Interruptions in the Expanded ATTCT Repeat of Spinocerebellar Ataxia Type 10: Repeat Purity as a Disease Modifier?

Spinocerebellar ataxia type 10 (SCA10) is one of numerous genetic disorders that result from simple repeat expansions. SCA10 is caused by expansion of an intronic ATTCT pentanucleotide repeat tract. It is clinically characterized by progressive ataxia, seizures, and anticipation, which can vary within and between families. We report two SCA10 families showing distinct frequencies of seizures and correlations of repeat length with age at onset. One family displayed uninterrupted ATTCT expansions, whereas the other showed multiple interruptions of the repeat by nonconsensus repeat units, which differed both in the length and/or sequence of the repeat unit. Disease-causing microsatellite expansions have been assumed to be composed of uninterrupted pure repeats. Our findings for SCA10 challenge this convention and suggest that the purity of the expanded repeat element may be a disease modifier. Spinocerebellar ataxia type 10 (SCA10) is one of numerous genetic disorders that result from simple repeat expansions. SCA10 is caused by expansion of an intronic ATTCT pentanucleotide repeat tract. It is clinically characterized by progressive ataxia, seizures, and anticipation, which can vary within and between families. We report two SCA10 families showing distinct frequencies of seizures and correlations of repeat length with age at onset. One family displayed uninterrupted ATTCT expansions, whereas the other showed multiple interruptions of the repeat by nonconsensus repeat units, which differed both in the length and/or sequence of the repeat unit. Disease-causing microsatellite expansions have been assumed to be composed of uninterrupted pure repeats. Our findings for SCA10 challenge this convention and suggest that the purity of the expanded repeat element may be a disease modifier. Unstable microsatellite repeats in disease-associated genes cause a large number of inherited neuromuscular and neurological disorders on expansion to abnormal sizes (Zoghbi and Orr Zoghbi and Orr, 2000Zoghbi HY Orr HT Glutamine repeats and neurodegeneration.Annu Rev Neurosci. 2000; 23: 217-230Crossref PubMed Scopus (1053) Google Scholar; Ranum and Day Ranum and Day, 2002Ranum LPW Day JW Dominantly inherited non-coding microsatellite expansion disorders.Curr Opin Genet Dev. 2002; 12: 266-271Crossref PubMed Scopus (101) Google Scholar). Expansion mutations can result in disease through a variety of mechanisms including suppression of gene expression, as in fragile X syndrome (FMR1 [MIM 309550]) and Friedreich ataxia (FRDA [MIM 229300]). Alternatively, normal protein function may be altered, as in Huntington disease (HD [MIM 143100]), Kennedy disease (SBMA [MIM 313200]), dentatorubropallidoluysian atrophy (DRPLA [MIM 125370]), and a number of spinocerebellar ataxias (SCA) caused by the expansion of CAG trinucleotide repeats coding polyglutamine tracts. A third mechanism appears to involve generation of toxic RNAs containing expanded repeats, as in myotonic dystrophy types 1 (DM1 [MIM 160900]) and 2 (DM2 [MIM 602668]) and fragile X–associated tremor/ataxia syndrome (FXTAS) (Ranum and Day Ranum and Day, 2002Ranum LPW Day JW Dominantly inherited non-coding microsatellite expansion disorders.Curr Opin Genet Dev. 2002; 12: 266-271Crossref PubMed Scopus (101) Google Scholar). SCA10 (MIM 603516) is a rare, dominantly inherited neurodegenerative disorder characterized by a unique combination of progressive ataxia, seizure, and anticipation (Grewal et al. Grewal et al., 1998Grewal RP Tayag E Figueroa KP Zu L Durazo A Nunez C Pulst SM Clinical and genetic analysis of a distinct autosomal dominant spinocerebellar ataxia.Neurology. 1998; 51: 1423-1426Crossref PubMed Scopus (62) Google Scholar; Matsuura et al. Matsuura et al., 1999Matsuura T Achari M Khajavi M Bachinski LL Zoghbi HY Ashizawa T Mapping of the gene for a novel spinocerebellar ataxia with pure cerebellar signs and epilepsy.Ann Neurol. 1999; 45: 407-411Crossref PubMed Scopus (78) Google Scholar; Zu et al. Zu et al., 1999Zu L Figueroa KP Grewal R Pulst SM Mapping of a new autosomal dominant spinocerebellar ataxia to chromosome 22.Am J Hum Genet. 1999; 64: 594-599Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The mutation of SCA10 is an unstable and massive expansion of an ATTCT repeat in intron 9 of the SCA10 gene mapped to chromosome 22q13.3. SCA10 is the only disease known to be caused by an expansion of a pentanucleotide repeat and, in its mutant form, is among the largest expansion mutations found to date in human genetic diseases (Matsuura et al. Matsuura et al., 2000Matsuura T Yamagata T Burgess DL Rasmussen A Grewal RP Watase K Khajavi M McCall AE Davis CF Zu L Achari M Pulst SM Alonso E Noebels JL Nelson DL Zoghbi HY Ashizawa T Large expansion of ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10.Nat Genet. 2000; 26: 191-194Crossref PubMed Scopus (396) Google Scholar). There is a large gap between the documented normal (10–22) and mutated repeat lengths (800–4,500) associated with SCA10. The presence of "premutation" or "reduced penetrance mutation" allele ranges is not yet known for SCA10, in contrast to the polyglutamine diseases including several types of SCAs. The SCA10 ATTCT expansion has unique characteristics of instability in both somatic and germline tissues, distinct from the other repeat expansions (Matsuura et al. Matsuura et al., 2004Matsuura T Fang P Lin X Khajavi M Tsuji K Rasmussen A Grewal RP Achari M Alonso ME Pulst SM Zoghbi HY Nelson DL Roa BB Ashizawa T Somatic and germline instability of the ATTCT repeat in spinocerebellar ataxia type 10.Am J Hum Genet. 2004; 74: 1216-1224Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Phenotype and instability patterns are also variable, both within and between families (Rasmussen et al. Rasmussen et al., 2001Rasmussen A Matsuura T Ruano L Yescas P Ochoa A Ashizawa T Alonso E Clinical and genetic analysis of four Mexican families with spinocerebellar ataxia type 10 (SCA10).Ann Neurol. 2001; 50: 234-239Crossref PubMed Scopus (107) Google Scholar; Grewal et al. Grewal et al., 2002Grewal RP Achari M Matsuura T Durazo A Tayag E Zu L Pulst SM Ashizawa T Clinical features and ATTCT repeat expansion in spinocerebellar ataxia type 10.Arch Neurol. 2002; 59: 1285-1290Crossref PubMed Scopus (55) Google Scholar); however, the underlying molecular basis is currently unknown. One possibility is that the configuration of the expanded alleles may vary with phenotype and genetic stability; however, such characterization of large expansions has been hindered by difficult cloning, propagating, and sequencing needs. We recently identified an early-onset patient (onset at age 14 years) with ataxia who was found to have an unusual SCA10 allele of 280 ATTCT repeats, which is much smaller than expansions previously identified in SCA10 families (fig. 1a). In this small pedigree, the patient's asymptomatic mother has the same 280-repeat expansion, whereas her asymptomatic father has normal ATTCT alleles. This suggests that repeat lengths <800 can be pathogenic and that a 280-repeat expansion may be within a size range with reduced penetrance. Long-range PCR (LP) successfully amplified the expanded allele as well as the normal allele. The expanded allele was agarose gel–purified and was sequenced from both ends. We were surprised to find interruptions within the ATTCT repeat tract. The configuration of this small expansion was quite complex. The most proximal part (5′ end) of the expansion is interrupted by multiple repetitive ATGCT repeats (fig. 1b) and the latter part (3′ end) by ATTCTAT septanucleotide repeats. Although it was not possible to obtain the whole sequence throughout the expansion, we did note a long stretch of pure ATTCT repeats in the middle of the expanded tract. The expanded alleles from both the affected daughter and her asymptomatic mother were identical in sequence through the readable portions. We hypothesized that interruptions might play a role in disease presentation and penetrance in this pedigree. This observation stimulated us to search for interruptions in other SCA10 patients. It is not possible, by use of current methods, to sequence through all other expansions, which, to date, exceed 800 repeats. To overcome this technical hurdle, we assessed the repeat configuration, using an ATTCT repeat-primed PCR (RP) that we previously developed to detect SCA10 expansions (Matsuura and Ashizawa Matsuura and Ashizawa, 2002Matsuura T Ashizawa T Polymerase chain reaction amplification of expanded ATTCT repeat in spinocerebellar ataxia type 10.Ann Neurol. 2002; 51: 271-272Crossref PubMed Scopus (34) Google Scholar). Here, the reverse primer with a repeat sequence complementary to ATTCT repeats, randomly binds at multiple sites within the ATTCT repeat tract, and generates a mixture of products containing a variable number of repeats with the forward primer in the unique sequence proximal to the repeat. To determine whether there are interruptions in SCA10 expansions, RP products of SCA10 patients were size separated by electrophoresis at a higher resolution than is typical for diagnostic purposes. In principle, a pure uninterrupted ATTCT expansion would be predicted to show a continuous ladder of products, as the reverse primer anneals randomly within the pure ATTCT repeat tract. Interruptions in repeat purity would expectedly yield a stuttered ladder of products, with stutter locations coincident with interruptions. We studied the two initially identified large families, since both are clinically and genetically well characterized (Grewal et al. Grewal et al., 1998Grewal RP Tayag E Figueroa KP Zu L Durazo A Nunez C Pulst SM Clinical and genetic analysis of a distinct autosomal dominant spinocerebellar ataxia.Neurology. 1998; 51: 1423-1426Crossref PubMed Scopus (62) Google Scholar, Grewal et al., 2002Grewal RP Achari M Matsuura T Durazo A Tayag E Zu L Pulst SM Ashizawa T Clinical features and ATTCT repeat expansion in spinocerebellar ataxia type 10.Arch Neurol. 2002; 59: 1285-1290Crossref PubMed Scopus (55) Google Scholar; Matsuura et al. Matsuura et al., 1999Matsuura T Achari M Khajavi M Bachinski LL Zoghbi HY Ashizawa T Mapping of the gene for a novel spinocerebellar ataxia with pure cerebellar signs and epilepsy.Ann Neurol. 1999; 45: 407-411Crossref PubMed Scopus (78) Google Scholar, Matsuura et al., 2000Matsuura T Yamagata T Burgess DL Rasmussen A Grewal RP Watase K Khajavi M McCall AE Davis CF Zu L Achari M Pulst SM Alonso E Noebels JL Nelson DL Zoghbi HY Ashizawa T Large expansion of ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10.Nat Genet. 2000; 26: 191-194Crossref PubMed Scopus (396) Google Scholar; Zu et al. Zu et al., 1999Zu L Figueroa KP Grewal R Pulst SM Mapping of a new autosomal dominant spinocerebellar ataxia to chromosome 22.Am J Hum Genet. 1999; 64: 594-599Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). All patients in family 1 showed continuous ladders, whereas those in family 2 showed a complex interruption pattern (fig. 2a). This pattern was similar in all the affected individuals in family 2 through three generations, although the location of interruptions was variable. The largest PCR products were gel purified, cloned, and sequenced. Expanded alleles from family 2 had two different septarepeat interruptions, ATTTTCT and ATATTCT, which are duplicated (fig. 2b), whereas family 1 had only pure repeats. The interrupted sequence configuration in family 2 was preserved in the affected members of three generations. We isolated no clones with >80 pentanucleotides, suggesting a limit to the capacity of Escherichia coli systems for propagating these sequences. The data indicate that the expanded ATTCT repeat tract is highly mutable for both length and sequence content in human patients with variable sequence changes. Sequence interruptions in normal alleles at SCA1 (MIM 164400), SCA2 (MIM 183090), FMR1, and FRDA have been well characterized (Chung et al. Chung et al., 1993Chung MY Ranum LP Duvick LA Servadio A Zoghbi HY Orr HT Evidence for a mechanism predisposing to intergenerational CAG repeat instability in spinocerebellar ataxia type I.Nat Genet. 1993; 5: 254-258Crossref PubMed Scopus (419) Google Scholar; Kunst and Warren Kunst and Warren, 1994Kunst CB Warren ST Cryptic and polar variation of the fragile X repeat could result in predisposing normal alleles.Cell. 1994; 77: 853-861Abstract Full Text PDF PubMed Scopus (318) Google Scholar; Imbert et al. Imbert et al., 1996Imbert G Saudou F Yvert G Devys D Trottier Y Garnier JM Weber C Mandel JL Cancel G Abbas N Durr A Didierjean O Stevanin G Agid Y Brice A Cloning of the gene for spinocerebellar ataxia 2 reveals a locus with high sensitivity to expanded CAG/glutamine repeats.Nat Genet. 1996; 14: 285-291Crossref PubMed Scopus (747) Google Scholar; Pulst et al. 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Cossee et al., 1997Cossee M Schmitt M Campuzano V Reutenauer L Moutou C Mandel JL Koenig M Evolution of the Freidreich's ataxia trinucleotide repeat expansion: founder effect and premutations.Proc Natl Acad Sci USA. 1997; 94: 7452-7457Crossref PubMed Scopus (262) Google Scholar; Montermini et al. Montermini et al., 1997Montermini L Andermann E Labuda M Richter A Pandolfo M Cavalcanti F Pianese L Iodice L Farina G Monticelli A Turano M Filla A De Michele G Cocozza S The Friedreich ataxia GAA triplet repeat: premutation and normal alleles.Hum Mol Genet. 1997; 6: 1261-1266Crossref PubMed Scopus (177) Google Scholar; Gunter et al. Gunter et al., 1998Gunter C Paradee W Crawford DC Meadows KA Newman J Kunst CB Nelson DL Schwartz C Murray A Macpherson JN Sherman SL Warren ST Re-examination of factors associated with expansion of CGG repeats using a single nucleotide polymorphism in FMR1.Hum Mol Genet. 1998; 7: 1935-1946Crossref PubMed Scopus (57) Google Scholar). They appear to stabilize their repeat tracts, and the loss of interruptions is associated with instability and repeat expansion. Most normal SCA1, SCA2, and FMR1 alleles carry one or more CAT, CAA, or AGG interruption, whereas GAGGAA hexanucleotide interruptions seen in FRDA are confined to premutations or larger normal alleles of FRDA. Interrupting sequences in a portion of large normal FRDA alleles are also thought to act as anchors to prevent further expansions. In stark contrast with these triplet-repeat diseases, SCA8 (MIM 603680) has normal alleles without interruptions and expanded alleles with interruptions (Moseley et al. Moseley et al., 2000Moseley ML Schut LJ Bird TD Koob MD Day JW Ranum LP SCA8 CTG repeat: en masse contractions in sperm and intergenerational sequence changes may play a role in reduced penetrance.Hum Mol Genet. 2000; 9: 2125-2130Crossref PubMed Scopus (92) Google Scholar). In our initial study (Matsuura et al. Matsuura et al., 2000Matsuura T Yamagata T Burgess DL Rasmussen A Grewal RP Watase K Khajavi M McCall AE Davis CF Zu L Achari M Pulst SM Alonso E Noebels JL Nelson DL Zoghbi HY Ashizawa T Large expansion of ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10.Nat Genet. 2000; 26: 191-194Crossref PubMed Scopus (396) Google Scholar), sequence analysis of 40 SCA10 alleles from 20 normal individuals who were homozygous for alleles ranging from 11 to 16 repeats showed tandem ATTCT repeats without interruptions. Considering this situation, we reevaluated the sequencing of a wider range of normal allele sizes, focusing especially on large normal alleles (≥17 repeats), which are found at a frequency of 7.1% (43/604 alleles) (Matsuura et al. Matsuura et al., 2000Matsuura T Yamagata T Burgess DL Rasmussen A Grewal RP Watase K Khajavi M McCall AE Davis CF Zu L Achari M Pulst SM Alonso E Noebels JL Nelson DL Zoghbi HY Ashizawa T Large expansion of ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10.Nat Genet. 2000; 26: 191-194Crossref PubMed Scopus (396) Google Scholar). As shown in table 1, more than half of large normal alleles have ATTGT-TTTCT or TTTCT interruption (71.1% of 38 examined), whereas all 78 normal alleles of 11–16 repeats showed an uninterrupted pentanucleotide. Interestingly, the location of all interruptions in normal alleles is confined to the second to the last (most distal) repeat. This is similar to the FRDA case, where most GAGGAA interruptions are restricted to the fifth from the last repeat (Cossee et al. Cossee et al., 1997Cossee M Schmitt M Campuzano V Reutenauer L Moutou C Mandel JL Koenig M Evolution of the Freidreich's ataxia trinucleotide repeat expansion: founder effect and premutations.Proc Natl Acad Sci USA. 1997; 94: 7452-7457Crossref PubMed Scopus (262) Google Scholar; Montermini et al. Montermini et al., 1997Montermini L Andermann E Labuda M Richter A Pandolfo M Cavalcanti F Pianese L Iodice L Farina G Monticelli A Turano M Filla A De Michele G Cocozza S The Friedreich ataxia GAA triplet repeat: premutation and normal alleles.Hum Mol Genet. 1997; 6: 1261-1266Crossref PubMed Scopus (177) Google Scholar). From these data, it is difficult to predict the consequences of ATTGT-TTTCT, TTTCT interruptions in the instability or stability of the normal length repeat tract. Interruptions have been observed in the majority of large normal alleles of ≥17 repeats. At the same time, cloned expansion alleles from two SCA10 families have shown uninterrupted, as well as interrupted, mutant repeats.Table 1Sequence Configuration of ATTCT Large Normal Alleles (≥17 Repeats)Allele SizePure Motifn-AB-1n-B-1Total17664161829112191135201102210011220011291001 Total11171038Note.—A = ATTGT and B = TTTCT. Open table in a new tab Note.— A = ATTGT and B = TTTCT. As reported elsewhere (Grewal et al. Grewal et al., 2002Grewal RP Achari M Matsuura T Durazo A Tayag E Zu L Pulst SM Ashizawa T Clinical features and ATTCT repeat expansion in spinocerebellar ataxia type 10.Arch Neurol. 2002; 59: 1285-1290Crossref PubMed Scopus (55) Google Scholar), the presence of seizures was found to be significantly different in the two families described above. Some 25% of affected members in family 1 exhibited seizure, whereas 80% in family 2 suffered from this SCA10-associated phenotype. Correlation between age at onset and repeat number also differs between the families despite clinically observed anticipation; we found a strong inverse correlation (r2=0.79, P=.001) in family 1 and no significance in family 2. Although we could not apply this RP technique to amplify the 3′ end of the expansion (since the genomic sequence downstream of the ATTCT repeat is immediately followed by a long stretch of AluSg, MER11A, and MER11B repeats) to confirm that the ATTCT tracts are uninterrupted in the whole expansion in family 1, differences in the sequence interruption patterns may well explain their variable effect on phenotype and patterns of instability. As seen in SCA1, pathogenicity in the overlapping zone between normal and disease ranges is directly related to the presence/absence of interruptions (Zühlke et al. Zühlke et al., 2002Zühlke C Dalski A Hellenbroich Y Bubel S Schwinger E Burk K Spinocerebellar ataxia type 1 (SCA1): phenotype-genotype correlation studies in intermediate alleles.Eur J Hum Genet. 2002; 10: 204-209Crossref PubMed Scopus (44) Google Scholar) and to their locations (Sobczak and Krzyzosiak Sobczak and Krzyzosiak, 2004Sobczak K Krzyzosiak WJ Imperfect CAG repeats form diverse structures in SCA1 transcripts.J Biol Chem. 2004; 279: 41563-41572Crossref PubMed Scopus (41) Google Scholar). Although it will require analysis of additional families to confirm this finding, our data strongly suggest that the sequence configuration itself can be a disease modifier in this noncoding repeat expansion disease. This may be explained by the notion that these pathogenic repeat expansions act as toxic RNA species, which might have different effects dependent on sequence and/or RNA structure. It has been assumed that noncoding microsatellite expansions contain uninterrupted pure repeats, on the basis of the finding that loss of interruptions appears to be a precursor to repeat instability. However, large expanded repeats have not typically been cloned and sequenced; thus, there is no direct evidence for this notion. We have demonstrated that this is not the case for SCA10, and we recommend caution regarding assumptions about sequence configurations in other disorders. Indeed, interruptions may be the basis for the complex relationship between repeat length and disease severity seen in other noncoding repeat expansions, including DM1, DM2, SCA8, SCA12 (MIM 604326), and FXTAS. It is also possible that interruptions with divergent effects on phenotype may also serve to further distinguish these diseases from the polyglutamine disorders. This work was supported by National Ataxia Foundation and National Organization for Rare Disorders grants (to T.M.); National Institutes of Health (NIH) National Institute of Child Health and Human Development grants HD38038 and HD29256 (to D.L.N.); NIH National Institute of Neurological Disorders and Stroke grant NS41547 (to T.A.); by the FRAXA Research Foundation and the BCM Mental Retardation Research Center (to D.L.N.); and by the Canadian Institutes of Health Research (CHIR) and the Fragile X Research Foundation of Canada (to C.E.P). C.E.P. is a CHIR scholar and a Canadian Genetic Disease Network scholar. The study was approved by the Baylor College of Medicine institutional review board, and the appropriate informed consent was obtained from human subjects. DNA was extracted from whole blood by conventional methods. The ATTCT region was PCR amplified using the primers attct-L (5′-AGAAAACAGATGGCAGAATGA-3′) and attct-R6 (5′-GCCTGGGCAACATAGAGAGA-3′), as described previously. DNA samples with a single normal allele by PCR underwent Southern blot analysis to assess large expansions, by use of 10 μg of EcoRI-digested genomic DNA and the 800-bp SCA10 probe (Matsuura et al. Matsuura et al., 2000Matsuura T Yamagata T Burgess DL Rasmussen A Grewal RP Watase K Khajavi M McCall AE Davis CF Zu L Achari M Pulst SM Alonso E Noebels JL Nelson DL Zoghbi HY Ashizawa T Large expansion of ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10.Nat Genet. 2000; 26: 191-194Crossref PubMed Scopus (396) Google Scholar). We amplified the ATTCT repeat region from genomic DNA, by using the primers L1 (5′-ACAAAGGATCAGAATCCCTGGAAAAGTTAAATATATGGG-3′) and R1 (5′-AGCATATAATTGAGTCTTGCCTTTCAAAATCCAGT-3′). Reactions were 50−μl volumes, 1× reaction buffer, 200−μM dNTPs, and 5 units KOD XL DNA polymerase (Novagen). The PCR conditions consisted of an initial denaturing at 94°C for 2 min, 30 PCR cycles (94°C for 15 s, 67°C for 5 s, and 70°C for 4 min), and an additional extension at 74°C for 10 min. We could successfully amplify the expanded allele (3.4 kb) equally with the normal allele (2.0 kb), corresponding to the Southern blot data. We then sequenced the expanded allele, using an Applied Biosystems (ABI) prism 377 automated sequencer with fluorescent dideoxynucleotides, after separation on a 1.0% agarose gel. The reaction was performed with 1 μM of the 32P-end–labeled forward primer P1 (5′-GAAGACAAATAGAAAACAGATGGCACA-3′) corresponding to a unique sequence of the ATTCT repeat, 0.1 μM of the reverse primer P2 (TACGCATCCCAGTTTGAGACGG(AATAG)8−3′) consisting of a repeat sequence with a hanging tail at its 5′ end, and 1 μM of the hanging primer P3 (TACGCATCCCAGTTTGAGACG) modified from that described elsewhere (Matsuura and Ashizawa Matsuura and Ashizawa, 2002Matsuura T Ashizawa T Polymerase chain reaction amplification of expanded ATTCT repeat in spinocerebellar ataxia type 10.Ann Neurol. 2002; 51: 271-272Crossref PubMed Scopus (34) Google Scholar). The RP products were electrophoresed on 6% denaturing polyacrylamide gels for >4 h for the investigation of patterns within the repeat tracts and were visualized by autoradiography. We gel-extracted RP products of SCA10 patients, with lengths corresponding to 80–100 repeats, after electrophoresis on 3% agarose gels. Our experience suggested that repeat lengths above ∼100 cannot be stably maintained in E. coli; thus, this is the upper limit of fragment lengths that can be isolated. If the RP products from an individual showed the pattern indicative of interruptions, we gel-purified the upper bands above the first interruption, to allow determination of the sequence involved in the interruption. RP products were cloned into pSMART LC vector (Lucigen). Colonies were size selected by preliminary PCR with the use of P1 and P3 and were subsequently sequenced. Because of the inherent instability of the ATTCT repeat when propagated in E. coli, the plasmids were grown at 30°C and with minimal agitation, which we found successfully reduced the deletion frequency of the repeat in clones. At least two independent clones from each PCR product were bidirectionally sequenced using an ABI prism 377 automated DNA sequencer with the two sequencing primers of the pSMART LC vector, following miniprep purification of plasmid DNA. If sequence data derived from clones was not consistent (presumably because of polymerase errors), then additional independent clones were sequenced. We amplified the ATTCT region from normal individuals or unassigned ataxia patients, whose number of repeats was already determined. We selected 38 individuals (23 Caucasian, 9 Mexican, and 6 Japanese) who had >17 repeats in either allele. PCR was performed in the reaction of 20−μl HotStarTaq Master Mix (Qiagen), with use of 0.2−μM primers L2 (5′-AATTGCCCAAAGATTAAAC-3′) and R1 (5′-TTCAGAGACTGCAGTAAAGACAGG-3′). PCR conditions were 95°C for 15 min, 30 PCR cycles (94°C for 30 s, 55°C for 30 s, and 72°C for 1 min), and an additional extension at 72°C for 10 min. Amplified products were cloned into pCRII-TOPO vector (Invitrogen). After PCR size selection using primers attct-L and attct-R6, colonies with different sizes were sequenced respectively. At least two independent clones of the same size for each PCR product were sequenced for the verification of sequence accuracy. We added the previously obtained data of 40 normal alleles (20 homozygous individuals) (Matsuura et al. Matsuura et al., 2000Matsuura T Yamagata T Burgess DL Rasmussen A Grewal RP Watase K Khajavi M McCall AE Davis CF Zu L Achari M Pulst SM Alonso E Noebels JL Nelson DL Zoghbi HY Ashizawa T Large expansion of ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10.Nat Genet. 2000; 26: 191-194Crossref PubMed Scopus (396) Google Scholar) and analyzed all the data together.