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Variation in the number of CAG repeats in the Machado–Joseph disease gene (MJD1) in the Japanese population
Journal of the Neurological Sciences 166 (1999) 71–73
Variation in the number of CAG repeats in the Machado–Joseph disease gene (MJD1 ) in the Japanese population a,
b
b
c
Zenjiro Matsuyama *, Hideshi Kawakami , Hirofumi Maruyama , Hiromichi Harada , Kazumasa Nakata c , Yumiko Yamaguchi c , Shigenobu Nakamura b a
Laboratories of Humoral Information, Department of Information Physiology, National Institute for Physiological Sciences, Okazaki 444, Japan b Third Department of Internal Medicine, Hiroshima University School of Medicine, Hiroshima 734, Japan c Japanese Red Cross Hiroshima Blood Center, Hiroshima 734, Japan Received 9 December 1998; received in revised form 29 April 1999; accepted 3 May 1999
Abstract Variation in the number of CAG repeats in the Machado–Joseph disease gene (MJD1 ) was examined by polymerase chain reaction and denaturing polyacrylamide gel electrophoresis analysis of 2134 normal and 135 affected chromosomes of Japanese individuals. The number of repeats ranged from 14 to 47 in normal alleles and from 61 to 84 in disease-associated alleles. The most frequent and lowest number of repeats was 14. The size distribution of normal MJD1 alleles did not fit a normal distribution curve, but was tetramodal. Repeats from 14 to 17, 18 to 23, 24 to 25, and 26 to 47 units were designated groups A through D, respectively. When examined Hardy–Weinberg equilibrium by chi-square analysis of goodness of fit; no evidence of significant deviation from the Hardy–Weinberg equilibrium was observed [x 2 54.248,16.919 (P50.05), df59]. The observed distribution peak of normal MJD1 alleles corresponding to peptides containing 10, 15, 20, and 24 glutamine suggests that stretches of 5 and 10 glutamine might constitute a functional domain of human MJD1. 1999 Elsevier Science B.V. All rights reserved. Keywords: Machado–Joseph disease (MJD); CAG repeat; Normal population; Polyglutamine; Spinocerebellar degeneration (SCD); Hardy–Weinberg equilibrium
1. Introduction Machado–Joseph disease (MJD) is an autosomal-dominant neurodegenerative disorder caused by a gene (MJD1 ) that is located at chromosome 14q32.1 and contains CAG trinucleotide repeats [1]. We have previously showed that the number of CAG repeats in MJD1 ranges from 14 to 34 in normal chromosomes and from 61 to 84 in diseaseassociated chromosomes, without any overlap, and that the severity of clinical symptoms increases and the age of disease onset decreases as the number of CAG repeat increases [2].
To elucidate the distribution of the number of CAG repeats in MJD1 by more many samples, we examined 2134 normal and 135 affected chromosomes from Japanese individuals, using polymerase chain reaction (PCR) and denaturing polyacrylamide gel electrophoresis [3]. The resulting data was subjected to statistical analysis with the chi-square test.
2. Materials and methods
2.1. Materials *Corresponding author. Tel.: 181-564-55-7851; fax: 181-564-557853. E-mail address: [email protected] (Z. Matsuyama)
Genomic DNA was extracted from buffy coats of 1067 general Japanese Red Cross blood donors aged 16–64
0022-510X / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0022-510X( 99 )00109-4
72
Z. Matsuyama et al. / Journal of the Neurological Sciences 166 (1999) 71 – 73
years, none of whom were type B and C hepatitis virus carriers, HIV virus carriers, or suffering from heart disease, neurodegenerative disease, cerebral infarction, epilepsy, renal disease, cancer, diabetes, hypertension, asthma, allergic diseases, leukemia, anemia, syphilis, or tuberculosis, as previously described [4]. High molecular weight DNA was extracted from leukocytes of 135 individuals with MJD [5]. We obtained informed consents from all MJD patients. And the donors of normal controls were informed by a bulletin board that their blood samples might be used for the purpose of public research and health.
2.2. Amplification and determination of the number of CAG repeats The CAG repeat region of MJD1 was amplified by PCR with the primers MJ-N (59-TCGTGAAACAATGTATTTTCCTTATG-39) and MJ-RN (59GATGTGAACTCTGTCCTGAT-39). Amplification was performed for 30 cycles (denaturation for 1 min at 958C, annealing for 1 min at 558C, and elongation for 1 min at 728C), and the 59-terminus of primer MJ-N was labeled, employing bacteriophage T4 polynucleotide kinase (Takara, Kyoto, Japan) and [r- 32 P]ATP at 378C. The PCR mixture (20 ml) contained 200 ng of genomic DNA, 50 mM Tris–HCl (pH 9.2), 14 mM (NH 4 ) 2 SO 4 , 1.75 mM MgCl 2 , 350 mM each of dCTP, dATP, and dTTP, 87.5 mM dGTP, 262.5 mM 7-deaza-dGTP, 200 ng of MJ-RN primer, 180 ng of unlabeled MJ-N primer, 20 ng of 32 P-labeled MJ-N primer, and 2.6 U of Taq and Pwo DNA polymerases (Boehringer Mannheim, Mannheim, Germany). PCR products (4 ml) were mixed with 1 ml of formamide loading buffer [98% (v / v) formamide, 10 mM EDTA (pH 8.0), 0.025% xylene cyanol FF, and 0.025% bromophenol blue], denatured at 998C for 10 min, and then placed on ice. Electrophoresis was performed on 8% HydroLink Long Ranger (AT Biochem, Malvern, PA) polyacrylamide gels containing 7 M urea and 42% formamide [3]. CAG repeat length was determined by comparison with a standard M13 sequencing ladder.
Fig. 1. Distribution of CAG repeat lengths of 2134 normal alleles of MJD1 in the Japanese population. The number of repeats ranged from 14 to 47, and repeats from 14 to 17, 18 to 23, 24 to 25, and 26 to 47 units were assigned as groups A through D, respectively.
to 84 in 135 affected chromosomes. There were no expanded alleles in 1067 general Red Cross blood donors examined. The distribution of repeat lengths in normal alleles did not fit a normal distribution curve. The most frequent repeat length was 14 in normal chromosomes. The repeat length of normal chromosomes showed a tetramodal distribution, grouped into repeats from 14 to 17, 18 to 23, 24 to 25, and 26 to 47 units which were designated as groups A through D, respectively. The peak in each group corresponded to 14 (777 alleles) repeats in group A, 19 (191 alleles) in B, 24 (70 alleles) in C, and 28 (279 alleles) in D (Fig. 1). The total number of alleles in groups A through D was 781, 355, 94, and 904, respectively (Table 1). No evidence of significant deviation from the Hardy– Weinberg equilibrium was observed [x 2 54.248,16.919 (P50.05), df59] (Table2). Chi-square analysis of independence revealed no significant difference in frequency of alleles in each group between sex [x 2 54.11 (P50.2178), df53].
2.3. Statistical analysis A P value of ,0.05 was considered statistically significant for all statistical methods applied (see Results). Data were analyzed using the computer software package JMP 3.0 (SAS Institute Inc., Cary, NC). Hardy–Weinberg equilibrium was tested by chi-square analysis of goodness of fit. The difference of the frequency of alleles in each group between sex was tested by chi-square analysis of independence.
3. Results The number of CAG repeats in the MJD1 gene ranged from 14 to 47 in 2134 normal chromosomes and from 61
4. Discussion Normal distributions have been demonstrated for the allele of genes associated with various trinucleotide disTable 1 The number of CAG repeat of MJD1 allele frequencies in the Japanese population: number of alleles observed / number of total alleles, with the frequency given in parentheses Groups
Number of CAG repeat
Frequency
A B C D
14–17 18–23 24–25 26–47
781 / 2134 (0.3660) 355 / 2134 (0.1664) 94 / 2134 (0.0440) 904 / 2134 (0.4236)
Z. Matsuyama et al. / Journal of the Neurological Sciences 166 (1999) 71 – 73 Table 2 Check for Hardy–Weinberg equilibrium Genotype
A–A A–B A–C A–D B–B B–C B–D C–C C–D D–D
Observed
Expected
N
%
N
%
146 125 31 333 29 13 159 1 48 182
13.7 11.7 2.91 31.2 2.72 1.22 14.9 0.0937 4.50 17.6
142.9 129.9 34.40 330.8 29.53 15.62 150.4 2.070 39.82 191.5
13.39 12.18 3.224 31.00 2.767 1.466 14.09 0.1940 3.732 17.95
eases [6–8]. Normal alleles of the myotonic dystrophy (DM) gene show a trimodal distribution [8]. We have now shown that the distribution of normal MJD1 alleles is also tetramodal. Although gorillas possess only 8–11 repeated glutamine in peptide produced by MJD1 gene [9], the lowest and most frequent numbers of CAG repeats in MJD1 of humans is 14 [2,9–11], which contains 10 continuous CAG repeat resulting in a stretch of 10 glutamine residues in the MJD1 protein. The peak of allele with 19, 24, or 28 repeats yields stretches of 15, 20, or 24 glutamine, respectively. The distribution peak of normal MJD1 alleles corresponding to peptides with 10, 15, 20, and 24 glutamine suggests that stretches of 5 and 10 glutamine might constitute a functional domain of human MJD1. We have previously shown that three factors influence the age of onset of MJD: (1) the number of CAG repeats in MJD1, (2) gender, and (3) dosage of alleles with expanded CAG repeats [12]. The normal Huntington’s disease allele, or a closely linked gene, has been reported to influence the age of disease onset [13]. We examined the affect of the normal MJD allele on the age of onset (data not shown) but there is no relation between the normal MJD allele and the age of onset. Although the normal MJD1 allele could also influence the age of onset of MJD, such an effect would be masked by the greater influence of the expanded allele. Analysis of the haplotype of the myotonic dystrophy (DM) gene has indicated that the larger alleles arose from the 5-repeat allele by a small number (from one to four) of mutational events. Subsequent mutation of this allele yields the longest normal alleles, which encode 19–30 CTG repeats and serve as a source of expanded alleles [6]. Although similar haplotype analysis remains to be performed for MJD1 and the content of our study is restricted to the description of the tetramodal distribution of the CAG repeat size in MJD gene, like DM gene, several ancestral mutational events might give rise to the shortest
73
and most frequent allele, (CAG) 14 , subsequently mutated to the allele in group D, (CAG) 26 – 47 , which, in turn, serves as a source of expanded alleles. To prove this hypothesis we are necessary to analyse the haplotype of MJD gene of normal controls and patients.
Acknowledgements We thank the Exploring Research Center for Molecular Medicine, Hiroshima University School of Medicine, for the use of their facilities. This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan.
References [1] Kawaguchi Y, Okamoto T, Taniwaki M et al. CAG expansions in a novel gene for Machado–Joseph disease at chromosome 14q32.1. Nature Genet 1994;8:221–8. [2] Maruyama H, Nakamura S, Matsuyama Z et al. Molecular features of the CAG repeats and clinical manifestation of Machado–Joseph disease. Hum Mol Genet 1995;4:807–12. [3] Maruyama H, Kawakami H, Nakamura S. Reevaluation of the exact CAG repeat length in hereditary cerebellar ataxias using highly denaturing conditions and long PCR. Hum Genet 1996;97:591–5. [4] Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1989;16:1215. [5] Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning. A Laboratory Manual, 2nd ed, New York: Cold Spring Harbor Laboratory Press, 1989. [6] Imbert G, Kretz C, Johnson K, Mandel JL. Origin of the expansion mutation in myotonic dystrophy. Nature Genet 1993;4:72–6. [7] Watkins WS, Bamshad M, Jorde LB. Population genetics of trinucleotide repeat polymorphisms. Hum Mol Genet 1995;4:1485– 91. [8] Zerylnick C, Torroni A, Sherman SL, Warren ST. Normal variation at the myotonic dystrophy locus in global human populations. Am J Hum Genet 1995;56:123–30. [9] Rubinsztein DC, Leggo J, Coetzee GA, Irvine RA, Buckley M, Ferguson-Smith MA. Sequence variation and size ranges of CAG repeats in the Machado–Joseph disease, spinocerebellar ataxia type 1 and androgen receptor genes. Hum Mol Genet 1995;4:1585–90. [10] Matilla T, McCall A, Subramony SH, Zoghbi HY. Molecular and clinical correlations in spinocerebellar ataxia type 3 and Machado– Joseph disease. Ann Neurol 1995;38:68–71. [11] Schols L, Vieira-Saecker AMM, Schols S, Przuntek H, Epplen JT, Riess O. Trinucleotide expansion within the MJD1 gene presents clinically as spinocerebellar ataxia and occurs most frequently in German SCA patients. Hum Mol Genet 1995;4:1001–5. [12] Kawakami H, Maruyama H, Nakamura S, Kawaguchi Y, Kakizuka A, Doyu M, Sobue G. Unique features of the CAG repeats in Machado–Joseph disease. Nature Genet 1995;9:334–5. [13] Farrer LA, Cupples LA, Wiater P, Conneally PM, Gusella JF, Myers RH. The normal Huntington disease (HD) allele, or a closely linked gene, influences age at onset of HD. Am J Hum Genet 1993;53:125– 30.
Variation in the number of CAG repeats in the Machado–Joseph disease gene (MJD1 ) in the Japanese population a,
b
b
c
Zenjiro Matsuyama *, Hideshi Kawakami , Hirofumi Maruyama , Hiromichi Harada , Kazumasa Nakata c , Yumiko Yamaguchi c , Shigenobu Nakamura b a
Laboratories of Humoral Information, Department of Information Physiology, National Institute for Physiological Sciences, Okazaki 444, Japan b Third Department of Internal Medicine, Hiroshima University School of Medicine, Hiroshima 734, Japan c Japanese Red Cross Hiroshima Blood Center, Hiroshima 734, Japan Received 9 December 1998; received in revised form 29 April 1999; accepted 3 May 1999
Abstract Variation in the number of CAG repeats in the Machado–Joseph disease gene (MJD1 ) was examined by polymerase chain reaction and denaturing polyacrylamide gel electrophoresis analysis of 2134 normal and 135 affected chromosomes of Japanese individuals. The number of repeats ranged from 14 to 47 in normal alleles and from 61 to 84 in disease-associated alleles. The most frequent and lowest number of repeats was 14. The size distribution of normal MJD1 alleles did not fit a normal distribution curve, but was tetramodal. Repeats from 14 to 17, 18 to 23, 24 to 25, and 26 to 47 units were designated groups A through D, respectively. When examined Hardy–Weinberg equilibrium by chi-square analysis of goodness of fit; no evidence of significant deviation from the Hardy–Weinberg equilibrium was observed [x 2 54.248,16.919 (P50.05), df59]. The observed distribution peak of normal MJD1 alleles corresponding to peptides containing 10, 15, 20, and 24 glutamine suggests that stretches of 5 and 10 glutamine might constitute a functional domain of human MJD1. 1999 Elsevier Science B.V. All rights reserved. Keywords: Machado–Joseph disease (MJD); CAG repeat; Normal population; Polyglutamine; Spinocerebellar degeneration (SCD); Hardy–Weinberg equilibrium
1. Introduction Machado–Joseph disease (MJD) is an autosomal-dominant neurodegenerative disorder caused by a gene (MJD1 ) that is located at chromosome 14q32.1 and contains CAG trinucleotide repeats [1]. We have previously showed that the number of CAG repeats in MJD1 ranges from 14 to 34 in normal chromosomes and from 61 to 84 in diseaseassociated chromosomes, without any overlap, and that the severity of clinical symptoms increases and the age of disease onset decreases as the number of CAG repeat increases [2].
To elucidate the distribution of the number of CAG repeats in MJD1 by more many samples, we examined 2134 normal and 135 affected chromosomes from Japanese individuals, using polymerase chain reaction (PCR) and denaturing polyacrylamide gel electrophoresis [3]. The resulting data was subjected to statistical analysis with the chi-square test.
2. Materials and methods
2.1. Materials *Corresponding author. Tel.: 181-564-55-7851; fax: 181-564-557853. E-mail address: [email protected] (Z. Matsuyama)
Genomic DNA was extracted from buffy coats of 1067 general Japanese Red Cross blood donors aged 16–64
0022-510X / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0022-510X( 99 )00109-4
72
Z. Matsuyama et al. / Journal of the Neurological Sciences 166 (1999) 71 – 73
years, none of whom were type B and C hepatitis virus carriers, HIV virus carriers, or suffering from heart disease, neurodegenerative disease, cerebral infarction, epilepsy, renal disease, cancer, diabetes, hypertension, asthma, allergic diseases, leukemia, anemia, syphilis, or tuberculosis, as previously described [4]. High molecular weight DNA was extracted from leukocytes of 135 individuals with MJD [5]. We obtained informed consents from all MJD patients. And the donors of normal controls were informed by a bulletin board that their blood samples might be used for the purpose of public research and health.
2.2. Amplification and determination of the number of CAG repeats The CAG repeat region of MJD1 was amplified by PCR with the primers MJ-N (59-TCGTGAAACAATGTATTTTCCTTATG-39) and MJ-RN (59GATGTGAACTCTGTCCTGAT-39). Amplification was performed for 30 cycles (denaturation for 1 min at 958C, annealing for 1 min at 558C, and elongation for 1 min at 728C), and the 59-terminus of primer MJ-N was labeled, employing bacteriophage T4 polynucleotide kinase (Takara, Kyoto, Japan) and [r- 32 P]ATP at 378C. The PCR mixture (20 ml) contained 200 ng of genomic DNA, 50 mM Tris–HCl (pH 9.2), 14 mM (NH 4 ) 2 SO 4 , 1.75 mM MgCl 2 , 350 mM each of dCTP, dATP, and dTTP, 87.5 mM dGTP, 262.5 mM 7-deaza-dGTP, 200 ng of MJ-RN primer, 180 ng of unlabeled MJ-N primer, 20 ng of 32 P-labeled MJ-N primer, and 2.6 U of Taq and Pwo DNA polymerases (Boehringer Mannheim, Mannheim, Germany). PCR products (4 ml) were mixed with 1 ml of formamide loading buffer [98% (v / v) formamide, 10 mM EDTA (pH 8.0), 0.025% xylene cyanol FF, and 0.025% bromophenol blue], denatured at 998C for 10 min, and then placed on ice. Electrophoresis was performed on 8% HydroLink Long Ranger (AT Biochem, Malvern, PA) polyacrylamide gels containing 7 M urea and 42% formamide [3]. CAG repeat length was determined by comparison with a standard M13 sequencing ladder.
Fig. 1. Distribution of CAG repeat lengths of 2134 normal alleles of MJD1 in the Japanese population. The number of repeats ranged from 14 to 47, and repeats from 14 to 17, 18 to 23, 24 to 25, and 26 to 47 units were assigned as groups A through D, respectively.
to 84 in 135 affected chromosomes. There were no expanded alleles in 1067 general Red Cross blood donors examined. The distribution of repeat lengths in normal alleles did not fit a normal distribution curve. The most frequent repeat length was 14 in normal chromosomes. The repeat length of normal chromosomes showed a tetramodal distribution, grouped into repeats from 14 to 17, 18 to 23, 24 to 25, and 26 to 47 units which were designated as groups A through D, respectively. The peak in each group corresponded to 14 (777 alleles) repeats in group A, 19 (191 alleles) in B, 24 (70 alleles) in C, and 28 (279 alleles) in D (Fig. 1). The total number of alleles in groups A through D was 781, 355, 94, and 904, respectively (Table 1). No evidence of significant deviation from the Hardy– Weinberg equilibrium was observed [x 2 54.248,16.919 (P50.05), df59] (Table2). Chi-square analysis of independence revealed no significant difference in frequency of alleles in each group between sex [x 2 54.11 (P50.2178), df53].
2.3. Statistical analysis A P value of ,0.05 was considered statistically significant for all statistical methods applied (see Results). Data were analyzed using the computer software package JMP 3.0 (SAS Institute Inc., Cary, NC). Hardy–Weinberg equilibrium was tested by chi-square analysis of goodness of fit. The difference of the frequency of alleles in each group between sex was tested by chi-square analysis of independence.
3. Results The number of CAG repeats in the MJD1 gene ranged from 14 to 47 in 2134 normal chromosomes and from 61
4. Discussion Normal distributions have been demonstrated for the allele of genes associated with various trinucleotide disTable 1 The number of CAG repeat of MJD1 allele frequencies in the Japanese population: number of alleles observed / number of total alleles, with the frequency given in parentheses Groups
Number of CAG repeat
Frequency
A B C D
14–17 18–23 24–25 26–47
781 / 2134 (0.3660) 355 / 2134 (0.1664) 94 / 2134 (0.0440) 904 / 2134 (0.4236)
Z. Matsuyama et al. / Journal of the Neurological Sciences 166 (1999) 71 – 73 Table 2 Check for Hardy–Weinberg equilibrium Genotype
A–A A–B A–C A–D B–B B–C B–D C–C C–D D–D
Observed
Expected
N
%
N
%
146 125 31 333 29 13 159 1 48 182
13.7 11.7 2.91 31.2 2.72 1.22 14.9 0.0937 4.50 17.6
142.9 129.9 34.40 330.8 29.53 15.62 150.4 2.070 39.82 191.5
13.39 12.18 3.224 31.00 2.767 1.466 14.09 0.1940 3.732 17.95
eases [6–8]. Normal alleles of the myotonic dystrophy (DM) gene show a trimodal distribution [8]. We have now shown that the distribution of normal MJD1 alleles is also tetramodal. Although gorillas possess only 8–11 repeated glutamine in peptide produced by MJD1 gene [9], the lowest and most frequent numbers of CAG repeats in MJD1 of humans is 14 [2,9–11], which contains 10 continuous CAG repeat resulting in a stretch of 10 glutamine residues in the MJD1 protein. The peak of allele with 19, 24, or 28 repeats yields stretches of 15, 20, or 24 glutamine, respectively. The distribution peak of normal MJD1 alleles corresponding to peptides with 10, 15, 20, and 24 glutamine suggests that stretches of 5 and 10 glutamine might constitute a functional domain of human MJD1. We have previously shown that three factors influence the age of onset of MJD: (1) the number of CAG repeats in MJD1, (2) gender, and (3) dosage of alleles with expanded CAG repeats [12]. The normal Huntington’s disease allele, or a closely linked gene, has been reported to influence the age of disease onset [13]. We examined the affect of the normal MJD allele on the age of onset (data not shown) but there is no relation between the normal MJD allele and the age of onset. Although the normal MJD1 allele could also influence the age of onset of MJD, such an effect would be masked by the greater influence of the expanded allele. Analysis of the haplotype of the myotonic dystrophy (DM) gene has indicated that the larger alleles arose from the 5-repeat allele by a small number (from one to four) of mutational events. Subsequent mutation of this allele yields the longest normal alleles, which encode 19–30 CTG repeats and serve as a source of expanded alleles [6]. Although similar haplotype analysis remains to be performed for MJD1 and the content of our study is restricted to the description of the tetramodal distribution of the CAG repeat size in MJD gene, like DM gene, several ancestral mutational events might give rise to the shortest
73
and most frequent allele, (CAG) 14 , subsequently mutated to the allele in group D, (CAG) 26 – 47 , which, in turn, serves as a source of expanded alleles. To prove this hypothesis we are necessary to analyse the haplotype of MJD gene of normal controls and patients.
Acknowledgements We thank the Exploring Research Center for Molecular Medicine, Hiroshima University School of Medicine, for the use of their facilities. This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan.
References [1] Kawaguchi Y, Okamoto T, Taniwaki M et al. CAG expansions in a novel gene for Machado–Joseph disease at chromosome 14q32.1. Nature Genet 1994;8:221–8. [2] Maruyama H, Nakamura S, Matsuyama Z et al. Molecular features of the CAG repeats and clinical manifestation of Machado–Joseph disease. Hum Mol Genet 1995;4:807–12. [3] Maruyama H, Kawakami H, Nakamura S. Reevaluation of the exact CAG repeat length in hereditary cerebellar ataxias using highly denaturing conditions and long PCR. Hum Genet 1996;97:591–5. [4] Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1989;16:1215. [5] Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning. A Laboratory Manual, 2nd ed, New York: Cold Spring Harbor Laboratory Press, 1989. [6] Imbert G, Kretz C, Johnson K, Mandel JL. Origin of the expansion mutation in myotonic dystrophy. Nature Genet 1993;4:72–6. [7] Watkins WS, Bamshad M, Jorde LB. Population genetics of trinucleotide repeat polymorphisms. Hum Mol Genet 1995;4:1485– 91. [8] Zerylnick C, Torroni A, Sherman SL, Warren ST. Normal variation at the myotonic dystrophy locus in global human populations. Am J Hum Genet 1995;56:123–30. [9] Rubinsztein DC, Leggo J, Coetzee GA, Irvine RA, Buckley M, Ferguson-Smith MA. Sequence variation and size ranges of CAG repeats in the Machado–Joseph disease, spinocerebellar ataxia type 1 and androgen receptor genes. Hum Mol Genet 1995;4:1585–90. [10] Matilla T, McCall A, Subramony SH, Zoghbi HY. Molecular and clinical correlations in spinocerebellar ataxia type 3 and Machado– Joseph disease. Ann Neurol 1995;38:68–71. [11] Schols L, Vieira-Saecker AMM, Schols S, Przuntek H, Epplen JT, Riess O. Trinucleotide expansion within the MJD1 gene presents clinically as spinocerebellar ataxia and occurs most frequently in German SCA patients. Hum Mol Genet 1995;4:1001–5. [12] Kawakami H, Maruyama H, Nakamura S, Kawaguchi Y, Kakizuka A, Doyu M, Sobue G. Unique features of the CAG repeats in Machado–Joseph disease. Nature Genet 1995;9:334–5. [13] Farrer LA, Cupples LA, Wiater P, Conneally PM, Gusella JF, Myers RH. The normal Huntington disease (HD) allele, or a closely linked gene, influences age at onset of HD. Am J Hum Genet 1993;53:125– 30.