School of Medicine, Sapporo 060, Japan, 6Department of Pathology, ... Sapporo 060, Japan, 7Department of Neurology, China-Japan Friendship Hospital, ...
1996 Oxford University Press
Human Molecular Genetics, 1996, Vol. 5, No. 7
923–932
ARTICLE
Intergenerational instability of the CAG repeat of the gene for Machado-Joseph disease (MJD1) is affected by the genotype of the normal chromosome: implications for the molecular mechanisms of the instability of the CAG repeat S. Igarashi1, Y. Takiyama1,2, G. Cancel3, E. A. Rogaeva4, H. Sasaki5, A. Wakisaka6, Y.-X. Zhou7, H. Takano1, K. Endo1, K. Sanpei1, M. Oyake1, H. Tanaka1, G. Stevanin3, N. Abbas3, A. Dürr3, E. I. Rogaev4, R. Sherrington4, T. Tsuda4, M. Ikeda4, E. Cassa8, M. Nishizawa2, A. Benomar9, J. Julien10, J. Weissenbach11, G.-X. Wang7, Y. Agid3, P. H. St. George-Hyslop4, A. Brice3 and S. Tsuji1,* 1Department
of Neurology, Brain Research Institute, Niigata University, 1 Asahimachi, Niigata 951, Japan, of Neurology, Jichi Medical School, Minamikawachi, Tochigi 32904, Japan, 3INSERM Unité 289, and Federation de Neurologie, Hopital de la Salpêtrière, Paris, France, 4Center for Research in Neurodegenerative Disease, University of Toronto and Department of Medicine, Division of Neurology, The Toronto Hospital, 6 Queen’s Park Crescent, Toronto, Ontario, Canada, M5S 1A8, 5Department of Neurology, Hokkaido University School of Medicine, Sapporo 060, Japan, 6Department of Pathology, Hokkaido University School of Medicine, Sapporo 060, Japan, 7Department of Neurology, China-Japan Friendship Hospital, Hepingli, Beijing, 100029, China, 8Departament de Neurologia, Hospital das Clinicas, Universidade de Sao Paulo, Sao Paulo, Brazil, 9Service de Neurologie de l’Hopital des Spécialités, Rabat, Morocco, 10Service de Neurologie, CHU de Bordeaux, Pessac, France, 11Généthon, 1 rue de l’lnternationale, 91000 Evry, and Unité de Genetique Moleculaire Human, CNRS URA 1445, Institut Pasteur, 75724 Paris Cedex, France 2Department
Received March 6, 1996; Revised and Accepted May 2, 1996
Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder caused by unstable expansion of a CAG repeat in the MJD1 gene at 14q32.1. 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) 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.
*To whom correspondence should be addressed
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Human Molecular Genetics, 1996, Vol. 5, No. 7
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 (1–8). 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 (11–13). 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 (14–21). As has been observed in other diseases caused by CAG repeat expansions such as Huntington disease (HD) (22–25), spinocerebellar ataxia (SCA1) (26–29), and dentatorubral-pallidoluysian atrophy (DRPLA) (30–32), 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 (33–35), fragile X syndrome (36,37) and myotonic dystrophy (DM) (38–40). 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 (38–40). 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-specific 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 41C for MJDP1, 47C for MJDP2, and 51C 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 (Table 11). 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 (Table 1). 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
925 Human Acids Molecular Genetics, Vol.No. 5, No. Nucleic Research, 1994,1996, Vol. 22, 1 7 Table 1.
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Human Molecular Genetics, 1996, Vol. 5, No. 7 Table 2. Influence of CGG/GGG polymorphism of the expanded CAG repeat on the intergenerational instability of the CAG repeat
*number of parent-offspring pairs (%)
to the CGG allele frequency in the normal chromosomes (39.0%) in the overall data set (Table 1). 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
