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Genomics 81 (2003) 78 – 84

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Evolution of the cystatin B gene: implications for the origin of its
variable dodecamer tandem repeat in humans夞
Motoki Osawa,a,* Mika Kaneko,a Hidekazu Horiuchi,a Takashi Kitano,b Yoshi Kawamoto,cNaruya Saitou,b and Kazuo UmetsuaaDepartment of Forensic Medicine, Yamagata University School of Medicine, Yamagata 990-9585, Japanb
Laboratory of Evolutionary Genetics, National Institute of Genetics, Mishima 411-8540, Japanc
Laboratory Primate Center, Primate Research Institute, Kyoto University, Inuyama 484-8506, Japan
Received 28 May 2002; accepted 21 October 2002

Abstract
The human cystatin B gene contains a variable number of 12-bp tandem repeats in its promoter region, of which the common alleles
contain two or three copies and unusual expansion causes progressive myoclonus epilepsy of the Unverricht–Lundborg type. We undertook
a comprehensive analysis of the genomic sequence to address the evolutionary events of this variable repeat. By examination of a contiguous
genome sequence spanning 5.0 kb and linkage analysis of detected polymorphic changes, we identified six major intragenic haplotypes in
unrelated Japanese subjects. The number of normal repeats was closely correlated with these alleles, indicating that changes in the arrayshould be comparatively rare events during human evolution. To examine the origin of the repeat array further, we also analyzed five primate
genomes. Repetitive polymorphism was unlikely in hominoids, and the array originated with the dodecamer itself in the course of primate
evolution. The variability conceivably developed after the separation to humans.
© 2003 Elsevier Science (USA). All rights reserved.
Keywords: Cystatin B; Primates; VNTR; Molecular evolution

Introduction
Cystatin B (CSTB), also called stefin B, is a 98-aminoacid single polypeptide that is a member of the cystatinsuperfamily, which includes fetuin and kininogen [1].
CSTB is a cytoplasmic and nuclear component with ubiquitous expression [2]. Cystatins are tightly binding reversible inhibitors of cathepsin family cysteine proteinases, inwhich their interacting sites are located in the amino-terminal half of the molecule [3]. Its role is thought to be that ofan inhibitor acting against proteinases leaking from lysosomes.
Progressive myoclonus epilepsy (EPM1, Unverricht–

夞 Sequence data from this article have been deposited with the DDBJ/
EMBL/GenBank Data Libraries under Accession Nos. AB083085 to
AB083089, AB083416, and AB083417.
* Corresponding author. Fax: ⫹81-23-628-5273.
E-mail address: mosawa@med.id.yamagata-u.ac.jp (M. Osawa).

Lundborg type) is an autosomal recessive disease characterized by stimulus-sensitive myoclonic seizures and slowly
progressive cerebellar ataxia [4]. Linkage analysis and disequilibrium studies mapped the gene responsible for thisdisease to chromosome 21q22.3 [5,6], and by Northern blot
analysis Pennacchio and colleagues [7] showed reduced
levels of CSTB mRNA in affected individuals. Missense
mutations initially appeared to account for the disease, but
further studies showed that the most common disorders
correlate with unstable insertions of approximately 600 to
900 bp in the promoter region of CSTB. These insertions
were found to consist of large repetitive expansions of a
GC-rich dodecamer unit, the motif CCCCGCCCCGCG [8].
An extensive repeat formation of more than 30 copies remarkably reduced the transcription rate of the gene [9].
Mice lacking CSTB by means of targeted gene disruption
developed ataxia and myoclonic seizures similar to thesymptoms observed for EPM1 patients [10]. Pathologicalfeatures of these mice displayed degradation of cerebellar

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M. Osawa et al. / Genomics 81 (2003) 78 – 84

granule cells, in which characteristic signs of apoptosis,
such as condensed nuclei and fragmented DNA, were evident.
In unaffected individuals, Lalioti et al. [11] identified thedodecamer tandem repeat as a polymorphism, of which
normal alleles contained two or three copies. The frequencyof the triple-repeat allele was 0.66 in the normal Caucasian
population. Polymorphic microsatellites consisting of simple units of less than 5 bp are widely dispersed over the
mammalian genome. In contrast, minisatellite arrays, which
consist of 10- to 100-bp-long elementary units, are differentiated from microsatellites by several properties such as
cluster at the telomeric side [12]. Although it is classified asa minisatellite, this dodecamer repeat in CSTB is unique ina couple of respects. The normal repeat array is very shortcompared with that of minisatellites, for which the common
overall size ranges from 0.5 to 30 kb [13]. Second, minisatellites usually exhibit internal variation among constituent repeat units, but the repeats in the dodecamer arrayappear to be uniform [11]. Since this repeat array moreclosely resembles pathogenic microsatellites rather thantypical minisatellites, we prefer the term “variable numberof tandem repeats” (VNTR) for it. One approach to investigating the significance of VNTR is to assess its evolutionary origin and sequence divergence by comparing intra- andinterspecific sequence differences [14]. In the present study,
we performed intragenic haplotyping through an analysis of
the whole genomic sequence of CSTB to examine the relationship of the VNTR to human alleles. After comparing thehuman sequence with those of five related primates, we
speculated from an evolutionary perspective how VNTR
arose and developed at this locus.

Results
VNTR analysis
The variable dodecamer tandem repeat is located from175 to 210 bp upstream of the ATG translation initiation
codon. Amplification of the region using the DF1 and DR1primers yielded products of 176 and 188 bp (Fig. 1). For
heterozygotes, the smaller of the two bands became predominant, but heterozygosity was clearly evident, as judged
by the appearance of heteroduplex bands in the polyacrylamide gels. The study of 189 unrelated Japanese subjects
revealed complete consistency, including 15 individuals homozygous for the three-copy repeat, 73 heterozygotes, and
101 homozygotes for the two-copy repeat. Otherwise, noapparent variants were observed. Frequencies of the threeand two-copy alleles, as calculated from the genotype frequencies, were 0.27 and 0.73, respectively. This populationgroup is statistically in Hardy–Weinberg equilibrium withregard to the copy number of the CSTB VNTR (␹2 ⫽ 0.126,
P ⬍ 0.05).

79

Fig. 1. Genotyping of the CSTB tandem dodecamer repeat by PCR using
the primers DF1 and DR1. After nondenaturing electrophoresis in a 7.5%
polyacrylamide gel, amplified products were stained with SYBR gold.
Lanes: M, molecular markers; 1, the homozygous triple repeat in a humansubject; 2, the heterozygote (human); 3, the homozygous double repeat(human); 4, the corresponding region in chimpanzee; 5, gorilla; and 6,
bonobo.

Haplotype analysis
To find nucleotide changes in the human CSTB locus, wesequenced the gene, which spans 2.7 kb in length andcontains three small exons. The analyzed sequence was
extended to the 5⬘-flanking region because only a small
number of variations were observed in the initial region. A
full sequence of 5.0 kb was determined for a total of 15
subjects (30 chromosomes) chosen from individuals homozygous for either of the two VNTR types. In the target
region, 16 single-nucleotide variation sites were found,none of which is located inside an open reading frame or at
an intron splicing junction (Fig. 2). Differences encountered
more than twice were classified as nonunique polymorphic
changes, which accounted for 10 sites, including 4 transition-type substitutions and 6 transversions. The other 6changes were regarded as unique singletons that represented
a rarer single change. To type 8 of the 10 polymorphic sites,
we performed PCR-RFLP on the samples of 189 subjects
and examined their patterns of linkage disequilibrium (Table 1). Most of the polymorphic variations were in complete
linkage disequilibrium with one another. Variability of the
VNTR was found to be closely linked to these variations as
well.
From the results obtained, we verified that CSTB could
be subdivided into six distinct intragenic haplotypes anddesignated these alleles CSTB*1 to *6 (Table 2). Homozygotes for CSTB*2 to *6 alleles were involved in our samples, which assisted us in the assignment of haplotypes. The
genotypes of 182 subjects could be described as combinations of the six alleles. The genotypes of the remaining
seven likely consist of distinct minor alleles. When theallele frequencies were estimated, we found that the
CSTB*3 allele was predominant in the test group. A test of
the Hardy–Weinberg equilibrium for the six alleles from the80

M. Osawa et al. / Genomics 81 (2003) 78 – 84

The VNTR sequence in primate CSTB

Fig. 2. Schematic diagram of the human CSTB and locations of identified
nucleotide changes. The numbers on the right indicate nucleotide positionsof variations detected in an analysis of 30 chromosomes, in which polymorphic changes are italicized. The variable positions in the 5⬘-flanking
region are numbered by reference to the position of the ATG translationinitiating codon. The structure of human CSTB, which includes transposable elements, is shown in the middle.

examined samples showed that their frequencies do notsignificantly depart from the expected proportions (␹2 ⫽11.98, P ⬍ 0.05). The overall nucleotide diversity (␲)
across the entire gene was estimated to be 0.0007.
When the relationship between the CSTB alleles and the
VNTR was examined, the number of repeat units was
closely related to a given allele. CSTB*1 to *4 alleles have
only the two-copy array, and the triple repeat was observed
only in CSTB*5 and *6 alleles. In particular, more than 90%
of CSTB*6 alleles had the triple repeat. In a preliminary
experiment using Caucasian samples, CSTB*6 and its derivative alleles were predominant, which is associated withthe higher frequency of the triple repeat.

To compare the human DNA sequence with those of
closely related primates, we also determined the orthologous sequence for the chimpanzee, bonobo, gorilla, orangutan, and Japanese macaque genomes. As shown in Fig. 3,
the VNTR sequence of the chimpanzee, gorilla, and orangutan genomes consists of an incomplete three-time repeatthat includes a C to T substitution in the second repeat unit,
which was identical among the hominoids. While we examined nine distinct chimpanzee subjects (18 chromosomes), the sequence was consistent, indicating that therepeat region is likely monomorphic. Also of interest is thefact that the complete two-time array was observed to behomozygous in the bonobo genome (Fig. 1, lane 6). As only
one bonobo subject (two chromosomes) was available, we
do not know if intraspecific variation exists. In the macaque,the corresponding region showed a more incomplete threetime array, in which the third repetitive part was of theconsensus motif and a total of four nucleotide substitutions
and an indel were observed in the first and middle repeats.
These organizations in the primate genomes presumably
indicate a stable ancestral precursor state at this locus.
Evolutionary relationshipIn the phylogenetic network (Fig. 4), constructed for thehuman CSTB alleles, we observed complex branching in theevolution from the chimpanzee. It includes possible parallel
substitutions or recombination events at this locus. The
primate CSTB sequences indicate which of the nucleotides
segregating in the human alleles is ancestral, as indicated inTable 2. Judging from conserved nucleotides at the corresponding positions, CSTB*1 allele appears to be more ancestral than the others in the course of human evolution.
When the full CSTB sequences of the four primate genomes are compared with the human sequence, those of thechimpanzee, bonobo, gorilla, and orangutan differ by 1.3,
1.2, 2.1, and 4.0%, respectively, from the human sequence.
In the open reading frame, one or two substitutions areevident among the human and four primates, one of which
is nonsynonymous at a position corresponding to aminoacid residue 63 (Asp to Glu) only in the chimpanzee. The

Table 1
Pairwise linkage-disequilibrium between CSTB polymorphic sites

G⫺2371C
C⫺2265G
A⫺2203C
T⫺801CG⫺794A
VNTR
T155C
G1055T

T⫺2406C

G⫺2371C

C⫺2265G

A⫺2203C

T⫺801CG⫺794A

VNTR

T155C

1.00
1.00
⫺0.77
0.69
0.69
0.80
⫺1.00
0.97

1.00
⫺1.00
0.95
0.95
0.90
⫺1.00
1.00

1.00
⫺0.47
⫺0.47
⫺0.28
⫺1.00
1.00

⫺0.71
⫺0.71
0.67
⫺1.00
⫺0.96

1.00
0.98
1.00
0.95

0.98
1.00
0.95

⫺1.00
0.96

⫺1.00

M. Osawa et al. / Genomics 81 (2003) 78 – 84

81

Table 2
Human CSTB alleles and their frequency based on 10 SNPs, accompanied by the corresponding nucleotides in the chimpanzee gene
Position

*1

*2

*3

*4

*5

*6

Chimp

⫺2406
⫺2371
⫺2265
⫺2203
⫺801
⫺794
155
1032a
1040a
1055
No. (%)VNTR 3/2c

T
G
C
A
T
G
T
G
G
G
21 (5.8)
0/17

T
G
C
A
C
A
C
G
G
G
53 (14.6)
0/40

T
G
G
C
T
G
T
G
G
G
159 (43.7)
0/110

C
G
G
C
T
G
T
G
G
G
12 (3.3)
0/8

T
G
G
C
C
A
T
G
G
G
45 (12.4)
9/15

C
C
G
A
C
A
T
T
T
T
74 (20.3)
21/2

T
G
C
A
T
G
T
NDb
NDb
G

a
The nucleotides at positions 1032 and 1040 were estimated by direct sequencing of 30 DNA specimens in which all were completely linked to the
nucleotide at position 1055b
ND, not determined. Since the vicinities of these sites included indels, it was difficult to identify the corresponding nucleotide in the chimpanzee sequence.
c
The number in each allele was judged from 111 subjects homozygous for either repeat of the VNTR.

phylogenetic tree is constructed from the primate sequences, as shown in Fig. 3. The open reading frame of themacaque gene revealed that 14 nucleotide substitutions, ofwhich 5 are nonsynonymous changes at amino acid positions 9, 18, 71, 84, and 96, had occurred.

Discussion
Trinucleotide repeat expansion has been identified as
causing a number of neurodegenerative disorders, including
Huntington disease and fragile X syndrome. EPM1 represents the first disorder attributed to the instability of asimple repeat unit—a dodecamer— other than a triplet,
which is often CAG or CGG [15]. Riggins et al. [16]reported that several genes expressed in the brain containGC-rich repeats upstream of the transcription start site. The
expansion of such repeats usually results in a reduction oftranscription. Normal and pathogenic ranges of repeat size,
and their allelic distributions in population groups, differ
among loci. The normal repeat number of 2 or 3 copies

observed in the CSTB locus is smaller than that for pathogenic triplets, which can range widely: for example, thenumber of repeats at the fragile X syndrome locus is 6 to
⬃40 copies [17]. The gene frequency of the triple repeat
accounted for 0.27 in a normal Asian group, as found in thisseries of experiments. Lalioti et al. [11] reported that thisfrequency in Africans and Caucasians is 0.53 and 0.66,
respectively. Being commonly variable, the number of repeats in the normal array appears to vary among ethnic
groups.
Analysis of a contiguous sequence of CSTB and of linkage of the observed variations comprehensively demonstrated the extent of allelic divergence and the relationshipof allelic variations with the copy number of the repeat. Six
distinct alleles, CSTB*1 to *6, were defined by intragenic
haplotyping of 10 closely linked polymorphic sites observed
over 5.0 kb, and we found a close relationship between
these alleles and the VNTR. The persistence of this association might indicate that changes in the repeat number arecomparatively rare events in human evolution. Moreover,when the human alleles are compared with that of theFig. 3. Multiple sequence alignment of the VNTR sequence and dendrogram of CSTB in humans and five related primates. Nucleotide substitutions areunderlined, and a gap relative to the human sequence is indicated by a dash. In the chimpanzee, another nucleotide change from G to A, indicated with R,
was observed in only one chromosome. The phylogenetic tree constructed by the neighbor-joining method is shown at left. The scale bar corresponds to 0.01
substitutions per site.

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M. Osawa et al. / Genomics 81 (2003) 78 – 84

Fig. 4. Estimated phylogenetic network for human CSTB. To simplify the
network, we constructed it from the six nonunique variable sites in the 5⬘
flanking region of 2.5 kb. The edge length corresponds to a nucleotide
difference that is denoted by the position number beside it. Grids indicate
possible parallel substitutions or recombination events. Circles indicate theobserved alleles whose designations are shown in Table 2. The filled-inblack and empty parts in the circles represent the proportions of triple anddouble repeats, respectively, of the alleles.

chimpanzee gene, CSTB*1 appears to be the most ancestralallele. The finding that this allele has only two copies in therepeat array is possibly interpreted to mean that the double
repeat is older in the human lineage. The increase in repeat
number to 3 may have separately occurred in CSTB*5 and*6 alleles with dispersion into populations.
The repeat segment of the chimpanzee gene was monomorphic in the search of nine subjects and that of themacaque sequence contained five changes that were likely
static as well. Although the smaller population size of hominoids may affect the result [18], these findings conclusively
suggest that the VNTR arose after the human– chimpanzee
split. Concerning the evolutionary origin of repetitive DNA,a stepwise mutation model has been proposed for microsatellites [19]. It is, however, unclear how the VNTR originated, or if variability was intrinsic to the 12-bp unit fromthe beginning. Since the basic sequence motif resides in thelast part of the segment in the macaque sequence, its simple
repeat structure should begin with the dodecamer itself inthe course of primate evolution. Deletion of the middle partfrom the incomplete triple repeat observed in the chimpanzee, gorilla, and orangutan genes, possibly by unequalcrossing over in meiosis, may have resulted in a set of two
motifs, which is strongly supported by the appearance of theperfect two-copy repeat in the bonobo sequence. While
microsatellite variability already existed in the early hominoid ancestor [20], the incidence of dodecamer repetitiveness is evolutionarily a recent event. Gray and Jeffreys [21]reported a similar human-specific polymorphism for theMS32 minisatellite. At the MS32 locus, an enlarged hypervariable array is observed in humans in contrast to theseveral stable copies observed in hominoids.
Lalioti et al. [11] identified alleles with 12 and 13 copiesof the dodecamer that were transmitted unstably to offspring, a situation called the “premutational state.” These
intermediate alleles are not associated with the clinicalsymptoms of EPM1. Expansion takes place over genera-tions to produce approximately 30 to 75 copies, with clinicalconsequences. The mechanism of array enlargement has notbeen elucidated, but it is likely due to strand slippage or
unequal crossing over [22]. Recent investigations of thesecondary substructure of the dodecamer DNA showed thatit can engage in tetraplex formation, with the production offolded hairpins, which causes hypervariability in tripletrepeat disease genes [23,24]. Errors should occur by unfolding of the hairpin structure during discontinuous DNA replication at the 3⬘ end. As shown by extensive studies, thenormal repeat number is seemingly restricted as a minimum
to 2 or 3 copies. We speculate that once the VNTR gained
additional repeat units, the allele became unstable beyond a
critical threshold due to the tendency toward tetraplex formation and was finally lost from the human DNA pool.
In conclusion, based on a comparative sequence analysisof the CSTB locus, we proposed a model to explain how
VNTR has evolved. Such analyses of the genome will
provide further insight into the evolutionary origins of humans and various diseases.

Materials and methods
Materials
Peripheral blood was obtained from 189 unrelated Japanese individuals. All volunteers gave informed and writtenconsent to this DNA analysis. DNA of one chimpanzee(Pan troglodytes), one bonobo (also called the pygmy chimpanzee, Pan paniscus), one orangutan (Pongo pygmaeus),and one Japanese macaque (Macaca fuscata) was obtained
from the Primate Research Institute, Kyoto University. The
DNA of one gorilla (Gorilla gorilla) was obtained from the
Ueno Zoo, Tokyo. The other eight DNA samples for chimpanzee were obtained from the Kumamoto Laboratory,
Sanwa Kagaku Kenkyusho, Kumamoto. Restriction enzymes were purchased from MBI Fermentas and Toyobo.
PCR amplification and tandem repeat genotyping
To obtain the region containing VNTR, we performed
PCR using the following oligonucleotides: DF1, CCCGGAAAGACGATACCAG (5⬘ to 3⬘ for all primers shown),
and DR1, GGCACTTTGGCTTCGGAG. Amplificationfrom 20 ng DNA was performed in a volume of 20 ␮l with
HotStar Taq polymerase (Qiagen) in a supplemented Q
solution mixture. The reaction proceeded in a GeneAmp
9700 cycler (PE Biosystems) at 95°C for 15 min for denaturation, followed by 35 rounds of temperature cycling, at
95°C for 20 s, 55°C for 20 s, and 72°C for 40 s. The
products were electrophoresed in a 7.5% nondenaturing
polyacrylamide gel, followed by staining with SYBR gold
(Molecular Probes).

M. Osawa et al. / Genomics 81 (2003) 78 – 84

Contiguous sequencing
Overlapping segments of CSTB were amplified using six
pairs of oligonucleotide primers designed according to thesequence (AF208234) published by Pennacchio et al. [7]: CGGTTCAAACTGTCTGCTGC and CACACAGTCCCTTTGGTTTAGG, GCAGGTGGTCCAGTCTAACAGAA andGAAGGCAGGGTTCTCTGGTG, GGCTCACACCTGTAATCCCAG and GGCTGGTATCGTCTTTCCG, DF1 and
CCTTATCACTGGGGACCTCTTG, CCGACTACAGTTTGGCCAAG and TCAAGCTGGGACCTCACCTAG, and
GGGAATCCAAGAAGCCACTG and CTGGAGGGGAAGGACCTG. This contiguous region spans a total of 5012 bp,
including exons, introns, and 5⬘ flanking region. After purification by means of a spin column (Qiagen), the PCR
products were labeled with a BigDye terminator cycle sequencing kit and then sequenced using an ABI Prism 310
autoanalyzer (PE Biosystems). A total of 15 DNA specimens were fully sequenced by this approach. The genomic
sequence was also determined for the chimpanzee, bonobo,
gorilla, orangutan, and Japanese macaque by this system.
Several additional primers were necessary for the analysisof the orangutan and macaque genes.
Detection of polymorphic substitutions
To detect polymorphic changes in the sequenced region, we employed the restriction fragment length polymorphism (RFLP) method. Nucleotide changes at nucleotide positions ⫺2406, ⫺2265, ⫺801, and 1055 weredetected by digestion of the PCR products with FokI,
HinfI, AluI, and HinfI, respectively, followed by polyacrylamide gel electrophoresis. To examine four otherspecific changes at nucleotide positions ⫺2371, ⫺2203,⫺794, and 155, we subsequently performed nested PCRusing the restriction site-incorporated oligonucleotide
primers GTAAAGCAAGTCCTGAATAGA, TGTACGGGTCCTGCCGCT, GAGGCGGAGCTTGCGG, andCAGGGGTGCGCAGCGTGG, respectively, in combination with the appropriate opposite-strand primer, as described above. The products were then digested by MboI,BseLI, HphI, and BalI, respectively. Since changes atnucleotide positions 1032 and 1040 were not detectableby the RFLP method, these were determined by direct
DNA sequencing of up to 30 DNA specimens. Haplotypes were constructed from nucleotide changes identified among the samples as previously described [25].
Statistical and phylogenetic analysis
Hardy–Weinberg tests were performed on the observed VNTR and allele frequencies with significancebased on a standard observed– expected ␹2 with df ⫽ 1
and 25, respectively. Pairwise linkage disequilibrium(D⬘) was estimated according to the maximum-likelihood
procedure of Thompson et al. [26], in which D⬘ was83

calculated by D/Dmax or D/Dmin. Nucleotide diversities(␲) were estimated by the method of Nei [27], available
at http://www.bio.ub.es/⬃julio/DnaSP.html. A phylogenetic tree for primate CSTB was constructed by the neighbor-joining method using the orangutan sequence as an
outgroup [28]. A phylogenetic network of human CSTB
alleles was constructed by the procedure of Saitou andYamamoto [29]. Because of the complexity of the network constructed from all 10 substitution events, theanalysis was simplified by using only changes in the 5⬘
flanking region of 2505 bp in this study.

References[1] V. Turk, W. Bode, The cystatins: protein inhibitors of cysteine proteinases, FEBS Lett. 285 (1991) 213–219.
[2] M. Riccio, Nuclear localization of cystatin B, the cathepsin inhibitor
implicated in myoclonus epilepsy (EPM1), Exp. Cell Res. 262 (2001)
84 –94.
[3] M.T. Stubbs, The refined 2.4 A X-ray crystal structure of recombinanthuman stefin B in complex with the cysteine proteinase papain: anovel type of proteinase inhibitor interaction, EMBO J. 9 (1990)
1939 –1947.
[4] R. Norio, M. Koskiniemi, Progressive myoclonus epilepsy: genetic
and nosological aspects with special reference to 107 Finnish patients,
Clin. Genet. 15 (1979) 382–398.
[5] A.E. Lehesjoki, Localization of the EPM1 gene for progressive myoclonus epilepsy on chromosome 21: linkage disequilibrium allows
high resolution mapping, Hum. Mol. Genet. 2 (1993) 1229 –1234.
[6] R.G. Lafrenière, P.J. de Jong, G.A. Rouleau, A 405-kb cosmid contig
and HindIII restriction map of the progressive myoclonus epilepsy
type 1 (EPM1) candidate region in 21q22.3, Genomics 29 (1995)
288 –290.
[7] L.A. Pennacchio, Mutations in the gene encoding cystatin B in progressive myoclonus epilepsy (EPM1), Science 271 (1996) 1731–
1734.
[8] M.D. Lalioti, Identification of mutations in cystatin B, the gene
responsible for the Unverricht–Lundborg type of progressive myoclonus epilepsy (EPM1), Am. J. Hum. Genet. 60 (1997) 342–351.
[9] M.D. Lalioti, H.S. Scott, S.E. Antonarakis, Altered spacing of promoter elements due to the dodecamer repeat expansion contributes toreduced expression of the cystatin B gene in EPM1, Hum. Mol.
Genet. 8 (1999) 1791–1798.
[10] L.A. Pennacchio, Progressive ataxia, myoclonic epilepsy and cerebellar apoptosis in cystatin B-deficient mice, Nat. Genet. 20 (1998)
251–258.
[11] M.D. Lalioti, Dodecamer repeat expansion in cystatin B gene inprogressive myoclonus epilepsy, Nature 386 (1997) 847– 851.
[12] G. Vergnaud, The use of synthetic tandem repeats to isolate new
VNTR loci: cloning of a human hypermutable sequence, Genomics
11 (1991) 135–144.
[13] A.J. Jeffreys, R. Neumann, V. Wilson, Repeat unit sequence variationin minisatellites: a novel source of DNA polymorphism for studying
variation and mutation by single molecule analysis, Cell 60 (1990)
473– 485.
[14] A. Fujiyama, Construction and analysis of a human– chimpanzee
comparative clone map, Science 295 (2002) 131–134.
[15] C.T. Ashley, S.T. Warren, Trinucleotide repeat expansion and humandisease, Annu. Rev. Genet. 29 (1995) 703–728.
[16] G.J. Riggins, Human genes containing polymorphic trinucleotide
repeats, Nat. Genet. 2 (1992) 186 –191.

84

M. Osawa et al. / Genomics 81 (2003) 78 – 84

[17] K. Snow, Analysis of a CGG sequence at the FMR-1 locus in fragile
X families and in the general population, Am. J. Hum. Genet. 53(1993) 1217–1228.
[18] D.C. Rubinsztein, Microsatellite evolution— evidence for directionality
and variation in rate between species, Nat. Genet. 10 (1995) 337–343.
[19] A.M. Valdes, M. Slatkin, N.B. Freimer, Allele frequencies at microsatellite loci: the stepwise mutation model revisited, Genetics 133(1993) 737–749.
[20] E.E. Eichler, Evolution of the cryptic FMR1 CGG repeat, Nat. Genet.
11 (1995) 301–308.
[21] I.C. Gray, A.J. Jeffreys, Evolutionary transience of hypervariable
minisatellites in man and the primates, Proc. R. Soc. London B 243(1991) 241–253.
[22] B. Charlesworth, P. Sniegowski, W. Stephan, The evolutionary dynamics of repetitive DNA in eukaryotes, Nature 371 (1994) 215–220.
[23] T. Saha, K. Usdin, Tetraplex formation by the progressive myoclonus
epilepsy type-1 repeat: implications for instability in the repeat expansion diseases, FEBS Lett. 491 (2001) 184 –187.

[24] P.V. Jithesh, P. Singh, R. Joshi, Molecular dynamics studies oftrinucleotide repeat DNA involved in neurodegenerative disorders,J. Biomol. Struct. Dyn. 19 (2001) 479 – 495.
[25] M. Osawa, Haplotype analysis of the human ␣2-HS glycoprotein(fetuin) gene, Ann. Hum. Genet. 65 (2001) 27–34.
[26] E.A. Thompson, S. Deeb, D. Walker, A.G. Motulsky, The detectionof linkage disequilibrium between closely linked markers: RFLPs at
the AI-CIII apolipoprotein genes, Am. J. Hum. Genet. 42 (1988)
113–124.
[27] M. Nei, Molecular Evolutionary Genetics (1987, Columbia Univ.
Press, New York.
[28] N. Saitou, M. Nei, The neighbor-joining method: a new method forreconstructing phylogenetic trees, Mol. Biol. Evol. 4 (1987) 406 –425.
[29] N. Saitou, F. Yamamoto, Evolution of primate ABO blood group
genes and their homologous genes, Mol. Biol. Evol. 14 (1997) 399 –411.