNA fragment: (b) they can be easily separated
from bacterial nucleic acids and purified; and (c) they
contain regions of DNA that are not essential for
propagation in bacteria: foreign DNA inserted into
these regions is replicated and propagated as if it werea normal component of the vector [M32].
121. Plasmids. Plasmids are extrachromosomal genetic
elements found in a variety of bacterial (and yeast)
species. They are small circular molecules of doublestranded DNA that range in size from I kb (kilobase)to greater than 200 kb. They replicate as independent
units as the host cell proliferates. 1 Plasmids often
contain genes coding for enzymes that. under certain
circumstances. are advantageous to the host bacterium.
Among the phenotypes conferred by the different
plasmids are: resistance to antibiotics. production of
antibiotics and production of colicin. Under normal
conditions, many plasmids are transmitted to newhosts by a process similar to bacterial conjugation. In
the laboratory, they can be transferred to bacteria byan artificial process known as transformation (see next
section).
122. Maniatis et al. [M32] list a number of properties
that a plasmid must have to be useful as a cloning
vector: (a) it should be relatively small and should
replicate in a relaxed fashion (to ensure high copy
numbers): (b) it should carry one or more selectable
markers to allow identification of transformants andto maintain the plasmid in the bacterial population:and (c) it should contain a single restriction site forone or more restriction enzymes in regions that are
not essential for plasmid replication: preferably. theyshould be located within genes coding for selectable
markers so chat insertion of a foreign fragment
inactivates the gene. In terms of insert size, plasmid
vectors can accommodate up to 5 to 6 kb of foreign
DNA. The most widely used vector is pBR322, a
plasmid under relaxed control that contains both
ampicillin and tetracycline genes and a number ofconvenient restriction sites.
'In general, replication of plasmid DNA is carried out by thesame set of enzymes involved in the replication of the bacterial
chromosome. Some plasmids are under stringent control, i.e., theirreplication is coupled to that of the host so that only one or at mosta few copies of the plasmid will be present in the bacterial celi
[N 19]. Plasmids under relaxed control, however, have copy numbersof 10-200; the copy number of the relaxed plasmids can be increased
by several thousands per cell if host protein synthesis is arrested(e.g., by treatment with chloramphenicol [C33]). In the absence ofprotein synthesis, replication of relaxed plasmids continues whereas
replication of chromosomal DNA and of stringent plasmids ceases[M32].

123. Bacteriophage lambda. Ever since the first
demonstration of the feasibility of using bacteriophage
lambda as a cloning vehicle [M33. R21. T21] a large
variety of vectors have been constructed [W 18).
Bacteriophage lambda is a double-stranded DNA
virus with a genomic size of about 50 kb: only about
60% of its genome is necessary for lytic propagationof the phage, the middle one-third (the stuffer portion)
being not essential. The maximum size of the insert
tolerated by lambda derivatives is about 24 kb.
124. Cosmid. A cosmid [C34] is a type of artificiallyconstructed small plasmid vector that can be packaged
in vitro in lambda phage. Typically, it contains: (a) adrug resistance marker and a plasmid origin of
replication: (b) one or more unique restriction sites forcloning: (c) a DNA fragment that contains the ligatedcohesive (cos) end site of the phage. The cosmid is ofsmall size, so that eukaryotic DNA fragments up to
50 kb in length can be accommodated.
125. Ml3 phage. Ml3 is a single-stranded DNA
bacteriophage with its circular DNA genome being
about 6500 nucleotides in length [D28]. The primaryadvantage of Ml3 as a cloning vehicle derives fromthe fact that the phage particles released from thebacterial cell contain single-stranded DNA that ishomologous to only one of the strands of the cloned
DNA fragment and can therefore be used as atemplate for DNA sequencing. Ml3 vectors andspecific primer DNAs have been developed to allow
sequencing of up to 350 bases from a single clone.

3.

Cloning and the production of multiple copiesof the inserted DNA fragment126. After a fragment of foreign DNA is inserted
into a suitable vector (plasmid. lambda phage. cosmid.
etc.), the next step is to introduce the vector into ahost organism. Host organisms that have been used inrecombinant DNA technology include E. coli, strain
K-12 (the most favoured), Bacillus subtilis and theyeast Saccharomyces cerevisiae. In the case of plasmid
vectors, in order to generate recombinant plasmids,
the plasmids are first isolated from a bacterial host by
disruption and centrifugation and the bacterial DNA
and debris are discarded. The plasmid DNA andforeign DNA are then exposed to an appropriate
restriction enzyme, mixed together and treated withligase. To reduce the likelihood of the plasmid
recircularizing without incorporating the foreign DNA,
the former is treated with the enzyme alkaline
phosphatase. The recombinant plasmid DNA is thenintroduced into a bacterial cell (by treating the latterwith calcium chloride, which renders its cell membrane
permeable to the plasmid DNA), a process referred toas transformation. The transformed bacteria are thengrown on nutrient agar in petri dishes. Each colony ofcells represents the progeny of a single cell andtherefore all the cells in a colony have the same
genetic constitution and all the cells in a clone willcontain the same inserted segment of foreign DNA.
lndi\'idual colonies can be subsequently sub-cloned togenerate multiple copies of a particular recombinant
DNA sequence.

127. Cloning in lambda vectors involves the following
steps: (a) the vector DNA is digested to completion
with the appropriate restriction enzyme. and in thecase of replacement vectors/ the left and right arms
are separated from the central stuffer fragments byvelocity gradient centrifugation or gel electrophoresis;(b) the two arms are then ligated in the presence offragments of foreign DNA having termini compatible
with those of the arms; and (c) the resulting recombinant DNAs are packaged in vitro into viable phage
particles that form plaques on the appropriate hosts.
The strategies used with other vectors are basicallysimilar.

4.

Selection and characterization ofrecombinant clones

128. If a clone has become transformed by a plasmidcarrying resistance to a particular antibiotic. then this
could be detected by the ability of this clone to growin the presence of the antibiotic: in the case of thephage, this will be revealed by the formation ofplaques. The next step is to determine whether thevector has acquired foreign DNA. In the case of aplasmid, this can be recognized by an altered antibiotic resistance, using replica plating techniques. For
instance, in the case of the plasmid pBR322. if foreign
DNA has been incorporated at the Pstl site (thusdestroying the ampicillin resistance gene). colonies will
not grow in the presence of ampicillin (though they
will remain resistant to tetracycline) and can therefore
be identified. picked off and cultured separately. In
the case of phage vectors, advantage is taken of thefact that certain strains normally produce blue plaques
when plated in the presence of a particular chromogenic substance (Xgal). but if foreign DNA is successfully inserted in the gene responsible for this colourchange, then they form colourless plaques.
129. A number of ingenious techniques have beendeveloped to characterize the recombinants that contain a specific DNA sequence (genetic. hybridization
with an appropriate probe, hybrid-arrested proteintranslation/: electron microscopy. etc.). A probe is asegment of single-stranded DNA or RNA that has
been labeled either radioactively (e.g.. 32P) or withsome biochemical marker such as biotin [L25]. A
gene-specific probe may be produced in several ways
from its constituent nucleotides. either because these
are already known or can be inferred from theaminoacid composition of the gene product, or by
first isolating mRNA from a relevant tissue and, using
this as a template. and with reverse transcriptase.
synthesizing the complementary copy of the DNA(cDNA). The important point here is that a probe will
search out and detect (hybridize with) complementary
sequences in the presence of a large amount of noncomplementary ON A. Hybridization of DNA extracted/Lambda derivatives having a single target site at which foreign
DNA is inserted are known as insertion vectors and those having apair of sites spanning a segment that can be removed and replacedby foreign DNA are known as replacement or substitution vectors.
kHybrid-arrested translation (HARn is a 1echnique used to
identify DNA sequences by hybridization with RNA and then study
the products of in vitro protein synthesis [P32l.

47

from a clone can also be carried out on an electrophoresis gel using the method introduced by Southern
and therefore referred to as a Southern blot (S42].
130. In the Southern blotting method, the DNA
extracted from a clone is first exposed to an appropriate restriction enzyme and the resultant DNA
fragments subjected to electrophoresis on agarose gelsand separated according to size. The DNA is thendenatured with alkali (to render it capable of hybridization with the complementary sequences of the probe)
and transferred to a nitrocellulose filter (Southernblotting). The DNA or RNA probe is labelled with 32p
and hybridized to the filter bound DNA. The sequence
complementary to the probe used would appear
as bands on autoradiographs. When the sequences
analysed are less than I kb. polyacrylamide gels may
be used [M34] for their separation, as used forsequencing DNA [M35. M36]. The DNA fragments inthis case may be transferred from the gel to the filter
electrically.
131. The Southern blotting method cannot. however.
be applied to RNA fragments separated by gelelectrophoresis because RNA does not bind to nitrocellulose. A modification of the procedure, called
the Northern blotting method, has therefore beendeveloped for use with RNA [A 7].

B.

CONSTRUCTION OF LIBRARIES

132. Through the use of techniques briefly discussed
in the preceding paragraphs. it has been possible to
make libraries of cloned DNA fragments [WI I]. The
advances in this area have been so rapid andphenomenal that, currently, gene libraries, somechromosome-specific libraries and genomic libraries
are available (see [015] for details). A cDNA (copyDNA or complementary DNA) library is constructedby synthesizing a double-stranded copy of the mRNA
population (using RNA-dcpendent DNA polymeraseor reverse transcriptasc) and integrating these cDNA
molecules into a restriction site in a plasmid [WI I].
The cDNA library is representative of mRNA sequences expressed in the particular cell type from which itis derived and is useful for studying tissue- or stagespecific gene expression. It contains fewer repetitive
DNA sequences than a genomic library and is asource of the coding sequences of the genome. A
genomic library contains all the coding and noncoding sequences [M22, FI I]. including single copy
sequences and repetitive sequences. It has also beenpossible to construct libraries of clones of at least
some individual human chromosomes (chromosomespecific libraries), mostly from hybrid cell lines,
following various enrichment procedures, such as
chromosome sorting [D 15).

C.

RESTRICTION FRAGMENT LENGTHPOLYMORPHISMS (RFLPs)

133. As mentioned earlier, the DNA restriction
enzymes recognize specific sequences in the DNA andcleave them, yielding fragments of definite lengths.
48

These fragments can be separated according to molecular size by electrophoresis on agarose gels. Differences among individuals in the length of a particular
restriction fragment (RFLP) can be due to several
kinds of genotypic differences: differences in one ormore nucleotide bases (resulting in Joss of a cleavage
site or formation of a new one) or insertion ordeletion of a stretch of DNA within a fragment(resulting in an alteration of fragment size). Thesechanges, which seem to occur approximately every
100-200 base pairs in the normal population, areapparently without any phenotypic effects. are inherited
in a Mcndelian co-dominant manner and thus can beused in family linkage studies to localize specific genes
of interest [Bl9. DI6-Dl8. Hl8. Hl9, Kl2. M23, 06.
S40. S4 J, W 12]. Cooper and Schmidtke [C35] havepublished a list of DNA polymorphisms found in thehuman genome by restriction enzyme analysis.

D.

RECOMBINANT DNA TECHNOLOGY IN
HUMAN GENETICS AND MEDICINE

134. Over the last six years or so, recombinant DNA
technology has revolutionized the study of the humangenome. It has led to the successful cloning of cDNAfor a large number (well over a hundred) of humanstructural genes, among which are several protooncogcncs, genes with known functions, as well as
unique sequences whose functions will ultimately
become known (B45. D19, El8, S40, S43, S44]. It has
permitted greater insights to be made into the finestructural organization of genes, the nature of molecular
defects that lead to disease states. gene regulation andgene expression and has helped in the pre-natal
diagnosis of certain heritable disorders. It has provided a powerful tool for gene mapping and fordelineating linkage relationships. Table 15 is takenfrom the paper of Cooper and Schmidtke [C35] andprovides a useful summary of these advances (see also
[J 14]). Some illustrative examples are discussed below.

I.

Direct analysis of genes

135. Globin gene clusters. The genetic analysis ofhuman globin genes and mutations that affect theirstructure and synthesis of haemoglobin (and thus leadto diseased states. the haemoglobinopathies) has
frequently set the pace and established precedents forstudies in the area of normal gene structure andfunction, as well as mechanisms of abnormal gene
expression leading to specific diseases [B46. W 17,
W42]. There are at least two reasons for this: (a) on a
global scale. the haemoglobinopathies cause far more
illness than Mendelian conditions, since about aquarter of a million severely affected individuals areborn every year with one or more of these disorders[Wl7]: and (b) the study of haemoglobinopathies has
been made easier by the fact that cells in which gene
expression is limited to the synthesis of globin chainsof haemoglobin can be obtained relatively easily(peripheral blood reticulocytes).
136. Haemoglobin is a tetramer consisting of twoalpha-like and two beta-like globin subunits. Thesesubunits are encoded by two clusters of globin genes,
the alpha and beta gene clusters. the former locatedon the short arm of chromosome 16 and the latter on
the short arm of chromosome 11. The alpha genecluster consists of two active alpha genes and a zeta
gene.
beta gene cluster consists of an epsilon gene,two different gamma genes, a delta gene and a beta
gene. The zeta and epsilon genes are only active in theembryo. the gamma genes normally only in the fetus
and the beta and delta genes only in the adult.

!he137. The advances in understanding of the structure
and organization of these genes, which have resulted
from the application of recombinant DNA methods
have been reviewed in a number of publications (e.g.,[AI8, Fl2, M61, 07, 014). Among the importantresults are: (a) the delineation of linkage relationships
between the alpha [EI9, L26. 015) and between thebeia genes [B47. F31, F32, L27. L28, M62, P33, R51,
T32]: (b) the identification of non-expressed globin
pseudogenes 1 v.·ithin the globin gene clusters [F33,121, P34]: (c) the discovery of intervening sequences(introns) which separate the coding regions [F38].
similar to those already known from studies of rabbit
and mouse globin genes [122, T33]: (d) the identification of repetitive sequence elements within globin geneclusters [F33, M61]: (e) the detection of DNA polymorphisms in the globin gene clusters and theircharacterization (reviewed in [AJ8, 014]) with theirapplications for pre-natal detection of haemoglobinopathies: and (f) the establishment of phylogenetic
relationships among mammalian and human globin
genes [E20).
138. Haemoglobinopathies. These belong to two
major groups: the first group includes those in which
there is a structural alteration in one of the globin
peptide chains: the second includes those in which thebasic abnormality is a defect in globin chain synthesis,
rather than a structural abnormality in the globin
chain itself. The classical example for the first group issickle cell anaemia and for the second, the thalassaemias. Sickle cell anaemia is a consequence of apoint mutation (transversion) at the sixth codon fromthe 5' end of the beta globin gene, which results in thereplacement of glutamic acid by valine [M25]:(glutamic acid)ftA ..... CCT

GAG

GAG ....

ps ..... CCT

GTG

GAG ....

l(valine)139. It is now possible to detect direct Iv the above
mutation by restriction enzyme analysis.· The A - T
substitution in codon 6 alters the recognition site forrestriction enzymes MnlL Ddel and Mstll [C21, 019,
Kl 4, Wl5]. For strictly practical reasons, only the lastis useful when standard Southern blot analysis isperformed. Loss of the cleavage site at codon 6
produces a 1.3 kb beta-gene fragment, compared witha 1. I kb normal fragment. This method has been
!These are ~NA sequences which share sequence homology withthe well-estabh_shed globin loci (in this case) but do not correspondto kn?wn ?lob1~ polypeptide chains. Globin pseudogenes have also
been 1denufied m other animal species such as rabbit, mouse. goatand sheep [FJ2).

successfully used in pre-natal detection studies [C21Kl4. 014).
'
140. Another approach that has been used for direct
detection of the /JS-mutation (and also of several
different beta thalassaemia defects) is the use of
sy?thetic oligonucleotides as probes. Under appropnate experimental conditions, short synthetic DNA
fragments (oligonucleotides, oligomers) can hybridize
to their homologous sequences but not to heterologous
sequences, i.e .. those with any degree of mismatch. A
probe 19-nucleotide Jong ( 19-mers) directed to thenormal beta globin sequence in the region of the/JS-mutation can hybridize efficiently with the normal
gene, but not with DNA containing the fi 5-mutation.
Conversely. a 19-mers probe strictly homologous to
the altered sequence will hybridize only with the ps_containing DNA [C38].
141. With respect to thalassaemias, the whole arrayof recombinant DNA techniques has been used
including the screening of genomic libraries with
cDNA probes, the isolation, characterization andsequencing of globin genes. and in vitro translationand protein synthesis. As a result of these studies, it is
now known that the molecular defects in /J 0 -thalassaemia (complete absence of beta globin synthesis)
include nonsense mutations due to base substitutions,nonsense mutations due to base deletions or insertions
causing frameshift, base substitutions at junctions
between introns and exons totally preventing normal
precursor mRNA processing and partial beta globin
gene deletion. Likewise, in JJ+-thalassaemia (wherebeta globin synthesis is present but markedly reduced),the lesions include base substirntions in introns
causing defects in precursor mRNA processing andbase substitutions in the 5' -flanking DNA that cause
reduced transcription of the beta globin gene. The
majority of beta globin defects, however, are the resultof point mutations (reviewed in [Fl2, M61. 07, 014];
see also [OIO and P51] for the use of synthetic
oligonucleotides for the direct detection of fJ+ ·thalassaemia and /1°-thalassaemia in pre-natal diagnosis).
142. Other examples. Alpha-1-antitrypsin deficiency(an autosomal recessive syndrome) predisposes theindividuals to the development of pulmonary emphysema and. for reasons still not clear, such a deficiency
may also present as infantile liver cirrhosis. The alpha1-antitrypsin cDNA clone has been constructed [Kl5]and m.1cleotide sequence analvsis has shown that the
deficiency is due to a point m~tation involving a G to
A transition, which leads to a single aminoacid
substitution from glutamic acid to lysine at residue
342 [Wl4]. a situation analogous to the sickle cellhaemoglobin. Since this point mutation does notcreate or destroy a restriction enzyme recognition site,
probes of oligonucleotides complementary to thenormal and mutant genes were synthesized andemployed to identify homozygous recessive individuals.
The probe specific for the normal gene sequence
yielded significant hybridization signals only with thechromosomal DNAs of normal individuals. Since onlyth~ recessive homozygotes will manifest the disease,this methodology will permit the identification of
fetuses lacking the normal gene sequence and allow
for pre-natal diagnosis of the deficiency syndrome.

49143. Studies with the arginosuccinate synthetaselocus (deficiency of this enzyme causes the disease
citrullinaemia characterized by ammonia intoxication,
mental retardation and early death) have now shownthat this locus involves an expressed gene on chromosome 9 and a family of related pseudogenes dispersed
to at least nine other chromosomes, including 6, X
and Y. Analysis of the genomic DNA suggests thatthe expressed gene is larger (> 50 kb) with at leastnine introns and the presumptive pseudogenes are
smaller(< 6 kb). Analysis of cultured fibroblasts from
citrullinaemia patients demonstrated the presence ofhybridizable mRNA which was shown to be defective[B20].
144. Hypoxanthine-guanine phosphoribosyl transferase (HPRT) which functions in the metabolic
salvage pathway of purines. is encoded by an X-linked
gene in man. Partial HPRT deficiencies are associatedwith gouty arthritis, while the absence of activity
results in Lesch-Nyhan syndrome. Although a complete characterization of the human H PRT gene has
not yet been achieved, the complete structure of themouse HPRT gene has been determined by restriction
mapping of lambda recombinants and selective DNA
sequencing: the gene was shown to be 33 kb long andto be split into 9 exons [M37]. From this study. thehuman gene appears to be of similar length. Thisestimate is in agreement with that of Jolly et al. [J 15]
based on analysis of HPRT-deficient mouse celltransformants that carried a functional human HPRT
gene. The coding regions of the mouse, hamster andhuman HPRT genes share a substantial (> 95%)
homology. with a divergence in the 3' and 5' untranslated regions [Fl3, N 15, N 16]. The mature mRNA forthe human HPRT is only about 1.6 kb in length [829,115, Ll3]: thus the functional mRNA is only about
5% of the genomic DNA that codes for HPRT andthis discrepancy in length is explained in part by thepresence of long non-coding introns that interrupt thecoding exons [W20]. There is evidence for the existenceof four autosomal cross-hybridizing sequences in thehuman genome [N 16], located on chromosomes 3(one}, 5 (one) and 11 (two) [P22]. The hybridization of
specific cDNA fragments to the aurosomal sequences,
as well as partial sequence analysis, suggest that these
HPRT-Iike sequences represent processed or intronless pseudogenes.
145. A characterization of mutant genes of 28 LeschNyhan patients using recombinant DNA techniques
showed that, in 23 of them. the Southern blot patterns
were identical to that found among normal controls.
while five showed patterns suggestive of major genealterations: one of them showed a total deletion of the
HPRT gene (exons 1-9), one a partial deletion (exons4-9), one a smaller partial deletion (exons 7-9), one apartial duplication (in the region staning with the 5'
end and extending to exon 4) and, finally, one analtered pattern (exons 4-6). which could not bedelineated [P22, Y9]. A similar conclusion, namely,that the nature of the alterations was different indifferent individuals. was reached by Wilson [W20]from studies of 5 unrelated patients, first by comparative mapping of tryptic peptides with the use of highpressure liquid chromatography and more recently by
restriction enzyme analysis.

50

146. Rees et al. [R62] have obtained evidence that, ina patient with a severe form of haemophilia 8, the only
significant difference from the normal factor IX gene(the clotting factor gene) is a point mutation within
the donor splice junction (one that is essential for thenormal splicing of pre-mRNA to mRNA). whichchanges the obligatory (and highly conserved) GT to
TT. In addition, the authors used oligonucleotide
probes specific for this mutation to demonstrate thefeasibility of carrier detection and pre-natal diagnosis
for relatives of the patient.
2. Indirect analysis of genetic disease using
gene probes to detect linked polymorphisms
147. The techniques of restriction endonuclease analysis of DNA have allowed the identification of anumber of polymorphisms (RFLPs), which in turnhave been found to have a number of applications inclinical practice and human genetics. In general, if an
RFLP is closely linked to a locus for a serious genetic
disease. this can be used for pre-natal diagnosis, forthe detection of dominant disorders before symptoms
appear or for the detection of female carriers of Xlinked diseases. Thus, for instance. Kan and Dozy[K 13] detected a Hpal site polymorphism located atthe 3' nanking segment of the beta globin gene that
was adjacent to the gene for sickle cell ·anaemia. This
has enabled its use in pre-natal diagnosis. As has been
mentioned earlier, direct detection of the ps gene has
now become possible using the endonucleases Mstll
and Ode! [C21, GI9, Kl4, 08, WJ5].
148. There is general agreement that several different
genes are probably involved in rendering an individual
susceptible to atherosclerosis and coronary arterydisease. additional important environmental factors
being cigarette smoking and overeating. The genetic
control of lipoproteins is clearly important since there
is an association of atherosclerosis with raised plasma
levels of low-density lipoproteins (LDL) and reduced
plasma levels of high-density lipoproteins (HDL). It
seems that while LDL may predispose the individual
to atherosclerosis, HDL affords protection against thedisease. Apolipoprotein A-I (apoA-1) is the major
protein constituent of HDL. The normal gene for
apoA-1 has now been isolated, cloned and characterized. the coding region of the mature apoA-1
mRNA consisting of 804 nucleotides [K39]. It has
been found using apoA-1 probes that, at least in someindividuals. premature atherosclerosis is associatedwith a variant apoA-1 gene with an insertion in thecoding region [K20. K21].
149. Using a cDNA clone for human phenylalaninehydroxylase (PH). which is responsible for phenylketonuria (PKU), Woo et al. [W21] demonstrated that
the PH gene is present in the cellular DNA of PKU
patients. suggesting that the classical PKU is notcaused by a deletion of the entire PH gene. Since PH
is a hepatic enzyme not readily detectable in pre-na ta!
diagnosis, the detection of 8 distinct polymorphisms at
this locus allows for pre-natal diagnosis of classicalPKU and identification of carriers of the trait [W21].
Likewise, discoveries of multiple restriction site polymorphisms detected within the 60 kb of DNA of thebeta globin gene cluster have permitted the detectionof about 85% of the pregnancies at risk for Pthalassaemia, using amniocytes alone [A6. D22, J6,
Kl4. Tl3].
150. Some progress has been made in understanding
the genetic basis of susceptibility to diabetes mellitus[type I or insulin-dependent diabetes mellims (IDDM)
or juvenile onset diabetes mellitus]. The human insulin
gene is located at l lp!S about 14 cM from thep-globin gene [L29] and has been isolated. cloned andsequenced (848). It is 1430 bp (base pair) in lengthwith two introns. About 360 bp upstream from thestarting point of transcription is a region with a very
high frequency of ON A length polymorphisms. referred
to as a hypervariable region. Taking advantage of thispolymorphism and the availability of cDNA insulinprobes, attempts have been made to determine thesusceptibility to IDDM diabetes by studying thefrequency of DNA polymorphisms in this region in
affected individuals. Depending on the fragment sizes
generated by restriction enzymes. Bell and Karam[849] found three classes of alleles defined by theirdifferent lengths due to the numbers and arrangements of tandem repeats at the polymorphic locus.
Class I and class 3 alleles are the most frequent, whileclass 2 alleles are rare, at least in Caucasians. In
Caucasians it appears that class I alleles occur
significantly more frequently in those with IDDM
than in healthy controls [B50].

3. Indirect analysis of genetic disease using cloned
D~A segments to detect linked DNA polymorphisms151. Since RFLPs can be used simply as genetic
markers, any disorder caused by a major locus andsegregating in pedigree can be mapped. The advantage
of such a procedure is that it does not require
knowledge of the biochemical basis of the disorder inquestion or the isolation of a specific gene [Bl7, D15.
W44]. Disorders that fall into this category include,
among others, Huntington disease (HO) (autosomal
dominant). myotonic dystrophy (autosomal dominant)and the X-Iinked Duchenne and Becker muscular
dystrophies. It should be realized, however. that the
chance of detecting a linkage between a randomly
selected probe and a particular RFLP is very small.
The statistical aspects of the problem-how manymarkers are needed. how polymorphic each marker
must be, how many informative families are needed,etc.-have been dealt with, among others, by Botstein[B 17]. Bishop and Skolnik [B 19]. Southern [S46].
Skolnik and White [S4 l] and Lange and Boehnke[LS].
152. The location of the gene for Huntington disease
was unknown until recently. Gusella et al. [G27] have
now discovered a polymorphic region in close linkage
to the gene. The probe used in this case. termed GS,
maps to chromosome 4; its close linkage to HO
indicates that the locus (or one of the loci) for thisdegenerative disorder lies on chromosome 4. A promising aspect of GS is that it is likely to make possible
the identification of the HO gene itself and thus
clarify the mechanism of this neurological disease.

153. Myotonic dystrophy is another autosomal disorder for which the age of onset and the degree ofseverity are highly variable; this can make it very
difficult to determine whether an individual at risk iscarrying the gene [H45]. The biochemical basis of thisdisorder is not understood, but the gene is known to
be on chromosome 19 (E21, W43) and shows close
linkage to a very infrequent peptidase D protein
polymorphism (016) as well as loose linkage to thecomplement [C3], secretor and Lutherean loci [D 16,
E21]. Apolipoprotein CII is one of a group of genes
coding for apolipoproteins, which has also been
mapped to chromosome 19, and the cDNA probe forthis gene detects an RFLP due to a variable TaqI site
about 2 kb downstream of the gene itself [M63]. In a
recent study. Shaw et al. [S92] examined the possibilityof linkage of this polymorphism to the myotonic
dystrophy locus in families and found that the twoloci are in fact closely linked (only one recombination
between the disease and marker loci in a total of
53 informative meioses) but pointed out that further
family studies are required to increase the precision of
their estimate of genetic distance.
154. Duchenne muscular dystrophy (DMD) is asevere X-linked disorder normally affecting only males
and leading to their early death; its biochemical basis
is also unknown at present [M64]. The disease has
also been found in a few girls so far, and in someinstances. an X-autosome translocation is present inwhich the exchange point in the X is in band Xp2 J
near the middle of the short arm (see [D44) for
citations of the relevant literature). The concurrenceof the repeated translocation breakpoint with DMD
phenotype suggests that band Xp21 may be the site of
the DMD gene. This map location is consistent withfamily studies that have shown linkage of the DMD
locus to two RFLPs (recognized by probes Ll.28 andRC8), flanking the Xp21 region [D17, D18. Hl9.
M23]. In order to achieve a more refined localizationof the DMD gene. De Martinville et al. [D44]analysed panels of somatic cells of hybrid lines
carrying various structural rearrangements of thehuman X-chromosome short arm v.'ith 21 X-chromosome-specific cloned DNA fragments. The finding ofinterest was that the ornithine transcarbamylase geneand four anonymous DNA sequences mapped withinband Xp2 l. flanking the presumed DMD locus.
155. In a detailed study on the isolation of probes
detecting RFLPs from X-chromosome-specific libraries,
Hofker et al. [H46] found that two of the isolated
probes detected a high frequency of RFLP. One. No.
754, maps between Xpll.3 and Xp21 and the other.
termed 782, maps between Xp22.2 and Xp22.3. In apilot study of families at risk for DMD, the authors
found that the recombination fraction between DMD
and 754 is less than 3 cM (between DMD and 782, it is
17 cM). These results suggest that probe 754 is
probably the most powerful probe currently available
for linkage studies with DMD. This suggestion bas
received support from the work of Francke et al.
[F34]. In a male patient who manifested four different
disorders (DMD, retinitis pigmentosa (RP), chronic
granulomatous disease and McLeod phenotype), theseinvestigators found a subtle interstitial deletion of part51

of band Xp21. Not only were they able to confirm thedeletion by somatic cell and molecular studies. butthey also showed that the probe 754 recognizes an
RFLP of high frequency; their linkage data suggest agenetic distance of about 3 cM between the OMO
locus and 7 54, with 95% confidence limits of 1-12 cM.
(For a similar attempt to map DNA sequences in ahuman Xp interstitial deletion that extends across theregion of the OMO locus, see Ingle et al. [I 10).)159. Through the use of a recombinant clone containing a sequence that occurs only in human chromosome 21, Davies et al. [029] were able co find a twoallele RFLP showing Mendelian inheritance. Studies
of DNA from Down syndrome patients and fromtheir parents enabled the confirmation of trisomy-21
by dosage hybridization to Southern blots and thedetermination of the origin of the extra chromosome 21.

156. In other studies, linkage between LI .28 and anon-lethal form of X-linked muscular dystrophy, theBecker muscular dystrophy (BMD) (which had Jongbeen thought to be a different clinical entity), wasdemonstrated. On the basis of data from analysis of
10 X-linked DNA polymorphisms (5 mapping on the
short arm and five on the Jong arm). Roncuzzi et al.
[R52] confirmed linkage of the Becker gene to L 1.28
and also showed that the locus is located between
Ll.28 and B24. It has been surmised that the locus for
Becker muscular dystrophy and that for OMO may be
the same [Kl2, K22]. Brown et al. [B69] used the p754
probe to study 14 OMO and 8 BMD kindreds; theirlinkage data suggest that both the disease loci are in
p21 (short arm of the X) at a distance of about
15-20 cM from the 754 locus. Data from families
informative for both the p754 and L 1.28 polymorphisms
suggest that p754 is closer than Ll.28 to the diseaseloci.

160. In a recent paper, Antonarakis et al. [Al9]tested the hypothesis that there is a genetic predisposition to non-disjunction and trisomy-21 associatedwith DNA sequences on chromosome 21. They used
DNA polymorphism haplotypes for chromosome 21
to examine the distribution of different chromosomes 21
in Down syndrome and control families from thesame ethnic group in Greece. Four common polymorphic sites adjacent to two closely Jinked singlecopy DNA sequences (namely pW228C and pW236B),
which map on the proximal long arm of chromosome 21, were found. One particular haplotype wasmuch more commonly associated with chromosomes 21
that underwent non-disjunction in the Down syndrome
families. suggesting that an increased tendency for nondisjunction may be due to DNA sequences associatedwith a subset of chromosome 21 bearing this haplotype.

157. The X-Iinked variety of retmrns pigmentosa(RP) found in 14-22% of RP families in the United
Kingdom, is one of the most severe clinical forms of
RP with onset in males in the first decade andprogressive blindness by the third or fourth decade.
Female carriers are difficult to identify because of
only partial manifestation due to random X-inactivation. Bhattacharya et al. [828] have reported close
genetic linkage between this X-Iinked disorder and a
RFLP identified by L 1.28. This probe is potentially
useful for carrier detection and early diagnosis,
though only approximately 40% of the cases (heterozygotes) appear informative for this probe, provided
genetic heterogeneity can be excluded by further
family studies.

4.

Other applications of recombinant DNA technology

158. Molecular studies on Down syndrome. Some
progress towards understanding of the molecularbiology of Down syndrome has been made in recent
years. The gene locus for superoxide dism utase (SO 0-1)
is located on 2lq22 which is very near, at least, to theregion of chromosome 21 involved in Down syndrome,
since trisomy for this smaJI segment is sufficient tocause Down syndrome (e.g., [H47, N27, P35, S93.
W45]). The gene for SOD-I has been cloned [L30.
L31] and may well contain the DNA sequences
involved in the pathogenesis of Down syndrome, or atleast provide a convenient starting point for chromosome analysis. Work currently under way on regional
mapping of DNA sequences in chromosome 21,
construction of a linkage map of this chromosome,etc., has recently been presented [E22].
52

161. Fetal sexing. In X-linked disorders, whichcannot as yet be diagnosed pre-natally, selective
abortion of any male fetus may be acceptable to amother at risk of having an affected son. In suchcircumstances. feta! sexing is usually carried out by
karyotyping cultured amniotic fluid cells. or more
recently. chorionic tissue. However, probes are now
being developed that contain DNA sequences specificfor the human Y chromosome, which can also be used
for feta! sexing [B5 l, G40].
162. The immune system. Recombinant DNA techniques have provided detailed information on thegenetic organization of the major histocompatibility
complex (MHC) or the HLA system. While theassociation of specific serologically detectable allelesat these loci to a number of diseases (reviewed in[020. Rl8]) has long been known. the availability ofmolecular clones for the different genes of the HLA
system is enabling the detection of a variety ofpolymorphisms that distinguish allelic forms of thevarious segments of MHC and may prove useful instudies correlating MHC haplotypes to propensity tocertain diseases [021, El2, Pl4). Likewise, the application of ON A methods to the study of genes controllingthe synthesis of immunoglobulins has been extremely
useful in understanding how antibody diversity is
generated [El8, L32, L33, R53, T35, Z5] and is likely
to provide insights into the nature of defects that may
underlie the generation of specific antibodies known
in certain inherited sensitivities (atopies).
163. Diagnosis and typing of tumours and lymphomas. Recombinant DNA techniques have potential
in providing additional information for diagnosis andtyping of tumours and lymphomas. Thus. for instance,Cleary et al. [C39] recently described the use of theSouthern blot hybridization technique to diagnose Bcell lymphoma by detecting clonal immunoglobulin
gene rearrangements in lymphnodes and other biopsy
tissues. The analysis of immunoglobulin gene rearrangements, which characterize B-cell lymphoma.
offers several advantages over conventional diagnostic
methods.
164. Biosynthesis of valuable hormones and proteins.
One of the major applications of recombinant DNA
technology is in the synthesis of medically important
drugs. In 1977, Itakura and colleagues [II I] reported
the first successful synthesis of somatostatin, a peptide
inhibiting the growth hormone, used in the treatmentof children with excessive growth. Since then, anumber of other valuable hormones and proteins havebeen synthesized (insulin [G41]. growth hormone[G42]. F-interferon [T34], L-interferon [N28, G43]).
165. Radiation genetics and genetic risk assessments.
In the 1972 U~SCEAR report [U3], the Committee
stated " ... the past decade has witnessed an almost
explosive growth of molecular biology ... impressive
as these developments are, molecular biology has not
yet provided information on the relationship between
the damage from ionizing radiation at the DNA leveland mutational events to explain or to predict thearray of mutational responses observed with different
dose-rates, cell stages, etc .. in mammals". As will beevident from the preceding discussions, the situation is
rapidly changing. Molecular methods have been greatly
refined and applied to the study of gene structure,organization and function and the nature of spontaneously arising mutations. Applied to human genetic
disorders, these studies have exposed considerable
genetic heterogeneity in many of these disorders at
the molecular level. This unmasking of genetic heterogeneity may have implications in the context of
estimating the additional burden of these disorders
due to radiation exposures. Furthermore, molecularmethods are currently being extended to investigate themolecular basis of induced mutations (see chapter VI)
and to examine differences in the spectra of mutations(including radiations of different quality). Theseadvances are bound to add more precision. both informulating questions and obtaining answers that are
relevant for genetic risk assessment.

E.

SUMMARY AND CONCLUSIONS

166. Recombinant DNA technology has provided aformidable array of new opportunities to study thehuman genome; it has increased the speed andprecision of gene mapping and has helped to define
the nature of genetic lesions and to analyse the actionof specific genes in health and disease. The methods
consist of generating DNA fragments in vitro by
restriction endonucleases, incorporating these fragments into a suitable vector. introducing the vector
into a host organism. producing multiple copies of theincorporated fragments and. finally, selecting. harvesting and characterizing the cellular clones containing
the fragments of interest.

167. Restriction endonucleases-of which over 500
are currently known-are DNAses that recognize
specific oligonucleotide sequences in the DNA andbreak the DNA within or near the sequences. generating DNA fragments of defined length and sequence.
Some of the fragments generated have free protruding
5' or 3' ends (cohesive or sticky ends) while others
have blunt ends. The vectors used for cloning in E.
coli are plasmids, bacteriophage lambda derivatives,
cosmids and bacteriophage M 13. In terms of insert
size. plasmids can accommodate up to 5-6 kb, bacteriophage lambda derivatives up to 15 kb and cosmids up
to 50 kb of foreign DNA.
168. The use of recombinant DNA techniques has
permitted the establishment of gene-specific. chromosome-specific and genomic libraries. A cDNA libraryis constructed by synthesizing double-stranded copy ofthe mRNA population using reverse transcriptase andintegrating these cDNA molecules into a restriction
site of a plasmid; it is a source of the coding sequencesof the genome. A genomic library consists of allcoding and non-coding sequences. The chromosomespecific library, as the term implies, is specific forindividual chromosomes.

I69. The DNA fragments generated by restriction
endonucleases differ in length among individuals(restriction fragment length polymorphism or RFLP):
these differences are apparently without any phenotypic effects, are inherited in a Mendelian co-dominantmanner and thus can be used in family linkage studies
to localize specific genes of interest.
170. The Southern blotting method is a very important technique for locating specific DNA fragments onelectrophoresis gels: the analysis of defects in theprocessing of RNA molecules is possible by Northernblotting.
17 l. The major advances achieved during the past
few years using recombinant DNA techniques in the
study of gene structure and organization and mutational alterations leading to disease states in man canbe broadly grouped under three headings: (a) direct
analysis of the normal and mutant genes using geneprobes or synthetic oligonucleotides (e.g .. globin geneclusters. thalassaemias, sickle cell anaemia. HLA gene
system. diabetes, haemophilia B. HPRT. antithrombin
III deficiency. alpha antitrypsin deficiency): (b) indirect
analysis of genetic disease (i.e .. mutant states) using
gene probes to detect closely linked polymorphisms(sickle cell anaemia. atherosclerosis. hypei-triglyceridaemia, phenylketonuria. osteogenesis imperfecta,etc.); and (c) indirect analysis of genetic disease using
cloned DNA fragments to detect linked polymorphisms(fragile-X, Huntington disease. muscular dystrophies.
myotonic dystrophy. retinitis pigmentosa, etc.). Several
findings emerging from these studies have applicationsin pre-natal diagnosis and in the diagnosis and typingof tumours and lymphomas. At the level of disease
management, recombinant DNA methods are proving
to be very useful in th•; synthesis of medicallyimportant drugs.
53172. It is hoped that the application of the recombinant DNA methods to radiation- and chemicallyinduced mutations will help to unravel differences inmutational spectra and to gain more precise insights
into repair processes, dose-rate and dose-fractionation
effects in experimental systems, aspects that are
pertinent to the evaluation of genetic radiation hazards
in man.

175. The product of at least 8 of these oncogenes is a
protein kinase, a phosphorylating enzyme. Six of thesekinases have so far been shown to phosphorylate theaminoacid tyrosine and at least five of the kinases
bind to the plasma membrane [BI. 813, H48]; thepp6ov-src of the Rous sarcoma virus and the p20 of the
Abelson leukemia virus are among the best known
examples of the protein products of these genes.

B.

Ill. GENES. CHROMOSOMES AND CANCER
173. In the 1982 UNSCEAR report, several Mendeliandisorders with predisposition to neoplasia and anumber of specific chromosomal defects associatedwith cancer in man were considered in some detail (seealso reviews by Neri [N20], Radman et al. [R25] andSasaki (S62]). During the last few years, some major
breakthroughs in this area have been reported andthese have significantly advanced knowledge about thegenetic basis of cancer. Paradoxically. these advances
have come about from studies of tumour viruses(although most naturally occurring tumours, particularly in man. do not appear to be caused by viruses)
and through the convergence of thought and techniques from viral oncology, cell genetics and molecularbiology. These advances have been reviewed in several
publications [BI, 83. 813, B52. C3, C40, C6I, DI I,
D45, H48. K3. K23, K40, Ll5, Rl2, S71. VI3, W4,
W25. W30, Y4] and thus only some of the salient
aspects are briefly summarized below.

A.

THE ONCOGENES OF RETROVIRUSES(v-oncs)174. The fact that certain viruses can cause raremalignant tumours in chicken, mice, rats, cats and avariety of other species bas been known for a longtime. Studies with retrovirusesm and particularly withsome members of this group, the acute transforming
viruses.n showed that their genomes contain specificgenes (viral oncogenes or v-oncs) responsible foroncogenicity as well as unrelated sequences requiredfor viral replication. Currently. about 20 different onespecific sequences have been identified in retro\'iruses[BI3, C5, Dll, Dl2, VI3. W4]. Table 16 lists most ofthese oncogenes and the viral prototypes in which they
were first diagnosed, with a brief description of their
oncogenic properties. Some of these genes regularly
cause multiple types of tumours (e.g .. the one geneof MC29 causes myelocytomatosis. carcinoma andsarcoma) while other genes like src of the Rous
sarcoma virus or the one gene of the avian myeloblastosis virus AMV cause one predominant type oftumour.
mRetroviruscs are a family of RNA viruses that replicate by wayof a DNA provirus integrated in cellular DNA.
ll'fhe acute transforming viruses are highly oncogenic; theyefficiently transform cells in culture and induce neoplastic disease
with short latent periods in infected animals. Approximately
20 viruses of this type isolated from chickens, mice, cats, rats andmonkeys have been characterized.

54

PROTO-ONCOGENES (proto ones) OR
CELLULAR ONCOGENES (c-oncs)

176. The biochemical definition of several v-oncs
was subsequently followed by the development ofcDNA probes made from the RNAs of Kirsten andHarvey sarcoma viruses [S29. S30, TIO]. Rous sarcoma
virus [S31] and Maloney sarcoma virus [F8] and theirhybridization (in molecular hybridization experiments)
to cellular DNA. The data that emerged from theseand other studies [AI3, CJ4, Cl5, F9, S32, W5, W29.
Z4] led to the discovery that avian and mammalian
genomes contain nucleotide sequences related toretroviral oncogenes. Homologues of over a dozen
other viral one genes have since been found in normal
cells of vertebrates [W4]. In addition, nucleotide
sequences similar to the ones of Rous sacroma virus,
MC29. Abelson and feline sarcoma viruses have evenbeen found in Drosophila [L6. S33]. Likewise,
sequences similar to the Harvey and Kirsten sarcoma
viruses have been found in the yeast Saccharomyces
cerevisiae [D46, G44].
177. These and other findings lend support to theview that some of these sequences emerged quite earlyand have been conserved over extensive periods in thecourse of evolution. It therefore seems unlikely thatthe vertebrate sequences in question represent genes ofendogenous retroviruses. The use of restriction endonucleases and molecular cloning have given substanceto this view. The cellular prototypes of most known
viral ones have since been cloned and compared withviral counterparts by heteroduplex analysis. endonucleases site mapping, fingerprinting and completesequence analysis. Such comparisons have revealed
that: (a) those parts of viral ones that are unrelated toessential genes of retroviruses have closely related
counterparts in normal cells: and (b) these counterparts. which are not linked to any of the genes
essential to retroviruses, display minor structuralpolymorphisms of the sort found in recognized cellular
genes and have (at least several of them) intervening
sequences (introns) that interrupt the coding sequences(exons) [Bl. 813. Dll]. Consequently. it has beensuggested that retroviral transforming genes are present
in normal cells in a latent form. Recent structural
analyses have indicated that the viral ones and cellular
genes, which share specific sequences, are not isogenic
and that they differ from each other in scattered point
mutations and in unique coding regions [DI I].

178. A variety of experimental results support theview that the cellular homologues of retrovirus oncogenes constitute a group of normal genes. The latter
have been designated by the generic name protooncogenes. The wealth of data currently availablesuggests that these cellular genes have oncogenic
potential and thus can be referred to as cellular
oncogenes or c-oncs. The function of proto-oncogenes
is unknown. However. they are commonly expressedin most or all tissues and sometimes regulated in
temporal or lineage-specific fashion [813], implyingthat they might play a central role in growth anddevelopment, processes that seem particularly likely to
be altered during neoplasia. The latter idea hasreceived confirmation by the findings that c-sis appears
to encode the platelet-derived growth factor [D33, W28]
and c-erbB, the epidermal growth factor receptor[D60].

C.

CELLULAR GENE ONCOGENESIS

I 79. Studies on the potential oncogenic activity ofcellular genes are dependent on an assay for determination of the biological activity of purified cellularDNA. In this assay (termed transfection). recipient
cell cultures are exposed to donor DNA in the form ofa calcium phosphate precipitate [ G2]. The efficiencyof transformation obtained varies with the cells used
as recipients. The established mouse cell line (fibroblasts) NIH/3T3 is most commonly used as a source
for recipient cells.
180. The potential of normal cellular genes in
inducing transformation has been investigated in a
number of studies. High molecular weight DNAs(> 30 kilobases, kb) from normal cells (chicken
embryonic fibroblasts, non-transformed mouse celllines and normal human embryonic lung fibroblasts)
were found to be ineffective in inducing transformation in transfection assays. However, after fragmentation to sizes of 0.5-5 kb, normal cell DNAs induced
transformation with efficiencies (0.003 transformant
per microgram of DNA) that were similar to subgenomic src-containing fragments of DNA of RSVtransformed cells [C5]. DNAs of cells transformed bynormal cell DNA fragments induced transformation
with high efficiencies (0.1-1.0 transformant per microgram of DNA) in secondary transfection assays,
indicating that these transformed cells contained
activated transforming genes that could be efficiently
transmitted by transfection [C5].
181. If carcinogenesis involves dominant genetic
changes (either by mutations or by gene rearrangements) affecting the structure or regulation of suchpotential transforming genes, it is possible that the
DNAs of some neoplasms contain activated transforming genes and therefore induce transformation
with high efficiencies upon infection. Several groups
have reported that the DNAs of a variety of tumours
are indeed able to induce transformation of NIH/3T3
mouse fibroblasts. In a number of cases. the transforming activities have been shown to be localized todiscrete DNA segments present in the tumour cell
genome and several of these have been isolated using
molecular cloning techniques (Table 17); a highproportion of those have proved to be mutant
members of the ras gene family. It was found that the
DNA of normal cells contains sequences closely
related to those of the isolated tumour oncogenes; thissuggests that the latter are probably derived fromnormal cellular sequences, presumably by a process ofsomatic mutation or rearrangement occurring in thetarget tissue during carcinogenesis [G 17. P 11. S34.
Vl3, W6].
182. DNAs of approximately 80-90% of chemicallyinduced and spontaneously arising tumours, however,
do not induce efficient transformation on transfection[Vl3]. Carcinogenesis in these neoplasms may involve
epigenetic changes or recessive genetic alterations thatwould not be detectable by transfection of tumourDNAs. Alternatively, these neoplasms may containdominant transforming genes, which do not induce
transformation of the NIH/3T3 cells used as recipientsin transfection assays.

D. RELATIONSHIPS BETWEEN CELLULARONCOGENES DETECTED IN TRANSFECTIONASSAYS AND THE TRANSFORMING GENESOF ACUTE TRANSFORMING RETROVIRUSES
183. As discussed above, cellular transforming genes
have been defined either by their affiliation withretroviruses or by transfection of DNAs of tumoursthat were not. in large part, induced by viruses. The
relationships between different cellular transforming
genes detected by transfection of NIH/3T3 cells havebeen clarified by the discoveries (Table 17) that manyof these genes represent cellular homologues of theoncogene v-Ha-ras of the Harvey murine sarcoma
virus or the oncogene v-Ki-ras of the Kirsten murine
sarcoma virus [D4, PI, S7. S35]. For example. thetransforming activity associated with the T24(EJ)
human bladder carcinoma line [04, PI, S7] andseveral chemically induced mouse squamous cell
carcinomas [B 14) corresponds to an activated versionof the cellular homologue c-Ha-ras l of v-Ha-ras.
Transforming genes corresponding to the cellularhomologue c-Ki-ras2 of v-Ki-ras are found in humancell lines established from bladder. lung, colon andgall bladder carcinomas. as well as biopsy tissue fromlung, pancreas and colon carcinomas and a rhabdomyosarcoma [P2, 04, S35, M 13]. Other cases of homology
between ras genes and transfected tumour genes arecoming to light [W6]. The most obvious consequenceof these newly discovered homologies is a realizationthat at least some of the cellular sequences canbecome activated via one of two routes: by recombination with a retroviral sequence or by non-viralsomatic mutational events.

E.

OTHER TRANSFORMING GENES

184. Shimizu et al. [S64] first described a newtransforming gene termed N-ras in a human neuroblastoma cell line, which was only weakly homologous
to v-Ha-ras and v-Ki-ras. Subsequently, transforming
genes of the N-ras type were also found in a numberof other cell lines (Table 17). No retroviral oncogene
corresponding to N-ras has been described. By transfection of chicken B lymphoma DNA into NIH/3T3
cells, Goubrin et al. [G 18] detected a new transforming gene unrelated to known oncogenes of acute55

transforming viruses. Subsequently. Diamond et al.
[D 13] showed that the same type of transforming gene
could be isolated from six Burkitt lymphoma celllines. Thus it was called B-lym-1. Human B-lym-1.
like the chicken 8-lym-l, was not found to behomologous to molecular clones of any retroviraltransforming genes tested. Cooper et al. [C43] reported
on the molecular cloning of a new transforming genefrom a chemically transformed human osteosarcomaderived cell line and showed that this is unrelated toknown oncogenes (chromosomal location: 7pl l.47qter). Eva and Aaronson [E23] published the firstreport of another new transforming gene associatedwith a human diffuse 8-cell lymphoma and havedesignated it as dbl.

F.

HOW MANY ONCOGENES?

185. Currently, at least I7 distinct retrovirus oncogenes are known (Table 16), each with a corresponding
cellular c-onc. Transfecrion with tumour DNA has
also uncovered 6- 7 new oncogenes. The number of
new oncogenes will increase with continued efforts inthis direction. However, it appears that the number ofcellular oncogenes may not be very large. The factthat most of the oncogenes first found in retroviruseshave been identified more than once in the same hostspecies. that several have turned up in more than one
species and that some of the oncogenes isolated fromtumour DNA had already been encountered in the
study of retroviruses. all suggest that the majority ofcellular oncogenes may already be in view. Bishop[B 13) has ventured the guess that there may be no
more than 50-100 oncogenes. Some of the 20 or socurrently recognized oncogenes are assignable to atleast two distinct gene families: the ras family. with atleast four distinct oncogenes. and the src-yes-mos
family, with another three [W6].

G.

MECHANISMS OF ACTIVATION OF
PROTO-ONCOGENES

186. Proto-oncogenes do not transform NIH/3T3
cells: In contrast, 10-20% of DNAs from different
tumour cells lead to neoplastic transformation of
NIH/3T3 cells after transfection. Cellular transforming
genes are derived from proto-oncogenes by activation
steps leading to qualitatiYe or quantitative changes in
the corresponding gene product. There are several
possible mechanisms for activation of proto-oncogenes(see [W25, Vl3] and [G32] for reviews); some of thesehave been shown to occur in human tumours andothers have been found in model systems.

1.

Point mutations in proto-oncogenes

187. Nucleotide substitutions and concomitant aminoacid change in members of the ras gene family is thegenetic alteration that most commonly explains thetransforming activity of tumour cell DNA. Table 18
presents a summary of the available data and showsthat changes affecting the same codons ( 12 and 61)
56

have been encountered in multiple mutant alleles ofc-Ha-ras 1. c-Ki-ras2 and N-ras. The question ofwhether repeated lesions at the same site imply a hot
spot for mutations or selection for random mutations
that confer rransforming activity upon a protooncogene has not yet been resolved. There are,however, different lines of evidence from in vitro
studies, suggesting that positions 12, 59 and 61 arc
functionally important in p2Jc-Ha-ras and that it isprobable that random mutations at these sites are selectable (see [V 13] for details). Of particular importancein this context is the work of Fasano et al. [F35] who
used sodium bisulphite to produce C to T transitions
at many positions in c-Ha-rasl gene and then tested
cloned mutants for their ability to transform NIH/
3T3 cells. Oncogenic mutants occurred only at a
restricted number of sites, including codons 12, 13. 59,
61 and 63. Three of these sites (12, 59 and 61) conform
precisely to the positions of mutations encountered in
spontaneous tumours and in viral ras oncogenes(Table 18) and all are close to such sites. The change
observed at position 59, ala to thr, is particularlyinteresting because it is one of three differences inamino acid sequence between c-Ha-ras I and v-Ha-ras.
implying that the viral oncogene has acquired twoalterations independently sufficient to induce cellulartransformation.
188. Further evidence for non-random nucleotide
substitutions at a specific site come from the work of
Bos et al. [B53]. These authors tested DNAs from
4 patients with acute myeloid leukaemia (AML) by anin vivo selection assay in nude mice using transfectedmouse NIH/3T3 cells: in addition. the DNA from oneother AML patient was tested in the focus-forming
assay. All five were found to contain an activated N-ras
oncogene. With synthetic oligonucleotide probes. it
was possible to demonstrate that. in all of them, theactivation was due to a point mutation at codon 13.
resulting in a replacement of gly to either val or asp;
there was no evidence for the activation of any otherras oncogenes. The question of why N-ras should be
preferentially activated in these leukaemias has notbeen answered and the authors have speculated thatN-ras. rather than other ras genes, may be criticallyinvolved in the control of proliferation and differentiation of cells in the haematopoietic lineage.
189. There are now several papers (e.g .. [Bl4. E23,
G4S, S94. Z6]) documenting the thesis that specificoncogenes are activated by exposure of experimental
mammals to carcinogens in vivo. For instance, inmice. Guerrero et al. [G45] induced thymomas withmethylnitrosourea [MNU] (i.p. injection. 30 mg/kg
body weight, once a week for five consecutive weeks)or gamma irradiation (4 weekly doses of 1.5 Gy each)
and used the DNA from the tumours to transfect
NIH/3T3 cells. A high percentage of the tumours
transformed the recipient cells and the oncogenic
phenotype was found to be due to the activation of
N-ras oncogene (MNU experiment) or the K-ras oncogene (gamma-ray experiment). Although the authors
did not clone the genes in question, they found that
there was no evidence for any rearrangements in theSouthern blot analysis. They therefore concluded thatthe presumed changes were point mutations.

190. Sukumar et al. [S94] used MNU (single i.p.
injection, 30 pg/g body weight) to induce mammary
carcinomas in Buf/N female rats and examined thetransforming activity of the DNA from these carcinomas in transfection experiments with NIH/3T3
cells. Each of the nine mammary carcinoma DNA
tested induced the appearance of foci of morphologically transformed cells. Upon analysis of whether
the transforming activity was related to the altered rat
ras sequences. it was found that. in all cases. transformed H-rasl oncogenes were present. Molecularcharacterization of these revealed a nucleotide change
at the 12th codon (GGA to GAA) of the normal genewith the result that there was an aminoacid change
from glycine to glutamic acid. As was mentioned
earlier, codon number 12 is one of the most importantsites at which mutations lead to activation of the
proto-oncogene. These studies were subsequently extended [26] using restriction enzyme analysis and it
was shown that in 48 out of 58 DNA samples from
MNU-induced carcinomas studied, the Ha-ras I had
been activated by the same G to A transition, the typeof mutations preferentially induced by MNU.

2.

Attachment of strong promotors/enhancers
to proto-oncogenes

19 l. Another mechanism of proto-oncogene activation has been studied in two other experimental
systems. Blair et al. [830] covalently linked themolecularly cloned long terminal repeat sequence(L TR) of Moloney murine sarcoma provirus DNA(Mo-MSV) to mouse c-mos, the proto-oncogene
homologous to v-mos. These constructs transformed
NIH/3T3 cells as efficiently as cloned subgenomic
Mo-MSV fragments containing both v-mos and LTR.
This is explained by transcriptional activation ofc-mos caused by promotor and/or enhancer elementsin the L TR DNA sequence. The resulting overproduction of the c-mos gene product is apparently sufficientfor malignant transformation of NIH/3T3 cells. The
above observation was confirmed in a related experimental system by Chang et al. [C4 J]. In this work,
viral L TR sequences were ligated to the human c-Hasras gene. After transfection of the constructs into
NIH/3T3 cells, the recipient cells became highly
tumorigenic and expressed high levels of the p21 geneproduct. (For other examples. see [045).)

192. It is worth noting here that, unlike the mousec-mos gene, the human c-mos gene could not be
activated by insertion of Mo-MSV elements at the
5' end of the coding sequence [W26]. Duesberg [D 11]
points out that promotor-activated c-mos and c-ras
genes have not been found in naturally occurring
murine tumours. although leukaemia viruses that
could provide LTR sequences are ubiquitous in mice.
Thus, increased expression of proto-oncogenes due topromotor attachment or insertion may not be sufficient
to transform normal mammalian cells in vivo. althoughestablished cell lines like NIH/3T3 can be transformedin this way by promotor-activated mouse c-mos andhuman c-Ha-ras.

3.

Promotor (enhancer) insertion near proto-oncogenes

193. This mechanism may be similar or identical tothat discussed above. In contrast to the transformation of NIH/3T3 cells by hybrid LTR-oncogene
constructs, however, activation of pro to-oncogenes
can also be achieved by insertion of a slow transforming virus into DNA flanking the proto-oncogene, but
not immediately adjacent to it. Hayward et al. [H33]reported that avian leucosis virus (ALV. a slow
transforming retrovirus lacking a known oncogene),
which causes B-cell lymphomas in the chicken after alatent period of more than 6 months. was found to be
integrated near (within 5 kb) the c-myc gene in thecells of some but not all 8 lymphomas. Fung et al.
[F21] found that ALV-LTR had integrated at least
0.5 kb upstream of c-erb-B gene locus in the genomeof chicken erythroblastosis cells. Since it is now
known that enhancers of transcription act over longer
distances than promotors [K24]. it is possible that theobserved effects are due to enhancer activity ofretroviral L TR sequences. Although the possibility
envisaged above may sometimes contribute to thetumorigenicity of slow transforming viruses, arguments can be advanced against the generalization ofthis hypothesis [D 11 ]: (a) promotor (enhancer) insertion and elevated expression of c-myc are not found in
all chicken B lymphomas: and (b) the insertion
hypothesis would call for a more rapid induction oftumours than is reflected by the long latency period
actually observed for the appearance of B-lymphoma
or erythroblastosis in AL V-infected chicken. (See also
[Vl3].)

4.

Amplification of proto-oncogcnes

194. Amplification of proto-myc DNA sequences(up to 20-fold) has been found in human myeloid
leukaemia cell line HL60 [C42. 030] and in thehuman colon cancer cell line Colo 320 [Al I]. The
Ki-ras proto-oncogene was amplified 30- to 60-foldin cells of the mouse adrenocortical tumour cell line
YI [S66]. The amplified Ki-ras proto-oncogene was
localized in double minute chromosomes in homogeneously staining regions (HSR). i.e., in cytologicalstructures characteristic of amplified genes in cultured
cells. In none of these cases has it been shown that
amplification of the corresponding oncogene is an
obligatory event in tumorigenesis. although it is
tempting to speculate that such amplification may be
involved at some stage of the multi-step process ofmalignant transformation of normal diploid cells([L34. 854]: see also [P37, S96, Vl3]).

5.

DNA transposition and rearrangements

195. Rechavi et al. [RI3] have reported on the firstcase of proto-oncogene activation in a non-virally
induced tumour by a process of DNA transposition.
The oncogene studied was the c-mos, the progenitor
of the transforming information in the Moloneymurine sarcoma virus [F8. CI8]. Previously. it had
been shown that the molecularly cloned 14-kb c-mos
gene was inactive when assayed for its ability to57

transform NIH/3T3 cells in transfection experiments.
To demonstrate the biological activity of this gene. it
was necessary to ligate a viral long terminal repeat(LTR) upstream from the 5' end of the gene [04].
In screening the DNAs of several BALB/c mouse
myelomas by Sourhern blot analysis, Rechavi et al.
detected an additional hybridization band, XRPC24,
in one of the tumours. Further studies revealed thatthe c-mos gene in this case has undergone DNA
rearrangement at the 5' end of the gene including theinsertion of a 159 bp DNA fragment having thestructure of an insertion element (IS). This gene,designated as re-mos (rearranged c-mos) was active intransfection assays with NIH/3T3 cells.
196. Other instances of proto-oncogene activation asa result of DNA rearrangements stemming fromchromosomal rearrangements, as well as those involving joint action of cellular transforming genes, areconsidered in the next section.

H. HUMAN ONCOGENE LOCALIZATIONSAND THE ACTIVATION OF C-ONCOGENES
THROUGH CHROMOSOMAL ABERRATIONS
197. During the past few years. a number of cellularhomologues (c-oncs) of retroviral oncogenes havebeen localized to specific chromosomes or chromosomal regions of the human complement (Table 19).
Although karyotypic abnormalities have been known
to exist in human tumours for quite some time [M 15,
U I, Y 4), the remarkable similarity between the chromosomal location of some of the c-oncs and thebreakpoints involved in some specific chromosomal
aberrations are noteworthy [C61. F2, G29. Rl2, R55,
Y4].
198. The best example studied is c-myc (an oncogene
originally found in B-cell avian myeloblastoma).
which is now assigned to 8q24. The translocationcommon in Burkitt's lymphoma (between the longarms of chromosomes 8 and 14) involves a break inchromosome 8 (at the 5' site of the c-myc gene) and a
break in chromosome 14 (at band q32) within thelocus for the immunoglobulin heavy chain. which isactive in these lymphoma cells [Kll]; the break inchromosome 14 seems to occur in different nucleotide
sequences in different Burkitt lymphoma lines. Dalla
Favera et al. [D 14] reported that the break is in thevariable region. but Taub et al. [Tl2] assigned it to theswitch region of the gene. In either case. the net result
is translocation of the c-myc gene from its normal
position on chromosome 8 ro a new location adjacentto the immunoglobulin heavy chain locus on chromosome 14. The c-myc transcription has been found to
be increased (up to 20 times) over the normal level insome patients. while there is no clear increasedtranscription in others. but rather an altered geneproduct [B15, Dl4. MIS, S37, Tl2]. It is not without
significance that the other chromosomes involved withchromosome 8 in translocation events in Burkitt
lymphoma (chromosomes 2 and 22) contain the sitesof the immunoglobulin kappa and lambda chains,respectively: the lymphomas express the kappa chain

58

in the case of t(8:2) and the lambda chain in the caseof t(8;22) [C 19. K3, Kl I. MI 9).
199. Recently, Rabbits et al [R27] have provided
evidence suggesting that c-myc oncogene activation in
Burkitt lymphoma occurs by disruption of a normal
control mechanism in which the c-myc protein itself is
involved. A translocation similar to that in Burkitt's
lymphoma occurs in mouse and rat plasmocytomas[K3]. In the mouse, the terminal segment of chromosome 15, carrying the c-myc gene, rearranges withchromosome 12 at the switch region of the heavy
chain constant-region gene [C20, 014. S37, Tl2].
200. Another interesting correlation is between c-abl
and the translocation of chromosomes 9 and 22 inchronic myeloid leukaemia (CML). The chromosomal
regions involved are 9q34 and 22q 11. The oncogenec-abl has been localized to 9q34, and it was possible todemonstrate that. in CML cases with the Philadelphia
Ph I chromosome, the c-abl sequences have beentranslocated from chromosome 9 to chromosome 22
at a position distal to AKI (adenylate kinase-1) locus
on chromosome 9, resulting in the activation of c-abl
expression (05].
201. Mapping of the break-points on chromosome 9indicated variable distances 5' (upstream) of the first
known exon of c-abl: in one individual. the breakpoint was 14 kb 5' of the oncogene; and in others it
occurred further upstream and has not yet been
accurately mapped [H49]. The situation on chromosome 22 is different: the break-points in different
CM Ls occurred in a restricted region of about 5.8 kb:
this region was termed break-point cluster region or
bcr [G46, H50] and its consistent rearrangement in
CML patients strongly implicates it in the pathogenesis of CML.
202. Shtivelman et al. (S97] recently cloned andcharacterized complementary DNA of the normal andCML-specific c-abl RNA and showed that the latter isa hybrid molecule, a fused transcript of bcr and c-abl
sequences. Nucleotide sequence analysis indicated thatthe protein translated from this 8-kb long fused RNA
has bcr information at its amino terminus and that it
retains most but not all of the normal c-abl protein.
The authors were able to show that the sequence at
the RNA function point is derived from the 3' end of a
78-bp bcr exon and the 5' end of the 174-bp abl exon
II. Therefore. the normal splice signals of the twogenes are used to form the hybrid transcript.
203. Although the information summarized aboveshows good correlation between the break-points
associated with consistent translocations or deletions
and the bands containing the oncogenes. no generalizations are as yet possible. For instance. c-fes. whichmaps at 15q24-25, has been correlated with the breakpoint in chromosome 15 which is involved in the transJocation t( 15; 17) associated with ANLL-M3. However.
recent evidence [Rl2, Y4] indicates that the breakpoint in chromosome 15. is at q22, i.e., at somedistance away from c-fes (a single chromosome bandmay contain 2000-3000 kb): furthermore. Sheer et al.

[S38, cited in RI2) have shown that the !Sq+
chromosome does not contain c-fes, which presumably
has been translocated to chromosome 17.
204. Likewise, the assignment of c-Ha-ras I to
I !pi I - pl5 raised the possibility that this oncogene
might be involved in the predisposition to nephroblastoma (Wilms' tumour. WT) seen in the aniridia-WT
association (A WT A) that is frequently caused by aninterstitial deletion of band I Ip 13. However. the results
reported by Martinville and Francke [M20] and Huerre
et al. [H 17] appeared to rule out such a possibility andalso showed that the c-Ha-rasl is located at Ilpl 1.12
or llpl4.2 [M20] or llpl5.I - Ilp15.5 [Hl7]. in any
case outside the region deleted in these patients (seealso [F23]).
205. Evidence is accumulating to suggest that themechanism(s) of tumorigenesis in Wilms' tumour
may be heterogeneous, as was demonstrated forretinoblastoma, another childhood tumour. In thelatter. the locus has been assigned to chromosome 13(13ql4). Although the hereditary pattern in familial
retinoblastoma is that of an autosomal dominant
mutation. there is evidence that the defect is recessiveat the cellular level [B3 l, C37]. At least four mechanisms for the development ofhornozygosity of recessive
alleles in the region of 13ql4 have been documented inretinoblastoma cells in culture: loss of an entire
chromosome [B3 l. C37], loss of one chromosome 13
homologue. with the reduplication of the remaining
chromosome 13 homologue [C37], somatic recombination with subsequent segregation resulting in homozygosity at all loci distal to the recombination site
[C37, 032], and deletion of a segment of chromosome
13 including 13ql4 [B31]. Data consistent with thesemechanisms have also been reported for Wilms'
tumour [F24. K25, 012, R28).
206. More recent results of Reeve et al. [R28], basedon DNA analysis of tumour DNA from patients withsporadic Wilms' tumour, show that. in both cases
studied, one of the two c-Ha-ras I allele was absent.
One tumour had a reciprocal translocation between
the short arm of chromosome 11 (at I lpl3) and thelong arm of chromosome 12, with no visible loss ofchromosome material. The other tumour had two cell
types: one showing marked hypo-diploidy with very
contracted chromosomes (so that no deletions could
be detected) and the other with apparently normal
karyotype. The authors point out that: (a) theirdemonstration of the loss of the c-Ha-ras I allele may
reflect events affecting the Wilms' tumour locus andthe quite distinct distally placed c-Ha-ras I gene; and(b) although the consensus of a gene mapping meeting
placed c-Ha-rasl at llpl5 [G30]. the localization ofc-Ha-rasl by Jhanwar et al. [Jl7] at I lpl4.l (using insitu hybridization studies with meiotic chromosomes)
places the above locus immediately adjacent to thetranslocation break-point (l lpl3) in one of the twotumours they studied. In their view, until the chromosome location of c-Ha-ras I has been determined withcertainty, one cannot exclude the possible functional
involvement of c-Ha-rasl in Wilms' tumour development. (See also [M65].)

I. ONCOGENIC TRANSFORMATION OF CELLSIN VITRO (IN TRANSFECTION EXPERIMENTS)
VERSUS ONCOGENIC ACTIVATION AND
PATHOGENESIS OF NEOPLASMS IN VIVO
207. As discussed earlier. the established mouse
fibroblast cell line NIH/3T3 has been used withremarkable success over the past few years, as aprincipal in vitro transformation assay to demonstrate
the presence of transfectable transforming genes in the
DNA of certain human and rodent tumour cell lines.
However. as is well known, carcinogenesis in vivo is amulti-stage process and it was thought. therefore, that
the NIH/3T3 cells-chosen because of their particularcompetence in taking up and expressing exogenous
DNAs-which are well adapted to grow indefinitely inmonolayer cultures, probably deviate substantiallyfrom normal target cells for oncogenesis in vivo. In
other words, the NIH/3T3 cells may already haveacquired several alterations usually developed by a cell
during its tumorigenic progression that make themsusceptible to tumorigenic conversion by a single-hit
event. The problem has been addressed bv threegroups of investigators, in three different k.inds ofstudy [L7. NI3. Rl5].
208. Newbold and Overall [N 13] found that Syrian
hamster dermal fibroblasts immortalized bv treatment
with chemical carcinogens (benzopyren;, dimethyl
sulphate and others) can be transformed to anchorageindependent growth by the human bladder carcinoma
EJ-c-Ha-rasl transforming gene and that the transforming activity was elicited only in newly immortalized cells. From these and other results. the authors
concluded that fibroblast immortality is a prerequisite
for transformation by the EJ-c-Ha-rasl gene.
209. Land et al. [L 7] reported that the EJ-c-Ha-ras I
gene transformed rat embryo fibroblasts only tolimited proliferation in semi-solid agar medium. However, in combination with the v-myc gene. or a geneconstruct consisting of an early simian virus-40
transcriptional promotor attached to c-myc. or certain
genes from DNA tumour viruses (middle and large T
antigen gene from polyoma virus 0 isolated as separate
molecular clones used either alone or in combination
with one another, or the EIA gene/' from adenovirus),
the EJ-c-Ha-rasl gene transformed rat embryo fibroblasts to tumorigenicity. Furthermore. Land et al.
found that EJ-c-Ha-ras 1, together .with the large T
polyoma antigen gene. or the EIA gene led to·unlimited tumour growth. whereas. EJ-c-Ha-rasl in
combination with the v-myc gene transfected into
diploid rat fibroblasts produced tumours of a limited
size in the nude mice. The authors concluded that the
myc gene greatly enhanced the transformed phenotype
created by the EJ-c-Ha-rasl but did not lead to fullytransformed cells after co-transfection with the genes
of DNA tumour viruses.
"The middle· T antigen induces morphological alteration andanchorage independence while the large-T antigen affects serum
dependency and cell immortalization.
?fhe adenovirus early region IA is responsible for establishment
function, i.e., the ability of cells to grow idefinitely in culture.

59210. The work of Ruley et al. [R 15] involved the useof primary baby rat kidney cells as recipients fortransfection with EJ-c-Ha-ras I, the polyoma virus
middle-T antigen and the adenovirus EIA gene. It
showed that the polyoma virus middle-T and EJ-cHa-ras I genes are individually unable to transform therat kidney cells. However, the adenovirus EIA provides
the functions required by these genes to transform
such cells following DNA-mediated gene transfer.
These results. like those of Newbold and Overall[N 13] and of Land et al. [L 7) mentioned above,strongly support the notion that carcinogenesis is atleast a two-step process to which different transforming genes must contribute and that separate
establishment and transforming functions are requiredfor oncogenic transformation of primary cells in
culture [C45, L2].
21 J. Further evidence that many neoplasms involve
activation of at least two distinct oncogenes, suggestingthat different oncogenes may function at different
stages of neoplasm development have been summarized
by Cooper (C40].

J. GROWTH FACTORS: MECHANISM OF
ACTION AND RELATION TO ONCOGENES
212. The proliferation of normal diploid cells in
culture is under the control of exogenous growth
factors. In the absence of the proper mitogens, cells
leave the cell cycle and become reversibly arrested in
the G /Go phase. Transformed cells, on the otherhand. have relaxed cell cycle control and may go
through the cell cycle in the absence of growth factors.
The lack of growth factor requirement thus seems to
be an important feature of transformed cells which
contribute to their defective growth control. The viewthat has gained increasing currency in recent years isthat proto-oncogenes are involved in cell cycle regulation and that any regulatory component in the chainof events linked to normal growth. if inappropriately
expressed or activated, may have oncogenic properties.
The subject has been extensively reviewed by Heldin
and Westmark [H32] and therefore only a few
essential aspects are pointed out below.
213. In addition to nerve growth factor [Yl2] there
are three well-characterized families of growth factors:
platelet-derived growth factor family (PDGF), epidermal growth factor family (EG F) and insulin-like growth
factor family (IGF) (see [C46, S67, W27 and Z3] forreviews). Evidence that a viral oncogene may code fora growth factor comes from a number of recent
studies. For example, the sis oncogene of the simiansarcoma virus displays striking homology to the
PDGF [033, W28], a small polypeptide that stimulates
growth of connective tissue cells [S67]. A portion of
the sequence of the receptor for EGF is virtually
identical to another oncogene (erb-B) carried by avian
erythroblastosis virus (034). Moreover. several growth
factor receptors. including those for PDGF [C47, EJ5.
N22], EGF [H34, U7] and insulin [K26. P24] as well
as 10 different oncogene protein products [B 13) sharefunctional homology in that they are all associatedwith tyrosine kinase activity (see Table 16).
60

214. Information on growth factors and their receptors has been obtained in studies in which BALB/c3T3
fibroblasts, arrested in their growth by serum starvation,
have been allowed to undergo synchronous progression
through the cell cycle, by the addition of serum. In theabsence of serum. 3T3 cells enter G 0 in which cell
division and growth stop and many metabolic processes are arrested. The addition of serum initiates acomplex series of events (G 1). which culminates 14-20 hlater in DNA synthesis. The PDGF is the serum
component that renders 3T3 cells competent toundergo DNA synthesis [Al2, D35. H35], althoughadditional components such as EGF and the somatomedins (progression factors) seem to be necessary tomaintain cell viability [P25, S68-S70. V7].
215. The availability of cloned DNA probes for avariety of cellular genes has made it possible toundertake studies on transcriptional events resulting
from growth factor stimulation [C48, G3L K27]. For
instance, Kelly et al. [K27] showed that PDGF (fibroblast mitogen). lipopolysaccharide (B-cell lymphocyte
mitogen) and concanavalin-A (T-cell lymphocyte
mitogen) stimulate the expression of c-myc (whose
abnormal expression is implicated in a wide variety ofnaturally occurring tumours) in those cells that
normally respond to them: within 1-3 hours after the
addition of these mitogens to the appropriate cells,c-myc mRNA concentration was increased between
10- and 40-fold with a return to baseline levels wellbefore the onset of DNA synthesis. The activation ofc-myc by PDGF is particularly noteworthy in the lightof the very close relationship between the sequence of
one chain of PDGF and that of the product of thec-sis oncogene [033, W28].
216. Greenberg and Ziff [G31] measured the effectof a mitogenic signal on the transcription of a range ofdifferent c-onc genes using isolated nuclei from
BALB/c3T3 cells. Within 15 minutes of serum stimulation. there was a striking ( 15-fold) increase in thetranscription of c-fos (the normal cellular homologueof the transforming gene of FBJ-osteosacroma virus).
which returned to its initial levels within 30 minutes.
No further changes in c-fos transcription were observed
as the cells progressed from G I to S. The authors also
observed a small but reproducible increase in c-myc
transcription after serum stimulation. which peaked
approximately 2 hours after stimulation and decreased
significantly within 4 hours. Under similar conditions.
many other c-oncs displayed barely detectable levelsof transcription at all times after serum stimulation.
217. Campisi et al. [C48] measured c-myc expression
during growth and quiescence in three cell lines: thenon-tumorigenic mouse fibroblast line A3 l and twotumorigenic derivatives, BPA3 I and DA3 I. bothtransformed by chemical carcinogens. The growth of
all these lines can be arrested by starvation (though
the tumorigenic cells are slower to respond). Whilearrested A3 l cells cease to express c-myc. the transformed cells continue to express c-myc at the samelevel after arrest. It would thus seem that c-myc
expression, and thus growth competence, is no longer
subject to regulation by serum growth factors after
transformation. The authors point out that theseobservations are consistent with the existing evidence
that the activation of c-myc depends on the disruptionof the genetic control of its expression and not with
mutations affecting the function of the product (asseems to be the case with respect to c-oncs of the ras
family).

discovered homologies is the realization that at least
some of the cellular sequences can become activatedthrough certain mechanisms. leading to qualitativeand quantitative changes in the corresponding geneproduct.

218. These and other results support the view thatthe proto-oncogenes play an active role in the regulation and control of cellular proliferation and thatgrowth factor independence and autonomous growthof transformed cells might be due to the constitutive
expression of any of the controlling elements along thenormal mitogenic pathway. The constitutively expressed factors. which function as transforming proteinsin the malignant cell, may be encoded by oncogenes.
or, alternatively. their expression may be under thecontrol of oncogenes.

223. The discovery that the genetic lesion leading to
the activation of the c-Ha-ras I proto-oncogene inhuman bladder carcinoma is a point mutation resulting
in the replacement of glycine by valine at the12th aminoacid in the protein product of the gene. led
to other discoveries: the c-Ki-ras2 and its acdvatedversion in certain lung and colon carcinomas differ by
the same G - A transversion at the 12th codon: incertain other cell lines derived from other tumours,
mutations at position 61 were responsible for thetransforming activity elicited in NIH/3T3 cells. It
would thus appear that codons 12 and 61 are themajor hot spots for activation of proto-oncogenes(members of the ras family) studied so far.

K.

SUMMARY AND CONCLUSIONS

219. This section contains a discussion of the exciting
advances that have been made during the last few
years in the study of the genetic basis of cancer: theseadvances have come about from convergent researchon retroviruses. DNA-mediated gene transfer, application of recombinant DNA methods and cell genetics.
220. The genomes of retroviruses contain specificgenes (viral oncogenes or v-oncs) responsible for the
production of tumours in a number of avian andmammalian species. The discovery that mammalianand other genomes contain nucleotide sequences
related to v-oncs and that these cellular protooncogenes have oncogenic potential, and the identification of the activated forms of the proto-oncogenes(transforming genes) in tumour cells. have helped tocatalyse and accelerate research in this area. bringing
the understanding of the genetic basis of cancer a step
closer.
221. The potential transforming activity of cellular
genes has been studied in transfection assays. In theseexperiments. normal cellular DNA fragmented to sizesof about 0.5-5 kb and transfected to NIH/3T3 cells(recipients) induced transformation with relatively low
efficiencies. However. DNAs of cells transformed bynormal cellular DNA fragments induced transformation with high efficiencies in secondary transfection
assays. The transforming activities were found to belocalized to discrete DNA segments present in thetumour cell genome. Several of these have been
isolated using molecular cloning techniques.
222. The relationships between different cellulartransforming genes detected in transfection assays
have been clarified by the discovery that many ofthese represent cellular homologues of the oncogene
c-Ha-ras of the Harvey murine sarcoma virus or the
oncogene v-Ki-ras of the Kirsten murine sarcoma
virus. A number of new transforming genes notbelonging to the ras family have also come to light.
Currently, we know of at least 17 distinct cellular
oncogenes, each with a corresponding retroviral oncogene. The most obvious consequence of these newly

224. The first case of proto-oncogene activation in a
non-virally induced tumour by a process of DNA
transposition has been reported. There are now anumber of instances in which proto-oncogene activation seems to be mediated through chromosomal
rearrangements. In these the remarkable concordances
between the chromosomal localization of the cellular
oncogenes and the break-points involved in somespecific chromosomal aberrations are noteworthy(Burkitt's lymphoma, chronic myeloid leukaemia,
acute lymphoblastic leukaemia, etc.). These findings
do not necessarily mean that all non-random chromosomal changes involved in several kinds of tumours
are associated with events in which the activation of
proto-oncogenes occurs through chromosomal changes
involving the proto-oncogenes.
225. Other possible mechanisms for proto-oncogene
activation have been explored in experimental systems,
which include: attaching of strong promotors/
enhancers to proto-oncogenes, inserting promotors/
enhancers near proto-oncogenes and then using these
constructs to study transformation in NIH/3T3 cells.
The evidence that there is amplification of. protooncogenes in certain tumour cell lines has Jed to
speculation that such amplification may be involved atsome stage in carcinogenesis.
226. The apparent conceptual conflict between cellulartransformation as studied in transfection assays andthe multi-step nature of carcinogenesis in vivo has
been the subject of research in a number of laboratories. It is clear that fibroblast immortality is a prerequisite for transformation in vitro and that while
NIH/3T3 cells are a useful tooL they deviate fromnormal cells in their ability to grow indefinitely inmonolayer cultures and thus probably contain alreadyseveral alterations that make them susceptible totumorigenic conversion in vitro. Furthermore, evidence
is emerging to suggest that different transforming
genes must contribute to the carcinogenic process andthat they function in different stages of neoplastic
development.
227. The question of the normal cellular functions ofproto-oncogenes has been investigated in a number of61

studies. The findings that several protein products ofviral oncogenes share homologies with growth factors
and/ or receptors support the view that proto-oncogenes
are actively involved in the regulation and control ofcellular proliferation and that growth factor independence and autonomous growth of transformed
cells probably result from the inappropriate expression
or activation of these controlling elements.

IV.

MOVABLE GENETIC ELEMENTS ANDSPONTANEOUS MUTATION RATES

228. The conceptual framework for one of the mostactive areas of current genetic research-the one
dealing with movable genetic elements-was establishedby McClintock over three decades ago [M3-M7].
Meticulous analysis of the results of crosses between
certain generically marked maize plants Jed her topostulate the existence of what she called controlling
elements. which had the capacity to move from one
chromosomal location to another. When a controlling
element was inserted next to a gene. it inhibited thatgene's activity and, conversely, when the element
moved away from a gene, the activity of that genereappeared. However. the realization that the phenomenon observed by McClintock and the effects
generated as a consequence (induction of novel
variation at particular loci, localized high mutability,
localized chromosome breakage) are not genetic oddities but represent events of a more general nature, had
to await confirmation in other organisms and theavailability of means to explore the problem more
fully.
229. Movable genetic elements have now been discovered in a number of species including bacteria,
blue green algae, yeast and Drosophila. The possibility
exists that they will eventually be discovered in man.
Some of these movable genetic elements have beencharacterized at the molecular level and there isevidence for some similarities between these and theintegrated proviruses of some vertebrate retroviruses.
The current state of the art with respect to movable
genetic elements has been the subject of a recent
symposium [C6] and a book edited by Shapiro [S72].
Some salient aspects of these elements are briefly
discussed below, focusing on bacteria, yeast, Drosophila and the mouse and on some speculations
concerning their possible presence in man.

A.

BACTERIA

230. Some of the first major insights into movable
genetic elements came from bacterial genetics in thelate 1960s and early I970s with the discovery of small,transposable DNA sequences that can insert themselves into bacterial chromosomes or into DNA oftemperate bacteriophages or plasmids [F3, J2, SI30].
Some of these DNA sequences are devoid of arecognizable phenotype, and contain no detectablegenes unrelated to insertion functions [C49, C50J.
These are called insertional sequences (IS). Other
62

insertions carry additional detectable gene(s), unrelated
to insertion functions and are called transposons (Tn).
The properties of these elements have been extensively
reviewed (B4, C62, H36, I8. K4, K28, K29, S9, SlO].
231. Insertional sequence elements were first detected
as a cause of spontaneous mutations in E. coli
(reviewed in [S9]). Insertion of discrete segments
0.8-1.5 kb Jong led to interruption of structural genes
or strong polar effects. Reversion to the original
phenotype was observed. Occasionally, insertion of an
IS element in front of a gene resulted in an activationof gene expression.
232. Recent developments in rapid DNA sequencing
techniques have allowed the elucidation of the structure of several IS elements. The IS elements range
from 0.8 to about 1.5 kb in size. The termini of thesecarry perfect or near perfect inverted repeats of about
10-40 base pairs; they are believed to serve as
recognition sequences for the transposition enzymes intheir role of fusing the ends of the IS elements withthe target DNA. The number of IS elements varies
from I to 40 and on the basis of the copy number of
the known IS elements (over lO have been wellcharacterized in E. coli), it is possible to calculate thatat least l-2% of the E. coli K 12 chromosome consistsof various IS elements [18).
233. Integration of the IS elements shows a certainsite specificity and the degree of specificity varies forthe different insertion elements. Their insertion into a
gene abolishes its functions. leading to a mutation.
Most of these mutations revere to wild type, indicating
the exact integration and excision of the IS element.
Reversion cannot be enhanced by mutagens. Bothintegration and excision are independent of RecA [J3,J4]. In addition to exact excision, secondary chromosomal aberrations (deletions, duplications) are often
detected in the vicinity of IS elements. The presence ofan inserted segment of DNA can be demonstrated bya number of biophysical techniques.
234. Insertion of an IS element is accompanied by ashort duplication of target DNA such that thetransposed element is flanked by direct repeats oftarget DNA. The length of the duplication, but not its
nucleotide sequence, is usually specific for the particular IS element. Different IS elements transpose atdifferent rates. depending on the genotype and thephysiological state of the host organism, as well as on
the particular structures of the donor and target
sequences. Rates varying between 10-3 (or perhaps
more usually 10- 5) and 10-9 per cell division per IS
element. have been reported.
235. Many of the notions and concepts about theproperties of IS elements are those extrapolated fromthe results of transposons (Tns). which contain one ormore genes in addition to those involved in insertionfunctions, rendering them more amenable to study.
Among the transposons. the properties of the membersof the Tn3 family and Tn 10 have been extensively investigated [H36, K28]. About 20 transposons belonging tothe Tn3 family have been isolated from at least
50 different bacterial genera. Most of these genera are
gram-negative bacteria but Tn551 (encoding erythromycin resistance) was isolated from Staphylococcus
aureus [K41 ]. Other members of the Tn3 family
encode tetracycline resistance. and several others
encode multiple antibiotic resistance.
236. Tn3 and its immediate relatives Tn 1 and Tn2,
carry the most common penicillinase gene found ingram-negative bacteria. These transposons are foundin plasmids from antibiotic-resistant bacteria, but may
transpose to bacteriophages and to the chromosomesof E. coli and many other bacteria. The Tn3-likeelements have a similar structure and are transposedby a common mechanism. Tn3 is 4957 kb long andhas a short inverted sequence of 38-bp at its end. The
presence of such short repeated sequences is acharacteristic feature of the members of the Tn3
family, other common features including the following:(a) they encode a related high molecular weightprotein that is essential for transposition: (b) they aretransposed by a two-step mechanism (in the first step,
the donor and recipient DNA molecules become fused
via a duplication of the transposon (co-integrate). andin the second. the duplicated copies undergo recombination to yield a precise transposition): (c) mostinsertions are clustered in narrow regions of the target
DNA, which are A+ T rich [H36]: (d) they exhibit aphenomenon known as transposition immunity. which
limits multiple insertions of the same transposon intoa plasmid [R29]: and (e) there is evidence that hostfunctions are required for transposition.
237. The ampicillin transposon Tn I transposes fromplasmid RP4 into E.coli chromosome with a frequency
of about ( 1-3) 10- 4/cell. Treatment of the bacteriawith UV light. mitomycin-C or nitrosoguanidine leads
to an increase in the frequency of transposition.
Mutation in lexA and RecA genes blocks the inductionof transposition: chloramphenicol (protein synthesis
inhibitor} and rifampicin (inhibitor of transcription)
decrease the effect of UV-induced transposition [K42,S98]. Mutants that lead to high efficiency of transpositions (het mutants) have been isolated [Il2]. (Seealso [ G4 7].}238. The intact TnlO transposon is 9300-bp long andhas 1400-bp inverted repeats at its ends. The 6500-bp
of non-repeated material include the 2500-bp tetracycline-resistance determinant and (probably) other
determinants the functions of which are not known[123. K43]. The 1400-bp repeats at the ends are closely
related but non-identical IS elements, IS 10-right andIS 10-left, that encode the sites and functions responsible
for Tn 10 transposition and that co-operate with one
another to mediate transposition of the intervening
tetracycline determinant [F36]. Of these two ISs (rightand left). the IS 10-right is the functional transposition
module.in TnlO. Insertion of a transposon into a genecauses a mutation in that gene and inexact excisionleads to deletions adjacent to the transposon. For
details of these and other effects. as well as molecularmodels that have been proposed, see [H36] and [K28].

B.

YEAST

239. The Ty (transposon yeast) elements were firstdescribed by Cameron et al. [Cl5]. who characterizeda Ty sequence (the Ty I) present at the yeast SUP4
locus (tyrosine-inserting suppressor locus: one of theeight yeast tyrosine transfer RNA genes). In thisstudy, the evidence for the transposability of Tyl was
obtained both from the observation of the location of
Tyl in different isolates and from the observation ofsuch changes during growth of a single isolate S288c
of Saccharomyces cerevisiae. The Tys are a family ofdispersed repetitive DNA sequences [R30]. Eachmember consists of a central region of about 5.6 kb
pairs of DNA flanked by direct repeats of approximately 330 bp sequence called delta: there are 30-35 Ty
elements per haploid yeast genome. In addition to thedelta elements associated with Ty sequences, the yeast
genome contains at least 100 solo delta sequences. The
presence of terminally repeated sequences indicatesstructural similarities to the prokaryotic transposons.
The Ty elements show considerable sequence divergence and differ from each other by simple base pairchanges and also by insertion. deletion and substitution mutations. Likewise, the delta sequences associatedwith the Ty elements show considerable sequence
heterogeneity.
240. Mutants that arose as a result of transposition
of Tys have been isolated at the histidine-4 locus.
alcohol dehydrogenase locus and cytochrome-c locus[E6. R31, R32. W3l]. Cloning of these genes. restriction mapping and sequence analysis indicated thatthese mutations resulted from the insertion of an
intact Ty element into the 5' -non-coding region of theaffected gene. Insertions into structural genes appear
to be rare, but more data are needed before generalizations can be made [R30, S99].
241. As in the case of bacterial transposons, Tytransposition results in a duplication of the target
DNA at the site of transposition. In addition to theability of Tys to transpose. they have been implicatedin a variety of genomic rearrangements. including
deletions. tandem duplications. inversions and chromosome translocations [R30]. These various rearrangements appear to be the result of general recombination
between different homologous Ty elements and not
the result of the transpositional activities associatedwith Tys.

C.

DROSOPHILA

242. Presumptive evidence for the occurrence ofmovable genetic elements in the Drosophila genome
first came from genetic studies of mutable or unstablegenes ([G8-G 10): reviewed in [G3, G4 and G7]). Suchmutations were found to revert to wild type at highrates and generate chromosomal rearrangements. By
analogy with what is known about the genetic
properties of IS-mediated mutations in bacteria, theabove observations were interpreted on the assumption of involvement of IS elements of unknown origin.
63

I.

MR-chromosomes243. In 1971, Hiraizumi [H6] reported low but
significant levels of spontaneous recombination incertain types of hybrid males whose second chromosomes were derived from flies captured in nature.
Such chromosomes were designated as MR (for malerecombination). It soon became clear that MR chromosomes are present in all the natural populations ofDrosophila studied in this respect. sometimes at highfrequencies (reviewed in [T6]). In studies of Drosophila
populations caught at a number of widely separated
sites in the Soviet Union, Berg [B6] found that:(a) males with the recessive singed bristles (sn) occurred
relatively frequently in collection of wild flies; and (b) an
inordinately high frequency of sn mutants occurredamong the descendants of wild-caught males whenbred in the laboratory. A subsequent detailed analysisof the sn mutants derived from wild-caught males
showed them to be mutationally unstable: they reverted
to wild type frequently and/or generated deletions at
the sn locus. It was hypothesized that the sn mutants
were the product of MR action. The test of thishypothesis [G6. G7] demonstrated its validity: MR
chromosomes of diverse geographical origins generate
unstable sn mutants at very high frequencies (100-200
mutants/10 5 chromosomes). Concurrent tests of MR
chromosome on several X-linked loci demonstrated
that three of them (ras. raspberry eye colour; y, yellow
body colour: and cm. carmine eye colour) were also
susceptible to MR action. The ras and y mutants, aswith the sn mutants, proved to be mutationallyunstable, whereas a few of the cm mutants tested werestable (see also [E3]).
244. There is evidence that MR element(s) may
themselves transpose [M8. Sl2, YI]. Transposition of
the MR element implies that, generally, the element
can be mapped to a specific chromosomal location.
For the second chromosome, a major MR element
exhibiting both male recombination-inducing andmutator capacities has been mapped to the left arm of
this chromosome [M8. S 13. S 14]. The analysis of thenature and distribution of MR-induced mutations[E26] showed that: (a) most of these mutations (10 out.of 13 tested) are due to the insertion of a particularmobile element (P-element): and (b) the distribution ofthese mutations [E26] is not identical with thatreported for mutations [L58].
245. In experiments on the effects of MR on radiationinduced genetic damage in males. Sobels and Eeken[S 15] found that the frequencies of autosomal translocations in the MR-containing males were not significantly different from those in males that did notcontain the MR: however, the frequencies of sexlinked recessive lethals were higher in the MR than in
the non-MR males. A more specific interaction of xrays and the mobile element P was observed whenrecessive lethals were induced by x irradiation in achromosome that carried P-elements at known sites.
The cytological analysis. using polytene chromosomes.
showed that among these lethals, there were specificbreakage events at the site of the inserted P-elements[E3I].
64

246. In other studies, Eeken and Sobels [E4] focused
attention on the reversion of a specific insertion
mutation (sn~IR. which was originally induced by MR
and which reverts spontaneously to sn+ under the
influence of MR) by two chemical mutagens, ethyl
nitrosourea (ENU) and methyl methanesulphonate(MMS). Since MR acts primarily in gonia. the two
chemicals were fed to first instar larvae. With ENU(I mM and 3 mM). the reversion of sn to sn+ waseither unaffected ( I mM) or increased by a factor of
2 (3 mM), suggesting a possible threshold. With MMS,
however. there were no demonstrable increases inreversion frequencies in the MR+ MMS group relative
to the MR group.
247. Eeken and Sobels [E5] also examined the extent
to which excision and incorporation of DNA sequencesby MR is affected by enzymatic pathways involved in
DNA repair. MR chromosomes were introduced into
males deficient for excision repair (mei-9 mutant) or
post-replication repair (mei-41 mutant) or into males
carrying both repair-deficient mutations. MR activity
was recorded using the induction of sn and ras
mutants as indicators. The interesting finding wasthat. in the double mutant with MR, there was a
striking enhancement (by a factor of 5-10) of thefrequencies of these mutational events. relative to the
double mutant without the MR. With the single
mutants mei-9 or mei-41, there was also an enhancement of the frequencies of sn and ras mutants in thepresence of MR, but this effect was of borderline
significance. In another study [E6], repair deficiency(again, mei-9, mei-41 or both) reduced the reversion ofsn to sn+ by more than 50%, suggesting that DNA
repair defects do interfere with the action of MR.

2.

The P-M system of hybrid dysgenesis

248. In the mid-1970s, Kidwell and Kidwell [K44],
Kidwell et al. [K5] and Sved [SlOO] advanced aunifying general hypothesis to account for the varietyof unusual phenomena that have been reported in theliterature on Drosophila, such as those associated with
the MR chromosomes. and introduced the term
hybrid dysgenesis. This was defined as ''a syndrome ofcorrelated genetic traits that is spontaneously induced
in hybrids between certain mutually interacting strains,usually in one direction only" [K5, K44]. These
include such phenomena as male recombination. maleand female sterility, transmission ratio distortion.
chromosomal aberration and high mutability, which
occur only in certain classes of outcrossed flies (andthus never within homogeneous strains). By examining
the F I hybrids from a series of inter-strain crosses.
Kidwell et al. [KS] found that each strain could beunambiguously classified as either P (paternally contributing) or M (maternally contributing). The traits
were most pronounced in the progeny of crosses
between M females and P males, greatly reduced in
the reciprocal cross and virtually ·absent in theprogeny of all P x P or M X M crosses. In most cases,
the P strains were those recently derived from wild
populations and the M strains were long established{lO years or longer) laboratory stocks. The traits
themselves showed common features, such as pre-ferential occurrence in the germline. The subject ofhybrid dysgenesis has recently been reviewed [85, 856,E25].
249. Further investigations into the inheritance of
these traits revealed that the causative factors could bemapped to multiple locations in the genome of
P strains [E24. SlOO]. The number and genomic locations of these factors varied among the different
P strains and not in the M strains. Such elements
operationally defined by their dysgenic traits were
designated as P factors. Considerable heterogeneity
among P factors was implied by the observation thatthe relative magnitude of each dysgenic trait was afunction of the particular P strain involved. In its
simplest form. the hypothesis of hybrid dysgenesis
states that P factors are present in P strains where
their transposition is repressed and are absent from M
strains. When chromosomes carrying P factors areplaced in the M cytotype. these elements become derepressed and transpose at high rates. Among othereffects, P factors would then induce mutations byinserting into and disrupting genetic loci. Such dysgenesis-induced mutations would be expected to bestable in the P cytotype. where P factor transposition
is repressed, but unstable in the M cytotype, where
they could revert by excision of the P factor. Biochemical evidence for this hypothesis has now beenobtained. Using molecularly cloned ?-elements fromhybrid dysgenesis-induced mutations at the w (white)
locus. Rubin et al. [R56] and Bingham et al. (855]
were able to show that such mutations are due toinsertions of ?-elements; these elements bore homology
to one another in sequence, although heterogeneous insize (0.5-1.4 kb in length). They were present in about
50 dispersed copies in P strains (as seen by in situ
hybridization of labelled P element sequences topolytene chromosomes) and, as would be expected.
absent in M strains.
250. O'Hare and Rubin [O 17) have now sequenced
the DNA of what they consider the primary Pelement. It is 2907 bp long with precise inverted
repeats of ·31 bp at the termini: unlike many othertransposable elements, it has no long direct repeats. In
addition to the 2.9 kb element, there are many smaller
elements, and sequence studies have revealed that they
can all be derived from the major one by internaldeletions that always leave the 31 bp repeats intact.
The size of the deletions range from a few hundred bp
to more than 2000.
251. A mutation caused by the presence of a Pelement can be considered an insertion mutation if theparental chromosome is known to lack the element atthat locus. Such is the case for four independent
mutations at the w locus [R56, SlOI]. In addition,dght independent mutations at the singed (sn) locus
and one at the yellow (y) locus were examined by insitu hybridization techniques and each had a P site at
the expected cytological position that was not present
in the parental chromosome [E25]. Examination of
the structures of the wild-type phenotypic revertantsof four different P-element insertion mutations inwhite (w) indicated that phenotypic reversion was
accompanied by excision of the P-element [R56]; theexc1s1on appeared to be precise within the limits ofresolution of the analysis, which was estimated to beless than about 50 bp. There is evidence for a \'arietyof partial excisions at they locus (for details see [E25]and [R33]). Three of the P-element insertions at the w
locus were found to occur at tightly clustered or
identical sites within the w locus, suggesting someinsertional site specificity. Site preference for insertionof ?-elements could account for the observed differences in the frequencies at which mutations arise
at various genetic loci in dysgenic individuals.

3.

The 1-R system

252. Another independent system of interacting
strains is that denoted by 1-R (for inducer andreactive) described by Picard [P5) and discovered
through its effect on female sterility. The interaction
postulated here is between chromosomally locatedinducer factors (I) and a cytoplasmic state called
reactive (R). The combination in a female fly of astrong I factor, which can be linked to any of the fourchromosomes, and an R cytoplasm leads to female
sterility. In females heterozygous for the I factor,
a phenomenon called chromosomal contamination
occurs. ln such females, homologous or non-homologous chromosomes lacking I acquire it. Genetic
evidence favours the interpretation that chromosomal
contamination involves the transposition of 1 fromone chromosome to another [B5, PS]. One otherfeature of the I-R system is its mutator activity. Afterthe transiently sterile females recover fertility, sexlinked recessive lethal and visible mutations occur
among their progeny at very high frequencies; therecessive lethals do not map randomly and, among thevisibles. some occur in clusters and some are unstable,
reverting at high rates [P5). The mutator effects
parallel those observed for MR and suggest that theunstable mutants are probably insertions.

4.

Interrelationships between the MR, P-M and1-R systems

253. The designations of the three mutator systems
discussed above reflect the mode of origin of themutator. Thus, MR reflects the increased frequency of
mitotic crossing-over occurring in males carrying
particular second chromosomes isolated from wildflies [H6]: hybrid dysgenesis refers to the highfrequency of occurrence of dysgenic traits among theprogeny of wild type (natural populations) males
crossed to laboratory strains of females: and the I-R
refers to a type of transitory sterility observed amongfemales obtained by crossing particular laboratory
strains (all these in Drosophila). All these mutatorsystems are of interest. not only because they generatehigh frequencies of mutations at various loci (mutants
which to a large extent appear to be insertion events)
but also because they produce all types of chromosomal aberrations. It is worth noting that both the
MR and P-M systems involve chromosomes derived
from wild flies.
65

254. The question of whether there exists some interrelationships between these mutator systems. or whether
they are all different, has been raised (see Green [G48]for a recent review). As may be recalled. I-R as amutator functions only in females, in contrast to MR
and P-M where mutator activity occurs in males andfemales. Thus it is reasonable to conclude that 1-R
represents a discrete mutator system, distinct fromMR and P-M. The MR and P-M systems share a numberof features including the following: (a) mutator
activity is manifest only when the chromosomes (MR)or chromosomes (P-M) are patroclinously inherited;(b) there is an inordinately high frequency of unstablemutants induced at the X-chromosome locus, sn; and(c) there are elevated frequencies of male mitotic
crossing-over. However. comparison of results ofstudies with MR and P-M system show that mutator
action is not a predictor of hybrid dysgenesis [G4S].
255. Biochemical evidence substantiating the thesis
that. in the P-M system. the P-elements are involved
has already been discussed. It was also shown, in
experiments of two kinds, that MR activity is associatedwith the ?-elements. In one experiment [G4S]. Southernblot analysis for the presence of l .2 and 3 kb
P-elements in DNA extracted from larvae of MR
strains was carried out. In the other [E26], sex-Jinked
recessive lethals isolated using an MR chromosome
were mapped genetically and further examined in
hybridization experiments in situ using a cloned
P-element, described as p6.1 by Rubin et al. [R56]. In
the first study. all the six strains examined carried theP-elements: in the second, in I O out of 13 cases,
hybridization was observed over the region where thelethals were genetically localized. It thus seems clearthat the MR and P-M systems are "part and parcel ofthe same mutator system" [G48]. The absence ofgonadal dysgenesis in the MR system needs further
study.

5.

Mobile genetic elements inferred from biochemical
studies

256. The existence of the mobile genetic elements
discussed so far was inferred from genetic studies,
although molecular methods were subsequently used
to identify them. Quite independently of these studies.
students of Drosophila molecular genetics have identified the presence, in Drosophila DNA, of several
dispersed moderately repetitive DNA sequencesq [R5,
R33, S 102]. In fact. such repeated gene families
compose nearly one-sixth of the Drosophila chromosomal DNA and most of the fly's middle repetitive
DNA. The genes that characterize these families aredesignated as 412, copia and 197 among others [A4.
F4, I3, R5].
257. The first biochemical evidence that elements ofthese repeated gene families transpose came fromobservations of polymorphisms in the number andqEukaryotic DNAs contain different sequence classes whose
constituent members can be broadly classified as: (a) unique(approximately one copy per haploid genome); (b) moderately
repetitive (- 106 copies per haploid genome); and (c) highly
repetitive [B60].

66

positions of several dispersed, moderately repet1t1ve
DNA sequences between different stocks of Drosophila[13, SI 03]. between cell lines and their parental fly
stocks [P38) and between Drosophila melanogasterand its sibling species. Drosophila simulans [L35].
TI1ese observations were soon extended to include alarge number of other moderately repetitive DNA
sequences [048, M66, P39, Y2, Y3]. The biochemicaland genetic properties of these have been recently
reviewed [G49, R33] and only a few important points
are mentioned below.
258. On the basis primarily of internal structures, thedispersed moderately repetitive sequences have beenclassified by Rubin [R33] into: (a) copia-like elements,
including copia, 412, 297, mdgl (mobile dispersed
genetic elements) (the fragments described in earlier
publications as Orn 225 and Dm 234 are part of thecomplete mdgl element [B104]), gypsy and a numberof others not yet well-characterized; (b) fold-back(FB) elements; (c) P-elements: and (d) other dispersed
repetitive families.
259. The copia-like elements range in size from 5 to
8.5 kb. carry long direct terminal repeats (169-571 bp)
and occur at approximately 10-100 copies in widely
scattered locations in the chromosome arms as well as
the centric heterochromatin. There is now direct
evidence that several Drosophila mutations (e.g.,
white-apricot (wa), bithorax (bx)) result from theinsertion of copia-Iike elements [B57, G 11, G50, L36].
Rubin et al. [R56] obtained evidence for the transposition of a copia element into the w locus, following
a cross between two strains known to produce hybrid
dysgenesis. It is possible that hybrid dysgenesis, inaddition to the dramatic increase it produces in thetransposition rate of ?-elements, also increases therate of transposition of copia-like elements.
260. The FB elements described by Potter andcolleagues [P40] comprise a family of heterogeneous,
but cross-homologous sequences ranging in size froma few hundred base pairs to several kilobases. Each
FB element carries Jong terminal inverted repeats; insome cases, the entire element consists of these
inverted repeats and in other cases. a central sequence
is located between these inverted repeats. The genetic
properties of mutations caused by the insertions of FB
elements are revealed by the behaviour of two suchmutations, we (white-crimson) and w0 ZL (white dominant zeste-like) [K45, L37]. A large transposable
element of the X-chromosome called the TE. whichusually carries w and an adjacent gene (roughest). has
FB elements flanking it [G50] contrary to the original
hypothesis of Gehring and Paro [G 11] that the copia
transposable element is present at both ends of TE,
261. The properties of P-elements have already been
described. Other dispersed repetitive families include
insertions ranging from 0.5 to 5 kb in length found inribosomal genes, as well as what are referred to as"scrambled clustered arrays of moderately repetitive
DNA sequences", which are 0.3-1 kb in length. Theirproperties have been reviewed [R33].

6.

Similarities between Drosophila transposable
elements and those in other systems

262. The possibility that the transpositions of discreteelements are analogous phenomena in prokaryotes
and eukaryotes such as Drosophila or yeast has been
discussed. For the element copia in Drosophila, thisanalogy holds at the molecular level [R6]. Rubin et al.
[R6] have listed the following common features
between copia and bacterial transposons: (a) theelements are inserted within many different target
sequences that share no obvious homology to eachother or to the ends of the transposable elements:(b) upon insertion, the elements generate a duplication,in direct orientation. of a few base pairs of the target
sequence found immediately adjacent to the ends ofthe element; (c) an element has the same terminal
sequence when inserted at different chromosomal
locations: and (d) the element contains a terminal
repetition binding the main body of the element.
These features are also shared by the Ty I element in
the yeast Saccharomyces cerevisiae [F5. G 12] and theOrn 297 in Drosophila. The integrated proviruses of
the avian retrovirus SNV (Spleen Necrosis Virus,
an avian reticuloendotheliosis virus) [S 17] and the
Moloney murine sarcoma provirus (M-MuSV) [D6.
M9] also share the structural features mentioned
above. although they are not known to transpose.
Although at present their significance is not clear. thestructural analogies are striking.
263. There is now evidence that among eukaryotes,
the transposition of copia-like elements in Drosophila
and of Ty I in yeast may involve reverse transcription.
i.e., their transposition is mediated through an RNA
intermediate ([B58. S 104]. also reviewed in [B59] and[G49]). Such transposable elements dependent onreverse transcriptase ha\'e been termed retroposons[R57] or retrotransposons [B59].

D.

MOUSE

264. The fact that the chromosomal DNA of inbred
and wild-type mice contains one or more copies
of endogenous murine leukaemia virus' (MuL V)
sequences and that the MuL V expression follows
Mendelian segregation in many strains. has been
known for some time (see Chan et al. [C22] andChattopadhyay et al. [C23] and references citedtherein). Several high leukaemic strains such as AKR,
C58, C3H/Fg, etc .. carry multiple copies (3-5 per
haploid genome) of ecotropic viral DNA sequences,whereas many low leukaemic strains such as OBA,BALB/c and C3H/He contain a single copy per
haploid genome. The structure of most ecotropic
MuLV genomes in high and low Jeukaemic strains issimilar to the prototype Gross-AKR MuL V. but theendogenous ecotropic MuL Vs are integrated at multiple
sites in mouse chromosomes in different strains,though possibly at a constant site within a strain.
'Endogenous MuL Vs have been divided into three related classeson the basis of their host ranges which are determined by the viralenvelope glycoprotein: ecotropic, which infect only murine cells,
xenotropic which infect primarily heterologous cells and amphotropic which replicate efficiently in cells of both types.

265. Jenkins et al [J7] studied the chromosomal
location of the DBA/2J ecotropic provirus Emv-3 bySouthern blotting and hybridization with an ecotropicspecific DNA probe in a number of inbred and speciallyconstructed hybrid mouse strains. It was found that
the Emv locus was near the d (dilute coat colour)
locus on chromosome 9 and that it co-segregated withthe d allele, suggesting close linkage. Analysis ofDNAs prepared from inbred strains and substrainsthat carried the wild-type allele of d. showed that they
did not carry the proviral sequence. Furthermore. a
spontaneous revertant (d - d+) in the DBA/2J strain,
was associated with the concurrent loss of most of theproviral sequence.
266. Further work [C76] demonstrated that, at least
for two germline revertants of d to d+, reversion was
accompanied by excision of the Emv provirus. by aprocess of homologous recombination between theproviral LTRs. Furthermore. this recombination process left behind exactly one LTR at the integration site
[H64] in d+ revertants. Thus, the proviral insertion
itself probably induced the d mutation by integrating
into non-coding regions in or around d. Theseobservations have been interpreted by the authors asproviding evidence for the thesis that the d locus
mutation in the DBA/2J mice is associated with the
integration of the provirus. The authors have evidence
for a close association between a unique MuLV
sequence and another coat colour mutation (the A>
locus). The question of whether the relatively highspontaneous reversion rates associated with the a andd loci (reversion rates about 3 times the forward
mutation rate) recorded in the earlier work of Schlager
and Dickie [S47] is in any way related to theintegration and excision of MuL V sequences is still
unclear, but. at least for d, proviral excision due tohomologous recombination may be responsible for theincrease in reversion rate.
267. The possibility of inactivation of cellular genes
through insertion of retroviral sequences in chromosomal DNA in vivo was investigated in the mouse by
Jaenisch et al. [J8]. Mouse embryos were exposed to
Moloney murine leukaemia virus [M-MuL V] and13 substrains were obtained in which the virus wasstably integrated into the germ line chromosomal
DNA, but at different locations or integration sites(the Mov loci). Twelve of these sub-strains weregenerated in experiments involving infection of preimplantation embryos and one was from infection of
post-implantation embryos. The substrains were maintained by mating normal females with males heterozygous for a given Mov locus. The virus-carrying
offspring were identified by testing for viraernia or forthe presence of virus-specific sequences in DNA fromliver biopsies. Each of the substrains carried one copyof the virus and the pattern of inheritance followed
Mendelian rules. For instance. in crosses involving
heterozygotes for a given Mov gene, the expected
number of Mov/Mov homozygous embryos at term
were observed. However. in one substrain that carried
the Mov-13 gene (this strain was the one derived from
infection of post-implantation stages), homozygosity
was lethal. Examination of pregnant females (thecross: +/Mov 13 X +/Mov 13) revealed that about6725% of the embryos were degenerated. DNA extracted
from these embryos invariably revealed homozygosity
at the Mov 13 locus. Schniecke et al. [SI33] haveshown that the Mov 13 provirus integrates into theal(l) collagen gene and embryos homozygous for thismutation begin to die at approximately day 12 whena I (I) collagen transcription normally becomes predominant in wild type.
268. There is one group of stable inserted elementsin the mouse. the intra-cisternal A-particle genes(IAPs). The IAPs are retroviral-like elements, but
appear to have no infectious or recognized extracellularphase; they are similar to the virus-like copia RNA
particle in Drosophila and are present in about 1000
copies per genome. Recently, Kuff et al. [K30] haveshown that these particles are responsible, by insertion.
for mutations involving the kappa immunoglobulin
light chain gene in mouse hybridoma cells andemphasize that the family of IAP-related genes may beconsidered as potential movable elements and a sourceof insertional mutations in the mouse.
269. Suggestions exist that other dispersed repeats(human Alu-type. see below) in the mouse-the BI
and B2 sequences-may also be candidates for transposable genetic elements. since they usually areflanked by short direct repeats, specific for each copyof the repeat. For details, see (G49, 19, K46. K47).

E.

MAN

270. The human genome contains between 300.000
and 500,000 copies of a 300 bp repeat sequence thatact as substrate for the Alu restriction enzymes andare referred to as Alu repeats [19. 124. S73]. Since thegenome contains 3 X 10 9 bp, these repeats must be oneof the most predominant groups of repetitive sequencesin the human genome and account for approximately
4% of the total DNA. The Alu repeats are present inall mammalian genomes and a 40 bp sequence appears
to have been conserved throughout mammalian evolution. Of particular interest is that most Alu elements
are flanked by direct repeats of sequences of some
7-20 nucleotides long [G33, H37], a feature incommon with mornble genetic elements: they are
therefore considered prime candidates for mobile
genetic elements within the genome.
271. There is the possibility that pairs of Alu
sequences might be able to act as pairs of insertion
sequences in transferring intervening DNA segments,
inter-Alu DNA around the genome, in other words.
acting the same way as bacterial transposons. Recently,
Calabretta et al. [C24] have found that both chromosomal and extra-chromosomal forms of inter-Alu
DNA in normal and human neoplastic tissues show
considerable inter-tissue and inter-individual variation(polymorphisms). From these and other results, theauthors have suggested that such chromosomal andextra-chromosomal segments of inter-Alu DNA reflect-'The name Alu derives from the fact that these sequences havebeen identified using restriction endonuclease Alul (from Arthrobacter luleus) which recognizes A G C T sequences in the DNA andcleaves them between G and C.

68

the presence of transposable elements. that they may
be involved in genetic rearrangements, and that theinterspersion of Alu repeats may arise from sequential
excision of the Alu sequence and their subsequent
reinsertion elsewhere in the genome.
F.

RELEVANCE IN THE CONTEXT OF RISKEVALUATION

272. The findings that transposable genetic elements
so far studied in various species are capable of causing
appreciable alterations in spontaneous mutation frequencies have documented the concept that, far frombeing interesting curiosities in a background of genomic
stability, the transposable genetic elements are important factors in the genetic control of mutation
phenomena in general. The question of whether theirmobility can also be substantially altered by mutagenic
agents such as x rays has not yet been resolved since thecurrently available data are very limited and do not lendthemselves to generalizations.
273. The possible relevance ofmobile-genetic-elementmediated spontaneous mutations in the context of risk
evaluations using the doubling dose method has
recently been discussed by Sankaranarayanan [S 132]and can be best illustrated by the following assumptions (which are not exhaustive) and the consequencesof these assumptions:(I)

All spontaneous mutations arise throughmechanisms that have been envisaged before
the advent of the .. mobile genetic element era"and represent rare errors made during DNA
replication and/or repair (mechanism A). The
contribution of transposable-genetic-elementmediated spontaneous mutations (originatingby mechanism 8) to the total is zero. There are
no qualitative differences between spontaneous
mutations and those induced by irradiation andradiation merely increases the frequency andtypes of events that lead to spontaneous mutations. If these assumptions are valid, then it issafe to use the doubling dose method for risk
estimation.

(2)

Spontaneous mutations are of two kinds. namely,
those that arise by mechanism A and those bymechanism 8 mentioned above (i.e., thoseindependent of mobile genetic elements andthose that are dependent on these). The frequencies of both kinds of mutations can beenhanced by irradiation. If these assumptions
are true. the consequences would be indistinguishable from those obtained for situation (I).

(3)

This is the same as situation (2), except here it
is assumed that only the frequencies of mutations that arise by mechanism A (i.e .. those thatare independent of movable genetic elements)
can be enhanced by radiation exposure while
those that arise by mechanism B are not. If
these assumptions are true, it is proper toestimate the doubling dose only for mutations
that arise by mechanism A. If one lets the
proportion of spontaneous mutations that arise
by mechanism A vary from I to zero (and thus

the B-type mutations from zero to I). and since
the induction rate one estimates is applicable
only to A-type mutations, it is easy to see that.
as the proportion of A-type mutations decreases,
the doubling dose will decrease. i.e., the relative
mutation risk will increase. When all spontaneous mutations are caused by mechanism B.
it is not correct to estimate the doubling dose asa ratio of the average spontaneous and induction
rates of mutations, since the numerator will be
zero.
274. The prediction for situation (3) can be illustrated
by the following example. In this, PA is the spontaneousprevalence of autosomal dominant and X-linked
disorders that have arisen by mechanism A (expressedas the number of cases per 10 6 live births): P 8 is thesame for the disorders that arise by mechanism B; m 1
is the average spontaneous rate of A-type mutations;m 2 is the average rate of induction per unit dose; c isthe doubling dose in Gy: 1/c is the relative mutation
risk; and E, the expected increase in the number ofcases (per 106) at equilibrium following irradiation at
a rate of 1 Gy per generation. The results ofcalculations summarized below show that: (a) as
the proportion of spontaneous mutations arising bymechanism A decreases, the estimate of the doublingdose also decreases (since the induction rate measured
is assumed to be applicable only to those mutations
that arise by mechanism A and therefore held constant):and (b) from the standpoint of the population, thetotal number of induced mutations will remain thesame (the E values given below).

Ps

PA
10,000
i,500
5,000
2,500

rn,

10-s
0
2,500 0.75 10-s
5,000 0.50 10- 5
7,500 0.25 10-5

rn"

C

1/c

E

10-s
10-s
10-5
10-5

I
0.75
0.50
0.25

I
1/0.75
1/0.501/0.25

10,00010,00010,00010,000

It is worth reiterating that there is as yet no hard
evidence as to whether the transposition rate of thetransposable elements is increased under conditions ofradiation exposure and there is no evidence on the
proportion of spontaneous mutations in man that
could reliably be attributed to the transposable elements. Consequently. it is fruitless to make further
speculations at the present time.

G.

SUMMARY AND CONCLUSIONS

275. The classical studies of transposable controlling
elements in maize were the first to indicate thepresence of chromosomally located mobile and transmissible elements in the genome that induce chromosome breaks. duplications, a variety of other structuralchanges, as well as changes in gene expression at manyloci. These observations were made before the first
demonstration of mobile DNAs in bacteria in theform of plasmids and bacterial viruses (bacteriophage).
which could insert themselves into the genome, butalso detach themselves completely and replicate independently in the cell.

276. Movable genetic elements have now been discovered in a number of other species studied in thisrespect (bacteria. blue-green algae. yeast and Drosophila) and there are indications that they may bepresent in mammals including man, although they
have not yet been demonstrated to transpose.
277. Bacterial transposable elements are of two
kinds: IS (insertion sequences; these contain only
genes related to insertion functions) and Tns (containing other detectable genes unrelated to insertionfunctions). The IS range in size from 0.8 to 1.5 kb andcarry perfect or near-perfect repeats of about 10-40 basepairs at their termini. It has been estimated that atleast 1-2% of the genome of E. coli Kl2 consists ofvarious insertion elements. Integration of the IS elements shows a certain specificity of sites. Theirtransposition into a gene abolishes its functions.
leading to a mutation. Insertion of an IS element isaccompanied by a short duplication of target DNA.
Different IS elements transpose at different rates.
278. Among the bacterial transposons, those belonging to the Tn3 family and Tn JO have been extensively
studied. Transposons are generally somewhat longer
than IS elements although their properties are basicallysimilar.
279. There is evidence for the thesis that treatmentof bacteria with UV, mitomycin-C, or nitrosoguanidine
may increase the rate of transposition of at least one
transposon (Tn I).
280. The transposable elements in yeast (the Tys) area family of dispersed repetitjve sequences consisting ofa central region of about 5.6 kb of DNA flanked bydirect repeats of approximately 330 bp sequences
called delta. There are about 30-35 Tys per haploid
genome. The evidence that Tys are transposable
comes from the the analysis of mutations at a numberof genetic loci. Most of the Ty insertion mutations
studied so far have occurred in the regulatory and notin the structural genes.
281. The first evidence for mobile DNA elements in
Drosophila was provided by the behaviour of a small
number of unstable mutations. These mutations display genetic properties analogous in many respects to
those of mutations caused by the insertion of transposable elements in maize and prokaryotes. Suchunstable mutations revert to wild type at unusuallyhigh rates and generate deletions and other chromosomal rearrangements. Three mutator systems thatgenerate unstable mutations, the MR. the P-M and theI-R. have been the subject of a number of genetic
studies.
282. In experiments in which the effect of MR onradiation-induced genetic damage in males was studied,it was found that the frequencies of autosomaltranslocations in MR-containing males were not significantly different from those in non-MR males: thefrequencies of sex-linked recessive lethals were about
50% higher in the MR-bearing males. There is someevidence that DNA repair defects may interfere withthe action of MR. In one case there appears to be
direct interaction between a P-element and x rays.
resulting in site-specific chromosome breakage events.
69283. The occurrence of hybrid dysgenesis, "a syndrome of correlated genetic traits that is spontaneously
induced in hybrids between certain mutually interacting
strains, usually in one direction only" and that
includes phenomena such as male and female sterility,
high mutability, chromosomal aberrations and malerecombination, has been extensively documented in
Drosophila. In the so-called P-M system. the strains
could be classified as either P (paternally contributing)or M (maternally contributing). The traits mentioned
above occur in crosses between P strain males and M
strain females, are much less pronounced in thereciprocal cross and are absent in PX P or M X M
crosses. The hypothesis that the occurrence of hybrid
dysgenesis in the P-M system is due to the insertion of?-elements (insertion sequences) has been tested biochemically and found to be correct. These P-elements
are present in about 50 dispersed copies in P strains
and, as expected. absent in M strains.
284. The question of whether the P-elements in the
P-M system and the MR factors discussed earlier are
similar has been examined in a few studies, using
genetic and molecular techniques. It has become clearthat both systems are integral parts of the same
mutator system, although they were originally identified by different experimental approaches.
285. Another independent system of interacting
strains in Drosophila is that designated as I-R (forinducer and reactive), discovered originally through its
effect on female sterility. Genetic evidence favours theinterpretation that the mutator effects observed in thissystem are also due to DNA insertions.
286. In addition to the transposable elements in
Drosophila. which were originally identified in genetic
studies (with a molecular follow-up). there is extensivee\'idence for the presence of several moderately
repetitive DNA sequences that are capable of transposition from one chromosome location to another.
These include the copia-like elements, fold-back elements, ?-elements (already described) and several
other dispersed repetitive families as yet not fully
characterized. Their insertion into genes results inmutations and a number of such insertion mutations
have been described.
287. In the mouse, there is evidence for unique
MuL V (murine leukaemic virus) sequences being
closely associated with two coat colour loci. called aand d; in the case of the d locus. a spontaneous
revertant from d to d+ was associated with theconcurrent Joss of the proviral sequence. Exposure ofmouse embryos to exogenous Moloney Jeukaemic
virus results in the stable incorporation of the virus
into the germ line chromosomal DNA, but at different
locations or integration sites (the Mov loci); homozygosity for one such locus seems to disrupt a genefunction essential for normal development and consequently to produce lethality of the homozygotes.
288. One group of stable inserted elements in themouse is constituted by the intra-cisternal-A-particle
genes (IAPs), which are retroviral-like elements. They
are responsible, by insertion, for mutations involving
70

the kappa immunoglobulin light chain in mousehybridoma cells. Furthermore. dispersed repeats suchas the 8 I and 82 sequences may be potential
candidates for movable genetic elements in the mouse.
289. The human genome contains between 300,000
and 500,000 copies of a 300 base-pair repeat sequencethat act as substrate for the Alu restriction enzyme(the Alu repeats): these repeat sequences constituteabout 4% of the human DNA. They are flanked bydirect repeats of sequences of some 7-20 nucleotides
Jong. a feature they share with movable genetic
elements studied in other species. There is evidence
that the Alu repeat sequences may be transposable
within the human genome.
290. The finding that a sizeable proportion of
spontaneous mutations in experimental organisms
studied in this respect can be caused by transposable
genetic elements may have implications for theevaluation of genetic radiation hazards in man using
the doubling dose method. Such implications havebeen outlined.

V. EXPERIMENTAL DATA ON THE
MECHANISMS INVOLVED IN THE
PRODUCTION OF CHROMOSOMALABERRATIONS IN CELLS IN VITRO
291. The 1982 UNSCEAR report reviewed the progress in studies on the relationship between DNA
repair processes and chromosomal aberration phenomena in mammalian cell systems. Among the aspects
covered were: (a) the molecular mechanisms involvedin the production of chromosomal aberrations; (b) the
isolation of mammalian cell lines hyper- or hyposensitive to DNA damaging agents; and (c) thedelineation of complementation groups for repairdeficient syndromes in man. New data that havebecome available since then shed further light on themolecular mechanisms. particularly on the nature ofprimary lesions in the DNA that lead to chromosomal
aberrations and on the role of DNA repair processes.
The aim of this chapter is to summarize some of theprincipal findings. For recent reviews. see Collins etal. [C52]. Miwa et al. [M38] and Ishihara et al. [19).
A.

NATURE OF THE PRIMARY DNA LESIONSINVOLVED IN THE FORMATION OF
CHROMOSOMAL ABERRATIONS

292. Among the wide variety of lesions that are
induced in cellular DNA by x rays and othermutagens (single-strand breaks (SSBs). double-strand
breaks (DS8s), base damage. mis-matches, DNADNA cross-Jinks, DNA-protein cross-links. etc.), DSBs
seem to be the principal ones involved in the
production of chromosomal aberrations. Direct evidence for this was provided by Natarajan and colleagues[N3. N4, N23, 02) and this was reviewed in the 1982UNSCEAR report. Briefly, these authors treatedx-irradiated CHO cells with an enzyme-Neurospora

endonuclease (NE)-that recognizes SSBs in the DNA
and converts them into DSBs. The prediction wasthat, should the DSBs be the principal lesions involvedin the formation of chromosomal aberrations. thenthe frequencies of aberrations should be increased in
the x rays+ NE group relative to the x rays alonegroup. This increase was in fact observed. Biochemical
evidence for the conversion of SSBs to DSBs by NE
was subsequently provided [N3]. However, when cells
are irradiated with neutrons-a treatment that produced predominantly DSBs-no potentiating effect of
NE would be expected: this possibility was also tested
and found to be true [02].

produce cohesive-ended DSBs. Though there wassome indication for an increase in frequencies withincreasing concentration of the enzymes. no clear-cut
dose-response relationships could be demonstrated. In
experiments on the cell-cycle stage dependence ofaberrations induction. it was found that cells in G 1
and G 2 are relatively resistant to treatment and S
phase cells are more sensitive. In several respects. thepattern of aberrations induced was similar to thatinduced by ionizing radiation. namely, sub-chromatid
and chromatid-type aberrations in 0 2, chromatid-type
in S, chromosome and chromatid-type in late 0 1 andearly S and chromosome-type in G 1•

293. Another possible way to inquire into the natureof the lesions responsible for the formation ofchromosomal aberrations is to choose agents that will
induce only one class of specific lesions in cellular
DNA and then study their efficiency in inducing
chromosomal aberrations. Restriction endonucleases(discussed in chapter II) represent such a class ofagents; they produce either blunt-ended or cohesiveended DSBs in the DNA. This approach was utilized
in independent studies by Bryant [B32], Natarajan
and Obe [N24] and Obe et al. [021].

296. In independent experiments, Winegar and
Preston [W53] reported linear dose-response relationships for aberrations induced by Alul, RsaI and Mspl.
In addition. they found that for Rsal (blunt-end) and
Mspl (cohesive-end) the frequency of aberrations was
the same for equivalent restriction enzyme doses.
The calculated frequency of cutting in CHO cells was
the same for these two enzymes. The authors suggested
that an improved cell permeabilization technique,which they used, could account for the differences
between their results and those of other authors.

294. In the work of Bryant. two restriction enzymes,
Puvll and BamHI, which generate blunt-ended andcohesive-ended DSBs. respectively. were used to treat
permeabilized V-79 Chinese hamster cells. Under theconditions of their experiments, PuvII induced bluntended DSBs resulted in both chromosome- andchromatid-type aberrations (exchanges and deletions)
including a high frequency of triradials (the latter wereassumed to arise from exchanges between isochromatid
and chromatid breaks). The total frequency of aberrations was proportional to the amount of enzyme used
up to 100 units per treatment flask (at the highest
enzyme concentration, the aberration frequency was
10 times the control frequency). Further increases inenzyme concentration produced no further increase inaberration frequencies. In contrast with these results,
BamHI (even at concentrations higher than thehighest used in the PuvII experiments) did not induce
aberrations above the spontaneous level. The ineffectiveness of DSBs with cohesive ends in causing
aberrations has been explained as being due to thestabilizing influence of the hydrogen bonding, andconsequent ease with which restitution can occur.
Because of a lack of such stabilization. DSBs induced
by Puvll may behave as true DSBs and are probably
more easily brought out of alignment and renderedirreparable or have a higher chance of interacting
illegitimately with other DNA strands. leading tochromosome exchanges.

297. In those experiments in which treatment with
ara-C (cytosine arabinoside, a specific inhibitor of
DNA polymerase alpha) or caffeine (an inhibitor of
DNA repair and one that is known to abolish theblock in G 2 stage induced by different mutagens) wasused in addition to Puvll, there was a doubling of the
breakage rate (except that. with caffeine. this was only
evident at low induction rates). Though ara-C is a
specific inhibitor of DNA polymerase alpha. it is
known to inhibit excision repair; the ultimate effect ofara-C on excision repair is deduced to be theinhibition of the ligation step. The finding that thepresence of ara-C during treatment with endon ucleases
leads to an increase in the frequency of aberrations
strongly suggested that ara-C affects ligation of DNA
double-strand breaks.

295. In the experiments of Natarajan and Obe [N24]three restriction enzymes that produce blunt-ended
breaks (EcoRV. Puvll and Smal) and three that
produce cohesive-ended breaks (BamHI, Asulll andNunlll) were used to treat CHO cells or PG-19 mouse
fibroblasts. The results showed that both kinds ofendonucleases induced chromosome-type (the mostfrequent class) and chromatid-type aberrations: however, the endonucleases that produce blunt-ended
DSBs were clearly more efficient than those that298. Further data on the induction of chromosomal
aberrations in CHO cells by restriction endonucleases
have been presented by Obe et al. [021). The ·authors
found that the G I stage of the cell cycle is particularly
sensitive in this regard. The differential yields ofaberrations recorded with different restriction endonucleases appeared to be correlated with the numberof recognition sites in the genome.
299. From entirely different kinds of experiments,
but all involving ara-C treatment. Preston [P7, P26]
has favoured the involvement of DNA base damage inthe origin of chromosomal aberrations induced by
x irradiation. The premiss and the approach were thefollowing: (a) ara-C has been shown to inhibit the
repair of DNA damage induced by a variety of agents:(b) ara-C inhibits repair replication. leaving a singlestrand gap in the DNA; and (c) this effect of ara-C
can be reversed by the addition of deoxycytidine. The
general approach has been to treat human lymphocytes
in various stages of the cell cycle with x rays or
chemical mutagens and to incubate the ceJls fordifferent periods of time with ara-C. In this way,71

single-strand gaps can be accumulated by the inhibitionof repair replication. If these accumulated gaps can beconverted to aberrations on reversing the ara-C
inhibition with deoxycytidine. then increased aberration
frequencies will result, compared with those in cells
given no ara-C treatment.
300. When human lymphocytes were x irradiated in
G 0 and then incubated with ara-C for I. 2. or 3 hours,
the yields of dicentrics. rings and acentric fragments
were all increased. compared with cells that received
only x irradiation [P7]. There was an approximately
linear increase in aberration frequency as a function ofara-C incubation time. The dependence of the increasein aberration yield with the duration of ara-C posttreatment suggested to Preston that misrepair of basedamage was most likely to be involved in the
induction ,of aberrations since most of the DNA
strand breaks would be rapidly repaired.
30 I. Natarajan [N25] pointed out that there are atleast three arguments that are not in support of thisline of reasoning: (a) Bender and Preston [B9] foundthat the maximum increase in aberration frequencies(80%) occurred during the first one hour of ara-C
post-treatment of x-irradiated (G 0) lymphocytes, aperiod when most of the strand breaks are repaired;(b) when irradiated G 0 lymphocytes are challenged
with ara-C after one hour of incubation in normal
medium, the potentiating effect of ara-C is greatly
reduced [N25]; and (c) while there is a profoundoxygen effect for x-ray- or gamma-ray-induced chromosomal aberrations and DNA strand breaks, the oxygen
effect is minimal for base damage [P27]. The finding
in the more recent studies of Natarajan and Obe [N24]with restriction endonucleases (ara-C post-treatmentof restriction endonuclease-treated cells leads to anincrease in aberration frequency and this effect is most
likely a consequence of ara-C-mediated inhibition ofligation of DSBs) provides one additional argument
against the hypothesis of Preston. These different
points of view need to be resolved by further
experiments.

B.

THE ROLE OF DNA REPAIR PROCESSES IN
THE FORMATION OF CHROMOSOMALA BERRA TI ONS INDUCED BY IONIZINGRADIATION

302. The approach of treating mutagenized cells withselective inhibitors of DNA repair/replication to gain
insights into the different stages of the repair pathways has been gaining increasing momentum. Amongthe studies in this area that have been made over the
past few years. several are focused primarily on thebiochemistry of repair processes, while some haveattempted to elucidate the role of such repair processes
in the formation of chromosomal aberrations inducedby ionizing radiation. Only the latter kind of study is
considered in the present section (see [N2-N5, N25
and K6] for recent reviews).
303. The inhibitors that are commonly used canarbitrarily be divided into those that inhibit DNA
synthesis (e.g., 5-fluorodeoxyuridine, FdUrd; 2' -de72

oxyadenosine. 2' Ado; hydroxyurea, HU; aphidicolin.
APC) and those that inhibit DNA repair (e.g .. ara-C;
3-aminobenzamide, 3-AB; benzamide, B).This distinction, however, is not an absolute one since compounds
such as HU. ara-C and aphidicolin can all inhibit both
replication and repair [D7]. and APC was first
discovered as a specific inhibitor of DNA polymerasealpha (and thus of DNA synthesis) and subsequently
found to inhibit DNA repair as well [K6].

I.

Inhibitors of DNA synthesis(a)Effects at the molecular level

304. The three inhibitors to be discussed here are
FdUrd, 2' Ado and HU. A common property of these
inhibitors is that they act at a late stage in thesynthesis of the immediate precursors of DNA. Theiraction leads to a disturbance of the balance within the
DNA precursor pool and a consequent reduction in
DNA synthesis. After phosphorylation to 5-fluorodeoxyuridilic acid, FdUrd is a powerful and specificinhibitor of the enzyme thymidylate synthetase. whichcatalyses the synthesis of thymidylic acid from deoxyuridilic acid [C7, H7]. The inhibition is irreversible.
Both HU and the triphosphate of 2'Ado are inhibitorsof the enzyme ribonucleotide reductase, which catalyses
the reduction of ribonucleoside diphosphates to thecorresponding deoxyribonucleoside diphosphates. The
inhibition is reversible (see [K6] for a listing of therelevant references).

(b)

Production of chromosome aberrations

305. All three substances, FdUrd, 'l Ado and HU,
are capable of producing chromosomal aberrations
themselves in both plant and cultured animal cells. In
cells treated in early interphase. exchange aberrations
predominate. whereas those treated in late interphaseshow mainly breaks and large gaps. It is believed thatthese compounds produce aberrations by disturbing
the normal semi-conservative DNA synthesis in the Sphase.
306. Similarly, all three inhibitors significantly
enhance the chromosomal damage produced by agents
such as x rays; this is true for Vicia faba root tips,
Chinese hamster cells and human lymphocytes. For
instance, in Chinese hamster cells and human lymphocytes, post-treatments with HU or 2' Ado during G 2
have a strong potentiating effect on the chromosomal
damage produced by S-independent agents in S or G:
and by S-dependent agents in G 0 • G I or S [HS, H9,
K7, N5].' The types of aberrations increased by'Ionizing radiation produces aberrations in all stages of the cell
cycle; these can be recovered at the firs! mitosis following
irradia1ion (i.e., their formaiion is S-(DNA synthcsis)-independent).
Amibiotics belonging 10 1hc bleomycin group also act via S-independent mechanisms. However, for UV and most chemical mutagens,which are capable of inducing lesions in chromosomes in all stagesof the cell cycle, the lesions require DNA and chromosome
replications (i.e., they are S-dependent) to be transformed intoaberrations that are always of the chromatid type, irrespective of
the stage of cell cycle treated.

HU post-treatments are gaps and breaks, whereas
exchange-type aberrations are unaltered or even decreased. In human lymphocytes. the most marked
potentiation of x-ray-induced chromosomal damage is
obtained when HU is given 2 hours before harvesting[K6).
307. It has been assumed that the enhancement of
the frequencies of chromosomal aberrations is aconsequence of the suppression of some form of DNA
synthesis that takes place late in G 2 when chromosomal DNA is damaged by mutagenic agents. Presumably, the DNA synthesis affected by the inhibitors
is required for the repair of the induced chromosomal
damage.
2.

{a)Inhibitors of DNA repair

lnhibi10rs of DNA polymerases

308. There are several inhibitors that are relatively
specific to the activity of alpha, beta and gamma
DNA polymerases. This specificity. however. seems todepend on the cell type and the state of the cell during
treatment (stationary versus exponentially growing
cells). Of these inhibitors. APC, which is absolutely
specific for polymerase alpha, and ara-C, which
preferentially inhibits polymerase alpha, have beenused in experiments to study their influence on
spontaneous and induced chromosomal aberrations[K6].
309. Ara-C inhibits DNA replication as well as
repair replication in cells following treatment with avariety of mutagenic agents. The probable mechanisms
by which ara-C inhibits DNA repair have been
discussed by Fram and Kufe [F25]. There is evidence
for the incorporation of ara-C into DNA rather thanRNA and this incorporation is both concentration andtime dependent. Furthermore. the degree of inhibition
of DNA synthesis is correlated with the extent of its
incorporation into DNA [F25]. While the mechanisms
underlying the effect of ara-C on DNA repair are notknown, both the competitive inhibition of DNApolymerase alpha and the incorporation of ara-C into
DNA segments may play a role in drug action.
{b)

Effects on chromosomal aberrations

310. Ara-C is capable of inducing chromosomal
aberrations not only in the DNA synthetic (S) phaseof the cell cycle but also in cells in the pre- ( Go or G 1)
and in the post-DNA synthetic phases of the cell cycle[B7-B9, KS. N5. P7]. The experiments of Preston[P7] on post-treatment effects of ara-C (following
x irradiation of human lymphocytes) were discussed
earlier. In another study. Preston and Gooch [PS]treated G 1 lymphocytes with 4-nitroquinoline-N-oxide(4-NQO) and. as would be expected of an S-dependent
agent, there was no increase in aberration frequency
compared with untreated controls. However, if 4-NQOtreated cells were incubated with ara-C for 6 hours,
aberrations were observed, but they were of the chromosome type. The conclusions were that: (a) chromosome-type aberrations can be induced in G I bychemicals that normally do not induce them. if repair
is inhibited by ara-C; and (b) in order to produce asignificant frequency of aberrations. rather long (atleast relative to x rays) ara-C incubation times are
required. The latter conclusion suggests that the repairof the 4-NQO damage that is converted into aberrations
is rather slow, compared with the repair of the x-ray
damage involved in aberration production. This leads
to the general conclusion that the frequency ofaberrations. and in many instances the spectrum ofaberration types. is determined by the rate of repair of
the DNA damage responsible for aberration production.
311. In a more recent paper. Heartlein and Preston[H5 l] advanced and tested the hypothesis that ara-Cinhibitable DNA damage provides a measure of interspecific difference in the sensitivities of lymphocytes tothe induction of chromosomal aberrations by x rays.
Human. rabbit, marmoset and pig lymphocytes were
irradiated in G 0 (0.5-3.0 Gy). incubated with ara-C for
0-4 h and processed for cytogenetic analysis. In all
four species. the frequencies of dicentrics increased
with incubation time (up to 3-4 hours). but the rates
of increase were different and approximately proportional to the ratios of x-ray-induced aberrations
observed in the absence of ara-C. Thus. for example,
human lymphocytes are about twice as sensitive as
rabbit lymphocytes to the induction of dicentrics by
x rays and the rate of increase of aberrations in thepresence of ara-C was about twice as great in humanrelative to rabbit lymphocytes. The authors suggestthat these findings are consistent with the existence ofpossible inter-specific differences in excision or resynthesis associated with the repair of damaged bases andthat this approach may be useful in explaining
differences in sensitivity to aberration induction (byx rays) among species or cell types and differences ofdose-response curves.
312. The experiments of Natarajan et al. [N29] were
aimed at a further clarification of the effects of ara-Con the repair of x-ray-induced lesions in human
G 0 lymphocytes. Attention was focused on the following aspects: (a) inhibition of repair by ara-C during 15
to 60 min following x irradiation and its effects on
chromosome aberration yields: (b) the effect of delayedara-C treatment on aberration yields: (c) estimation of
the rate of strand-break repair following x irradiationin the presence of ara-C. by nucleoid sedimentation
technique; and (d) the effects of ara-C during the first
30 min following x irradiation. using the technique ofprematurely condensed chromosomes.
313. The results showed that treatment with ara-C
for a time as short as 15 min following x irradiation
increased the yields of chromosomal aberrations in avery striking manner: longer durations (up to 2 hours)
also led to further increases in aberration yields, butthese were quite small. relative to the increase observedwith a 15 min treatment. These data thus suggest that
ara-C may inhibit, predominantly, the repair of shortlived x-ray-induced lesions. If this interpretation werecorrect, with ara-C administered I hour after x irradiation, the potentiating effect of ara-C would be
expected to be greatly reduced and this was in fact

73

what was observed. Biochemical studies (nucleoid
sedimentation) revealed that ara-C prevented rejoiningof breaks very effectively and this inhibition was
maximal already with a 30 min ara-C treatment: withdelayed ara-C treatment (given I hour after x irradiation), the effect on repair of strand breaks was
negligible. as would be expected. On the basis of theseresults and their earlier data with restriction endonucleases, those of Iliakis and Bryant [I 13] and of
Holmberg and Gumauskas [H52], Natarajan et al.
[N29] concluded that ara-C can inhibit the repair of
directly induced single- and double-strand DNA breaks
and this accounts for the increased yields of chromosomal aberrations. The results obtained with thepremature chromosome condensation technique (discussed in the next section) are in line with thisinterpretation.
314. Aphidicolin (APC). another inhibitor of DNApolymerase alpha. does not induce chromosomal
aberrations in human lymphocytes when treated in G 0[N5]. The chromatid-type aberrations observed by
Bender and Preston [89] after treatment of G 0lymphocytes with APC are probably the result of
residual APC present in the medium [Zl]. APC.
however, induces chromatid breaks and gaps whenhuman lymphocytes or CHO cells are treated in G:[B9. N5], indicating the occurrence of a residual DNA
synthesis during the G 2 stage.
315. The frequencies of chromosome-type aberrations
are potentiated when x-irradiated G 0 lymphocytes are
post-treated with APC [89]: both breaks and exchangetype aberrations increase with the duration of posttreatment. However, the finding that APC induces
lesions in G 0 which synergistically interact with x-rayinduced lesions to produce an increase in chromatidtype aberrations [B9] appears to be due to residual
APC in the medium [Zl]. When G 0 lymphocytes are
x-irradiatcd and post-treated with APC, there is a 2to 3-fold increase in the frequencies of chromatid
breaks but either no increase or a slight decrease in
the frequencies of exchanges, indicating an inhibitory
effect of APC on the rejoining of DNA strand breaks.
With CHO cells in G 1, however, post-treatment with
APC after x irradiation produces only a slightpotentiation of the chromosome damage. This may be
due to the possibility that the inhibition of repair ofdamaged DNA by APC depends on the metabolicstate of the cells. inhibition being more effective mstationary than in proliferating cells [N5, Zl].

(c)

Inhibitors of ADP-ribosyl transferase (ADPRT)

316. ADP-ribosyl transferase (ADPRT), which is also
known as poly-(ADP-ribose) polymerase or synthetase,is a DNA-dependent nuclear enzyme on which a
considerable amount of work has been done during
the last few years. This enzyme covalently attaches
ADP-ribose moieties derived from NAO to proteins
to form mono- or poly-ADP-ribosyl derivatives [Hll,S21]. ADPRT activity is strongly stimulated by breaks
in DNA [BIO] and the enzyme is required for efficient
DNA repair [C8, D8]. The evidence so far available
indicates that. in most cases. the ADPRT activity is74

not required for the incision, excision or repair
synthesis steps. but is required for the strand-rejoining
step. particularly that mediated by DNA ligase-II[S21,S76].
317. Exposure of cells to alkylating agents or toradiation results in an inhibition of glycolysis, due to a
drastic fall in cellular NAO levels. This drop in
cellular NAO induced by DNA damaging agents, inparticular by streptozotocin, a derivative of MNU,
methyl nitrosourea. is due to activation of ADPRTand not to inhibition of NAO biosynthesis or toactivation of NAO glycohydrolase [W32]. Furthermore, cytotoxic agents that do not directly affect the
integrity of DNA, such as colchicine or FdUrd. do nothave this effect on cellular NAO [S21]. In addition to
the clear evidence suggesting that ADPRT is required
for efficient DNA repair, there is also evidence that
ADPRT activity is required for certain types of celldifferentiation [F6. 15].
318. Inhibitors of ADPRT activity such as 3-aminobenzamide (3-AB) and benzamide (8) prevent the
depletion of cellular NAO that is caused by DNAdamaging agents. These inhibitors also retard DNA
strand rejoining in some cell types after certain kindsof DNA damage. such as simple alkylating agents inpermanent cell lines like Ll210 lymphoid cells. in cellstrains such as human skin fibroblasts and in protozoa[S2 l]. There is a small retardation of DNA strand
rejoining after ionizing radiation. but no evident effectof ADPRT inhibitors after ultraviolet radiation [S21,C9].
319. A number of investigators have studied theeffects of inhibitors of ADPRT activity on the
spontaneously arising and mutagen-induced chromosomal aberrations. sister chromatid exchanges (SCEs)
and point mutations [N2, N5, N6, HJ2, 03]. Under
normal culture conditions (FIO medium with 15%
feta! calf serum) when human lymphocytes or CHO
cells are incubated in the presence of 3-AB or B evenat high concentrations (JO mM). there is no evidence
for an increase in the frequency of chromosomal
aberrations [N5, N6]. However. if CHO cells aregrown in Eagle's MEM or in a medium deficient innicotinamide and in the presence of JO mM of
BrdUrd. 3-AB induces chromatid aberrations, especiallyof the exchange type [I-I 12], suggesting that the
metabolic condition of the cells is an important factor
for the manifestation of the effects of 3-AB. Fibroblasts derived from human chromosome instability
syndromes such as ataxia telangiectasia or Fanconi's
anemia (which have low but significant levelsof spontaneously arising chromosomal aberrations)
respond to 3-AB with increased frequencies of aberrations in a dose-dependent manner [N2]. These observations have been interpreted as an interference of
3-AB on the repair of spontaneously arising DNA
breaks in these cells.
320. When human lymphocytes are x-irradiated in
G 0 and incubated in a medium containing 3-AB for
6 hours prior to mitogenic stimulation, or during theentire period of 48 hours of culture, the frequencies ofchromosomal aberrations increase 2- to 3-fold [N6].

The increase due to 3-AH post-treatment is similar,
irrespective of the duration of post-treatment, indicatingthat most of the aberrations induced by x rays are
fixed immediately after irradiation. The observed
increases in aberration yields are interpreted as due tothe slowing down or partial prevention of restitutionof DSHs in DNA by this inhibitor, thus allowing for
an increased possibility for the formation of exchanges.
321. Treatment of CHO cells in G~ with 3-AH
following x irradiation also increases th~e frequencies
of induced aberrations (breaks and exchanges) by afactor of 2-3. As will be recalled. the other inhibitorso'. DNA repair such as APC and HU which act bydifferent mechanisms when given immediately after x
irradiation of G 2 cells increase only chromatid breaks
and not exchanges [K7, N6, Zl]. Thus, 3-AH isdifferent in its mode of action in that it does notcompletely inhibit the rejoining of breaks but only
causes a delay in the repair of breaks.
322. The results obtained with short-wave UV show.
however, that 3-AH post-treatment does not affect theyields of induced chromosomal aberrations [N6].
unlike ara-C, HU and APC, which have marked
effects (enhancement of the yields of chromosomal
aberrations).
323. It is now established that several inhibitors of
ADPRT induce SCEs [N6. 03]. Interestingly, underconditions in which very high frequencies of SCEs
were obtained, there was no increase in the spontaneous
frequencies of chromosomal aberrations. From detailed
studies on the mechanism of induction of SCE induction by 3-AH. Natarajan et al. [N2] have concluded
that the replication using HU-containing strand as
template is responsible for the increased frequencies of
SCEs in cells grown in a medium containing 3-AH.
The rationale was that HU-containing DNA is fragileand during replication is susceptible to spontaneous
breaks. The repair of such breaks is slowed down bv
3-AB. thus allowing the possibility to form SCE;.
Shiraishi et al. [S48, S49] reported similar results with
Bloom's syndrome [HS] cells. They demonstrated that,in HS cells (which. as is well known. are characterized
by a high spontaneous frequency of SCEs). most of
the SCEs occur during the second cell cycle when BUcontaining DNA is used as a template for replication.
324. When cells are treated with a mutagen in thepresence of 3-AB or H. the response to the inductionof SCEs varies. depending on the mutagen and theinhibitor used. Thus, in experiments with CHO cells.
there is no, or only very weak, potentiation of SCE
induction by short-wave UV or mitomycin-C: with
EMS (ethyl methanesuiphonate) and MMS (methyl
methanesulphonate), there is strong potentiation andwith ENU (ethyl nitrosourea), there is again only
weak potentiation [N2].
325. Neither 3-AH nor B induce mutations at the
HPRT locus in CHO cells, either when treated alone
or in the presence of HrdUrd for two cell cvcles. a
condition that induces about a 10-fold increas~ in thefrequency of SCEs [N6]. Post-treatment with 3-AB

after EMS, ENU and short-wave UV treatment ofcells had no effect on the frequencies of HPRT
mutations. Yet. as mentioned in the preceding paragraph, for SCEs there is strong potentiation effect for
EMS.

C. PREMATURELY CONDENSED
CHROMOSOMES: A MODEL SYSTEM FOR
VISUALIZING EFFECTS OF DNA DAMAGE,REPAIR AND INHIBITION OF REPAIR AT THE
LEVEL OF CHROMOSOME STRUCTURE
326. In most in vitro cytogenetic studies on theeffects of mutagens, the cells treated at a given stage inthe cell cycle are allowed to proceed to mitosis toevaluate chromosomal damage. In 1970. Johnson andRao [J18] described a phenomenon whereby if mitotic
and interphase cells were fused. factors from themitotic component would cause nuclear membrane
breakdown and premature condensation of the interphase chromatin. The resulting chromosome units
were called prematurely condensed chromosomes(PCC). Thus. one can visualize the interphase
chromosomes as discrete units. Their morphology
reflects the stage of the cell in the cell cycle at the timeof fusion. Cells from G 1 phase yield PCC with a single
chromatid per chromosome. S cells show a fragmented
appearance and G 2 cells give rise to PCC with two
closely aligned chromatids per chromosome. The
number of PCCs per cell equals the number ofchromosomes observed at mitosis and G 1 and G 2
PCCs approximate mitotic chromosomes in size andlocation of the centromere.
327. The formation of PCCs occurs very quickly
after fusion of mitotic and interphase cells and cantake place across species barriers (e.g., mitotic CHO
cells can induce PCC in fish or human cells). The factthat this technique allows the visualization of interphase chromosome means that structural damage canbe assessed directly in interphase cells. Waldren andJohnson [W33] found that chromosome damage after
x irradiation could be immediately assayed in G 1 cellsby counting the number of fragments of G 1 PCC.
Since the number of G 1 PCCs prior to treatment
equals the number of chromosomes in the cell. achromosome break would result in the formation ofan additional chromosome piece. Using this technique,Waldren and Johnson observed a linear dose-response
curve for G 1 He La cells after x irradiation. Similarly,
Hittelman and Rao found that chromosome damage
could be immediately visualized in the G, PCCs of
CHO cells after gamma irradiation. bleo~ycin andadriamycin treatment [H38-H40].
328. During the past few years. the potential of thistechnique has been exploited to study the induction ofchromosomal aberration in mammalian cells andprobe into the molecular mechanisms operating during
DNA and chromatin repair. The use of DNA repair
inhibitors in these studies have been particularly
helpful in unravelling the consequences of repair
inhibition at the chromosomal level. These and otherthemes have recently been reviewed by Hittelman[H41].

75

329. In the experiments of Natarajan et al. [N29]mentioned earlier. the effects of inhibition of repair by
ara-C was studied in irradiated (2.0 Gy) human G 0lymphocytes using the PCC technique. Irradiatedlymphocytes, after appropriate post-treatments with
ara-C were fused with mitotic CHO cells and subsequemly processed for chromosome analysis. It was
found that: (a) the yield of fragments in cells
irradiated and fused immediately was similar to thatin irradiated cells treated with ara-C for 30 min before
fusion: and (b) irradiated cells treated with ara-C
30 min after irradiation responded with somewhat
lower frequencies of fragments. These observations
have been interpreted by the authors as indicating theinhibitory effect of ara-C on x-ray-induced short-lived
lesions. Since, in these studies, only fragments werescored, the question of whether the changes in thenumbers of fragments reflect corresponding changes
in the frequencies of exchange aberrations and/or
simple fragments as such could not be answered.
330. Pantelias and Wolff [P41] examined the effects
of ara-C treatment on irradiated and unirradiated G 0
human lymphocytes using the PCC technique andshowed that, with long treatment durations (up to
8.5 hours), ara-C alone induced a significant increasein the frequencies of fragments per cell, but during thefirst hour, there was no increase. X rays alone induced
chromosome fragments, which decreased during thefirst hour in the presence of ara-C; then, however. thenumber of fragments increased, so that by the end of
the 8.5 hours treatment period, the yield was increased
by about 30% over the starting period of time zero. In
the absence of ara-C, the yield of fragments did notincrease. but continued to decrease for about 5 hours.
The total yield of fragments in the ara-C + x-raygroup was simply the sum of yields induced by ara-C
and x rays individually. The use of very long
treatment durations with ara-C, the finding that theclastogenic activity of ara-C was expressed in only
about 50% of cells and the fact that it is not possible
to discriminate between ara-C effects on x-ray-induced
exchange aberrations or fragments. make comparisonswith other data obtained through metaphase analysis
difficult.

D.

SUMMARY AND CONCLUSIONS

331. A number of recent studies have shed further
light on the nature of the lesions in DNA that lead tochromosomal aberrations in somatic cells in vitro andthe processes of DNA repair associated with theformation of these aberrations. Particularly interesting
are the new data obtained with the use of DNArestriction enzymes. which provide further direct
evidence for the thesis that double-strand breaks in
DNA are the principal lesions involved in aberration
formation.
332. The role of the DNA repair processes inchromosome aberration formation has been extensively
studied during the past few years using specificinhibitors of replication and/or repair. All these
inhibitors have in common the property of enhancing
the frequencies of chromosomal aberrations induced by76

physical or chemical agents. although the mechanisms
and the magnitude of the potentiation effect varies,
depending on the metabolic state of the treated cells,
the mutagen and the specific inhibitors used.
333. One of the principal repair enzymes that is
activated in mutagenized cells is ADP-ribosyl transferase; this is a nuclear enzyme that is sensitive to
ON A breaks and it may modulate the rate of theirrejoining. possibly by regulating the activity of DNA
ligase II. Inhibition of the activity of this enzyme byinhibitors such as 3-aminobenzamide leads to an
enhancement in the frequency of radiation-induced
chromosomal aberrations. Fibroblasts derived fromcarriers of human chromosome instability syndromes
such as ataxia telangiectasia or Fanconi's anaemia(which have low but significant levels of spontaneouschromosomal aberrations) respond to inhibitors suchas 3-aminobenzamide with increased frequencies ofaberrations.
334. It is clear that several inhibitors of ADP-ribosyltransferase induce sister chromatid exchanges (SCEs).
Under conditions in which high frequencies of SCEs
are obtained. there is no increase in the frequencies ofchromosomal aberrations. An examination of thefunction of bromodeoxyuridine used in the techniquefor detecting SCEs has provided evidence showingthat these inhibitors are more effective at enhancing
SCEs when the template DNA strand during S phase
contains bromodeoxyuridine.
335. The potential of the technique of prematurechromosome condensation by artificial fusion of
mitotic and interphase cells is being exploited to study
the induction of chromosomal aberrations and theassociated molecular mechanisms.

VI.

OTHER RELEVANT DATAA.

STUDIES IN EXPERIMENT AL MAMMALSINCLUDING PRIMATE SPECIES

I.

Production of heritable tumours in mice and rats
following irradiation and/or chemical treatment

336. The earlier work of Nomura [N9] demonstratingthat congenital anomalies and tumours could be
detected in the progeny of irradiated ICR mice wasdiscussed in the 1982 UNSCEAR report. In a subsequent paper, Nomura presented the detailed resultsof this and other work with urethane [N7]. Of
particular interest is the finding that both x-irradiatedand urethane-treated male or female mice produced
progeny with a much increased tumour incidence,with a clear dose-response relationship for treatedpost-meiotic stages in males and less clear-cut results
for spermatogonial treatment: oocytes at late follicular
stages were resistant to x rays in the range of
0.36-1.08 Gy, but very sensitive to higher doses. At thehighest dose of 5.04 Gy, the tumour incidence in theoffspring was around 30% following irradiation ofspcrmatids in the male or oocytes in the female, afrequency that is 6 times that in the controls. The
spectrum of tumours in controls and in offspring oftreated parents was similar. with about 90% being
papillary adenomas of the lung and the remainder
various malignant tumours of other organs. Matings
continued down to the F 3 generation clearly demonstrated heritability of the induced high tumour incidence
and the pattern of inheritance resembled that of adominant trait with 40% penetrance, on average.
Although x irradiation of males resulted in dominantlethals and cytologically demonstrable translocations.
no such effects were observed in animals treated withurethane. Nomura has argued that the mutations that
result in excess tumours are probably not grosschromosomal rearrangements.
337. In further studies. Nomura [N30] extended
these results to LT and N5 strains of mice. In theseexperiments, adult males were exposed to 0.504 Gy ofx irradiation, mated to unirradiated females and theF 1 progeny scored for tumours. Since the LT mice
were very sensitive to x irradiation and fertility wasnever recovered. only the progeny derived from
irradiated post-meiotic germ cells were available forscoring. The data showed that the frequencies oftumour-bearing progeny as well as of tumours werehigher following irradiation. In the LT mice. thesefrequencies were the following: tumour-bearing
progeny, 21.3% (16/75) in the x-ray group versus
9.0% (37/411) in the controls: and frequency of lungtumours. 16% (12/75) in the x-ray group, and 5.4%
(22/411) in controls. In addition, 5.3% of the progeny
in the x-ray group had leukaemia, whereas thisfrequency was only I% in the controls. In the N5
strain. following spermatogonial irradiation, the frequencies of tumour-bearing progeny and of tumours
were likewise higher than in controls: tumour-bearing
progeny. 33.2% (76/229) in the x-ray group and
23.0% (56/244) in the controls; lung tumours, 21.0%(48/229) in the x-ray group and 14.3% (35/224) in thecontrols. In experiments with N5 mice, 3.9% of theprogeny had leukaemia (relative to 0.4% in controls)and. among the female progeny, 28 out of 123 (22.8%)
in the x-ray group had ovarian tumours compared to
18/116 (15.5%) in the controls. It was noted that thefrequencies of lung and other tumours in theseexperiments were in the same general range as those in
the ICR strain. Nomura's results with the N5 strain
also indicated that induced lung tumours showed adominant pattern of inheritance with an average
penetrance of about 40%.
338. In the same paper, Nomura [N30) presented
results of studies aimed at examining whether the
induced tumours were benign or malignant throughtransplantability of induced tumours. Among 26 induced tumours in the N5 strain (11 lung tumours,
2 fibrosarcomas, 3 undifferentiated tumours, 2 hepatomas, etc.) only 3 (I lung tumour and 2 hepatomas) were
not transplantable, suggesting that most of the induced
tumours were malignant.
339. In other experiments, Nomura [N8] focused on
the problem of whether or not the manifestation ofsuch presumed tumour mutations could be enhanced.

The rationale was the following: if germ-line mutations
lead to heritable tumours. then all the cells comprising
the organ in question (in this case the lung) must carry
the mutations and have the potential to develop
tumours. However. most often, not more than one
tumour nodule was present in the lung of the tumourbearing progeny (experiments discussed above) and. as
already mentioned. the average penetrance of thepresumed tumour mutations is only about 40%.
Consequently. if the progeny of irradiated parents(some of which must carry the tumour-inducing
mutations) are given an exposure to a carcinogen suchas urethane, whose mode of action. at least undercertain conditions, is promotion of carcinogenesis[B 12. NIO], then it should be possible to increase thelevel of expression of tumours (i.e .. both the numberof tumour-bearing animals and the number of tumours
per animal).
340. As in earlier experiments, mice of the ICR strain
were used. In some experiments, mature (60-65 dayold) male or female mice were irradiated with 2.16 Gyof x rays and mated to unirradiated animals of theother sex to produce F I progeny. In others, pregnant
females were irradiated on day 9 or 15 after theappearance of the vaginal plug. The F I progeny weredivided into two groups: one group \vas killed eightmonths after birth: the other group received a single
subcutaneous injection of 5µmollg body weight ofurethane at 21 days of age and was killed 5 months
later. Offspring of non-irradiated parents were similarly
treated with urethane. The numbers of tumour nodules
in the lungs were counted.
341. The main results were the following: (a) x irradiation significantly increased the incidence of lungtumours in the progeny, regardless of which sex was
irradiated and 88 out of the 93 tumour-bearing
offspring developed only one tumour nodule in thelung while the remainder developed two nodules:(b) in the group treated with x rays and urethane(x irradiation of parental post-meiotic stages or latefollicular stages). the number of progeny developing
large clusters of tumour nodules was strikingly enhanced: (c) in the group treated with urethane alone.
2 out of 71 animals developed clusters of tumour
nodules; (d) with in utero irradiation. there was aslight non-significant increase in tumour frequency,
but additional treatment with urethane similarly enhanced the incidence of animals with large clusters:(e) when all the data were considered together, theproportion of affected progeny with large clusters oftumour nodules was 18% (34/1889) in the grouptreated with x rays and urethane: when corrected forthe control incidence (2.8%, in the group treated withurethane alone), this figure became 15.6%. which ismore than twice the frequency (6.3%) in the grouptreated with x ray alone. It is instructive to note thatin the experiments of Russell [R58] no specific locus
mutations were recovered in 13,315 F I offspring derived
from urethane-treated (1750 mg/kg body weight)
spermatogonia. Neither did Nomura [N9] observe theinduction of mutations causing tumours after spermatogonial treatment with urethane, although a large
increase in somatic mutations was observed in melanocytes [N41].
77342. In rats, Strel'tsova et al. [S 106] found thatx irradiation ( I Gy) 5 days before mating Jed toincreased incidence of breast tumours in the female
progeny. irradiation of females being more effective inthis regard. Following 90Sr injection (14.8 kBq g- 1) tofemale rats, there was a higher incidence of sarcomasin the progeny, than in controls [N31]. Likewise.
injection of a single intraperitoneal dose of 238pu(185 kBq kg- 1) to female rats 5 days before mating.
led to a significantly higher incidence of tumours in
the progeny conceived 300 days after injection; theexperimental progeny exhibited certain malignancies(osteosarcoma. liver tumours) not observed in controls[L39]. The same dose of 238 Pu injected into male rats
also produced tumours in the progeny, but thefrequencies were lower [L39].
2.

Radiation-induced congenital anomalies in mice

343. In its 1982 report, UNSCEAR discussed theinformation available at that time [K I6. N9] on the
induction of congenital anomalies in the progeny ofirradiated mice. Kirk and Lyon [Kl7] have now
obtained further data from two experiments. In thefirst, male mice were exposed to x-ray doses of 1.08.
2.16, 3.60 and 5.04 Gy and mated to unirradiated female
mice at various intervals ( 1-7. 8-14, 15-21 and 64-80 days
post-irradiation) so as to sample both post-meiotic
stages and spermatogonial stem cells. In the second.
only spermatogonial stem cells were sampled following
a single x-ray exposure to 5.0 Gy and a fractionatedexposure to 10 Gy (5 Gy + 5 Gy. 24 hours interval
between the fractions). Contemporaneous controls weremaintained. Pregnant mice were killed on day 19
following the appearance of vaginal plug and theuterine contents examined for the presence of congenitally abnormal fetuses.
344. The results showed that: (a) in the first experiment. while the frequencies of congenital anomalies in
the irradiated series were higher than in controls. there
were no statistically demonstrable differences. either
between the effects of different doses or between
different intervals, except in week 3 (treated spermatids), at which sampling interval the frequencies
increased with increasing dose (from 1% to 5%):(b) the overall frequency of congenital anomalies was
2.0 ± 0.3% (40 abnormal fetuses among 1950 examined)
in the irradiated series (summed over all doses andthe first three sampling intervals}, which is significantly higher than that in contemporaneous controls(0.24 ± 0.17%, 2/831 ), the lowest frequency found in
the authors' various experiments): (c) the data on the
induction of congenital anomalies following spermatogonial irradiation in experiment 1 were equivocal,
as the frequencies varied from 1.1 to 2.0% between
the different doses, and were not significantly
different from that observed in the corresponding
controls (2.1 %); (d) in the second experiment (spermatogonial irradiation), however. the frequencies recorded(2.2 ± 0.5% after 5 Gy and 3.1 ± 0.6% after 5 + 5 Gy)
were not significantly different from each other. but
higher than the control frequency of 0.7 ± 0.3%: inany case. the enhancing effect of dose fractionation(expected on the basis of results with specific locus
78

mutations and reciprocal translocations) was notobserved: and (e) dwarfism (i.e., less than 75% of theaverage body weight of the rest of the litter) andexencephaly were the most commonly observed malformations in all series.
345. ln studies aimed at testing whether irradiationof post-implantation maternal environment would
have any effect on the induction of congenital
anomalies by irradiation. West et al. [W46] transferred
pre-implantation embryos surgically between females
that had been irradiated (3.6 Gy. x rays) before
conception or left unirradiated. The results wereequivocal: in the group in which the embryos (3.5 days
post-coitum) were transferred from untreated donors
to irradiated recipients (group D). the frequency ofcongenital anomalies was not significantly different
from that in which the transfer was from untreateddonors to untreated recipients (group E. negative
control) and that in which the females were irradiatedprior to conception and no embryo transfer was made(group A, positive control). In the group in which theembryo transfer was from irradiated donors to untreated recipients (group C), the frequency was not
significantly lower than in group A. although thefrequency in group C was higher than in group E. The
embryo transfer experiments also showed that irradiation of the uterus had no significant effect on thefrequency of subsequent post-implantation mortality
or mean feta! weight.
346. In further experiments. West et al. [W47]
showed that when mouse ovaries were surgicallyexteriorized and selectively irradiated or shielded in a
specially constructed apparatus. and the abdomen(and uterus) irradiated (3.7 Gy of x rays) 15-21 days
prior to conception. there was no significant effect oneither post-implantation mortality or congenital anomalies attributable to irradiation of the abdomen (anduterus) alone. Exposure of the ovaries to 3.27 Gy ofx irradiation, however, during the same period increased
the frequency of both post-implantation mortality andcongenital anomalies.

3.

Further data on spontaneous and radiation-induced
non-disjunction and chromosome loss in mice

347. The subject of non-disjunction and chromosome Joss in experimental mammals has been reviewed
in the earlier reports of the Committee. including the1982 UNSCEAR report. Recent reviews have been
provided by de Boer and Tates [D36], Lyon [L16] andRussell [R36]. The fact that maternal age plays animportant role in the aetiology of human aneuploidy
is well established. In the mouse also. there is evidence
for an increase in the frequency of aneuploid embryos
in aged females of some strains [G34. F26. T17].
Several hypotheses have been put forward to explain
this age effect. For instance. Po!ani et al. [P28]suggested that, in human females. the failure of
dissolution of the nucleolus in aged oocytes may be apossible mechanism leading to non-disjunction of the
D and G group acrocentric pairs. Henderson and
Edwards [H42] proposed the so-called production line
hypothesis according to which: (a) developmental andnutritional gradients may occur in the fetal ovary:(b) these gradients could give rise to a reduction inchiasma frequency in the late-formed. relative to theearly-formed. oocytes in the meiotic sequence; (c) the
first-formed oocytes with high chiasma frequencies are
the first that are ovulated in the adult female; thoseformed later with lower chiasma frequencies andperhaps some achiasmatic chromosome pairs would
be ovulated later in life: and (d) the loss of chiasrnata
may affect the small chromosome pairs more than thelarge and, as univalents. they may subsequently
undergo irregular segregation at anaphase I. leading
to aneuploidy.

(a)

Cytogenetic methods

348. Speed and Chandley [S77] tested the production
line hypothesis using the random-bred Swiss albino
and the inbred CBA/Ca mouse strains. In the first setof experiments. surface-spread oocyte preparations
were made from fetal ovaries by the method of Speed[S78J. Examination under the light microscope wascarried out on oocyte preparations from day 15 ofgestation and day I post-partum (day 20). A total of1150 oocytes from 71 ovaries were analysed in the
Swiss strain and 676 oocytes from 50 ovaries in the
CBA strain.
349. The results showed that in neither of the twostrains was there any evidence of gradients in oocyte
development or production line; errors in synapsis
were rare and oocytes containing univalents were evenrarer. \\'hen such abnormalities occurred. they were
evenly distributed over several gestational days. being
no more common in the late than in the early stages ofoocyte maturation. Furthermore, there was no evidence
that nucleolar material interfered in any way withprophase pairing. One observation of some significancewas that of a premature desynapsis of the smallest
chromosome pair ( 19) in late pachytene oocytes; thiswas observed in about 6% of CBA oocytes analysedover days 17-19 and in about 8% of the Swiss strain
oocytes analysed over the same period. The authors
believe that the premature desynapsis of bivalent 19 isa normal feature of late oocyte development in themouse.
350. In another set of experiments. Brook et al.
[833] obtained further data that were inconsistent
with the production line hypothesis but that supported
the notion that biological and not chronological ageof the female is an important factor in the genesis of
aneuploidy. In these experiments, inbred CBA/Ca
mice 6-8 weeks old were allocated at random to twogroups: one group was unilaterally ovaricctomized(uni-ovx) and the other sham operated. They weremated to males of known fertility at different ages
(63-91 days. 154-182 days, 245-280 days, and308-350 days) and the pregnant females were killed on
day 4 for counts of corpora lutca and uterine analysis
of pre-implantation embryos to determine the incidenceof aneuploidy. In addition. the oestrous cycle of
20 uni-ovx and sham-operated mice was determined
between 60 and 390 days. A further group of about
60 females was used to study the rate of utilization ofprimordial follicles.

351. The results were the following: (a) the uni-ovx
mice had fewer subsequent oestrous cycles (a mean of
15.3 over the period between 90 and 390 days versus
20.4 in the sham-operated mice) and an earlier onsetof acyclicity (at 330 days of age versus some 40 days
later in the sham-operated ones): (b) the mean numberof embryos obtained per animal was higher in shamoperated animals at all ages. though the difference wasmost marked among the oldest groups: (c) trisomic
embryos were not found in either sub-group at ages
63-91 days. whereas monosomics were present in bothsub-groups (11.9% and 6.1%. in uni-ovx and shamoperated groups, respectively): in the intermediate agegroup ( 154-182 days). the proportion of monosomic
embryos was virtually unchanged but three trisomic
embryos (4.0%) were found in the uni-ovx group (theoverall incidence of monosomic and trisomic together
was 17 .3% in the uni-ovx group relative to 4.3% in the
sham-operated one): (d) the highest incidence ofaneuploidy was found in the uni-ovx animals at
245-280 days (22.0% versus 12. l %. but the numbers
are small): and (e) quantitative histological studies
showed that the rate of primordial follicle utilization
was not increased by unilateral ovariectomy, but therate of their depletion was comparable with that in the
sham-operated animals.
352. The authors pointed out that their observations
are not amenable to interpretation on the basis of an
earlier utilization of the last remaining oocytes which,according to the production line hypothesis, are most
likely to be defective: neither can they be explained byany factor that is determined strictly by the chronological age of the female. Rather. they seem to suggestthat abnormal meiotic chromosome segregation is an
epiphenomenon of the process of physiological ageingof the reproductive system. They further suggest that
their results may have implications for human aneuploidy and Down syndrome in particular; they imply
that unilateral ovariectomy may be an additional risk
factor for Down syndrome. Furthermore. any factor
that depletes the oocyte population could advance thematernal age effect for aneuploidy.
353. Tease [TI 7] investigated the x-ray induction of
non-disjunction in young and old mice. Females
10-14 week or 43-54 weeks old were super-ovulatedwith 5 i.u. of pregnant mare's serum gonadotrophin.
followed 45 hours later by 5 i.u. of human chorionicgonadotrophin. Three hours after the latter treatment,
they were irradiated with 0.10. 0.25 or 0.50 Gy of
high-dose-rate x rays. They were mated overnight tomales and fertilized eggs were recovered the following
day. The eggs were cultured in vitro to metaphase of
the first cleavage division. Under this regimen. maternaland paternal pronuclei remain separate and canbe distinguished through differences in chromosome
staining and contraction. Only the numbers of hyperhaploid pronuclei were used as measures of chromosome non-disjunction. The results showed that:(a) spontaneous non-disjunction occurred more frequently in oocytes of old females (1.54%) compared toyoung females (0.19% ): (b) after x irradiation, bothyoung and old females displayed significant linear
increases in hyperhaploidy with absorbed dose, but
the slopes of the regressions did not differ significantly:
79

(c) the numbers of structural aberrations in maternal
pronuclei showed significant linear relationships toabsorbed dose in young and old females, but there
were no significant differences between young and old
females in this respect: and (d) there was no evidence
for an increase in sensitivity to radiation induction ofeither numerical or structural aberrations as a functionof maternal age.
354. In a subsequent study, Tease [T38] used higher
x-ray doses (1-6 Gy) to various dictyate oocyte stages(sampled at 3.5. 9.5. 16.5 and 23.5 days after
irradiation of females 10-14 weeks old) and studied
the induction of hyperhaploidy using the same techniques as described in the preceding paragraph.
Significant increases were found in the irradiatedgroups relative to the controls: the frequencies ofhyperhaploidy increased with dose and the latter wasconsistent with either a dose-squared or a quadratic
dose-effect relationship. Furthermore, oocytes sampled
3.5 days after irradiation were less responsive than theothers, the order of increase in sensitivity being
3.5 days. 9.5 days, 23.5 days and 16.5 days. Structural
chromosomal anomalies also increased with dose andcould be described either by a dose-squared or aquadratic relationship: the general order of increase insensitivity for the induction of structural anomalies
was roughly similar to that for hyperhaploidy.
355. In other experiments, Tease [Tl8] examined
whether the use of gonadotrophic hormones to induce
ovulation altered the radiosensitivity of the oocytes tothe induction of structural or numerical aberrations.
Young mice were stimulated to ovulate with either low
or high doses of hormones and then irradiated (0.5 Gyof x rays) followed by analysis of the first cleavage
embryos. as in earlier work [TJ7]. There was noevidence for a hormonal dose-related difference inspontaneous or radiation-induced chromosome nondisjunction. Radiation-induced chromosome fragments
were observed at a higher frequency in maternal
pronuclei of I-cell embryos from females treated withlarge amounts of hormones: this increase, however,
was not statistically significant.
356. Tease and Fisher [T42] have now investigatedthe extent to which radiation-induced non-disjunction
may be related to the clastogenic properties of x rays.
Acute x-ray doses between 0.1 and 1.0 Gy were given
to preovulatory oocytes (with the same regime as thatgiven in [TI 7]) and chromosomal aberrations werescored at metaphase I. The yield was markedly higher
than after irradiation of dictyate oocytes, but thedose-response was similar, ~·ith best fit to a quadratic
curve. By comparing these results with those for nondisjunction frequencies after the same doses to thesame oocyte stage, Tease and Fisher concluded that:(a) visible damage to the centromere is not the
principal mechanism of radiation-induced non-disjunction; and (b) the involvement of induced multivalents as the principal mechanism is quite likely, inview of their high frequency and of the fact that they
may generate tertiary trisomic embryos.u However.
1/"fhe model of chromatid~interchange-mediated non-disjunction
was proposed more than 15 years ago from the results observed onradiation-induced non-disjunction in Drosophila immature oocytes(see [P52] for a review).

80

the relative proportions of primary and tertiary
trisomic embryos following x-ray-induced non-disjunction have yet to be determined.
(b)

Genetic methods

357. The numerical sex-chromosome-anomaly (NSA)
method was employed by Russell [R34] in an experiment designed to test whether advancing age increases
the incidence of either spontaneous or radiationinduced non-disjunction or chromosome loss in female
mice. The NSA method [R35. R36] permits detectionof maternal or paternal non-disjunction or chromosome loss by way of genetic markers that affectexternal phenotype. Irradiated (200 R, x rays) orcontrol Ta/+ females of various ages were mated tole/Y males (Ta and le are X-linked markers). A
number of abnormal sex-chromosome genotypes cantheoretically result from maternal or paternal nondisjunction (each occurring in first or second division).
maternal or paternal chromosome loss. mosaics involving any of these. or certain sex-linked translocations. Some of these are unequivocally diagnosableby phenotype. Others require genetic and/or cytological verification.
358. Old females ( 11.5-12 months at irradiation),
divided into virgins (1289 offspring) and pluripara(1103 offspring), were compared with young females(2.5-3 months at irradiation, 2451 offspring) irradiatedconcurrently. There were 6 groups of contemporary
controls. aged up to 15 months at time of mating. withthe 11.5-12 month group divided into virgins andpluripara. as was the case for the irradiated females.
Altogether, 15,986 offspring were observed. Amongthese were 115 possible exceptionals. of which 45 were
tested genetically, 4 cytologically. and 38 both
genetically and cytologically. These tests yielded only
2 exceptional sons. one Ta/le/Y (due to paternal nondisjunction) and one Ta/+/Y// /+/Y (i.e .. mosaic
resulting from maternal non-disjunction). There were
41 certain and 13 questionable exceptionals among
daughters. with most of them being due to paternal
non-disjunction of sex chromosomes and thus not
obviously resulting from the variable parameters of
the experiment. Neither maternal non-disjunction nor
maternal chromosome Joss was increased by advancing
age of female in either the treated or untreated group.
At any one age, radiation did not increase theincidence of maternal events. The XMXMY male.
albeit a mosaic. is the first proven case of maternal
sex-chromosome non-disjunction on record for themouse.
359. Searle and Beechey [SI05] have discussed thevarious genetic approaches to studying autosomal nondisjunction in mice through the use of Robertsoniantranslocations. including the one devised by Lyon et al.
[L38]. Cattanach et al. [C53] described two conceptually similar genetic methods involving the use of
marker genes and Robertsonian translocations, thelatter present only in tester parents, to detect events inchromosomally normal mice. In the first. designatedas the Rb method, normal mice are mated to tested
stock animals that are heterozygous for one or more

Robertsonian translocations with appropriate markers
on the chromosomes to enable detection of nondisjunction or loss of the autosomes under study. In
the second method, termed the MBH method, normal
mice are again mated to tester stock animals doubly
heterozygous for two different Robertsonian translocations that share a common arm (monobrachialhomology or MBH) and with appropriate markers.
360. In their experiments using the Rb method.
Cattanach et al. chose the Rb I Robertsonian translocation (a translocation between chromosomes I and 3)
and in those using the MBH method. the Robertsoniantranslocations were those involving chromosomes I
and 3 and I and 15. The chromosome I markers wereIn (leaden) and fz (fuzzy). The Rb method was used to
study: (a) loss of chromosome 1 in female mice
attributable to either non-disjunction or other mechanisms following absorbed x-ray doses of 0. I. 2. 3 and4 Gy; (b) chromosome I gain attributable to nondisjunction in female mice following a single absorbed
x-ray dose of 4 Gy; and (c) maternal chromosome I
loss following x irradiation (1 Gy) of I-cell zygotes.
The MBH method was used to study chromosome loss
following x irradiation of spermatozoa and spermatids(3.0 Gy). and for determining non-disjunction ofchromosome 1 after irradiation (0.75 and I Gy) of
spermatocytes. Appropriate cytological analysis andbreeding tests were carried out on the exceptional
animals.
361. The results were the following: (a) with the Rb
method. the frequencies of chromosome I losses
showed an approximately linear increase with increasing
dose (mature oocytes, slope of(6.44± 1.94) 10- 4 ay- 1);{b) at the dose of 4 Gy, there was a single non-disjunctional case among 3353 progeny (mature oocytes):and (c) the frequency of maternal chromosome loss
following irradiation of zygotes was 0.69% at I Gy.
362. In the MBH experiments (irradiation of postmeiotic male germ cells). 9 exceptional (loss ofchromosome I) progeny were found among a total of
2586 (0.35%). which was significantly higher than in
the controls (2/4150 or 0.05%). No exceptional
progeny were recovered following irradiation of spermatocytes. The authors pointed out that: (a) the two
new tester methods have potential for screening forchromosome loss and non-disjunction; (b) the one
non-disjunctional exception found after irradiation of
mature oocvtes does not establish that this was
induced by \ rays, but provides the first definitive
genetic evidence of autosomal non-disjunction: (c) while
the NSA method onlv detects chromosome loss
among female progeny: with the Rb method both
sexes of progeny are informative regarding nondisjunction; the estimated frequency of x-ray-induced
chromosome I loss is about 16 times higher than thatof X-chromosome Joss at near equivalent doses(mature oocytes. zygotes): and (d) with the MBH
method. the spontaneous and x-ray-induced frequencies of loss of paternal chromosome 1 are similar tocorresponding frequencies of sex chromosome loss. asscored by the NSA method.
363. All the various genetic methods used forthe detection of non-disjunction with the aid ofRobertsonian translocations depend on successful
complementation of a gamete with an extra chromosome from one parent and a gamete with corresponding
loss of a chromosome from the other. Searle andBeechey [SI34] pointed out that, for certain mouse
chromosomes, there is defective complementation,with lethality of the expected complementation product. This is especially true of complementation where
there is maternal chromosome duplication combinedwith paternal deficiency and is known to involve atleast mouse chromosome numbers 2. 6, 7 and 8.
Cattanach and Kirk [C77] described a similar phenomenon in mouse chromosome 11. in which maternal
duplication and paternal deficiency led to offspringthat were only half the normal size at birth, while thereciprocal cross led to abnormally large offspring.
Thus. for the affected chromosomes. genetic tests
for non-disjunction may give misleading results, bysuggesting absence of non-disjunction where there isreally absence of normal complementation.

4.

Further data on the induction of reciprocaltranslocations in mouse spermatogonia

364. The 1977 UNSCEAR report [U2) considered
the data available then [G 15] on the induction ofheritable reciprocal translocations following spermatogonial irradiation in mice. It was found that: (a) the
frequency of F I males heterozygous for reciprocaltranslocations (genetic tests) increased with exposure
up to 600 R {although the difference between theyields after 150 R (0.6%) and 300 R (0.88%) was not
significant); {b) at exposure levels of 300 Rand above.
the ratio of translocations was roughly 1/8 of thatfound earlier by Ford et al. [F7]; and {c) at the lower
exposure of 150 R, this ratio was 1/4 (although notsignificantly different from 1/8 in view of the small
number of F I males tested).
365. Generoso et al. [G 16] have now obtained more
extensive data (summarized in Table 20) from which it
can be seen that. at all radiation exposures, thefrequencies are higher than in the controls. The shapeof the exposure-frequency curve is humped and theascending portion of the curve is consistent withlinearity. The coefficients per R per gamete at 150 R,
300 R and 600 R are verv close to one another; theaverage coefficient (weighted by the reciprocal of thevariances. on the basis of data collected after acute aswell as fractionated exposures) can be estimated as(3.89 ± 0.25) 10-5/R/gamete. A comparison of theabsolute frequencies at the different exposure levels
with the frequencies expected on the basis of cytological results (last column, the latter were calculatedusing the same assumptions as those employed by
Ford et al. [F7]) shows that these are in agreement at
150 R: at higher exposures. the observed frequencies
are lower than expected.
366. The finding of Ford et al. [F7] that. after
spermatogonial x irradiation (600 + 600 R, 8-weekinterval) of male mice, the frequency of semi-sterilemales in the F I was only one-half of that expected on thebasis of multivalent configurations diagnosed cytogenetically in spermatocytes (descended from irradiated81spermatogonia) of the irradiated males themselves was
discussed in the 1972 report. Ford et al. [F7] suggested
that this differential transmission to the F 1: (a) may be
due to selection acting against translocation-carrying
cells after meta phase I: and (b) that such a selection
could be related to the occurrence of male sterility inheterozygotes for translocations that cause spermatogenic arrest both before and after metaphase I (seealso [C63] and [Sl07]). If the disparity between theyields of translocations in cytogenetic and genetic
experiments is due to the latter cause, then one would
expect a progressive reduction in frequency throughmale meiosis both before and after meiosis. However,
if it were due to the possibility that spermatocytes
carrying translocations tend to take longer to passthrough metaphase I (sec for instance [N32]), then thefrequency of translocations estimated on the basis ofmetaphase analysis would represent an overestimateof the true frequency. Crocker [C64] conducted
experiments to t.est these possibilities.
367. In these experiments, male mice homozygous
for certain Robertsonian translocations (the MC andRIB stocks) and normal mice carrying only aerocentric chromosomes were exposed to 500 R of x rays.
From 12 to 16 weeks later. the animals were killed andtestes preparations made for spermatocyte analysis by
two methods: (a) scoring of the proportion of translocations that involved metacentrics (for comparison
with the proportion expected to be induced inspermatogonia on the assumption of random exchange
between metacentrics and acrocentrics): and (b) scoringof the number and sizes of clones of acrocentric andmetacentric translocations from individual tubules(since each clone represents a single induction event,factors such as meiotic delay or selection acting after
induction should affect clone size, but not the numberof clones). It was found that. with both these methods,
the frequency of metacentric translocations was enhanced at metaphase I. Furthermore, the clone size ofthe metacentric translocations, was higher relative tothat of clones from acrocentric translocations but the
number of clones of metacentric translocations was as
expected. These observations therefore suggest thatthe enhancement was due to events occurring after
induction. Since selection would be expected to
produce the opposite effect, Crocker concluded that
his results provide evidence in favour of the delay
hypothesis. He further suggested that, if delay iscapable of enhancing the frequency of metacentric
over acrocentric translocations, it is possible that asimilar difference in retardation between normal cells
and those carrying acrocentric translocations could
account for the differential transmission to the F 1•
368. In an earlier cytogenetic study involving unequally fractionated x-ray exposures to mouse spermatogonia, van Buul and Leonard [V8] obtained someindications for a possible threshold effect for spermatogonial sensitization between 75 and 100 R. In thiswork, discussed in the 1982 UNSCEAR report, a total
exposure of 1000 R was administered in two unequal
fractions (separated by 24 hours) of 100 + 900 R,
75 + 925 R, 50 + 950 R and 25 + 975 R. Appropriate
single exposure controls were run concurrently. It was
found that, when the conditioning exposure was below

82

JOO R, there was no enhancement of the translocation
frequencies in the fractionation experiments relative tothe single-exposure controls.
369. According to the authors [V8]. the results
supported the idea that depletion of the stem cell
population is the important cause in triggering survivors into the more active cycle (as has been
postulated by Cattanach and Crocker [C54]) because
at exposures below 100 R, stem cell depletion would
be relatively slight though there would be major
killing effects upon differentiating spermatogonia.
Van Buul and Leonard [V9] conducted another set ofexperiments using BALB/c and Swiss random-bred
mice, with conditioning exposures as low as 12.5 R(the total exposure was always 1000 R). However,contrary to earlier results, the present ones showed
considerable variation between experiments. in spiteof the same or very similar fractionation regimes andmouse strains (in one experiment there was noenhancement effect of the 100 + 900 R regime, whereas
this showed a striking increase in the earlier study) sothat the earlier assumption of a threshold for spermatogonial sensitization could not be upheld. These andother data have recently been reviewed by van Buul
[VIO].
370. Pomerantseva et al. [P42] compared the cytogenetic effects of chronic versus acute 13iCs gamma
irradiation on the induction of reciprocal translocations in mouse spermatogonia. Doses of 1.5-4.5 Gy
were delivered at four different rates (i.e., 2. 7 10- 6 ,5.8 10- 6 , 9.4 10- 6 and 4.5 Gy per min). The results
showed that the frequencies of translocations at thedose rate of 9.4 J0-6 Gy/min were, on the average.
only about one-tenth of those at the highest dose rate.
in conformity with earlier results from their laboratory
and other laboratories. Further reduction in dose
rate did not lead to a concomitant reduction inyields. However, at a dose of 3 Gy, delivered at
2.7 10- 6 Gy/min, the yield is about twice if the resultsfrom one mouse with a high frequency of translocations (15/79 or 19%) are excluded; if not, the yield
is about 4 times higher than that at dose rates of
5.8 10- 6 and 9.4 10-6 Gy/min (1.18 ± 0.29% versus
0.56 ± 0.16% and 0.42 ± 0.13%: then umbers ofanalysed
cells were, respectively, 1679 or 1600, 2514 and 2400).

5. Elimination of x-ray-induced chromosomal
aberrations induced in pre-ovulatory mouse oocytes
by early embryonic death371. Reichert et al. [R37] re-examined the questionof the elimination of chromosomal damage induced
by x irradiation of pre-ovulatory oocytes in vivo,
through cytogenetic analysis of M II oocytes or embryos
at the 2-cell stage. In one set of experiments, NMRI
female mice 10-12 weeks old were superovulated(treated with pregnant mare's serum and humanchorionic gonadotrophin), irradiated with x rays
(0.2-6 Gy) and the oocytes were prepared for MII
analysis: in another experiment, pre-implantationembryos (mainly 2-cell stages) were examined after an
x-ray dose of 2 Gy; and in a third experiment,treatment was identical to the second, but the animals
were killed 13.5 days after appearance of the vaginal
plug for intra-uterine analysis and chromosomal
preparation from the liver of the embryos. About 87%of the MII oocytes showed structural chromosomal
aberrations after irradiation with 2 Gy: in contrast.
among the 2-cell stages, only 69% carried aberrations.
The 13.5-day-old fetuses were practically aberration
free. These results suggest an almost complete elimination of chromosomally damaged oocytes and zygotes
already before birth. The structural aberrations included
breaks. translocations and deletion and numericalaberrations were mostly hypoploidies.

6.

Aneuploidy and structural aberrations (spontaneousand radiation-induced) in the germ cells of rodents

372. Mikamo and colleagues carried out a number ofstudies using the Chinese hamster (in some. using rats)
to examine the primary incidence of spontaneouschromosomal anomalies in oocytes and zygotes, theirmechanisms of production and radiosensitivity profiles. Some of these data were discussed in the 1982UNSCEAR report. lllustrative recent results are briefly
considered below.

(a) Effect of pre-ovulatory overripeness of rat primaryoocytes on the incidence of chromosomal anomalies
373. The artificial arrest of the normal sequence ofoogenesis is known to exert a deleterious effect on theovum when the delay exceeds a certain time limit (seeMikamo and Hamaguchi [M39] and the references
cited therein). Since Mikamo [M40] first described ateratogenic effect of pre-ovulatory overripeness of the
primary oocytes on the embryos of Xenopus, it has
been recognized that overripeness causes developmental and chromosomal anomalies [M67]. In the work of
Kamiguchi et al. [K3 l], sexually mature female rats
with consistent 4-day oestrous cycles were intraperitoneally injected with sodium pentobarbital on
two successive days (once on the day of proestrous
and twice on the second day) to delay ovulation. On
the third day, the treated females were mated to males
and 2-cell embryos were collected from oviducts
52 hours after fertilization for scoring of morphologicalabnormalities. Chromosome analysis was carried out onmorphologically normal embryos (a great majority ofthe morphologically abnormal ones did not reach
metaphase of the second cleavage).
374. It was found in the treated group that: (a) the
frequency of developmental abnormalities was increased by a factor of over 2 ( 15. 1% versus 6.4%) andthe frequency of unfertilized eggs was also higher(13.5% versus 1.6%); and (b) there was a striking
increase in the frequency of aneuploid and mosaic
embryos (10.1% versus 4.0%). suggesting that delayed
ovulation induced non-disjunction and anaphase lagof chromosomes during both the meiotic and firstcleavage divisions. There was also a significant increaseof polyploidy. a consequence of polyspermy in thetreated group.

(b) Incidence of spomaneous chromosomal anomalies
in Chinese hamster and mouse oocytes and early zygotes
375. Using an improved cytogenetic technique [K32.
M41] Mikamo and Kamiguchi [M42] examined thespontaneous incidence of chromosomal anomalies in
Chinese hamster oocytes and early zygotes. Secondary
oocytes were collected from the ampullary region of
oviducts of female hamsters 5-8 months old and used
for chromosome preparations. To arrest mitosis of
I-cell and 2-cell zygotes, colchicine was injected into
copulated females and the zygotes were subsequently
collected by flushing of the oviducts. In secondary
oocytes, 1.6% (31/1970) were aneuploid (0.6% hyperand l % hypo-haploidy): 0.4% were diploid (giant
diploid oocytes twice the normal size) and there were
no diploid oocytes caused by a suppression of the firstpolar body. A further 1.8% carried structural aberrations.
376. In I-cell embryos. 3.2% (37/1146) were aneuploid with approximately equal numbers in the hyperand hypo-haploidy groups, 1.5% of the embryos weretriploid (0.9% of diandry, 0.3% of digyny and 0.3% of
uncertain cases), and 2.8% carried structural anomalies. In 2-cell embryos, the incidence of triploidy(1.6%) and haploidy (0.4%) were similar to those in
I-cell zygotes. However, the incidence of aneuploidy
was lower (2.0%), suggesting selective death of someaneuploids. The frequencies of structural anomalies
were higher in the embryos (2.8%. in I-cell zygotes
and 3. 7% in 2-cell zygotes) relative to those in oocytes.
The authors found that: (a) the incidence of aneuploidyin 1-cell zygotes of maternal origin (2.1%) was 3 times
that of paternal origin: (b) first-division meiotic errors
were also 3 times higher than second-division errors(1.6% versus 0.5%); and (c) the incidence of femalederived structural aberrations was the same as that of
the male-derived ones (1.3% and 1.4%, respectively).
377. In another study, Sugawara and Mikamo [S79]analysed the effect of maternal aging on the configuration of chiasmata. formation of univalents andsegregation of first meiotic chromosomes in young (58 months) and old (16-19 months) Chinese hamsters.
Primary oocytes were collected from mature follicles
approximately 10 hours before ovulation and secondary
oocytes were obtained from the oviducts 5 hours after
spontaneous ovulation. It was found that the average
number of chiasmata per oocyte was significantly
lower in the old hamsters than in the young ones, but
terminal chiasmata were more frequent in the oldanimals. results reminiscent of those of Henderson
and Edwards [H42]. Since the 11 meiotic chromosomes could be divided into four morphologicallydistinguishable sub-groups. it was possible to determine whether the same bivalent forming univalents at
MI actually underwent non-disjunction in the following meiotic division. The incidence of both MI oocyteswith a univalent pair (2.0% versus 9.1 %) and aneuploid MII oocytes (1.5% versus 3.6%) due to firstmeiotic non-disjunction was significantly higher in theaged group than in the young group; however.
univalents occurred almost exclusively in the small
metacentric chromosomes (96%) whereas non-disjunction took place nearly equally in each chromosomal
83sub-group. These results demonstrate no correlation
between the univalents seen at MI and non-disjunction
during the first meiotic division and this is true ofboth young and old hamsters.
378. In a further study, Sugawara and Mikamo [S80]adduced evidence for a striking induction of nondisjunction in Chinese hamster primary oocytes by intraperitoneal colchicine injection (3 pg/g body weight) at
the onset of formation of the first meiotic spindle inoocytes of females with a normal oestrous cycle. The
incidence of aneuploid MII oocytes was 25.9% in theexperimental group, while it was 2.0% in the controls.
Their results support the notion that maternal ageeffects known for aneuploidy may in part be associatedwith incomplete formation of spindle microtubules,
which in turn can lead to increased incidence of
meiotic chromosome non-disjunction in oocytes ofaged females.
379. Tease and Fisher [T43] used lower doses ofcolchicine (0.25 and 0.5 µgig) in similar work withfemale mice and obtained only a 10% increase overcontrols in hyperhaploidy and a 20% increase in
bypohaploidy. However. they found a marked age
effect of colchicine in that old (50-56 weeks) female
mice had a higher sensitivity to metaphase I arrest at
the smaller dose, as compared with young (9-12 weeks)
females. but a lower sensitivity at the higher dose. The
authors consider that the maternal age effect on nondisjunction may be related to a difference in spindle
behaviour in young and old females.
(c) X-ray induction of structural chromosome
aberrations in Chinese hamster and mouse oocytes

380. The earlier data of Mikamo et al. [M43] onstage-dependent changes in chromosomal radiosensiti vity of primary oocytes in Chinese hamster werediscussed in the 1982 UNSCEAR report. These results
demonstrated that early diakinesis was the mostradiosensitive phase, the incidence of metaphase II
oocytes with structural aberration being about 15 times
that after treatment of early dictyotene stages ofdioestrus (43% versus about 3%) (see also Mikamo[M44]). Irradiation of subsequent stages, (i.e .. lateMl-anaphase I. telophase I. polar body emission),
however, yielded fewer chromosomal aberrations, andmore dominant lethals and might be related to theoccurrence of unrepaired lesions and a reduction in
DNA repair capacity, for which there is some evidence
from mouse studies [M68] after UV irradiation.
381. In subsequent experiments. Kamiguchi and
Mikamo [K33] determined the dose-frequency relationship for structural chromosome aberrations in early
diakinesis (over a range of x-ray doses from 0.1 to
4.0 Gy). The types of aberrations found were breaks,
deletions, fragments and exchanges and the metaphase
II aberrations were mainly of the chromatid type. The
frequencies were found to increase with dose (from0.0033/cell after 0.1 Gy to 0.814 in the 2.0 Gy group)
the data being consistent with a quadratic model. A
comparison of these results with those published by
Reichert et al. [R38] for the mouse (in both studies.
84

the meiotic stage of the oocytes and chromosome
analysis were similar) shows that the rate of inductionin the hamster is only about one-half of that in themouse. This is in contrast with earlier results where
the spermatogonia and .lymphocytes of both these
species were found to have similar radiosensitivities[B34. B35].
382. Tease and Fisher [T42] obtained much higher
aberration yields than Reichert et al. [R37] after
similar radiation regimes with mouse oocytes. but theaberrations were scored at metaphase I rather than at
metaphase II. They considered that these differences
most probably reflect the greater accuracy of the metaphase I method for assessing chromosomal damage.
Similarly to Reichert et al.. however, they reported
that the data on chromatid interchanges best fitted aquadratic model.
383. Tease [T44] also investigated whether dictyate
oocytes of old (42-52 weeks) female mice were moreradiosensitive than those of young ( 10-15 weeks) ones
when metaphase I chromosome preparations weremade from superovulated females 3.5 or 12.5 days
after 0.5 Gy or 2.0 Gy acute x irradiation. Very few
aberrations were found after 0.5 Gy. but, as expectedfrom previous work. after 2.0 Gy the aberration
frequencies were significantly higher at 12.5 days thanat 3.5 days of sampling. At this latter dose level, there
were more chromosomal aberrations in metaphase I
cells from old females than from young ones. but thedifferences were not significant.
384. Searle and Beechey [S 135] also comparedchromosome aberration yields in young (8-12 weeks)and old (about 48 weeks) female mice after the same
x-ray doses of 0.5 Gy and 2.0 Gy. but with a slightly
longer interval of 2-3 v.·eeks between irradiation andmetaphase I chromosome preparations. Although theinterchange frequencies were not significantly higher
in the old females, the overall frequencies of theaberrations scored (interchanges + fragments) weresignificantly higher after 2.0 Gy in oocytes of old
females (35.5%) than of the young ones (12.5%).
Thus. there is some evidence that maturing oocytes ofolder females are more sensitive to aberration induction, although data are limited and more work isneeded.
(d) Synaptonemal complexes as indicators of induced
structural change in chromosomes of Syrian hamster
spermatogonia and of post-meiotic germ cells
in the mouse385. The conventional cytogenetic method of studying induction of chromosomal damage in sperrnatogonia of experimental mammals involves the analysisof spermatocytes (descended from treated spermatogonia) at diakinesis/metaphase I. The translocations
give rise to multivalent configurations. which, given
good cytological preparations, are relatively simple todetect at diakinesis/metaphase I. This technique has
been very successfully used over the years in quantitative radiation cytogenetic studies, particularly those
aimed at ascertaining the frequencies of reciprocalatranslocations. However, for multivalent associations
to persist until diakinesis/metaphase L homologous
segments must synapse and there must be chiasma
formation in at least one of the translocated segments.
as well as in the non-translocated segment. As Evans[EI7] pointed out. this requirement for chiasma
formation may mean that rearrangements of small
segments may be missed unless pachytene cells areanalysed.

7

,t
I

/
I
I

8

/

/'man
/

/
5

386. Cawood and Breckon [C55] compared theefficiency of the conventional method mentioned
above with that involving the analysis of pachytene
cells using synaptonemal complexes as visible markers(the SC method) and Syrian hamsters as experimental
material. Following testicular irradiation with 2.6 Gy,
the animals were kept for 5-15 weeks before they were
killed. One testis from each of 7 animals was used formaking cytological preparations with the conventional
and the SC methods. The results showed that, despite
variation in the frequencies of abnormal cells (i.e.,
those carrying multivalent configurations) between
animals. the overall frequencies with the SC method
were about twice as high as those with the conventional methods (8.6% versus 4.6%). The authors
considered that the analysis of diakinesis/metaphase I
cells alone may lead to a serious underestimation of
the amount of structural change induced in spermatogonia after irradiation and that the SC method canprovide information not obtainable by any othermethod. Kalikinskaya et al. [K55] reached similar
conclusions in their experiments. They also pointed
out that the SC method could be useful in the analysisof the causes of sterility of F I males descended from
irradiated P I males.
7. Induction of reciprocal translocations inspermatogonia of non-human primate species
387. Cytogcnetic data on the induction of reciprocaltranslocations in stem cell spermatogonia of nonhuman primates and in those of man were reviewed in
the l 97i and 1982 UNSCEAR reports. The overall
conclusions were that: (a) the sensitivity of the
spermatogonia of the marmoset Saguinus fuscicollis ishigher than that of the rhesus monkey. Macaca
mulatta: (b) at the dose levels studied, the yields oftranslocations from irradiated human spermatogonia
are roughly comparable to those in the marmoset
species mentioned above: (c) following acute x irradiation. the dose-effect relationship in all the three
species is humped with a maximum around I Gy; and(d) the dose-effect relationship up to the dose of peakyield is consistent with linearity. Some additional data
have now become available from studies with therhesus monkey at low dose rates and after fractionatedexposures [VI4, VJ5] and with two new primate
species. namely, the marmoset Callithrix jacchus [V5,VI I] and the crab-eating monkey, Macaca fascicularis[M27, M69, T45]. All the currently available primate
data are summarized in Table 21 (see also Figure I).
388. Van Buul [Vl4] and Van Buul et al. [VI5]found (Table 21) that, when an x-ray dose of I Gy isdelivered to rhesus monkey spermatogonia at a dose

4

.

c
u

•

3

0..

__ ..... • ..........crab-eating monkey..
(Calllthrl1)
I

OOH (Gy)

2

3

Figure I. Dose-response relationships for the induction ofreclprocal translocalions in stem-cell spermatogonia of someprimate species by high dose-rate x or gamma irradiation.

rate of 0.002 Gy/min, the yield of translocation isabout one-half of that at the same x-ray dose delivered
at 0.3 Gy/min. A gamma-ray dose of I Gy deliveredat either 0. 14 or 0.0002 Gy/min gave similar yields,
however, and these were about the same as thatobtained at an x-ray dose of I Gy delivered at
0.002 Gy/min. When an x-ray dose of 4 Gy wasdelivered into two equal fractions separated by aninterval of 24 hours. the yield was higher relative tothat after a single dose of 4 Gy. and less than one-halfof that expected on the basis of additivity of yields;
the latter observation is in contrast with that obtainedin mouse experiments [L57. M80) in which. withsimilar fractionation regimes. the yields continued toincrease linearly with dose.
389. The data for Callithrix jacchus suggest that thesensitivity of this species to the induction of translocations by acute x or gamma irradiation may besimilar to that of the rhesus monkey. but clearly lower
than that ofSaguinus fuscicollis studied by Brewen et al.
[B6l]. However, Macaca fascicularis responds with frequencies that are intermediate between those observedin the rhesus monkey on the one hand and Saguinus
fuscicollis on the other [M27. M26]. The possible basis
for these differences is discussed below (chapter VI,
section C). Comparing their data for Macaca fascicularis with those for different rodent species publishedin the literature, Matsuda et al. (M69) noted that, overthe dose range below I Gy, the sensitivity of Macaca
fascicularis (slope of 1.79 10-2 Gy- 1) is similar to thatof the mouse. the Chinese hamster and the rabbit(slopes per Gy: mouse. (1.39-2.56) 10- 2; Chinese
hamster, 2.15 10-2: rabbit, 1.39 10- 2). The values forthe guinea pig arc different in two different studies:
0.98 10- 2 [L41] and 2.75 10-2 (B62]: consequently, it
is difficult to make rigorous comparisons.
85390. Tobari et al. [T45] have a study in progress
involving the induction of translocations in spermatogonia of the crab-eating monkey by chronic gamma
irradiation ( 1.8 10- 5 Gy/min: about 0.024 Gy/22 h/day). The total doses are: 1.0, 1.5 and 2.0 Gy. The
results available so far (at 1.0 and 1.5 Gy) areconsistent with a linear dose-effect relationship definedby the equation
Y = 0.91 10- 3 + 0.16 10-2 D

where Y is the yield of translocations in percentage
and D is the dose in Gy. When these data arecompared with those obtained at high dose rates by
the same authors (Table 21) it is clear that the slope
after chronic gamma irradiation is only about onetenth of that after high-dose-rate irradiation. Thus,
there is evidence for a pronounced dose-rate effect inthis primate species. in contrast with the situation
recorded for the rhesus monkey (see paragraph 388).

8.

Qualitath·e comparison between spontaneousand radiation-induced mutations in mice

391. Detailed genetic analysis of mutations recoveredfrom irradiated and control parents in mouse specificlocus experiments indicates that the mechanisms bywhich spontaneous mutations arise may differ fromthose involved in the radiation induction of mutations[R39]. The frequency of fractional (mosaic) mutants
at all 7 of the marked loci was roughly similar in
offspring of irradiated and control mice [R40], and thesame was true in a large sample in which the study
was restricted to the c locus [R4 I]. Thus. radiation
does not induce fractionals, and the observed fractional mutants in irradiated as well as control groups
are presumably of spontaneous origin. At the c locus,
the large majority of spontaneous mutations werefractionals, and, at d, almost one-half were also
fractionals. (Fractionals would probably be nondetectable at se and more poorly detectable at d thanat c.) Analysis of segregation ratios derived from
16 c-locus fr'actionals [R71] Jed to the conclusion thatthe mutations had occurred in one strand of thegamete DNA, or in a daughter chromosome derived
from pronuclear DNA synthesis of the zygote. or in
one of the first two blastomeres prior to replication.
392. Another mechanism implicated in the production of certain spontaneous mutations is the so-called
double non-disjunction, whereby the offspring receives
two copies of the marked chromosome and no copiesof the wild-type chromosome. Several spontaneous dse mutants [R42] as well as an Hbbd c+ mutant areapparently of this type.'' The event is detectable only
where two or more markers are present on thechromosome involved. When only one marker ispresent. the result is indistinguishable from a repeat
mutation to the marker allele. Since there is no''However, the results of translocation intercrosses involving
chromosome 7 (on which the c and Hbb loci are located), as well as
the lack of simultaneous cchp mutant offspring in specific locus
experiments, indicate non-complementation of gametes with gains
and losses of chromosomes [S134]. It seems probable, therefore,that the Hbb mutant arose by a different mechanism.

86

evidence that either mosaic or double non-disjunction
mutants are induced by radiation exposure of thegerm cells, Russell [R39] has proposed that the
population of spontaneous mutants might have astrong admixture of types not present in the population of induced mutants and has, therefore, raisedquestions on the appropriateness of the doubling-dose
approach in the calculation of genetic risk.
393. It is possible to make a rough calculation of the
maximum extent to which the spontaneous mutation
frequency, and therefore the doubling dose, would bechanged if all double non-disjunctional events were
excluded in specific locus data in males. In all. thespectrum and viability of 32 specific locus mutations
recovered from unirradiated male mice have beenreported [E33, S 136]. There were no simultaneous cchp
mutations among these, but one out of 2 d se mutations
was homozygous viable and therefore probably the
result of double non-disjunction. There were no
mutations at the a locus while only one out of 9 at thes locus might have been viable in the homozygote. The
9 mutations at the b locus included a cluster of 2,
which seems much more likely to be the result of aspontaneous mutation early in gametogenesis than adouble occurrence of double non-disjunction. All but
one of the others which could be fully tested seem
viable repeats of b. Thus, between I and 8 out of the
32 mutations or 3-25% might have been the resultof double non-disjunction and therefore should be
excluded from doubling dose calculations.
394. Since, in all, 60 specific locus mutations in
1,005,363 offspring of control males have been reported[E33, R69a, R70], i.e., a mutation frequency per locus
of 8.5 10- 6. the exclusion of possible non-disjunctional
events would lead to a corrected control frequency
within the range of 8.2 10-6 and 6.4 10- 6 per locus.
Russell and Kelly [R70] derived a regression coefficient of 7.34 10- 6 per Gy from all the specific locus
spermatogonial data for chronic gamma-ray exposureof male mice. Use of the corrected control rate range
and the induction rate mentioned above gives acorrected doubling dose estimate of between 1.12 Gy
and 0.87 Gy. Clearly, the currently used doubling dose
estimate for chronic gamma-ray exposures of I Gy
would remain appropriate.

9.

l\folecular analysis of mouse specific locus mutations

395. The original spontaneous d (dilute) mutationand the closely linked se (short ear) are used among
the seven markers in mouse specific-locus mutationrate studies. Mutations involving the wild-type alleleof these markers can lead to altered melanocyte
morphology. abnormal cartilage development, central
nervous system disorders, or embryonic death. A large
number of radiation-induced mutations involving d.
se, or both loci were genetically analysed by complementation and deficiency mapping [R43]. These analyses resulted in the characterization of at least
16 complementation groups and the identification of
6 pre-natally lethal and 2 neo-natally lethal locibetween and around d and se. Since the originald mutation was caused by the integration of MuLV

into chromosome 9 (see chapter IV), this provirus canbe used as a marker to gain molecular access to thed-se region. Cellular DNA sequences flanking theprovirus have been cloned and used to probe genomic
DNA from several of the genetically characterized
mutants in this region [R44). Specific DNA fragments
have been correlated with specific deletions. and aphysical map of one deletion break-point has been
constructed [R7I]. The molecular analysis also provides physical proof that many of the mutations aredeletions. confirming what had earlier been surmisedon the basis of genetic evidence.

10.

Genetic and cytogenetic effects of incorporated
radioacti~·e isotopes in mice

396. Data on genetic and cytogenetic effects ofincorporated radioactive isotopes were reviewed in theearlier reports of the Committee and more recently by
Searle [S8I]. This section focuses on data that became
available since the publication of Searle's review.
397. Balonov and Kudritskaya [B36] compared theeffects of tritium, administered as tritiated water(3HOH), and chronic gamma irradiation on the
induction of dominant lethality in post-meiotic andmeiotic male germ cell stages. Spermatids were foundto be more radiosensitive than either spermatocytes orspermatozoa. The effect did not depend on the dose
rate in the range from I 10- 3 to I.7 Gy/min (total
doses from 0.5 to 3.7 Gy). The RBE of tritium(relative to chronic gamma irradiation) increased withdecreasing dose, reaching a value of about 2.5 at doses
less than 0.1 Gy. These studies were subsequently
extended to include the induction of reciprocal translocations in spermatogonia [P43]: they showed that
the RBE of tritium increased from I at a dose of l Gy
to 2 at a dose of 2 Gy.
398. In another study, Pomerantseva et al. [P29]examined the effects of incorporated 14 C after single.
long-term (33 days) and chronic (6 and 12 months)
treatment of male mice ( (CBA X C57BL) F 1) with
14 C-glucose. The frequencies of dominant lethals in
post- and pre-meiotic germ cells, of translocations inspermatogonia (analysis of spermatocytes) and of
sperm abnormalities were determined. The estimatedabsorbed gonadal doses were the following: 0.22, 0.50
and l.01 Gy (single-exposure experiments). 0.74 and1.47 Gy (long-term exposures). 0.006 and 0.031 Gy(chronic exposures for 6 months). 0.031 and 0.066 Gy( 12-month chronic exposure).
399. In the single-exposure groups, the frequencies
of dominant lethals (expressed as the ratio of live
embryos per female in the experimental group to thatin the control group) increased approximately linearlywith increasing dose in post-meiotic cells: in premeiotic cells. there was no induction. In the Jong-term
exposure group. the frequencies were somewhat lower
than in the single-exposure groups (post-meiotic aswell as pre-meiotic cells). In the 6-month chronic
group, the frequencies were again low and there wasno measurable induction in the 12-months chronic
group. The frequencies of translocations induced inspermatogonia were low (0.34-0.84% in single-exposuregroups; 0.20-0.60% in the long-term group: and0.06-0.10% in the chronic groups) and only in the
single-exposure and long-term-exposure groups were
the frequencies higher than in controls. Sperm abnormalities were about 1% in all the groups, except in the
13-month exposure group where they ·were between
3.4 and 4.6% (control, 1.8%). On the basis of theirearlier results with external chronic gamma irradiation.
the authors concluded that 14c is no more effective
than chronic gamma rays.
400. Balonov et al. [863] showed that tritiatedglucose (3.3 MBq/g. corresponding approximately toan absorbed dose of I Gy) administered to malemice induced dominant lethals (in post-meiotic cells)
and reciprocal translocations (in spermatogonia)
with a pattern similar to that of tritiated water. In
other studies, Pomerantseva and Ramaiya [P44]found that intraperitoneal injection of 238 Pu-nitrate( 18.5 Ios-0.28 J05 Bq/g) with estimated doses of between
0.06 and 0.47 Gy induced dominant lethals in postmeiotic cells and translocations in spermatogonia.
Relative to chronic gamma irradiation, the absorbed
dose from 238Pu (alpha irradiation) was about 20 times
more effective. confirming earlier results [U2].
401. In the experiments by Generoso et al. [G55]. a
2 39Pu-citrate solution (about 379 kBq/kg body weight)
was injected into the tail vein of male mice(101 X C3H/F 1), which were then mated to T stock
females. Two series of experiments were carried out.
In the first series. the intervals from treatment to midpoints of mating periods were 23.5 and 52.5 weeks(with estimated spermatogonial stem cell doses of
0.54-1.07 Gy for the first mating period and 1.42-2.85 Gy
for the second). In the second series, these intervals
were 29 and 56.5 weeks (with estimated spermatogonialstem-cell doses of 0. 70-1.41 Gy and 1.55-3.09 Gy). The
male progeny were screened for the presence oftranslocations in genetic tests and those that were
found to be sterile or partially sterile were examined
cytologically.
402. In the first series. the frequencies of translocations were 0.20% (2/987. first interval) and 0.39%(6/1560, second interval). In the second series. thecorresponding frequencies were 0.23% (6/2633) and
0.56% (14/2505). Thus. there was an increase infrequency with time (i.e., accumulated dose). Withcorrection for control frequency (from another study
carried out with a different strain: 3/8095 or 3. 7 10- 4),
the rate of induction per Gy has been estimated to be
between 1.45 and 2.91 10-3. The effectiveness of alphairradiation does not appear to be markedly different
from that of acute x rays (rates of 3.89 10- 2 per Gy[GI6] and 3.42 10-3 per Gy [L50]). (Since there are no
genetic data on translocation induction after chronic
gamma rays. the effectiveness at low dose rates could
not be calculated.)
11. Sensitivity of the mouse, Chinese hamster andprimate oocytes to the killing effects of radiation

403. Data on the radiosensitivity changes between
oocytes in different stages of development and in87

different mammalian species were discussed in theearlier UNSCEAR reports and more recently byDobson and Felton [D37] and Dobson et al. [D49].
The new data on the radiosensitivity of mouseprimordial oocytes have raised some questions on thevalidity of extrapolation of mouse radiation mutagenesis results to human females. Primordial oocytesof two primate species studied show also striking
differences and oocytes of neo-natal Chinese hamsters
show some important differences relative to thesituation in the mouse and rats. This section isdevoted to a discussion of these results.

(a)

The mouse404. The fact that primordial mouse oocytes are very
sensiti\'e to the killing effects of x or gamma radiation
is well known. For instance. if a young female mouse
is exposed to a gamma-ray dose of 0.4 Gy and theovaries are examined a few days later, the remarkablefinding is that no immature primordial oocytes (whichconstitute about 90% of the oocyte population) arepresent: only growing and maturing oocytes (which
are about 10% of the oocyte population) remain [D52,013]. The LD 50 is of the order of 0.06 Gy. Similar
results were obtained following single intra-peritoneal
injections of 3HOH, reflecting the generally very highradiosensitivity of mouse oocytes to radiation-induced
killing [D39, D51, D52]. When the animals arechronically exposed to low levels of 3HOH in body
water during early life (from conception to 14 days after
birth), there is a dose-dependent germ cell destruction at
all tritium levels examined in a more than 100-fold rangeof concentrations. from 0.4 MBq/ml to 31 kBq/mL
without any evidence of a threshold (exponential
decrease in oocyte survival with an LD 50 of about
74 kBq/ml). When these results are compared withthose obtained after similar chronic low-level exposure
to gamma rays, the RBE of tritium increases fromabout 1.5 (at about 0.035 Gy/day; dose 0.5-0.6 Gy) tonearly 3 at low dose rates and low doses (less than
0.005 Gy/day and an effective dose of less than
0.05 Gy) ([D39, D40]: see Figure II). The increase of
RBE with decreasing dose is not due to any increased
effectiveness of tritium, but to a decreased effectivenessof.gamma rays at low doses.
405. The exceptionally high sensitivity of mouse
oocytes to the killing effects of lHOH and gamma
rays. together with the finding that 3H-TdR incorporated in the chromosomes is much Jess effective inthis regard [B64]. have prompted the suggestion thatthe target for killing effects in these cells is not in the
nucleus but at some distance away from it. The
realization that the target most likely involves cytoplasmic structures. adjacent follicle cells or the plasma
membrane prompted studies with neutrons for information on target cross section. These studies [S82]indicated that the target area. assumed to be circular.
had a diameter larger than the nucleus but smaller
than any combination of oocyte plus follicle cells.
Together with an estimated target volume (determinedfrom gamma-ray data) of about 4 µm 3, these results
suggest that the plasma membrane or somethingsimilar to it in geometry and location is the likely
target.
88

100
50
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0
0

1
0

0.1

0.2

0.3

0.4

0.5

EFFECTIVE DOSE (Gy)

3

1

0

0.01

0.02

0.03

GAMMA-RAY DOSE RATE (Gy/day)Figure II. Survival of primordial oocytes (as a function of doseand dose rate) In mice chronically exposed from conception to
14 days of age to tritlated water or to cobalt-60 gamma rays,
and the RBE of tritium compared with gamma radiation.
[D40]

406. Assuming that the membrane is the target. thedata for tritium, neutrons and gamma rays can bereconciled, and in each case the D 37 dose corresponds
to a deposition in the plasma membrane of about 500
to 1000 eV. Monte-Carlo calculations show that for an
x-ray exposure of 50 R to female mice. the predicted
oocyte survival (cells with mean plasma membranedose not exceeding 0 37) was about 1%, which is inagreement with observations [041]. The authors statethat in the experiments with exposure of 50 R done by
Russell, the nuclear dose to the surviving oocytes
could be much Jess, too low '"to give scorable yields ofmutations". They argue that this could explain thenegative findings, and that this has implications forthe estimation of genetic risk.
407. The possibility that the nuclear dose to surviving
oocytes might be less than the exposure given was firstsuggested by Russell in 1965 [R 72] as one of the threepossible explanations for the absence of mutations
induced in early oocytes by fission neutrons. He statedthat "the mechanism could conceivably be the survivalof only those oocytes that escaped having a proton
track pass through their genetic material". However.
he went on to argue that, if this were the explanation.
"the effect would not be seen with x or gamma rays".
In a large experiment with 50 R of x rays, the effectwas seen, namely, a very low or zero induced
mutation rate in early oocytes [R73]. Russell's argument that a reduced dose to the nucleus could not be
invoked to account for this was based on the view thatthe distribution of ionizations in the cell with x rays
would be so diffuse that the dose to the large nucleus
could not differ appreciably from the exposure of 50 R
administered. Current concepts of microdosimetry stillagree with this view.

(b)

Chinese hamsters

408. Tateno and Mikamo [T25] studied the responseof the neo-natal Chinese hamster ovaries (ovaries
exposed to l Gy on days 0, 2, 5, 6, 8, 10, 12 and 14
after birth) to the killing effects of x irradiation.
The ovaries were excised 48 hours after irradiation.
examined histologically and the results compared withthose obtained with unirradiated controls. The important findings are the following: (a) the Chinese
hamster oocytes are highly resistant to radiation
during the pachytene stage (at birth all ooctyes are atpachytene) and become extremely susceptible to thekilling effects of x rays when the oocyte maturation
advances further, especially during diplotene and thevery early dictyate phases (on day 4 the majority areat diplotene and by day 6 nearly all oocytes reach the
diplotene or dictyate phase): there is an almost
complete killing by I Gy at these stages: (b) with theonset of the resting phase (day JO onwards) the
oocytes become increasingly resistant. reaching levels
similar to pachytene: these observations are in contrast
with those in rats where the increase in resistance ismore gradual and the pachytcne levels are neverreached [M45]. Furthermore. whereas high sensitivity
is restricted in the Chinese hamster to days 4-8(diplotene-dictyate). after which the oocytes becomeprogessively more resistant. high sensitivity persists inthe mouse for a longer period (neo-nates from 8 to
26 days old). (See [T25] for a discussion.)(c)Primate species

409. Pregnant squirrel monkeys (Saimiri sciureus)
were exposed continuously to 3H in drinking waterfrom conception to birth of the young (153 days) andthe ovaries of the offspring were examined at birth[D37, D50. D52]. Concentrations of 3HOH in maternal
drinking water ranged from 204 down to 2.6 kBq/ml
and corresponding body-water levels were measured
at 116 down to 1.9 kBq/ml, delivering doses of about
6.8-0.1 mGy/day. The surprising finding was that there
was a massive oocyte loss which increased exponentially with dose. The LD 50 level was 18.5 kBq/ml(giving l mGy/day). which is about one-fourth of thatfound in mice. With other primates (the rhesus
monkey. Macaca mulatta, and the bonnet monkey.
Macaca radiata). and with similar treatment schedules(from the time of proof of pregnancy, day 25, to
parturition. day 164: lHOH in maternal drinking
water at 185 kBq/ml; 3H body-water concentrations
level by day 60 at 144 kBq/ml: dose rate of 8.6 mGy/day
during the second and third trimesters}, the ovaries of
the new-born showed about a 20% loss of oocytes(and thus much Jess than in squirrel monkeys). By 6
months, however. more fully expressed losses were
striking: 59% in the rhesus monkey and 95% in
bonnet monkeys.
4!0. Dobson [D50] suggests that. judging from these
data, early germ-cell losses exceeding 50% may lead topremature menopause in women. Since effective oocyte
LD 50 values for pre-natal rhesus monkey. bonnet
monkey and squirrel monkey were about 600, 200
and 100 mGy. respectively, and if such doses wereaccumulated over the second half of human gestation(160 days), doses of 3.8. l.2 or even 0.6 mGy/d may
already suffice to produce early menopause.
12.

Summary and conclusions

41 l. Further data have become available on theradiation induction of heritable tumours in mice.
Spermatids in males and maturing oocytes in females
seem to be the most sensitive stages for the inductionof genetic changes leading to tumours in the progeny.
The spectrum of tumours in controls and in theoffspring of irradiated parents is similar, with about
90% being papillary adenomas of the lung. The
pattern of transmission of these tumours is consistent
with a dominant mode of inheritance and a penetranceof about 40%. These data, originally obtained with the
ICR strain. have now been extended to include twoothers (the LT and N5 strains). Furthermore, evidence
showing that most of the tumours were malignant has
been obtained.
412. Treatment of the F 1 progeny descended from
irradiated parents (or in utero irradiation of pregnant
mice with the known carcinogen urethane) led to an
enhancement of tumour incidence relative to those in
the progeny that were not treated with urethane,suggesting that urethane may promote carcinogenesis.
413. X irradiation of rats or treatment with radioactive isotopes ( 90Sr. 238Pu) increased the frequency oftumours in the progeny.
414. Further data on the radiation induction ofcongenital anomalies in the offspring of irradiatedmice have been obtained. In these experiments. males
were irradiated and mated to unirradiated female miceand the progeny screened. by intra-uterine examinations. for the presence of congenital anomalies. The
overall frequencies of these anomalies (ignoring thedifferent radiation doses of between I and about
5 Gy) were significantly higher following irradiationof post-meiotic germ cells. The results of spermatogonial irradiation were variable: some experiments
showed significant increases over controls. whileothers did not show such differences. Dwarfism andexencephaly were the most commonly observed malformations.
89415. The question of whether irradiation of postimplantation maternal environment would have any
effect on the induction of congenital anomalies wasstudied in experiments involving embryo transfers andin others where the ovaries were surgically exteriorized
and the abdomen selectively irradiated. No significant
effects were found.
416. A number of recent studies on spontaneous andradiation-induced non-disjunction and chromosome
loss in mice have been reviewed. The so-called
production line hypothesis advanced to explain theeffect of maternal age on spontaneous non-disjunction
was tested using two different approaches. These data
showed that the hypothesis could not be confirmed
and that abnormal meiotic chromosome segregation isassociated with the process of physiological aging of
the reproductive system. A comparison of the frequencies of chromosomal aberrations and of nondisjunction after irradiation of pre-ovulatory oocytes
suggested the possibility that the induction of chromosomal exchanges might be the principal mechanism ofradiation-induced non-disjunction. This would be inline with previous findings in Drosophila.
417. New cytogenetic (examination of the fertilized
oocytes in vitro at the first cleavage divisions) andgenetic (use of the numerical sex-chromosomal anomaly
method) data have been reported concerning theeffects of maternal irradiation on the incidence of
non-disjunction. In the cytogenetic experiments. incontrols. the frequency of hyper-haploid female pronuclei was higher in the oocytes of old, as comparedwith young, females: but after irradiation, the increases
that were found did not show a relationship to age. In
the genetic experiments. however. neither maternal
non-disjunction nor maternal chromosome loss wasincreased by advancing age of the females in either theirradiated or the unirradiated group.

carrying translocations taking longer to pass throughmetaphase I.
420. Cytogenetic studies designed to examine the lossof radiation-induced chromosomal aberrations in preovulatory oocytes, 2-cell embryos and fetuses show thatmost of the structural aberrations induced in the oocytes
are efficiently eliminated by cell death before development proceeds to a feta! age of 13.5 days. Most of theelimination already occurs by the 2-cell embryonicstage.
421. In rats. there is now evidence that pre-ovulatory
overripeness of primary oocytes (prevention ofripening
through the use of pentobarbital) leads to a striking
increase in the frequency of aneuploid embryos derived
from them.
422. The frequencies of spontaneously occurring
chromosomal anomalies in Chinese hamster oocytes
and early zygotes have been determined using an
improved chromosome preparation technique. The data
support the notion that the incidence of aneuploidy ofmaternal origin (2.1 %) is three times higher than that ofpaternal origin and first division meiotic errors areabout three times more frequent than second division
errors. In experiments in which the effect of maternal
age on chiasma configuration. formation of univalents
and segregational errors at meiosis I of Chinese
hamster oocytes were studied. there was no evidence
for any correlation between univalents seen in metaphase I and non-disjunction during the first meiotic
division.
423. There is evidence to suggest that. in Chinese
hamster females, the early diakinesis stage is mostsensitive to the induction of structural aberrations ofchromosomes. A comparison of the radiation data
available for this stage in Chinese hamsters and miceshows that the former species is only one-half as
sensitive as the latter.

418. Two new methods have been designed to study
autosomal non-disjunction and chromosome loss innormal mice. making use of tester stocks containing
Robertsonian translocations and appropriate genetic
markers. While the evidence for radiation-induced
chromosomal loss was strong. confirming earlier
results for sex chromosomes, that for induced nondisjunction was. at best. weak.

424. New data using a technique for the analysis ofpachytene cells and the associated synaptonemal
complexes for the study of radiation-induced translocations in spermatogonia of Syrian hamsters haveprovided evidence for the thesis that the conventional
method of analysing diakinesis/metaphase I cells
underestimates the frequency by a factor of 2.

419. Further data on the radiation induction ofheritable reciprocal translocations in male mice (following spermatogonial irradiation) have been presented.
These show that up to 600 R, there is an exposuredependent increase in frequency, the slope being(3.89 ± 0.25) 10-s per R. A comparison of theabsolute frequencies at the different exposures obtainedin this study with those from cytogenetic studies at thesame exposure levels shows that the frequencies after
150 Rare consistent with expectations (i.e., one-quarterof the frequencies observed in spermatocytes): at higher
exposures, the observed frequencies of heritable translocations are only one-half of the expected ones. Data
have been obtained from cytogenetic studies suggestingthat these discrepancies may be due to spermatocytes

425. Further cytogenetic data on the induction ofreciprocal translocations by x or gamma rays in someprimate species have now been published (acute highdose-rate exposures). The marmoset Callithrix jacchus
has been found to have a sensitivity similar to that of
the rhesus monkey: both these species are much lesssensitive than the other marmoset species, Saguinus
fuscicollis. The crab-eating monkey, Macaca fascicularis, however, is more sensitive than the rhesus
monkey and Callithrix jacchus but less sensitive than
Saguinus fuscicollis in this regard. Furthermore. there
is a pronounced dose-rate effect after chronic gamma
irradiation of the crab-eating monkey (spermatogonial
exposure), the yield being about one-tenth of thatobtained at high dose rates.

90426. The finding that the frequencies of fractional
mutations recovered at the specific loci studied inmouse experiments are similar in control and after
irradiation suggests that fractionals are not induced byirradiation. Furthermore, there is some evidence thatspontaneous mutations, but not radiation-induced
mutations. may arise via double non-disjunction.
Since the population of spontaneous mutations may
thus represent an admixture of types not occurring inradiation experiments, it has been suggested that thevalidity of using the doubling dose method for risk
evaluations may become questionable; however. theinfluence of the doubling dose estimate is likely to besmall.

427. The recent demonstration that, in the mouse,spontaneous d locus mutations are associated with the
integration of murine leukaemic virus and the cloningof cellular DNA sequences flanking the provirus. has
now opened up the possibility of molecular dissectionof genetically characterized mutants at this locus.

428. The most recent data on the genetic andcytogenetic effects of incorporated radioactive isotopes
in mammals relate to the effects of 3H (given as
tritiated water to male mice) on the induction of
dominant lethals (post-meiotic germ cell sampling)
and to those of 14 C (given as 14 C-labelled glucose tomale mice) on the induction of dominant lethals (postmeiotic and pre-meiotic cells) and of reciprocal translocations in spermatogonia. The 3H results suggestthat the RBE (relative to chronic gamma rays)
increases with decreasing dose reaching a value ofabout 2.5 at doses of gamma rays lower than 0.1 Gy.
The 14 C results show that radiocarbon and chronic
gamma rays are nearly equal in producing the effects
mentioned. The effects of tritiated glucose are similar
to those of tritiated water. The cytogenetic dataobtained with 238 Pu are confirmatory of those reviewed
in the 1977 UNSCEAR report. The genetic data on
reciprocal translocations show that alpha-ray doses
from 239 Pu are no more effective than from acute x rays.

429. A detailed study on the radiosensitivity ofoocytes of neo-natal Chinese hamsters has been
carried out. The results show that oocytes in thepachytene stage (most of the oocytes present at birth)
are very resistant; this resistance abruptly decreases at
diplotene or early dictyotene, remains at the samelevel until day 8 (nearly all the oocytes reach diplotene
or dictyate phase) followed by a steep rise thereafter.
These changes in radiosensitivity profile in the Chinese
hamster are more marked than in the mouse or rat.

430. The three primate species studied from thestandpoint of the sensitivity of their primordial
oocytes to the killing effects of 3H show that thesquirrel monkey is even more sensitive than themouse. The other two. the rhesus monkey and thebonnet monkey, do not manifest this unusually highsensitivity.

B.

I.

STUDIES WITH IN VITRO SYSTEMS

Spontaneous and radiation-induced thioguanine
resistant cells in human lymphocytes

431. In the 1982 UNSCEAR report, the data ofAlbertini [A6] and Strauss and Albertini [S23J on
spontaneous 6-thioguanine resistant (6-TGR) variants
in peripheral blood lymphocytes were presented. As
may be recalled, these authors made use of thecytotoxicity of 6-TG to human lymphocytes possessing
normal HPRT levels to distinguish, and select for.
viable 6-TGR variants. In autoradiographic studies,such variants were shown to undergo DNA synthesis(incorporation of 3H-labelled thymidine) and survive
in the presence of normally toxic levels of 6-TG
following PHA stimulation.
432. These studies have now been extended by
Vijayalaxmi et al. [V2, Vl6] to lymphocytes from
Bloom syndrome (BS) and Fanconi anaemia (FA)patients. In the 7 BS patients, the corrected (i.e.,corrected for cycling S-phase cells) variant frequencies
were in the range 8.5-24.9 10- 4 with a mean of
17 .3 10- 4• In the age- and sex-matched controls. therange was 1.6-2.8 10- 4 (mean. 2.1 10- 4), indicatingthat. in BS patients. the variant frequencies are clearlyhigher (by about 8 times) than in controls. Likewise.
in the 2 FA patients studied [V 15], the correctedvariant frequencies were 11.4 10- 4 and 17. 7 10-4 ,which are higher than in controls ( 1.5 10- 4 and1.8 10- 4 ). It is instructive to note that even the control
frequencies determined by the autoradiographic method
are higher than those recorded in fibroblast work
(around 1.0 10- 5) [C69, D62].
433. The autoradiographic technique has the disadvantage that the 6-TGR cells are killed during theprocedure and the evidence that they are mutants istherefore circumstantial. Recently, it has become
possible to clone human lymphocytes from peripheral
blood [M46, M47] and the high efficiency of thismethod has made it possible to detect [A 14] andenumerate [M48] 6-TGR lymphocyte clones. The
frequency of 6-TGR lymphocyte clones that could be
detected by this technique is 10-6 to 10- 5 in healthy
young adults, and the frequency increases with age[T26]. Following expansion of the clones, it has beenshown that they are unable to incorporate hypoxanthine and direct measurements show little or no
HPRT activity [Al 4. M48].
434. In a recent paper. Albertini [A20] reviewed theautoradiographic and cloning assays from the selection of 6-TGR mutants in human lymphocytes. The
TGR mutant frequencies determined by the clonal
assays were found to be in the range from 10- 6 to 10-s
for normal adults: autoradiographically determinedvariant frequencies were also in the same range fornormal adults. when the lymphocytes were cryopreserved before study to remove phenocopies. Thus,
the generally higher frequencies recorded with theautoradiographic method without the use of cryopreservation, as was done in earlier studies (see also
[Vl5]). is due to the presence of phenocopies in the
mutant samples.
91435. Vijayalaxmi and Evans [V4] used the T-cellcloning technique to select and clone 6-TGR cells
present in human peripheral lymphocytes in viva. It
was found that the incidence of such cells ranged from0.83 10-5 to 2.53 10-s (mean: 1.48 10- 5) in healthy
individuals aged between 19 and 79 years. There were
no sex differences. However, the frequencies increased
with age at a rate of 2.4 cells/ I0 7 lymphocytes/year.
Exposure of G 0 lymphocytes in vitro to x-ray doses of
up to 2 Gy resulted in a dose-dependent increase in
the TGR cell frequencies; the rate of increase wasapproximately proportional to the square of the dose
although statistically a linear dose-effect relationshipcould not be excluded. With the latter model. theestimated rate of increase was 1.95 10- 5/cell/Gy. Boththe spontaneous and induced rates were similar to
those recorded for fibroblasts in a number of earlier
srndies and were from JO- to 20-fold lower than thoseobserved with the autoradiographic method.
436. A similar but independent study has been
carried out by Sanderson et al. [S83]. Mutationinduction to 6-TGR resistance was studied in freshly
isolated lymphocytes from healthy individuals. using
x rays and UV irradiation. In 8 of the 11 individuals
whose G 0 lymphocytes were studied using x irradiation,
proliferating lymphocytes on day 3 of culture werealso studied in the x-ray experiments. In addition to
studying mutation induction, data on survival andexpression time were also collected. The results
showed that: (a) the expression time was between 3
and 7 days; (b) with both kinds of radiation, there wasa dose-dependent increase in mutation frequency,
measured on day 10 after irradiation. For the G 0lymphocytes, for instance. the frequencies were 2.3 10- 5
after 2 Gy and 9.2 10- 5 after 4 Gy: lymphocytes
irradiated on day 3, i.e., proliferating ones. showed alower mutation induction and a higher mortality
relative to G 0 lymphocytes. The frequency at 2 Gy is
quite similar to that recorded in the work of Vijayalaxmi
and Evans [V4] in whose studies a 48 hours expression
time was used.
437. In the study of Vijayalaxmi et al. [Vl6] mentioned earlier, the T-cell cloning technique was also
used to determine the spontaneous frequencies of TGR
mutants in 5 FA patients. 5 FA heterozygotes andtheir age- and sex-matched controls. In the FA patients.
the frequencies were in the range 0.41-1.54 10- 5 (mean,
0.98 10- 5) compared to frequencies for controls, whichwere in the range 0.34-0.44 10-5 (mean, 0.40 10- 5).
Two of the FA patients had frequencies similar tocontrols, whereas in the other three there was a 3-foldelevation. There were no significant differences between
FA heterozygotes and controls. in all of whom therange was 1.2-1.9 10- 5 • which is similar to thatobserved in an earlier study of 24 healthy controls[V4]. It is instructive to note that the controls for FA
patients had lower spontaneous frequencies than thosefor FA heterozygotes. This is interpreted by theauthors as a reflection of the difference in their ages
(controls for FA patients ranged between 8 and17 years old and controls for FA heterozygotes from36 to 51 years old) and is in line with the earlier
observation [V4] that the incidence of TGR lymphocytes increases with increasing age.

92438. Messing and Bradley [M73] also used the T-cellcloning technique to study the frequency of TGR
mutants in lymphocytes of 12 breast cancer patients
before and after radiotherapy (gamma rays) and inpatients with heart disease unexposed to radiation. In
the normal controls (n = 12, mean age 35 years), theaverage mutant frequency was I. I 10- 6. which is wellwithin the range found by Morley et al. [M48]. The
frequency of mutants in the heart patients (n = 12.
mean age 52.4) was 2.4 10-6. which is not significantly
different from the above. In the cancer patients before
radiotherapy. however, the mutant frequency was
higher, being on average 8.6 10-6 (range, 0.1-54. 7 10- 6 ).
Although the radiotherapy patients were older thanthe controls. the authors point out that it is unlikelythat age alone accounts for the difference, since theolder heart patients had a frequency close to that of
the controls. After exposure of cancer patients to
50 Gy (2 Gy per working day for 5 weeks to the chestwall and surrounding lymph nodes: the treatment
began 1-2 months after surgery), or an estimated 5 Gyreceived by lymphocytes. the average frequency was
36.8 10- 6 (range, 7.6-102.2 10- 6). It was also foundthat sampling after 6 Gy revealed no effect on the
average mutant frequency. since blood was usuallydrawn from these patients 3 days after the start of thetreatment, a period insufficient to permit phenotypic
expression [S83].

2.

Induction of mutations and chromosome aberrations
in mammalian (including human) somatic cells
at low doses and dose rates

439. Making use of a special line of Chinese hamster
fibroblasts (V79S) capable of exponential growth insuspension culture. Crompton et al. [C27] examined themutational response of the cells to low-dose-rate 6<1Co
gamma radiation (0.032-0.04 Gy/hour). administeredover a period of 2-3 weeks in continuous (chemostat)cultures. At periodic intervals, cells were removed,
tested for viability and allowed to multiply. Following
an expression period of 6-10 days, the cells were again
tested for viability and for the induction of 6-TGR
mutants. In addition. the mutational responses of cells
to acute x irradiation ( 1.9 Gy/min) following growthin unirradiated continuous culture for 3-6 days werealso determined. The results showed a clear-cut
increase in mutation frequencies under conditions oflong-term exposure to low-level irradiation and there
was no indication of a reduced effectiveness of chronic
exposures. The authors suggested several possible
explanations for their findings. including the following:(a) differences in the distribution of cell cycle phases incontinuously irradiated cultures relative to normal ones:(b) differences in doubling times between irradiated andunirradiated cultures; (c) induction of error-prone
repair processes; and (d) build-up of mutagenic
intermediates in cultures during the extensive irradiation periods.
440. Pohl-Rilling et al. [PI6] have now published theresults of a co-ordinated research programme of theInternational Atomic Energy Agency in which the invitro responses of human peripheral blood lymphocytes to low doses of x irradiation (0.004, 0.01, 0.02,

0.03. 0.05, 0.1 and 0.3 Gy) were studied. Blood from
2 donors was used to conduct one master experimentand the culture time was 48 hours. The coded slides
were scored by investigators from IO participating
laboratories. The main findings, based on over 60.000
cells scored were the following: (a) the frequencies of
all types of chromosomal aberrations at 0.004 Gy
were, according to the authors· analysis, significantly
lower than the control values: (b) there was noincrease in the frequencies of dicentrics up to 0.02 Gy
and in those of terminal deletions up to 0.05 Gy;(c) the mean frequencies of all aberrations considered
together were not significantly different from one
another at 0.01, 0.02, and 0.03 Gy: and (d) over theentire dose range the dose-effect relationship is clearly
non-linear. A fit of these data to a linear-quadraticmodel showed that the observed total aberration
frequencies at doses of 0.0 I, 0.02, 0.03. 0.04 and0.05 Gy were below the curve defined by the model.
The authors explained these observations on the basisof possible altered kinetics of aberration induction atvery low doses. probably due to DNA repair mechanisms operating in these cells.

3.

;\folecular approaches to the stud)· of mutations
and D:"IA repair in mammalian somatic cells

441. The molecular approaches that are currently
being used to study mutations and DNA repair in
mammalian somatic cells, the results so far obtained
and the prospects for the future have recently beenreviewed by Lehmann [LI 7] and Thacker [T28]. Some
illustrative examples are discussed below.
442. Thacker et al. [TISJ introduced the E. coliXGPRT gene"· into HPRT deficient V79 Chinese
hamster cells by transfection (those expressing XGPRT
activity were selected by their resistance to the purine
synthesis inhibitor azaserine). Two sublines designatedas XGPRT+6 and XGPRT+Jo, which showed notendency to lose their resistance to azaserine despiteover 40 generations in non-selective conditions. wereselected for further analysis. Restriction enzyme andSouthern blot analyses revealed that the XGPRT genehas been incorporated in each subline as a single copy.
Since bacterial XG PRT will also use 6-thioguanine(6-TG) as a substrate. the authors carried out experiments in which XGPRT+JO cells were x-irradiated andmutants deficient in XGPRT activity (XGPRY-) wereselected using 6-TG resistance as a marker. The results
showed a linear increase of mutant frequencies with
x-ray dose. and higher spontaneous and induced
frequencies of these mutants over the corresponding
frequencies for HPRT deficiency in the parental V79
line. In other experiments in which the hamster
HGPRT gene was introduced into HG-PRT- cells.
x irradiation also led to the recovery of high frequencies
of mutants. All these data thus suggest that this highmutability is a property of the chromosome sites atwhich the transferred DNA integrates. Of particularinterest is the finding that the majority of 6-TG"'XGPRT: the bacterial gene coding for xanthine-guanine phosphoribosyl 1ransfcra.sc, which has now been cloned. The bacterial
XGPRT enzyme has functional similarity to the mammalian HPRT,
but XGPRT can utilize xanthine as a substrate, while HPRT (andmammalian cells in general) cannot.

resistant mutants of XGPRT+Jo show deletions of theintroduced gene (as defined in hybridization experiments).
443. In a similar study. Tindall et al. [T27] introduced
the E. coli XGPRT gene into a CHO line deficient in
HPRT activity and used the transformant cell line in
x-ray and UV mutagenesis studies. The results showeda dose-dependent induction of TGR mutants withmaximal recovery of UV-induced mutants after 7-9 daysof expression time. While this cell line (designated asgpt+) showed a cell-killing response comparable to thenormal CHO-HPRT+ cell line following irradiation.
there is a 10-25-fold increase in mutation induction at
the gpt+ locus versus the HPRT locus for a given doseof radiation. Two TGR clones (one spontaneous andone x-ray induced) so far analysed by Southern blot
hybridization were found to contain deletions involving
at least a portion of the gpt+ gene.
444. Thompson and colleagues [T29, T39] haveisolated a number of UV-sensitive CHO cell~ deficientin excision repair: these lines fell into five complementation groups. Two of these lines were sensitive toboth UV and mitomycin-C. Using human DNA as
donor and these cell lines as recipients in DNA
transfer experiments. Rubin et al. [R47J obtained anumber of transfectants that were no longer sensitive
to UV and mitomycin-C. Molecular analysis suggested
that the transfected human sequences contained thewild-type alleles of the defective gene. UV -resistanttransfectants were also obtained by Maclnnes et al.
[M49] by transferring wild-type hamster DNA into a
different repair-deficient recipient.

445. Using another repair-deficient CHO cell line(complementation group 2) as recipient for humanDNA. Westerveld et al. [W34] obtained evidence forthe correction of the repair defect in the CHO cells:
these authors subsequently cloned (in a cosmid vector)
the complete human gene. which corrects the defect in
the hamster mutant. This gene, designated as ERCC-1.
is about 17 kb long. This work represents the firstsuccessful attempt at cloning a human repair gene andis likely to be of use in studies devoted to the nature.
control and function of the ERCC-1 gene product inexcision repair.
446. In contrast with the relative ease of correcting
repair defects in Chinese hamster cell lines, studies
using human cells (xeroderma pigmentosum (XP).
ataxia telangiectasia (AT)) as recipients have had only
limited success (see [LI 7]). However, indications havebeen obtained that at least one AT cell line may havea defect in the cellular equilibrium between ligationand exonuclease digestion of DSBs in DNA [C56] andthat. despite the postulated DNA repair defect in AT,
these cells are competent recipients in DNA transfer
experiments [D42].
447. Meuth and colleagues [G35, M50. N26] analysed
mutations induced in the hamster adenine phosphoribosyltransferase (APRT) gene using the cloned APRT geneprobe [L18]. It was found that most EMS-induced
mutations in Chinese hamster cells resulted fromchanges below the level of detection and could besingle base-pair changes or deletions of less than93

50 base pairs; most of the spontaneous mutations werealso point mutations. Mutations induced under conditions of deoxynucleotide pool imbalances were also
predominantly point mutations. Two of the nine
gamma-ray-induced mutations at the dihydrofolate
reductase locus (studied with the aid of a cDNA
probe) contained altered restriction patterns consistent
with their being deletions, insertions or rearrangements [G36].
448. Spontaneous and UV-induced mutations at the
HPRT locus in Chinese hamster cells have been
analysed using HPRT gene probes. One out of
10 spontaneous mutations and one out of 9 UVinduced ones had large deletions in the HPRT gene[F27]. Fenwick at al. [F28] studied a HPRT mutant ofChinese hamster cells and its wild-type revenants.
There was no evidence for an altered restriction
pattern in the mutant cell line, suggesting that it
contained a pqint mutation. Some of the spontaneous
revertants produced a much stronger hybridization
signal, suggesting that the gene had been amplified up
to 20 times, with a corresponding overproduction ofmRNA.
449. Vrieling et al. [V 17) examined 19 x-ray-induced(600 R) HPRT-deficient mutants in V79 Chinese
hamster cells with Southern blotting using a mouse
cDNA probe. Twelve of the mutants had large
deletions (> 10 kb) of the HPRT DNA sequences.
Cytological studies of chromosome preparations ofthese 12 showed that. in at least 3 of them. part of thelong arm of the X-chromosome was lost. The authors
estimate that at least 70-80% of x-ray-induced mutants
at this exposure arc caused by large deletions. In thework of Thacker [T40] 43 gamma-ray-induced (5 Gy)
and 15 alpha-particle-induced ( 1.2 Gy) HPRT deficientmutants in V79 Chinese hamster cells were studied
with Southern blotting using a full length hamster
HPRT cDNA. Twenty out of the 43 gamma-rayinduced mutants were found to lack the functional
HPRT gene and another 10 showed various partial
gene deletions and/or rearrangements; the remaining
13 showed no detectable changes. In the alpha series.
6 out of the 15 were total gene deletions, 5 had partial
deletions and/or rearrangements and 4 had no detectable changes. Thus, it would appear that about 70%
or more of radiation-induced HPRT deficient murants
arise through large changes in the HPRT gene,
especially deletion.
450. Albertini et al. [A2 l] reported that 3 out of
11 TGR-Jymphocytes derived from a single normal
individual (by a T-cell cloning technique) showeddefinite changes by Southern blot analyses with a
947-bp full length HPRT cDNA clone. 4 showed
uncertain changes and in the remaining 4, there were
no detectable changes in the genomic DNA afterdigestion with 5 restriction endonucleases. These
results provide unequivocal evidence that the 6-TGR
mutants are in fact mutants. Similar results werereported by Turner et al. lT41]. Twelve of the
21 mutant clones (57%) had alterations not evident
cytogenetically; these involved major changes including
deletions, exon amplification and novel, sometimes
amplified, bands on Southern analysis.
94

4.

Use of somatic cell hybrids to identify repair genes
and their chromosome locations in human cells

451. Mouse cells repair UV-induced DNA damage
with about 5-10% of the efficiency of human cells. Lin
and Ruddle [L9] reported that somatic cell hybrids
between primary mouse cells and SV-40-transformed
XP cells (complementation group A) exhibited wildtype (human) levels of DNA repair capacity, as judged
by the levels of unscheduled DNA synthesis; they also
demonstrated that mouse chromosome 4 was involvedin compensating the DNA repair defect in XP cells.
Lalley et al. [LIO] isolated human x mouse somatic
cell hybrid clones, employing human cells normal for
UV repair, UV-irradiated them and examined theirrepair profiles using the BrdUrd photolysis assay. It
was found that some of the hybrid clones had apattern of repair similar to that of human cells, some
had a pattern similar to that of mouse and some fell inbetween. Since the numbers of dimers per 10 6 dalton
were the same in mouse, human and hybrid cells, theyexamined hybrid clones for the presence or absence ofindividual human chromosomes. A strong correlation
between the presence or absence of human chromosome 3 and the presence or absence of UV repair was
found. In these three clones, there was also a
correlation between human chromosome 14 and excision repair capacity, although there was one clonethat was positive for chromosome 14 but negative forrepair.
452. Lalley et al. [LIO) also examined whether
mouse cells could contribute one or more steps
required for excision repair of UV damage in XP cells,
Fusion experiments were carried out between cells
from 5 of the XP complementation groups (A-E) andmouse cells. The resultant hybrid clones segregatedhuman chromosomes. When the XP x mouse hybrid
cells were analysed for repair ability, it was found thatmouse cells reduced the level of defective repair ofXP cells in complementation groups A-D but not in
E. When the XP x mouse hybrids lost certain humanchromosomes (the chromosomes not specified), theylost the ability to be complemented by the mouse
genome. suggesting that these hybrids had lost thegene(s) coding for the repair step that is rate-limitingin the mouse.
453. An essentially similar approach has been usedby Hori et al. [I-!21] to study repair in mouse cells.
These authors examined somatic cell hybrids produced
by fusion between the UV-sensitive, excision-repairdeficient Q3 ITG3 cell line (derived from mouse
lymphoma L5178Y cells) and PHA-stimulated human
lymphocytes for their UV sensitivity and unscheduled
DNA synthesis (UDS). From the initial two hybrid
clones, subclones were isolated at various intervals
and segregation of UV-sensitive clones was tested.
Most of the UV-resistant clones exhibited nearly thesame resistance as the parental L5 l 78Y cells. whereas
most of the UV-sensitive clones showed UV hypersensitivity similar to that of the Q31 mutant; the UDS
capacity of the UV-resistant clones was similar to thatof human lymphocytes. Analysis of the distribution of
specific human chromosomes in each subclone showed
a positive correlation between UV sensitivity and thepresence or absence of human chromosome 13.

5.

Inducible (SOS-like) responses to DNA damage andadapth·e responses in mammalian cells(a)Inducible SOS-like responses

454. The fact that E.coli and other bacteria respond tovarious adverse conditions by inducing SOS responses-'is well established [K48, L42, R59. W48]. Theseobservations and indications that inducible functions
may be associated with the tolerance of DNA damage
in living cells catalyzed a search for analogous
phenomena in eukaryotic cells. Although there aredata suggesting that mammalian cells respond tophysiological perturbations (which in E. coli act as
inducing signals for SOS responses), there is as yet nounequivocal evidence for the de-repression of previously
repressed genetic functions and no indication thateukaryotic cells respond to inducing signals by expressing a battery of genes under the control of acommon regulatory system, as is true in E. coli [F37].
Thus the evidence for SOS-like responses in mammalian cells is largely phenomenological. It has been
recently reviewed [H53. F37] and only a few illustrative examples are discussed here.
455. Viruses have been widely used as probes tostudy a variety of cell functions since they offer the
possibility to separate the effects of cell treatment
from those resulting from the treatment of the viruses.
Table 22 taken from the paper of Herrlich et al. [H53]
provides a summary of results obtained using viruses.
In these studies. the virus is UV-irradiated andallowed to infect UV-irradiated or chemically treatedhost cells (or unirradiated or untreated host cells).
Evidence for an enhanced reactivation (ER: survivalof irradiated virus on treated host/survival of irradiatedvirus on non-treated host) and enhanced mutagenesis(EM: mutation frequency of the irradiated virus intreated host/mutation frequency of the irradiatedvirus in non-treated host) was sought. As can be seenin Table 22. UV-irradiated virus such as Herpes. SV40
or adenovirus or transfected viral DNA survived
better in host cells that had been pre-treated withmutagen than in control cells. The ER factor increases
as the time interval between cell treatment andinfection increases. until a maximum is reached after
which the expression of ER decreases. The ER factors
are in most cases on the order of between I and 5 andcertainly Jess than those observed with lambda phage
after infection of E. coli. The kinetics suggests thatinducible repair processes might be involved in ER.
The UV reactivation of HSV (herpes simplex virus)
and of Kilham virus (a single-stranded parvovirus) is
inhibited in the presence of cycloheximide [L46],
suggesting a requirement for active protein synthesis.
Cycloheximide does not block protein synthesis selectively in mammalian cells, however, and DNA synthesis is also arrested. In general. ER is associatedwith treatments that cause breaks or gaps in cellular·'The term SOS responses in bacteria denotes the initiation of aregulatory signal causing a simultaneous de-repression of a numberof genes. The products of some or all such genes enhance thesurvival of the bacterium or of its normal resident phages, prophage
induction, phage mutagenesis, cell filamentation, cellular responses
to DNA damage (including excision repair and DNA damage
tolerance) and genetic recombination [F37].

DNA or that inhibit DNA synthesis [B68]. Its
expression in human cells appears to be independentof excision repair, since the phenomenon can also be
shown in XP cells that are excision-repair deficient[L48).
456. With respect to EM (enhanced mutagenesis),the process presumed to be activated or induced
appears to be mutagenic, or partly mutagenic [D54,SI09. SI 10]. The rate of reversion to the wild type ofUV-irradiated SV-40 temperature-sensitive mutantsgrown in UV-irradiated monkey kidney cells is increased
relative to that in unirradiated cells [G52. S 110].
Sequence analysis in one case suggested that thereversion occurred at a site opposite to a possible
thymine dimer. indicating that the mutation wasprobably targetedY [S 110. S 111]. The same conclusionof targeted mutagenesis can be deduced from the factthat the degree of mutagenesis depended largely on
irradiating the virus prior to infection [C65. C66].
457. Some laboratories have reported increased mutagenesis of non-treated virus in mutagen pre-treated cells[C65. D54], but others [D55, Sl09, SI 10] have failed
to see such an increase. In cases where untargetedmutagenesis has been recorded, this could be theresult of an error-prone damage tolerance mechanismthat is activated by host-cell DNA damage. an
observation that is in line with untargeted mutagenesis
recorded in phage-bacterial systems.
458. Integrated DNA from DNA or RNA viruses
can be activated to produce viral particles by treatment of cells with a variety of DNA-damaging agents(reviewed in [F37]). This process bears at least asuperficial resemblance to prophage induction inlysogenic bacteria. An interesting parallelism in doseresponse curves has been found for UV induction of
SV-40 provirus and enhanced reactivation of irradiated
HSV [Z7]. and for the induction of SV-40 provirus
and the formation of SCEs following exposure to
ethylmethanesulphonate or mitomycin-C [K49]. But it
remains to be established whether these correlations
signify a common regulation of the expression of thethree functions.
459. Another approach that has been used to study
induction of repair in mammalian cells is the use of
split-dose protocols. in which a first relatively small
dose is given (and is presumed to be the inducing
stimulus), followed by a large DNA damaging dose.
When CHO cells are exposed to a small UV doseseveral hours prior to a larger dose, and are subsequently pulse labelled and analysed in alkaline
sucrose gradients, the DNA synthesized after exposure
to the second dose is smaller than that observed for
DNA from cells that did not receive the first small
dose. However, the DNA increases in size when thecells are subsequently incubated in unlabelled medium;
YA mutation is targeted if it occurs at the site of the original
mutagenic interaction, e.g., UV-induced pyrimidine dimer. ln
contrast, the term untargeted mutation occurs in an unirradiatedphage when SOS proficient host cells arc previously induced. These
could be divided into mutations opposite spontaneous depurinations
and into truly untargeted replication errors. These cannot be
distinguished at present [M70].

95this apparent enhanced rate of chain elongation is notobserved in cells incubated with cycloheximide between
the two UV treatments. suggesting the need for de novo
protein synthesis. These results have been interpreted
in terms of a possible mechanism for enhanced DNA
synthesis that facilitates replication past dimers [D57].
460. Studies of the overall rate of DNA synthesis
through autoradiography of cells labelled with
3H-thymidine also show enhancement in split-dose
protocols [M7 I). However, in mutation experimentswith CHO cells, in which they were first irradiatedwith x rays at times O and 17 hours before being
irradiated with UV. there was no evidence for anenhanced recovery of ouabain-resistant or 6-TGR
mutations (x rays in these experiments is a putative
inducer of error-prone cellular replicative response,
since it is known that they do not induce ouabainresistant mutations) [C67].
461. Cornelis et al. [C66] studied the problem ofwhether the expression of inducible error-prone repair
processes could be correlated with an increased
sensitivity to oncogenic transformation. In these experiments, the authors determined the transformation
frequency in unirradiated and UV-irradiated human
VH12 cells, using a deletion mutant of SV40 Jacking
the origin of DNA replication. The results showedthat UV-irradiated cells were transformed more efficiently than unirradiated cells. This enhanced transforming activity (defined as the ratio of the efficiencyof transformation per cell in UV-irradiated cultures tothat in unirradiated cultures) increased as the interval
between cell irradiation and transfection with viral
DNA increased. Susceptibility to transformation decreased again after a maximum was reached when thecells were infected three days after irradiation. Sincetransformation with SV40 is thought to be due to theinsertion of the virus genome into host sequences by aprocess involving recombination. this observation
provides evidence for the existence in human cells ofa UV-inducible recombination process.
462. The data discussed in the foregoing paragraphs(and others discussed in [F37] and (H53]) strengthen
the possibility that SOS-like responses are inducible inmammalian cells, all following DNA damage or arrestof replication. Formal proof. however. requires knowledge of the type of reactions involved or of the genes
coding for repair enzymes involved in this pathway.
both of which are not available at present.

(b)

Adaptive responses

463. The SOS-like responses in mammalian cells
discussed so far resull in increased survival as well as
increased yields of mutations. ln 1977. Samson and
Cairns [SI I 2) reported that chronic exposure of
E. coli cells to very low non-toxic levels of the simple
alkylating agent N-methyl-N' -nitro-nitrosoguanidine(MNNG) results in the de novo induction of an
adaptive DNA repair pathway. Once induced, this96

pathway affords the cells considerable protection
against both the killing and the mutagenic effects ofalkylating agents. i.e., it is antimutagenic and thus isdifferent from SOS-like responses. Adaptation is
specific for alkylation damage; it can be induced by,
and provide protection against. all the simple alkylating
agents that have been so far tested (methyl-nitrosourea,
methyl-methanesulphonate, ethyl-methanesulphonate.
N-ethyl-N' -nitrosoguanidine ). Adaptation cannot be
induced by. nor protect against, UV light or 4-nitroquinoline-1-oxide damage [.127, SI 13].

464. Since the resistance of MNNG did not develop
when protein synthesis was inhibited by chloramphenicol. it was proposed that a protein was responsible forthe (error-free) repair of mutagenic lesions induced by
the exposure to a low level of MNNG. Subsequent
work confirmed that such an explanation was indeed
correct. Of the several adducts formed in the DNA
upon exposure to alkylating agents, 0 6-alkylguanine is
implicated as premutagenic [G53. L47] and precarcinogenic [G54]: if left unrepaired, Q6-alkylguanine isrecognized as adenine during DNA replication and so
causes GC AT transitions. Schendel et al. [SI 14]
and Robins and Cairns [R61] demonstrated that. in
the MNNG experiments, the adapted E. coli cells are
exceedingly efficient at removing Q6-methylguaninefrom their DNA. This removal is extraordinarily rapid[C68] and achieved by a hitherto undescribed DNA
repair mechanism, which is now termed as theadaptive response mechanism. Surprisingly, the
06-methylguanine is not eliminated by nucleotide
excision repair but rather by a direct transfer of themethyl group from the 0 6 position of guanine to acysteine residue, which is probably the adaptive
enzyme itself (DNA methyltransferase). The enzyme
has been recently purified [D58]. It became clear thatthe adaptive enzyme can act only once, and istherefore consumed during the DNA methyltransferase reaction [L48. R61]. The mutagenic protection
provided by the adaptive response therefore shows anunusual and characteristic saturation at very highdoses of the alkylating agent [S 114, SI 15].

465. Studies have been carried out to examine
whether adaptive responses could be demonstrated
with mammalian cells; they represent a logical extension of those in bacteria. Most of these studies, carriedout with alkylating agents, have been recently reviewed
by Samson and Schwartz [SI 13] and Frosina and
Abbondandolo [F38] and are beyond the scope of thepresent section. There is. however. one study withradiation in which presumptive evidence for an
adaptive response has been obtained and this isconsidered below.
466. Olivieri et al. [O 18] cultured human peripheral
blood lymphocytes in 3H-thymidine-containing medium(3H-thymidine was added 4-6 hours after culture
initiation and was the source of low-level chronic
radiation) and irradiated them with 1.5 Gy of x rays at
7. 9 or I I hours before fixation. Appropriate controls
were maintained. It was found that in theH-thymidine + x-ray group, the yield of chromatidaberrations (chromatid and iso-chromatid deletions
and chromatid interchanges) was less than the sum of
the yields of aberrations induced by 3H-thymidine andx rays separately. At the same fixation times. nonradioactive thymidine did not affect the yield of x-rayinduced aberrations. In another experiment, the effectsof continuous treatment with 3H-thymidine on che
frequency of chromatid aberrations treated with 0.4 Gyof x rays 50 hours after stimulation (and fixed 3 hours
later) were studied. The findings were similar. The
authors suggest that these responses are analogous to
the adaptive response to alkylating agents. whereby
prior treatment with small doses for a long period oftime reduces the damage occurring after larger dosesof similar agents given for a short time.
3

6.

Summary and conclusions

467. New data on the incidence of spontaneous
6-thioguanine resistant variants (studied with theautoradiographic method) in human lymphocytes
from Bloom syndrome and Fanconi anaemia patients
have been published: they show that these are about
eight times higher than in controls. The frequencies
determined by the autoradiographic methods are
generally higher than those known for mutants studied
at the same locus with the same selective agent in
human fibroblasts. However, if the lymphocytes are
cryopreserved before study to eliminate phenocopies.
the frequencies become lower and comparable tothose recorded in fibroblast work and in T-cell cloning
work (see below).
468. The newly developed T-cell cloning technique
has been successfully used in studies on the determination of spontaneous as well as radiation-induced
6-thioguanine resistant mutations in human peripheral
blood T-lymphocytes. The spontaneous frequencies
are in the range between 10- 6 and 10-s in healthy
young adults and the frequency increases with age.
With x irradiation. there is a dose-dependent increasein mutation frequency. Both the spontaneous andinduced frequencies are comparable with those known
from work on fibroblasts and from autoradiographic
work on lymphocytes in which the lymphocytes are
first cryopreserved before study.
469. The spontaneous frequencies of 6-thioguanine
resistant mutations determined by the T-cell cloning
technique, show that, in Fanconi anaemia patients,
they are about 2-3 times higher than in controls. With
Fanconi anaemia heterozygotes, there is no suchdifference.
470. Breast cancer patients who have undergone
radiotherapy show elevated levels of 6-thioguanine
resistant mutants in their lymphocytes (T-cell cloning
method) and these are higher than those in the same
patients before therapy. in controls and in heart
patients.
471. New data on the induction of 6-thioguanine
resistant mutations by chronic gamma irradiation ofChinese hamster fibroblasts (over a period of 2-3 weeks
in chemostat culture) suggest that there is no reduced
effectiveness of chronic versus acute irradiation.
472. Recent results of a collaborative study on the
x-ray induction of chromosomal aberrations (over therange from 0.004 to 0.3 Gy) in human peripheral
lymphocytes in vitro (G 0 irradiation) show no increasein aberration yields up to 0.05 Gy, beyond which theincrease is linear with dose.
473. Molecular approaches are being used to study
mutation and DNA repair in mammalian cells. Amongthe possibilities explored are: (a) introduction of theE. coli XGPRT gene (by transfection) into HPRTChinese hamster fibroblasts and their use in radiation
mutagenesis experiments: {b) correction of excision
repair deficiency of certain excision repair Chinese
hamster cell lines through introduction of wild-typehuman DNA: (c) cloning of the first human repair
gene; (d) similar attempts to correct repair defects inhuman cells (xeroderma pigmentosum, ataxia telangiectasia) through trasfection of suitable donor DNA; and(e) molecular characterization of spontaneous andradiation-induced mutations using appropriate cDNA
probes. The currently available data suggest that amajority of radiation-induced 6-thioguanine resistantmutants in Chinese hamster cells are large deletions.
474. Somatic cell hybrids (mouse X human) havealso been used to identify repair genes and theirchromosomal locations in human cells. Indications
that human chromosomes 13 and 14 may carry genes
involved in the correction of excision repair deficiency
in XP cells have been obtained.
475. A number of studies have been carried out toexamine whether inducible SOS-like repair processes(known from earlier work with bacteria) could bedemonstrated in mammalian cells following DNA
damage. Most of these data have been collected inexperiments using viruses as probes. They provide
evidence for the occurrence of such repair processes.
However, there is as yet no unequivocal evidence forthe de-repression of previously repressed genetic functions and no indication that eukaryotic cells respondto inducing signals by expressing a battery of genes
under the control of a common regulatory system, as
is the case in E. coli.
476. Most of the studies that bear on the problem ofadaptive responses-cellular responses to mutagenic
treatments that result in higher survival and lower
yield of mutations-carried out in mammalian cells
have used chemical mutagens and carcinogens. In one
study with human lymphocytes, in which low-level3
H-irradiation was used to stimulate the putative
repair process involved in adaptive response before
giving doses of 0.4 or 1.5 Gy of x radiation. evidence
for a possible adaptive response was obtained: theyield of chromosomal aberrations is lower in the3
H + x-ray group than in the group given x rays alone.

97

C. INTERSPECIFIC COMPARISONS OFKARYOTYPES AMONG PRIMATE SPECIES:RELEVANCE FOR THE CHOICE OF PRIMATE
SPECIES CLOSE TO MAN FOR RADIATIONCYTOGENETIC STUDIES
477. The work of Dutrillaux et al. [D59] on thechromosomal evolution in primates was discussed in
the 1982 UNSCEAR report. From this work, theauthors concluded that: (a) the pattern of chromosomal evolution is not the same in the different
branches of the evolutionary tree, i.e .. the types ofrearrangements that have occurred vary from one
group to another (suborder, family, genus); (b) there isa very close similarity between the chromosome
banding patterns of the simians studied and man; and(c) all the quantitative and some of the qualitative
variations detected involve the heterochromatin. Fromthe standpoint of comparisons of chromosomal sensitivity of radiation (or other mutagens) to the
induction of different kinds of aberrations. two
principal questions remained unresolved: firstly, canthe evolutionary tree provide any clue with respect tothe inducibility of a given kind of chromosomal
aberration in different species and in man and thepathological consequences that may derive from such
induced aberrations; and, secondly. can quantitativeand qualitative comparisons based on the induction of
one kind of aberration in different species provide aproper perspective and a basis for predicting therelative radiosensitivity of human chromosomes todamage that is relevant in the context of induced
pathological states?
478. Dutrillaux and colleagues have now devised ascheme [D60] for comparing the karyotypes of different primate species that is potentially capable ofproviding some answers to the above questions. In
this scheme, the karyotype of each species is transformed into two histograms. one on the basis of
distribution of distances to telomeres (or more precisely,
the half-length of the chromosomes) and the other, on
the basis of distances to centromeres (= arm length).
To draw the first histogram, the individual chromosomes are ordered in terms of decreasing length (inR-banded preparations) and the relative half-lengths
are expressed as a percentage of the total length of the
genome. Each chromosome is assigned a constantwidth of 2 units so that the relative half-length andwidth define an area for each chromosome and the ·total area for each genome is 100 units. The second
histogram makes use of distance to the centromeres(arm length), ordered as above in terms of decreasing
length (i.e .. relative length of the chromosome arms).
which, together with a constant width of 1 unit,
likewise define a relative area for each chromosome
arm. Here too. the total area for all the chromosome
arms is 100 units. The heterochromatin at the telomericregions and in the short arms of acrocentrics is
considered separately from the euchromatin and thecentromeric heterochromatin. The amount of each ofthese is arbitrarily counted as 0.25% of the haploidkaryotypes.
479. The karyotypes of the different species characterized as above (each as two histograms) were used to98

discern similarities and differences between them.
Furthermore, since the histograms represent the distribution of all the chromosome segments of the
karyotype, they can be used as a basis to predict therelative sensitivities of chromosomes of the different
species to a given clastogenic agent. For instance. if
the chromosome breaks (involved in the different
kinds of rearrangements) are randomly distributed
among the different chromosomes and the chromosome arms (i.e.. show a proportionality to thechromosome lengths and arm lengths). the breakpoint distribution profiles should be superimposable
on the above histograms. However. if breaks are not
located at random (e.g., excessive number of breaks
in centromeric or telomeric regions or preferential
involvement of short or long arms), the break-point
profile will not be superimposable on the histograms
representing the species karyotype.
480. Among the eight primate species (including man)characterized as indicated above. the orangutan (Pongo
pygmaeus) and the marmoset (Callithrix jacchus) havekaryotypes most similar to man. followed by thegorilla (Gorilla gorilla). chimpanzee (Pan troglodytes),
the crab-eating monkey (Macaca fascicularis) and thesquirrel monkey (Saimiri sciureus). The profile of theAfrican green monkey (Cercopithecus aethiops) isclearly different from all the rest and. finally. thechimpanzee and gorilla have karyotypes most similar
to each other. When the comparisons are restricted tothe distributions of the relative lengths of the chromosomes only, the marmoset is clearly the closest to
man, followed by the gorilla and chimpanzee. If.
however, only the distributions of the relative lengthsof the chromosome arms are considered, the orangutanis the species closest to man. followed by the crabeating monkey (if acrocentric short arms of humanchromosomes are neglected) and the gorilla. If theheterochromatin distributions are taken into account.
the karyotype of orangutan appears closest to that ofman.
481. In order to test their model with respect to breakpoint distributions after irradiation, Muleris et al. [M72]
and Paravatou-Petsota et al. [P45] performed experiments in which peripheral blood lymphocytes of the
crab-eating monkey and of the chimpanzee were
gamma-irradiated in vitro (2 and 3 Gy). The breakpoints involved in the different rearrangements were
carefully mapped and the profiles so obtained compared with those expected on the basis of a random
distribution of break-points. In the work with the
crab-eating monkey, from the sample of 319 rearrangements, a total of 603 break-points could be accurately
mapped. The results were the following: (a) the overall
distribution of break-points showed a significant
excess in smaller chromosomes, as had been foundearlier in work with human lymphocytes and fibroblasts [D9, DlOJ: (b) in the case of reciprocal translocations, smaller chromosomes were more frequently
involved than expected on a random basis; the breakpoints showed a slight but non-significant tendency to
be located near the telomeres and the overall patternof break-point distribution could be characterized as
random; (c) for dicentrics, there was a significant
excess of breaks in the juxta-telomeric regions, but nosignificant excess near the centromeres; (d) for pericentric inversions, there was a significant deficit ofjuxta-telomeric breaks, an excess of breaks far fromtelomeres (i.e., near to centromeres) and a greater
involvement of longer chromosomes: and (e) for paracentric inversions, there was a slight non-significant
excess of juxta-telomeric breaks and of breaks near
the centromcrcs and a non-random involvement of
longer chromosomes and longer chromosome arms.
482. In the work on chimpanzees [P45]. the situation
was different. From a sample of 460 cell karyotypes
after R-banding, 1047 rearrangements were detected
and the break-points mapped. It is important to notethat the chimpanzee has terminal heterochromatin inmany of its chromosome arms. A comparison of thenumber of breaks in the euchromatin (1008) and inheterochromatin (138) in relation to their relative sizes( 100 units and 7 units, respectively) showed a large
excess of breaks in the heterochromatin. and thesewere located both in the terminal heterochromatin as
well as in the short arms of the acrocentrics. In thecase of reciprocal translocations, again, there was asignificant excess of breaks in the juxta-telomericregions and in the short arms of the acrocentrics. For
dicentrics. there was a significant excess of breaks.
again. in heterochromatin and a significant lack of
breaks in the euchromatin, except in the distal regions:the terminal Q-bands were even more affected than
the short arms of acrocentrics. In the case of pericentric
inversions. there was a tendency for chromosomes withrelatively small arms to be preferentially involved. (Infact. chromosomes 9, 22 and 16. which are relatively
small metacentrics. were among the most frequently
affected chromosomes in relation to size.) Finally, forparacentric inversions. almost all the breaks were in
the euchromatin (29/30) and no such inversions weredetected in the short arms of the acrocentrics.
483. Paravatou-Petsota et al. [P45] have pointed outthat the high sensitivity of chimpanzee lymphocytes tothe x-ray induction of dicentrics (2.2-fold higher thanwith human lymphocytes at a dose of 2 Gy) recordedin the work of Leonard et al. [L49] may in fact be
related to the presence of terminal heterochromatin in
about half of the chromosome arms. Among the
primates. the only other species that has terminal
heterochromatin is the gorilla and its lymphocytes
would be expected to be more sensitive than those of
man to the induction of dicentrics: this in fact had
been found to be the case ([061. L49]; 1.6 times moresensitive than human lymphocytes). From all thesefindings, the authors concluded that: (a) there are realdifferences in break-point distribution. between different rearrangements in the same species and between
different species; (b) the presence of juxta-telomericheterochromatin may be an important factor in thesensitivity of the lymphocytes of the different species
to dicentric induction: and (c) the differences in breakpoint distributions and differential involvement ofchromosomes in the various types of rearrangement
suggest that dicentrics may not be representative of
the sensitivity of induction for other rearrangements
and. therefore. dicentrics may not be the appropriate
cytological end-points in inter-specific comparisons ofradiosensi ti vi ty.

VII. RE-EXAMINATION OF AND FURTHERAPPROACHES TO RISK ASSESSMENTSA.

MUTATIONAL DAMAGE

1. Consistency of the genetic findings in Hiroshima
and Nagasaki studies with those expected from radiation
genetic studies with mice

484. As may be recalled. in its 1977 [U2] and 1982
[Ul] UNSCEAR reports. the Committee extrapolated
from the data on the induction of dominant skeletaland dominant cataract mutations in mice to risks
from the induction of mutations having dominant
effects in the first generation progeny. Furthermore, inthe 1982 report the results of continuing genetic
studies [Sl28] on the progeny of the survivors of theHiroshima and Nagasaki bombings were discussed. In
these studies. Schull et al. [S 128] used four indicatorsof genetic effects. namely. untoward pregnancy outcomes (i.e .. still birth. major congenital defects anddeath during the first post-natal week). occurrence ofdeath in live-born during the first 17 years of life.
frequency of children with sex-chromosomal aneuploidy
and frequency of children with mutations resulting inelectrophoretic variants. As Schull et al. stated," ... in
no instance is there a statistically significant effect ofparental exposure . . . but for all indicators. theobserved effect is in the direction suggested by the
hypothesis that genetic damage resulted from theradiation exposure ... ".
485. Ebling [E29. E30] examined the question ofwhether the above conclusion is consistent withexpectations based on mouse results with dominantcataract and dominant skeletal mutations. He used theestimate of the genetically significant dose (sustainedby the survivors of Hiroshima and Nagasaki) of
I.I 10 4 man Sv arrived at by WHO in its report [W52)on the "Effects of nuclear war on health and healthservices". The expected increases in the frequencies ofdominant mutations in 19.000 children were derived
as follows:

Cararacrs(I) Mutations/gamete/Sv(2) Genetically significant dose(man-Sv)(3) Expected number of cases ofcataracts/skeletal mutations[= (I) X (2))(4) Multiplication factors forthe overall dominant
mutation rate(5) Total number of expected
dominant mutations on thebasis of the cataract/skeletal
mutation data[= (3) X (4))

Skeleralmurarions0.45-0.55 10- 4 10.l 10- 4

I. I J04

1.1 J04

0.5-0.6

11

41

5

20-25

56

486. It is clear that, in the sample of 19,000 children
born to Hiroshima and Nagasaki survivors. less than
1 cataract mutation and about 11 dominant skeletal
99

mutations would be expected; the total number of
expected dominant mutations will be in the range
20-25 (on the basis of the cataract data) or about 56(on the basis of the skeletal mutation data). Eblingargues that with the kinds of end-points used in the
Hiroshima and Nagasaki studies: (a) it is not surprisingthat no clear-cut positive evidence for the induction ofgenetic damage could be demonstrated; and (b) the
Hiroshima and Nagasaki results are not inconsistent
with the expectations based on the cataract anddominant skeletal mutation data.
487. Abrahamson [A24) and the report of the UnitedStates Nuclear Regulatory Commission [E32] arrived
at similar conclusions. In their analysis of the Japanesedata, they used: (a) the paper of Schull et al. [S 131]
(specifically Table 7 of this paper in which mortality
up to age 17 for 16,713 children of exposed parents isgiven according to the distribution of parental doses)
as a basis; (b) the linear-quadratic equation for the
induction of gene mutations and of chromosomal
aberrations developed independently of the Japanesedata on the basis of experimental results (with themouse for mutations and with the marmoset Saguinus
fuscicollis for translocations): and (c) average doses
for each exposure group {parents). i.e., 0.05 Gy(0.01-0.09 Gy group), 0.295 Gy (0.10-0.49 Gy group).
0.745 Gy (0.50-0.99 Gy group) and 200 Gy (> 1.00 Gy
group). These values were introduced into the equations to project the number of cases of each genetic
event relative to the child sample size in each of the 32
sectors of exposure in the matrix by Schull et al.
488. As summarized in the United States Nuclear
Regulatory Commission report " ... among the 16.713
children born to parents. one or both of whom were
exposed, we conclude that there should have been
about 50 total cases of genetic defects distributed asfollows: 24 dominant. 5 X-linked. 4 aneuploid and
15 unbalanced translocations (early deaths) plus 55 casesof balanced translocation (detectable in otherwise
normal individuals). In addition. the lower limit
prediction is about 8 additional cases of genetic
defects plus 6 individuals with balanced translocations
and the upper limit prediction is approximately 170
additional cases of genetic defects plus 137 individuals
with balanced translocations. It should be obviousthat the central estimate prediction of cases should
lead to a statistically insignificant, that is. undetectable
increase in genetic disorders among the 16.713 progenyof irradiated parents. For example. there were 1040
deaths in this group of 16.713 progeny up to the ageof 17 (6.22%). In the unexposed groups. there were
2.191 deaths of 33.976 progeny produced (6.45%) andthe two frequencies are not significantly different. nor
would they have been even if 50 additional cases wereadded to the exposed group."
2.

Risks from the induction of recessive mutations

489. In the 1977 and 1982 UNSCEAR reports, therisk from the induction by radiation of recessive
mutations was considered negligible and no attempt
was made to quantify this risk. Instead, attention was
focused on the induction of mutations having harmJOO

ful dominant effects. and the risk from the inductionof recessives having heterozygous effects was considered as one component of the risks (see also [879,B80. 022]). Searle and Edwards [S 126] have now
provided a numerical estimate of risks for recessive
disorders. the principal considerations being that:firstly. harmful recessive mutations presumably canarise at all loci coding for essential proteins (perhaps
at least 10,000). and mutations to dominant alleles
may be a property of relatively few loci; and, secondly.
while many recessives doubtless remain to be discovered. those known at present tend to have earlier andmore severe effects than dominants. Induced recessive
mutations can cause harm by: (a) partnership with adefective allele already established in the population:(b) partnership with another recessive mutation induced
at the same locus: (c) the formation of homozygous
descendants, i.e., identity by descent; and (d) heterozygous effects.
490. Searle and Edwards based their calculations on
a combination of data from observations on humanpopulations and from mouse experiments and found
that: (a) a genetically significant dose of 0.0 I Gy ofx or gamma radiation received by each parent once ina stable population with a million live-born would
induce up to 1200 extra recessive mutations: (b) frompartnership effects. about 1 extra case of recessive
disease would be expected in the following ten
generations: (c) homozygosity resulting from identity
by descent would not normally occur until the fourth
generation after exposure. but, under certain assumptions. about JO extra cases of recessive diseases would
be expected from this cause by the tenth generation:and (d} in the same period, about 250 recessive alleles
would be eliminated in heterozygotes. on the assumption of a 2.5% heterozygous disadvantage. Searle and
Edwards have argued that these deleterious heterozygous effects should not be combined with those of
dominants. However, this would only apply to slightly
deleterious effects: more serious ones would probably
be included in the dominant category.
3. First-generation litter size reduction·following
irradiation of spermatogonial stem cells in male mice:a measure of deleterious effects of induced mutations(including deletions) and translocations
491. Selby and Russell [S 127] used the data on Ii ttersize reductions routinely obtained in the course of
specific locus experiments of Russell and colleagues
between I 950 and 1961 to study the effects ofirradiation of spermatogonial germ cells for thisendpoint of genetic damage. The data come from
14 experiments (carried out at different x-ray andgamma-ray exposures, both at high and low dose
rates) and involve a total of 158,490 litters. Litter sizes
were compared between experimental and control
groups at about three weeks after birth. In order toreduce variability. comparisons were made only withconcurrent controls and between groups of litters
having mothers of approximately the same age.
492. The main results were the following: (a) at thehigh exposure rate of 90 R/rnin. litter size reduction

(LSR) at weaning showed a humped exposure-frequency
curve (similar to those recorded earlier for the
induction of specific locus mutations and of translocations), being significantly lower after 1000 R thanafter 300 R or 600 R: (b) there was a pronounced
effect of exposure rate, the responses being much lessat rates of 0.009 R/min and 0.001 R/min. and. at
300 R. the exposure-rate reduction factor could be
estimated as about 6.7; and (c) for the nine chronic
gamma-irradiation experiments, the slope (defining
the induction of LSR per R) was (0.00194 ± 0.00076)%.
On the basis of these results, it can be estimated that if
male mice were exposed to I R of low-LET. low-doserate irradiation. the total number of induced deaths
between conception and weaning age (i.e .. 3 weeks),
would be about 19 out of every million F I mice thatwould have lived to three weeks of age in the absenceof irradiation. The authors suggested that about halfof these deaths would be the consequence of induced
reciprocal translocations and the other half due togene mutations and small deficiencies.
493. In his population studies in which a testicular
x-ray exposure (high exposure rate) of 276 R was
administered to male mice in every generation, Luning[L59] demonstrated that the net productivity (i.e., thenumber of weaned young per female) in the irradiatedpopulation was reduced by about the same amountfrom the first to the 18th generation, i.e., there was noevidence for an accumulation of genetic damage
having adverse effects on productivity. His data andanalysis pertain to a total of 5034 litters and 30,995
progeny at birth in the irradiated population and 5693
litters and 36.430 progeny at birth in the control. The
total numbers of young at weaning were. respectively.
25. 766 and 30.484. Litters with no weaned offspringwere excluded from the analysis. The results showed
that: (a} the ratio of litter sizes at birth between theirradiated and the control population was 0.9629(average value based on tests in generations 7-10. 11-14
and 15-18). indicating a 3. 71 % reduction in litter size
at birth in the irradiated, relative to the control.
population: and {b) the ratios of the number ofweaned offspring to total births in the irradiated andcontrol populations were. respectively, 0.8368 (again
an average value based on tests in the generations
mentioned above): the ratio of these two figures(i.e., irradiated/control) is 0.9934. indicating a 0.66%
induced death between birth and weaning in theirradiated population. Thus, the total amount of
induced death between conception and weaning canbe calculated as 4.37% at 276 R (it should be notedthat 85% of this induced death occurs before birth andthe remainder between birth and weaning).
494. On the basis of these data and on the assumption of linearity. the rate of induction of genetic
changes ea using death between conception and weaning
can be estimated as 0.01583% per R. If the exposurerate reduction factor of 6.7 (estimated by Selby andRussell at an exposure of 300 R) is used to correct theabove figure for effects expected at low dose rates,
then the rate becomes 0.002363% per R. corresponding
to 24 cases of death between conception and weaning
per R per million. This figure is in close agreementwith the 19 cases per R per million estimated by Selby

and Russell. Furthermore, if the relative proportionsof deaths between conception and birth and between
birth and weaning estimated for high-dose-rate irradiation (i.e .. 0.85 and 0.15. respectively) are applicable.
then about 20 of the 24 induced deaths will occur
before birth and 4 between birth and weaning.
495. At the request of the Committee. Searle andPapworth [Sl37] have now carried out an analysis oflitter sizes at birth and at weaning in 48,165 offspringof male mice exposed over 12 weeks to fission
neutrons or gamma rays. In the analysis. litters inwhich no young was weaned were excluded. Meanlitter sizes at birth were 2.3% lower after paternal(stem-cell spermatogonial) exposure to 2.14 Gy fission
neutrons + 0.93 Gy gamma rays, as compared with
6.06 Gy gamma rays + 0.025 Gy fission neutrons. thedifference reaching a significant level in male offspring. Moreover. the proportion of offspring whichsurvived to weaning fell significantly by 1.9% in theneutron series. On the assumption of a Q value of 20
for neutrons for the induction of these genetic changes
having dominant sub-lethal effects (on the basis ofresults for dominant visible mutations. see [B81]) the
authors estimated that the extra pre- and post-natal
mortality was induced by an extra dose equivalent of
37.17 Sv. This is equivalent to 6 extra pre-natal and5 extra post-natal deaths per million offspring induced
by a dose equivalent of 0.01 Sv. i.e., a total of 11 extra
deaths in all. Thus. in this experiment, 55% of theinduced extra lethality acted at or before birth and
45% after birth (versus 85% and 15%, respectively, in
Liining's experiments).
496. It is thus clear that three entirely different setsof experiments lead to similar conclusions. namely,that low-LET irradiation of male mice at low doses
and low dose rates would be expected to result in
extra deaths in F I between conception and weaning.
totalling 19 per million per R in the experiments of
Selby and Russell, 24 per million per R in those of
Liining and about 11 per million per 0.01 Sv in theanalysis of Searle and Papworth. The relative proportions of post-natal mortalities in the studies of Liining
and of Searle and Papworth are. respectively. 0.15 and
0.45, i.e., 3 and 5 cases per million per R (or per
0.0 I Sv); if these relative proportions are applied tothe data of Selby and Russell. then the corresponding
estimates are about 3 or 9 per million per R. The
Committee favours the use of a range between 5 and
10 per million per R to avoid the impression of too
much precision in these estimates.
497. Considering now the relevance of these data inthe context of risk assessment, it is important to notethat. in the past, the methods used by the Committee
for estimating dominant genetic damage have reliedon data collected in mouse experiments in which theevents were scored at weaning or in adult mice:consequently the question remained open as to whether
induction of genetic changes having dominant sublethal effects had been overlooked. If the results
obtained in the mouse and discussed above areconsidered applicable in the human context. it can beconcluded that the risk of induction of genetic changes
with dominant sub-lethal effects is relatively small.

IOI

While it is not possible to be precise as to when theextra post-natal mortality (observed in the mouse) will
become manifest in the human beings, it would
probably occur sometime in early life after birth.
B.

CHROMOSOMAL DAMAGE

1. An estimate of the percentage of conceptions withan unbalanced reciprocal translocation that sunive birth498. In the 1972 report. in the context of estimating
risks from the induction of reciprocal translocations inman, UNSCEAR estimated that about 6% of all
human conceptions with a structurally unbalanced
chromosome complement may result in live births
with congenital anomalies. This figure was also usedin the 1977 and 1982 UNSCEAR reports and wasestimated on the basis of limited data on the incidenceof unbalanced structural rearrangements in cytogenetic
surveys of new-born children (0.03%; 7/21.996) andon that in spontaneous abortions ( 1.07%: 8/747) thenavailable and on the view expressed by Carr [C73]that about 45% of all conceptions spontaneously
terminate before birth. The proportion of conceptions
that have an unbalanced chromosome complement
and that survive to live birth should therefore be

where p 1 is the proportion of conceptions that resultin live birth (0.55); a 1 is the frequency of unbalanced
structural rearrangements in live-born (0.0003); p 2 isthe proportion of conceptions that result in abortions(0.45): and a 2 is the frequency of unbalanced structural
rearrangements in abortions. However, an arithmetic
error was made in the calculations, resulting in theestimate of 6%. as has recently been pointed out byRussell [R69]. The correct estimate of conceptions
with unbalanced structural rearrangements among allconceptions with unbalanced structural rearrangements. with the data then used. is 3.5%. As Russell
pointed out. the estimate of 6% instead of 3.5% canhardly be regarded as serious in view of the size of thesampling errors of the human data.
499. Leaving aside the arithmetic error that led to an
estimate of 6%, it is instructive to inquire whether a
new estimate can be derived that will be consistentv.·ith current knowledge. Important in this exercise are
the following considerations: (a) more extensive dataon the frequencies of chromosomal anomalies inspontaneous abortions and in new-borns have become
available since the publication of the 1972 report: and(b) in the 1972 report, the frequencies of all unbalanced
structural rearrangements were taken into consideration, whereas in fact the calculations should be
restricted to the presumed unbalanced products ofreciprocal translocations only, since radiation does
not appear to induce Robertsonian translocations.
500. In discussing the review of Hook [HJ] on thecontribution of chromosomal anomalies to spontaneousabortions (and still birth) in relation to gestational age(see LB), it was pointed out that an estimated 15% of

102

all conceptions recognized by 5 weeks of gestation
onwards terminate in spontaneous abortions. This
figure does not include early pre-implantation losses
occurring prior to the clinical recognition of pregnancy
or the early post-implantation losses. both of which
probably occur [H60]. Tests are now available thatallow the detection of pregnancies within a week of
implantation: the results of one study [M78] of earlypregnancy loss suggest that 33% of pregnant women
abort spontaneously. However, there are as yet nodata on the incidence of chromosomal anomalies inthese early pregnancy losses. It is therefore preferable
to use the commonly accepted figure of 15% for
spontaneous abortions in the human species, since thisestimate is backed by extensive data.
501. From an analysis of cytogenetic data frompublished surveys of spontaneous abortions (n = 5726)
and new-born (n = 59,454), Jacobs [JIO] estimatedthat the total frequency of unbalanced structural
rearrangements was 1.54% in the abortus materialand 0.050% in new-born. The frequencies of stablestructurally abnormal chromosomes (i.e., monocentricchromosomes that have missing and/or additional
material. equivalent to unbalanced products of reciprocal translocations) alone were 0.681% in theabortus material and 0.012% in new-born (for thelatter. the estimate based on 67,014 babies summarized
in the 1982 UNSCEAR report is 0.01% which isalmost identical to Jacobs' estimate).
502. With these data, it is possible to make a new
estimate of the proportion of conceptions with anunbalanced structural rearrangement (or with anunbalanced product of reciprocal translocations) thatwill result in live birth, relative co all conceptions thathave an unbalanced structural rearrangement (or an
unbalanced product of a reciprocal translocation). For
making the overall estimate. the values to be used are:
Pi=0.85, p 2 =0.15, a 1 =0.0005. and a 2 =0.0154.
With these. the estimated value is 15.5%. If only theunbalanced products of reciprocal translocations are
considered, the figures to be used in the equation are:P1 = 0.85, P2 = 0.15. a 1 = 0.00012, and a2 = 0.00681
and the estimated figure is 9.1 %. In other words,
15.5% of all conceptions with unbalanced structural
rearrangements will survive birth, and 9.1 % of allconceptions with an unbalanced product of reciprocaltranslocation will survive birth. The Committee believes
that it is preferable to use a round figure of 9% for thepercentage of unbalanced products of reciprocal translocations that will survive to birth. in the context ofrisk estimations.

2. Model radiation studies with human peripheral
blood lymphocytes and fibroblasts to estimate theproportion of balanced reciprocal translocations,which if induced in germ cells may generate viable
imbalances after meiosis
503. Genetic theory dictates that a carrier of abalanced reciprocal translocation will, after meiosis,
produce three kinds of gametes with respect to theirchromosomal constitution: normal (i.e., not carrying
the translocation), translocation carrying. and un-balanced (i.e., with various deletions and duplications
for defined chromosome segments). While usually noadverse viability or phenotypic effects are expected to
be associated with the zygotes (and the progeny). with
the normal chromosome complement or with thebalanced reciprocal translocation-carrying complement, the majority of the zygotes with the unbalanced
complements will not be viable and will be eliminatedthrough early embryonic losses or abortions. A small
proportion of them. however, may be viable despite
the chromosomal imbalance and may thus result inchildren with one or another kind of birth defect. In
general. the extent of imbalance that is likely to begenerated and its effects on viability depend, among
other things. on the location of the break-points in thechromosomes involved in the translocation, the lengthsof the chromosome segments translocated. the genie
content of these segments and the segregational
properties of the translocations in meiosis. Segregationand fertility analyses of familial reciprocal translocations (ascertained through balanced or unbalanced
probands) [A5. 066, 067, Il4, VI9] provide someclues about the nature. variety and the extent of
imbalances compatible with live birth, but there are
no data on the proportion of balanced reciprocaltranslocations induced in human germ cells that may
generate chromosome imbalances that are viable. This
information is an essential prerequisite for the realistic
appraisal of the risks stemming from the induction oftranslocations by radiation.
504. In order to provide at least a partial answer tothis question. Outrillaux and colleagues carried out
model experiments with human peripheral blood
lymphocytes [D9] and fibroblasts [D JO]. In thelymphocyte study. G 0 lymphocytes were irradiated invitro with gamma-ray doses of I and 3 Gy. cultured
for 48-96 hours. and processed for the scoring oftranslocations. All the mitoses were analysed inphotographs after R-banding. A total of 247 translocations, each resulting from two chromosome breaks,
were scored. The break-points of the translocations
were accurately determined and the segments involvedin the translocations were quantified by using the
band-length unit defined in earlier work [A5]. =505. In the fibroblast work. the cells were derived
from skin biopsies from individuals who underwent
cosmetic surgery or from growing cells from otherbiopsies. These were irradiated with doses of I. 3 or
6 Gy. A total of 124 independent translocations wereascertained. In addition, 90 other translocations weredetected in cultures developed from the skin of ayoung boy acutely irradiated in an accident with aniridium-92 source [M 10]. The break-points, lengths ofsegments involved. etc.. were defined in R-banded
preparations for all the 214 translocations. as in thecase of those derived from lymphocytes.
506. The authors used the information obtained
from the analysis of these translocations: (a) to inquire
into the minimum possible imbalance that each ofthese translocations will generate. should they occur in~otal length of the autosomes is 280 band-length units; one unit
is thus 1/280 of the total length [A5].

germ cells, by considering the different types ofsegregation: and (b) to compare these estimates withthose available from studies of cases of partialmonosomies and trisomies reported in the literatureand that are known to be compatible with live births.
507. The main conclusions are the following: withthe lymphocyte-derived translocations. I 16 out of the247 (47%) are capable of generating viable imbalances,
should they occur in germ cells: 25 of the 247 ( JO%)may give rise to imbalances regarded as possibly not
viable: and with the remaining 106 (43%), the imbalances would be of such a magnitude as to beincompatible with survival. The amounts of imbalance
expressed in units of band length are 4.5. 5.5 and 10.2,
respectively, for the viable. possibly not viable and not
viable categories. If the last two categories are
combined, the imbalance is 9.3 units. In 43 families
where the translocations had been ascertained throughabnormal probands (i.e .. individuals who carried theunbalanced forms of the translocations and who wereviable). calculations showed that the mean amount of
imbalance was 4.1 units, an imbalance that is close to
the 4.3 units classified as viable in the radiation
experiment. In families where the translocations had
been ascertained through balanced probands (24 families) and where the unbalanced products were known
to have led to spontaneous abortions. sterility, etc.,
the mean imbalance was 8.0 units. again not strikingly
different from 9.3 units mentioned above for the
possibly not viable and not viable categories. The
authors believe that the proportion of translocations(116/247) giving rise to viable imbalances in thelymphocyte-derived translocations may be a slightoverestimate and that about 2/5 may be morereasonable.
508. The conclusions were essentially the same forthe fibroblast-derived translocations, the breakdown
among the categories of viable. possibly not viable
and not viable being 83 out of 214 (39%), 24 out of
214 (11.5%) and 107 out of 214 (50%). respectively.
This remarkable similarity in the distributions oftranslocations despite the differences in the tissue oforigin suggest that it is possible to extrapolate from
one tissue to another at least qualitively (i.e., to infer
the nature of the translocations and their segregational
consequences). It also indicates that the mean lengthof the exchanged segments is probably very similar in
the two tissues and that it is unlikely that the responseof the germ cells will be any different.
509. The authors stress that what they have attempted
to establish is the proportion of translocations that
may give rise to viable chromosome imbalances in theprogeny, should they be induced in germ cells. Theirfinding that about 2/5 of the translocations may giverise to viable imbalances is only a first step. notsufficient in itself to allow estimation of the risk ofchromosomal imbalance in terms of abnormal progeny.
For the former. it should be pointed out that reciprocaltranslocations are known to be induced in mitoticallydividing germ cells (namely. spermatogonia) as well as
in meiotic and post-meiotic ones. For the latter,
information is particularly needed on: (a) the probability that a translocation-carrying germ cell that103

may give rise to viable imbalance will actually produce
a gamete of this particular karyotype; and (b) the
probability that a zygote derived from such a gamete
will actually survive to birth rather than leading to an
early embryonic loss or an abortion.
510. In the mouse, about one-half of the gametes
from male germ cells carrying a reciprocal translocation are chromosomally unbalanced; the two
common types of imbalance involve duplication of
one of the segments exchanged combined with thedeletion of the other. while in some translocations
tertiary trisomy for a small translocation product may
also be relatively common. On the basis of theseresults, Searle [SI 29] suggested that if about one-halfof the unbalanced gametes from a viable imbalance
type of translocation led to zygotes that may survive
to birth, then one-half of 40% (or 20%) of unbalanced
gametes might lead to malformations at birth. He
cautioned that, in view of the great variation in
expression of these chromosomal anomalies, it isdifficult to be certain how many of the zygotes would
actually sun1ive to birth. Furthermore, extrapolationfrom mouse results to man is fraught with uncertainty
and probably incorrect with respect to the risk of
producing unbalanced progeny, for the following
reasons: (a) the mouse has only acrocentric chromosomes whereas most human chromosomes are nonacrocentric: (b) translocations with two distal breakpoints (i.e .. close to telomeres) are those most likely
to be viable; and (c) a karyotype with n acrocentrics
has only half as many such regions as a karyotype
with n non-acrocentric chromosomes. This means that( 1/2) 2 = 1/4 of the number of exchanges between
telomeric regions will occur in the first type ofkaryotype compared with the second. Therefore, therisk of producing viable imbalances (from balanced
reciprocal translocations) may be much higher in man
than in the mouse.

C.

SUMMARY AND CONCLUSIONS

511. Ehling examined the principal conclusion of thecontinuing genetic studies on the children of survivorsof the atomic bombs in Hiroshima and Nagasaki,
namely, that there are no statistically significant effectsof parental exposure. In doing this. he took into accountthe sample sizes involved, the genetically significant
dose estimated to have been received by the parents, thesensitivity of the end-points (untoward pregnancy outcomes, sex-chromosomal anomalies, survival throughthe first 17 years of life and the frequency ofbiochemical variants) and calculated what would be
expected on the basis of mouse data. He concluded
that the absence of significant genetic effects in the
Hiroshima and Nagasaki studies is not inconsistent
with expectations based on the mouse data. Similar
conclusions were reached by Abrahamson and by theUnited States Nuclear Regulatory Commission.

512. Basing their calculations on a combination ofhuman and mouse data. Searle and Edwards attempted
to estimate the risk from the induction of recessive
mutations in man. in terms of recessive diseases.
According to them, if a genetically significant dose of104

0.0 I Gy of x or gamma radiation is received by eachparent once in a stable population with a million liveborn, there will be about one extra case of recessive
disease in the following ten generations, i.e .. only aminority of the recessive mutations actually induced
would become manifest as recessive diseases within
ten generations.
513. Three sets of data on litter size reduction inmice in the first generation following spermatogonial
irradiation of parental males were analysed andcompared with respect to the amount of pre-natal(between conception and birth) and post-natal (between
birth and weaning) mortality induced by irradiation.
All these data sets are consistent with each other inthat they provide evidence for the induction of genetic
changes that lead to pre- and post-natal mortality.
The rate for the latter has been estimated to be about
5-10 cases per million births per R of low-LET, lowdose-rate irradiation of males. If it is assumed that thesituation would be similar following irradiation ofhuman males, then such death would presumably
occur some time in early life after birth.
514. The question of whether the estimate of 6%
used in the 1972 and subsequent UNSCEAR reports
for the percentage of human conceptions with anunbalanced reciprocal translocation that survive birthneeds revision was re-examined in the light of new
data. It was concluded that these data permit anestimate of 9% to be made. and that this figure can
now be used in the context of estimating risks
stemming from the unbalanced products of induced
reciprocal translocations.

515. Model radiation studies with human peripheral
blood lymphocytes and fibroblasts were conducted toascertain the proportion of balanced reciprocal translocations (should they be induced in human germ
cells) that may generat; viable chromosome imbalances.
It was concluded that roughly 40% of the induced
translocations may do so, but this figure in itself is not
sufficient to allow estimation of risks of chromosomal
imbalance in terms of abnormal progeny.

VIII.

CURRENT RISK ASSESSMENTS

516. As will be evident from the data reYiewed in thepreceding chapters (see also [S 138]) human genetics
and experimental radiation genetics are now passing
through a period of rapid growth and development.
Several advances in mammalian radiation genetics
throw light on the magnitude of genetic risks andallow some gaps in our knowledge to be filled, at least
partially. The data concerned are discussed below.

517. Inducrion of genetic changes having dominant
sub-lethal effects in the first generation. In the 1982UNSCEAR report, the risk of induction of dominantdeleterious mutations was assessed mainly from dataon the induction of dominant mutations causing
hereditary cataracts or skeletal abnormalities in mice,
combined with information on known dominantdiseases in man. Jn the experiments, the mutations
were scored at weaning (cataract mutations) or inadult progeny (skeletal mutations) and the question
remained open, therefore, as to whether the inductionof genetic changes having dominant sub-lethal effects
had been overlooked. Analysis of three extensive setsof data on litter size reduction in mice (generated inexperiments involving spermatogonial irradiation of
the parental males: see paragraphs 491-497) lend
credence to the view that such risks are small. as
recapitulated below.
518. The data of Selby and Russell [S 127] allow an
estimate of 19 10- 6 per R for the rate of induction of
dominant genetic changes (acting between conception
and weaning) that lead to reduction in litter size. The
rate of 24 10-<> per R that could be obtained from
Lilning's [L59] data (see paragraph 492) is quite
comparable to that of Selby and Russell. Lilning's data.
however. permit a partitioning of the total mortality rate
into that occurring before birth (about 20 10-6 per R)
and that occurring between birth and weaning (4 10- 6
per R). From a comparison of litter sizes at birth and of
survival between birth and weaning in experimen.ts
involving chronic gamma-ray (6.06 Gy + 0.025 Gy neutrons) and fission-neutron (2.14 Gy + 0.93 Gy gammaray) exposures, Searle and Papworth [S 137] foundthat the extra mortality observed in the neutron series
could be attributed to an extra dose equivalent of
37.17 Sv (on the assumption of a Q value of 20 for
chronic neutrons relative to chronic gamma rays).
Their estimated rates are 6 10- 6 per 0.0 I Sv for prenatal mortality and 5 10- 6 per 0.01 Sv, for mortality
between birth and weaning. Although the results of
Selby and Russell do not permit the partitioning of thetotal mortality occurring before and after birth, byapplying the relative proportions for these estimatedin the other two studies (Lilning: 0.85: 0.15 and Searleand Papworth: 0.55 : 0.45) it may be inferred that therate of post-natal mortality in the work of Selby andRussell would be about 3-9 cases per 10 6 births per
0.01 Gy. Thus. when all the results are considered
together. the induction rate of dominant genetic
changes causing death between birth and weaning, canbe estimated to be about 5-10 cases per 10 6 births per
0.01 Gy. If these results can be considered applicableto man, they would point to about 5-10 cases of death
some time between birth and early life per 10 6 live
births per 0.01 Gy of low-LET. low-dose-rate irradiation of males in the parental generation.
519. Induction of heredi1ary tumours. Recent work by
Nomura ([N8, N30]. paragraphs 338-342) has revealed
that radiation can induce heritable pulmonary andother tumours (average of 40% penetrance) in the F 1
generation. Thus. in principle, this effect should also
be included among those for the estimation of
dominant genetic risks. However, these tumours show
a very mild expressivity. As it is not yet known
whether any serious health effects may be associatedwith these tumours, they have not been included in thepresent risk estimates.
520. Induction of recessive genetic diseases. It is now
possible to provide new quantitative estimates of riskof recessive disease, stemming from the induction ofrecessive mutations (paragraphs 489-490). These estimates show that a one-time exposure to 0.0 I Gy of
low-LET, low-dose-rate irradiation of the parental
generation is not associated with any appreciable riskof induced genetic disease in the first generation (thesame conclusion was reached in the 1982 UNSCEAR
report): however, this exposure could result in one
extra case per million live-born by the tenth generation. The most serious detrimental effects of recessive
mutations in heterozygous condition would probably
be included in the dominant category of risk estimatesby the direct method (Table 23).
52 l. Fragile-X-linked genetic disease. It has become
clear that the heritable fragile site on the human Xchromosome is a major case of X-linked mental
retardation, next only to Down syndrome (paragraphs
47-62). The prevalence of this condition is currently
estimated as 400 10-6. This means that the prevalenceof X-linked disorders in man may need to be revised
upwards from about I OOO 10-6 to 1400 10- 6• However,since it is not yet known whether the fragile site on the
X-chromosome is inducible by radiation, it remains
difficult to include this condition into the risk estimates.
522. Risk from the induction of reciprocal translocations. Data are available on the induction ofreciprocal translocations by chronic gamma irradiation of spermatogonia of both the rhesus and thecrab-eating monkeys ([V 15, T45]. paragraphs 387-390,
and Table 21). In the former species, the observed
frequency of 0.43% (in spermatocytes descended from
irradiated spermatogonia after 1 Gy of gamma irradiation at 0.0002 Gy/min) after correction for controls(0.08%) allows an induction rate estimate of 0.35 10- 4
per 0.0 I Gy. From this. the rate of balanced reciprocaltranslocations in the F I progeny could be estimated as
0.09 10- 4 per 0.01 Gy and that of unbalanced products,
0.18 10- 4 per 0.01 Gy (on the assumption of no
reduction in transmission). If. as indicated earlier(paragraph 502), 9% of the unbalanced zygotes result inchildren with congenital malformations, then the risk
for the latter can be estimated as 1.62 1o- 6 per 0.0 I Gy.
523. In the cytogenetic work with the crab-eatingmonkey, the dose-effect relationship after chronic
gamma irradiation was consistent with linearity, thecoefficient of induction being0.16 10- 4 per 0.0 I Gy. The
same calculation as that for the rhesus monkey data
leads to an estimate of 0.72 10-6 surviving offspring per
0.0 I Gy, reasonably close to the estimate of 1.62 10- 6
per 0.01 Gy made for the rhesus monkey data. It would
therefore seem that a combined estimate of I 10- 6 per
0.01 Gy can be used as the lower limit of risk (instead of
0.3 Io- 6 per 0.0 I Gy used in the 1982 UNSCEAR report)of malformed progeny, associated with the induction ofreciprocal translocations in human males. Since, atpresent. it is unknown which of the non-human primate
species may be the best model for man, the Committee
prefers to retain the earlier estimate (made in I 977 and1982. on the basis of the limited cytogenetic data in man
and in the marmoset species Saguinus fuscicollis) todefine a possible upper limit of risk, but with anappropriate correction introduced to take into accountthe newly estimated proportion of unbalanced zygotes(i.e .. 9%) that will survive and result in congenitally105

malformed children. This revised upper limit estimate ofrisk is 15 10- 6 per 0.01 Gy. Thus, the risk from theinduction of translocations, expressed as congenitally
malformed children in the F 1• is now estimated to lie in
the range of 1-15 cases per million live births per
0.01 Gy.
524. Induction ofcongenital anomalies in the progeny ofirradiated parents. Kirk and Lyon [K 17] and Nomura[N9] have demonstrated the induction of congenital
anomalies (scored shortly before birth) following
spermatogonial irradiation of mice (paragraphs 343344). However, neither the true nature of the dose-effectrelationship nor the extent to which mice with theseanomalies would survive to birth and thus overlap withother categories of genetic damage is yet clear.
Therefore. these data have not been used in the present
risk estimates.
525. Summary of risk estimates obtained using thedirect method. These are given in Table 23. It can be seen
that: {a) the estimates of the risk of induction of
mutations leading to dominant genetic disease (based on
the skeletal and cataract mutation data in mice) are thesame as those arrived at in the 1982 UNSCEAR report(i.e., about 10-20 cases of affected individuals per 10 6
live births per 0.0 I Gy of parental irradiation and about
0-9 cases of affected individuals per 10 6 live births per
0.01 Gy of maternal irradiation); (b) the risk estimate
from the induction of genetic changes with dominant
sub-lethal effects (based on the data on litter size
reduction in mice) is given in footnote b of Table 23.
since there is uncertainty about the extent to which suchdata can be extrapolated to the human situation(namely. the comparability of death between birth andweaning in mice with death in early extra-uterine
human life): and (c) the risk associated with theinduction of reciprocal translocations is now revised(relative to that given in the 1982 UNSCEAR report)
to encompass a range of 1-15 cases of affectedindividuals per 10 6 Jive births per 0.0 I Gy of low-LET
paternal irradiation (and about 0-5 cases per 106 live
births per 0.01 Gy under the same conditions forirradiation of females). This change was dictated by
the new data obtained in the rhesus and crab-eatingmonkeys and the new analysis on the percentage ofconceptions with unbalanced genomes that may resultin children with congenital anomalies. Other details
are given in the appropriate footnotes to Table 23.
526. Summary of risk estimates obtained using the
doubling dose method. At present, there are no reasons
to alter either the doubling dose estimate of l Gy
(for chronic low-LET irradiation conditions) or theestimates of spontaneous prevalence of autosomal
dominant, X-linked, autosomal recessive and chromosomal disorders. Consequently, the estimates of risk
for these disorders are the same as those made in 1982
and these are reproduced in Table 24. However. thesituation is different with respect to disorders ofcomplex aetiology (congenital anomalies and othermultifactorial disorders). Jn particular, new data on
the latter (these disorders are predominantly of late
onset) suggest that their spontaneous prevalence ismuch higher than was estimated in 1982 ( 1982: 4. 7%:
now estimated at about 60%). This difference is due to106

the fact that disorders that become manifest up to age
70 have now been included (in contrast with theinclusion of only those that become manifest up to age
21 in the 1982 report). Considerable uncertainty still
remains on the following questions: (a) whether the
doubling dose estimate of I Gy (based on clear-cut
genetic end-points in the mouse. such as specific locus
mutations, dominant visibles and reciprocal translocations) is applicable to disorders of complex
aetiology; and (b) whether the estimate of 5% mutation component used in the 1977 and 1982 reports isa realistic figure for these disorders. In the absence offurther information, and particularly on the mechanisms of maintenance of these disorders in the
population and the effect of radiation on theirprevalence, the Committee is not in a position toprovide risk estimates for these disorders.

IX. ESTIMATES OF DETRIMENT FOR
SPONTANEOUSLY ARISING MENDELIAN,
CHROMOSOMAL AND OTHER DISORDERS
527. The 1982 UNSCEAR report reviewed the information available then on the approaches to theestimation of detriment associated with spontaneously
arising Mendelian, chromosomal and other disorders
and provided some tentative estimates using years oflife lost and impaired as indicators. The results ofstudies that have since been carried out are summarized
below.
A. THE EFFECTS OF MENDELIANDISORDERS ON HUMAN HEAL TH
528. Costa et al. [C74] and Hayes et al. [H6I]
conducted a literature survey to estimate the impact ofMendelian disorders on human health and their
response to medical treatment. The asterisked entries(i.e .. those with a confirmed mode of inheritance) in
McKusick's 1978 catalogue [M79] were used as a basis
for disorder selection (every third asterisked entry).
From these (after exclusion of conditions due toneutral polymorphisms and of those for which insufficient information was available), the authors selected
a total of 351 conditions ( 147 autosomal dominants.
170 autosomal recessives and 34 X-linked ones) for
further analysis. Published information on each ofthese was collected and examined to determine age at
onset, impact on longevity. reproduction and social
handicap. However, no prevalence estimates are given
for any of these conditions.
529. About 75% of these conditions were found to
be disadaptive. impairing one or more of the following:life span. reproduction, development and/or social
adjustment. Of these, 25% manifest themselves atbirth. 70% by age three years and over. 90% by theend of puberty. With regard to age at onset, autosomal recessives and X-linked disorders show aunimodal distribution and almost all are expressed byreproductive age. Autosomal dominants, on the other
hand, showed a trimodal age distribution with modes
occurring during morphogenesis. infancy and early
adult life.

530. About 58% of the conditions affect more thantions showed disturbances and delay in the develone bodily system and these are most often recessives
opment of social behaviour: the XXX girls and XXY
and autosomal dominants and are, in general. assoboys had difficulty in forming relationships, the XXY
ciated with a single system involvement. Of the 351
and XYY boys had excess negative moods and XYY
conditions, 42.5% are compatible with a normal life
boys had a greater incidence of maladjustment thanspan, as reported in the source literature and thethe other two groups or controls. Nonetheless, noremainder lead to premature death, most often in theindividual in this series had any serious mental
pre- and intra-reproductive age groups. Autosomal
retardation or abnormality. Although the sample sizes
recessives and X-linked ones have a more severe effectare small, it is worth stressing that the group studied
on longevity than autosomal dominants. Prognosis
represents an unbiased sample (the children werealso differs with the system involved: among thethose included in the Edinburgh new-born cytogenetic
conditions selected by the authors, those affecting the , survey) and the conclusions. therefore, are of greater
immune system carry the most severe prognosis, none
general relevance than those derived from selected
being compatible with a normal life span and 64%
groups (e.g., cytogenetic surveys of mentally retarded
causing death before 20 years of age. Involvement of
individuals and inmates of penitentiaries).
the respiratory system, blood and blood-forming
tissues, the nervous system and intermediary meta534. In another paper, Tierney et al. [T14] reported
bolism follow the immune system (in descending
the results of a longitudinal study of growth andorder) for their degree of impact on longevity.
development on 30 (of the 36) children who wereidentified as having structural chromosome rearrange531. Of all the conditions examined, 69% are assoments in the Edinburgh new-born survey. In addition.
ciated with decreased reproductive capacity. the major
4 siblings who had structural chromosomal anomalies
contributors being autosomal recessives and X-linked
were included. The breakdown is: 7 de novo andconditions. The largest impact on reproductive capacity
7 familial reciprocal translocations. 3 de nova andis seen in those phenotypes that affect the immune
9 familial Robertsonian translocations, 3 Y-autosome
system, followed by the endocrine. the genitourinarytranslocations and 5 inversions of chromosomes otherand the nervous systems. About 75% of the conditions
than chromosome 9 (children with inversion of chromosome 9 were considered to have a normal variant}. Twocompatible with life beyond infancy affect schooling
and/or ability to work. A severe handicap is mosthundred and twenty children with normal karyotype
often associated with phenotypes involving (in(220) from the same new-born population were used
descending order of severity) the nervous system,
as controls.
metabolic function, the musculoskeletal system, ears
and eyes.
535. The principal findings were the following: (a) the
IQ scores of the 10 children with de novo Robertsonian532. With respect to amenability to medical treator reciprocal translocations were significantly lower
ment, Hayes et al. [H61] estimate that the response isthan those of 20 children with familial translocations,slight in the whole sample of 351 conditions: life span
but not lower than those of normal controls; however,
could be prolonged in 15% of the conditions, reprothere were 3 children in the de novo group of aboveductive capacity in 11 % and social adaptation in 6%.
average intelligence: (b) 7 children with familial
Among the 65 inborn errors of metabolism, treatment
reciprocal translocations had significantly higher IQ
gives relief in 12%. partial response in 40% and none
scores than those with familial Robertsonian transin the remaining 48%. The authors suggest that theselocations (n = 9) and control groups: (c) with thefindings have implications for prognosis, genetic
exception of one child with a de nova reciprocal
counselling and medical care of patients with Mendeliantranslocation who died (and who was characterized as
disease.
mentally retarded). all other children attended normal
schools. These limited data are consistent with theview that balanced structural rearrangements are
B. DETRIMENT ASSOCIATED WITH
probably not as harmful (based on cytogenetic studies
CHROMOSOMAL ANOMALIES ASSESSED IN
in primarily mentally retarded populations; reviewed
TERMS OF PSYCHOLOGICAL DISTURBANCES,
in the 1982 UNSCEAR report: see also chapter I) as
LEARNING PROBLEMS. GROWTH AND
previous reports have implied.
DEVELOPMENT
533. Bancroft et al. [B21]. Ratcliffe [R8]. Ratcliffe
and Field [R9) and Ratcliffe et al. [RIO. Rl I]
presented their results on the assessment of psychological disturbances and educational problems inchildren with sex-chromosomal anomalies. Eighteen
XYY, 23 XXY and 16 XXX children aged between 2
and 13.5 were examined as part of a long-term followup study of these children. All three conditions were
found to have a significant degree of language delay,
the XXY and XXX conditions being more affected.
Likewise, the children exhibited significant deficits incognitive ability with the XXX being more severely
affected in this respect. Furthermore, all three condi-

C. DETRIMENT ASSOCIATED WITHSPONTANEOUSLY ARISING CONGENITALANOMALIES IN MAN ESTIMATED USINGYEARS OF LIFE LOST OR IMPAIREDAS INDICATORS

536. In chapter I, the results of Hungarian studies[Cl3] on birth prevalences of isolated and multiple
congenital anomalies in man were presented andcompared with those for the United States andCanada. It was pointed out that the Hungarianprevalence of 597.4 per 10 4 live births (of which about
43% is due to congenital dislocation of the hip) may
107be an underestimate of the true prevalence rate andthat the underestimation relates primarily to the lessserious major congenital anomalies. However, the factthat in Hungary systematic records of the mortality of
individuals with congenital anomalies are kept andorganized according to ICD codes, provided thepossibility for their use. in conjunction with prevalencedata, to construct mortality profiles for each of thedifferent congenital anomalies and to make reliable
estimates of detriment.
537. Some of the essential aspects of the analysis
carried out by Czeizel and Sankaranarayanan [C 13]
are the following:
The birth prevalences for congenital anomalies(a)in conjunction with the total number of births(1,975,524) for the study period 1970-1981
permitted estimates of the total numbers ofindividuals with given congenital anomalies;
(b)

(c)

The Central Statistical Office. Budapest. provided data on the mortality of individuals withcongenital anomalies who died at specific ages/
age-intervals (a total of 23 age-groups/ageintervals); these data were used to construct amortality profile and to estimate mean ages atdeath. For the sake of brevity and conveniencein presentation. the mortality data were grouped
into three age groups: infant death (from day 0
to 365th day). early death (ages 1-19). late death(ages 20-69);
In calculations on the mean number of years oflost life for any given congenital anomaly. theaverage life expectancy for the general population was assumed to be 70 years (in fact. for the
study period. the mean life expectancy for thegeneral population was 66.8 years for males and73.1 years for females in Hungary);(d)Since. in the case of congenital anomalies. one
is dealing with birth defects, individuals withcongenital anomalies were considered to havepotentially impaired life for the duration of
their life span;(e)

Since the degree of impairment depends on
the nature of the handicap and the efficacy
of medical procedures, actual life impairment
was considered to exist if the proportion ofhandicapped individuals (even after appropriate
medical help) with a given congenital anomaly
was equal to or more than 15%: this was anarbitrary cut-off point. based on consultations
with a number of specialists in Hungary;(f)

The total number of years of lost life wascomputed by multiplying live birth prevalenceby the average number of years of life lost:
similarly, the total number of potentially impaired years of life for each kind of congenital
anomaly was calculated by multiplying the live
birth prevalence by the mean life expectancy of
individuals;(g)

The total number of actually impaired years oflife was calculated by multiplying the estimate
for potentially impaired life years (entry (f)above) by the percentage of individuals with thepersistent handicap.

108538. Table 25 presents an overall summary of theresults (see [Cl3] for details). It can be seen that. witha total prevalence of about 600 per 10 4 live births (i.e ..
600 individuals affected with one or another kind of
isolated or multiple congenital anomaly per 10 4 live
births) used in the calculations, about 4800 years oflife are lost. 37.000 years of life are potentially
impaired (of which about one-half is due to congenital
dislocation of the hip) and 4500 years of life are
actually impaired per 104 live births. In terms of theaverage number of years of lost life (an index of
detriment at the individual level), the CNS anomalies
cause the greatest amount of detriment. followedby anomalies of the respiratory and cardiovascularsystems. chromosomal anomalies, and others. Anomalies of the ear. face and neck, of genital organs and of
the musculoskeletal system and cleft lip. with orwithout cleft palate, have a small or negligible effect inthis regard. When the ranking is done according to thetotal number of years of life lost (an index of
detriment at the population level), however, anomaliesof the cardiovascular system are associated with thehighest amount of detriment. followed by those of thecentral nervous system and others.
539. The total number of potentially impaired yearsof life-a measure of the upper limit of impairmentthat would be expected when suitable medical facilities
are unavailable and a reflection of the contribution ofsome of the less serious anomalies-is highest formusculoskcletal anomalies: this is due to the fact thatin Hungary congenital dislocation of the hip has avery high birth frequency (257.7 per 10 4 as compared
to about 10 per I 0 4 for many other European andEuropean-derived populations: see Carter [C25]).au If
the latter figure is used, the number of potentiallyaaczeizel and Tusnady [ C32) have extensively discussed thepossible reasons for the high prevalence of congenital dislocation of
the hip in Hungary. Among these, the following four deserve
mention: (a) a genuinely high rate of incidence in the Hungarian
population; (b) owing to the low birth rate in Hungary. theproportion of first-born is high (in Budapest, 64.3% of live births in
1962-1967 were first-born) and this condition is more common
among first-born; (c) relaxation of selection: in earlier generations. asevere congenital dislocation of the hip was a serious obstacle. formarriage, pregnancy and childbirth and thus interfered with thetransmission of genetic liability. However, this condition can now
be successfully treated with no adverse effects on fitness; this meansthat the genes involved are being transmilled without hindrance andtherefore the liability for this condition may increase in the future;and (d) the high prevalence is most probably a reflection ofwidespread adoption of neo-natal and infantile orthopaedic screening
and the consequent high rate of detection. Because of this early andextensive screening, a large number of cases of diagnosed dysplasiaof the hip joint of lesser severity are subjected to treatment together
v.ith true congenital dislocation of the hip. While undoubtedly aconsiderable proportion of congenital dislocation of hip cases oflesser severity heal spontaneously, there is evidence [F19) that, in acertain percentage of these cases, osteo-arthrosis of the hip develops
in adult life. Thus, the large number of cases of congenital
dislocation of the hip that undergo treatment may lead to a
reduction in the prevalence of osteo-anhrosis in adults. However, it
is likely that some infants with merely an unstable hip or withadduction contracture are unnecessarily subjected to treatment. It isworse, of course, not to recognize and treat congenital dislocation of
the hip than to place a presumably normal child in the Pavlik stirrup.
Sometimes, it is impossible to draw a sharp line between conditions
requiring treatment and those likely to recover spontaneously
among the dysplasias observed in infants 3-4 months old. Furthermore, unjustified delay may mean that one misses the mostadequate time for treatment and by treating a less severe dysplasia
one may prevent osteo-anhrosis in adult life.

impaired life years for musculoskeletal conditions will
be reduced from about I 8,000 to 3100 years. (Thetotal birth prevalence of all congenital anomalies will
also be reduced from 597.4 per 10 4 to 349.7 per 104 ).
540. An index of detriment based on years ofactually impaired life provides a good perspective of
the situation under modern medical conditions. As
will be clear from Table 25, with this criterion
anomalies of the cardiovascular system are associatedwith the highest amount of detriment. followed bythose involving the genital organs, chromosomal
anomalies and others.
541. There are at least two reasons why the estimatesof detriment presented in Table 25 may be underestimates. Firstly, it is clear that these figures areheavily dependent on the Hungarian birth prevalences.
which, as mentioned earlier. may be underestimates.
Secondly, the frequencies of chromosomal disorders
are certainly underestimated in Hungary. at least by afactor of 3. This is clear not only from the results ofchromosome surveys of new-born children (summarizedin [Ul]) but also from the fact that partial deletions
and duplications involving every chromosome of thehuman complement are now known (but we do not
yet have estimates of prevalences). and from theongoing studies on the relationship between mental
retardation and the fragile site on the X-chromosome(see chapter I).

D.

COMPARISON OF THE ESTIMATES OF
DETRIMENT FOR MENDELIAN.
CHROMOSOMAL AND OTHER DISORDERS
542. Table 26 compares the estimates of detrimentfor Mendelian and chromosomal disorders made byCarter [C26] (see also (Ul]) with those made by
Czeizel and Sankaranarayanan [C 13] for congenital
anomalies. It is clear that monogenic disorders with a
total birth prevalence of 125 per 10 4 live births
account for about 1900 years of lost life. 3000 years ofpotentially impaired life and 1500 years of actually
impaired life, per 10 4 live births. Congenital anomalies.
on the other hand, account for over 4500 years of lost
life, 37,000 years of potentially impaired life (about
half of which is due to congenital dislocation of thehip) and 4000 years of actually impaired life per 104
live births. The estimates of Carter for chromosomal
disorders per 10 4 live births (prevalence. 40: lost lifeyears, 900: potentially impaired life-years, 1800: andactually impaired life-years, 900) are probably morereliable, for reasons mentioned earlier. although, here
too, partial deletions and duplications of chromosomes
and X-linked mental retardation due to fragile-X havenot been included.

E. FREQUENCY AND ECONOMIC BURDEN
OF GENETIC DISEASE IN A PAEDIATRICHOSPITAL
543. Carnevale et al. [C75] surveyed a total of 2945
admissions to the National Institute of Paediatrics in
Mexico City to assess the frequency of admission andthe economic burden due to disorders with a genetic
basis. The patients were classified into five categories:(a) single-gene disorders; (b) chromosomal disorders;(c) complex genetic aetiology: (d) unknown aetiology;
and (e) non-genetic disorders. Of all the admissions
studied, 126 or 4.3% fell into the first two categories(61 autosomal dominants, 20 autosomal recessives,
20 X-linked recessives and 25 chromosomal), 998(33.5%) into the complex aetiology group, 406 (13.8%)
into the group with unknown causes and 1425 (48.4%)
into the group of non-genetic disorders. In terms of
economic burden. patients of categories (a)-(c) came
more frequently from outside the city and had moreadmissions for longer durations with a higher numberof surgeries. On the basis of all the data, the authors
suggest that Mendelian and chromosomal disorders
and those of complex aetiology, although less frequent
than environmentally caused diseases. impose a considerable financial burden on the hospital and affectedfamilies.

F.

SUMMARY AND CONCLUSIONS

544. A literature survey has been conducted toestimate the impact of Mendelian disorders on humanhealth and their response to medical treatment. About
75% of the 351 conditions (autosomal dominant,autosomal recessive and X-linked) included in theanalysis were found to be harmful. impairing one ormore of the following: life span. reproduction. development and/or social adjustment. About 42% arecompatible with a normal life span and the remainder
lead to premature death. most often in the pre- andintra-reproductive age groups. Autosomal recessives
and X-linked disorders have a more severe effect onlongevity than autosomal dominants. Medical treatment can help to prolong life span in 15% of theconditions. reproductive capacity in 11 % and social
adaptation in 6%.
545. In a long-term follow-up of children withnumerical sex-chromosomal anomalies and autosomal
structural anomalies (ascertained in the Edinburgh
new-born survey), the detriment associated with theseanomalies was assessed using psychological development, learning and cognitive abilities, social behaviour
and growth and development as end-points. While
children with sex-chromosomal anomalies (XXY. XXX.
XYY) had some problems (language delays, deficits incognitive ability, higher incidence of maladjustment,etc.), none of them had any serious mental retardationor other handicap that could be characterized as really
adverse. Likewise. there were no really adverse effects
associated with balanced autosomal translocations.
Children with familial translocations had significantly
higher IQ scores than those with de novo ones and all
children attended normal schools. The sample sizes
involved in these studies. however, are small.
546. The detriment associated with spontaneously
arising isolated and multiple congenital anomalies has
been assessed on the basis of data available from
Hungary. In these calculations, the average life expectancy for the general population was assumed to be
70 years. It has been found that, with a total estimated109

prevalence of about 600 per 104 live births, thesecongenital anomalies may cause. per 10 4 live births,
about 4800 years of life loss. about 37,000 years ofpotentially impaired life and about 4500 years ofactually impaired life. However. one-half of the total
potentially impaired years of life is due to congenital
dislocation of the hip, which is known to have a highbirth prevalence in Hungary.

T a b l547. Estimates of detriment for congenital anomalies
made in the present report arc much higher than thosefor Mendelian and chromosomal disorders (in termsof years of lost life or impaired life) made in the 1982UNSCEAR report. Mendelian and chromosomal disorders as well as those of complex aetiology impose aconsiderable burden on medical care facilities and on
affected families.

e

Cytogenet1c abnormalities and embryonic and fetal deathby gestational age( from Hook [ Hl))

Gestational age In completed weeks
5-7

8-11

12-15

16-19

20-27

All known(pooled)

Proportion abnormal
among those
488/965
1342/3363
24/137625/1330 156/475
karyotyped ~/49-45650.6
47.0
Per cent abnormal !I
17 .5
39.9
32.8
10.7
95" confldence
lnterval b/
12.4-23.8 47. 7-53.S 44.6-49.4 29.2-36.7 B.4-13.4
Range (In "lamong studies
14.0-25.8 40.4-64.4 39.1-56.2 18.5-69.6 6.6-27.0 r;_/Calculated from data from 5 speclf1c studies (In Honolulu, Geneva, Hiroshima,
London, New York) presented by Warburton et al. [Wl).
~/ Assuming homogeneity among populations studied. In fact the ranges suggest thatat least for the 16-19 week group thls Is unlikely.
r;_/ Excluding extremes of O and 4°" In two studies In which only small numbersof fetuses were studied.

!I

I JO

Ta b i e2

Proport1on of recognlzed conceptuseswith cytogenetlc abnormalltles by lnterval(from Hook [Hl])Gestatlonallnterval(weeks)Per cent
cytogenetlcallyabnormal
1n those lost
In lntervals ~/

5-7
8-11
12-15
16-10
20-27
>28 (st1llb1rths)
Total

17.5
50.6
47.0
32.8
10.7
5.7

Proportlon ofrecognlzed
concept usesxlost 1nInterval (%)
6.3
3.3
2.5
1.0
1.4
1.0

X
X
X
X
X
X

=

Proportlon ofembryos or fetuses
that have cytogenetlc
abnormality detected
durlng Interval (%)
1.1
1. 7

=

15.5

1.2
0.3
0.14
0.06
4.5

Total of all recogn1zed pregnancles that(a) result In fetal or embryonic death(b) have cytogenet1c abnormality

4. 5%

Live blrths wlth recognized chromosome abnormality(0.6%) x proportlon of eventual live births among
recognized conceptions (84.5%)
=

0. 5%

Total = estimated proportion of recogn\zable
concept\ons w\th chromosome abnormality

5.

~/

o"

Calculated uslng data from Harlap et al. [H62] on fetal and embryonic
death rates from 5 to 27 weeks, and assuming a 1% fetal death rate(still birth rate) thereafter, and using data from Warburton et al. [W2]on abnormalities In those from 5 to 28 weeks; from Sutherland et al. [Sl]on still b1rth; and from Hook and Hamerton [H2) on live births.
Note that ages are 1n completed weeks, so Interval 20-27, for example,
is from day 140 to day 195 Inclusive.

111

T a b 1 e

3

A comparisonof the frequencies of chromosomal anomalies In new-borns(anomalies detected using chromosome banding methods}
ln surveys carried out In Norway [!:!.!l] and Denmark (!il.)with those In other surveys (sulTJTlarlzed In [!!l)l
In which banding techniques were not used

Denmark

NorwayAberration type

( 1830children;bandedpreparationsofchromosomes)(3658
children;bandedpreparat lonsofchromosomes)( 11 , 148
children;nobandingofchromosomes)(59,465
children;nobandingofchromosomes)

No. frequency

No. frequency

No. frequency

No. frequency

( ")

{")

Sex-chromosomal
anoma 1i es
5 !l/ 0.27
Autosomal trlsomles 5 f_/ 0.27
Robertson Ian
translocatlons
4 !!_/ 0.22
Ree I proca 1
0.27translocatlons
5Autosomal Inversions
0.05
excluding thoseof chromosome 9Others
4 g/ 0.22

18

0.26
0. 16

134
88

0.23
0.15

3 1/

D.08

17

0. 15

57

0 .10

7

0. 19

15

0. 13

61

0 10

2

0.05

0.01

9

0.02

5 f./

0. 13

13 j/

o. 12

23

0.04

93

0.83

372

0.64

93

0.83

372

0.64

1.30

39

1.05

Inversions,chromosome 9

12

0.66

35

0.96

Total

36

1.96

74

2.01

t/
!!_/

~/

f/
g/
h/

I1

1/

112

0. 41
0. 19

24

lil

( ")

(")

15 g/
7 h/

Subtotala/

Pooled studies
al

29

•

Results of 7 surveys; see [Ul) for details.
One case of 47,XYY, one case of 47,XXY and 3 Inversions of Y.
One case of +18, 3 cases of +21 and one case of +21 mosaic.
2 cases of t(l3q;l4q) and 1 case of t(l4q;2lq).
2 cases of small supernumerary chromosomes and 2 cases of small
supernumerary/mosaics.
l case of +mar, 2 cases of +ring and 2 cases of duplications.
3 cases of 47,XXY, 2 cases of 47,XYY. 3 cases of 47,XXX, 4 cases of
45,X and 2 cases of Inversion Y.
One case of +18, 5 cases of +21 and 1 case of +B.
2 cases of t(l3;14) and 1 case of t(l4;21).
6 cases of +mar, l deletion and 6 cases of unbalanced Y-autosometranslocatlons.

Ta b l e

4

Com11arlson of the results of survexs of mentallX retarded 11atlents(From Rasmussen et al. [Rl])Total
examlnations(%)

Autosoll'a lother than
Down's( "lSexchromosomal
anomalies
( "l

449

9.7
8.2
12.0
7.3

1.3
1.9
0.8
0.6

0.9
1.0
0.8
0.2

11.8
11. l
13.6
8.2

7880

9.7

1.2

0.8

11. 7

1905 !/

14.7

2.4

1.8

18.8

Down'ssyndrome

6163
512
756

Total

Ref.

( "l
[ Jl)
[Al)

en J

[ K2)

[Rl)

In banded chromosome preparations.

!I

Ta b l e5Ma]or chromosome aberrations In 11atlents with
mult111le congenital anomalies (MCA}/ mental retardat\on (MR} sxndromes(from Coco and Penchaszadeh [Cl))Sex
chromosome
aberrations

Autosomal
aneuploldy(excl .21)

Deletionsand rings

Extramarkersor r\ngs

Structural
rearrangement !I

No.

No.

"

No.

No.

No.

"

2.0

l

2.0

2
1
1
5

4.0

2

4.0

4

2.4

50
50
170
50
121
15!1/
50
50
90
51
199£/
200
200
Total:
1296

"
2.0
0.6
0.8

1

1.1
2.0
2.0

2
9

1.2
7.2

2.0

l
2
2
2

4.5

14

"
0.6
2.0

3

2.0
2.0

4.0

2.5

11

5.5

10

5.0

14

0.8

33

l. 7

41

2.8

11

"
2.0

4

5

1
9

1.8

4.1

2.2
3.9
7.0

No.

2.0

1
1
1
3

l

4

"

2
2
3
1

3.3
13.3

4.0

6.7

6.0
1.1

13

6.0
2.0
6.5

0.8

47

3.7

4

1.2

4.0

2.0
0.5
0.5
l. 5

12

2
2

1. 1

7

Ref.

Total

Other

l. 2

No.

"

4

8.0
10.0

[S3)
[ Dl )

7.6[A2,A3)
[W3)

5
13
3
19
3
3
6
5
5
40
5
42

6.0
15. 7
20.0
6.0
12.0
5.5
9.8
20. l
2.5
21.0

153

11.D

[C2]
[ L1)

[ T2)
[112)

[D2)
[[2]
[ 01 )[Tl][ Cl ]

unbalanced rearrangements (duplications and/or deflc\encles) and apparently balanced
Interchanges.
b/ HR and MCA 1nd\vlduals from a total of 54 patients with mental retardation studied.
£/ After sorting out Individuals w\th Down syndrome and other known condlt\ons among 384 patients w\thMCA/MR syndromes and not tak\ng Into account two lnd\vlduals with minor famll\al chromosome variants.

!I Includes

113

Ta b l e

Ii

Chromosomal anomal1es 1n 958 1nfert1le males[D24]

Sex chromosome anomal 1es

Autosome anoma 11 es
Type

!I

Type

HumberNumber1

47, XXV

21

446, XX
47, XXV/46, XX

3

45, XV, t(DqDq)
45, xv, t( l 3ql4q)45, xv. t(l4q2lq)
45, xv. t(l4q22Q)
46, xv. t(ll ;22)(q23;qll)
46, XV, t ( 3; 11 )( Q24; q24)
46, XV, t(li;ll)(Q26;q22)
46, XV, t(6;20)(ql6;pl3)
46, xv. t(5;8)(pl4;qll)
46, xv. t(l0;14)(q25;ql3)
46, XV, 1nv(l )(p22;q32)
46, XV, 1nv(7)(p22;q21)
46, XV, 1nv(10)(pll ;q21)
47, xv. +mar
48, xv. +mar, +mar
46, xv. llqfra

l
l
3
l
l
l
l
1
l
l
l
l
2
l

Total

22

x. 1nv(V)46, X, ter rea(V)46, X, del(Vq)46, V, t(X;l )(q27;q31)
46, X, t(V;5)(qll ;pl5)

l
l
5
l
2
l
l

Total

36

47, XVV46,

del = delet1on; ter rea a end to end translocat1on; 1nv = 1nvers1on; ttranslocat1on; p = short arm of chromosome; q = long arm of chromosome;
fra = frag1le s1te; +mar = add1t1onal small marker chromosome. Except
for 3 cases of 1nv( V), a 11 sex-chromosome anoma 11 es were found amongazoosperm1cs. Among autosome anomal1es, only two cases of t(l3ql4q),
the t(3;11) and the 1nv(l) were found 1n azoosperm1cs.
Table

7

A comoar1sonof the prevalence of chromosomal anomalies 1n subfert1le men
as a whole and 1n azoosperm1cs alone w1th those 1n surveys of new-borns[D24]

Prevalence per 103 males 1n
Anomaly
Newborns

XXV
XX (males)
XVV
In( V)
Robertson1antranslocat1ons f/
Reciprocaltranslocat1ons £/Autosomal 1nvers1ons
Supernumeraries
!/

b/

£/
d/

ii
114

All
subfert1les
M

Azoosperm1cs

1.24 !l./
0.05
0.92
0.24

34
l
l

129
8
3

0.92

7.2 ( X 8)

2.7 ( x3)

l .05 !!/
0.62
0.32

8.3 ( X 8) fl
3.5 ( X 6) !!/1.9 (x 6)

3.6 ( x3) f/

(x30)
(x20)

l. 4 ( X 5)!I

4

( xl 00)(xl60)( x3)( xl 6)

2.4 (x4) !!/2.7 (x9)

Prevalence est1mates based on all studies surrmar1zed by Dutr1llaux etal., unless otherw1se stated. The factor by wh1ch these are higher 1n
the first two groups relative to the new-barns Is g1ven In parenthes1s.
Includes mosaics with an XXV cell l1ne.
Note that the s1tuat1on Is different from that of sex-chromosomal
anomal1es 1n wh1ch the magnitude of increase (relat1ve to new-borns) ls
h1gher among azoosperm1cs than among subfertlles as a whole.
Includes only studies done w1th chromosome banding.
Includes only the data of Dutr11laux et al. s1nce th1s study 1s thelargest one carr1ed out us1ng chromosome banding.

Table8

Chromosomal anomal1es 1n 820 1nfert1le males

Type

Patlents
Sperm count/ml

No.

"

21
1
4
1
1

2.5
0.1
0.5
0.1
0.1

Azoospermla
Azoospermla
Azoospermla
Azoospermla
Azoospermla

13/14
14/21
1/3 (p34/q21)
3/5 ( q24/q13)
6/10 (q14/q22)

4
2
l
1
l

0.5
0.2
0.1
0. 1
0. 1

(0.8-12) 106(1.0-3) 106
Azoospermla
1 ,0 106
Azoosperm\aInversion 9 (p11q12)

22

2.7

9 azoospermla
13 ollgozoospermla

0. 1

6 106

27

3.3

Yq-

17

2. 1

21h+

12

1.5

22h+
lh+
13h+
14h+
15h+
16h+

6
2

0.7
0.2
0.4
0.2
0.7
0.2

18 azoospermla
9 ollgozoospermla
7 azoosperml a
10 ollgozoospermla
7 azoospermla
5 ollgozoospermla
6 ollgozoospermla
2 ollgozoospermla
3 ollgozoospermla
1/1 azoo/ollgozoospermla
1/5 azoo/ollgozoospermla
1/1 azoo/ollgozoospermla

Sex chromosomal
abnormallt1es (3.4")
47,XXY
47 ,XYY
46,XY/47,XXY
47,XXY/48,XXXY
46,XX
Autosomal translocat1ons(1.°")

Invers1on 9 (p23/q33)
Chromosomal
variants (9.3")
Yq+

3

2
6

2

115

Ta b l e

9

The ~revalence of fra(Xl In different grou~s of Individuals studied

Hales

Refs.
Numberfra(XJ

Neonates (Australia)

1810

0

Institutionalized
retarded (Australia)

444

7

1.6

Hales with mental retardatlon of unknown origin
In an Institution(Finland)

150

6

4.0

Institutionalized
retarded (moderate tosevere) (Belgium)

354

57

16. l

Severely retarded boys
(IQ< 50) from various
registers (Sweden)

96

7

7.3

Less severely retarded
children from various
registers (Sweden)

110

s

4.5

Sheltered workshops formentally handicapped(Austra l\a)84

0Special schools formentally handicapped(Aust r a 11 a)

328

11

Retarded Individuals
In community placement(Hawaii, United States)

274

Referral patients(Austral\a)

2283

~/

116

females

Authors'
group
designation"

Numberfra(X)

1648

0[SS, S86]

80

0

[SS]

"
[KlO]

30

3.3

[ r29 ]~/

[B2, 825]

61

0

[B25]

44

0

[S86]

3.4

174
128

2
5

5

1.8

278

13

5.3

1890

3

l. 1
3.9

[S86]
[T31]

0.4

[Jl2, H54]

1.6

[S86]

The 58 fra(X) positive Individuals were found to belong to 37 different
faml 1 les.

Table10

Break-polnts ln constltutlonal chromosome rearrangements: a surmiary[H2B, H29]

Break-polnts ln rearrangements~/
Orlgln ofrearrangement

In bands w\th fraglle s\teRatio ofobservedtoexpected

NumberObserved

Expected

Materna 1
Paternal
Unknown QI

269
202
701

56
40

l2B

29.6
22.2
77.1

1.9
1.8
l. 7

Tota 1

1172

224

128.9

1.8

~/
QI

Includes data from amnlocentesls studles [H28] and thosefrom spontaneous abortions, stlll births and new-borns.
Includes de novo rearrangements.

117

Table11

Speclflc chromosomal changes assoclated wlth partlcular types of cancers
1nvolv1ng bands where fraglle sltes have been asslgned
or located near such bands(General references (843, Lll, Y4, Yll))

Chromosome
and fragl le

Chromosomal abnormal1ty
and brealc-po1nts

Cancer

References

s He

2q13
Jp14

6p23

t(2;8)
t(3;1B)(p21 ;q12)t(J;ll)(pl3-14;pl5)t(3;9)(p21 ;q24)
del(J)(pl4;p23)
del(J)(pl4)t(6;9)(p2J;q34)

7p11

del(7q)Bq22t(8;21 )(q22;Q22)9p21llql 3t(9;11 )(p21 ;q23)
del(9)(p21)t(ll;l4)(qlJ;q32)t(llq); del(llq)l lq23t( 11: 12)( q23 or q24: ql2)t(9;11 )(p21 ;q23)t(4;11 )(Q21 ;q23)

l2ql3

+12

16q22

t(9;12)(p12;ql3)
1nv(l6)(pl3;q22)

Burlcltt's lymphoma

H1xed sal1vary gland tumour

Renal cell carc1noma
Renal cell carc1noma
Small cell lung carc1noma
Breast carc1noma
Acute non-lymphocyt1cleulcaem1aAcute non-lymphocyt1cleulcaemla
Acute myeloblast1c
leul:aem1a-H2
Acute monoblast\c leulcaemla
Acute lymphocyt\c leulcaemla
Non-Hodglc\n lymphoma
Acute myelo1d and lymphoid
leul:aem1as and lymphomas
Ewlng's sarcoma
Acute myeloblast1c
leul:aem1a-H5
Acute lymphocyt1c leulcaem\aChron1c lymphocyt1cleulcaem1aH\xed sallvary gland tumour
Acute myeloblast1c[Lll, Y4)
[H55)

[ P31]
[C60]
[W3B)
[Al6]

($88)
[Lll, Y4]
[ R49, S89]
[H44]

[K36]
[fJO]
[Lll, Y4)
[ L1 l. Y4)
[H44]
[Vl2]

[Lll, Y4]
[M56]
[ L11]

leulcaem1a-H4

del(l6)(q22)17p12t(15;17); 1nv(l7q)

20pll

del(20q)

Acute non-lymphocyt1cleulcaemla
Acute promyelocyt1cleulcaemla; chronlc
myelogenous leulcaemla(blast cr1s1s)
Hyeloprol1ferat1ve dlsorder[A17)[Lll, Y4]

[Lll, Y4)

Note: some frag1le s1tes (2ql3, 7pll, 12q13, 17pl2 and 20pll) may or may notco1nc1de w1th the brealc-po1nts of the chromosomal changes \nvolved 1n thedlfferent cancers shown 1n the Table.

I 18

Ta b l e

12

A comQarison of birth Qrevalences of congenital anomalies
In the United States (M12], Hungar~ [C13] and British Columbia (T9](based on ref. [Cl3])
Prevalence~/ per 1o4
ICD
code

Congenital anomaly

UnitedStatesTotalbirths740-741 Anencephaly and splna
17.4 ( 42) !l.l
blflda742
Other anoma 11 es of the35.2 ( B5)
nervous system
23.6 ( 57)743 Anomalies of the eye
744
Anomalies of the ear,
14.9 ( 36)face and neckB6.5 (209)745-747 Cardiovascular anomalies
748
Anomalies of the
14. 1 ( 34)
respiratory system
749
Cleft palate and27.3 ( 66)cleft lip
750-751 Anomalies of the61.7 (149)digestive system752-753 Anomalies of the genitalorgans and urinary system 115.5 ( 279)754-756 Husculoskeletal and364.7 ( 881)
skeletal anomalies
excluding congenital
dislocation of the hip
757 Anomalies of the
101 .4 ( 245)
Integument
16. l ( 39)758 Chromosomal anomalies
759 Other and unspecified
12.8 ( 31)anoma 11 es
Subtotal CAs

BrltlshColumbia

HungaryTotalbirthsLlvebirthsLlvebirths

Hi.6

10.3

9.2

14.6
3.2

11.4

7.9
7.B

3.2

4.7
80.8

4.6

6.0

79.2

42.6

2.8

2.8

1.5

14.8

14.5

17.5

27 .8

27.8

17.6

93.7

90.9

27.5

56.5

55.0

52.0

7.6
12.6

7.4

2.4

12.6

14 .1

21.5

20.0

1.9

357.2

339.7

208.0 !!/

891.2(2153)

Subtotal, affected births 632 .1-775.0 f /119

Table 12, contlnued
Prevalenceil per 104
ICD

Congenital anomaly

code

754.l

BrltlshColumbla

Hungary

Total
b1rthsTotalbirthsUve
b1rthslive
b1rthsCongenital dlslocatlonof the hlp

73.3 (177)257. 7257. 7

14.2

Total CAs (ICD 740-759)
Total, affected b1rths(ICD 740-759)

964.5(2330)
704.0-846.l ~/

614.9

597.4

222.0 f/
374.D g/428.0 hi

110.4
1. 2

11D.4
B.B
1.2

3.2
9.6

735.9

717.8

236.4

Inguinal hernia
550
553 Umbll lcal hernla
227-228 Congen\tal tumours

Grand total, CAs
Grand total,
affected b\rthsii

Un ltedStates127.9 (309)l O. 8 ( 26)
6.2 ...illl( 350)
1109 .6(2680)
849.2(2051) ii

9.4

1. 4

Unless otherwise stated, the United States prevalence f\gures refer to thenumber of congenital anomalies (and not to the number of affected blrths);
In the other two studies, the figures are based on the numbers of affected
Individuals.
!1_1 The figures given ln parentheses (column 4) refer to the number ofcongenltal anomal les on which the prevalence estimates g\ven \n column 3
are based.
r;_I The estimate of 632.1 \s arr\ved at by assum\ng that the number of births
with congenital d\slocatlon of the h\p, hern\as and tumours \s the same as
the number of these CAs (= 527) and that a total of 2051-527 = 1524 b\rthshave 2153 CAs or roughly 1.41 CAs per affected births.
Therefore,
891.211.41 = 632. l births are affected by CAs. The est\mate of 775.0 Is
arrived at by a similar procedure, assuming that the affected births =2051-177 = 1874. These had 2153 CAs or roughly 1.15 CAs per affectedbirth; 891.2/1.15 = 775.0.
d_/ The f1gure of 208.0/104 \s for the per\od 1952-1972 (excluding
congenital dlslocat\on of the hip),fll The estimate of 704.0 1s arrived at by assum\ng that the number of cases
affected w1th hernias and congenltal tumours ls the same as the number ofthese CAs ( = 350) and that a total of 2051-350 = 1701 cases had 2330 CAs
or 1.37 CAs per affected case. Therefore, 946.511.37 = 704.0. The valueof 846. l ls arrlved at by assuming that 2051 affected blrths had 2330 CAs
or roughly 1.14 per affected birth. Thus the number of affected births ls
964. 511 . 14 = 846. 1 .
f./ The figure of 222.0 \s appl\cable to the total per\od of 1962-1972,
Including the congenital dislocation of the hlp.
g/ The minimal estimate of Trimble and Doughty [T9) for the total prevalenceof CAs (ICD entr\es 740-759).
hi The corrected or adjusted estimate of Trimble and Doughty [T9] for the
total prevalence of CAs (!CD entries 740-759).
gl The number of malformed b1rths In a total of 24,153 [Ml2].

120

Ta b 1 e

13

Some e~ldemlologlcal features of selected multlfactorlal disorders In man~I

Population
prevalence per 10•!CD

Disorder

250.0
250.1
274
295
296

Hungarianstudies

100 !!./(25-500) £/

1 :4

42

13-62

0.47

[Sl 16][B70, 019]

480 (190-1600)

1:2

60

20-

0.65

(B71]

[K50, W49, 1134,
R63, 1:51 J

20 ( 8-56)

1:1

11

1-19

0.30

30 ( 13-37)
100 (70-200)

15:1
1:1

25
25

18-55
15-45

0.50
0.80

[N74]

131 (40-500)

1 :2

40

20-60

0.80

[ P46 ,U8a/[R64]

5 ( 1-13)

1:2

33

10-50

0.58

[ P48]

60
160
850
359

170 ( 33-429)
160 ( 80-220)
SOO (200-1000)
SOO (300-2000)

1:1
1:1
1:1
3:1

4
55

60
50

0-70
201030-

0.50
0.32
0.75
0.65

[l51]
[U9]
[C71,G59][C70][ G56, 1:52, P47,
S117]
[A22, [27, G57,
S117, W50]
[872, Sll8,
Sl19, S120][A23, 063, G58, H35]
[f39, H55 J[H36, N37]
[1475, Sl 21]

1250

500 (200-3560)

1 :3

30

20-

0.70

[l52]

[H56]

360

400 ( 300-500)
500 ( 50-900)
500 (150-900)

1:1
1:1
2: 1

25
35

0.70
0.65

[S122]

[R66]

45

5-50
1-70
30-60

0.48

249
460

[N38]

(064, R63]3

5 ( 4-10)

1:1

35

20-60

0.60

[ P49]

(873)

94

13
90

100 (40-120)
12 ( 3-25)
60 (10-150)

1 :3
1: 1
2: 1

35
1

0.63
0.80
0. 70

[H57, R67) (V18, W51]
[D65]

45

251-1030-60

60

70 (50-80)

1:1

18

10-25

0.50

[N76]

[H77]

39

130 ( 10-400)

1: 1

20

10-60

0.75

[875]

[R68]

4

4 ( 2- 7)

1:9

34

13-45

0.90

[N39,S123] [H58, 1:53, l53]131

179 ( 50-500)

1 :2

40

35-64

0.83

[S124]

Ankyloslng spondylltls
Juvenile osteochondroslsof spine
Adolescent Idiopathic
scollos Is

19
1100

18 ( 5-23)
900 ( 400-1 SOO)

5:1
1 :2

23
12

18-35
8-18

0.79
0.56

[G60]
[876]

41

33 (13-64)

1 :2

13

10-18

0.88

[877, C72] [S125]

Total

6051

Thyrotoxlcosls with or65
without goitre(Grave's disease)
Diabetes mellltus (adult407
onset; non-Insulin
dependent, type II)
Diabetes mellltus
20(juvenile onset; Insulindependent, type I)
Gout18
Schizophrenic psychoses
85

345
365

Epilepsy
Gl~UCO!Da401
Essential hypertension
410-441 Acute and sub-acute
myocardial Infarction
454
Varicose veins of lower
extre111t les
477
Allergic rhinitis
493
Asthma531-533 Gastric ulcer, duodenal
ulcer, peptic ulcer,
site unspecified
556
Idiopathic proctocolltls(colitis ulcerosa)
Cholellthlasls
574
579.0 Coellac disease
592
Calculus of kidney andureter691
Atoplc dermatitis andrelated conditions,mainly eczema
Psoriasis and related696
d I sorders710.0 Systemic lupus
erythematosus
714.0 Rheumatoid arthritis
720.0
732.0
737.3

OthersHerlta-Affective psychoses (uni- 110
and bi-polar depression)Multiple sclerosis
4

340

Sexratio

References" : r Hean Range bll lty

Hungary
242

Onset (yrs)Othercountries

[R65]

[874]

[l54, L55, l56,
020, P50]([28][H59, HO]

5607

a/

Preliminary data (Czelzel et al.).

£I

Range of prevalences reported In different studies.

b/ Best estimate.

121

Ta b1 e

14

Some restrlctlon endonucleases and thelr recognltlon and cleavage sHes { *}(from [ElB]; a more exhaustlve 11st ls glven \n (Rl9))

EnzymeOrganl sm

Cleavage sHe ( *)3'

5'

TETRANUCLEOTIDES (4)Alu!
Arthrobacter luteus
Haemophllus aegyptlus
Hae III
Haemoph1lus para1nfluenzae
Hpall
Moraxella bovls
Mbol
Taql
Thermus aquat1cus
Moraxella specles
Mspl
PENTANUCLEOTIDES (5)Anabaena var1ab111s
Ava II
Desulfovlbrlo desulfurlcans
Ddel
EcoRII
Hlnfl

Escherlchla coll R.245
Haemoph11us lnfluenzae Rf

A G * C T
G G * C CC * C G G
* G A T C
T * C G A

C * C G G
G * G AT C C
C * T N A GAC C T G G
G * A N T C*

HEXANUCLEOTIDES (6)Anabaena var1ab111s
Aval
Brevlbacterlum albldum
Ball
Baclllus amylollquefaclens H
BamHI
Baclllus glob1g11
Bgl II
Escherlchla coll RY13
EcoRI
Haemophllus aegyptlus
Hae II
Haemophllus 1nfluenzae Re
HlncII
Haemophllus lnfluenzae Rd
H1nd1II
Haemophllus paralnfluenzae
Hpal
Haemophllus suls
Hsul
Prov1denc1a stuartll
PstI
Streptomyces achromogenes
SacI
Streptomyces albus
Sall
Serratla marcescens
Smal
Xanthomonas malvacearum
Xmal
Streptomyces phaeochromogenes
Sphl

C * Py C G Pu G
G G * C C A
G * G A T C C
A * G A T C T
G * A A T T C
Pu G C G C * Py
G T Py * Pu A C
A * A G C T T
G T T * A A C
A * A G C T T
C T G C A * G
G A G C T * C
G * T C G A CC C C * G G G
C * C C G G G
G C A T G * C

HEPTANUCLEOTIDES (7)Mlcrocoleus specles

C C * T N A G G

MstII

T

Note: some enzymes (lsoschlzomers) recognlze the same sequence(e.g., H1nd1II and Hsul: HpaII and Mspl).
Py= pyrlmldlne, Pu= purlne, N = any base.

122

Table15

Analysis of human genetic disease by means of recombinant DNA technology(based on [C35] with some additions)

DiseaseGene probe

Ref.

A. DIRECT ANALYSIS OF GENETIC DISEASEUSING GENE PROBES TO DETECT INTRAGENIC
DEFECTS
Antlthrombln III deficiency
a-Antltrypsln deficiency
Atherosclerosis
Diabetes
Ehlers-Danlos syndrome
Growth hormone def1c1ency
Haemophilia B
Hereditary perslstance of fetal
haemoglobin
Hypoxanthlne-guanlne phosphorlbosyltransferase (HPRT) deficiency
Lesch-Nyhan syndrome
Osteogenesls lmperfecta
Retlnoblastoma
Sickle cell anaemia
Thalassaemla

Antlthrombln III[Pl7]Synthetic ollgonucleotlde [K19]
Apollpoproteln A-1[K20]
Insulin[H31]
a(l) Collagen[ Pl 8]Growth hormone[P19)
Factor IX[G26]
13-Globln[F20,T22)
·HPRT

[W20)

HPRT
[Y7, N16]
Pro al(l) collagen[C36, P18)
Chromosome 13 DNA segments [C37]13-Globln[ G19)
Synthetic ollgonucleotlde [C38]a- and 13-globln[011, L12]
Synthetic ollgonucleotlde [010]

B. INDIRECT ANALYSIS or GENETIC DISEASEUSING GENE PROBES TO DETECT
LINKED POLYMORPHISMS
Atherosclerosis
Diabetes (type II)
Growth hormone deficiency type I
Hypertrlglycerldaemla
Sickle cell anaemia

Apollpoproteln A-1
Insulin
Growth hormone
Apollpoproteln A-1
13-Globln

Lesch-Nyhan syndrome
Osteogenesls lmperfecta
Phenylketonurla
Thalassaemla

HPRT
pro a2(1) Collagen
Phenylalanine hydroxylase
13-Globln

Disease

Probe

Distance between
probe and disease
locus (cM)

[K21]
[R22,R23]
[P20]
[R24]
[K13, P21,
B27]
[N16, Y7)
[T23]
[W21]
[B27]

Ref.

c. INDIRECT ANALYSIS or GENETIC DISEASEUSING CLONED DNA SEGMENTS
TO DETECT LINKED DNA POLYMORPHISMS

Fraglle-X mental retardation
syndrome
Huntlngton's chorea
Henkes kinky hairMuscular dystrophy
BeckerDuchenne
Myotonlc dystrophy[D16]
Retlnoschlsls
Sterold-sulphatase-X-llnked
1chthyosls
Retinitis plgmentosa

Factor IX
GB
LI.28

<12
<10
16

[C29]
[G27]
[W22]

Ll.28
LI.28
RC8
tDP34
RC8

16
19
20
38
17

[K12]

LI .2817

Complement C3 gene

[K22]
[D18]
[D1 B]
7

UCB
UCB

15
25

[W24]
[W23]

LI .28

3

[B2B]

123

Ta b1 e

Hi

Ident1f1ed oncogenes and the1r v1ral ~rotot~~es(from references [Bl3, CJ, 011))

Protein products

Cancer 1n animals

Gene ~I

Prototype v1rus

Isolation
sourceB1ochemicalfunction

Subcellularlocation

Sarcomav-src

Rous sarcoma vl rus

ChickenPK(tyr)Q/

v-fps rjFuj lnaml sarcoma vl rus

Ch lclcenPK( tyr)Cat

PK(tyr)Plasmamembrane
Plasmamembrane
Plasmamembrane

ChickenPK( tyr)?

++

ChickenPK(tyr)?

++Chicken

DNA blnd1ng

Nucleus+++

Chicken

?

t

+

+

t

Chicken?

Cytoplasm/
membranes
Nucleus+t

Turkey

?

?

+

+

House

?

Cytoplasm

v-fes ,;_/ Snyder-Thell In feline
sarcoma vl rus
v-yes
Y-73 Yamaguchi sarcoma
v1rus
v-ros
Rochester-2 sarcoma
virus
v-myc
Hyelocytomatosls v1rus
strain HC29
v-erb-A
Avian erythroblastos1s
v-erb-b v1rus
v-111yb
Av1an myeloblastosls
virus
v-rel
Av1an ret1culoendothellosls v1rus strain T
Moloney mur1ne sarcoma
v-mos
v1rus
Abelson leukaemia v1rus
v-abl
v-fos
v-Ha-rasv-bas ~/
v-K 1-ras
v-fms
v-sls

~/
b/

c/
d/

'i./
f/

124

House,cat

FBJ 111Ur1ne osteosarcoma
v1rus
Harvey rat sarcoma virus

House

Kirsten rat sarcoma
v1rus
SH feline sarcoma v1rus
S1m1an sarcoma virus

Rat

Rat/mousePK( tyr)

Plasmamembrane?

1

B1nds GTP: PlasmaPK(thr)f/ membrane
PlasmaCat?

Membranes

Woolly77F1broblasts

Blood
cells

+

+

7

+

+

+

Care 1- Acutenoma leukaemia

Trans format 1on
1n cell culture

+

+

+

+

+

+

t

•!!/

+

?

+

+

+

?

+

+

+

1

+
+

+

+
+

monkey/cat
The names of viral genes are treated here as generic terms, although, 1n most Instances, several
separate v1ral isolates are known and the proteins encoded by the homologous oncogenes 1n the different
isolates may differ 1n size.
PK denotes protein k1nase; the amlnoac1d subject to phosphorylatlon 1s g1ven In parentheses.
The genes £Qi and~ are homologous genes of viruses from different species.
Osteosarcoma.
Names assigned before adoption of a standard nomenclature: homologous genes transduced from different
species are now given the same name. Examples include abl from mice and cats and ill from woollymonkeys and cats.
Binds guanine nucleotides and carries out autophosphorylatlon on threonlne.

Tab 1 e

17

Cand1date oncogenes detected as transforming genes
in the NIHl3T3 cells[V13 ~I)

Genes

Examples of cell types 1n wh1ch transformlng act1v1ty ls foundc-Ha-rasl

Bladder carcinoma cell l1ne (human)
Urlnary tract tumours (human)
Lung carclnoma cell 11ne (human)
DMBA and MNU-lnduced mallJ1lary carclnomas (rat)DMBAITPA-1nduced sk1n pap111omas, benign and mallgnant (mlce)
Melanoma cell llne (human)
Malll1lilry carc1nosarcoma 11ne (human)
MC, BP, DEN and HNNG-transformed pr1mary cells (gu1nea p1g)
Hyelo1d tumour cell 11ne (mouse)c-K1-ras2

Lung carc1nomas and cell 11nes (human)
Colon carc1nomas and cell 11nes (human)
Pancreat1c carc1noma cell 11ne (human)
Gall bladder carc1noma cell 11ne (human)
Rhabdomyosarcoma cell 11ne (human)
Ovar1an carcinoma, pr1mary (human)
Acute lymphocytlc leukem1a llne (human)
MC-lnduced f1brosarcomas and MC-transformed fibroblasts(mouse)y-1rrad1at1on 1nduced thymoma (mouse)
MC-1nduced thym1c lymphoma and macrophage tumour 11nes(mouse)BP-~nduced f1brosarcoma (mouse)

N-ras

Neuroblastoma cell 11ne (human)
Burkitt lymphoma 11ne (human)
f1brosarcoma and rhabdomyosarcoma cell 11nes (human)
Promyelocyt1c leukaem1a 11ne (human)
T cell leukaem1a 11ne (human)
Melanoma cell 11nes (human)
Teratocarc1noma 11ne (human, late passage)
Lung carcinoma 11ne (human)
Acute and chron1c myeloblast1c leukaemia (human)
Lung carc1noma cell 11ne (mouse)
Carc1nogen-1nduced thymoma (mouse)

c-mosPlasmocytoma cell 11nes (mouse)c-erbB-related[NU-related neuroblastoma cell l1nes (rat)Blym

ALV-1nduced bursal lymphomas (ch1ckens)
Burkitt lymphoma 11nes (human)

Tlym

Intermed1ate T cell lymphoma 11nes (human)
HLV-1nduced T cell lymphomas (mouse)

tx-1

HallJ1lary carc1noma cell 11ne (human)
HHTV and DHBA-1nduced rnalll1lilry carc1nomas (mouse)

tx-2

Pre B tumour cell 11nes (human)
AbMLV-1nduced pre B tumour cell 11nes (f'DOuse)

tx-3

Hyeloma and plasmocytorna cell 11nes (human, mouse)

tx-4

Mature T cell lymphoma l1nes (human, mouse)

UnnamedUnnamedPre B cell leukaem1a cell 11ne (human)
HNNG-treated osteosarcoma cell 11ne

QI

To be consulted for detailed references.
QI Abbrevlat1ons: MC, methylcholanthrene; DHBA, dlmethylbenzanthracene;
TPA, tetradecanoyl-13-phorbol acetate; MNU, methyln1trosourea;
HNNG, N-methyl-N-n1tro-n1troguan1d1ne; BP, benz(a)pyrene;
DEN, d1ethylnltrosam1ne; ENU, ethylnltrosourea.

~I

125

Ta b1 e18

Hutat\ons \n transform\ng ras genes[Vl3)

Codons
Source of allele

ras allele

References
12

59

61

Norma 1

Human c-Ha-rasl

gTy ala gln

GGC

GCC

CA G

[Clf>]

Norma 1

Rat

GGA
gly

?

?

[Sg4]

A C A

C AA

[ D47 J

c-Ha-rasl

Ha-HSYv-Ha-ras[lliJ gTn

RaSY

v-Ra-ras ~/aTa gTn

GCA

CAA

[R54J

Bladder Ca l\nes

Human EJ/T24-Ha-rasl

aTa

GCC

CA G
"""gTn[T5, S65, R2,
Cl6, Tll]

Lung Ca 11ne

Human HS242-Ha-ras1

aTa [filJ

G C C

cfi'lG( YS J

Kanmary Ca

Rat

?

?

[S94J

NormalHuman c-K1-ras2

GCA

K\-MSY

v-K1-ras

[T36)

a1a

CAA
gln
CAA
gln
CAA
gTn

GCA

CAA

[Cl7]

NHU-Ha-rasl

aTa

~
GC
A

[S63, Cl7]

Lung Ca 11ne

Human Calu-K1-ras2

Colon Ca 11ne

Human SW480-Kl-ras2

Lung Ca

Human LL-10-K1-ras2

?

7

[S95]

Lung Ca 1 \neHuman A2182-K1-ras2

?

?

[S95]

Bladder Ca l\neHuman Al698-Kl-ras2

?

?

[S95]

Lung Ca 11 ne

Human PR371-Kl-ras2

?

7

[N21]

Lung Ca 11ne

Human PR310-Kl-ras2

gTy

ala

GCA

u.rn
!!_/
his

Norma 1

Human H-ras

GGT

GCT

C AA

Neuroblastoma l\neHuman SK-H-ra s

giy aia gin
GGT G C T
glyala

Teratocarc1noma
11ne, late passage

Human PAl-N-ras.i.lAl.I.
asp~ [T24]?

flbrosarcoma 11ne

Human HTlOBO-N-ras?

?

Mal1gnantmelanoma line

Human Mel H-ras

~

aia

GCT

!!./

Lung Ca 11ne

Human SW1271 H-ras

GGT
g1y

aia

GCT

[YlO]

aTa gTn

GGT

GGT

?

a/ Amino acid positions aligned with those of other ras proteins.
~/ Personal co11111unlcatlons to [Yl3].

126

[Ml3]

[T24]

[T37]

!!./

Table19

Locallzatlon of cellular oncogenes to human chromosomes(Based on [B13, G29, R12, Y4])

Cellularoncogene

Chromosomal
locatlon

Ref.

H-ras
c-src
c-fos
c-myb

lp11 ..p13
lp34 ..p36[ll 4]

17[B13][014, H16)
[22]
[05)
[Nl2, P12][014, N12, Tl2)
(05, H5, J16]
[H15, 05, f22)
[J17]
[031]
[05)
[05, S36][H5, H16, 014, J16)
[G29 W25]

20ql 2-+13
22ql3.l[J16]

2

6q22-+24
6q21-+qter

c-Kl-rasl
c-mos
c-myc
c-abl
c-Ha-rasl
c-ets ~/
c-Ha-ras2
c-Kl-ras2
c-fes
c-erb-A
c-src
c-sls
~/

[H15, Hl4, Rl4, R26J

6

8q22
8q24
9q34. 1
llpll-+15
11p14.1
11 q23-+24X

12
15q26.1[ L14]

Cellular counterpart located 3' to v-myb ln
the acute E26 acute leukaemla avlan vlrus.

Ta b 1 e

20

Inductlon b!f: X ra!f:S of herltable rec ll!roca 1 translocatlons
ln mouse Sl!ermatogonla[G16]

Exposure( R)

Number of
maleprogenytestedNumber oftranslocatlon
heterozygotes

frequency
(%)

~/
0
150
300
600
1200
500x4 s/600x2 ~/
~/

!1_/

£/

~/

3078

l
19

3342

46

1075
1038
1135
1198

20
6

5433

84

64

0.018
0.62
1.38
l.86
0.58
7.40
5.34

Expected
frequency
(%)
!1_/

0.63
2.04
4 .18
0.94
10. 42
7.23

The frequencies for the irradlated groups are based on
all partially sterile males plus the sterlles that havebeen cytologically confirmed; in the controls, the frequency
ls based on partially steriles only.
The expected frequencies were estimated from cytogenetlc
data uslng the assumptions employed by ford et al [f7].
Fractionated exposures. Interval between doses, 4 weeks.
fractlonated exposures. Interval between doses, 8 weeks.
127Ta b l e

21

Induction of rec\Qrocal translocatlons In Qrlmate SQec\es,studied c~togenet1ca11~ through SQermatoc~te anal~s1s of 1rrad1ated males

Trans location yield
SpeciesNumberofanimals

Rad1at1on

Dose
( Gy)

Hacaca mulatta(Rhesus monkey)
Hacaca mulatta(Rhesus monkey)

1
2
2
22
6
6
7

128

Not given.

478

1. 4
1.5
1.0

0.8
1. 2
1.0

none

[L40]
[L40]

[L40)

(861)

X

600
0.25
600
!I
X
0.50
600
!I
X
1.00
600
!I
X
2.00
200
!I
X
3.00
600
!I
(Rate of induction up to
X

4

none

2

X

4
4

X

6
3
3
4

3
3

!I

1815
1130

Definite

none

6
3
3
3

Human

1.00 0.63
2.00 0.63
3.00 0.63

Ref.
Definiteplus
possible

2
2
2
2
1
2

4

Macacafasclcular1s( Crab-eat 1ng
monkey)

(Gy/
mln)

( ")

[Vl4]

4

Call1thrlx
jacchus(marmoset)

X

Numberofce 11 s3600
0.08
0.25 0.30
5940
0. 19
X
0.50 0.30
2750
0.36
X
1.00 0.304650
0.86(Rate of 1nduct1on up to
Gy: 0.86 10-2/Gy)
X
2.00 0.30
3350
0.99
X
3.00 0.30
1475
0.68
X
1.00 0.002 3870
0.34
y1.00 0.0002 15900
0.43
y1.00 0.147700
0.43
4.00 0.30
X
1450
0
X
0.30
560
0. 71
2.00+2.00 Gy (24 h Interval)

7

Sagulnus
fusclcoll Is(marmoset)

X
X

Dose
rate

0
2.80
3.30
7.80
7.50
7.00
Gy: 7 .44 10-2/Gy)

and
[Y15]

230
260
420
880

0
0.80
1.20
0.80

4700
0.25 0.25
4500
0.50 0.25
4500
1.00 0.25
3500
2.00 0.25
3000
3.00 0.25
3000(Rate of Induction up to

0.09
0.56
0.53
1. 16
1.07
1.91
1.86
2.67
2.47
1.50
1.33
Gy: l . 79 10-2/Gy)

[H27]

4000

0.28

[T45]

X

0.50 0.35
1.00 0.35
2.00 0.35

none
y
y
y
y
y

1.00

2

y

2

4000
0.33
0.18 10-4( Rate of 1nduct1on up to 1.5 Gy: o. 16 10-2/Gy)

2
2
2
2

none

y

0.18 10-4

and
[M69]

1.50

200
0
0.7B
371
4.00
!I
X
2.00
al
300
7.00
X
6.00
~/
180
6 .10
(Rate of 1nduct1on up to 2 Gy: 3.41 10-2/Gy)(Rate of 1nduct1on up to l Gy and1ncludlng Sa~u1nus fusc1coll1s data:
7.7 10- /Gy)

X

[VS]

and
[ Yl I)[B61]

Table22

Mutagen-Induced survival and mutagenesls (experiments using a viral probe)[H53]

MaximalVI rus ( ONA)

~
~

~(r)
HSY
HSV
HSY
HSY

Host

Host
treatment

reactivation
treatment

E.coli
L co 11E.coli
Honkey kidney (CV-1)
Monkey kidney (CV-1)
Honkey kidney (CV-1)
Monkey kidney (CV-1)

26
UV
UV
20
4
UV
UV
2
y ray
10(0 d), 4(3 d)HU
3Aflatoxln 813(0 h)-10(40 h)

Induced mutagenesls

Ref.

8-10 (clear plaques)
8-10 (clear plaques)
4 (clear plaques)

[053]
[865]
[865]
[866]
[ 8£>7][L43][L44 J

2.7 (tk~tk-)(16 h)[l][ 054 J
[G51]
[l45]

AAAF

HSY
HSY
HSY
HSY

Monkey kidney (Vero)UV
Human kidney (N8K)
UV
Normal human flbroblasts(KO)UVUV
Xeroderma plgmentosum
UV
Honkey kidney (CV-1)
Monkey kidney (CY-lP)
UV
UV
Monkey kidney (CY-lP)
y ray
Monkey kidney (CY-1)
AAAF
Honkey kidney (CV-lP)
AAAF
Monkey kidney (CV-lP)
Aflatoxln
Bl
Monkey kidney (CV-lP)

SV40
SV40

SV40
SV40
SV40
SV40
SV40

2(16 h)2(36 h)2(4 d)
2-3(4 d)2.4(0 d, 4 d)

7(1 d)[J0-2]
10(1 d)6-8(0 d, 4 d)
6( 1 d)Monkey kidney ( CV-1 P)
Honkey kidney (8SC-1)
Monkey kidney (CV-1 P)
Honkey kidney (CV-1 P)
SV40-DHA
Monkey kidney ( 8SC-1)
Simian adenovlrus 7 Monkey kidney (CV-1)
Simian adenovlrus 7 Monkey kidney (CY-1)
Adeno 5
Normal human f I brob 1as ts
Adeno 2
Norma 1 human fibroblasts
Adeno 2 (y) f/
Normal human fibroblasts
Minute-virusof miced/House A9
Parvovlrus Lull I
Human kldney (N8K)
Kllha11 rat virus
Rat cells
Vacclnla
Monkey kidney (CV-1)
Pollo
Monkey kidney (CV-1)

Hltomycln C
UV
UV
Hltomycln C
UV
UV
y ray
UV
y ray
y ray
UV
UV
UV
UV
UV

10-100 (ts-t)(l d)[ 10-4 J

20-25( ts-t )( 1 d)[l 10-31
27[5 10-41
4-20(1 d-3 d)[10-21 _

AAAF
HMF
EMS
SV40
SV40
SV40-0NA
SV40-0NA[l45]100(1 d)3(3 d)
4.9(1 d)3(1 d)
5(3 d)
2(0 d)-15(4 d)1.3(0 d)-10(5 d)
3- 5 b/5-15 ~/

8-16(ts-owt:)(1 d)[6 10- 41l(ts-t)(3 d)[J0-3] !Il(ts--wt)(l d) !I1( ts-owt)( 1 d) !Il(ts-owt:)20 h [1] !I

4(30 h)[D.5-30]
2(0 d, 2 d)
2(0 d, 2 d)
1(0 d, 4 d)
1(0 d, 4 d)

[ 8£>7][Sl08][Sl09][867][ Sll O][055]
[Sl08l[S110l
[C65]
[G52]
[G52]
[ Cf>5][8671[867][ D56][J25][J2f>][R60][ GS I][ G51][B67][B67)

Survival of Irradiated virus on treated host
Survival of Irradiated virus on non-treated host
Mutation frequency of the Irradiated virus In treated hostInduced mutagenesls:
Mutation frequency of the Irradiated virus In non-treated host
The virus Is UV-Irradiated, except when stated otherwise. In SOClle experlnients, the time elapsed between host cell
treatment and Infection Is Indicated In parentheses, the multiplicity of Infection Is given In square brackets. The
type of mutation screened for Is also Indicated. The list Is Incomplete, particularly for the bacterial systems·
desc r I bed.
HU, hydroxyurea: AAAF, acetoxyacetylamlnofluorene: HMS, methylmethanesulfonate: EMS, ethylmethanesulfonate
Reactivation factor:a/Irradiated viral DNA could Itself fully Induce a mutation mechanism In the host cell.

b/ Here survival Is calculated from V antigen positive cells.

c/ Extremely high y dose of 3 Hrad.
~/

Single-strand DNA virus.

129T a b l e

23

R1sk of 1nduct1on of qenet1c damage 1n man per 0.01 Gy
at low dose rates of low-LET 1rrad1at1on, accord1ng to the d1rect method(see Table 24 for r1sk est1mates us1ng the doubl1ng dose method)

Genet1c damage

Expected frequency (per 106 11ve b1rth)of genet1cally abnormal ch11dren
1n the f1rst generation
after Irradiation
Males

Mutations having dom1nant effects~/ !1_/

-

10 to

Recessive mutat1ons t/
Unbalanced products of rec1procaltranslocat1ons

remales
20 f./

0 to - 9 Q/

0

l to

0

15 f./

0 to

5 g/~/ Includes r1sk from the Induction of dominant mutations, deletions andbalanced rec1procal translocat1ons w1th dominant effects and from that of
the most detrimental effects of recessives.
!1_/ Does not Include the risk of Induction of genetic changes wlth dominant
sub-lethal effects wh1ch may k111 between b1rth and early 11fe (between
5 and 10 cases per 106 live births per 0.01 Gy of paternal 1rradlat1on;
estimated on the bas1s of data on litter size reduction In mice; see
VII.A.3 for deta\ls). ror maternal 1rrad1atlon, a comparable estimate 1s
not ava1lable.
c/ The lower 1 lmlt of - l O Is der1 ved from the data on dom1 nant cataract- mutat 1ons and the upper l 1m1t of - 20 from those on doml nant skel eta 1
mutat1ons (both 1n m1ce). The latter 1s the same as that arrived at 1n the
1977 report [U2].
A mult1p11catlon factor of 2 has been used 1n the
est1mate based on skeletal data, but not 1n the one based on cataractdata.
Th1s factor 1s an attempt to allow for the 11kel1hood that many
dominant mutat1ons (especially those affecting bodlly systems other than
the skeleton) remain to be detected. A correction factor of 0.5, whichallows for skeletal mutat1ons that are not cl\nlcally slgnHlcant, 1s not
required for the estimate based on cataract data. See the 1982 report [Ul]for deta 11 s.
&/ The lower l\mlt of zero Is based on the assumption that the mutational
sens1tlv1ty of human l01nature oocytes Is similar to that of mouse Immature
oocytes; the upper llmlt of - 9 1s based on the assumption that thesens ltl vlty of the human oocytes Is s 1mllar to that of the mature andmaturing mouse oocytes and that the latter Is 0.44 times that ofspermatogonla. See the 1982 report [Ul] for details.
t/ Although the risk of recessive disease from the Induction of recess1ve
mutations 1s zero In the first generation, about l extra case per million
11ve b1rths would be expected In the following ten generations frompartnership effects and, on certain assumptions, about 10 extra cases per
mll 11 on would be expected by the tenth generat1 on from effects due to
Identity by descent. See [S126] for further deta1ls.
f./ The lower llmlt of - l per m1111on Is based on comb1ned cytogenetlc datafrom chronic low-LET lrrad1at1on experiments 1nvolv1ng the rhesus monkey
and the crab-eating monkey (see paragraphs 522-523) and the upper llm1t of- 15 per mlll1on 1s based on the comb1ned human and marmoset Sagu1nus
fusc1collls cytogenet1c data [U2).
It has been assumed that 9% of
unbalanced products of reciprocal translocat1ons wlll result 1n the b1rthof congen1tally malformed ch1ldren (paragraph 502), wh11e the rema1n1ng 91%
lead to lntra-uter1ne death, often at a very early embryonic stage.
i/ The lower 11mlt of zero 1s based on the assumption that the sens1t1v1ty of
the human l01nature oocytes to the Induction of heritable reciprocaltranslocat1ons w111 be slm\lar to that of the mouse 101nature oocytes w1th
respect to the Induction of chrolllOsome aberration phenomena.
The upper
limit of - 5 per m1111on Is based on the assumptions: (a) that thesenslt1v1ty of the human l01nature oocytes to the 1nduct1on of rec1procaltranslocatlons will be one-half of that of human and marmoset spermatogon1a(based on results w1th m1ce on heritable translocatlons); (b) that thefrequency of unbalanced products wl 11 be s h t Imes that of recoverable
balanced translocat1ons; and (c) that about 9% of unbalanced products w1ll
result In congenitally malformed ch\ldren (paragraph 502). See the 1982
report [Ul] for deta1ls.

130Table24

Estimated effect of 0.01 Gy per generation of low doseor low dose rate low-LET Irradiation
on a population of one m1111on 11ve-born, according to the doubling dose method[The doubling dose assumed 1n these calculations ls 1 Gy(see Table 23 for risk estimates using the direct method)]

Disease class1ficat1onCurrent1nc1dence

Effect of 0.01 Gy per generation
First generation

Autosomal dominant and X-linked
diseases d/
Autosomal recessive diseases
Chromosomal diseaes due to:
Structural anomalies
Numerical anomalies

!l./

f/

10000
2500 ~I

15
Sl1ght

400 f /
3400 g/

Equ111br1u111

100
Slow Increase

2.4

4

Probably
very small

Probably
very small!I Follows

that given in the BEIR Report [B79], except that chro1110somal
diseases are div1ded Into those w1th structural and those with numericalanoma l les.
!l_/ Based on the results of the Br1tish Columbia survey and other stud\es; fordetails see the 1977 Report [U2); note that the new data on congenital
anoma 11 es and other multHactoria l disorders are not used ( see paragraphs
73-93 and 526).
f./ The first generation increment is assumed to be 15% of the equ\libr\um
value for autosomal dominant and X-linked diseases and about 3/5 of theequ111br1um value for structural anomalies. See the 1977 and 1982 reports[U2, Ul) for details.
~/ Includes diseases with both early and late onset, but excludes thefragile-X syndrome.
~/ Also includes diseases maintained by heterozygous advantage.
[I Unbalanced structural rearrangements.
g/ Excludes mosaics.

131Ta b1 e

25

Sunmary of blrth 1irevalences and detrlment e~tlmates for congenltal anoma 1les(from [Cl3))

ICD code

Prevalenceper 104
11ve b1rths

Congen1tal anomaly

Averageyears oflost 1lfe

Total years per 104
11 ve b1 rths

Total years
lost per10~11ve blrths

Potent 1a 11 y Actually
1mpa 1red
1mpa1red
21. 7
3.2

740-742

CNS anoma 11 es
Eye anomalles
744
Anoma11es of ear, face and neck745-747
Card1ovascular system anomalies
748
Anomalles of respiratory system
749
Cleft palate, cleft 11p t cleft
palate750-751
Anoma11es of the alimentary system
752
Anoma l 1es of gen Ha 1 organs
753
Anoma 1les of the urinary organs
754
Anoma11es of the muscoloskeletal
system (excluding congenital
dlslocat1on of the hip)754.3
Congenital d1slocat1on of the hip
755-756.5 Anomal1es of skeletal system
758
Chromosomal anomalies
Mlscellaneous (1ncludlng multlple)
756.6757.9
congenltal anomalles
and 759
743

55.l
13.4
0
23.5

4.6

79.2
2.8
14.5
27 .B
75.2
15.7
34.3

24.5
2. 1

0
1838.5
65.9
33.3

327.2
180.6
322.0
3722.2
130.1
981.7

287
100
97
981
72

16. l
0
13.2
0

433. l0207.1
01512. l5264.0891.9
2399.9

69
720
399
101

0
13.0

0
268. l
312.7
434.4

18039.0
11 BO. 9569.3
1483. 6

180
268
560
432

4828.337004.5

4490

257.7
20. 7
12.6
27.4

15.9

597 .4

8. 1

Tab 1 e24.8

1191.843.4

26

Sunmary of detrlment estimates for spontaneously-arising
Mendellan, chromosomal and other dlsorders
All estimates are per 104 Individuals and an assumed life expectancy
at birth of 70 years for the general population.
{The estimates for monogenlc and chromosomal dlsorders are thoseof Carter dlscussed In the 1982 Report [Ul)and those for congenltal anomalies, of Czelzel and Sankaranarayanan, [Cl3])

Category

Prevalence

Total years
of
lost
life

Total years
ofpotentially
Impairedlife

Total years
ofactually
Impaired
l 1fe

125
40
585

1900
890
4515

3000
1800
37000 <;./

1500
900
3930

Monogenlc
Chromosomal a/
Congenital anomalies Q/
~/
~/
£/
132

Does not Include the fragile site on the X-chromosome which Is
associated with mental retardation.
Exclud1ng chromosomal anomalies.
One-half of this Is due to congenital dislocation of the hip.

224

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