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
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 repeatS. 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, 2Department 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
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 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.
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 -3 5 ), 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 ,16 ).
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.
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 ).
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 (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 [chi]2 analysis (P <0.0001, P <0.05, P <0.0001 and P <0.0001 respectively).
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).
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.
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 ([chi]2 = 4.19, P = 0.38), for the paternal transmission ([chi]2 = 7.45, P = 0.11) or for the maternal transmission ([chi]2 = 2.48, P = 0.65) (Table 2 ).*number of parent-offspring pairs (%)
Table 2 Influence of CGG/GGG polymorphism of the expanded CAG repeat on the intergenerational instability of the 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.
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*[Ex-C]: expanded (CAG)n-CGG; [Ex-G]: expanded (CAG)n-GGG; [N-C]: normal (CAG)n-CGG; [N-G]: normal (CAG)n-GGG.
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 (Table1) 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 CAG 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 (42 -44 ). 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.
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.
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 ).
PCR was performed in a total volume of 20 [mu]l 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 [mu]M each of dATP, dGTP dCTP and TTP, 1 [mu]M 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 95oC for 2 min, the PCR was carried out for 32 cycles consisting of 1 min denaturation at 95oC, 1 min annealing at 60oC, and 1 min extension at 72oC, followed by a final extension at 72oC for 7 min. 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 [mu]g/ml sheared salmon sperm DNA with a probe concentration of 1*106 c.p.m./ml (49 ). Hybridization temperatures were 41oC for MJDP1, 47oC for MJDP2, and 51oC 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 44oC for MJDP1, at 50oC for MJDP2, or at 54oC for MJDP3 and MJDP4. The membranes were autoradiographed to Fuji RX films at -80oC using an intensifying screen.
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 [chi]2 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.
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. Fenelon 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.
1 Nakano,K.K., Dawson,D.M. and Spence,A. (1972) Machado disease: a hereditary ataxia in Portuguese emigrants to Massachusetts. Neurol. 22,49-55.
2 Woods,B.T. and Schaumburg,H.H. (1972) Nigro-spino-dentatal degeneration with nuclear ophthalmoplegia: a unique and partially treatable clinicopathological entity. J.Neurol. Sci. 17, 149-166.
3 Rosenberg,R.N., Nyhan,W.L., Bay,C. and Shore,P. (1976) Autosomal dominant striatonigral degeneration: a clinical, pathologic, and biochemical study of a new genetic disorder. Neurol. 26, 703-714.
4 Lima,L. and Coutinho,P. (1980) Clinical criteria for diagnosis of Machado-Joseph disease: report of a non-Azorean Portuguese family. Neurol. 30,319-322.
5 Barbeau,A., Roy,M., Cunha,L., de Vincente,A.N., Rosenberg,R.N., Nyhan,W.L., MacLeod,P.L., Chazot,G., Langston,L.B., Dawson,D.M. and Coutinho,P. (1984) The natural history of Machado-Joseph disease: an analysis of 138 personally examined cases. Can. S. Neurol. Sci. 11,510-525.
6 Sakai,T., Ohta,M. and Ishino,H. (1983) Joseph disease in a non-Portuguese family. Neurol. 33,74-80.
7 Yuasa,T., Ohama,E., Harayama,H., Yamada,M., Kawase,Y., Wakabayashi, M., Atsumi,T. and Miyatake,T. (1986) Joseph's disease: clinical and pathological studies in a Japanese family. Ann. Neurol. 19, 152-157.MEDLINE Abstract
8 Takiyama,Y., Oyanagi,S., Kawashima,S., Sakamoto,H., Saito,K., Yoshida,M., Tsuji,S., Mizuno,Y. and Nishizawa,M. (1994) A clinical and pathologic study of a large Japanese family with Machado-Joseph disease tightly linked to the DNA markers on chromosome 14q. Neurol. 44, 1302-1308.
9 Takiyama,Y., Nishizawa,M., Tanaka,H., Kawashima,S., Sakamoto,H., Karube,Y., Shimazaki,H., Soutome,M., Endo,K., Ohta,S., Kagawa,Y., Kanazawa,l., Mizuno,Y., Yoshida,M., Yuasa,T., Horikawa,Y., Oyanagi,K., Nagai,H., Kondo,T., Inuzuka,T., Onodera,O. and Tsuji,S. (1993) The gene for Machado-Joseph disease is mapped to human chromosome 14q. Nature Genet. 4,300-304.MEDLINE Abstract
10 Sasaki,H., Wakisaka,A., Takada,A., Yoshiki,T., Ihara,T., Suzuki,Y., Hamada,T., Iwabuchi,K., Onari,K., Tada,J., Suzuki,T. and Tashiro,K. (1995) Mapping of the gene for Machado-Joseph disease within a 3.6-cM interval flanked by D14S291/ D14S280 and D14S81, on the basis of studies of linkage and linkage disequilibrium in 24 Japanese families. Am. J. HumGenet. 56,231-242.
11 St. George-Hyslop,P., Rogaeva,E., Huterer,J., Tsuda,T., Santos,J., Haines,J.L., Schlumpf,K., Rogaev,E.I., Liang,Y., McLachlan,D.R.C., Kennedy,J., Wessenbach,J., Billingsley,G.D., Cox,D.W., Lang,A.E. and Wherrett,R. (1994) Machado-Joseph disease in pedigrees of Azorean descent is linked to chromosome 14. Am. J. Hum. Genet. 55,120-125.MEDLINE Abstract
12 Sequeiros,J., Silveira,l., Maciel,P., Coutinho,P., Manaia,A., Gaspar,C., Burlet,P., Loureiro,L., Guimaraes,J., Tanaka,H., Takiyama,Y., Sakamoto,H., Nisizagawa,M., Nomura,Y., Segawa,M., Tsuji,S., Melki,J. and Munnich,A. (1994) Genetic linkage studies of Machado-Joseph disease with chromosome 14q STRPs in 16 Portuguese-Azorean kindreds. Genomics 21,645-648.MEDLINE Abstract
13 Stevanin,G., Cancel,G., Durr,A., Chneiweiss,H., Dubourg,O., Weissenbach,J., Cann,H.M., Agid,Y. and Brice,A. (1995) The gene for spinal cerebellar ataxia 3 (SCA3) is located in a region of ~3 cM on chromosome 14q24.3-q32.2. Am. J. Hum. Genet. 56,193-201.MEDLINE Abstract
14 Kawaguchi,Y., Okamoto,T., Taniwaki,M., Aizawa,M., Inoue,M., Katayama,S., Kawakami,H., Nakamura,S., Nishimura,M., Akiguchi,l., Kimura,J.,Narumiya,S. and Kakizaki,A. (1994) CAG expansions in a novel gene for Machado-Joseph disease at chromosome 14q32.1. Nature Genet.8, 221 -227.MEDLINE Abstract
15 Maruyama,H., Nakamura,S., Matsuyama,Z., Sakai,T., Doyu,M., Sobue,G., Seto,M., Tsujihata,M., Oh-i,T., Nishino,T., Sunohara,N., Takahashi,R., Hayashi,M., Nishino,l., Ohtake,T., Oda,T., Nishimura,M., Saida,T., Matsumoto,H., Baba,M., Kawaguchi,Y., Kakizuka,A. and Kawakami,H. (1995) Molecular features of the CAG repeats and clinical manifestation of MachadoJoseph disease. Hum. Mol. Genet. 4,807-812.MEDLINE Abstract
16 Takiyama,Y., Igarashi,S., Rogaeva,E.A., Endo,K., Rogaev,E.I., Tanaka,H., Sherrington,R., Sanpei,K., Liang,Y., Saito,M., Tsuda,T., Takano,H., Ikeda,M., Lin,C., Chi,H., Kennedy,J.L., Lang,A.E., Wherrett,J.R., Segawa,M., Nomura,Y., Yuasa,T., Weissenbach,J., Yoshida,M., Kidd,K.K., Tsuji,S. and St GeorgeHyslop,P. (1995) Evidence for inter-generational instability in the CAG repeat in the MJD1 gene and for conserved hapiotypes at flanking markers amongst Japanese and Caucasian subjects with Machado-Joseph disease. Hum. Mol. Genet.4,1137-1146.MEDLINE Abstract
17 Stevanin, G. et al. (1996)Characterization of the unstable expanded CAG repeat in the MJD1 gene in 4 Brazilian families of Portuguese descent with Machad-Joseph disease. J.Med. Genet. 32, 827-830.
18 Cancel,G., Abbas,N., Stevanin,G., Durr,A., Chneiweiss,H., Neri,C., Duyckaerts,C., Penet,C., Cann,H.M., Agid,Y. and Brice,A. (1995) Marked phenotypic heterogeneity associated with expansion of a CAG repeat sequence at the spinocerebellar ataxia 3/Machado-Joseph disease locus. Am. J. Hum. Genet. 57,809-816.MEDLINE Abstract
19 Durr,A., Stevanin,G., Cancel,G., Duyckaerrs,C., Abbas,N., Didierjean,O., Chneiweiss,H., Benomar,A., Lyon-Caen,O., Julien J., Serdaru,M., Penet,C., Agid,Y. and Brice,A. (1996) Spinocerebellar ataxia 3 and Machado-Joseph disease: clinical, molecular, and neuropathological features. Ann. Neurol. 39,490-499.MEDLINE Abstract
20 Macil,P., Gaspar,C., DeStefano,A.L, Silveira,L., Coutinho,P., Radvany,J., Dawson,D. M., Sudarsky,L., Guimaraes,J., Loureiro,J.E.L., Nezarati,M.M., Corwin,L.I., LopesCendes,l., Rooke,K., Rosenberg,R., MacLeod,P., Farrer,L.A., Sequeiros,J. and Rouleau,G.A. (1995) Correlation between CAG repeat length and clinical features in Machado-Joseph disease. Am. J. Hum. Genet. 57,54-61.
21 Twist,E.C., Casaubon,L.K., RuKledge,M.H., Rao,V.A., MacLeod,P.M., Radvany,J., Zhao,Z., Rosenberg,R.N., Farrer,L.A. and Rouleau,G.A. (1995) Machado Joseph disease maps to the same region of chromosome 14 as the spinocerebellar ataxia type3 locus. J. Med. Genet. 43, 25-31.
22 The Huntington's Disease Collaborative Research Group. (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell 72, 971-983.
23 Duyao,M., Ambrose,C., Myers,R., Novelletto,A., Persichetti,F., Frontari,M., Folstein,S., Ross,C., Franz,M., Abbott,M., Gray,J., Conneally,P., Young,A., Penney,J., Hollingsworth,Z., Shoulson,l., Lazzarini,A., Falek,A., Koroshetz,W., Sax,D., Bird,E., Vonsattel,J., Bonilla,E., Alvir,J., Conde,J.B., Cha,J.-H., Dure,L., Gomez,F., Ramos,M., Sanchez-Ramos,J., Snodgrass,S., de Young,M., Wexler,N., Moscowitz,C., Penchaszadeh,G., MacFarlane,H., Jenkins,B., S rinidhi,J., Barnes,G., Gussela,J. and MacDonald,M. (1993) Trinucleotide repeat length instability and age of onset in Huntington's disease. Nature Genet. 4, 387-392.MEDLINE Abstract
24 Snell,R.G., MacMillan,J.C., Cheadle,J.P., Fenton,l., Lazarou,L.P., Davies,P., MacDonald,M.E., Gusella,J.F., Harper,P.S. and Shaw,D.J. (1993) Relationship between trinucleotide repeat expansion and phenotypic variation in Huntington's disease. Nature Genet. 4,393-397.MEDLINE Abstract
25 Andrew,S.E., Goldberg,Y.P., Kremer,B., Telenius,H., Theilmann,J., Adam,S., Starr,E., Squitieri,F., Lin,B., Kalchman,M.A., Graham,R.K. and Hayden,M.R. (1993) The relationship between trinucleotide (CAG) repeat length and clinical features of Huntington's disease. Nature Genet. 4, 398-403.MEDLINE Abstract
26 Orr,H,T., Chung,M.-Y., Banfi,S., Kwiatkowski,T.J. Jr., Servadio,A., Beaudet,A.L., McCall,A.E., Duvick,L.A., Ranum,L.P.W. and Zoghbi,H.Y. (1993) Expansion of an unstable trinucleotide CAG repeat in spinocerebellar ataxia type 1. Nature Genet. 4, 221-226.MEDLINE Abstract
27 Chung,M.-Y., Ranum,L.P.W., Duvick,L.A., Servadio,A., Zoghbi,H.Y. and Orr,H.T. (1993) Evidence for a mechanism predisposing to intergenerational CAG repeat instability in spinocerebellar ataxia type 1. Nature Genet. 5, 254-258.MEDLINE Abstract
28 Ranum,L.P.W., Chung, M.-Y., Banfi,S., Brayer,A., Schut,L.J., Ramesar,R., Duvick,L.A., McCall,A., Subramony,S.H., Goldfarb,L., Gomez,C., Sandkuijl,L.A., Orr,H.T. and Zoghbi,H.Y. (1994) Molecular and clinical correlations in spinocerebellar ataxia type 1 (SCA1): evidence for familial effects on age at onset. Am. J. Hum. Genet. 55, 244-252.
29 Ranum,L.P.W., Lundgren,J.K., Schut,L.J., Ahrens,M.J., Perlman,S., Aita,J., Bird,T.D., Gomez,C. and Orr,H.T. (1995) Spinocerebellar ataxia type 1 and Machado-Joseph disease: Incidence of CAG expansions among adult-onset ataxia patients from 311 families with dominant, recessive or sporadic ataxia. Am.J. Hum. Genet. 57, 603-608.
31 Nagafuchi,S., Yanagisawa,H., Sato,K., Shirayama,T., Ohsaki,E., Bunda,M., Takeda,T., Tadokoro,K., Kondo,l., Murayama,N., Tanaka,Y., Kikushima,H., Umino,K., Kurosawa,H., Furukawa,T., Nihei,K., Inoue,T., Sano,A., Komure,O., Takahashi,M., Yoshizawa,T., Kanazawa,l. and Yamada,M. (1994) Dentatorubral and pallidoluysian atrophy expansion of an unstable CAG trinucleotide on chromosome 12p. Nature Genet. 6, 14-18.MEDLINE Abstract
32 Ikeuchi,T., Koide,R., Tanaka,H., Onodera,O., Igarashi,S., Takahashi,H., Kondo,R., Ishikawa,A., Tomoda,A., Miike,T., Sato,K., Ihara,Y., Hayabara,T., Isa,F., Tanabe,H., Tokiguchi,S., Hayashi,M., Shimizu,N., Ikuta,F., Naito,H. and Tsuji,S. (1995) Dentatorubral-pallidoluysian atrophy (DRPLA): clinical features are closely related to unstable expansions of trinucleotide (CAG) repeat. Ann. Neurol. 37, 769-775.MEDLINE Abstract
33 Squitieri,F., Andrew,S.E., Goldberg,Y.P., Kremer,B., Spence,N., Zeisler,J., Nichol,K., Theilmann,J., Greenberg,J., Goto,J., Kanazawa,l., Vesa,J., Peltonen,L., Almqvist,E., Anvret,M., Telenius,H., Lin,B., Morgan,K. and Hayden,M.R. (1994) DNA haplotype analysis of Huntington disease reveals clues to the origins and mechanisms of CAG expansion and reasons for geographic variations of prevalence. Hum. Mol. Genet. 3,2103-2114.MEDLINE Abstract
34 Almqvist,E., Spence,N., Nichol,K., Andrew,S.E., Vesa,J., Peltonen,L., Anvret,M., Goto,J., Kanazawa,I., Goldberg,Y.P., and Hayden,M.R. (1995) Ancestral differences in the distribution of the A2642 glutamic acid polymorphism are associated with varying CAG repeat lengths on norrnal chromosomes: insights into the genetic evolution of Huntington disease. Hum. Mol. Genet. 4,207-214.MEDLINE Abstract
35 Rubinsztein,D.C., Leggo,J., Goodburn,S., Barton,D.E. and Ferguson-Smith,M.A. (1995) Haplotype analysis of the A2642 and (CAG)n polymorphism in the Huntington's disease (HD) gene provides an explanation for an apparent founder HD haplotype. Hum. Mol. Genet. 4,203-206.MEDLINE Abstract
36 Snow,K.,Tester,D.J., Kruckeberg,K.E., Schaid,D.J. and Thibodeau,S.N. (1994) Sequence analysis of the fragile X trinucleotide repeat: implications for the origin of the fragile X mutation. Hum. Mol. Genet. 3,1543-1551.MEDLINE Abstract
37 Kunst,C.B. and Warren,S.T. (1994) Cryptic and polar variation of the fragile X repeat could result in predisposing normal alleles. Cell 77,853-861.MEDLINE Abstract
38 Imbert,G., Kretz,C., Johnson,K. and Mandel,J.L. (1993) Origin of the expansion mutation in myotonic dystrophy. Nature Genet. 4,72-76.MEDLINE Abstract
39 Neville,C.E., Mahadevan,M.S., Barcelo,J.M. and Korneluk,R.G. (1994) High resolution genetic analysis suggests one ancestral predisposing haplotype for the origin of the myotonic dystrophy mutation. Hum. Mol. Genet. 3,45-51.MEDLINE Abstract
40 Rubinsztein,D.C., Leggo,J., Amos,W., Barton,D.E. and Ferguson-Smith,M.A. (1994) Myotonic dystrophy CTG repeat and associated insertion/deletion polymorphism in human and primate populations. Hum. Mol. Genet.3,2031-2035.MEDLINE Abstract
41 Stevanin, G.; Cancel,G., Didierjean,O., Durr,A., Abbas,N., Cassa,E., Feingold,J., Agid,Y and Brice,A. (1995) Linkage disequilibrrium at the Machado-Joseph disease/spinal cerebellar ataxia 3 locus: evidence for a commom founder effect in French and Portuguese-Brazilian families as well as a second ancestral Portuguese Azorean mutation. Am. J. Hum. Genet. 57, 1247-1250.MEDLINE Abstract
42 Jeffreys,A.J., MacLeod,A., Tamaki,K., Neil,D.L. and Monckton,D.G. (1991) Minisatellite repeat coding as a digital approach to DNA typing. Nature 354,204-209.MEDLINE Abstract
43 Monckton,D.G., Neumann,R., Guram,T., Fretwell,N., Tamaki,K., MacLeod,A., and Jeffreys,A.J. (1994) Minisatellite mutation rate variation associated with a flanking DNA sequence polymorphism. Nature Genet. 8, 162-170.MEDLINE Abstract
44 Jeffreys,A.J., Tamaki,K., MacLeod,A., Monckton,D.G., Neil,D.L. and Armour,J.A.L. (1994) Complex gene conversion events in germline mutation at human minisatellites. Nature Genet. 6, 136-145.MEDLINE Abstract
45 O'Hoy,K.L., Tsilfidis,C., Mahadevan,M.S., Neville,C.E., Barcelo,J., Hunter,A.G.W. and Korneluk,R.G. (1993) Reduction in size of the myotonic dystrophy trinucleotide repeat mutation during transmission. Science 259, 809-812.MEDLINE Abstract
46 Hunter,A.G.W., Jacob,P., O'Hoy,K.L., MacDonald,l., Matter,G., Tsilfidis,C. and Korneluk,R.G. (1993) Am. J. Med. Genet. 45, 401-407.
47 Ouweland,A.M.W., Deelen,W.H., Kunst,C.B., Uzielli,M.-L.G., Nelson,D.L., Warren,S.T., Oostra,B.A. and Halley,D.J.J. (1994) Loss of mutation at the FMR1 locus through multiple exchanges between maternal X chromosomes. Hum.Mol. Genet. 3,1823-1827.
48 Maniatis,T., Fritsch,E.F. and Sambrook,J. (1989) Molecular cloning: A laboratory manual 2nd edn. Cold Spring Harbor.
49 Tsuji,S., Martin,B.M., Barranger,J.A., Stubblefield,B.K., LaMarca,M.E. and Ginns,E.I. (1988) Genetic heterogeneity in type 1 Gaucher disease: Multiple genotypes in Ashkenazic and non-Ashkenazic individuals. Proc. Natl. Acad. Sci. USA 85,2349-2352.MEDLINE Abstract
*To whom correspondence should be addressed
This page is maintained by OUP admin. Last updated Thu Oct 31 15:25:15 GMT 1996. Part of the OUP Journals World Wide Web service.Copyright Oxford University Press, 1996
