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The SCA12 Mutation as a Rare Cause of Spinocerebellar Ataxia
Jeremy A. Cholfin, BS;
María-Jesús Sobrido, MD, PhD;
Susan Perlman, MD;
Stefan M. Pulst, MD;
Daniel H. Geschwind, MD, PhD
Arch Neurol. 2001;58:1833-1835.
ABSTRACT
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Background Spinocerebellar ataxias are a group of
phenotypically and genetically heterogeneous disorders characterized by
progressive degeneration of the cerebellum. The expansion of a CAG
repeat upstream of the PP2APR55ß gene has been recently
reported as a novel cause of a dominantly inherited ataxia (SCA12) in a
kindred with limb tremor as an early feature.
Objective To explore the relative frequency of SCA12
among familial and sporadic spinocerebellar ataxias in an ethnically
diverse patient population.
Methods We used polymerase chain reaction to analyze
CAG repeat size in a series of patients presenting to an ataxia clinic
in California.
Results The SCA12 expansion was not detected in any of the
cases investigated. The largest allele found had 22 repeats, a finding
within the proposed nonpathogenic range. Distribution of repeat size
and heterozygosity were similar to that described previously.
Conclusions These results, coupled with findings in
other populations, indicate that the SCA12 mutation is a rare cause of
spinocerebellar degeneration. Diagnostic testing for SCA12 should be
considered in patients with cerebellum disorders and an atypical
clinical phenotype, especially when tremor is initially present.
INTRODUCTION
THE SPINOCEREBELLAR ataxias (SCAs) are a heterogeneous group of inherited neurodegenerative
disorders that primarily affect the cerebellum, brainstem, and spinal
tracts.1 At least 13 genetic loci and 7 different SCA genes
have been identified. SCA1, SCA2, SCA3/MJD,
SCA6, and SCA7 are caused by expansions of CAG
nucleotide repeats in coding regions of the corresponding
genes.2 However, the genetic defect underlying a
significant subset of SCAs remains to be elucidated.
Recently, Holmes et al3 described a large pedigree (family
R) with a novel form of autosomal dominant ataxia (SCA12) that was
associated with the expansion of a CAG repeat that lies upstream of the
gene encoding a brain-specific regulatory subunit of protein
phosphatase 2A (PPP2R2ß) on chromosome 5q31-33. The number of CAG
repeats was 7 to 28 in healthy controls and 66 to 78 in affected family
members. Although patients with the SCA12 mutation displayed clinical
features similar to those found in many SCAs, the syndrome was somewhat
unusual in that it presented initially with upper extremity tremor and
led to dementia in some cases.3, 4 No instance of expansion
was detected in 394 neurologically normal subjects and 1099 individuals
with neurologic disease, including 748 subjects with ataxia, suggesting
that SCA12 is uncommon among spinocerebellar
degenerations.3 Most subjects studied were of European
ancestry.3 It is, therefore, important to screen additional
series of patients for expansions to determine the relative prevalence
of SCA12 among ataxias. Herein, we investigated the SCA12 mutation in
an ethnically heterogeneous group of patients with
ataxia.
METHODS
We examined 211 patients from 180 families of diverse ethnic
background with inherited or sporadic ataxia.5 All subjects
were ascertained for spinocerebellar degeneration in the University of
California, Los Angeles, Neurological Services ataxia clinic. Cases
were classified by mode of inheritance and subdivided according to
clinical features; those with an autosomal dominant pattern of
inheritance were categorized using Harding's
classification.1 After obtaining informed consent, genomic
DNA was purified from peripheral blood leukocytes using the Puregene
kit (Gentra Systems, Plymouth, Minn). The SCA12 repeat was
amplified by polymerase chain reaction (PCR) in 20 µL of total volume
reactions containing 40 ng of DNA; 1pM of IRD-700–labeled primer
(5'-TGCTGGGAAAGAGTCGTG-3'); 10pM of unlabeled primer
(5'-GCCAGCGCACTCACCCTC-3'); 250µM of
deoxyguanosine triphosphate, deoxycytidine triphosphate, deoxythymidine
triphosphate, and deoxyadenosine triphosphate; 1x PCR
buffer containing 1.5mM magnesium chloride; 1x Q-Solution
(Qiagen, Valencia, Calif); and 1.0 U of Taq DNA polymerase
(Qiagen). After an initial denaturation step of 5 minutes at
95°C, 33 cycles of PCR were performed at 95°C for 45 seconds,
60°C for 30 seconds, and 72°C for 45 seconds, followed by a
final extension at 72°C for 10 minutes. The PCR products were loaded
on 6% acrylamide gels and electrophoresed in an automated sequencer
(LI-COR, Inc, Lincoln, Neb) at 1500 V for 4 hours at 50°C. Fragment
sizes were determined with GeneImagIR computer software version 3.0
(LI-COR, Inc). The number of CAG repeats was calculated using
the following formula:
RESULTS
One hundred eighty kindreds were screened for the SCA12 mutation in the
present study, including families of Chinese, Japanese, Southeast
Asian, East Indian, Middle Eastern, Hispanic, African American, and
European ethnic origin. There were 96 patients (45%) with a
dominant family history, 97 patients (46%) with a
recessive or sporadic ataxia, and 18 patients (9%) in whom the mode of
inheritance could not be determined. Table 1 shows the patient phenotypic
distribution.
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Classification of Ataxia Patients*
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The PCR amplification of the SCA12 locus gave a pattern of 2
distinct alleles in most cases analyzed. Under the conditions used, the
presence of spurious stutter bands was minimized and did not interfere
with genotype interpretation. Allele sizes ranged from 9 to 22 CAG
repeats, with (CAG)10 being the most frequently encountered
(Figure 1). Heterozygosity was
61.6%.
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Distribution of CAG repeat size at the SCA12 locus in patients
with ataxia. All 211 patients in our sample are included (422
chromosomes). No expansions were detected.
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The SCA12 expansion was not detected in any of the patients studied. A
control sample with known allele sizes of 10 and 78 CAG repeats was
included in all experiments. The genotype of this sample showed clearly
visible alleles of the expected size, therefore minimizing the
possibility that expanded alleles in this size range were missed
because of inefficient amplification.
COMMENT
An expansion of a novel CAG repeat on chromosome 5 has been
associated with spinocerebellar ataxia in a kindred of German
descent.3 Recently, a second family of different ethnic
origin with a similar phenotype was identified as having SCA12
expansions between 55 and 61 triplet repeats.6 In the
present study, we did not detect the SCA12 mutation in 96 subjects with
dominant cerebellar ataxia, which includes a diverse, unselected
representation of ethnic groups. Similarly, in a recent
report,7 no expansions were found in 392 unrelated patients
from the United Kingdom, including 99 with a dominant family history.
Furthermore, no expanded SCA12 alleles were detected in
recessive or sporadic cases of ataxia in the present study or in
previous reports.3, 7, 8 The unusual clinical presentation in
affected members of family R and the rarity of CAG repeat expansion at
the SCA12 locus among patients with ataxia suggest
that SCA12 should be considered in cases with atypical phenotype,
especially if limb tremor is present in the initial clinical
picture. However, expansions at the SCA12 locus are rare in
more typical dominant ataxias and account for very few cases of
spinocerebellar degeneration.
The SCA12 allele distribution and heterozygosity index in our
patients were similar to those previously described.3, 7 A
(CAG)10 repeat was the most frequently detected allele
among patients with ataxia in the present study. The largest allele
found had 22 repeats, which is
within the proposed nonpathogenic range
suggested by other reports.3, 7 Additional studies of
patients with expansions are required to better define the pathogenic
range of SCA12 and to investigate whether a correlation exists between
repeat size and severity of the disease.
Recent functional genomic studies have shown that the SCA12 mutation
affects the expression of a downstream reporter gene in
vitro,9 suggesting that the CAG expansion near the 5' end
of the PPP2R2ß gene is pathogenic and not a benign
polymorphism in linkage disequilibrium with a causal mutation. However,
the molecular and cellular effects of the SCA12 mutation remain to be
characterized.
SCA12 and some of the other mutations recently associated with ataxia
are rare,10 suggesting that a number of as yet unidentified
genetic defects underlie spinocerebellar degeneration of unknown
origin.
AUTHOR INFORMATION
Accepted for publication March 30, 2001.
This study was supported by a fellowship from the Fundación Pedro
Barrié de la Maza, La Coruña, Spain (Dr Sobrido),
and by grant NS33123 from the National Institutes of Health,
Bethesda, Md (Dr Pulst).
We thank the patients and their families for their participation and
Russell Margolis, MD, for providing control SCA12 patient DNA. We
gratefully acknowledge the Smith family for their generous donation in
memory of Waverly Smith, and the National Ataxia Foundation,
Minneapolis, Minn, for providing pilot grant suport for the University
of California, Los Angeles, ataxia research program.
Reprints not available from the authors.
From the Neurogenetics Program, Department of
Neurology, University of California, Los Angeles School of Medicine (Mr
Cholfin and Drs Sobrido, Perlman, and Geschwind), and Division of
Neurology, Cedars-Sinai Medical Center, Los Angeles (Dr Pulst).
REFERENCES
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ABSTRACT
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