Abstract

Machado-Joseph disease (MJD) is an autosomal dominant multisystem neurodegenerative disorder caused by unstable expansion of a CAG repeat in the MJD1 gene at 14q432.1. To identify elements affecting the intergenerational instability of the CAG repeat, we investigated whether the CGG/GGG polymorphism at the 3′ end of the CAG repeat affects intergenerational instability of the CAG repeat. The [expanded (CAG)n-CGG]/[normal (CAG)n-GGG] haplotypes were found to result in significantly greater instability of the CAG repeat compared to the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] or [expanded (CAG)nGGG]/[normal (CAG)n-GGG] haplotypes. Multiple stepwise logistic regression analysis revealed that the relative risk for a large intergenerational change in the number of CAG repeat units (←2 or >2) is 7.7-fold (95% CI: 2.5–23.9) higher in the case of paternal transmission than in that of maternal transmission and 7.4-fold (95% CI: 2.4–23.3) higher in the case of transmission from a parent with the [expanded (CAG)n-CGG]/[normal (CAG)n-GGG] haplotypes than in that of transmission from a parent with the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] or [expanded (CAG)n-GGG]/[normal (CAG)n-GGG] haplotypes. The combination of paternal transmission and the [expanded (CAG)n-CGG]/[normal (CAG)n-GGG] haplotypes resulted in a 75.2-fold (95% CI: 9.0–625.0) increase in the relative risk compared with that of maternal transmission and the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] or [expanded (CAG)n-GGG]/[normal (CAG)n-GGG] haplotypes. The results suggest that an inter-allelic interaction is involved in the intergenerational instability of the expanded CAG repeat.

Introduction

Machado-Joseph disease (MJD) is an autosomal dominant multisystem neurodegenerative disorder characterized by cerebellar ataxia, spasticity, progressive external ophthalmoplegia, bulging eyes, dystonia and peripheral amyotrophy (18). We mapped the gene for MJD to chromosome 14q24.3–32.1 by linkage analyses of five Japanese MJD families (9), which was subsequently confirmed by others in linkage analyses of not only Japanese (10) but also Portuguese-Azorean kindreds (1113). Unstable expansion of a CAG repeat in the MJD1 gene at 14q32.1 has recently been discovered to be the causative mutation (14). The number of CAG repeat units in expanded alleles ranges from 60 to 84, while that in normal alleles ranges from 14 to 44 (1421). As has been observed in other diseases caused by CAG repeat expansions such as Huntington disease (HD) (2225), spinocerebellar ataxia (SCA1) (2629), and dentatorubral-pallidoluysian atrophy (DRPLA) (3032), the intergenerational increase in the number of CAG repeat units has been shown to result in genetic anticipation of MJD, which is more prominent in paternal transmission than in maternal transmission (15,16).

Results of recent investigations suggest the presence of cis-acting elements which may lead to instability of trinucleotide repeats in SCA1 (27), HD (3335), fragile X syndrome (36,37) and myotonic dystrophy (DM) (3840). The presence of cryptic AGG triplets within the CGG repeat in the gene for fragile X syndrome (FMR1) (37), interruption of the CAG repeat in the SCA1 gene (27) and the 1 kb insertion/deletion polymorphism 5 kb upstream of the CTG repeat in the DM gene have been found to be closely associated with the instability of the trinucleotide repeats (3840). Analysis of a three-base deletion/insertion polymorphism at nucleotide positions 2642–2645 of the HD gene, which is located ∼150 kb downstream of the CAG repeat, showed that the deletion alleles are associated with larger CAG repeats of normal chromosomes than the insertion alleles, suggesting that the expansion of the larger CAG repeats of normal chromosomes with the deletion allele to a full mutation is the source of HD mutations (33,34). Despite these studies, the molecular mechanisms of the CAG repeat instability remain to be elucidated.

In detailed haplotype analyses of MJD chromosomes, we found a strong linkage disequilibrium of MJD chromosomes at AFM343vf1 and found a common haplotype that is frequently shared by Japanese and Azorean MJD chromosomes, which suggests either a founder effect or the presence of predisposing chromosomes prone to expansions of the CAG repeat (16,41).

Interestingly, a CAG/CAA polymorphism within the CAG repeat and a CGG/GGG polymorphism at the 3′ end of the CAG array in the MJD1 gene have been described (14). Since these polymorphisms are located within or near the CAG repeat, we speculated that analysis of these polymorphisms would reveal clues to the molecular mechanisms of the intergenerational instability of the expanded CAG repeat. Therefore, we investigated how these polymorphisms are associated with the intergenerational instability of the expanded CAG repeat of the MJD1 gene. Our results strongly suggest that an interallelic interaction between normal and expanded alleles is involved in the intergenerational instability of the expanded CAG repeat.

Figure 1.

Determination of the CAA/CAG and the CGG/GGG polymorphisms by allele-specfic oligonucleotide hybridization. PCR products amplified from genomic DNA from eight MJD patients, which were obtained using MJD52 and MJD25 as primers, were electrophoresed through 2% agarose gels and transferred to nitrocellulose membranes. The four membranes carrying the PCR products were hybridized to MJDP1 (5′-TGCTGCTGCTTTTG-3′), MJDP2 (5′-AAGCAGCAACAGCAGCA-3′), MJDP3 (5′-AGCAGCAGCGGGACCTA-3′) or MJDP4 (5′-AGCAGCAGGGGGACCTA-3′). Hybridization was performed at 41°C for MJDP1, 47°C for MJDP2, and 51°C for MJDP3 and MJDP4.

Figure 1.

Determination of the CAA/CAG and the CGG/GGG polymorphisms by allele-specfic oligonucleotide hybridization. PCR products amplified from genomic DNA from eight MJD patients, which were obtained using MJD52 and MJD25 as primers, were electrophoresed through 2% agarose gels and transferred to nitrocellulose membranes. The four membranes carrying the PCR products were hybridized to MJDP1 (5′-TGCTGCTGCTTTTG-3′), MJDP2 (5′-AAGCAGCAACAGCAGCA-3′), MJDP3 (5′-AGCAGCAGCGGGACCTA-3′) or MJDP4 (5′-AGCAGCAGGGGGACCTA-3′). Hybridization was performed at 41°C for MJDP1, 47°C for MJDP2, and 51°C for MJDP3 and MJDP4.

Results

Normal chromosomes with the CGG allele are more frequently associated with larger CAG repeats than those with the GGG allele

The genomic segment containing the CAG repeat of the MJD1 gene was amplified by polymerase chain reaction (PCR), and the PCR products were subjected to agarose gel or polyacrylamide gel electrophoresis and blotted onto nitrocellulose or nylon membranes. The single base substitutions of the CAG/CAA and the CGG/GGG polymorphisms were identified by allele-specific oligonucleotide hybridization (Fig. 1).

Regarding the CAA/CAG polymorphism within the CAG repeat, the CAA allele was far more abundant than the CAG allele, with the overall frequency of the former being 92.9%. Little variation in the frequencies of these alleles was observed amongst the different ethnic populations.

The CGG/GGG polymorphism, however, showed quite characteristic allelic frequency distributions among normal chromosomes in each ethnic population. The GGG allele was consistently associated with smaller CAG repeats than the CGG allele (Fig. 2). Interestingly, the allele with 14 repeat units, which was the smallest CAG repeat, was exclusively associated with the GGG allele. On the other hand, the CGG allele was consistently associated with a larger range of numbers of repeat units in all the ethnic populations. The difference was statistically significant in the Japanese, Chinese and non-Portuguese Western European populations. Although the trends were the same, the Portuguese-Azorean and Russian populations did not show significant differences in the CAG repeat size, which may have been due to the small sample size (Fig. 2).

CGG allele is more frequently associated with pathologically expanded CAG repeat of the MJD1 gene than the GGG allele

Eighty of the 88 independent MJD chromosomes (91.0%) in the overall data set had the CGG allele, which is in striking contrast to the CGG allele frequency in the normal chromosomes (39.0%) in the overall data set (Fig. 2). All 41 of the Japanese pedigrees, all four of the Chinese pedigrees, 25 of the 27 non-Portuguese Western European pedigrees (92.6%) and 10 of the 16 Portuguese-Azorean pedigrees (62.5%) had the CGG allele. These results are also in striking contrast to those for the normal chromosomes in the corresponding ethnic populations (53.5, 48.3, 25.3 and 10.9% respectively), with statistically significant differences as determined by χ2 analysis (P <0.0001, P <0.05, P <0.0001 and P <0.0001 respectively).

Table 2.

Influence of CGG/GGG polymorphism of the expanded CAG repeat on the intergenerational instability of the CAG repeat

With regard to the CAA/CAG polymorphism within the CAG repeat of the MJD1 gene, all of the expanded CAG repeats contained the CAA allele. However, it should be noted that the CAA allele was also very frequently present on normal chromosomes (93.2% of overall normal chromosomes).

Influence of gender of affected parent on intergenerational instability of expanded CAG repeat

For the analysis of intergenerational instability of the expanded CAG repeat, we analyzed 101 parent-offspring pairs. The absolute value of intergenerational change in the number of CAG repeat units was 2.0 ± 0.2 (mean ± SEM) (range:−8 to +9, variance = 6.6) in the 101 MJD parent-offspring pairs. There was a statistically significant difference in the intergenerational change in the number of CAG repeat units between paternal and maternal transmissions (paternal transmission: mean ± SEM = 2.8 ± 0.3, range = − 7 to +8, variance = 9.5, n = 46; maternal transmission: mean ± SEM = 1.3 ± 0.2, range = − 8 to +6, variance = 3.9, n = 55, P <0.001 by MannWhitney U test and P <0.005 by F test), which indicated that the expanded CAG repeats were less stable in paternal transmission than in maternal transmission.

Influence of CGG/GGG polymorphism in cis to the expanded CAG repeat on the intergenerational instability of the expanded CAG repeat

Based on the observations that the CGG allele is more frequently associated with the expanded CAG repeat on the MJD chromosome and with larger CAG repeats on normal chromosomes than the GGG allele, we then investigated whether the CGG allele is associated with greater intergenerational instability of the CAG repeat than the GGG allele by analyzing the 101 parent-offspring pairs. Although the intergenerational change in the number of CAG repeat units of the expanded CAG repeat in cis to the CGG allele (mean ± SEM: 2.1 ± 0.2, variance: 7.2) was larger than that of the expanded CAG repeat in cis to the GGG allele (mean ± SEM: 1.1 ± 0.2, variance: 1.9), the difference was not statistically significant as determined using the Mann-Whitney U test (P = 0.085). Comparison of variances, however, indicated that the expanded CAG repeat in cis to the CGG allele is associated with a larger variance than that in cis to the GGG allele (P <0.05, F test).

To compare the distributions of the intergenerational changes in the number of CAG repeat units, the subjects were divided into five groups according to the degree of the intergenerational change in the number of CAG repeat units (Table 2). In this analysis, there were no statistically significant differences in the distributions between the expanded CAG repeat in cis to the CGG allele and that in cis to the GGG allele in the combined data set (χ2 = 4.19, P = 0.38), for the paternal transmission (χ2 = 7.45, P = 0.11) or for the maternal transmission (χ2 = 2.48, P = 0.65) (Table 2).

CGG/GGG polymorphism in trans to the expanded CAG repeat affects the intergenerational instability of the expanded CAG repeat

To investigate whether the CGG/GGG polymorphism on normal chromosomes of MJD-affected parents affects the intergenerational instability of the CAG repeat, we analyzed 65 affected parent-offspring pairs for which we were able to determine the CGG/GGG polymorphism on both the expanded allele-carrying and the normal chromosomes. To compare the distributions of intergenerational changes in the CAG repeat size, the subjects were divided into the five groups according to the degree of intergenerational change in the CAG repeat size as described above (Fig. 2). Haplotype designations were made as follows: [Ex-C], expanded (CAG)n-CGG; [Ex-G], expanded (CAG)n-GGG; [N-C], normal (CAG)n-CGG; [N-G], normal (CAG)n-GGG.

Figure 2.

Comparison of intergenerational changes in the number of CAG repeat units between transmission from MJD-affected parents with the [Ex-CN-G] haplotypes and transmission from those with the [Ex-CN-C] haplotypes or the [Ex-G/N-G] haplotypes. We analyzed a total of 65 pairs of affected parents and their offspring for which we were able to determine the CGG/GGG polymorphism on both expanded and normal alleles. The subjects were divided into five groups according to the degree of the intergenerational change in the number of CAG repeat units (contraction by more than 2, contraction by 1 or 2, no change, expansion by 1 or 2 and expansion by more than 2 repeat units). Haplotype designations are as follows: [Ex-C], expanded (CAG)n-CGG; [Ex-G, expanded (CAG)n-GGG; [N-C], normal (CAG)n-CGG; [N-G], (CAG)n-GGG.

Figure 2.

Comparison of intergenerational changes in the number of CAG repeat units between transmission from MJD-affected parents with the [Ex-CN-G] haplotypes and transmission from those with the [Ex-CN-C] haplotypes or the [Ex-G/N-G] haplotypes. We analyzed a total of 65 pairs of affected parents and their offspring for which we were able to determine the CGG/GGG polymorphism on both expanded and normal alleles. The subjects were divided into five groups according to the degree of the intergenerational change in the number of CAG repeat units (contraction by more than 2, contraction by 1 or 2, no change, expansion by 1 or 2 and expansion by more than 2 repeat units). Haplotype designations are as follows: [Ex-C], expanded (CAG)n-CGG; [Ex-G, expanded (CAG)n-GGG; [N-C], normal (CAG)n-CGG; [N-G], (CAG)n-GGG.

Analysis of the combined data set of paternal and maternal transmissions revealed that the pairs in which the parent had the [Ex-C/N-G] haplotypes showed significantly larger intergenerational changes in the number of CAG repeat units than those in which the parent had the [Ex-C/N-C] (P = 0.0035) or the [Ex-G/N-G] haplotypes (P = 0.023). Sixty-one percent of transmissions in which the parent had the [Ex-C/N-G] haplotypes resulted in a large intergenerational change (defined as an intergenerational change of ←2 or >2), while only 18% of transmissions in which the parent had the [Ex-C/N-C] or the [Ex-G/N-G] haplotypes resulted in such a large intergenerational change. There was no statistically significant difference in the intergenerational change in the number of CAG repeat units between transmissions in which the parent had the [Ex-C/N-C] haplotypes and these in which the parent had the [Ex-G/N-G] haplotypes (P = 0.39).

Analysis of the maternal transmission data set revealed that the pairs in which the mother had the [Ex-C/N-G] haplotypes exhibited a significantly larger intergenerational change in the number of CAG repeat units than those in which the mother had the [Ex-G/N-G] haplotypes (χ2 analysis; P = 0.0091), but did not show a significantly different intergenerational change compared to those in which the mother had the [Ex-C/N-C] haplotypes (χ2 analysis; P = 0.23). Although the pairs in which the father had the [Ex-C/N-G] haplotypes showed a larger intergenerational change in the number of CAG repeat units than those in which the father had other haplotypes, the difference was not statistically significant (χ2 = 10.4, P = 0.24).

Gender of affected parent and combination of haplotypes on expanded and normal chromosomes are the major determinants for the greater intergenerational instability of the expanded CAG repeat

In the multiple stepwise logistic regression analysis, only the gender of the affected parent and the interaction term of CGG on the expanded allele and GGG on the normal allele were entered to explain the large intergenerational change. Once the two factors had been entered, no other factors including the genotypes of expanded or normal alleles, or the size of the expanded or normal alleles were entered.

As shown in Table 3, the relative risk for the large intergenerational change in the number of CAG repeat units in paternal transmission adjusted for other factors was significantly higher (OR = 7.7, 95% CI = 2.5–23.9) than that in maternal transmission. The relative risk in the case of transmission from a parent with the [Ex-C/N-G] haplotypes adjusted for other factors was also significantly higher (OR= 7.4, 95% CI = 2.4–23.3) than that in the case of transmission from a parent with the [Ex-C/N-C] or [Ex-G/N-G] haplotypes. The combination of paternal transmission and the [Ex-C/N-G] haplotypes resulted in an even greater relative risk for the large intergenerational change (OR = 75.2, 95% CI = 9.0–625.0) (Table 3).

Table 3.

Relative risks for a large intergenerational change (<−2 or >2) in the number of CAG repeat units conferred by the gender of the affected parent and the interaction between normal and expanded alleles

Discussion

The distributions of the sizes of the CAG repeat on normal chromosomes were found to exhibit characteristic variations depending on the CGG/GGG polymorphism located at the 3′ end of the CAG repeat and on the ethnic background (Table 1). Interestingly, the CGG allele of the normal chromosomes was associated with larger CAG repeats than the GGG allele of the normal chromosomes. We also found that the CGG allele was more frequently associated with MJD chromosomes (91.0%) than the GGG allele, which is in striking contrast to the frequency of the CGG allele in the normal chromosomes (39.0%). These observations raise the possibility that the CGG element in cis to the expanded CAG repeat confers the instability of the expanded CAG repeat. Alternatively, a founder effect may underlie the frequent association of the CGG allele with MJD chromosomes in all the populations analyzed in the present study. The finding that all the Japanese MJD chromosomes carried the CGG allele (Table 1) and the fact that a common haplotype of the markers flanking the MJD1 gene is frequently observed in Japanese MJD chromosomes (16), support the presence of a founder effect in Japanese MJD.

To determine whether the CGG/GGG polymorphism on the expanded or normal Chromosomes affects the intergenerational instability of the expanded CAG repeat, we analyzed in detail the intergenerational changes in the size of the CAG repeats of parent-offspring pairs with various combinations of genotypes. As shown in Table 2, though the expanded CAG repeat with the CGG allele tended to undergo larger intergenerational changes than that with the GGG allele, the difference was found to be statistically significant using the F test (P <0.05), but not using the Mann-Whitney U test (P = 0.063). Therefore, we could not unequivocally conclude that the CGG allele in cis to the expanded repeat confers the instability of the expanded CAG repeat based on the present results, though the possibility remains.

The most striking observation in the present study is that the CGG/GGG polymorphism on the normal alleles of MJD-affected parents strongly influenced the intergenerational instability of the expanded CAG repeat. As shown in Figure 2, the expanded CAG repeat with the CGG allele of the affected parents exhibited significantly different degrees of intergenerational instability depending on the genotype of the normal chromosome. The results strongly suggest that there is an inter-allelic interaction between the MJD chromosomes with an expanded CAG repeat and the normal chromosomes. Multiple stepwise logistic regression analysis clearly revealed that the gender of the affected parent and the combination of the haplotypes of the normal and expanded chromosomes are the major determinants for the large intergenerational instability of the expanded CAG repeat. This analysis also revealed that the relative risk for the large intergenerational change in the number of CAG repeat units is 7.7-fold (95% CI: 2.5–23.9) higher in paternal transmission than in maternal transmission, and 7.4-fold (95% CI: 2.4–23.3) higher in the case of transmission from a parent with the [Ex-C/N-G] haplotypes than in that of transmission from a parent with the other haplotypes. The combination of paternal transmission and the [Ex-C/N-G] haplotypes resulted in a 75.2-fold (95% CI: 9.0–625.0) increase in the relative risk compared with that of maternal transmission and the [Ex-C/N-C] or [Ex-G/N-G] haplotypes. Although it is an intriguing question whether transmission from a parent with the [Ex-G/N-C] haplotypes is associated with greater intergenerational instability, parent-offspring pairs in which the parent had these haplotypes were unavailable apparently due to the extremely low frequency of the GGG allele on the MJD chromosome.

Although the instability of the expanded CAG repeat has been well documented, the molecular mechanisms of the instability remain unknown. The present study unexpectedly revealed that the genotype of the normal chromosome substantially affects the degree of intergenerational instability of the expanded CAG repeat. Intriguing observations, which are quite similar to our findings, have been made on the mechanisms of mutation of the human MS32 minisatellite (4244). Results of detailed structural analysis of mutant alleles showed that the mutation frequently involved complex inter-allelic gene conversion events restricted to one end of a tandem repeat array near the site of a C/G polymorphism (43,44). In the case of C/G heterozygotes, the incoming repeats were mostly derived from the corresponding position of the C allele with the G allele acting as an acceptor and the C allele acting as a donor, which indicates that the gene conversion events are substantially influenced by the C/G polymorphism near the tandem repeat array of the MS32 minisatellite. Since none of the mutants showed exchange of the C/G polymorphism flanking the tandem repeat array of the MS32 minisatellite, the gene conversion events were considered to be restricted to within the tandem repeat array of the MS32 minisatellite. Occurrence of gene conversion events was also suggested in the case of contraction of expanded trinucleotide repeats to the normal size ranges in myotonic dystrophy (45,46) and fragile X syndrome (47).

Taken together, these findings suggest that gene conversion events are also involved in the intergenerational instability of the expanded CAG repeat in MJD. Nucleotide sequence analysis of the expanded CAG repeat as well as the flanking sequences of the affected parent-offspring pairs revealed that the CGG/GGG polymorphism was not converted in association with intergenerational changes in the CAG repeat size as was similarly observed in the case of the MS32 minisatellite. Although the present study did not provide direct evidence of the occurrence of gene conversion events, the findings that an inter-allelic interaction is involved in the intergenerational instability should provide a clue to the molecular mechanisms of the instability of the CAG repeat. To confirm that gene conversion events are involved in the meiotic instability of the CAG repeat, extensive structural analyses of sperm DNA from MJD-affected individuals with various genotypes are required. Moreover, other polymorphisms flanking the expanded CAG repeat should also be analyzed to elucidate in detail the molecular mechanisms of the instability of the expanded CAG repeat.

Materials and Methods

DNA samples

Blood samples were obtained with informed consent from 107 affected individuals of 41 Japanese MJD families, 79 affected individuals of 27 non-Portuguese western European MJD families (25 French families, one Spanish family and one Belgian family), 56 affected individuals of 16 Portuguese-Azorean MJD families, 20 affected individuals of four Chinese MJD families, six affected individuals of three North African MJD families (two Moroccan families and one Algerian family), two affected individuals of one Black African MJD family (Guyanan family), four affected individuals of two African American MJD families, two affected individuals of one Yemeni MJD family and two affected individuals of one American Caucasian MJD family. Blood samples were also obtained from 101 Japanese, 97 non-Portuguese western European (French), 46 Portuguese-Azorean, 23 Russian and 29 Chinese unrelated healthy normal volunteers with informed consent. High molecular weight genomic DNA was extracted from peripheral blood as described (48).

For the analysis of the effect of CGG/GGG polymorphism in cis to the expanded CAG repeat, we analyzed 101 affected parent-affected offspring pairs from Japanese (n = 30), Chinese (n = 8), non-Portuguese western European (n = 32), Portuguese Azorean (n = 24), African American (n = 2), American Caucasian (n = 1), North African (n = 3) and Black African (n = 1) pedigrees.

For the analysis of the effect of CGG/GGG polymorphism in trans to the expanded CAG repeat (on normal chromosome of affected parent), we needed to determine both haplotypes on normal as well as MJD chromosomes. This analysis was performed for 65 affected parent-affected offspring pairs from Japanese (n = 30), Chinese (n = 8), non-Portuguese western European (n = 15), Portuguese Azorean (n = 9), African American (n = 2), American Caucasian (n = 1), North African (n = 3) and Black African (n = 1) pedigrees. The data sets were comprised of 31 pairs in which the affected parent had the [Ex-C/N-G] haplotypes, 24 pairs in which the affected parent had the [Ex-C/N-C] haplotypes and 10 pairs in which the affected parent had the [Ex-G/N-G] haplotypes; ([Ex-C], expanded (CAG)n-CGG; [Ex-G], expanded (CAG)n-GGG; [N-C], normal (CAG)n-CGG; and [N-G], normal (CAG)n-GGG).

We found no affected parents with the [Ex-G/N-C] haplotypes in our data set apparently due to the extremely low frequency of the GGG allele on the MJD chromosome.

Determination of number of CAG repeat units

The primer sequences used for PCR analyses of genomic DNA were those described by Kawaguchi et al. (14). PCR and determination of the number of CAG repeat units were performed as described (16).

Analysis of CAA/CAG and CGG/GGG polymorphisms

PCR was performed in a total volume of 20 µl containing 200 ng of genomic DNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 10% DMSO, 200 µM each of dATP, dGTP dCTP and TTP, 1 µM of each primer and 5 U of Taq polymerase (Takara, Otsu). The primer sequences used for the PCR were those described by Kawaguchi et al. (MJD52 and MJD25) (14). After the initial denaturation at 95°C for 2 min, the PCR was carried out for 32 cycles consisting of 1 min denaturation at 95°C, 1 min annealing at 60°C, and 1 min extension at 72°C, followed by a final extension at 72°C for 7. Determination of the CAA/CAG and CGG/GGG polymorphisms was performed by separation of the PCR products by either agarose gel electrophoresis or polyacrylamide gel electrophoresis, blotting onto membranes and allele-specific oligonucleotide hybridization. PCR products were first electrophoresed through 2% agarose gels and blotted onto nitrocellulose membranes. PCR products whose normal alleles were difficult to separate by agarose gel electrophoresis were electrophoresed through 5% polyacrylamide denaturing gels and blotted onto nylon membranes (Hybond-N+, Amersham, Buckinghamshire) by capillary blotting. To determine the CAA/CAG and CGG/GGG polymorphisms, four allele-specific oligonucleotides were synthesized for each site. For the CAA/CAG polymorphism, MJDP1 (5′-TGCTGCTGCTTTTG-3′) and MJDP2 (5′-AAGCAGCAACAGCAGCA-3′) were used. For the CGG/GGG polymorphism, MJDP3 (5′-AGCAGCAGCGGGACCTA-3′) and MJDP4 (5′AGCAGCAGGGGGACCTA-3′) were used (the sites for the single-base mismatches are underlined). We designed the sites for the single-base mismatches in the center of 17-mer oligonucleotides except in the case of MJDP1. Since the CAA/CAG polymorphism is located within the CAG array, the presence of the CAG allele results in a continuous CAG repeat, which makes the design of a primer specific for the CAG allele difficult. Based on preliminary experiments, we found that the 14mer (MJDP1) containing a flanking sequence of [5′-CTTTTG] gave the best results for discriminating the CAG allele from the CAA allele. Oligonucleotides were end-labeled using polynucleotide kinase, and hybridization was performed in 6× SSC, 10× Denhardt's, 0.05% Na pyrophosphate and 50 µg/ml sheared salmon sperm DNA with a probe concentration of 1×10 concentration of 1×106 c.p.m./ml (49). Hybridization temperatures were 41°C for MJDP1, 47°C for MJDP2, and 51°C for MJDP3 and MJDP4. The filters were washed in 1× SSC, 0.5% SDS for 2 h at room temperature, and finally for 0.5 h at 44°C for MJDP1, at 50°C for MJDP2, or at 54°C for MJDP3 and MJDP4. The membranes were autoradiographed to Fuji RX films at −80°C using an intensifying screen.

Statistical analysis

Differences in intergenerational changes in the number of CAG repeat units were analyzed using the Mann-Whitney U test. Comparisons of allele distribution and distributions of intergenerational changes were performed using the χ2 test. Differences in variances of intergenerational changes were analyzed using the F test. Differences were considered to be statistically significant if P <0.05.

To identify factors affecting the intergenerational instability of the expanded CAG repeat, multiple stepwise logistic regression analysis was performed using (i) the genotype of the CGG/GGG polymorphism of the expanded allele of the affected parent, (ii) the genotype of the CGG/GGG polymorphism of the normal allele of the affected parent, (iii) the interaction term between the expanded allele and normal allele of the affected parent ([Ex-C/N-G] = 1; other combinations = 0), (iv) the gender of the affected parent, (v) the size of the expanded CAG repeat of the affected parent and (vi) the size of the normal CAG repeat of the affected parent as the explanatory variables and the status of the intergenerational change [large intergenerational change (←2 or >2) =1, and small or no intergenerational change (−2< and <2) =0] as the response variable. A significance level for entry into the model was set to 0.05. The multiple stepwise logistic regression analysis was performed using SAS software release 6.08.

Acknowledgments

We are very grateful to the members of the MJD families for their cooperation. We would like to thank Dr L.P.W. Ranum for providing us with DNA samples of MJD patients and their offspring (two affected parent-offspring pairs of two African American MJD families and one affected parent-offspring pair of an American Caucasian MJD family). We also thank Dr K. Sakimura for his valuable advice. We would like to thank Drs H. Chneiweiss, P. Damier, D. Grid, D. Laplane, M. Haguenau, J. Cassiman, C. Legum, A.D. Korzcyn, P. Sousa, A. Toutain, G. and P. Labauge for providing us with blood samples from some of the families and the Association Francaise contre les Myopathies, the Groupement d' Etudes et de Recherches sur les Genomes and the VERUM Foundation, the Medical Research Council of Canada and the Alzheimer Association of Ontario for their financial support. This study was also supported in part by a Grant-in-Aid for Scientific Research on Priority Areas and a Grant-in-Aid for Creative Basic Research (Human Genome Program) from the Ministry of Education, Science and Culture, Japan, a grant from the Research Committee for Ataxic Disease, the Ministry of Health and Welfare, Japan, a Research Fellowship grant from the Japan Society for the Promotion of Science for Young Scientists, special coordination funds from the Japanese Science and Technology Agency and a grant from the Uehara Memorial Foundation.

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