2001 Oxford University Press

Human Molecular Genetics, 2001, Vol. 10, No. 21 24372446

CAG repeat instability at SCA2 locus: anchoring CAA

interruptions and linked single nucleotide
polymorphisms
Shweta Choudhry1, Mitali Mukerji1, Achal K. Srivastava2, Satish Jain2 and
Samir K. Brahmachari1,3,*
1Functional

Genomics Unit, Centre for Biochemical Technology (CSIR), Mall Road, Delhi, India, 2Department of
Neurology, Neurosciences Center, All India Institute of Medical Sciences, New Delhi, India and 3Molecular Biophysics
Unit, Indian Institute of Science, Bangalore, India

Received June 20, 2001; Revised and Accepted August 7, 2001

Spinocerebellar ataxia 2 (SCA2) is an autosomal

dominant neurodegenerative disorder that results
from the expansion of a cryptic CAG repeat within the
exon 1 of the SCA2 gene. The CAG repeat in normal
individuals varies in length from 14 to 31 repeats and
is frequently interrupted by one or more CAA triplets,
whereas the expanded alleles contain a pure uninterrupted stretch of 34 to 59 CAG repeats. We have
previously reported the presence of a limited pool of
ancestral or at risk haplotypes for the expanded
SCA2 alleles in the Indian population. We now report
the identification of two novel single nucleotide polymorphisms (SNPs) in exon 1 of the SCA2 gene and
their characterization in 215 normal and 64 expanded
chromosomes. The two biallelic SNPs distinguished
two haplotypes, GT and CC, each of which formed a
predominant haplotype associated with normal and
expanded SCA2 alleles. All the expanded alleles
segregated with CC haplotype, which otherwise was
associated with only 29.3% of the normal chromosomes. CAA interspersion analysis revealed that
majority of the normal alleles with CC haplotype were
either pure or lacked the most proximal 5 CAA interruption. The repeat length variation at SCA2 locus
also appeared to be polar with changes occurring
mostly at the 5 end of the repeat. Our results demonstrate that CAA interruptions play an important role in
conferring stability to SCA2 repeat and their absence
predisposes alleles towards instability and pathological expansion. Our study also provides new haplotypes associated with SCA2 that should prove useful
in further understanding the mutational history and
mechanism of repeat instability at the SCA2 locus.
INTRODUCTION
Spinocerebellar ataxias (SCAs) are a clinically heterogeneous
group of autosomal dominant neurodegenerative disorders

DDBJ/EMBL/GenBank accession nos AF330028AF330033

characterized by progressive deterioration in balance and coordination. The symptoms occur due to progressive neuronal loss
primarily in the cerebellum, but also in other parts of the
central nervous system. Eight disease loci have been identified
to date as causing this phenotype: spinocerebellar ataxia 1
(SCA1), SCA2, MachadoJoseph disease (MJD)/SCA3, SCA6,
SCA7, SCA8, SCA10 and SCA12 (110). The causative mutation associated with all these disease types is abnormal expansion of a trinucleotide repeat motif in their corresponding gene,
except for SCA10, which is due to a pentanucleotide (ATTCT)
repeat expansion. These repeats at different SCA loci are
highly polymorphic in normal individuals, but once the repeat
number crosses a particular threshold, specific to an individual
locus, instability is observed leading to the manifestation of the
disease (110).
SCA2, which was initially described in a Cuban population
(11), has now been reported worldwide and is a quite frequent
form of autosomal dominant cerebellar ataxia in various populations (1216). The molecular basis for the disease is an
expansion of a CAG repeat tract in exon 1 of the SCA2 gene
located on chromosome 12q24.1 (24). In normal individuals
the CAG repeat is not only polymorphic in length, ranging
from 14 to 31 repeats, but also cryptic in nature, having one or
more interruptions of CAA triplets (24,1216). In contrast,
the expanded SCA2 alleles contain a pure, continuous stretch
of 34 to 59 CAG repeats (24,1216). Despite the presence of
CAA interruptions and variability in their number, most studies
of SCA2 CAG repeat in human populations have focused on
repeat length alone (1216). Sequence interruptions within
human di- and trinucleotide repeat tracts have been shown to
play an important role in conferring increased genetic stability
to the repeat tracts (1720). The absence of these interruptions
at least in fragile X syndrome and SCA1 predisposes alleles to
expansion and eventually to disease status (2124). It has been
postulated that interruptions provide genetic stability to the
repeat tracts by inhibiting strand slippage, such that upon
transmission the interrupted tracts are less likely to expand
(20,25). The observation that the majority of the normal SCA2
alleles possess an interrupted repeat configuration, whereas the
disease alleles consist of pure CAG repeat tracts, suggests that
it is important to determine both the length and the internal

*To whom correspondence should be addressed at: Functional Genomics Unit, Centre for Biochemical Technology (CSIR), Delhi University Campus,
Mall Road, Delhi 110 007, India. Tel: +91 11 7416489; Fax: +91 11 7667471; Email: [email protected]

2438 Human Molecular Genetics, 2001, Vol. 10, No. 21

Figure 1. Schematic presentation of microsatellite markers and SNPs used for haplotype analysis. The top line depicts the positions of 25 exons of the SCA2 gene
(34) and three microsatellite markers, D12S1333, D12S1672 and D12S1332. The second line shows the relative locations of two novel SNPs (A and B) and CAG
repeat tract in exon 1 of the SCA2 gene. The first SNP (A) is located 177 bp upstream of the CAG repeat and is scored as either a G or C. The second SNP (B) is
present 106 bp upstream of the SCA2 repeat tract and contains either a T or a C base. Both the polymorphisms are also shown in sequence context below the gene.

structure of SCA2 repeat alleles to evaluate their tendency for

instability.
SCA2 has been shown to be the most frequent cause of
dominantly inherited ataxia in the Indian population
(16,26,27). Haplotype studies carried out using microsatellite
markers flanking the SCA2 repeat have provided the support
for a limited pool of ancestral or at risk haplotypes from
which SCA2 disease chromosomes are derived in our population (16,28). In this paper, we report the identification and
characterization of two novel single nucleotide polymorphisms
(SNPs) in exon 1 of the human SCA2 gene. Using these polymorphic markers, we have extended our previous study of
SCA2 in the Indian population (16) and investigated the role of
various genetic factors such as repeat length, CAA interruption
pattern and haplotype background in predisposing repeats
towards instability and pathological expansion at the SCA2
locus. Based on our results we demonstrate that the instability
of SCA2 CAG repeat alleles is likely to result from the absence
of anchoring CAA interruptions.
RESULTS
Polymorphic markers around the SCA2 CAG repeat
In order to amplify the SCA2 CAG repeat containing region by
PCR we had designed and tested three primer sets on normal
and SCA2 positive samples. Sequence analysis of the PCR
products obtained using one of these primer sets, SCA2-FP3
and SCA2-RP3 (Materials and Methods), revealed two novel
intragenic, biallelic SNPs in exon 1 of the human SCA2 gene.
The two SNPs were at nucleotide positions 481 and 552 in the

human SCA2 mRNA sequence (GenBank accession no.

U70323; www.ncbi.nlm.nih.gov/Genbank/). The first polymorphic site, as shown in Figure 1A, had either a G or a C base
and was 177 bp upstream of the polymorphic CAG repeat
stretch (SNP database reference SNP Id no. 695871;
www.ncbi.nlm.nih.gov/SNP/). The second polymorphic site
(Fig. 1B) was situated 106 bp upstream of the CAG repeat tract
and contained either a T or a C base (SNP database reference
SNP Id no. 695872). While the first substitution changes the
amino acid sequence from valine to leucine, the second substitution is neutral.
SNPs and SCA2 CAG repeat length
We analyzed the two SNPs and the SCA2 CAG repeat length
polymorphism in 215 normal and 64 expanded chromosomes.
The phases of the two SNP alleles were determined based on
their segregation pattern (Materials and Methods). Although
four haplotypes are possible with two biallelic polymorphic
systems, only two were observed: GT or CC haplotype. No GC
or CT haplotype was detected in any sample, suggesting that
either these haplotypes are very rare or G, T and C, C are
exclusively linked to each other. The frequency of each SNP
haplotype and its association with CAG repeat number is
summarized in Table 1. In 215 normal chromosomes analyzed,
the GT and the CC haplotypes were represented in 70.7 and
29.3%, respectively. Since only 8% of the normal chromosomes at SCA2 locus contain CAG repeat number other than
22, we also compared the frequency distribution of the SNP
haplotypes in chromosomes with 22 repeats and chromosomes
having other than 22 repeats. Both these CAG repeat
categories were significantly different (Fishers exact test,

Human Molecular Genetics, 2001, Vol. 10, No. 21 2439

Table 1. Frequency distribution of SNP haplotypes in normal and expanded

SCA2 chromosomes
CAG repeat length

Percentage GT
haplotype (n)

Table 2. Haplotype analysis of SCA2 pedigrees

Pedigree

SNP haplotype D12S1333

D12S1672

D12S1332

Percentage CC
haplotype (n)

AT006

CC

AT009

CC

AT010

CC

AT024

CC

AT027

CC

AT031

CC

AT038

CC

AT048

CC

AT049

CC

AT056

CC

AT092

CC

AT017

CC

AT029

CC

AT052

CC

AT077

CC

AT041

CC

AT064

CC

Normal (1830 repeats)

215

70.7% (152)

29.3% (63)

Repeats (= 22)

175

70.9% (124)

29.1% (51)

Repeats (other than 22)

31

22.6% (7)

77.4% (24)

Expanded (3548 repeats)

64

0.0% (0)

100% (64)

P = 0.0000) for the two SNP haplotypes, the chromosomes

with CC haplotype being much more frequent in the group
with repeat other than 22 (77.4%) than with 22 repeats (29.1%)
(Table 1). Further studies revealed a highly significant difference (2 = 99.40, P < 0.0001) in the distribution of the two SNP
haplotypes between the normal and the expanded SCA2
chromosomes (Table 1). All the expanded chromosomes
segregated with CC allele showing that disease chromosomes
are in complete association with CC haplotype.
Association of SNP haplotype and flanking microsatellite
markers
A common disease haplotype has been observed for Indian
SCA2 pedigrees using flanking microsatellite markers
D12S1333, D12S1672 and D12S1332 (locations shown in
Fig. 1) (16,28). In order to examine the association between
the microsatellite markers and the SNP alleles, the haplotype
analysis was carried out in 17 SCA2 pedigrees and 200 normal
control chromosomes. The disease haplotype segregating in
each of the 17 pedigrees is shown in Table 2. A marked association was observed between the disease locus and the SNP
haplotype, with all the disease chromosomes segregating with
CC haplotype. Strong association was also observed between
SCA2 and allele 1 (225 bp) at D12S1333 locus, alleles 3
(281 bp) and 4 (283 bp) at D12S1672 locus and allele 8
(208 bp) at D12S1332 locus (Table 3). The haplotype 1-3-8 for
markers D12S1333, D12S1672 and D12S1332 was associated
with SCA2 expansion in six and the truncated haplotype 1-3
for markers D12S1333 and D12S1672 in 11 out of 17 SCA2
families studied (Table 3).
A significant difference in the frequency distribution of the
microsatellite alleles associated with SCA2 expansion mutation was observed for the normal chromosomes when grouped
by SNP haplotype (Table 4). For example, allele 1 of
D12S1333 marker was significantly (Fishers exact test,
P = 0.0033) over-represented in normal chromosomes with CC
haplotype (25%) than with GT haplotype (7%). Similarly allele
3 of D12S1672 marker was present in much higher frequency
in CC chromosomes (44%) compared with GT (3%). Despite a
significant association of allele 8 at D12S1332 locus with
disease chromosomes, no apparent difference was observed
between the frequency distribution of this allele in GT (8%)
and CC (7%) chromosomes. The truncated haplotype 1-3,
which accounted for 65% of the SCA2 families, was associated with only two (2%) control chromosomes, both having
CC haplotype (Table 4 ).

Seventeen unrelated SCA2 pedigrees of Indian origin were typed using SNPs
and microsatellite markers. Some parts of this table are derived from previously
reported studies (16).
D12S1333: allele 1 = 225 bp, allele 3 = 229 bp; D12S1672: allele 3 = 281 bp,
allele 4 = 283 bp, allele 5 = 285 bp; D12S1332: allele 5 = 202 bp, allele 6 = 204 bp,
allele 7 = 206, allele 8 = 208 bp.

SNP haplotype and CAA interruption pattern

In order to assess the allelic diversity at SCA2 locus and to
determine the effect of CAA interruptions on repeat stability,
215 normal and 64 expanded chromosomes were analyzed for
the interruption pattern of CAA triplets within the SCA2 CAG
repeat tract. Considerable variation was observed in the cryptic
nature of the SCA2 repeat in normal chromosomes (Table 5).
Two interruption patterns (CAG)8CAA(CAG)4CAA(CAG)8 or
8+4+8 and (CAG)13CAA(CAG)8 or 13+8 (Materials and
Methods for nomenclature) were observed most frequently,
accounting for 70.7% (152/215) and 20.5% (44/215) alleles,
respectively. Among 215 control chromosomes, 157 (73.0%)
had two CAAs, 53 (24.7%) had one CAA, four (1.8%) were
devoid of interruptions and one (0.5%) had three CAA interruptions. When both the repeat length and the CAA interruption pattern were considered, the heterozygosity at SCA2 locus
was much higher (51%) for our sample set compared with 13%
based on repeat length alone. While 98% (211/215) of the
normal chromosomes had an interrupted repeat configuration,
sequence analysis of 64 expanded SCA2 alleles revealed a
homogeneous, uninterrupted CAG repeat stretch.
A marked split was observed in the number and the pattern
of CAA interruptions between the chromosomes with GT and
CC haplotype (Fig. 2). Ninety-eight percent (149/152) of the
chromosomes with GT haplotype had two or more CAA interruptions while 86% (54/63) of the CC chromosomes had either

2440 Human Molecular Genetics, 2001, Vol. 10, No. 21

Figure 2. Distribution of CAA triplets in the SCA2 CAG repeat tract of 215 normal chromosomes. CAG repeats are represented by open circles and CAA
interruptions are represented by closed circles. Alleles are grouped by SNP haplotype and are arranged in ascending order of the repeat length.

one or were devoid of CAA interruption (Fig. 2). This

difference in the number of interruptions present within the

CAG repeat tracts of chromosomes with GT and CC haplotype

is statistically quite significant (2 = 150.122, P < 0.0001). The

Human Molecular Genetics, 2001, Vol. 10, No. 21 2441

Table 3. Linkage disequilibrium between polymorphic markers and SCA2 mutation

Marker

Allele

Patients (frequency)

D12S1333

16/17 (0.94)

1/17 (0.06)

11/17 (0.65)

30/200 (0.15)

0.0013

Positive

5/17 (0.29)

10/200 (0.05)

0.0077

Positive

D12S1672

Controls (frequency)

P-value

Association

25/200 (0.13)

0.0000

Positive

2/200 (0.01)

0.2269

1/17 (0.06)

12/200 (0.06)

1.0000

7/17 (0.41)

61/200 (0.30)

0.6164

2/17 (0.12)

25/200 (0.12)

1.0000

1/17 (0.06)

43/200 (0.22)

0.3248

7/17 (0.41)

16/200 (0.08)

0.0035

SNP haplotype

CC

17/17 (1.00)

63/215 (0.29)

0.0014

Positive

D12S1333-SNP

1-CC

16/17 (0.94)

6/83 (0.07)

0.0000

Positive

D12S1333-SNP-D12S1672

1-CC-3

11/17 (0.65)

2/83 (0.02)

0.0000

Positive

D12S1333-SNP-D12S1672-D12S1332

1-CC-3-8

6/17 (0.35)

1/83 (0.01)

0.0003

Positive

D12S1332

Positive

Truncated haplotypes

Association between marker alleles and SCA2 mutation was determined by Fishers exact test. Alleles (or haplotypes) showing linkage disequilibrium with SCA2
mutation are indicated as positive (P-value < 0.01).

Table 4. Associations between microsatellite alleles and SNP haplotypes

Marker

Allele

Controls with GT
Controls with CC
P-value
haplotype (frequency) haplotype (frequency)

D12S1333

10/141 (0.07)

15/59 (0.25)

0.0033

1/141 (0.007)

1/59 (0.02)

0.5069

4/141 (0.03)

26/59 (0.44)

0.0000

3/141 (0.02)

6/59 (0.10)

0.0274

D12S1672

D12S1332

2/141 (0.01)

10/59 (0.17)

0.0003

45/141 (0.32)

16/59 (0.27)

0.7468

19/141 (0.13)

6/59 (0.10)

0.6466

28/141 (0.20)

15/59 (0.25)

0.4721

12/141 (0.08)

4/59 (0.07)

0.7507

The frequency distribution of normal control chromosomes with GT and CC

haplotypes for the microsatellite marker alleles associated with expanded
chromosomes in SCA2 pedigrees (Table 3). The alleles, which showed strong
linkage disequilibrium with SCA2 expansion mutation, are indicated in bold
and are discussed in the text.

bordered by two interruptions and a 3 tract, distal to the last

interruption. For alleles with a single CAA interruption, the
number of uninterrupted CAG repeat tracts was reduced to
two: a 5 and a 3 portion. When the number of interruptions
was greater than two, alleles were considered to possess two or
more middle tracts of CAG repeats.
In order to determine which of these repeat tracts display the
length polymorphism seen in normal chromosomes, we
sequence analyzed SCA2 normal chromosomes containing a
spectrum of repeat sizes. A total of 22 distinct allele types were
identified based on repeat length and CAA interruption pattern
(Fig. 3). A continuous, uninterrupted CAG repeat configuration was observed on six allele types with CAG repeat length
ranging from 16 to 29 and all having CC haplotype. For the
remaining 16 interrupted allele types, the 5 tract of pure
repeats was much more variable (range 522 repeats) than
either the middle (range 47) or the 3 tract (range 813
repeats), indicating that for different size SCA2 alleles the vast
majority of repeat length variability has occurred at the 5 end
of the repeat (Fig. 3).
Origin of the SNP alleles

first 5 CAA interruption was observed at the triplet position 9

and the second at position 14 in 97.4% (148/152) of the GT
chromosomes. In contrast, 73.0% (46/63) of the chromosomes
with CC haplotype had their first 5 interruption at position 14
indicating the absence of the most proximal 5 CAA interruption.
Polar variation within the SCA2 repeat
Based on the most common SCA2 repeat substructure
(CAG)8CAA(CAG)4CAA(CAG)8, the SCA2 repeat stretch
was divided into three continuous CAG repeat tracts: a 5 CAG
repeat tract proximal to the first interruption, a middle tract

To further determine the ancestral status of the two SNPs and

to compare the SCA2 CAG repeat organization, we tested
them in chimpanzee (GenBank accession no. AF330028;
www.ncbi.nlm.nih.gov/Genbank/), gorilla (accession no.
AF330029) and various Old World monkeys: langur (accession no. AF330030), rhesus monkey (accession no.
AF330031), baboon (accession no. AF330032) and bonnet
macaque (accession no. AF330033). The region containing the
SNPs and the CAG repeat tract was conserved in evolution.
The primer set used to detect these polymorphic sites in the
human genome also amplified comparative DNA fragments

2442 Human Molecular Genetics, 2001, Vol. 10, No. 21

Figure 3. CAA interruption pattern of various lengths of SCA2 repeat. Open circles represent CAG triplets while closed circles represent CAA interruptions. Alleles
are grouped by SNP haplotype and are arranged by increasing overall repeat length.

from the genomes of other non-human primates. The CAG

repeat length, the repeat substructure and the SNP haplotype of
all the samples analyzed is shown in Figure 4. The CAG repeat
tracts were not only polymorphic in length but were also interrupted by varying number of CAA triplets. The CCG triplets
adjacent to the CAG repeats were also found to be polymorphic and their number varied from one to six in various
species examined. All the samples studied had CC haplotype
(Fig. 4), suggesting that CC represents the ancestral form in
mammalian evolution.
DISCUSSION
We have identified and characterized two novel SNPs in exon
1 of the human SCA2 gene. Two haplotypes distinguished by
these two SNP markers, GT and CC, were represented in 70.7
and 29.3% of the normal SCA2 chromosomes, respectively. In
contrast, all the disease chromosomes examined in our sample
set shared a common CC haplotype. Haplotype studies
performed using these SNP markers and linked microsatellites
indicated major founder effects in the origin of expanded
SCA2 alleles in the Indian population, with a subset of normal
chromosomes with CC haplotype predisposed to undergo
subsequent expansion. This linkage disequilibrium of SCA2
expanded alleles to CC haplotype suggests that these SNPs are
likely to be linked to cis acting factors that directly influence
repeat stability. Therefore, we used these SNPs to examine
various genetic factors, such as total repeat length, CAA inter-

ruption pattern and haplotype background, which might

contribute to repeat instability and expansion at SCA2 locus.
Sequence analysis of SCA2 CAG repeat for the presence of
interrupting CAA triplets revealed that 98% of the normal
alleles are interrupted by one or more CAA interruptions,
whereas all the expanded alleles contain uninterrupted CAG
repeats. Ninety-eight percent of the normal chromosomes with
GT haplotype had two or more CAA interruptions with 8+4+8
as the common interspersion pattern. Interestingly, the CC
haplotype seen on 100% of the expanded chromosomes was
present on normal chromosomes, 86% having single or no
CAA interruptions and 13+8 as the most common repeat
configuration. This data suggests that the chromosomes with
CC haplotype either lack the most proximal CAA interruption
or have a tendency for recurrent loss of 5 CAA triplet by a
mechanism which tends to preserve the overall length of the
repeat. It has been postulated that a minimal length of pure
repeats is required to initiate instability at a repeat locus. The
presence of interruptions breaks the repeat into smaller repeat
tracts and thus protects it from instability by reducing the
length of continuous uninterrupted repeats. There is evidence
in the case of SCA1 and fragile X syndrome that larger uninterrupted repeats are more likely to expand than cryptic repeats
(2124). This is also true for dinucleotide repeats where the
degree of polymorphism for a repeat locus is generally proportional to the length of the perfect repeat (20). The protective
mechanism of interruptions may very well be due to their
ability to impair slippage between the complementary strands

Human Molecular Genetics, 2001, Vol. 10, No. 21 2443

Figure 4. CAG repeat length, repeat substructure and SNP haplotype in non-human primates. Open circles represent CAG triplets; dark circles represent CAA
interruptions and hatched circles represent CCG triplets adjacent to the CAG repeat tract.

(25). Thus, absence of 5 CAA interruption in chromosomes

with CC haplotype might represent one of the factors contributing to repeat instability presumably because only a single
event will be required to create a perfect CAG repeat and hence
predispose to expansion. A recent report by Costanzi-Porrini et
al. (29) of two patients with SCA2 phenotype and having an
interrupted 34 CAG repeat allele with configuration 24+8,
suggests that the loss of all interruptions is not absolutely
necessary for repeat expansion. A slow sequential lengthening
of 5 repeat tract in alleles lacking the most proximal CAA
interruption can also result in expansion to pathological range
at the SCA2 locus. Therefore, predisposition towards repeat

instability and expansion in SCA2 appears correlated not

strictly to the repeat length but rather to the purity of the repeat.
The polar variation in SCA2 repeat is similar to that
observed for CGG repeats in fragile X syndrome and in hypervariable human and murine minisatellite loci where allele
diversity is largely determined by sequence changes at one end
of the repeat array (22,23,30). Unlike fragile X syndrome
where the majority of the changes occur in the most 3 tract
(22,23), the 5 tract (relative to the orientation of transcription)
of SCA2 CAG repeat was found to be more variable. The
observed polar variation among SCA2 alleles demonstrates
that the replication slippage is a frequent event in the 5 portion

2444 Human Molecular Genetics, 2001, Vol. 10, No. 21

Table 5. SCA2 repeat interruption patterns

Repeat length

Repeat
substructure

No. of CAAs

No. of
chromosomes
(frequency)

18

9+8

1 (0.005)

21

12+8

1 (0.005)

22

8+4+8

152 (0.707)

22

13+8

44 (0.205)

23

9+4+8

1 (0.005)

23

8+4+9

2 (0.009)

23

13+9

3 (0.014)

23

14+8

3 (0.014)

23

23

1 (0.005)

24

24

2 (0.009)

29

8+4+4+10

1 (0.005)

29

20+8

1 (0.005)

29

29

1 (0.005)

30

13+7+8

2 (0.009)

Total number of chromosomes (n)

215

The interruption patterns of SCA2 CAG repeat alleles and the frequency of
their occurrence among 215 randomly selected normal chromosomes. CAA
interruption patterns have been abbreviated. A plus sign represents a CAA
and the number denotes the uninterrupted CAG repeats (Materials and
Methods). The alleles are arranged in ascending order of repeat length.

of the CAG repeat and supports the hypothesis that it is the lack
of the most proximal 5 CAA interruption that acts to destabilize the CAG repeat region and results in the highest variability
of pure repeat lengths at 5 end. The observed variability within
the continuous tract of CAG repeats and the fact that changes
involve differences in multiples of 3 bp clearly favors slipped
strand mispairing as the most likely mechanism of repeat
length variability at the SCA2 locus (31). Unequal sister chromatid exchange and gene conversions have also been proposed
as a mechanism of repeat length variability (32). However, if
these were the dominant mode of repeat length variation for
SCA2 repeats then one might expect larger alleles to contain
three or more CAAs as they are constructed in a cassette like
arrangement by conversion or unequal cross over. Such alleles
have been observed but appear to be rare (Table 5 and Fig. 2).
Analysis of comparative DNA fragments from the genome
of other non-human primates gave insight into the evolutionary
history of SCA2 CAG repeat with CC haplotype representing
the ancestral form in mammalian evolution. The absence of
two other haplotypes (GC and CT) among the four possible
combinations (GT, CC, GC and CT) suggests that the mutation
from C to G and C to T creating the GT allele might have
occurred simultaneously and this new haplotype GT, either
through selection (repeat stability) or genetic drift, became the
more common allele associated with normal SCA2 chromosomes in our population.
In summary, we report here a very comprehensive study of
the factors that may drive variability and ultimately repeat
instability at the SCA2 locus. Two newly characterized SNPs,

the closest of the surrounding markers to the SCA2 CAG

repeat, showed a complete association of expansion mutation
with CC haplotype in our population. The identification of a
common CC haplotype for disease chromosomes facilitated
the characterization of the factors involved in repeat instability
at the SCA2 locus. Based on our data we demonstrate that
CAA interruptions play a pivotal role in restricting mutability
at the SCA2 locus and that their absence predisposes the alleles
to expansion. However, larger surveys of SCA2 repeat
substructure and SNP haplotype in different ethnic groups will
be necessary to further confirm these findings.
MATERIALS AND METHODS
Subjects
The study was carried out on 22 SCA2 pedigrees comprising of
123 members including patients and family members. Some of
these pedigrees have been reported previously (16). To establish the distribution of SNPs and CAA interruption pattern, 215
normal chromosomes were analyzed as controls. These chromosomes were selected to be representative of overall distribution of repeat length that we have observed for the SCA2 locus
(16). All the subjects were of Indian origin and mixed
geographical derivation. Since only 8% of the normal SCA2
alleles possess other than 22 repeats, an additional 15 alleles
with repeat number other than 22 were also analyzed. Blood
samples were collected from all patients and normal individuals with informed consent. Ten apparently unrelated bonnet
macaques (Macaca radiata), two baboons (Papio hamadryas),
two rhesus monkeys (Macaca mulatta), two langurs (Presbytis
entellus), a gorilla (Gorilla gorilla) and a chimpanzee (Pan
troglodytes) sample were also investigated.
Amplification of SCA2 CAG repeat region
Genomic DNA was isolated from peripheral blood leukocytes
using a modification of the salting out procedure (33). The
region containing the SCA2 CAG repeat was PCR amplified
using previously published primers (3) and the exact size of the
repeat in the fluorescently labeled PCR product was determined by Gene Scan analysis using an ABI Prism 377 automated DNA sequencer (Perkin Elmer, Foster City, CA).
SNP detection and CAA interspersion analysis
Sequencing analysis was carried out for characterization of
two SNPs and CAA interruption pattern of SCA2 repeat. The
region containing the SNPs and the CAG repeat stretch was
amplified using primers SCA2-FP3 (5-CTCCGCCTCAGACTGTTTTGGTAG-3) and SCA2-RP3 (5-GTGGCCGAG
GACGAGGAGAC-3). Approximately 100 ng of genomic
DNA was amplified in a 50 l reaction volume containing a
final concentration of 5 mM Tris, 25 mM KCl, 0.75 mM
MgCl2, 0.05% gelatin, 20 pmol of each primer, 200 M dNTPs
and 0.5 U of Taq DNA polymerase. Samples were denatured at
94C for 3 min followed by 35 cycles of denaturation
(94C, 45 s), annealing (52C, 30 s), extension (72C, 45 s)
and a final extension of 7 min at 72C in a Perkin Elmer GeneAmp PCR System 9600. The PCR products were purified from
bands cut out of agarose gel using a QIAquick gel extraction
kit (Qiagen Inc., CA) and were directly sequenced using dye

Human Molecular Genetics, 2001, Vol. 10, No. 21 2445

terminator chemistry on an ABI Prism 377 automated DNA

sequencer with the PCR primers. For SCA2 positive and
normal control families, the phase of the two SNP alleles was
determined based on the segregation pattern. For normal
control individuals when sample showed homozygosity, phase
was easily assigned. For heterozygous samples, the phases
could be determined by performing sequencing on the parents
DNA samples and deducing the phase based on the segregation
pattern. The phases could not be resolved unambiguously for
some samples and were excluded from the study.
Nomenclature of CAG repeat configuration
CAG repeat interruption patterns are summarized as follows: a
plus sign (+) designates the position of a CAA interruption and
the number refers to the length of uninterrupted CAG repeats.
A 8+4+8 allele, for example, symbolizes the sequence of
(CAG)8CAA(CAG)4CAA(CAG)8. CAA interruptions are
described in the text as 5 or 3 relative to the orientation of
transcription of the SCA2 gene.

3.

4.

5.

6.

7.

8.

Haplotype analysis
Haplotypes were generated for the SCA2 positive families
using three microsatellite markers D12S1333, D12S1672 and
D12S1332. These markers span a region around the SCA2
CAG repeat in the following order: telomere-D12S1333200 kb-D12S1672-350 kb-D12S1332-centromere, with D12S1333
and D12S1332 flanking the CAG repeat (Fig. 1). D12S1672 is
in the first intron of the SCA2 gene and is 20 kb centromeric to
the CAG repeat stretch, which is in the first exon of the SCA2
gene (3,34). The frequency of these markers was also determined
in the normal control chromosomes. In all the analyses the
CEPH family member 1347-02 was used as a control for
verification of repeat size.
ACKNOWLEDGEMENTS
We thank Dr Nitai P.Bhattacharyya and Dr Partha P.Majumder
for their generous contribution of few normal and SCA2
samples for this study. We are grateful to Dr Q.Saleem for
helpful discussions and Dr C.B.Rao for assistance with statistical analysis, Ms R.Jaya, Ms M.Ruchi and Ms T.Sakshi for
help with Genescan and sequence analysis, and Ms Manju,
Ms Gurjit and Ms Anuradha for technical support. We would
like to acknowledge Ms Sanghamitra Roy for extraction of
patient DNA samples. We would also like to thank the Primate
Research Facilities of the National Institute of Immunology,
New Delhi, the Indian Institute of Science, Bangalore and
Dr Lalji Singh of the Centre for Cellular and Molecular
Biology, Hyderabad, for providing the primate samples. Financial
support from the Department of Biotechnology, Government
of India to the Program on Functional Genomics to S.K.B. is
duly acknowledged.
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