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Anew locus for spinocerebellar ataxia (SCA21) maps to chromosome 7p21.3-p15.1

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A New Locus for
Spinocerebellar Ataxia
(SCA21) Maps to
Chromosome 7p21.3-p15.1
Isabelle Vuillaume, MD,1 David Devos, MD,3
Susanna Schraen-Maschke, PharmD, PhD,1,2
Christian Dina, PhD,4 Arnaud Lemainque, PhD,5
Francis Vasseur, PhD,4 Guy Bocquillon, BS,1
Patrick Devos, BS,1 Carole Kocinski, BS,1
Christiane Marzys, BS,1 Alain Destée, MD, PhD3
and Bernard Sablonnière, MD, PhD1,2
We investigated a French family with a new type of autosomal dominant spinocerebellar ataxia that was excluded from all previously identified genes and loci. The
patients exhibited a slowly progressive gait and limb
ataxia variably associated with akinesia, rigidity, tremor,
and hyporeflexia. A mild cognitive impairment also was
observed in some cases. We performed a genomewide
search and found significant evidence for linkage to chromosome 7p21.3-p15.1. Analysis of key recombinants and
haplotype reconstruction traced this novel spinocerebellar
ataxia locus to a 24cM interval flanked by D7S2464 and
D7S516.
Ann Neurol 2002;52:666 – 670
The autosomal dominant cerebellar ataxias (ADCAs)
constitute a group of neurodegenerative diseases that
can be classified clinically into three categories.1 All
ADCAs are characterized by ataxia variably associated
with other neurological features. Clinical features are
variable among ADCAs, even in the same family, and
genetic studies have shown a great heterogeneity in
these diseases.2 Thus, classification based on the responsible genes or genetic loci is becoming prevalent.
Molecular genetic studies have shown that ADCAs are
genetically heterogeneous, and 15 different loci already
have been reported to date.2– 4 Among these loci, causative genes have been further identified as expansions of
trinucleotide (CAG) repeats for SCA1 (MIM 164400),
SCA2 (MIM 183090), SCA3 (MIM 109150), SCA6
(MIM 183086), SCA7 (MIM 164500), and more recently for SCA17 (MIM 600075),5 whereas untranslated
CTG or CAG tracts are identified in SCA8 (MIM
603680) or SCA12 (MIM 604326). In SCA10 (MIM
603516), the responsible gene has been identified as a
large expansion of a ATTCT repeat tract.6 However, the
genes or loci have not been identified in approximately
40% of French ADCA families,4 implying the presence
of other unidentified responsible genes. We recently
have identified a four-generation French family that segregates a new form of ADCA.7 All known ADCA genes
and loci were excluded by direct mutation or geneticlinkage analysis. Using a genomewide linkage analysis,
we mapped the locus for this family to chromosome
7p21.3-p15.1. This novel locus was registered as
SCA21, with approval from the Human Genome Nomenclature Committee.
Subjects and Methods
Subjects
The subjects were recruited from a four-generation French
family in which a new form of ADCA was ascertained, clinically characterized by gait and limb ataxia, akinesia, hyporeflexia, and mild cognitive impairment. The magnetic resonance imaging showed atrophy of the cerebellum without
involvement of the brainstem7 and basal ganglia (data not
shown). Twenty-three family members were examined by at
least one of us (D.D. and A.D.), and 15 met the disease
criteria defined as the presence of gait or limb ataxia. After
informed consent (approved by the institutional ethics committee of CHRU) was obtained, blood samples were drawn
from 29 subjects (Fig).
Genotype Analysis
From the 1Unité Fonctionnelle de Neurobiologie, Laboratoire de
Biochimie et Biologie moléculaire; 2Institut National de la Santé et
de la Recherche Médicale U422; 3Fédération de Neurologie, Hôpital R. Salengro, Faculté de Médecine and Centre Hospitalier Régional et Universitaire; 4CNRS UPRESA 8090, Institut de Biologie
de Lille, Lille; and 5Centre National de Génotypage, Evry, France.
Received Mar 18, 2002, and in revised form Jun 18. Accepted for
publication Jun 21, 2002.
Published online Aug 25, 2002, in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.10344
Address correspondence to Dr Sablonnière, Unité Fonctionnelle de
Neurobiologie, Laboratoire de Biochimie et Biologie moléculaire,
Hôpital R. Salengro, Centre Hospitalier Régional et Universitaire,
Bd du Professeur Leclerc, 59037 Lille Cedex, France.
E-mail: b-sablonniere@chru-lille.fr
666
© 2002 Wiley-Liss, Inc.
A genomewide screening was performed with the ABI
PRISM Linkage Map Set (version 2.0 PE; Applied Biosystems, Foster City, CA) consisting of 400 highly polymorphic
fluorescent-labeled microsatellite markers chosen from the
Généthon linkage map8 with an average spacing of 10 to
20cM. After a multiplex polymerase chain reaction (PCR)
protocol, the markers were coamplified with an average of
four markers per PCR. The amplifications were done with
the GeneAmp 9700 PCR system, with a total volume of
10␮l containing 10% PCR buffer, 3.0mM MgCl2, 0.25mM
each dNTP, between 0.65 and 4.5pmol each primer, 20ng
DNA, and 0.4 units AmpliTaq Gold. Samples were subjected to an initial denaturation step of 95°C for 12 minutes,
followed by 30 cycles of 94°C for 15 seconds, 55°C for 15
seconds, and 72°c for 30 seconds with a final extension of
72°C for 10 minutes. PCR products were pooled, purified,
Fig. Pedigree of the family in this study. (squares) Men; (circles) women. (filled symbols) Affected subjects; (open symbols) unaffected subjects; (symbol with a dot) possible carrier. (diagonal line) Deceased. Haplotypes are presented below symbols. Alleles corresponding to D7S481, D7S2514, D7S2464, D7S2557, D7S503, D7S673, D7S516, D7S2515, D7S2848, and D7S2252 are
shown from top to bottom. Black bars represent the disease haplotype. Informative recombination events place the SCA21 disease
locus between markers D7S2514 and D7S2515.
and separated with a MegaBace 1000 automated sequencer
(Amersham Biosciences, Arlington Heights, IL). Alleles were
sized using the GENETIC PROFILER Program (version
1.1).
Linkage Analysis
Linkage analysis was performed using the LINKAGE Program Package (version 5.2),9 assuming an autosomal dominant inheritance and a disease frequency of 1 to 100,000.
Pairwise logarithm of odds (LOD) scores and multipoint
LOD scores were calculated using programs from
FASTLINK 3.0P. Penetrance in each subject was assigned to
the four liability classes determined from the age at onset:
0.95 at age 40 years or more; 0.85 at age 31 to 40 years;
0.65 at age 21 to 30 years; and 0.50 at age 20 years or less.
The allele frequencies of each marker for LOD score analysis
were those defined by Généthon in the white population and
reported in the Genome Database.
Results
Clinical Features
A summary of the clinical profile of the 15 patients is given
in Table 1. Four patients (III-3, III- 5, III-7, and III-12)
classified as unaffected on initial examination 3 years ago further developed ataxia as well as hyporeflexia and thus were
classified as affected. At the time of examination, gait or limb
ataxia was present in all affected subjects. The most striking
features was a variable onset and a slow progression of the
disease. Average age at onset was 17.4 ⫾ 7.4 years (n ⫽ 14)
and ranged from 6 to 30 years. Age at onset tends to be
earlier through successive generations. In 14 informative
parent-child pairs, the mean anticipation was 10.6 ⫾ 4.6
years, ranging from 4 to 18 years.
Cerebellar testing disclosed gait and limb ataxia associated
with dysgraphia and dysarthria. Slight to mild akinesia of the
limbs was often present with inconstant rigidity, postural and
rest tremor of the limbs without response to dopa. There was
no motor deficit, no cranial nerve palsy, no pyramidal sign,
and no sensory loss, but there was hyporeflexia in all but two
Vuillaume et al: Spinocerebellar Ataxia (SCA21)
667
Table 1. Summary of Clinical Findings
Patient Characteristics
Age at examination (yr)
Age at onset (yr)
Gait ataxia
Limb ataxia
Dysarthria
Dysgraphia
Akinesia
Rigidity
Tremor
Hyporeflexia
Hyperreflexia
Mental status (MMS or IQ*)
I-2
II-3
II-5
II-8
II-10
II-12
II-13
II-15
III-2
III-3
III-5
III-7
III-10
III-12
III-13
65
30
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺
24/30
45
19
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺
28/30
42
25
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺
25/30
41
25
⫹
⫹
⫹
⫹
⫹
⫺
⫺
⫹
⫺
27/30
37
20
⫹
⫹
⫹
⫺
⫹
⫺
⫹
⫹
⫺
28/30
36
12
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫺
24/30
34
25
⫹
⫺
⫹
⫺
⫹
⫺
⫺
⫹
⫺
29/30
32
20
⫹
⫹
⫺
⫹
⫹
⫺
⫹
⫺
⫹
28/30
24
7
⫹
⫹
⫺
⫹
⫹
⫹
⫹
⫹
⫺
ND
21
20
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫺
ND
22
21
⫹
⫹
⫹
⫹
⫹
⫺
⫺
⫹
⫺
ND
11
10
⫹
⫺
⫹
⫺
⫺
⫺
⫺
⫹
⫺
ND
9
6
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫺
64*
13
13
⫹
⫹
⫺
⫺
⫺
⫺
⫺
⫹
⫺
ND
14
9
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫹
70*
MMSE ⫽ Mini-Mental State Examination; ND ⫽ not determined.
patients. The ophthalmological examination showed intermittent microsaccadic pursuit and square wave jerks without
nystagmus nor retinopathy. Neuropsychological examination
shows a cognitive impairment, moderate in two patients (I-2
and II-12) and more severe in two young children with IQs
of 64 and 70, respectively.7 Reevaluation of clinical data in
affected members of generation II showed no obvious worsening of neurological features 3 years later. Contrary to the
other family members, II-3 had a positive medical history of
neuroleptic treatment, carbon monoxide intoxication, and
early beginning alcoholism leading to several hospitalizations
and the loss of the custody of her children. The cerebellar
syndrome was unchanged, but there were orofacial and head
dyskinesia associated to a severe extrapyramidal syndrome
with axial and limbs rigidity associated to rest and action
tremor.
Genotypic and Linkage Data
The first convincing linkage evidence for the disease locus
was obtained from D7S516, for which we detected a positive LOD score of 1.93 at recombination fraction of 0.08.
We explored this result further by using 13 additional
markers from this region, selected from the Généthon linkage map8 and Cooperative Human Linkage Center database10 to confirm and refine the genetic localization. LOD
scores greater than 3.20 were obtained for six consecutive
markers around D7S503 (Zmax ⫽ 3.26 at ⌰ ⫽ 0.05), suggesting that the disease locus is linked to these markers (Table 2). Changing the allele frequencies did not have a significant effect on LOD scores. As expected by the results of
two-point LOD score analysis which suggest a candidate
interval with an identical Zmax value of 3.26 at ⌰ ⫽ 0.05
for six consecutive markers, multipoint analysis did not add
information on the localization of SCA21 locus inside this
interval (data not shown). Recombination events, visualized
by haplotype reconstruction, confirmed the results obtained
from linkage analysis in this family. Flanking markers were
defined by recombinations in affected Subjects II-15
(D7S2514, telomeric) and III-2 (D7S2515, centromeric),
delimiting an approximately 24cM region on chromosome
7p21.3-p15.1 (see Fig). Subject III-6, aged 19 years, had
the same haplotypes as the affected members but had not
presented with cerebellar ataxia nor dysmetria yet but with
an isolated hyporeflexia of the four limbs and could be an
asymptomatic carrier. Subject II-3 was classified as affected
on the basis of clinical criteria. She reported a significant
668
Annals of Neurology
Vol 52
No 5
November 2002
history of toxic exposure since the onset of symptoms at
age 19 years. Her cerebellar syndrome was generally more
severe than that of other members of similar age, with atypical associated features such as dyskinesia. All eight of her
children were examined, and none of them had ataxia nor
hyporeflexia. Subject II-3 essentially lacks the haplotype
shared by the affected, and is considered a phenocopy.
However, on the basis of her phenotype, assigned before
the genetic analysis, she is classified as affected in the LOD
score calculations.
Discussion
Clinical signs, age at onset, and duration of the disease vary
substantially between and within ADCA families.3,4,11 The
currently favored classification for late-onset ataxia is based
on genetics. As these diseases are mapped to specific genetic
loci, they are assigned specific numbers SCA1 to 17. Many
of these SCAs fall within Harding’s ADCA type I. Because
there is considerable clinical overlap among SCAs, most of
them do not truly fit into the Harding classification. It thus
appears wiser to identify them by their SCA designation.12
Patients in this family present a distinct and slowly progressive ataxia syndrome with extrapyramidal signs such a
tremor, akinesia, and rigidity that prompt us to classify this
kindred as ADCA type I. The signs that are specifically associated with other pure cerebellar ataxia such as seizures in
SCA10,13 mental retardation in SCA13,14 myoclonus in
SCA14,15 and essential head tremor in SCA1616 did not represent specific features of our disease.
Many of the known SCA mutations are unstable expansions of a CAG repeat, and intergenerational expansion of
the repeat has provided the basis for anticipation in SCA1, 2,
3, and 7. Our family shows earlier onset of the disease in
successive generations, although this apparent anticipation
may be attributable to ascertainment biases. The presence of
a dynamic mutation such as a trinucleotide repeat expansion
appeared unlikely because RED assays were inconclusive.7
Obviously, the possibility of a small trinucleotide repeat expansion or another type of expansion causing the disease
cannot be excluded.
In conclusion, we have mapped a locus on chromosome
7 responsible for a distinct slowly progressive ataxia
(SCA21) associated with a prominent extrapyramidal syndrome, a mild cognitive impairment, and near-normal eye
movements. Several genes have been identified in the
7p21.3-p15.1 region, and if none of them appeared to be
Table 2. Pairwise LOD Scores between the Disease Locus and Chromosome 7p Markers
Recombination Rate (␪)
Markers
0.00
0.01
0.05
0.1
0.2
0.3
0.4
Zmax
␪ max
D7S481
D7S2514
D7S2464
D7S513
D7S2557
D7S503
D7S493
D7S673
D7S2525
D7S516
D7S2515
D7S2848
D7S2252
D7S2250
2.56
⫺2.43
0.12
0.97
0.96
0.97
0.95
0.98
0.96
⫺0.50
⫺0.70
0.03
⫺1.25
⫺1.07
2.53
0.57
0.50
2.86
2.86
2.86
2.86
2.86
2.86
1.39
0.86
0.65
0.52
0.72
2.37
1.70
0.91
3.26
3.26
3.26
3.26
3.26
3.26
1.88
1.95
1.69
1.38
1.58
2.16
1.96
1.00
3.17
3.17
3.17
3.17
3.17
3.17
1.91
2.18
1.95
1.63
1.81
1.72
1.81
0.89
2.62
2.62
2.62
2.62
2.62
2.62
1.63
1.99
1.81
1.56
1.70
1.22
1.34
0.62
1.84
1.84
1.84
1.84
1.84
1.84
1.15
1.45
1.33
1.16
1.26
0.66
0.68
0.25
0.90
0.90
0.90
0.90
0.90
0.90
0.53
0.70
0.65
0.57
0.62
2.56
1.96
1.00
3.26
3.26
3.26
3.26
3.26
3.26
1.93
2.18
1.95
1.63
1.81
0.00
0.10
0.09
0.05
0.05
0.05
0.05
0.05
0.05
0.08
0.10
0.10
0.10
0.10
LOD ⫽ logarithm of odds.
an evident candidate for SCA21, three genes could represent potential candidates owing to their function or pattern of expression. SCIN (SCINDERIN) encodes a Ca⫹⫹
regulated member of the gelsolin superfamily of actin filaments severing proteins that appears to function in
the mechanism of release of neurotransmitters.17 SP4 transcription factor (MIM 600540) is a nuclear transcriptional
activator mainly expressed in brain which belongs to the
SP1 family of zinc-finger proteins.18 As found in TATAbinding protein, its transactivation domain contains
glutamine-rich regions suggesting the possibility of a trinucleotide repeat expansion mutation within these sequences.
A third gene, KOC1, encoding the IGF-II mRNA binding
protein 3 (MIM 146732), could represent a potential candidate, because its mouse homolog is known to regulate
neuronal differentiation.19 Identification of the specific mutation in the family in this study will contribute to the
diagnosis and genetic counseling of at-risk subjects, as well
as to our understanding of the molecular mechanisms of
SCA21. The defined candidate region (approximately
24cM) is still large and our current challenge is to narrow
this interval using additional families with linkage to this
locus, so that the search for disease-causing mutation is
more effective.
This work was supported in part by the University of Lille 2, and by
grants from the Centre Hospitalier Régional et Universitaire (Délégationà la Recherche), the Centre National de Génotypage, and the
Association pour l’Etude et la Prévention des Maladies du Système
Nerveux.
We are grateful to the family members for their participation and
Drs M. Delpech and M. Lathrop for constructive comments on
the manuscript. We are grateful to Dr P. Vermersch for referring
the first ataxia patient identified from this family to us.
References
1. Harding AE. Clinical features and classification of the inherited
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2. Subramony SH, Filla A. Autosomal dominant spinocerebellar
ataxias ad infinitum? Neurology 2001;56:287–289.
3. Klockgether T. Recent advances in degenerative ataxias. Curr
Opin Neurol 2000;13:451– 455.
4. Stevanin G, Dürr A, Brice A. Clinical and molecular advances
in autosomal dominant cerebellar ataxias: from genotype to
phenotype and physiopathology. Eur J Hum Genet 2000;8:
4 –18.
5. Nakamura K, Jeong SY, Uchihara T, et al. SCA17, a novel
autosomal dominant cerebellar ataxia caused by an expanded
polyglutamine in TATA-binding protein. Hum Mol Genet
2001;10:1441–1448.
6. Matsuura T, Yagamata T, Burgess DL, et al. Large expansion of
the ATTCT pentanucleotide repeat in spinocerebellar ataxia
type 10. Nat Genet 2000;26:191–194.
7. Devos D, Schraen-Maschke S, Vuillaume I, et al. Clinical features and genetic analysis of a new form of spinocerebellar
ataxia. Neurology 2001;56:234 –238.
8. Dib C, Faure S, Fizames C, et al. A comprehensive genetic map
of the human genome based on 5,264 microsatellites. Nature
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Mol Genet 1995;4:1837–1844.
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cerebellar ataxia: phenotypic differences in genetically defined
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type 10. Ann Neurol 2001;50:234 –239.
14. Herman-Bert A, Stevanin G, Netter JC, et al. Mapping of
spinocerebellar ataxia 13 to chromosome 19q13.3-13.4 in a
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15. Yamashita I, Sasaki H, Yabe I, et al. A novel locus for dominant
cerebellar ataxia (SCA14) maps to a 10.2-cM interval flanked
by D19S206 and D19S605 on chromosome 19q13.4-qter. Ann
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17. Trifaro JM. Scinderin and cortical F-actin are components of
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Oligodendrocytic
Polyglutamine Pathology
in DentatorubralPallidoluysian Atrophy
1
Subjects and Methods
Patients and Transgenic Mice
2
Mitsunori Yamada, MD, PhD , Toshiya Sato, MD,
Shoji Tsuji, MD, PhD,2
and Hitoshi Takahashi, MD, PhD1
White matter degeneration is one of the pathological
conditions of dentatorubral-pallidoluysian atrophy. Autopsy brains exhibited a reduced number of glial cells in
the lesions and an involvement of oligodendrocytes in
nuclear inclusion formation, which previously has been
recognized only as a pathological hallmark in neurons.
Dentatorubral-pallidoluysian atrophy transgenic mice
showed an increased number of affected glias with increasing age and with larger expansions of CAG repeats.
These findings suggest that glial cells in dentatorubralpallidoluysian atrophy also are involved in the polyglutamine pathogenesis.
Ann Neurol 2002;52:670 – 674
From the Department of 1Pathology and 2Neurology, Brain Research Institute, Niigata University, Niigata, Japan.
Received Mar 29, 2002, and in revised form Jun 24. Accepted for
publication Jun 30, 2002.
Published online Sep 25, 2002, in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.10352
Address correspondence to Dr Yamada, Department of Pathology,
Brain Research Institute, Niigata University, 1 Asahimachi, Niigata
951-8585, Japan. E-mail: nori@bri.niigata-u.ac.jp
670
© 2002 Wiley-Liss, Inc.
Dentatorubral-pallidoluysian atrophy (DRPLA)1 is a
hereditary neurodegenerative disease caused by the expansion of a CAG repeat.2,3 The major neuropathology of this disorder is a combined degeneration of the
dentatorubral and pallidoluysian systems1,4,5; however, it has shown that some DRPLA patients also
present with white matter lesions.6,7 The changes are
not correlated with the onset age, disease duration, or
disease severity6 but appear to be a function of patient age.7 Neuropathologically, the lesions showed
diffuse myelin pallor but lacked obvious ischemic
changes,5,6 and the pathogenesis remains unclear. In
this study, we conducted immunohistochemical and
morphometric analyses on human DRPLA brains to
determine whether glial cells in the white matter are
involved in the accumulation of expanded polyQ
stretches, which is a pathogenic abnormality common
to neurons of all the CAG repeat diseases. To explore
the chronological changes of glial cells and the influence of the CAG repeat size, we further extended the
study to DRPLA transgenic mice with 76 or 129 glutamines.
Twelve patients with DRPLA and six controls served as the
subjects. We divided our 12 DRPLA patients into three subgroups according to classifications by Naito.8 Clinical findings of the patients are summarized in Table 1. Ten of the
12 patients also were examined genetically under informed
consent. We also examined the transgenic mice harboring a
single copy of a full-length human mutant DRPLA gene
with 76 CAG repeats9 or with 129 CAG repeats.10 The Q76
mice were examined at 14 (n ⫽ 3) and more than 100 weeks
of age (n ⫽ 5), but because the Q129 mice died by 16 weeks
of age,10 examinations were restricted to 14 weeks of age
(n ⫽ 3). Instead of aged Q129 mouse brains, we examined a
brain of the mosaic mouse at 129 weeks of age carrying 76
and 129 CAG repeats.
Immunohistochemistry
Formalin-fixed, paraffin-embedded brain sections were
stained with hematoxylin and eosin and Klüver-Barrera
stains. For immunohistochemistry, sections were immunostained using mouse monoclonal antibodies against neurofilament (1:50; Sanbio, Uden, Netherlands) or expanded
polyQ stretches (1C2, 1:16,000; Chemicon, Tenecula, CA
as described previously.11 We used diaminobenzidine as the
chromogen. To characterize the 1C2-immunopositive cells,
we also subjected the sections to double immunostaining
combined with rabbit anti 䡠 sera against glial fibrillary
acidic protein (GFAP; DAKO, Glostrup, Denmark;
1:2,000) or against transferrin (TF, 1:2,000; DAKO) as the
marker of oligodendroglia.12 GFAP or TF was detected using a VECTASTAIN ABC-AP kit (Vector Laboratories,
Burlingame, CA) and Vector Blue as the chromogen. For
negative controls, the primary antibodies were replaced
with normal sera.
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ataxia, locus, maps, spinocerebellar, anew, p15, chromosome, 7p21, sca21
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