RADIATION RESEARCH 179, 293–303 (2013)0033-7587/13 $15.00
Ó2013 by Radiation Research Society.
All rights of reproduction in any form reserved.
DOI: 10.1667/RR3095.1

The Genetic Risk in Mice from Radiation: An Estimate of the Mutation
Induction Rate per Genome
Jun-ichi Asakawa,a,1 Mieko Kodaira,a Harry M. Cullings,b Hiroaki Katayamac and Nori Nakamuraa
Departments of aGenetics, b Statistics, and c Information Technology, Radiation Effects Research Foundation, 5-2 Hijiyama Park, Minami-ku,
Hiroshima 732-0815, JapanINTRODUCTION
Asakawa, J., Kodaira, M. N., Katayama, H., Cullings, H.
and Nakamura N. The Genetic Risk in Mice from Radiation:
An Estimate of the Mutation Induction Rate per Genome
Radiat. Res. 179, 293–303 (2013).

Exposure to ionizing radiation increases the risk ofdeveloping various diseases, including cancer and noncancer diseases (1–3). When germ cells are exposed toradiation, the risk of hereditary disorders may be increased.
As the genetic effects of radiation have been confirmed invarious species, it is unlikely that humans are an exception.
Nevertheless, no clear evidence has been obtained forradiation-induced germline mutations among the offspringof atomic-bomb (A-bomb) survivors (4), or of those ofchildhood cancer survivors (5, 6).
Among the past studies on the genetic effects of radiationin animals, specific-locus tests in mice are the mostcomprehensive. The experiments were primarily conductedby Russell’s group (7 loci) at the Oak Ridge National
Laboratory and to a lesser extent by Lyon’s group (6 loci) at
the Medical Research Council in Harwell. Through thestudies, it was recognized that the mean mutation induction
rate found by Russell’s group at the 7 loci was 2.76 3 105/locus per Gy (a, se, c, b, p, d and s) and was considerably
higher than the rate obtained by the Lyon’s group of 0.86 3
105/locus per Gy at the a, bp, pa, fz, ln and pe loci (7). Sucha difference in radiation sensitivity among loci makes it hardto estimate the mean mutation induction rate in the genome.
Summarizing the data, the current UNSCEAR Reportsuggests the mean mutation induction rate at 36 genes is
1.08 3 105/locus per Gy, which was nearly one-half of themean in the 7 loci study (7).
To better understand the mean mutation induction rate per
genome, it would be necessary to examine a large numberof genes per individual concurrently. For this purpose,previous studies have used the restriction landmark genome
scanning (RLGS) method for analysis of mouse offspringborn to X-irradiated parents (8). This method can visualize
and screen about 2,000 DNA fragments (end-labeled with
32
P after digestion of genomic DNA with specific restriction
enzymes) as spots on a two-dimensional autoradiogram.
This technique results in reproducible intensities and low
coefficients of variation (CV) of 0.12 (9), that allow adeletion mutation for a heterozygous fragment to be
detected as a decreased spot intensity of about 50%. The

Restriction Landmark Genome Scanning (RLGS) is amethod that uses end-labeled 32P NotI sites that are mostly
associated with coding genes to visualizes thousands of DNA
fragments as spots in two-dimensional autoradiograms. Thisapproach allows direct detection of autosomal deletions as
spots with half normal intensity. The method was applied tomouse offspring derived from spermatogonia exposed to 4 Gyof X rays. A genome-wide assessment of the mutationinduction rate was estimated from the detected deletions.
Examinations were made of 1,007 progeny (502 derived fromcontrol males and 505 from irradiated males) and 1,190
paternal and 1,240 maternal spots for each mouse. The
results showed one deletion mutation in the unirradiatedpaternal genomes of 502 offspring (0.2%) and 5 deletions in
the irradiated paternal genomes of 505 offspring (1%). The
difference was marginally significant, with the deletion sizes
ranged from 2–13 Mb. If the frequencies are taken at facevalue, the net increase was 0.8% after an exposure of 4 Gy, or
0.2% per Gy per individual if a linear dose response isassumed. Since the present RLGS analysis examined 1,190
NotI sites, while the mouse genome contains ;25,000 genes,
the genomic probability of any gene undergoing a deletion
mutation would be 253 0.2%, or 5% per Gy. Furthermore,since the present RLGS screened about 0.2% of the total
genome, the probability of detecting a deletion anywhere in
the total genome would be estimated to be 500 times 0.2% or100% (i.e., 1 deletion per Gy). These results are discussedwith reference to copy number variation in the humangenome. Ó 2013 by Radiation Research Society

Editor’s note. The online version of this article (DOI: 10.1667/
RR3095.1) contains supplementary information that is available to all
authorized users.
1Address for correspondence: Department of Genetics, Radiation
Effects Research Foundation, 5-2 Hijiyama Park, Minami-ku,
Hiroshima, 732-0815, Japan; e-mail: jun@rerf.or.jp.
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rate of false negatives (the probability of missing mutations)is estimated as about 3% (9), and the rate of false positives(the probability of detecting normal sequences as mutations)is kept very low, because nearly all of the mutation
candidates can be confirmed by additional means. The
method does not require probes for mutation detection,
while the nature of mutations can usually be studied bycloning and sequencing the wild-type allele or mutant allele.
In our previous study, the BALB/c strain of mice wasused for both parents, and NotI enzyme (GC/GGCCGC)
was used to end-label DNA to mark GC-rich sequences in
the genome (8). We considered it desirable to screen DNA
fragments that are primarily related to functional genes
rather than fragments with unknown functions. We
postulated that because mutations in functional genes would
be more likely to result in phenotypic changes, and hence
would be more significant as a hereditary risk after radiation
exposure. The NotI sites are disproportionally located at
unmethylated ‘‘CpG islands’’, that are common upstream ofgenes and are thought to be associated with functional
genes. Thus, we and others propose that the use of NotI as
the landmark enzyme in the RLGS will result in the delivery
of a high proportion of visualized spots from active genes(10, 11). Our study gave rise to only 4 deletion mutations
and the results indicated that the maximum induction rate ofdeletion mutations was 0.2 3 105/locus per Gy (8). This
result was close to the proposed rate of 0.3 3 105/locus per
Gy (7) with 30% of the specific-locus mutation comprised
large deletions.
To obtain more information on the mutation induction
rate at different parts of the genome, a second study was
conducted to measure the mutation rate at AT-richsequences in the genome (12). In this study, AflII(recognition sequence: C/TTAAG) was used as thelandmark enzyme for radiolabeling of the DNA fragments.
This method was found to specifically visualize AT-rich
DNA fragments as spots. Unfortunately, normal alleles ofmost of the mutated spots contained repeat sequences (e.g.,satellite, SINE, LINE and short tandem repeat sequences)and therefore presented as multiple copies in the genome,
which prevented us from characterizing the mutations. None
the less, the estimated mutation rate was 0.6 3 105/locus
per Gy, which is in the same range as that estimated at GCrich sequences. In addition, we found 3 possible deletions
free from the repeat sequences among 184 offspring (514spots per individual) derived from 5 Gy irradiatedspermatogonia with no corresponding mutations in thecontrol group. Unfortunately, since the same strain of mice
(BALB/c) was used for both parents in the first study,
parental origins of the mutations could not be determined.
Further, that study raised a possibility that the results
obtained might not represent conditions in wild-type
animals because the BALB/c strain bears a mutation in agene involved in DNA repair (DNA-PKcs) (13). To
overcome these caveats in the present study, we used male
hybrids of C57BL/6 and C3H/He strains (abbreviated as

B6C3) for irradiation, and females from the JF1 strain (astrain somewhat remote from laboratory mice). Mated theJF1 females with the irradiated B6C3 males allowed theparental origin of mutations to be determined.
MATERIALS AND METHODSMice
B6C3 male mice (8 weeks old) were purchased from Charles River
Japan. The JF1 strain was derived from a Japanese Fancy Mouse thatoriginated from a Japanese wild mouse (Mus musculus molossinus),
and which was established at the National Institute of Genetics (NIG,
Mishima, Japan) (14). The animals were kindly supplied by Dr. T.
Shiroishi at the NIG. Thirteen 10-week-old male mice were exposed to
4 Gy of X rays. During irradiation, each male mouse was immobilized
on a styrofoam plate (ventral side up), and the body, except for thetestes, was shielded with a 3-mm-thick lead plate. The X rays were
given vertically by a Shimadzu X-ray generator, operated at 220 kVp
and 8.0 mA through a 0.5 mm aluminum and 0.3 mm copper filter at adose rate of 1.19 Gy/min. After an 8-week recovery period, the mice
were individually mated with 3 JF1 females to collect offspring
derived from irradiated spermatogonia cells. The offspring from thesecrosses constituted the exposed group. The F1 mice born to theunirradiated B6C3 males (22 animals) and the JF1 females (66animals) constituted the control group. When the offspring reachedapproximately 6 weeks of age, they were sacrificed, and their spleens,livers and kidneys were removed and stored at 808C until use. A partof the spleen was used before cryopreservation to extract highmolecular-weight DNA for the screening. When putative mutations
were detected, kidney and liver DNA samples were used to confirm
the experimental findings. Experiments were carried out according to
the standard protocols employed at the Radiation Effects Research
Foundation (RERF). Mice were housed and cared for at the RERF
animal care facility and the study plan was approved by the in-house
Animal Care Committee.
Restriction Landmark Genome Scanning (RLGS)
DNA was prepared, labeled and electrophoresed as described (9, 15,16). Briefly, genomic DNA was double digested with NotI (GC/GGCCGC) and EcoRV (GAT/ATC) restriction enzymes. The 4-base
5 0 overhangs produced by the NotI digestion were end-labeled with
32
P-dCTP and 32P-dGTP with a fill-in reaction with a polymerase sothat only DNA fragments containing the cleaved NotI ends were
quantitatively visualized. The DNA digests were then separated by a
two-step electrophoresis. For the first-dimension electrophoresis, twotypes of 61-cm-long agarose gel casts were prepared, 0.9% and 0.8%
gel, for an optimal separation of 1–5 kb and 5–10 kb NotI-EcoRV
fragments, respectively. Subsequently, the NotI-EcoRV-digested DNA
fragments were further digested in situ with a third restriction enzyme,
HinfI (G/ANTC), and the agarose gels were placed on top of thesecond-dimensional polyacrylamide gels for separation of thefragments. The gels were then dried and subjected to autoradiography.
Measurements of Spot Intensities
Details of spot intensity measurements have been described
previously (9, 17). Briefly, autoradiograms were scanned and digitizedimages were obtained using a laser film scanner (Abe-Sekkei, Tokyo,
Japan). For each selected spot in a study sample, a mean intensity of
10 neighboring spots was used to calculate the ratio of spot intensities
between the study gel and the master gel to compensate locally for any
variation in the degree of background darkness of each autoradiogram.
For this calculation, the two largest and two smallest values wereignored, and the remaining 6 ratios were averaged. The raw value ofGENETIC RISK ESTIMATE OF RADIATION IN MICE

the spot intensity was divided by this local average to obtain theadjusted spot intensity (9, 17).
Molecular Analysis
Cloning and sequencing. To recover normal DNA fragments fromthe spots which had undergone mutations, genomic DNA from amouse that showed no mutations was first digested with NotI and
EcoRV. The digested DNA (the NotI sites were not filled in) were
subjected to selection procedures for enrichment of fragments that
contained NotI sites (9). The enriched nonlabeled DNA fragments
were then mixed with ordinarily radiolabeled fragments, and themixed DNA samples were subjected to standard two-dimensional gel
electrophoreses. After development of an autoradiogram on a semidried gel, the gel area corresponding to the spot on the autoradiogram
was punched out, and the DNA was recovered and cloned into a NotIHinfI vector (18). After selection of bacterial clones, base sequencesof the inserts were determined using ABI Prism 310 Genetic Analyzer(Applied Biosystems, Tokyo, Japan). Several clones were examinedfor each mutation and the consensus sequence reads were mapped on
the mouse reference sequence, NCBI 37/mm9, using a basic localalignment search tool (BLAST)-like alignment tool or BLAT at
Ensemble (http://www.ensembl.org/Mus_musculus/blastview). Chromosomal locations and upstream and downstream sequence information were obtained for each mutation.
Copy number estimation. SYBRt Green-based qPCR analyses
were carried out using a LightCycler PCR system (Roche, Tokyo,
Japan). The qPCR assays were performed according to the
manufacturer’s instructions. The assays were conducted in triplicatesfor each DNA sample of trio individuals (mutant offspring and theparents). The sequences of each primer are shown in Supplementary
Table 1 (http://dx.doi.org/RR3095.1.S1).
Deletion size estimation. The size of each deletion was estimatedby comparative genomic hybridization (CGH) analyses using Roche
NimbleGen microarray systems. Whole-genome tiling arrays with 720
k probes were used with a mean distance between probes of about 1.4
kb. CGH experiments were carried out according to the manufacturer’s instructions. The hybridized slides were scanned with a MS200
microarray scanner and the log2 fluorescent ratio of each probe set wascalculated with the NimbleScan and graphically plotted using
SignalMap software.
Statistical Analysis
All observed numbers of mutations were treated as realizations ofPoisson random variables characterized by a mutational rate expressedin terms of mutations per genome per generation, given here aspercentage for convenience due to the small numbers involved.
Confidence intervals for Table 3 were estimated by the ‘‘cii’’
procedure of Stata 11 statistical software (Stata Corporation, 2011).
P values for the comparison of control and irradiated mice andcomparisons of the BALB/c and B6C3 strains were calculated using
the method based on assuming that the number of mutations in theirradiated group is distributed binomially and is conditional on theobserved total number of mutations in both groups (19), with a ‘‘midP’’ correction for the discreteness of the observed values (20).

RESULTS

Selection of Spots for Mutation Analysis

In the present study, male B6C3 mice and female JF1mice were used. Since the JF1 strain is somewhat distantevolutionally from laboratory mice, nearly half of theoffsprings’ spots consisted of single-copy spots that wereunique to one parent. Consequently, the total number of295

spots appearing in the autoradiograms of the offspring
increased by nearly one-half (a total of about 2,000 spots)
when compared with results from each parental strain.
To select appropriate spots for screening, a clear
autoradiogram was chosen from both the 1–5 kb and 5–
10 kb images (fragment lengths indicated in the first
dimension gel run). These autoradiograms served as themaster images for comparison with the spots observed in the
autoradiogram images of the test samples: 958 (1–5 kb
image) and 1,204 spots (5–10 kb image) were initiallyconsidered suitable for the screening because they did not
overlap with the nearby spots, they were not located at amargin of the gel (where the appearance of such spot is notstable), and they did not consist of multiple copies (i.e., they
were not large spots).
After examination of spot images of 50 test samples,
those spots that were identified as polymorphic (i.e., either
absent in some offspring or had a variation of approximately
50% in density among the offspring) were excluded.
Finally, 864 spots (256 were unique single-copy spots
derived from JF1 strain, 254 were unique single-copy spots
from B6C3 strain and 354 were two-copy spots that werecommon to both strains) were selected for 1–5 kb images
and 973 spots (391 were unique and derived from JF1, 343
were from B6C3, and 239 were common to both strains)
were selected for the 5–10 kb images.
Crude Mutation Data

Two autoradiograms (1–5 kb and 5–10 kb images) fromone F1 mouse provided information on 1,190 (254 þ 354 þ
343 þ 239) paternal fragments and 1,240 (256 þ 354 þ 391
þ 239) maternal fragments (Fig. 1). The selected spots were
examined in DNA samples from a total of 1,007 F1 mice
(505 from irradiated and 502 from unirradiated spermatogonia). A total of 595,387 irradiated paternal spots (505 F1),
583,051 unirradiated paternal spots (502 F1), and 1,288,352
unirradiated maternal spots (1,007 F1) were finally subjected
to mutation screening. The actual numbers were slightly
smaller (by 1–2%) than expected since some parts of thegels were not suited for quantification of spot intensity.
A two-copy spot with normalized intensity below thechosen cut-off point (65% of mean value), or a single-copy
spot that disappeared in an image from an offspring (where
the parents displayed a spot within the normal intensity
range) were considered as a mutation candidate. Subsequently, to exclude the possibility of mosaic mutations,which is unlikely to be derived from mutations from
irradiated spermatogonia, kidney and liver DNA samples
were used to confirm the observations made on spleen DNA
samples. Two mutation candidates observed in experimentswith spleen DNA samples were found as normal when
DNA samples from kidney and liver of the same individuals
were used. Therefore, these two were regarded as nonmutants and were excluded from further examinations.
Also, the remaining candidate mutations were all confirmed

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TABLE 1
Characteristics of the Mutations Detected
Spot location
Mutation origin DosePaternal

Maternal

Mouse ID

Mutation type

Spot ID affected GenBank ID Spot size (bp) Chromosome Strand

0 Gy B6C3-0G-1 2.3-Mb deletion

S378
S893
4 Gy B6C3-4G-1 10.6-Mb deletion S280
B6C3-4G-2 4.7-Mb deletion S984
S1592
L261
L1483
B6C3-4G-3 1.9-Mb deletion L400*
L440*
B6C3-4G-4 13-Mb deletion
S390
S884
B6C3-4G-5 4.3-Mb deletion S1290*
S1315*
B6C3-4G-6 (CT)29 to (CT)32 S909
0 Gy JF-0G-1(GT)32 to (GT)14 L700

AB491227
AB491228
AB491229
AB491230
AB491231
AB491232
AB491233
AB491234
AB491235
AB491236
AB491237
AB491238
AB491239
AB491240
AB491242

749
515
910
443
287
968
338
845
715
860
519
345
366
513
634

þ
þ
þ
þ
–
þ
þ
þ
–
–
þ
þ
–
þ
þ

10
10
9
6
6
6
6
13
13
6
6
7
7
7
2

Start (NotI)
122,422,557
122,633,767
37,297,185
90,938,614
90,412,667
92,656,006
90,412,741
101,386,885
101,386,892
99,642,935
99,643,396
56,044,432
56,044,439
50,484,981
116,801,581* L400 and L440, and S1290 and S1315 were derived from consecutive 3 0 and 5 0 fragments which contain common NotI sites.

in kidney and liver DNA samples. Finally, a total of 15
spots were found to be mutated in 8 animals: specifically,
12 spots were found in 6 mice from the exposed group and3 spots were found in 2 mice from the control group (Table1). The locations of these spots are indicated with arrows in
Fig. 1, and patterns of normal and mutant spots are
displayed in Fig. 2.
Characterization of Mutations
Parental origins of mutations. Identification of parental
origins of deletions that occurred at single-copy spots (i.e.,
strain-specific spots) did not require any further molecularanalyses. In contrast, two deletions observed in mice B6C34G-4 and B6C3-4G-5 involved two-copy spots common toboth parental strains (Table 1) and hence determination of
their parental origins required single nucleotide polymorphism (SNP) information in the deleted (or nondeleted)regions of the genome. For this purpose, a set of PCRprimers were prepared at the 5 0 and 3 0 regions of thedeletion breakpoints so that only the deleted alleles would

be amplified (the primer distances were designed to be atleast several Mb apart so that PCR amplification would notwork on normal DNA). The amplified products weresequenced and the deletion breakpoints were specified.
Following a search of the Mouse Phenome Database at theJackson Laboratory (http://phenome.jax.org/), SNP information was obtained in the deleted regions; four SNPs in thedeletion of B6C3-4G-4 mouse and two in the deletion of
B6C3-4G-5 mouse. Those SNP sites were PCR amplified,sequenced and compared with the SNP database to showthat both deletions occurred in the irradiated paternal
genome (Suppl. Table 2).
Microsatellite mutations. In two mutations, both occurredat single-copy spots, loss of the spot at a normal position
was accompanied by the appearance of a novel single-copy
spot near the original position (Fig. 2D). The appearance ofsuch a new single-copy spot slightly above or below theoriginal position is a hallmark of mutations at microsatellite
sequences. In fact, sequencing of the DNA isolated from thenormal and mutant spots revealed that one mutation resulted
from a gain and the other from a loss of 2-base repeat units

TABLE 2
Sequences and the Locations of Breakpoints of Deletions Identified in the Exposed Group
Mouse ID (chromosome)
B6C3-4G-1 (9)B6C3-4G-2 (6)B6C3-4G-3 (13)
B6C3-4G-4 (6)B6C3-4G-5 (7)First breakpoint

Second breakpoint

Junction

TTCATGACTGcttc
31,956,359
GCACTCAGAGctat
89,709,524
CTGTACCATGtcta
100,816,543
TCTCTGAACTcggg
99,507,761
CAGACAACATttca
54,370,412

cagt AGAGACACCC
42,515,497
cttGGAAAGATATT
94,405,362
cagaAAGGGGATGC
102,748,640
gggTTATTGGATTT
112,539,830
ggggGGAAATGTAA
58,625,965

TTCATGACTGAAGAGACACCC
GCACTCAGAGAAAGATATTCTGTACCATGAAGGGGATGC
TCTCTGAACTTATTGGATTTCAGACAACATGGAAATGTAA

Note. Uppercase letters indicate retained nucleotides, lowercase letters indicate deleted nucleotides. Underlined bases were homologous
between the first and second breakpoints. Breakpoint positions were the last 3 0 and first 5 0 retained nucleotides.

GENETIC RISK ESTIMATE OF RADIATION IN MICE

297

FIG. 1. RLGS patterns of mouse DNA. Spots that had undergone mutations are marked with arrows.

in microsatellite sequences (JF-0G-1 and B6C3-4G-6 mice,
Table 1).
Deletion mutations. The remaining 13 spots showeddecreased spot intensities but without any new spots. They
were found among 6 mice, specifically, single spot waslost in 1 mouse, 2 spots each were lost in 4 mice, and 4
spots were lost in one mouse (Table 1). Since it seemed
likely that the multiple losses of spots in each mouse wasassociated with a single event (i.e., a large deletion),sequencing data of the wild-type allele (with nonmutatedspots) were used to determine their physical locations in
the genome after an Ensemble BLAT search. The results
showed that the reasoning was indeed correct. The two
spots that were lost in B6C3-4G-3 and B6C3-4G-5 mice
were 5 0 and 3 0 fragments originating from the same single
NotI sites. Another mutant mouse (B6C3-4Gy-4) had lost 2
spots, while their NotI sites were only 462 bp apart on thesame chromosome 6. Therefore, the 2 spots in thesemutations were most likely included in one deletion. The
remaining 2 mice (B6C3-0Gy-1 and B6C3-4Gy-2) had lost
2 and 4 spots, and the NotI sites were found to locate on
the same chromosomes 10 and 6, respectively. Thus, theselosses were also thought to be included in one large
deletion, respectively, although the locations of the NotI
sites were 210 kb and 2,245 kb apart from each other.

The copy numbers of such regions thought to be involvedin large deletions were estimated by the qPCR assays. As
shown in Fig. 3, the 6 candidate mutations were all found tocarry only single copies (primers were set to amplify DNA
fragments within the deleted spot), whereas all of thepaternal samples had two copies. In one mutation that
occurred in the B6C3-4G-5 mouse, DNA was not available
from the mother, which, however, does not change theconclusion because the mutation is determined as being
derived from the paternal genome by the SNP data.
To confirm that the simultaneous losses of multiple spots
which were mapped on a single chromosome were causedby single deletions, the high-density array CGH technique
was applied to three DNA samples harboring losses ofmultiple spots (B6C3-0Gy-1, B6C3-4Gy-2 and B6C3-4Gy4 individuals). The results clearly demonstrated that eachmutation was caused by a single large deletion (Fig. 4).
Application of array CGH analysis to the remaining DNA
samples allowed a rapid acquisition of approximate deletionsize, which facilitated the subsequent determination ofdeletion breakpoints. The estimated deletion sizes are
summarized in Table 1. It is clear that, regardless of thenumber of spots lost, deletions were on the order of
megabases except for the mutations at microsatellite
sequences.

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FIG. 2. Close-ups of RLGS patterns from normal and mutant DNA. Panel A: An example of the loss of one copy of DNA at a two copy spot
(spot intensity was reduced by half). Panels B and C: Examples of the loss of a spot (indicated by white squares) that occurred at single copy spots.
Panel D: An example of loss of a spot that accompanied a new spot (indicated by ‘‘l’’) near the normal position (indicated by a white arrow), an
indication of a mutation at a repeat sequence. The numbers indicated below each panel represent the spot IDs in Table 1.

Characterization of breakpoint sequences. Since the six
deletions found in the present study were all underheterozygous conditions, PCR primers were designed sothat normal alleles were too long to be amplified, but only
the mutated shorter alleles could undergo the amplification.
The base sequences of the 5 0 upstream and 3 0 downstreamof each deletion were also determined. We aligned theirsequences on the reference mouse genome by BLAT search(UCSC Genome Browser: http://genome.ucsc.edu/) or
ENSEMBL (Genome Browser: http://www.ensembl.org/
index.html). One mutation found in the control group(B6C3-0G-1 mouse carrying a 2.3 Mb deletion) had
truncated LINE-1 sequences of approximately 2 kb at bothbreakpoints. The results imply that the deletion occurred asa result of nonallelic homologous recombination (NAHR).
The sequence information at the breakpoints of theremaining five deletions in the exposed group is given in
Table 2. Two deletions had one-base homology between thetwo breakpoints, whereas the remaining three had no suchpartial homology, indicating that they arose throughnonhomologous end joining (NHEH).
Other Findings

In addition to the 9 mice bearing germline deletion
mutations, two mice had lost a number of single-copy spots.
Case 1. In one female mouse in the control group, 9
single-copy spots (all from the JF1 genome) were lost.
Sequence data of these spots revealed that they all mapped
to the X chromosome. Array CGH analysis indicated that
this animal had only one X chromosome and no Y
chromosome was present (i.e., monosomy X). It was
confirmed that the X chromosome present in the offspring
was derived from the B6C3 paternal mouse and hence it
was the entire maternal X chromosome that was lost either
before or after fertilization.
Case 2. Another female mouse also in the control group
had lost 38 single-copy spots (all maternal). At the sametime, 40 single-copy spots (all paternal) showed increased

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GENETIC RISK ESTIMATE OF RADIATION IN MICE

FIG. 3. Copy number estimates of the 6 deletion candidates. Paired PCR primers were set around the affected
NotI sites. The spot numbers indicated on top of each panel represent the spot IDs shown in Table 1. The meansof triplicate measures are shown. FA, F1 and MO stand for father, offspring and mother, respectively.

intensities similar to the level of two-copy spots. Cloning
and sequencing analyses of DNA samples from 15
randomly selected spots among the 40 showed that they
all mapped to the same chromosome 10. Array CGH
analysis showed that chromosome 10 looked normal (i.e.,
two-copy signals) through the entire chromosome. These
results indicated that the genome of this mouse consisted of
two copies of paternal chromosome 10 and that no maternal
chromosome 10 was present: specifically, this was a
uniparental disomy (UPD). Since these two mutations arechromosomal events, they are not included in the estimateof mutation induction rate described below.

small, the usual statistical theory for estimating a P value
based on normal theory approximation may not be accurate
in this case. Therefore, a number of methods were tried for
estimating the P value for a difference between control andirradiated mice in the current study, as described more fullyin ‘‘Statistical Analysis’’ in the Materials and Methods.
Resulting estimates for the P value of a one-sided test were
marginally significant, ranging from about 0.045–0.11,
depending on the method used.
DISCUSSION

How to Define the Mutation Rate
Radiation Effect

The present study found one paternally derived deletion
mutation among 502 F1 mice in the control group, whichsuggested a spontaneous mutation rate of 0.2% (95% CI,[0.028%, 1.4%]) under the present experimental conditions(screening of 1,190 paternally-derived spots per offspring).
As five deletion mutations were found in the 505 offspring
derived from the irradiated spermatogonia with 4 Gy, an
induced mutation rate was estimated as 0.2% per Gy, with
an approximate one-sided 95% CI, based on the assumptionthat the induced mutation rate cannot be ,0, of [0, 0.40%
per Gy]. Because the numbers of observed mutations were

In a previous study, the induced mutation rate wasestimated by considering each NotI site to be equivalent toa gene in the specific-locus tests (i.e., the test used the ratioof the number of the affected NotI sites over the total
number of NotI sites examined). However, several
deletions were found to involve two adjacent fragmentsthat shared the same NotI sites. Therefore, different
estimations were made based on either the number ofmutation events, the number of offspring, or the number ofaffected alleles (8). In the present study, two deletions
involved single spots, whereas four deletions involved 2
spots and one deletion involved 4 spots (Table 1). Under

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FIG. 4. Array CGH patterns of three mutations. The top panel represents the results of a B6C3-0G-1 mouse,the middle panel represents a B6C3-4G-2 mouse and the lower panel represents a B6C3-4G-4 mouse. Arrows
indicate locations of NotI sites in the deletions.

such conditions, it does not seem appropriate to define themutation induction rate in terms of the NotI site, because
the loss of a single NotI site n times independently is not
the equivalent of the loss of n NotI sites within one
deletion (i.e., a single event). Consequently, the present
study should be considered as a specific-locus test
involving 14 NotI sites that have undergone deletion
mutations, and the mutation induction rate should be
estimated as the number of deletion events per genome. In
the following discussions, mutations at microsatellite
sequences will be excluded from the radiation-induced
mutations. Likewise, insertions or deletions of transposon
sequences and uniparental disomy will also be excluded.

sets of data (Table 3). Although the results for the BALB/cstrain may look consistently higher than the results for the
B6C3 strain, the data sets are not large enough to indicate astatistical significance either in the unexposed or the exposedgroups. It should be mentioned that RLGS studies with mice
numbering about 1,000 are not sufficient to indicate that
BABL/c mice are more sensitive to radiation for mutationinduction in spermatogonia. These results are in line with theresults of studies at repair kinetics for radiation-induced DNA
double-strand breaks (observed as cH2AX foci): namely, thekinetics were only slightly slower in BALB/c-derived cells
compared with B6C3-derived cells (21).
Estimation of the Mutation Induction Rate

Possible Radiation Sensitivity of the BALB/c Strain

Since we used two strains for irradiation, BALB/c andB6C3, and the BALB/c strain is known to carry a mutation at
the DNA-PK gene (13), it was interesting to compare the two

Present results showed one deletion mutation in the control
group (n ¼ 502) and five deletion mutations in the exposedgroup (n ¼ 505, 4 Gy dose) (Table 1). Thus, the crude rate ofdetecting a deletion mutation is about 0.2% per Gy (i.e., one

301

GENETIC RISK ESTIMATE OF RADIATION IN MICETABLE 3
Summary of Previous Results (Experiment 1) (8) and Present Results (Experiment 2)Experiment

Irradiated strain

Dose

No. F1 mice

Number of deletions
relevant to the riskof paternal genome

1
1
1
2
2

BALB/cBALB/cBALB/c
B6C3
B6C3

0 Gy
3 Gy
5 Gy
0 Gy
4 Gy

190
237
79
502
505

1*
3
1
1
5

Estimated mutation frequency
in spermatogonia [CI]1/190 ¼ 0.53% [0.013%, 2.9%]
3/237 ¼ 1.3% [0.26%, 3.7%]
1/79 ¼ 1.3% [0.032%, 7.1%]
1/502 ¼ 0.20% [0.005%, 1.1%]
5/505 ¼ 0.99% [0.32%, 2.3%]* There were 2 identical deletions (clonal events), but the parental origins could not be determined. Therefore, the parental origins of the twodeletions were assumed to be derived equally from the paternal and maternal genomes.

animal among 500 examined). Since the estimated number ofcoding genes is about 25,000 per genome (22), or about 253
the number of the 1,190 NotI sites examined here, theprobability of a deletion mutation (detectable with the RLGS
method) that includes any coding gene of the genome isestimated by multiplying the induction rate (0.2%) by 25 or
0.05 (5%) per haploid genome. Another approach is based on
the estimation that the present RLGS technique screens about
0.2% of the genome (i.e., the mean fragment size in the first
dimension gel is 5 kb and 1,190 spots 3 5 kb ¼ 6,000 kb,
which is 0.2% of the haploid genome of 3,000 Mb).
Therefore, multiplying the mutation induction rate of 0.2%
by 500 (i.e., 1/0.002) gives a probability of 100% or 1, for the
probability of a deletion mutation occurring any place in the
genome following exposure of spermatogonia cells to 1 Gy.
In contrast, Russell’s 7 locus tests gave an estimatedmutation rate of ;2 3 105/locus per Gy, while UNSCEAR
estimated a mean of 1.08 3 105/locus per Gy following theinclusion of 29 additional genes (for a total of 36 genes) (7).
When the number of coding genes (i.e., 25,000) is multipliedby the mean mutation induction rate per locus, an estimate of
0.25 (25%) is obtained (i.e., 1 3 105/locus 3 25,000 genes/
genome), which represents the probability that a coding geneis involved in a mutation (not necessarily a large deletion)
after exposure of spermatogonia cells to 1 Gy.
The estimated probability of any gene of a genome
undergoing mutation appears roughly 53 larger when basedon the specific-locus data (25%) compared with that on the
RLGS data (5%). The difference may be simply due tostatistical fluctuation in the small number of mutants
observed in the present study, but can also be explainedby the higher sensitivity of the mutation detection in thespecific-locus test. Namely, this is a functional test of
specific genes, and therefore any kind of mutation thatinactivates the normal function can be detected, includingbase substitutions, deletions of a few bases and also
inversions, which are mostly not detectable with the RLGS
method. In contrast, the detection of deletions/insertions
with the RLGS method usually requires size alterations of
5% or larger of the DNA fragments during the second
dimension electrophoresis (fragments range between 300–
2,000 bp): namely, ;15 bp or larger.

The difference between the two estimates may become
even smaller if the analyses were restricted to possible large
deletions detected in the specific locus tests. Specifically, it isreported that about one-third (29/76 ¼ 0.38) of radiationinduced alleles are lethal under homozygous conditions,which is probably due to inclusion of adjacent essential
gene(s) in the deletion (Table XV of Searles’s review, see ref.
23). In this calculation, results for d and s loci were excluded
because their nonfunctional (but intragenic) alleles were later
found to be homozygous lethal (24, 25). As a consequence,
the above mentioned 53 difference in the estimated mutationinduction rate between the specific-locus test and the RLGS
method may be reduced to about 23 difference whenpossible deletions are compared. It is impressive that the two
totally different systems for detection of germline mutations
in mice provide estimated risks of mutations (or deletions) ina coding gene that are within 2–53 difference (5% by RLGS,
25% by specific locus tests and 7% when specific locus data
were restricted to homozygous lethal mutations). The major
finding following a genome-wide search of mutations with
the RLGS method is that deletion mutations are not easily
induced in the genome. This is potentially good news forhumans, concerned about radiation-induced germline mutations, but will require researchers to move ahead with further
sophisticated systems such as micro array-based CGH, andultimately whole genome sequencing to truly estimate themutation induction rate in mammalian germ cells.
Future Directions

The Human Genome Project was completed in 2003, andresearch interests have shifted toward analysis of individual
differences in the human genome. Studies to date indicatedthat the human genome has already accumulated a large
number of mutations, e.g., more than one million SNPs,
several hundred thousand deletions/insertions (copy number
variations, CNV) and several hundred deletion/insertion
events in coding exons. The genome of James Watson
contains dozens of genes that are likely to be nonfunctioning(26) however these apparently nonfunctioning genes could
possibly function under heterozygous conditions because thenormal alleles could have failed to be sequenced by chance
due to lower coverage of the sequences of their positions. Our

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ASAKAWA ET AL.

results using RLGS indicate that the number of new
mutational events (deletions) induced by an acute exposureof 1 Gy (which is rare, even assuming accidental exposure
conditions) is probably 1 or fewer per genome. Since thehuman genome already contains many CNVs, adding one
additional deletion to the genome per se may not be seemcritically detrimental. Rather, the nature of specific genes
involved in a deletion may be more important than the fact ofa deletion. In this context, it is important to know the
proportion of genes with and without health effects underhemizygous conditions (i.e., deletion heterozygotes). The
former are represented as mutations at haplo-insufficient
genes and the latter as haplo-sufficient genes. In addition,
recent genome sequencing studies indicated that an ordinary
individual carries 250–300 nonfunctioning alleles and 50–100
variations that are possibly disease related (27). Some studies
even indicated that each genome contained 30 to over 100
genes that appeared as mutation homozygotes (loss-offunction state) (e.g., 28, 29). Through such genome
sequencing studies of clinically normal individuals, it isexpected that a list of haplo-sufficient genes would increase
rapidly in the near future, while a list of potentially haploinsufficient genes would require more effort and time. The
proportion of the latter will be helpful for further evaluation ofgenetic risks of radiation.
In conclusion, the time has come to recognize the fact thatthe human genome has already accumulated a large numberof mutations of different kinds. Therefore, risk evaluationneeds to incorporate this important information (as abaseline) so that additional genetic risks from radiation
may be put into perspective, and thereby better understoodby the public.
ACKNOWLEDGMENT
We thank Y. Nakamoto, S. Kaneoka, T. Tsuji, A. Miura, S. Mishima,
K. Naito, J. Kaneko, M. Imanaka and H. Haba for their technicalassistance, and Dr. L. Kapp for his careful reading of the manuscript. We
thank Dr. T. Shiroishi at the National Institute of Genetics for providing
the JF1 mice. The Radiation Effects Research Foundation (RERF),
Hiroshima and Nagasaki, Japan is a private, nonprofit foundation fundedby the Japanese Ministry of Health, Labour and Welfare and U.S.
Department of Energy (DOE), the latter in part through DOE Award DEHS0000031 to the National Academy of Sciences. This publication was
supported by RERF Research Protocols RP 7–85 and RP 2–07. A part ofthis work was supported by MEXT KAKENHI 20312033 to JA. The
views of the authors do not necessarily reflect those of the two
governments.
Received: June 7, 2012; accepted: October 9, 2012; published online:
January 31, 2013

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