• © 1997 Nature Publishing Group http://www.nature.

com/naturegenetics

article

Cloning of the SCAl gene reveals a highly

unstable CAG repeat expansion
Gilles David 1, Nacer Abbas 1, Giovanni Stevaninl, Alexandra Diirr 1•2 , Gael Yvert3, Geraldine CanceP,
Chantal Weber\ Georges Imbert 3, Frederic Saudou3, Eric Antoniou\ Harry Drabkin4 , Robert Gemmill\
Paola Giunti5 , Ali Benomar6 , Nick Wood 5 , Merle Ruberg 1, Yves Agid 1•2 , Jean-Louis Mandel3 & Alexis Brice 1•2

The gene for spinocerebellar ataxia 7 (SCA7) has been mapped to chromosome 3p12-13. By positional cloning, we
have identified a new gene of unknown function containing a CAG repeat that is expanded in SCA7 patients. On
mutated alleles, CAG repeat size is highly variable, ranging from 38 to 130 repeats, whereas on normal alleles it
ranges from 7 to 17 repeats. Gonadal instability in SCA7 is greater than that observed in any of the seven known
neuro-degenerative diseases caused by translated CAG repeat expansions, and is markedly associated with paternal
transmissions. SCA7 is the first such disorder in which the degenerative process also affects the retina.

The autosomal dominant cerebellar ataxias (ADCAs) are a com-
plex group of hereditary neuro-degenerative disorders character-
ized by variable degeneration of the cerebellar cortex, the inferior
olivary, the pontine nuclei, the basal ganglia, the spinal cord and
peripheral nervesl.2. Clinical manifestations include cerebellar
ataxia that may be associated with ophthalmoplegia, loss of vision,
dysarthria, pyramidal and extrapyramidal signs, deep sensory loss
or dementia. Classification of the ADCAs was greatly simplified
by the recognition of three clinical subtypes designated ADCA
I-III (refs 3,4).
The association of cerebellar ataxia and progressive macular
dystrophy with autosomal dominant inheritance represents a dis-
tinct form of ADCA, classified as type II according to Harding3.4.
Several reports have provided detailed characterizations of the
phenotype associated with ADCA II (refs S-8). The gene for
ADCA II (SCA7) was mapped to the short arm of chromosome 3
(refs 7,9,10), and the localization confirmed in families of diverse
geographical origins· 11 •12 , suggesting that ADCA II is a genetically
homogeneous subtype of ADCA. Further reduction of the candi-
date region permitted the construction of an integrated map, based
on a yeast artificial chromosome (YAC) contig, which spanned
the entire candidate regions.
A strong anticipation in age at onset, particularly during pater-
nal transmission, has been observed in ADCA II, as in neuro-degen-
erative disorders such as Huntington's disease, spino-cerebellar
ataxia 1 (SCA1), SCA2, SCA3/MJD (Machado-Joseph disease) and
dentato-rubral-pallido-luysian atrophy (DRPLA). These disorders
share an underlying molecular mechanism: the expansion of a tr-
inucleotide CAG repeat in the coding sequence of the corresponding
genes 13- 20 . The detection of a specific 130-kD polyglutamine-con-
taining protein in SCA7 patients21 •22 and the cosegregation of the
disease with a CAG expansion visualized by the RED (repeat -expan-
sion detection) technique23 suggested that a CAG repeat expansion
was also implicated in ADCA II. We now report the positional
cloning of the SCA7 gene and evidence for the presence of a highly
unstable CAG expansion in mutated alleles.
Positional cloning of the SCA7 CAG repeat
SCA7 sequences from two overlapping YACs in a contig of the
SCA7 candidate regionS-801_a_8 and 882_d_9 (chimaeric)-
were subcloned into a bacteriophage lambda vector, and the result-
ing libraries were screened for the presence of both CAG repeats
and human repetitive sequences. Three CAG/human double-pos-
itive clones were isolated from YAC 801_a_8 and one from YAC
882_d_9. Sequence analysis of the subcloned CAG containing frag-
ments revealed two CAG repeats, one of which was an uninter-
rupted (CAG) 10 repeat derived from YAC 882_d_9 (Fig. 1). The
corresponding 1.7-kb EcoRI fragment, used to probe Southern
blots of Mboi!EcoRI-digested DNA, detected a 1.33-kb fragment in
all individuals and an additional larger fragment, ranging from
1.44 to 1.66 kb, specific to SCA7 patients (not shown). This result
was highly suggestive of the presence of the SCA7 CAG repeat in
the clone derived from YAC 882_d_9. Polymerase chain reaction
(PCR) analysis of a reduced YAC panel of the SCA7 regions
mapped the SCA7 CAG repeat to a S-cM region flanked by
microsatellite markers D3S 1600 and D3S3635 (not shown).
Sequence analysis of the 1. 7-kb EcoRI fragment revealed that
the CAG repeat was located in a 579-bp open reading frame (ORF)
in which two putative exons, 336 bp and 69 bp in size, were pre-
dicted by the GRAIL program 24 (Fig. 1). Subsequent determina-
tion of the eDNA (see below) confirmed these sequences to be the
first two coding exons.
Behaviour of the CAG repeat in SCA7 families
Five French families with SCA7 were selected for analysis of the
mutation. The clinical features of the 18 fully examined patients
are summarized in Table 1. Mean age at onset was 27.2± 16.0 years
(n= 19; range, 1-61) and decreased from generation to genera-
tion (generation I, 44.7± 11.1; generation II, 21.9±8.5; genera-
tion III, 3.5±3.5, P=0.001). In twelve parent/child pairs, the mean
anticipation was 23.8± 12.8 years per generation (range, 5-47)
and was greater in paternal±32.0 (1.2; n=6) than in maternal±
15.5 (8.4; n=6) transmissions (P<O.OS).

1
INSERM U289, Hopi tal de Ia Salpetriere, 47 bd. de I'H6pital, 75651 Paris Cedex 13, France. 2Federation de Neurologie, Hopi tal de Ia Salpetriere, 47 bd. de
I'H6pita~ 75651 Paris Cedex 13, France. 3Institut de Genetique etde Biologie Moleculaire et Cellulaire (IGBMC), CNRS, INSERM, ULP. B.P. 163, 67404 Illkirch
Cedex, C. U. de Strasbourg, France. 4 University ofColorado Health Sciences Center, 4200 East 9th Avenue, Denver, Colorado 80262, USA. 5Institute ofNeurology,
Queen Square, London WClN 3BG, UK. 6Service de Neurologie (Prof T. Chkili), Hopi tal des Specialites, Rabat, Morocco. G.D. & N.A. contributed equally to this
work. Correspondence should be addressed to A.B. e-mail: brice@u289.ext.infobiogen.fr

nature genetics volume 17 september 1997 65

article • © 1997 Nature Publishing Group http://www.nature.com/naturegenetics

Table 1 • Oinical characteristics of patients with SCA7 All patients and two asymptomatic at-risk subjects had expan-
sions (Fig. 2) ranging from 38 to about 130 repeats (mean, 51.8±
Number of families 5
19.8; median, 45; n=21). The size of the largest expansion, observed
Number of patients 18 in a juvenile case, could not be precisely determined by polyacry-
Men/women 11n lamide-gel electrophoresis, but if contained at least 130 CAG
Mean age at onset of visual failure 22±15 (1-45) years repeats. As observed in other SCAs25 •26 , the electrophoretic pattern
Mean age at onset of cerebellar ataxia 29±16 (1--£1)years of the expanded alleles (Fig. 2) suggested that significant somatic
Mean age at examination 38±18 (26--£7) years
Mean age at death (n = 5) 34±27 (4--£8) years mosaicism was present in SCA7 patients. There was a strong nega-
tive correlation (r=-0.93) between the age at onset and the CAG
Clinical signs repeat number (Fig. 4). We observed increases in the CAG repeat
Cerebellar ataxia and dysarthria 100% number in 13 of 14 parent/child transmissions (Fig. 5), with a mean
Increased reflexes in the lower limbs 78%
Extensor plantar reflex 44% of 13.1 ±22.8 additional CAGs per transmission (median, 4.5) . This
Lower limb spasticity 43% phenomenon was markedly greater in paternal (25.3±31.8; median,
12.5; n=6) than in maternal (3.9±3.4; median, 2.5; n=8) trans-
Decreased visual acuity (blindness) 83% (28%) missions (P<0.05). The two largest increases ofCAG repeat size
Optic atrophy 69%
43%
(37 and ~85) were transmitted by an affected father.
Pigmentary retinopathy
Supranuclear ophthalmoplegia 56%
Viscosity of eye movements 79% Cloning of the SCA7 eDNA and expression analysis
A 107-bp fragment located downstream from the CAG repeat in the
Dystonia 18% predicted ORF (Fig. 1) was used to screen three eDNA libraries (see
Myokymia 15%
below). Five clones were isolated, corresponding to three partly over-
Decreased vibration sense 62% lapping cDNAs (Fig 1). Sequence analysis allowed us to establish a
Dysphagia 77% 3,969-bp consensus sequence that contained a 2,727-bp ORF, flanked
Sphincter disturbances 67% by a 513-bp 5'-UTR and a 729-bp 3'-UTR (Fig. 1). The sequence
Hypoacusis 33% of the ORF was confirmed by analysis of at least two independent
Mental impairment 19% eDNA clones. The ATG codon at position 562 most probably rep-
resents the initiation codon, as it is the first ATG in the ORF and is
located in a good Kozak's consensus sequence 27 • The important
PCR analysis of CAG repeat length revealed a size ranging from nucleotides at positions -9 (G), -6 (G) and -3 (A) are conserved.
7 to 17 repeats (mean, 10.4± 1.3; median, 10} in controls from The 2,727-bp ORF starting at position 514 predicted a protein of
different geographical origins (n=58 chromosomes; Figs 2,3). 892 amino acids that contained a polyglutamine tract, encoded by
Most of the normal alleles (78%) had ten repeats, with an expected CAG repeats at codons 30-39 (Fig. 6). A nuclear localization signal
heterozygosity rate of 35%. (NLS) consensus 28 was predicted at amino-acid positions 378-394.

Fig. 1 Schematic representation of

c..__ _ _,___ _ _ _ _ _ _ _ _
-'---------------------------------------• AlO
Dl--------------------------------------------------~
the SCAl region on chromosome
3p12-13 . Top: The two overlapping
YACs, 801 _a_8 and 882_d_9, are
placed with respect to the genetic
maps. Polymorphic markers are rep·
resented by black circles, non-poly-
morphic markers by black squares.
The distance between each marker is
arbitrary. The genetic interval con-
Genomic DNA taining the SCAl CAG repeat is indi-
cated by a double black arrow. The
1---i
two isolated CAG repeats are repre-
JOObp sented with their corresponding
physical locations on the YACs (dot-
ted lines). Bottom: The open boxes
indicate the coding regions on
genomic DNA and eDNA consensus
sequences. Solid boxes represent the
position of the CAG repeat. The posi-
Al, All, A27 t ion of the 107-bp fragment used for
•eDNA clones the screening of the eDNA libraries is
1----1 indicated by a thick line above the
250bp genomic DNA. The size and position
of the eDNA clones are shown below
the SCA7 eDNA consensus sequence.

66 nature genetics volume 17 september 1997

 • © 1997 Nature Publishing Group http://www.nature.com/naturegenetics

SAL-313 AAD-104 Fig. 2 Detection of the SCAl CAG repeat by PCR analysis. Partial pedigrees
article

are represented at the top, and ages at onset (years) are indicated above
II each lane. Brackets indicate the normal (7-17) and the expanded (38-130)

~'?o
CAG repeat size range. Genotypes were 10/42, 10/10, 10/55, 10/44, 10/43,
10/12, 12/80, 10/43, 10/45 and 10/10 CAG repeats from left to right on each
lane. Notice the highly unstable expansion pattern. Patient SAL-313-6 trans-
mitted an expanded allele with 37 additional CAG repeat to his son, SAL-313-
16, resulting in a marked anticipation in age at onset.
16 10 11
55 - 10 26 28 - 8 45 35 ~·IS

lines and brain proteins from SCA7 patients21 •22 • It is known, how-
ever, that polyglutamine expansions reduce electrophoretic mobil-
ity. The four French patients in whom a 130-kD SCA7 protein was
detected by western blot carried expansions ranging from 45 to
80 CAG repeats 21 •22 • Comparisons with nucleotide and protein
sequences in the GenBank and the Swissprot databases revealed
E three human sequence tagged sites (U49879, X95831 and X98704)
and three human expressed sequence tags from colon, testis, and
fetal liver and spleen (AA099235, AA398030 and H78490) to be
identical to regions of the SCA7 eDNA. Northern-blot analysis
revealed a 7.5-kb transcript (Fig. 7), which was abundantly
expressed in heart, placenta, skeletal muscle and pancreas, with a
fainter signal in brain, lung, liver and kidney (not shown). Expres-
sion was ubiquitous in central nervous system (CNS) but appeared
to be higher in cerebellum than in the other structures analysed.
The gene appears to be conserved in mammals (human, monkey,
mouse, rat and cow); a clear cross-hybridization fragment was
observed on a zoo blot of EcoRI -digested DNA when the same
probe as for the northern blots was used (not shown).

Discussion
We have identified the SCA7 gene by positional cloning, and have
established that ADCA II is the eighth disease-in addition to
N SBMA, HD, DRPLA, SCAl, SCA2, SCA3/MJD and SCA6-asso-
ciated with the expansion of a trinucleotide CAG repeat in the
coding region of the responsible gene 29 • The common features of
this growing group of hereditary neurodegenerative disorders
(except for SCA6) are genetic anticipation, negative correlation
between the CAG repeat size and the age at onset, instability of
We propose that the protein encoded by the SCA7 gene be called the repeat and ubiquitous expression of the gene. In SCA6, the
ataxin-7, by analogy to other identified SCA gene products. The repeat is stable and expression selective30 . Instability is particu-
predicted size of ataxin-7, approximately 95 kD, is smaller than larly marked in ADCA II, however.
the 130-kD size observed on western blots oflymphoblastoid cell Normal SCA7 alleles contained seven to 17 repeats and are not
. .

~
45 controls
patients
40 .~ymptomatic

35

,., 30
]
...ft 25
.
C>
Cl>
.Q 20
a
z= 15
Fig. 3 Distribution of CAG repeat
sizes on normal and SCA7 chromo- 10
somes. Open bars indicate control
chromosomes (n =58) from unrelated
individuals. Black and hatched bars 5

~-'-L-L..L-L..L"-'-.L..L.J..U_.L.J IL'~' L' ' '

indicate expanded chromosomes
from 19 SCA7 patients and two at-
risk carriers, respectively. Notice the
0 I' J 'I' I' /l ._ili_ !/ _ili

high frequency of the normal allele 5 10 15 20 25 30 35 40 45 50 55 80 130

with ten CAG repeats (78%) and the
wide range of expansions. Number of CAG repeats

nature genetics volume 17 september 1997 67

article
70 1
• © 1997 Nature Publishing Group http://www.nature.com/naturegenetics

13or D

60 • ~
~
• 80
~ D
~

"'
Q)
50
• g ~
,.:, 40 ..c:
~
~
....
VI
55
D •
~ 30 •
••• "'a.
Q)
0
.... D •
• •
50
~ •• ~
• "u
20
~ • <(
•
10
I•• •
'+-
0
45
D ••
o L-~- · - --- --'- __ .. ..JL ~ Q:j
.0
D

35 55 75 95 115 135 E 40
::J
Number of CAG repeats z
Fig. 4 Negative correlation between the age at onset and the number of CAG 35 ~--~- ~ --'
repeats in expanded alleles from 19 SCA7 patients. Values in brackets indicate
the number of parent/child pairs. The coefficient was calculated for an expo- 35 40 45 50 55 80 130
nential regression (r=-0.93). Number of CAG repeats (parent)
· - - -- -- Fig. 5 Instability of the SCAl repeat in fourteen parent/child transmissions.
Note the absence of decreases in CAG repeat length and the presence of two
large increases, with 37 and 85 additional CAG repeats, among the paternal
highly polymorphic. The expected 35o/o heterozygosity level is lower transmissions. Black squares indicate paternal transmissions; black circles indi-
than that of the other loci containing polyglutamine expansions- cate maternal transmissions.
except for SCA2, in which normal CAG repeats are interrupted by
CAA trinucleotides thought to stabilize repeat length 15- 17 . The
expanded SCA7 alleles show a large range of sizes, 38 to about 130 reported until now. The smallest expansion is in the same range as
repeats, surpassing the 121 repeat allele observed in HD 13, the largest in SBMA, HD, SCAl and SCA2, but is notably smaller than in
SCA3/MJD and DRPLA, and larger than in SCA6 15-1 7,30,31 _ The
M S E 3 great variability in age at onset and severity of the disease in ADCA
R A A D D v R G E p R R A A A A A G
G A A A A A A R
p p p p Q p Q R
T R p E D G G p G A A s T s A A A M
p eenftll
IP II~ I. . ,p
21
39
57
75
II correlates well (r==-0.93) with the size of expanded alleles.
The electrophoretic patterns of the expanded alleles reveal sig-
nificant somatic mosaicism in blood, even for the smallest expan-
A T v G E R R p L p s p E v M L G Q 93 sions. Mosaicism in the brain remains to be investigated. Analysis
s WN L wv E A s K L p G K D G T E 111
L D E s F K E F G K N R E v MG L c 129 of twelve affected parent/child transmissions suggests that gonadal
R E D M p I F G F c p A H D D F y L 147 mosaicism is possibly greater than that in blood. This has not yet
v v c N D c N Q v v K pp Q A F Q s H 165 been quantified, however. None of the transmissions showed a
y E R R H s s s s K p L A V p p T 183
5 v F s F F p s L 5 K s K G G 5 A 5 201 reduction in CAG repeat length, and only one showed no change. A
G 5 N R 5 5 s G G v L s A s s s s s 219 mean increase of 13 CAG repeats per transmission was observed-
K L L K s p K E K L Q L R G N T R p 237
M H p I Q Q s R v p H G R I MT p s 255 significantly greater in paternal than in maternal transmissions.
v K v E K I H p K MD G T L L K s A 273 Consistent with the strong anticipation observed in ADCA II, par-
v G p T c p A T v s s L v K p G L N 291 ticularly during paternal transmissions 5•7•8•32 , two paternally trans-
c p s Ip p Kp pp T L p s p Gp Q I L N G 309
K G L A T L E K K E D N s N 327 mitted alleles, responsible for juvenile cases in two families ,
N R K F L N K R L s E R E F D p D I 345 contained 37 and more than 85 additional CAG repeats. The strik-
H c G v I D L D T K K p c T R s L T 363
li
c K T H s L T R R A V Q G -TR 381 ing anticipation with parental sex bias observed in SCA7 is similar
12 n1 -1s illtiii!I!RBI R E K E L I 399 to that in DRPLA33 . The degree of intergenerational instability of
R H P D ol o p P Q P L R D P H P A 417 the CAG repeat is, however, greater in SCA7 than in any of the seven
p p R T s Q E p H Q N p H G v I p s 435
E s K p F v A s K p K p H T p s L p 453 known neuro-degenerative diseases caused by translated CAG repeat
R p p G c p A Q Q G G s A p I D p p 471 expansion. It should be noted that the sizes of the two paternal alle-
p v H E s p H p p L p A T E p A 5 R 489
L s s E E G E G D D K E E s v E K L 507 les, 43 and 45 CAG repeats, that underwent large expansions were
D c H y s G H H p Q p A s F c T F G 525 within the range of most of the expanded alleles, suggesting that
5 R Q I G R G y y v F D 5 R WN R L 543 gonadal instability is not simply a function of repeat size.
R c A L N L MV E K H L N A Q L WK 561
K I p p v p s T T s p I 5 T R I p H 579 As postulated for HD, additional factors might explain indi-
R T N s v p T s Q c G v s y L A A A 597 vidual variations in the degree of sperm mosaicism 34 •35 . In
T v s T 5 p v L L s s T c I s p N s 615 SCA3/MJD, polymorphisms located close to the CAG repeats seem
K s v p A H G T T L N A Q p A A 5 G 633
A MD p v c s M Q S R Q v s s s s s 651 to influence the degree of instability3 6 •37 , but none were found in
s pp s T p 5 G L s s v p sp 5 p M 5 R 669 PCR products from seven SCA7 normal alleles that were
K Q K L K s s K s L R K E s s G 687
N s T N c Q N A s s s T s G G s G K 705 sequenced. Other factors, such as position (surrounding sequences
K R K N s s p L L v H s s s s s s s 723 in cis or trans, or location with respect to the origin of replica-
s s sp sp ys Hp s M E s F R K N c v A H 741 tion 38 ) or genetic background, may therefore contribute to inter-
s G s T v T 5 5 H 5 I G L N 759
c vp Tp N K A Np A V N v R H D Q s G R 777 individual variability in the stability of CAG repeats. It is also
G T G 5 A E s I K R M S v MV 795 interesting to note the high level of meiotic recombination in the
N 5 s D 5 T L s L G p F I H Q s N E 813
L p v N 5 H G s F s H s H T p L D K 831
L I G K K R K c s p s s s s I N N s 849
s s K p T K vp A K v p A V N N v H M 867
Fig. 6 Predicted amino-acid sequence of ataxin-7. The polyglutamine tract
K H T G T I G A Q G L M N s s L L 885
and the position of the putative NLS are indicated by shaded boxes.
H Q p K A R p * 893

68 nature genetics volume 17 september 1997

 a
• © 1997 Nature Publishing Group http://www.nature.com/naturegenetics
article
neuro-degenerative disorders. SCA7 is
the first such disorder in which the
degenerative process affects the retina in
addition to other brain structures. The
SCA7 mutation is also unusually unsta-
ble, indicating that locus specific factors
and inter-individual variability con-
Kb Kb tribute to the mutational process. Hap-
lotype studies should show whether
certain alleles are predisposed to expan-
sion, as in SCA3/MJD 36 •41 • Finally, the
9.5- 9.5- genetic homogeneity of ADCA II and the
7.5- 7.5- facility with which the expansion can be
detected by routine methods now per-
mit reliable diagnosis.
4.4- 4.4-
Methods

2.4- 2.4- YAC subcloning and screening. DNA from

YACs 80l_a_8 and 882_d_9 was partly digest-
ed with restriction enzyme Sau3A (Boeh-
ringer) and cloned into bacteriophage
1.35- 1.35- Lambda DASH-II!BamHl vector (Stratagene)
according to the manufacturer's protocol. A
total of 20,000 primary plaques per YAC
library were screened with both a 32 P end
labelled (CAG) 10 oligonucleotide and a 32 P-
containing random primed human genomic
DNA probe. Membranes were hybridized
Fig. 7 Northern-blot analysis of SCA7 expression in various human tissues. Hybridization signals obtained with a overnight at 65 oc in Church buffer4 2, washed
~-actin control probe were of similar intensity in each lane (data not shown). a, In human brain, a 7.5-kb RNA
twice in IX SSC, 0.1% SDS for 45 min at 65 oc
was widely expressed, with particularly high levels in cerebellum. b, An SCA7 transcript of the same size is also
detected in fetal tissues, including brain. and exposed overnight to X-ray film
(Amersham). After isolation of the double-
positive clones, phage DNA was extracted
according to standard protocols 43 .
SCA7 region, suggested by the large discrepancy between the esti- Phage DNA was digested with various enzymes, and the fragments, sep-
mated genetic and physical distances (Fig. 1), compared with the arated by electrophoresis on l o/o agarose gels, were blotted on Hybond N+
usuall-Mb/1-cM estimation. In addition, the SCA7CAG repeat, membranes and hybridized as described above. Fragments that hybridized
mapped between markers D3S1600 and D3S3635, is centromeric with both the human DNA and the (CAG) 10 probes, including a 1.7-kb
to the interval previously delimited by recombination events EcoR! fragment, were isolated and subcloned in the pUC18 vector.
observed in a 48-year-old woman, BRA-9015, with apparently typ- Plasmid DNA was sequenced with ABI dye primer and dye terminator kits
ical late-onset clinical features 8. Further analysis of the SCA7 CAG and protocols on an ABI-Prism 377 sequencer.
repeat in this patient, however, did not reveal the presence of an
SCA7 eDNA cloning. The 1.7-kb genomic sequence was analysed for puta-
expanded allele (Giunti et al., in preparation).
tive coding sequences with GRAIL Ia software24 . Primers (primer 5':
The sequence of the SCA7 eDNA shares no significant homolo- 5'-CGCAATGGCGACGGTCGG-3 ' and primer 3': 5'-CGTCCTTC-
gies with known genes or proteins that would give insight into the CCAGGAAGTTTG-3') corresponding to sequences in the putative exon
function of ataxin-7. However, the polyglutamine region, which containing the CAG repeat were used for the amplification of a !07-bp PCR
is followed by a small proline repeat, shows some resemblance to probe (nucleotides 780-886). Random primed eDNA libraries constructed
the polyglutamine/proline-rich regions in huntingtin and in some from lymphoblastoid cell lines of a SCA7 (DAN) or SCA2 (AAD) patient 17 ,
homeodomain-containing proteins (opa domain) and other tran- and an oligo-dT primed substantia nigra eDNA library constructed from a
scription factors 39 • Many transcription factors contain gluta- control subject44 were screened (see YAC screening), for a total of 2 x 106
mine/proline-rich activation domains, and it has been shown that plaques per library. Plasmids were excised according to manufacturers' pro-
tocols, and plasmid DNA was sequenced as described above.
homopolymeric stretches of these two amino acids can activate
transcription in vitra3 9 • Because ataxin-7 is present in the nuclear Northern-blot analysis. Human multiple tissue, fetal and brain northern
fraction of lymphoblasts 21 •22 and contains an NLS consensus blots (Clontech) were hybridized with a 979-bp Stul!Hindlii fragment
sequence, it may be a potential transcription factor. As in the other located downstream of the CAG repeat, isolated from the A2 eDNA clone
seven neurodegenerative diseases with the same type of mutation, (Fig. I), according to the manufacturer's protocol. The membranes were
it is postulated that the polyglutamine expansion causes acquisition then washed twice in IX SSC, 0.1 o/o SDS at 50 oc for 45 min and exposed
of a toxic property4°. The presence of the expansion is not dele- on X-ray film.
terious in itself, however, as the gene is expressed ubiquitously,
but only selective populations of cells die. Even within the CNS, Families and clinical analysis. Blood samples were taken from 35 consent-
there is no correlation between expression and neuro-degenera- ing individuals from five French families with ADCA II, including 19
patients. Thirty individuals, including 18 patients, were fully examined. In
tion. It has been suggested that binding to another protein, wi.th an
the largest family, SAL-313, linkage to the SCA7 locus was previously
affinity that varies with the length of the polyglutamine tract, shown 7• Age at onset, established by unsteady gait or visual failure due to
might determine the pattern of neuro-degeneration. A binding ascertained maculopathy, was determined for all patients. Comparisons of
protein for mutated ataxin -7 has yet to be identified. means were performed with non-parametric and Student's t-tests, and
In conclusion, the cloning of the SCA7 gene confirms the impor- x
comparisons of frequencies with the X2 test or the Yates corrected 2 test
tance of translated CAG repeat expansions in the pathogenesis of when necessary.

nature genetics volume 17 september 1997 69

article • © 1997 Nature Publishing Group http://www.nature.com/naturegenetics
Analysis of CAG repeats. Genotyping of trinucleotide repeats was per-
formed by PCR amplification, using primers 4Ul024 (S'-TGTTACATTG-
TAGGAGCGGAA-3') and 4U716 (S '-CACGACTGTCCCAGCAT-
CACTT-3'), as described elsewherel8.25 •
GenBank accession number. SCA7 eDNA; AJ000517.
Acknowledgements
We thank]. Bou, A. Camuzat, P. Dijan, ].M. Garnier, M. Holmberg, P. Michel,
C. Penet, G. Pietu, Y. Pothin, ]. Tassin, Y. Trottier, S. Vi~aire and]. Zlotogora for
their contributions to this study. This research was supported by the Association
Frantaise contre les Myopathies (A.F.M.), the Centre National de la Recherche
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Scientifique, the lnstitut National de Ia Sante et de Ia Recherche Medicale, the
VERUM Foundation, the Groupement d'Btudes et de Recherches sur les
G&wmes (N" GREG9794), the CHRU ofStrasbourg, the Association pour le
Developpement de Ia Recherche sur les Maladies Genetiques Neurologiques et
Psychiatriques, the Association Franfaise Retinitis Pigmentosa-Retina France,
Biomed (N" CEE BMH4-CT%0244) and Biomed Concerted Action (N" CEE
BMH1-CT94-1243 ). G.D. was supported by the Association Franfaise Retinitis
Pigmentosa-Retina France and the Federation des Aveugles et Handicapes
Visuels de France. G. C., G.l. and H. D. and R.G. were supported by the AFM, the
Association Huntington France and NIH grant ROI HG000358, respectively.
Received 30 May; accepted 23 July 1997.
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nature genetics volume 17 september 1997