Trinucleotide Repeats in 202 Families With Ataxia: Title and subTitle BreakA Small Expanded (CAG)_n Allele at the SCA17 Locus FREE

THE SPINOCEREBELLAR ataxias (SCAs) are neurodegenerative disorders that are clinically and genetically heterogeneous. Ten genetically different SCAs are known to be caused by trinucleotide repeat (TNR) expansions. In the dominant SCAs, the mutant proteins show an expanded polyglutamine tract in SCA1, SCA2, Machado-Joseph disease (MJD), SCA6, SCA7, and dentatorubropallidoluysian atrophy (DRPLA),^{1 - 9} whereas SCA8 and SCA12 are caused by untranslated (CTG)_n and (CAG)_n expansions, respectively.^{10 - 11} In Friedreich ataxia (FRDA), the mutant protein is deficient in homozygotes for a (GAA)_n expansion in intron 1 of the FRDA gene.¹² Recently, an expanded CAG repeat tract has been found at the TATA-binding protein gene (TBP) in an isolated patient with symptoms of ataxia and intellectual deterioration.¹³ TBP repeat expansions have since been described in 4 Japanese families affected by a new type of ataxia with dementia named SCA17.¹⁴ Matsuura et al¹⁵ found a large expansion of pentanucleotide (ATTCT)_n in intron 9 of the SCA10 gene in patients with spinocerebellar ataxia type 10, thus being the first to show the existence of a new class of dynamic mutations.

Function of the genes involved is known only for SCA6, SCA12, and SCA17. SCA6 is due to a small CAG expansion (21-33 CAGs) in the coding region of a voltage-gated calcium channel α-subunit gene (CACNA1A).⁹ SCA12 is caused by a CAG expansion in the 5′-untranslated region of a brain-specific regulatory subunit of the protein phosphatase PP2A gene.¹¹ The recently described SCA17 is caused by a (CAG)_n expansion in the coding region of the transcription factor TBP gene.¹³

Associations between prevalence of dominantly inherited SCAs and frequency of large normal (CAG)_n alleles have been found in Japanese and European populations, indicating that these may contribute to the generation of expanded alleles in the SCAs.¹⁶

In an attempt to improve our understanding of TNR expansions leading to SCA, we have (1) assessed repeat size at the SCA1, SCA2, MJD1,SCA6, SCA7, SCA8,SCA12,SCA17/TBP, DRPLA, and FRDA loci in a large group of Portuguese and Brazilian patients with ataxia; (2) examined TNR distributions of normal alleles at these loci; (3) determined the frequency of TNR expansions; (4) compared frequencies of large normal alleles with relative frequencies of SCA at each loci; and (5) looked for genotype-phenotype correlations.

Individuals with SCAs who were referred to UnIGENe, Instituto de Biologia Molecular e Celular (Porto, Portugal) between 1997 and 2000 were included on a consecutive basis. These individuals were referred to this laboratory for the molecular diagnosis of SCAs. Dominant inheritance was assumed based on the presence of at least 1 affected family member in 2 or more successive generations. Recessive transmission was presumed when the patient had a history of consanguinity or siblings affected without (aged) affected parents. An isolated occurrence was assumed in the absence of a family history. We studied 202 unrelated families: 145 lived in Portugal, and 57 in Brazil. Dominant inheritance was apparent in 106 families, whereas recessive transmission was suspected in 33 Portuguese families; 63 individuals had isolated cases of SCA.

Peripheral blood was collected from patients and their relatives after written informed consent was obtained. Genomic DNA was isolated from peripheral blood leukocytes using standard techniques.¹⁷

Molecular analysis of the CAG and CTG repeat loci were performed by polymerase chain reaction (PCR) amplification using the published primer sequences;^{1 ,4 ,6 ,8 - 11 ,18} the PCR was carried out with 1µM of each primer, 200µM of deoxynucleotides, 1.0mM of magnesium chloride, 10mM of Tris (pH 9.0), 50mM of potassium chloride, 1 U of Taq polymerase, and 2% formamide, in a final volume of 25 µL. Samples were processed as previously described.^{6 ,9 ,19 - 20} Polymerase chain reaction products were analyzed on 6% polyacrylamide gels. Allele sizes were determined by comparing migration relative to an M13 sequencing ladder. DNA sequencing was performed to accurately assess repeat size and the presence of interruptions. Sequencing reactions were performed using a ThermoSequenase DNA sequencing kit (USB, Cleveland, Ohio) with 5 µL of amplified DNA. Both the CTA and CTG repeats on the SCA8 gene are polymorphic, and PCR assay determines the combined size of the 2 repeats.

The analysis of the intronic (GAA)_n on the FRDA gene was performed by PCR using primers 2500F¹² and 104FGAA²¹ for expanded alleles, and primers GAA-R and GAA-F for normal alleles, following the conditions described.²² Expanded allele sizes were analyzed by comparing migration relative to molecular weight standards. Normal sizes were assessed by analysis of fluorescent-labeled PCR products in an automated DNA sequencer (model 4200; Li-COR, Lincoln, Neb) using 5.5% Long Ranger gels (FMC Bioproducts, Rockland, Me).

Possible differences between Portuguese and Brazilian groups in normal repeat frequency distributions were assessed using 2 nonparametric tests: the Kolmogorov-Smirnov 2-sample test and the Mann-Whitney U test. Allele frequencies at each locus were estimated by the gene count method, and heterozygosity (H) was calculated as

where X_i is the estimated frequency of the ith allele at the locus. Statistical analyses of differences in estimates of heterozygosity between Portuguese and Brazilian individuals, as well as differences in the frequency of large normal alleles for each locus, were performed with the Fisher exact test.

The frequency distributions of normal alleles in the dominant SCAs caused by translated TNR expansions in the Brazilian group were not significantly different from those in the Portuguese group (P>.05 using the Mann-Whitney U test and Kolmogorov-Smirnov 2-sample test) (Figure 1). In the case of the SCA2 locus, the normal (CAG)_n alleles ranged in size from 17 to 31 repeats in the Portuguese group and from 19 to 24 repeats in the Brazilian group; the allele with 22 repeats was the most frequent in both groups. No significant differences were found between the 2 groups at the most recently identified SCA8, SCA12, and SCA17/TBP ataxia loci (Figure 1). Interestingly, no SCA8 alleles smaller than 18 repeats and larger than 35 repeats in the Portuguese patients, or larger than 30 repeats in the Brazilian patients, were observed. A third class of extremely large normal alleles at the SCA8 locus, varying in size from 40 to 91 CTG repeats,²³ was not found in either group; this might be due to the small sample size. At the SCA12 locus, small normal alleles were closely distributed around an allele with 10 CAGs, which represented more than 65% of all normal alleles in each group; the other class of normal alleles comprised CAG repeat sizes of 12 to 18 units. A large normal SCA12 allele containing 28 CAG repeats was also observed in the Portuguese group. The recently described SCA17/TBP ataxia locus presented a distribution of normal allele sizes varying from 29 to 40 CAGs in both groups, with distributions peaking at the allele with 38 CAG units. At the FRDA gene, normal alleles can be subdivided into 2 classes depending on their GAA repeat length: short normal alleles, with 5 to 10 GAA triplets, and long normal alleles, with 12 to 60 GAA triplets.²⁴ At this locus, long normal alleles represented 18% and 22% of Portuguese and Brazilian individuals, respectively (Figure 2A). Although the Brazilian group had a slightly larger proportion of long normal alleles, this difference was not significant.

The most polymorphic loci were DRPLA, MJD1, SCA1, SCA8, SCA17/TBP, and SCA6, whereas SCA2, SCA7, and SCA12 were the least polymorphic ones (Table 1).

Analysis of loci involved in the SCAs showed that 110 unrelated patients (55%) had ataxia due to a TNR expansion. Analysis of loci with translated (CAG)_n tracts showed that 78 unrelated patients with ataxia had 1 allele in the expanded range. Expansions at the MJD1 locus were found in 114 individuals, 102 affected and 12 asymptomatic, from 67 families that had ataxia with dominant inheritance (63%), as well as in 4 isolated cases (6%). Six individuals (5 affected) from 3 families with dominant ataxia (3%) had an expanded allele at the SCA2 gene. An expanded allele at the DRPLA locus was found in 5 patients and 1 possibly affected individual, from 2 kindreds with dominant ataxia (2%). One patient from a family that had ataxia with dominant inheritance (1%) had an expanded allele at the SCA6 locus. Three patients from 1 kindred with dominant ataxia (1%) had 1 expanded allele at the SCA7 gene. Analysis of the untranslated (CTG)_n at the SCA8 gene showed that 4 patients from 2 unrelated families with dominant ataxia (2%), as well as 4 isolated cases (6%), exhibited 1 expanded allele. Expansions of the intronic (GAA)_n at the FRDA gene (Figure 2B) were found in both alleles in 31 patients from 21 families with recessively inherited ataxia (64%) and in 5 isolated cases (8%). Table 2 shows the frequency of TNR expansions for each SCA by geographic origin.

Evidence from several populations has suggested that the disease prevalence of many of these SCAs may be associated with the presence of large normal alleles at the respective loci.¹⁶ To study the frequency of TNR expansions in our 2 groups relative to the frequency of normal alleles of larger size at the various loci, we used the criteria of Takano et al¹⁶ (Table 3). Large normal alleles at the SCA1 (>34 repeats) and SCA6 (>13 repeats) loci were significantly more frequent in Brazilian individuals than in the Portuguese individuals. Normal alleles in the upper tail of the SCA2 distribution (>22 repeats) appeared to be overrepresented in the Portuguese group, but the P value obtained was of borderline statistical significance.

The clinical phenotypes were very heterogeneous. All of our affected individuals showed clinical symptoms of cerebellar ataxia, with or without other associated features. Epilepsy was present in patients with DRPLA from 1 family, only cognitive impairment was observed in some patients with DRPLA and SCA8, and visual impairment was seen in affected individuals with SCA7. Two of the 3 families with SCA2 had been thought to have MJD. The families with SCA6, SCA7, and SCA8 and 1 of the 2 kindreds with DRPLA did not have a previous clinical diagnosis of these disorders. Altogether, 13 patients with no family history tested positive for a TNR expansion: 4 isolated patients had a mutation for MJD (2 of them having a clinical diagnosis of possible MJD), 3 of the 5 isolated patients with FRDA expansions had a clinical diagnosis of the disease, and the (CTG)_nexpansion at the SCA8 gene was present in 4 isolated cases. Table 4 shows the mean age at onset and expanded allele size ranges for patients with TNR expansions.

The analysis of the SCA2 gene in our patients disclosed an allele with 32 CAGs in a 48-year-old isolated patient who had been clinically diagnosed as having atypical FRDA. Her age at onset of gait ataxia was 2 years; during the following years, she developed a spinocerebellar syndrome. The disease progressed rapidly as time passed, with evidence of neuropathy and a mild cognitive impairment; by age 30 years, she was nonambulatory. We first tested the FRDA gene and identified 2 normal alleles; thus, we have proceeded with the analysis of other known SCA mutations and detected an allele with 32 repeats at the SCA2 gene. Sequence analysis of this allele showed an interrupted CAA repeat configuration: therefore, this is probably not a pathogenic allele. Moreover, a recently performed muscle biopsy showed ragged-red and cytochrome oxidase–negative fibers as well as reduced activity of the mitochondrial respiratory chain on polarography.

Besides the mutations described previously, the analysis of the CAG repeat at the TBP gene showed an expanded allele with 43 units in a 64-year-old patient with ataxia. Sequence analysis of this allele showed an interrupted repeat configuration of (CAG)₃(CAA)₃(CAG)₉ CAACAGCAA(CAG)₂₃CAACAG, encoding 43 glutamines. This patient began experiencing symptoms of gait ataxia at age 52 years. During the last 5 years, he exhibited progressive mental deterioration and dementia. The family history indicated that his deceased mother presumably had the same disease. There are no other patients in this family. The normal allele range previously described for TBP was 25 to 42 CAG repeats.^{13 ,25 - 27}

The analysis of TNR sizes at the loci implicated in SCA in a large group of Portuguese and Brazilian patients allowed the identification of an expansion in more than half of them, including a significant number without a family history. This study showed that DRPLA disease, frequent in Asian populations²⁸ but rare in European individuals,¹⁶ was present in the Portuguese families. On the other hand, no expanded alleles were seen at the SCA12 locus, confirming previous impressions that they are very rare.¹¹ (GAA)_n expansions at the FRDA gene were also frequent among our recessive Portuguese families with ataxia (64%). The genetic basis of approximately 40% of families with ataxia remains unknown.

In most cases of SCAs, phenotypic features are helpful indicators of the underlying genotype. Dementia and seizures associated with symptoms of cerebellar ataxia are characteristic features of DRPLA²⁹ ; however, in 1 of our families with the DRPLA mutation, the only patient available had symptoms of cerebellar ataxia alone.

We identified the molecular basis in 13 patients with ataxia who had no family history. Eight (13%) did not have a clinical diagnosis of a specific SCA type. A recent study has also shown that 19% of cases of apparently idiopathic ataxia were due to TNR expansions.³⁰ We propose that all isolated individuals with ataxia of unknown cause should be investigated for TNR expansions at known SCA loci.

The normal CAG size range described for the TBP gene (in 2525 chromosomes belonging to individuals of European, Asian, African American, and Hispanic origin) was 25 to 42 repeat units.^{13 ,25 - 27} Previous findings have implicated a de novo expansion of 63 CAG repeats at the TBP gene in a 14-year-old Japanese patient, confined to a wheelchair at age 13 years with symptoms of ataxia and intellectual deterioration.¹³ Expansions in the SCA17/TBP gene have also been found in 4 Japanese families with ataxia and dementia in which expanded alleles were 47 to 55 CAG units.¹⁴ We found a TBP allele with 43 CAG units in a 64-year-old affected individual with mild ataxia and dementia; the late onset of disease as well as the mild clinical symptoms seem to correlate with the small size of the expanded allele (compared with the early onset observed in the patient with 63 glutamines).

Genetic diagnosis and counseling may sometimes be difficult with these diseases caused by TNR expansions. In SCA1, for instance, there is no gap between normal and pathological alleles, whereas SCA2 alleles with 32 and 33 CAGs have been considered of indeterminate significance. Normal SCA1 and SCA2 CAG repeats are interrupted by CAT or CAA triplets, respectively, whereas pathogenic expansions are pure, uninterrupted CAG repeats.^{6 ,29} Uninterrupted alleles with 32 CAGs³¹ and 33 repeat units³² have been found in young, at-risk subjects in families with SCA2, both resulting from the contraction of alleles with 40 repeats. Recently, another SCA2 allele with 33 uninterrupted CAG units was found in a patient with a mild balance problem; this patient had an onset at age 60 years.³³ We found an SCA2 allele with 32 CAGs interrupted by a CAA triplet in a patient with a childhood onset of severe ataxia. The severe phenotype, the altered function of the mitochondrial respiratory chain, and the presence of a CAA interruption suggest that this SCA2 allele is not the cause of the disease in this case. As in SCA1, in SCA2 there is no gap between normal and pathological alleles.

Our present findings indicate that for both SCA2 and SCA6 TNRs, large normal alleles may contribute to the generation of expanded alleles and thus to the relative frequency of these SCAs in the 2 groups. For the remaining loci, the frequency of normal alleles was similar in the 2 groups. Of interest is the fact that no association between the frequency of DRPLA and that of large normal alleles was found in the Portuguese group. This may be owing to the effect of a specific haplotype associated with large normal alleles, shared with patients who have DRPLA, that is prone to further expansion into the disease range. In a group of Japanese individuals, all expanded and intermediate DRPLA alleles shared a unique haplotype that is frequent in Asian populations and is usually associated with large normal alleles.²⁸ The same haplotype has also been found in European patients, although it rarely occurs in normal chromosomes in this population. This suggests that DRPLA expanded alleles in Japanese²⁸ and Portuguese patients may have evolved from a common founder chromosome. Haplotype analyses in our families with DRPLA are now being carried out to test this hypothesis.

Accepted for publication December 19, 2001.

Author contributions: Study concept and design (Drs Silveira, Januário, Margolis, Koide, Tsuji, and Holmes and Ms Guimarães); acquisition of data (Drs Pinto-Basto, Vale, Parreira, Januário, Barbot, Tuna, Barros, Holmes, Margolis, Jardim, Pandolfo, Coutinho, and Sequeiros, Messrs Miranda, Moreira, Mendonça, and Coelho, and Mss Alonso, Ferro, Ferreirinha, and Poirier); analysis and interpretation of data (Drs Barbot, Koide, Pandolfo, Tsuji, and Sequeiros, Mr Miranda, and Ms Poirier); drafting of the manuscript (Drs Silveira, Januário, Margolis, Koide, Tsuji, and Holmes and Ms Guimarães); critical revision of the manuscript for important intellectual content (Drs Pinto-Basto, Parreira, Vale, Januário, Barbot, Tuna, Barros, Jardim, Coutinho, and Sequeiros, Messrs Mendonça and Coelho, and Mss Moreira, Alonso, Ferro, Ferreirinha, and Poirier); statistical expertise (Ms Guimarães); obtained funding (Drs Barbot, Pandolfo, and Sequeiros); administrative, technical, and material support (Drs Pinto-Basto, Vale, Januário, Tuna, Barros, Koide, Tsuji, Holmes, Margolis, Pandolfo, and Sequieros, Messrs Miranda, Moreira, Mendonça, and Coelho, and Mss Alonso, Ferro, Ferreirinha, and Poirier); study supervision (Drs Silveira, Pandolfo, Coutinho, and Sequeiros).

This work was supported by research grant PRAXIS/SAU/13226/1998 and the Financiamento Plurianual de Unidades de Investigação from Fundação para a Ciência e Tecnologia (FCT), Lisbon, Portugal. Messrs Miranda and Mendonça and Mss Moreira, Alonso, Ferro, and Ferreirinha are the recipients of scholarships from the PRAXIS Programme, FCT.

We thank C. A. Ross, PhD, for collaboration, C. Costa, MD, for referring patients, and the family members for cooperation.

Corresponding author and reprints: Isabel Silveira, UnIGENe, IBMC, Rua do Campo Alegre 823, 4150-180, Porto, Portugal (e-mail: isilveir@ibmc.up.pt).