J Mol Evol (2004) 59:329–339
DOI: 10.1007/s00239-004-2628-5

Comparative Genetics of Functional Trinucleotide Tandem Repeatsin Humans and Apes
Aida M. Andrés,1 Marta Soldevila,1 Oscar Lao,1 Vı́ctor Volpini,2 Naruya Saitou,3 Howard T Jacobs,4Ikuo Hayasaka,5 Francesc Calafell,1 Jaume Bertranpetit1
1

Unitat de Biologia Evolutiva, Facultat de Ciències de la Salut i de la Vida, Universitat Pompeu Fabra, Barcelona, Spain
Molecular Genetics Department, Cancer Research Institute Barcelona, Spain
3
Division of Population Genetics, National Institute of Genetics, Mishima, Japan
4
Institute of Medical Technology and Tampere University Hospital, University of Tampere, Tampere, Finland
5
Sanwa Kagaku, Kenkyusho Kumamoto Primate Park, Misumi, Japan
2

Received: 28 November 2003 / Accepted: 21 January 2004

Abstract. Several human neurodegenerative disorders are caused by the expansion of polymorphic
trinucleotide repeat regions. Many of these loci arefunctional short tandem repeats (STRs) located inbrain-expressed genes, and their study is thus relevantfrom both a medical and an evolutionary point ofview. The aims of our study are to infer the comparative pattern of variation and evolution of this setof loci in order to show species-speciﬁc features inthis group of STRs and on their potential for expansion (therefore, an insight into evolutionary
medicine) and to unravel whether any human-speciﬁcfeature may be identiﬁed in brain-expressed genes
involved in human disease. We analyzed the variability of the normal range of seven expanding STR
CAG/CTG loci (SCA1, SCA2, SCA3-MJD, SCA6,
SCA8, SCA12, and DRPLA) and two nonexpanding
polymorphic CAG loci (KCNN3 and NCOA3) inhumans, chimpanzees, gorillas, and orangutans. The
study showed a general conservation of the repetitive
tract and of the polymorphism in the four species andhigh heterogeneity among loci distributions. Humans
present slightly larger alleles than the rest of species
but a more relevant diﬀerence appears in variability
levels: Humans are the species with the largest vari-

Correspondence to: Dr. Jaume Bertranpetit; email: jaume.bertranpetit@upf.edu

ance, although only for the expanding loci, suggestinga relationship between variability levels and expansion potential. The sequence analysis shows highlevels of sequence conservation among species, a lackof correspondence between interruption patterns andvariability levels, and signs of conservative selective
pressure for some of the STR loci. Only two loci
(SCA1 and SCA8) show a human speciﬁc distribution, with larger alleles than the rest of species. This
could account, at the same time, for a human-speciﬁc
trait and a predisposition to disease through expansion.
Key words: Spinocerebellar ataxia — Trinucleotide
repeat expansion — Short tandem repeat evolution— Primates

Introduction
The ﬁeld of comparative genetics has undergone a
recent boom due to its power as a tool for the understanding of the evolutionary and functional factors shaping a given genome region and for the searchof the genetic basis of species uniqueness. Despite thelarge eﬀort made to obtain comparative genetic information, little is still known about similarities anddiﬀerences between the human genome and that of

330
Table 1. Genetic characteristics of the nine loci examined in this study
Locus

MIM

Chromosome

RepeatRegionExpansion

SCA1
SCA2
SCA3
SCA6
SCA8
SCA12
DRPLA
KCNN3
NCOA3

601556601517607047601011603680604326125370602983601937

6p23
12q24
14q24-q32
19p13
13q21
5q31-33
12p13
20q12
1q21

CAG
CAG
CAG
CAG
CTG
CAG
CAG
CAG
CAG

CodingCodingCodingCoding
Untranslated
5¢ UTR
CodingCodingCoding

Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No

Note. Chromosome, gene chromosomal localization; repeat, repeat unit; region, location of the repetitive region within the gene; expansion,
expanding (yes) or nonexpanding (no) loci.

our closest phylogenetic relatives, the apes. In recent
years, several human-speciﬁc genetic traits have been
detected by a comparative analysis of human andprimate species, mostly from the analysis of genetic
regions in chimpanzees (for reviews, see Gagneux and
Varki 2001; Hacia 2001). Comparative information
has mainly been obtained for noncoding regions, andsuch data have been the key to the understanding ofhominoid phylogeny (Chen and Li 2001; see Ruvolo
1997 for a previous review). Comparative analysesof functional regions usually aim to understand thefunctional constraints on a particular genetic region,
including purifying, positive, and balancing selection.
In some cases, analysis may help to explain the appearance of a new genetic variant in a particularspecies, such as lysozyme enzymes in primates(Messier and Stewart 1997) or in the FOXP2 gene inhumans (Lai et al. 2001; Enard et al. 2002).
We focused our study on the comparative analysis
of a special group of genetic elements: functional
CAG/CTG repetitive tracts. These are functional
CAG/CTG short tandem repeats (STRs), mostly
coding for polyglutamine tracts; they are found ingenes that are highly expressed in the brain and their
expansion in repeat number causes neurodegenerative disorders. Many of the genes in this study cause
spinocerebellar ataxia (SCA) and are named, accordingly, SCAn loci, where n denotes a locus deﬁning number.
These type of loci share the mutation dynamics ofthe rest of STRs of the genome, that is, they mutate
by adding or subtracting one (or rarely more thanone) repeat unit, but diﬀer by expanding into abnormally long alleles that produce ataxia, dystrophy
or similar diseases. This group of genetic diseases has
not been detected in nonhuman species, a fact that
could just be attributed to the greater knowledge ofhuman disease but might also reﬂect a unique humanpathogenic trend. Beyond the mere description, thecomparison of the patterns of variability in the normal range of humans and apes can allow the testingof hypotheses on the causes of expansion and disease.
This study can therefore be viewed in the context of

evolutionary medicine, where the comprehension of
the natural history of a disease may lie in the particular characteristics (either of the locus or of thespecies) of disease predisposition.
The variability of expanding loci has been studiedin humans in order to determine population-speciﬁc
disease risk factors and to understand the evolutionary forces shaping this variability (Watkins et al.
1995; Jodice et al. 1997; Andrés et al. 2003). On theother hand, interspeciﬁc comparative studies on theseloci have focused on allele length comparison between humans and other primate species and havedealt mostly with a few species used as references in asingle locus approach. The special interest in allele
length diﬀerences among species is based on the observation that long alleles have an increased mutation
rate and higher probability of very long leaps (Webster et al. 2002) and the possibility of expansion into
the pathogenic range (as shown [Fu et al. 1991; Nolinet al. 2003] for fragile X).
We have analyzed, in four species (human, chimpanzee, gorilla, and orangutan), nine STR loci including those present on the SCA1 (spinocerebellar
ataxia 1 locus), SCA2, SCA3 (or Machado–Joseph
disease locus), SCA6, SCA8, SCA12, DRPLA (dentatorubral–pallidoluysian atrophy), KCNN3 (potassium intermediate/small conductance calciumactivated channel, subfamily N, member 3), andNCOA3 (nuclear receptor coactivator 3) genes (Table 1). They all are functional, have diﬀerent genomic
locations and functions, and share a high central
nervous system expression and the presence of a CAG/
CTG repetitive tract. Seven are expanding disease-related loci, while the remaining two are coding but donot seem to expand into pathogenic alleles. Variation
at KCNN3, other than expansion, has been proposed
as being associated with mental diseases and ataxia(Dror et al. 1999; Figueroa et al. 2001), although as apredisposing factor rather than as a single direct cause.
A strong correlation between expansion and variability in the normal range has been demonstrated by
the observation that expanding STRs are the mostvariable group of STRs of the human genome

331(Chakraborty et al. 1997; Jodice et al. 1997; Dekaet al. 1999). As these expansions have not been detected in apes, our aim is to determine whether theseloci show similar levels of variability in apes to thoseobserved in humans (and therefore diversity and expansion potential would not be directly related) orwhether the high levels of polymorphism are exclusive to the species for which the expanding disease has
been detected.
We have also determined whether shared factors
among loci (such as the existence of a coding poly[CAG] tract) or locus-speciﬁc ones (such as diﬀerential
mutation patterns or selective events) led to the diversity of observed allele distribution of expanding loci.
Ascertainment bias may be a relevant problem
when trying to infer general patterns from a group ofloci selected in one of the species or populations
compared. Nevertheless, this problem does not aﬀect
our study, as we are interested in determining theevolution of this speciﬁc group of loci (where theobserved tendencies are clear in terms of expansion
and disease) but do not try to generalize our observations to infer traits for the rest of STRs, which
would produce a strong ascertainment bias.

Materials and Methods
Allele Typing
Twenty common chimpanzees (Pan troglodytes subspecies troglodytes [one individual] and verus), 13 gorillas (Gorilla gorilla subspecies gorilla), and 4 to 6 orangutans (Pongo pygmaeus subspecies
abelii [two individuals] or unknown) were typed for the number of
repeats at the nine loci shown in Table 1. Data for SCA1 and SCA3
in chimpanzees (16 individuals) were obtained from the literature(Limprasert et al. 1996, 1997). In order to avoid sampling a single
primate population, we obtained the samples from very diﬀerent
sources: Coriell Cell Repositories (USA), European Collection Cell
Cultures (UK), Barcelona Zoo (Spain), Kumamoto Primate Park
(Japan), Institute of Zoology, London (UK), and Dr. Takafumi
Ishida, Tokyo (Japan). Although it is not possible to assume that
this sample set is representative of genetic variation of all apes, care
was taken to try to obtain a diverse sample set, whose heterogeneous origin reduces the limitations of studying a small population
sample. Subspecies of ape specimens were determined by ampliﬁcation by PCR of both hypervariable regions of the mtDNA
D-loop region and direct sequencing with internal primers. Subspecies identiﬁcation may not be crucial for our study, however, as
variation at nuclear loci does not cluster by subspecies, at least for
chimpanzees (Kaessmann et al. 1999).
All DNA samples were ampliﬁed for the region containing therepetitive element by PCR, with primers and conditions previously
described for human analysis (Table A1). In the KCNN3 gene,which contains two CAG repetitive segments, the most variable
repetitive tract (nucleotides 513 to 569 in NCB1 Reference Sequence NM_002249.3) was ampliﬁed in a fragment that did notinclude the other repetitive tract. Lengths of the ampliﬁed fragments were subsequently typed with GeneScan software version 3.7(Applied Biosystems) after electrophoresis on 6% denaturing gel
performed on an ABI Prism377 automatic sequencer (Applied Biosystems). A human sample of known genotype, previously se-quenced and typed, was used as a standard size control for all
GeneScan runs.
At least one individual per species and locus was directly sequenced to verify the correspondence between ampliﬁed fragment
length and number of repeats. Sequences were determined using
BigDyes sequencing kits, versions 2.0 and 3.0 (Applied Biosystems),
on automatic sequencers ABIPrism 377 and 3100 (Applied Biosystems). The only exception is DRPLA, for which ape sequencesfrom GenBank were used (accession number AJ133270 [Pan paniscus], AJ133271 [Gorilla gorilla], AJ133272 [Pongo pygmaeus]).
Sequences were assembled and analyzed with the SeqmanII program (Lasergene 1999 package; DNASTAR, Inc.). Homology between human and mouse sequences was obtained by BLAST,
additional alignment of sequences (Seqman II and Clustal programs), and manual determination of the conserved regions.

Allele Frequencies and Statistical Analysis
The correspondence between allele size and number of repeats forevery species allowed the estimation of allele frequencies by direct
allele counting. Human distributions for healthy individuals result
from pooling African, European, Indian, and East Asian samples for
all loci; the distributions were obtained from the literature or thetyping of healthy individuals when necessary (Andrés et al. 2003). As
they come from normal individuals, we do not expect to ﬁnd a signiﬁcant proportion of premutated alleles (those with an intermediate
allele length, i.e., between normal and pathogenic, and which havehigh expansion probability). Allele frequencies were averaged acrosspopulations without weighing by sample size, as the number ofchromosomes varied greatly among populations. All subsequent
analyses were performed with the pooled human distribution.
The diﬀerent parameters of allele size distributions for every
locus and species were determined. Mean, standard deviation,
variance, and variation coeﬃcient of repeat number were calculatedwith the SPSS statistical package. Expected heterozygosity wascalculated with the Arlequin 2.000 package (Schneider et al. 2000).
In order to analyze the possibility that variance was inﬂuenced by
diﬀerences in sample size, we computed it on an increasing sample
size, for every locus and species, with a program that takes apseudosample of 8–40 chromosomes (the range in size between oursmallest and our largest ape samples) from the original distribution
and calculates its variance. After 1000 random extractions for each
pseudosample size, the average variance for every pseudosample
size is plotted in a graph that shows how variance relates to
pseudosample size.
To determine whether variances observed in species with asmaller sample size could be obtained from a hypothetical humansample of eight chromosomes (our smallest sample size, that oforangutans), we performed a second resampling experiment. In thiscase, a pseudosample of eight chromosomes was obtained for every
human distribution and its variance was calculated; after 10,000
sample extractions, we obtained a distribution of variances frompseudosamples of eight human chromosomes. To determine thesigniﬁcance of our results, the variance was compared to the 95%
conﬁdence interval of the pseudosample variance distribution.
The four species were compared in terms of mean and varianceof repeat number. Two tests were performed for mean repeat
number with permutation tests: the comparison between the four
species for every individual locus (considering every species as adiﬀerent category) and the comparison between humans and therest of the species for every locus (considering ‘‘apes’’ a category,
which included all ape species, and comparing it with the ‘‘human’’category). The permutation test was performed as follows: Individual chromosomes were randomly shuﬄed between classes(species or species groups), maintaining the original sample sizes.
For every permutated data set, the diﬀerence in the average numberof repeats between the two classes was computed. This process was332

Fig. 1. Graphical representations of individual allele size distributions and average length and variability parameters for species
and loci. 1.1–1.9 Allele length distributions in the four species. The
distributions are the result of typing, for repeat number, chimpanzee (in red), gorilla (blue), and orangutan (yellow) chromo-

somes, and pooling human distributions from African, European,
Indian, and East Asian origin (black). 1.10 Mean allele size for
species and locus. 1.11 Variance of repeat number for species andlocus.

333
repeated 1000 times. The test is signiﬁcant if the probability ofobtaining a diﬀerence in average repeat number in the permutations as large as in the observed data set, in a one-tailed test, is<0.05 for the human–ape comparison or <0.0056 (after Bonferroni correction) for the four-species comparison.
Variance comparison among the four species, for every locus,
was performed with Scheﬀé–Box (log ANOVA) test for homogeneity of variances (Sokal and Rohlf 1995, p. 397); as this test is notavailable in statistical packages, an ad hoc program has been
written and is available on request (oscar.lao@upf.edu). In order toobtain a single overall signiﬁcance value for all loci, the individual
Scheﬀé–Box p values for every locus were combined with Fisher’s
test for probability combination (Sokal and Rohlf 1995, p. 795).
When a species showed a single allele for a given locus the test was
performed using the rest of the species.

Results
Data on nine CAG repeat loci, whose characteristics
are shown in Table 1, were obtained for humans (ofAfrican, European, Indian, and East Asian origin), for
20 chimpanzees, 13 gorillas, and 4 to 6 orangutans; allchromosomes analyzed were in the ‘‘normal,’’ nonexpanded range. Allele size distributions are plotted bylocus in Fig. 1, and statistical parameters of the distributions and expected heterozygosity are shown in
Table 2. The ampliﬁed fragment was sequenced in atleast one individual for species and locus; sequences of
all repetitive regions are shown in Fig. 2, and their accession numbers are available in Table A2, including
compared mouse loci. Comparative locus analysis was
carried out on diﬀerent levels, searching for general
species trends and for locus-speciﬁc trends.
General species-speciﬁc trends can be detected bycomparing the parameters of the distribution forevery species over all loci (Table 3). Humans show ahigher mean number of repeats than the other species, reaching statistical signiﬁcance (permutation test
humans vs. apes, p < 0.001; see Materials and Methods for the groups considered), showing that atrend exists in allele length among the diﬀerent species. Nevertheless, the trend is not followed by all
loci, as discussed below.
Variance and coeﬃcient of variation of repeat
number show a decreasing trend from humans to orangutans, as shown in Table 3. Statistically signiﬁcantdiﬀerences in variance exist between species, with allindividual loci showing signiﬁcant diﬀerences among
species (p < 0.05) and the combined p value also being
statistically signiﬁcant. Interestingly, for the seven expanding loci humans presented the largest variance.
The small sample size in the ape species may bias
the estimation of the dispersion parameters. As statedin Materials and Methods, the samples for nonhuman species are of very heterogeneous origin, which
reduces the possibility of underestimating variance bysampling from a single, localized population or inbred zoo collection. Furthermore, to explore to what
extent human variance is larger than that of the rest

Table 2. Sample size, statistical parameters, and expected
heterozygosity for each species and locus
Locus

Species

Var.

CV

H

SCA1
SCA1
SCA1
SCA1

Human
1098 29.29
Chimpanzee
32 23.09
Gorilla22 20.68
Orangutan
10 23.50

2.55
1.91
0.57
1.35

6.52
3.64
0.32
1.83

8.72
8.32
2.78
5.91

0.850
0.764
0.541
0.644

SCA2
SCA2
SCA2
SCA2

Human
1214 22.15
Chimpanzee
40 25.40
Gorilla22 17.55
Orangutan
08 16.25

1.10
0.98
0.86
0.46

1.22
0.96
0.74
0.21

4.98
3.89
4.94
2.94

0.291
0.680
0.368
0.429

SCA3
SCA3
SCA3
SCA3

Human
1164 22.72
Chimpanzee
32 17.75
Gorilla26 10.50
Orangutan
12 24.33

5.68 32.29 25.01 0.890
1.88 3.55 10.70 0.716
0.95 0.90 9.12 0.459
0.49 0.24 2.07 0.485

SCA6
SCA6
SCA6
SCA6

Human
1130 11.76
Chimpanzee
40 11.13
Gorilla24 9.00
Orangutan
8 11.50

2.02
0.94
0.78
0.76

SCA8
SCA8
SCA8
SCA8

Human
1824 23.59
Chimpanzee
38 16.13
Gorilla26 18.15
Orangutan
08 7.00

3.51 12.29 14.87 0.876
2.26 5.09 14.08 0.848
2.43 5.90 13.50 0.812
0.00 0.00 0.00 0.000

SCA12
SCA12
SCA12
SCA12

Human
3336 12.27
Chimpanzee
40 11.33
Gorilla24 8.38
Orangutan
8 9.50

3.01
2.02
0.49
1.31

DRPLA
DRPLA
DRPLA
DRPLA

Human
Chimpanzee
GorillaOrangutan

809 14.07
40 14.25
26 9.00
8 15.00

3.94 15.53 28.02 0.907
1.50 2.24 10.58 0.754
0.00 0.00 0.00 0.000
0.00 0.00 0.00 0.000

KCNN3
KCNN3
KCNN3
KCNN3

Human
Chimpanzee
GorillaOrangutan

968
40
24
8

17.88
20.05
18.29
12.50

2.31 5.32 12.90
2.16 4.66 10.84
3.36 11.26 18.54
1.31 1.71 10.80

0.840
0.831
0.754
0.821

NCOA3
NCOA3
NCOA3
NCOA3

Human
Chimpanzee
GorillaOrangutan

880
40
26
8

28.50
26.00
22.69
20.88

0.90
2.47
1.09
0.83

0.540
0.732
0.148
0.750

N

Mean SD

4.07 17.17 0.786
0.88 8.49 0.724
0.61 8.76 0.685
0.57 6.78 0.607

9.08 24.56 0.781
4.07 17.93 0.735
0.24 5.97 0.489
1.71 14.21 0.750

0.80
6.10
1.18
0.70

3.15
9.56
4.84
4.12

Note. N, sample size; mean, mean number of repeats; SD, standard
deviation; var., variance; CV, coeﬃcient of variation; H, expected
heterozygosity. Human parameters were calculated from thepooled distribution (see Materials and Methods).

of the species as a consequence of its larger sample
size, we performed two independent tests.
First, we studied whether variance increased withsample size by a permutation test on pseudosamples
of 8–40 chromosomes for the four species in every
locus. Results showed that reduction of sample size
does not lead to a reduction of variance (data notshown) and that the resampling average value remains within the range of the values obtained fromthe original distribution. Therefore, as expected, areduced sample does not determine lower variance334

Fig. 2. Sequence of the repetitive
region for the expanding loci in thefour species. The line over thesequences marks the STR sequence
region, and the total length of theoverlined sequence region isshown. Repetitive segments areindicated without detailing theexact number of repeats, andsegments containing six or more
repeat units, with higher
probabilities of slippage events
than shorter segments, aremarked in bold face.

value. A second resampling test was performed, inwhich we tested whether variances similar to thoseobtained for apes could be obtained from pseusosamples of eight human chromosomes from our human distributions (see Materials and Methods). Of
the seven loci with larger variance in humans than in
the rest of the species, four loci (SCA3, SCA8,SCA12, and DRPLA) showed large and statisticallysigniﬁcant diﬀerences (p < 0.05) between humans
and orangutans (the species with the lowest samplesize), and two of them (SCA3 and DRPLA) showedsigniﬁcantly larger variances in humans than in any
other species. This analysis suggests that beyond
some sample size inﬂuence, our results cannot be
exclusively explained for diﬀerences in sample size,and variance divergence for this group of loci seems
to be a species characteristic.
Heterozygosity is high and very similar between
humans and chimpanzees and low in gorillas andorangutans (Table 3). This observation is due to the335
Table 3. Parameters of allele size distributions for species: Pooled
sample size, statistical parameters, and expected heterozygosity foreach species in the diﬀerent lociSpecies

N

Mean

SD

Var.

CV

H

Human
Chimpanzee
GorillaOrangutan

12423
342
220
78

20.25
18.35
14.92
15.61

2.78
1.79
1.17
0.72

9.68
3.47
2.35
0.78

15.49
10.49
7.60
5.20

0.751
0.754
0.473
0.498

Note. Data for all loci were pooled for each species. Abbreviations
are the same as those used in Table 2; see Note there.

large inﬂuence of SCA2 and NCOA3, with higher
heterozygosities in chimpanzees than in humans;
without these two loci, mean heterozygosity is higher
in humans than in chimpanzees.
In order to determine which loci are mostly responsible for the species trends, mean repeat number(Fig. 1.10) and variance of repeat number (Fig. 1.11)
were compared for each species and locus. Humans do
not always show larger allele sizes at all loci or at all
expanding loci, and only three (SCA1, SCA8, andNCOA3) show statistically signiﬁcant larger mean allele length in humans than in any other species. On theother hand, variance is higher in humans than in the restof species for all expanding loci but not for the twononexpanding loci. Nonetheless, the low number ofnonexpanding loci analyzed prevents us from generalizing this interesting diﬀerence in variance.
The individual comparison among loci distributions
shows a strong heterogeneity in allele size distribution(Fig. 1), and sequences of the STR alleles over the four
species (Fig. 2) illustrate that repeat regions are very
complex and that diﬀerent loci and lineages show heterogeneous amounts and patterns of divergence. The
sequence distribution comparison clearly shows a lackof general patterns (in STR sequence and interrupting
complexity) that could explain the observed allele sizeand variability of expanding loci.
Interestingly, when comparing human and mouse,
only very short tracts were detected for almost allmouse loci, from the complete absence of the tract (inSCA6, SCA8, and SCA12 the repetitive region could
not be detected) to very short CAG/CAA repetitive
regions (four CAG repeats in SCA1, two in SCA2,
ﬁve in SCA3, four in DRPLA, and one in NCOA3).
Only KCNN3 keeps the repetitive tract over phylogenetically distant species, as a repetitive CAG/CAA
region exists in the mouse sequence, with the interrupting codons CAA (Gln) and TCG (Ser), which areabsent in primates.
Discussion
Our study shows that the ‘‘ﬂexible conservation’’ thatexists in human functional CAG/CTG tracts (highpolymorphism in number of repeats combined withsequence conservation) is also found in apes. The
conservation of the variable tracts in all species
strongly supports the presence of the repeat regions in
the common ancestor of humans and apes in all nineloci studied, and the presence of polymorphism inalmost all species suggests the existence of ancestralpolymorphism.
Previous studies comparing human and mouse
CAG/CAA tracts found a relationship between interrupting levels and conservation between very distantspecies (Albà et al. 1999), suggesting that older tracts
would be more frequently interrupted by non-CAG
codons. We failed to ﬁnd this relationship in our set of
repetitive tracts, as the most conserved locus (KCNN3,
with a long repetitive region in both humans and mice)is totally uninterrupted in primates, and many tracts
showing low conservation between distant species areprofoundly interrupted in primates.
Species-Speciﬁc Characteristics
The combined analysis of all loci points to speciesspeciﬁc trends. In allele length comparison, larger alleles in humans than in other species were reported for
nonfunctional STRs (Rubinsztein et al. 1995a; Crouau-Roy et al. 1996; Cooper et al. 1998), a controversialconclusion (Ellegren et al. 1995, 1997). Previous studies
found longer alleles in humans than in other species for
SCA1, SCA3, AR, and HD CAG repeats and in the FA(GAA) locus (Rubinsztein et al. 1995b; Djian et al.
1996; Limprasert et al. 1997; Choong et al. 1998;
Gonzalez-Cabo et al. 1999; Justice et al. 2001). Thissuggests a general increase in the number of repeatsfrom monkeys to apes and humans for expanding loci(although similar allele length was found by Limprasert
et al. [1996] in the SCA3 locus). On the other hand, a
study on functional nonexpanding CAG STR shows
shorter alleles in humans than in apes (Saleem et al.
2001). Therefore, a clear picture of the human speciﬁccharacteristics in functional STRs had not emerged
beyond single locus comparisons.
The data presented in this paper for nine functional trinucleotides show signiﬁcantly higher numberof repeats in humans, a trend that is unique to thislineage and shows that, beyond locus heterogeneity, aspeciﬁcity in allele length exists in this set of STRs.
The trend is not present in all loci and is mainly (butnot exclusively) due to SCA1, SCA8, and NCOA3.
In our set of samples and loci, we found diﬀerencesin variance among the four species, with the highest
values in humans. In the genomic regions studied sofar, DNA sequence diversity is lower in humans thanin chimpanzees and other apes (Crouau-Roy et al.
1996; Kaessmann et al. 2001; Noda et al. 2001),
possibly due to a demographic bottleneck in the human lineage (Jorde et al. 2000). Surprisingly, thepresent results show that humans are more diverse

336(measured as variance of allele distribution) than anyof the other species studied for an ample set of expanding functional trinucleotide tandem repeats. No
demographic factor (which would aﬀect the wholegenome) or ascertainment bias eﬀect for the selectionof the STRs in humans could explain this ﬁnding,which is not a general STR trend, but it is exclusive offunctional STRs that can expand and produce disease. This is not found in noncoding STRs (andprevious results [Crouau-Roy et al. 1996; Garza et al.
1995; Wise et al. 1997] are not concordant) or in othersimilar STRs (such as the nonexpanding KCNN3 andNCOA3, analyzed in this study).
The possible sampling error in apes has been reduced by choosing individuals of diﬀerent origins asmuch as possible (see Materials and Methods), andthe eﬀects of diﬀerent sample size for the diﬀerent
species have been proven to be small through resampling procedures (see Results). Moreover, variability levels may be investigated to test whether our
primate samples present lower variability levels thanhumans at other genetic loci. Seventeen of the chimpanzees typed in this study were previously analyzed
for 16S rRNA (Noda et al. 2001); the subset of individuals analyzed in both studies show variability
levels (p = 0.0014 ± 0.0003) comparable with thoseexisting for the 16S rRNA of the whole human species (p = 0.0016 ± 0.0004) (Ingman et al. 2000).
These results show that a part of our sample ofchimpanzees is as variable as humans at a global
scale, and thus the expected diversity of chimpanzees
in CAG repeats should be higher than humans.
Therefore, our results are not the consequence of asmall sample size or the selected sample of individuals, and locus-speciﬁc factors (related to CAG loci)acting in diﬀerent ways in diﬀerent species are needed
to explain the observation that the loci that can expand in humans are more variable in humans than in
any other species.
Human expanding STRs have previously beenshown to be more variable than other di-, tri-, or
tetranucleotides in the human genome (Chakraborty
et al. 1997; Jodice et al. 1997; Deka et al. 1999),
suggesting a relationship among variability, expansion, and disease. Moreover, our results show thatthese loci are more variable in humans than in any
ape, suggesting that this pattern could be related to ahuman speciﬁcally capacity for expansion. The relationship suggests that loci with increased variance
may be more likely to expand to pathogenic alleles,
and thus lead to disease.
Diﬀerent scenarios would be compatible with theincrease in variance in one species: the ﬁrst is a highmutation rate in the absence of strong selective constraints, which would increase variability of the STR(Di Rienzo et al. 1998), leading to new alleles that, if
long enough, would increase slippage probability to

expanded alleles. Humans do not have mean larger
alleles, and therefore allele length diﬀerences do notseem to explain the observation. Nevertheless, weobserved a statistically signiﬁcant relationship between the longest allele and variance, even correcting
for inﬂuence of mean allele size (r = 0.6552, p <0.0005). The observed relationship between varianceand longest allele can be explained for the nature ofvariance calculation (as alleles far away from themean allele size will strongly inﬂuence variance).
Thus normal but long alleles might aﬀect the STR
dynamics: as mutation rate increases with allele size,
very long alleles can contribute to the high mutation
rate and high variability levels. But this factor, although important for the production of pathogenic
alleles, would have a minor eﬀect on the overallvariance, as they are rare in the population, and its
increased mutation rate will probably not be large
enough to explain the huge diﬀerences observed invariance for the whole distribution. When computing
variances without the very long alleles (eliminating
the ﬁve longer alleles in a range of less than 40 repeats), variance decreases only 20% on average, farfrom the diﬀerences with other species, showing thatvery long alleles are not the main responsible alleles
for the large human variances in expanding loci. No
other factors seem to exist to explain diﬀerences inmutation rates.
A second possibility, given the functionality of theloci, is the existence of some kind of balancing selection that, as in the case of other human genes like
HLA (Hedrick and Thomson 1983; Hughes and Nei
1988) or CCR5 (Bamshad et al. 2002), would maintainthe high diversity by favoring the existence of diﬀerent
alleles in the population. This is a truly speculative
explanation, as no external evidence exists for thepresence of balancing selection acting over these loci,but the fact that they are functional require consideration of a nonneutral explanation for our ﬁndings.
A detailed knowledge both of the mutation rate
and pattern for each locus and of the functional behavior of the diﬀerent alleles may be necessary tointerpret these ﬁndings and to infer whether mutationor selection are the main causes of the increase ofvariability in humans.

Locus-Speciﬁc Characteristics
Diﬀerences in a single functional locus among species
may inﬂuence its function, and these diﬀerences areespecially interesting as these genes are expressed inbrain and their variation outside the normal range
has pathogenic eﬀects. These characteristics (functionality and variability) make poly(CAG) regions
attractive candidates for having brain-related functions, especially in the human lineage, which shows

337

many brain-speciﬁc phenotypic traits compared to
the rest of species.
The analysis of each locus demonstrates the existence of a strong evolutionary heterogeneity, showingthat speciﬁc evolutionary forces have been acting oneach gene, governing its diversiﬁcation and evolution.
No important diﬀerences in allele length exist between
humans and apes, the only exception being SCA1 and
SCA8, the two candidate loci to present functional
diﬀerences between humans and the other species if thelength of the repetitive tract inﬂuenced protein function. This possibility has been suggested for SCA1(Yue et al. 2001) and SCA8 (see Andrés et al. 2003 for a
detailed explanation of SCA8 interspeciﬁc diﬀerences).
The variability levels are also highly heterogeneousamong loci, ranging from loci with similar diversity inall species (as in SCA6 and SCA12) to those with extreme diﬀerences (SCA3 and DRPLA).
As in allele length distributions, there is a strong
heterogeneity in the repetitive sequence conservation
among loci for the diﬀerent species: in contrast to thetotal conservation of the CAG tract in some loci
(SCA6, SCA12, and KCNN3), others show important sequence diﬀerences among species, both in thepresence of interruptions and in the composition of

the repetitive segment (as SCA2, SCA3, SCA8, or
DRPLA), which results in diﬀerences in repeat
number. Nevertheless, the complexity in sequenceinterruption patterns could not be related to theamount of allele size diversity; and any simple patternof STR mutation depending on the repetitive sequence that could be inferred from the analysis of one
functional STRs locus does not seem to apply toothers. Heterogeneity is the main rule.
The comparative study of seven expanding andtwo nonexpanding brain-expressed functional STRs
has shown the overall maintenance of polymorphismwith a higher mean in humans for some loci and alarger amount of variability in humans for expandingloci. Comparative studies focused on the search of
species speciﬁcities on functional regions often do notdeal with intraspeciﬁc variability, trying to ﬁnd out
ﬁxed changes among species. Here we suggest that
diﬀerences in genetic variability in important brainexpressed genes should also be considered. Species
variability levels on phenotypic traits can also be a
species-unique characteristic, and the study of diversity in speciﬁc genes with important gene function
may give a clue toward understanding the speciesspeciﬁc evolution of these genes.

Appendix
Table A1. Primer sequences used in this study
Locus

PrimerSequence

Ref

SCA1

rep1
rep2
sca1bR
sca1cF *sca1cR *
sca2a
sca2b
sca2bR *sca2cF *mjd52
mjd70 *
sca3bF *
s-5f1
s-5r1
sca6bF *sca6cR *sca8-f3 *sca8-r4 *sca12mF *sca12mR *b37rb33f *
drplacF *drplacR *kcnn3mF *kcnn3mR *ncoa3mF *ncoa3mR *AACTGGAAATGTGGACGTACGCATGCAACATGGGCAGTCTGAGCGTACTGGTTCTGCTGGG
CACCAGTGCAGTGGCCTCGCGGCCGGTGTTCTGCGGAG
GGGCCCCTAACCATGTCG
AGGGCTTGCGGACATTGG
CTTGCGGACATTGGCAGCCGCGAGCCGGTGTATGGGCC
CCAGTGACTACTTTGATTCG
CTTACCTAGATCACTCCCAA
ACAATGTATTTTCCTTATGAATAGCACGTGTCCTATTCCCCTGTGATCCTGGGTACCTCCGAGGGCCGCTGGTG
CCTGTGATCCGTAAGGCCGG
GCCTGGGACCCGCCTCTCC
TTTGAGAAAGGCTTGTGAGGACTGAGAATGGGTCCTTCATGTTAGAAAACCTGGCTCCACTGCAGCAAAGAGCAGCTGCAGTGGCGAGATGGCAGG
CACCAGTCTCAAACAATCACCATG
CCTCCAGTGGGTGGGGAAATGCTCCCACCACCTCCTCCCTATGGGGAGACATGGCGTAAGGGTGTG
TCGCTGCAGCCTCAGCCTCGCCAGGCCCACTTGTAGCTG
GAGAGCTGCTAAGTCATCAC
GTTGATATGGAAACTGTTGCG

Orr et al. 1993
Orr et al. 1993
primers designed for this study
primers designed for this study
primers designed for this study
Pulst et al. 1996
Pulst et al. 1996
primers designed for this study
primers designed for this study
Kawaguchi et al. 1994
Kawaguchi et al. 1994
primers designed for this study
Zhuchenko et al. 1997
Zhuchenko et al. 1997
primers designed for this study
primers designed for this study
Koob et al. 1999
Koob et al. 1999
Andrés et al. 2003
Andrés et al. 2003
Nagafuchi et al. 1994
Nagafuchi et al. 1994
primers designed for this study
primers designed for this study
Andrés et al. 2003
Andrés et al. 2003
Andrés et al. 2003
Andrés et al. 2003

SCA2

SCA3

SCA6

SCA8
SCA12
DRPLA

KCNN3
NCOA3

Note. *, primers used for sequencing. Original reference for each primer is given.

338
Table A2. New nucleotide data accession numbers
Locus

Chimpanzee

GorillaOrangutan

SCA1
SCA2
SCA3
SCA6
SCA8
SCA12
KCNN3
NCOA3

AY225351
AY225366
AY225369
AY225372
AY225354
AY225357
AY225360
AY225363

AY225352
AY225367
AY225370
AY225373
AY225355
AY225358
AY225361
AY225364

AY225353
AY225368
AY225371
AY225374
AY225356
AY225359
AY225362
AY225365

Note. Mouse accession numbers: Mouse SCA1[NM_009124], SCA2 [NM_009125], SCA3 [XM_127081], SCA6 [NM_007578], SCA8
[AF252281], DRPLA [NM_007881], KCNN3 [AF357241], NCOA3 [NM_008679].

Acknowledgments.
We thank Monica Vallés for her technicalsupport and Anna Pérez-Lezaun for her technical help and feedback. The authors would like to especially thank Arcadi Navarro
for reading the manuscript and for his help with statistical comparisons, Michael Greenacre for statistical help, Lynn B. Jorde,
Pascal Gagneux, and Mar Albà for reading an early version of themanuscript, and Lynda Vigilant, Pascal Gagneux, and Anne C.
Stone for helpful advice on primate subspecies determination.
Tanzanian human samples were kindly supplied by Dr. Clara
Menéndez from the Unitat d’Epidemiologia i Bioestadı́stica(Hospital Clı́nic, Barcelona). Primate samples were supplied by theBarcelona Zoo (under the agreement of the Primate DNA Bank
with the Pompeu Fabra University), Ikuo Hayasaka, and Dr.
Takafumi Ishida of the University of Tokyo (Japan) or obtained
from the Institute of Zoology (London), Coriell Cell Repositories(CCR), and European Collection of Cell Cultures (ECACC). This
study was supported by the Dirección General de Investigación(Spanish Goverment), Grants SAF 2001-0772 to J.B. and
BOS2001-0794 to F.C., and Grants-in-Aids to Priority Area from
MEXT, Japan, to N.S. A.M.A. was ﬁnancially supported by afellowship from Generalitat de Catalunya, 2000FI 00686.

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