Single sperm analysis of the CAG repeats in the gene for Machado-Joseph disease (MJD1): evidence for non-Mendelian transmission of the MJD1 gene and for the effect of the intragenic CGG/GGG polymorphism on the intergenerational instability
Single sperm analysis of the CAG repeats in the gene for Machado-Joseph disease ( MJD1 ): evidence for non-Mendelian transmission of the MJD1 gene and for the effect of the intragenic <underline><i> C </i></underline> GG/ <underline><i> G </i></underline> GG polymorphism on the intergenerational instabilityY. Takiyama1, K. Sakoe1, M. Soutome1, M. Namekawa1, T. Ogawa1, I. Nakano1, S. Igarashi2, M. Oyake2, H. Tanaka2, S. Tsuji2 and M. Nishizawa1,*
1Department of Neurology, Jichi Medical School, Minamikawachi, Tochigi 329-04, Japan and 2Department of Neurology, Brain Research Institute, Niigata University, 1 Asahimachi, Niigata 951, Japan
Received January 13, 1997;Revised and Accepted April 7, 1997
To investigate the mechanism of the meiotic instability of expanded CAG repeats in the gene for Machado-Joseph disease (MJD1), we analyzed the CAG repeat sizes of 1036 single sperm from six individuals with Machado-Joseph disease (MJD). The segregation ratio between single sperm with an expanded allele and those with a normal allele is significantly different (P <0.0001) from the expected 1:1 segregation ratio, which demonstrates segregation distortion of expanded alleles in male meiosis. In single sperm from individuals with the [expanded (CAG)n-CGG]/[normal (CAG)n- GGG] genotype, significantly greater instability of the CAG repeat was observed compared with single sperm from individuals with the [expanded (CAG)n- CGG]/[normal (CAG)n-CGG] genotype (F-test, P <0.001). These findings in single sperm confirm non-Mendelian transmission of the MJD1 gene and the effect of the intragenic CGG/GGG polymorphism on the intergenerational instability of the CAG repeats in the MJD1 gene, which have been observed in clinical and genetic studies. Our results indicate similarities and dissimilarities between MJD and Huntington's disease or myotonic dystrophy in terms of the inter-allelic interaction, segregation distortions and size distribution of trinucleotide repeats in mutant alleles. Further study is required to determine whether there is a common mechanism underlying the instability of the triplet repeats in `triplet repeat diseases'.
Machado-Joseph disease (MJD) is an autosomal dominant, multisystem, neurodegenerative disorder characterized by a variable clinical phenotype which includes cerebellar ataxia, pyramidal signs, dystonic-rigid extrapyramidal syndrome, peripheral neuropathy, progressive external ophthalmoplegia, facial myokymia and bulging eyes (1 ). Although it was originally reported in Portuguese-Azorean descents (2 -4 ), MJD is now regarded as one of the most common autosomal dominant spinocerebellar degenerations in the world (5 ).
We mapped the gene for MJD to human chromosome 14q24.3-32.1 by linkage analyses of five Japanese families (6 ). Using a novel gene containing CAG repeats located on chromosome 14q32.1, as a candidate gene, Kawaguchi et al. discovered the causative gene for MJD (MJD1), and showed expansion of the CAG repeats in MJD patients (7 ). Like other neurodegenerative disorders with a trinucleotide repeat expansion, an inverse correlation of age at onset with the size of the expanded CAG repeats has been demonstrated in MJD (7 ,8 ).
Interestingly, clinical and genetic studies have shown that the CAG repeat in the MJD1 gene is unstable during parent-offspring transmissions (8 ). We found that two factors, paternal transmissions and a polymorphism in normal alleles, contribute to the intergenerational instability of the CAG repeats in the MJD1 gene (9 ,10 ). Of particular interest is that an intragenic CGG/GGG polymorphism at the 3' end of the CAG repeats in the MJD1 gene in normal alleles affects the intergenerational instability of the CAG repeats, suggesting the presence of inter-allelic interaction (9 ). In addition, we recently have found that significant segregation distortions in favor of transmission of the mutant alleles in male meiosis occur in MJD and dentatorubral pallidoluysian atrophy (DRPLA) (10 ).
To investigate directly the mechanism of the intergenerational instability of the CAG repeats in the MJD1 gene in male meiosis, we undertook an analysis of single sperm obtained from six MJD individuals. Here, we report that our results on single sperm confirm the previous clinical and genetic observations.
In single sperm from the six individuals with MJD, 629 expanded alleles and 407 normal alleles were detected, which is significantly different ([chi]2 = 47.57, P <0.0001) from the expected 1:1 segregation ratio. In individual cases, meiotic segregation distortions of expanded alleles were clearly observed in the six individuals, and were significant in four.
We determined the change in the size of the CAG repeats in the MJD1 gene from six MJD individuals by comparing the size of the CAG repeats from single sperm with the size of those from peripheral blood leukocyte DNA of the same individual, according to the method used previously in single sperm analysis of CAG repeats in the Huntington's disease gene (11 ).We refer to the cases in which the size of the CAG repeats is smaller in sperm than in leukocytes as `contractions', and the opposite cases as `expansions'. Table 1 showsthe results of typing 1036 sperm from the six individuals with MJD representing four different expanded allele sizes and four different normal allele sizes. Expanded alleles (74-80 repeats) showed a much higher mutation frequency (88-99%) than normal alleles (0-4%). Among the 629 sperm carrying expanded alleles, 92% differed in size from the individuals' leukocyte expanded alleles. Of these, 32% showed expansions and 60% contractions.
We also analyzed six normal alleles with CAG repeats ranging in size from 14 to 30 CAG repeats in 407 sperm. No mutations in the normal allele carrying 14 CAG repeats were detected in 115 sperm (Table 1 ). Among 292 sperm carrying the 28-30 repeats, only five expansions (+1 repeat) and four contractions (-1 or -2 repeat) were observed.
Single sperm typing data for six MJD-affected individuals
Size of CAG repeats(C or G)a
No. of sperm analyzed
Mean
Expanded (%)
Contracted (%)
Range
Variance
Expanded allele 1b (patient 1)
74 (C)
106
+0.39
54
34
58-93
24.16 (-16 to +19)
Expanded allele 2b (patient 2)
74 (C)
100
-1.20
37
51
58-82
27.82 (-21 to +8)
Expanded allele 3c (patient 3)
74 (C)
105
-2.21
41
50
53-98
55.67 (-21 to +24)
Expanded allele 4b (patient 4)
75 (C)
113
-1.66
27
62
59-85
18.12 (-16 to +10)
Expanded allele 5c (patient 5)
76 (C)
104
-3.63
22
77
55-99
72.90 (-21 to +22)
Expanded allele 6b (patient 5)
80 (C)
101
-6.62
8
90
56-94
32.18 (-24 to +14)
Normal allele 1c (patient 3)
14 (G)
64
0.00
0
0
Normal allele 2c (patient 5)
14 (G)
51
0.00
0
0
Normal allele 3b (patient 2)
28 (C)
54
+0.04
4
0
Normal allele 4b (patient 6)
29 (C)
77
+0.04
4
0
Normal allele 5b (patient 1)
30 (C)
69
0.00
0
0
Normal allele 6b (patient 4)
30 (C)
92
-0.07
0
4
a(C or G): [(CAG)n-CGG] or [(CAG)n-GGG]. bSingle sperm from individuals with [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] genotype. cSingle sperm from individuals with [expanded (CAG)n-CGG]/[normal (CAG)n-GGG] genotype. The variance of the change in size of the CAG repeats of single sperm from individuals with the [expanded (CAG)n-CGG]/[normal (CAG)n-GGG] genotype was significantly greater than that of single sperm from individuals with the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] genotype (P <0.001).
In the single sperm analysis, the mean changes in the size of the CAG repeats in the six expanded alleles ranged from +0.39 to -6.62 repeats (Table 1 ). We also examined the CAG repeats in the MJD1 gene in pooled sperm DNA from the same individuals. Figure 1 shows that the CAG repeats in pooled sperm DNA carrying expanded alleles became shorter with increase in repeat size than those in leukocyte DNA, which confirms the data in Table 1 . When we compared the size distribution of the CAG repeats in pooled sperm DNA carrying expanded alleles with that in leukocyte DNA carrying expanded alleles, we found that the degree of CAG repeat mosaicism in the expanded alleles in the pooled sperm DNA was greater than that in the expanded alleles in the leukocyte DNA from all six individuals.
Thesingle base substitutions of the CGG/GGG polymorphism in leukocyte DNA were identified by allele-specific oligonucleotide hybridization (9 ). The variance of the change in size of the CAG repeats in the MJD1 gene of single sperm from individuals with the [expanded (CAG)n-CGG]/[normal (CAG)n-GGG] genotype (55.67 and 72.90) was significantly greater than that in the MJD1 gene of single sperm from individuals with the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] genotype (18.12, 24.16, 27.82 and 32.18) as shown in Table 1 (F-test, P <0.001). The CGG/GGG polymorphism in normal alleles was not related to the mean change in the size of the CAG repeats or the mutation frequency.
The variance of the change in repeat size in sperm from individuals with the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] genotype was not correlated with the CAG repeat size of their leukocyte DNA (Table 1 ).
Our data from the single sperm analysis of the MJD1 gene confirm the previous clinical and genetic findings. First, our single sperm data confirm the occurrence of meiotic segregation distortion of expanded alleles, which we reported on previously (10 ). In MJD, the mutant alleles were transmitted to 73% of all offspring in paternal transmission (10 ). The segregation ratio between expanded and normal alleles in single sperm is ~6:4, which is consistent with the clinical data.
Second, with regard to whether the CGG/GGG polymorphism at the 3' end of the CAG repeat affects the intergenerational instability of the CAG repeat, we found in a parent-offspring study that the [expanded (CAG)n-CGG]/[normal (CAG)n- GGG] genotype resulted in significantly greater instability of the CAG repeat than did the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] or [expanded (CAG)n-GGG]/[normal (CAG)n- GGG] genotype (9 ). The data from the single sperm analysis of the MJD1 gene confirm this finding as shown in Table 1 , strongly suggesting that an inter-allelic interaction is involved in the intergenerational instability of the expanded CAG repeat. Since none of the Japanese MJD patients whose genotypes we have examined to date have the [expanded (CAG)n-GGG]/[normal (CAG)n-GGG] genotype (9 ), we could not analyze sperm from individuals with this genotype in the present study.
In this study, we defined the change in size of the CAG repeats in the MJD1 gene by comparing the size of the CAG repeats from single sperm with the size of those from peripheral blood leukocyte DNA of the same individuals, according to the method used in the previous single sperm analysis of the CAG repeats in the Huntington' disease (HD) gene (11 ). However, the difference in size between the CAG repeats from the leukocytes and those from the sperm could be due to (i) instability of the expanded repeat size during meiosis, and (ii) mitotic instability during somatic cell divisions, or a combination of both processes, and we have no way of distinguishing a contraction during spermatogenesis from an expansion during somatic cell divisions. As far as we know, there are no reports of studies of mitotic instability during somatic cell divisions in any of the known diseases associated with CAG repeat expansion.
With regard to the measure of the degree of meiotic instability, we used the variance of the change in repeat size in sperm from four individuals with the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] genotype. As shown in Table 1 , the CAG repeat size of leukocyte DNA was not correlated with the variance of the change in repeat size in sperm.
In the case of myotonic dystrophy (DM), comparison of individual patient samples collected at two different times revealed that the degree of somatic heterogeneity of the CTG repeat size in DM increases with age and that a subtle but definite increase in the size of the expanded CTG allele occurs (12 ). In one patient with DM, the expanded repeat increased from 400 repeats to 500 in a 5 year interval, whereas in another it increased from 154 repeats to 158 in the same time period (12 ). Further investigation is required to determine whether the postnatal expansion of the CAG repeats occurs in MJD.
Single sperm analyses of the CAG repeats in the causative genes of the CAG repeat diseases have to date been performed for only a very small number of patients: three for HD (11 ), one for spinal and bulbar muscular atrophy (SBMA) (13 ) and six for MJD in this study. Of course we must take these small numbers into consideration when interpreting our data. Nevertheless, our data from the single sperm analysis of the MJD1 gene differ from those for the HD gene (11 ) in the following respects; meiotic segregation distortions of expanded alleles were clearly observed in the MJD sperm, while the segregation ratio between normal and HD alleles was not statistically different from the expected 1:1 in HD sperm. Second, contractions (60%) were more frequent than expansions (32%) in the MJD1 gene in MJD sperm, while most HD alleles (93%) showed expansions in HD sperm.
Although only one example of analysis in one individual with 47 CAG repeats in the androgen receptor gene has been reported on for SBMA, SBMA alleles in single sperm of this patient revealed 66% expansion and 15% contraction mutations, similar to HD data (13 ). With regard to spinocerebellar ataxia type 1 (SCA1), the mutation frequency of the SCA1 gene in sperm has not been reported (14 ).
The differences in the frequencies of contraction and expansion mutations in the causative genes among MJD, HD and SBMA sperm might be explained by differences in the CAG repeat size. The CAG repeats of the expanded alleles in this study (74-80 repeats) were larger than those of the SBMA allele (47 repeats) (13 ) and the HD alleles (38, 49 and 51 repeats) (11 ). Alternatively, the DNA sequences of the CAG repeat region might influence the mutation frequency. In fact, an interruption of the CAG repeats, which is present in the MJD1 gene but not in the HD and SBMA genes, might be related to the differences in the mutation frequency.
There are some similarities in mutation characteristics between the MJD and DM loci. First, in analysis of sperm from DM patients, marked contractions were detected in sperm with large CTG repeats (15 ). Furthermore, Ashizawa et al. reported that small expansions in the causative gene in DM fathers resulted in larger expansions in the same gene in their offspring, while large expansions in the causative gene in DM fathers resulted in smaller expansions in the same gene in their offspring (16 ). Second, segregation distortion in favor of transmission of the mutant alleles in male meiosis was also reported to occur in DM (17 ,18 ). Third, inter-allelic interaction, which influences the instability of the triplet repeats, is suggested to be involved in male meiosis. In DM, gene conversion events are reported to play a role in contraction of the expanded triplet repeats (19 ,20 ), which have also been detected in fragile X syndrome (21 ). Extensive structural analyses of sperm DNA from individuals with MJD are required to confirm that gene conversion events are also involved in the meiotic instability of the CAG repeats in the MJD1 gene.
Our present study revealed similarities and dissimilarities in terms of inter-allelic interaction, segregation distortions and size distribution of trinucleotide repeats in mutant alleles in MJD, HD, SBMA and DM. Further study is required to determine whether a common mechanism underlies the instability of the triplet repeat in these `triplet repeat diseases'.
Samples of peripheral blood and semen were obtained with informed consent from six patients with MJD. DNA from peripheral blood leukocytes was extracted using standard DNA extraction protocols (22 ). Sperm DNA was obtained using the method of Jeffreys et al. (23 ). The primer sequences used for PCR analysis of genomic DNA were as described by Kawaguchi et al. (7 ). PCR and determination of the number of CAG repeats of genomic DNA were performed as described previously (8 ,9 ).
Single sperm cells were micromanipulated using a Trancell TS-008 (JEOL Trading Co. Ltd.) (24 ). A single sperm was placed into a 0.2 ml tube containing 2.5 [mu]l of alkaline lysis buffer. Prior to PCR, tubes were left at room temperature for 15 min and heated at 65oC for 10 min, followed by neutralization with 2.5 ml of a solution containing 300 mM KCl and 900 mM Tris-HCl, pH 8.5 (25 ).
Two rounds of PCR were used to amplify the MJD1 gene CAG repeat region using a nested PCR strategy. First-round PCR was performed in a total volume of 25 [mu]l containing the neutralized sample, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 10% dimethylsulfoxide (DMSO), 200 [mu]M each of dATP, dGTP, dCTP and TTP, 0.8 [mu]M of each primer and 5 U of Taq polymerase (Takara). The primers used for the PCR were MJD52 and MJD70 as described (7 ). After the initial denaturation at 95oC for 2 min, the PCR was carried out for six cycles consisting of 1 min at 95oC , 1 min at 57oC and 1 min at 72oC.
Second-round PCR was performed in a total volume of 20 [mu]l containing 2.5 [mu]l of the first PCR product, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 5% DMSO, 200 [mu]M each of dATP, dGTP and TTP, 30 mM dCTP, 0.26 MBeq of [[alpha]-32P]dCTP, 0.6 [mu]M of each primer and 2.5 U of Taq polymerase (Takara). One of the primers used for PCR was MJD60 (5'-TATACTTCACTTTTGAATGTTTCAG-3'), which was newly designed, and the other was MJD25, described elsewhere (7 ). After the initial denaturation at 95oC, the PCR was carried out for 40 cycles consisting of 1 min at 95oC, 1 min at 62oC and 1 min at 72oC, followed by a final extension at 72oC for 7 min. The PCR products were then electrophoresed on a 6% polyacrylamide denaturing gel, and the number of CAG repeats of the MJD1 gene in single sperm was determined.
If MJD1 PCR products were detected in any one of three no-sperm controls, the data set was discarded. Thus, the possibility of contamination from an exogenous source during isolation of single sperm and PCR amplification was excluded. It is easy to imagine how contraction might result from Taq polymerase artifacts with regard to small expansions, since smaller PCR products would have a selective amplification advantage (11 ). To test for the presence of Taq artifacts, we performed the same control PCR experiments as described in the method of single sperm analysis of the SBMA locus (26 ). Our results are consistent with the data, showing that the contractions were not PCR artifacts.
Differencesin the segregation ratio of a single sperm with an expanded allele and that with a normal allele were determined using the [chi]2 test.Differencesin the degree of the change in size of the CAG repeats of the MJD1 gene between sperm from individuals with the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] genotype and the [expanded (CAG)n- CGG]/[normal (CAG)n-GGG] genotype were analyzed using the F-test.
We are very grateful to those who have contributed tissues for this study. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas, a grant (08670722) from the Ministry of Education, Science, and Culture, Japan and grants from the Research Committee for Ataxic Diseases of the Ministry of Health and Welfare, Japan.
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*To whom correspondence should be addressed. Tel: +81 285 44 2111 ext. 3524; Fax: +81 285 44 5118; Email: mnishiz{at}ms.jichi.ac.jp
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