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Spinocerebellar ataxia type 31 (SCA31) (MIM 117210) is one of the most prevalent subtypes of autosomal dominant cerebellar ataxia in Japan.1, 2, 3, 4, 5, 6, 7 Several cross-sectional and prospective studies on Japanese patients have shown that SCA31 is a late-onset, relatively pure cerebellar form of ataxia.1, 2, 3, 4, 5, 6, 7, 8 It is caused by an intronic insertion of pathogenic pentanucleotide repeats, (TGGAA)n, in the BEAN and TK2 genes on chromosome 16q21–q22.9, 10 The length of the insertion varies between patients, ranging from 2.5 to 3.8 kb.7, 9 A weak inverse correlation between the length of the inserted repeats and age at onset has been reported;7, 9 however, little is known about the inter-generational instability of the inserted repeats during transmission. The aim of this study was to assess the genetic instability of the causative mutation during transmission in SCA31. For this purpose, we analyzed inter-generational differences of inserted repeat length and age at onset in parent-child pairs with SCA31.

We collected 17 parent-child pairs from 15 families in this study (32 patients in total, Table 1). We determined age at onset for each patient from their medical records. Nine pairs in eight families showed paternal transmission, while eight pairs in seven families were maternal transmission. Of the 17 parent-child pairs, reliable information on onset age for both generations was available in 14 pairs from 12 families (Table 1). The age at onset was lower in the child generation than in the parent generation (11 pairs, 78.6%) (Table 1). The difference in age at onset ranged from −20 to +7 years in the child generation. When age at onset was compared between the parent and child generations, the difference was statistically significant (P=0.007, paired t-test), indicating a younger age at onset in the child generation. In four pairs, age at onset was considerably lower (>10 years) in the affected child than in the parent. On average, the change of onset age was −6.4±7.3 years (mean±s.d.) in the child generation when compared with the parent generation.

Table 1 Age at onset and length of inserted repeats in SCA31 parent-child pairs
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Of the 17 parent-child pairs, genomic DNA was available for 11 pairs from 10 families (21 patients, Table 1). We performed PCR amplification of the fragment encompassing the insertion, purified the PCR product with a QIAquick PCR Purification Kit (QIAGEN, Tokyo, Japan), and digested it with HaeIII, as described previously.7, 9 After HaeIII digestion, one of the authors (AM) randomized and anonymized the samples so that the examiner (KY) was blind to the clinical information of the samples. Gel electrophoresis was performed at a constant 35 V overnight (20 h) and a photograph of the gel was taken with a ruler. The sizing of HaeIII-digested fragments was performed according to an approximate curve (Supplementary Fig. 1), which was drawn based on the migration distance of the molecular size standards from the origin. We sized the fragments five times independently for each patient and calculated the mean and a 95% confidence interval. The mean length of the HaeIII-digested fragment was substituted for that of the inserted repeats. Informed consent for genetic testing was obtained from all study participants.

Representative gel electrogram of HaeIII-digested fragments in eight parent–child pairs is shown in Figure 1. The mean length of the inserted repeats in this cohort (21 patients) is shown in Table 1. In 11 parent–child pairs, the length of the inserted repeats increased in six pairs (five paternal and one maternal) during transmission and decreased in five pairs (three paternal and two maternal). The difference in repeat length in the child generation ranged from −30.9 to +82.5 bp, when compared with the parent generation. On average, the inserted repeats expanded by 12.2±32.7 bp (mean±s.d.) during transmission. This change was within 5–10 repeats, when calculated as the number of pentanucleotide repeats.

Figure 1
figure 1

Representative gel electrogram of HaeIII-digested fragments. Eight parent-child pairs are shown side by side. The family and patient numbers correspond to those in Table 1 1–12: paternal transmission; 13–16: maternal transmission; M: 1 kb DNA size marker (Nippon Genetics).

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The sequence of the inserted repeats in SCA31 is too long and complicated to assess precisely.9 With the current method for analysis, we could not determine the exact size or detailed sequence of the inserted repeats, but their mean length (with a 95% confidence interval) was not significantly different between parent–child pairs. Clinically, it appears as if there is anticipation in onset age in SCA31. Therefore, the relatively younger age at onset in the child generation may be partly due to a bias of awareness of genetic risk. However, the precise effect of (TGGAA)n on disease onset needs to be clarified further.

In total, we analyzed the length of the inserted repeats in 26 patients with SCA31 in this study (Table 1). Of these 26 patients, age at onset was ambiguous in 3 (Patient No. 1, 6 and 14, Table 1). We found no significant correlation between the inserted repeat length and age at onset in 23 patients (Kendall τ Rank Correlation Coefficient: −0.141, P=0.353; Spearman ρ Rank Correlation Coefficient: −0.232, P=0.287) (Figure 2). This was not consistent with our previous observation that there was a weak inverse correlation between inserted repeat length and age of onset in 89 patients from 69 families.7 We consider this is mainly because the present study focused on genetic instability during transmission, and the total numbers of patients and families analyzed were much smaller than our previous study.

Figure 2
figure 2

Correlation between inserted repeat length and age at onset (n=23). Age at onset appeared to be weakly associated with inserted repeat length, but the correlation was not statistically significant.

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SCA31 has a strong founder effect in the Japanese,11, 12 but genetic testing has shown that the maximum difference in inserted repeat length reaches over 1 kb in SCA31 patients.7, 9 Our present data indicate that the inserted repeats in SCA31 are changeable in length during transmission, but that a single transmission may not alter their length so much. Thus, it may take a large number of transmissions to make a significant difference in the inserted repeat length.

The causative mutation for SCA31 is unique among autosomal dominant cerebellar ataxias because it is an intronic insertion of complicated pentanucleotide repeats.9 Among autosomal dominant cerebellar ataxias due to non-coding repeat expansion, it appears that SCA31 shares common features with SCA36, which is caused by a GGCCTG repeat expansion in the first intron of NOP56.13 Both diseases are characterized clinically as late-onset, cerebellar ataxias.1, 2, 3, 4, 5, 6, 7, 8, 13, 14 They have a strong founder effect and distinct ethnic predisposition for the Japanese, although patients genetically confirmed as SCA36 are much smaller in number than those with SCA31 and present with additional features of motor neuron involvement.11, 12, 13, 14, 15 With a remarkable expansion of intronic repeats, it is speculated that an RNA gain-of function mechanism is involved in the pathogenesis of both diseases.10, 13, 16 Interestingly, genetic anticipation is not observed in SCA36.14 It is likely that SCA31 and SCA36 resemble each other in the dynamism of their disease-causing mutations.

In conclusion, we consider that there is no drastic change in the length of the inserted repeats during transmission in SCA31. This may be the molecular basis for the relatively homogeneous clinical phenotype observed in the same family and between families. To our knowledge, our study is the first to examine the inter-generational instability of the inserted repeats during transmission in SCA31 in detail, but it contained only a small number of parent-child pairs; therefore, a nationwide, multicenter collaborative study is desirable to validate our results.