Machado–Joseph disease gene products carrying different carboxyl termini

Neuroscience Research 28 (1997) 373 – 377

Rapid communication

Machado– Joseph disease gene products carrying different carboxyl termini Jun Goto a,*, Masahiko Watanabe a, Yaeko Ichikawa a, Su-Bog Yee a,b, Noriyo Ihara a, Kotaro Endo c, Shuichi Igarashi c, Yoshihisa Takiyama c,d, Claudia Gaspar e, Patricia Maciel e, Shoji Tsuji c, Guy A. Rouleau e, Ichiro Kanazawa a a

Department of Neurology, Institute for Brain Research, Faculty of Medicine, Uni6ersity of Tokyo, 7 -3 -1 Hongo, Bunkyo-ku, Tokyo 113, Japan b Di6ision of Anthropology, Department of Biological Science, Graduate School of Science, Uni6ersity of Tokyo, 7 -3 -1 Hongo, Bunkyo-ku, Tokyo 113, Japan c Department of Neurology, Brain Research Institute, Niigata Uni6ersity, 1 Asahimachi, Niigata, Niigata 951, Japan d Department of Neurology, Jichi Medical School, Minamikawachi, Tochigi 329 -04, Japan e Department of Neurology, The Montreal General Hospital Research Institute, and Centre for Research in Neuroscience, McGill Uni6ersity, 1650 Cedar A6enue, Montreal, Quebec H3G 1A4, Canada Received 4 March 1997; accepted 2 May 1997

Abstract Three cDNA clones for the Machado–Joseph disease gene (MJD1 ) were isolated, two of which have a new exon sequence and a distinct 3% terminal nucleotide sequence resulting in a new carboxyl terminal domain in the translated product. The nucleotide sequence of the other one is similar to the previously published one except for five polymorphisms, one of which is a single nucleotide substitution resulting in a change from the stop codon (TAA; allele A) to a tyrosine residue (TAC; allele C). Genetic analysis results suggest that Japanese MJD mutations are associated with allele A. © 1997 Elsevier Science Ireland Ltd. Keywords: Machado–Joseph disease; cDNA; Stop codon; Polymorphism; Linkage disequilibrium; Spinocerebellar ataxia

Machado–Joseph disease (MJD) is a progressive neurodegenerative disorder characterized clinically by cerebellar dysfunction and various associated symptoms such as ophthalmoplegia, dystonic-rigid extrapyramidal syndrome and amyotrophy. The disease is inherited in an autosomal dominant manner and has been mapped to chromosome 14q24.3-q32.1 (Takiyama et al., 1993; St George-Hyslop et al., 1994). The MJD gene, designated MJD1, was cloned recently, and the disease was found to be associated with an unstable expansion of a CAG repeat in this gene (Kawaguchi et al., 1994). It has been shown that the expanded polyglutamine tract * Corresponding author. Tel.: + 81 3 58008672; fax: +81 3 38132129.

which is translated from the repeat induces cell death in cultured COS cells and Purkinje cells in MJD transgenic mice (Ikeda et al., 1996). The product of MJD1 was originally reported to be composed of 339 amino acid residues plus various numbers of glutamine repeats, with a molecular weight calculated to be 40–43 kDa in normal individuals (Kawaguchi et al., 1994). In addition to variation of the polyglutamine tract length, a polymorphic single amino acid substitution downstream of this tract was described. To study MJD on the molecular and cellular levels we performed isolation of cDNA clones for MJD1. Sequence analysis of them revealed carboxyl terminal variations of the MJD1 product, which are caused by alternative splicing and a stop codon polymorphism. We investigated whether

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Fig. 1. Amino acid sequence of the MJDI product. Dash (-) represents the same amino acid residue as that in the MJDla sequence (Kawaguchi et al., 1994). Blank is a deleted residue. Dot (0) indicates a polymorphic site accompanying amino acid substitution. GenBank accession numbers for the pMJDI-I, pMJD2-I and 5-l sequences are U64820, U64821 and U64822. respectively.

and how the polymorphicstopcodonis associated with the MJD mutations in Japanese families. Here we report on new variants of the MJDI product and the results of genetic analysis of the stop codon polymorphism. To obtain a cDNA clone for MJDl we screened a human brain cDNA library. Complementary DNA was synthesized from poly(A)+ RNA which was prepared from the caudate nucleus of the cadaver of an 87 year old female with no neurological disease. The i, ZAP XR vector (Stratagene) was used to construct the library. Approximately 2 x lo6 plaques of the amplified library were screened. As a probe, a 223 bp genomic DNA fragment of MJDl was amplified by polymerase chain reaction (PCR), and then reamplification was carried out for labeling with digoxigenin-dUTP (Boehringer Mannheim). The primers used were MJD1007F (TTCACATCCATGTGAAAGGCCA) and MJD1229R (ACCTTGCTCCTTAATCCAGG). Conditions for the first amplification were 30 thermal cycles consisting of 1 min at 94°C 1 min at 55°C and 3 min at 72°C. For the second amplification, we chose 51°C as the annealing temperature, and the other parameters were the same as in the first. Hybridizations were carried out at

65°C in 1% bovine serum albumin (fraction V), 100 mg/ml denatured salmon sperm DNA, 1 mM EDTA (pH 8.0), 0.5 M sodium phospahte buffer (pH 7.2) and 7% sodium lauryl sulfate (SDS), and membranes were finally washed at 65°C in 0.2 x standard saline citrate (SSC) and 0.1% SDS. Positive clones were identified by chemiluminescence of AMPPD (Tropix). Three cDNA clones were obtained, and after two rounds of plaque purification the plasmids were excised in vivo from the phage clones according to the instructions of the manufacturer (Stratagene); they were designated pMJDl-1, 2-l and 5-l. The nucleotide sequences of both strands of all three clones were determined using an automated fluorescence sequencer (ABI 373A). The insert sizes in pMJDl-1, 2-l and 5-l were 1899 bp, 1202 bp and 1381 bp, respectively. Sequence data (not shown) were submitted to GenBank (U64820, U64821 and U64822). The nucleotide sequences of 851 bp at the 3’ end of pMJDl-1 and 352 bp at the 3’ end of pMJD5-1 were found to be new. The clone pMJDl- 1 contains a 3’ untranslated region of 734 bp, a poly-A stretch, and two polyadenylation sites which are 510 bp apart. In pMJD5-1 a poly-A stretch is located 17 bp from the upstream polyadenylation signal sequence. The se-

J.Gotoetal. iNeuroscience Resrurch 28 (1997) 373-377

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Fig. 2. A; Nucleotide sequence of the region of MJDI containing the stop codon polymorphism. Dot (0) indicates the polymorphic nucleotide. Nucleotide numbers are according to Kawaguchi et al., 1994. B; PCR-SSCP analysis. Genotypes are A/A for individual # 1192, C/C for # 1543 and A/C for # 1573.

quence at the boundary between the new and the published sequence almost perfectly matches the consensus sequence of the splicing donor site (‘06’@GTAAG’“6XG), suggesting that the cDNA of pMJDI-1 and 5-l is derived from alternative splicing. We confirmed this by reverse transcriptase RT-PCR analysis of RNA extracted from brains of control and MJD individuals (data not shown). Fig. 1 shows the amino acid sequences predicted from the nucleotide sequences of pMJDl-1, 2-l and 5-1, together with that of MJDla (Kawaguchi et al., 1994). The carboxyl terminal domain of 17 amino acid residues in MJDla is replaced by a new domain of 30 amino acid residues in the PMJDl-1 and 5-l proteins. The MacVectorTM software package (IBI) was used for sequence analysis. The carboxyl terminal domain of MJDla is predicted to be hydrophobic and the new carboxyl terminal domain of the pMJDl- 1 and 5-l proteins is predicted to be hydrophilic. The secondary structure of the new carboxyl terminal domain is predicted by both Chou--Fasman and RobsonGarnier methods to be an n-helix. The difference in the secondary structure of the carboxyl terminal domains might provide a basis for differences in functions of the isoforms. Three single nucleotide substitutions accompanying changes identified in pMJD2-1: codon were 6hy~TG(Z’2Met) to GTG(Val), 987CGG(3’8Arg) to The GGG(Gly) and TA “1XA(36’Stop) to TAQTyr). substitution at nucleotide position 987 has been described previously (Kawaguchi et al., 1994). The A to C

substitution at nucleotide position 1118 which is the third position of the ochre stop codon (TAA) results in a change in the site of the termination of translation, and thus addition of 16 amino acid residues (YELHVIFALHYSSFPL) at the carboxyl terminus (Fig. 2A). To determine how the substitution at nucleotide position 1118 occurs, we sequenced genomic DNA fragments spanning from nucleotide position 1007- 1229 of four unrelated individuals. Direct sequencing of the PCRamplified genomic DNA fragments revealed that the base at nucleotide position 1118 was either an A or a C. We identified three genotypes: A/A, A/C and C/C. To determine the genotypes simply for screening of populations we developed a PCR-SSCP method (Fig. 2B). PCR amplification of a genomic DNA fragment containing the polymorphic site was performed using the primers MJD1007F and MJD1229R. The conditions of amplification were the same as those of the first PCR for the probe preparation described above. The amplified alleles were separated by 12.5% polyacrylamide gel electrophoresis using the PhastSystem (Pharmacia) and, thereafter, visualized by silver staining. The running conditions were 400 V, 10 mA, 2.5 W and 4°C. The genotypes of 62 unrelated healthy Japanese individuals were determined, and the observed frequencies of alleles A and C in these indivduals were 0.371 and 0.629, respectively. The genotype frequencies were in Hardy-Weinberg equilibrium (Table 2). This polymorphism brings about a drastic change in the predicted protein as mentioned above. Does this difference have any effect on A4JDI function? The product of either or

J. Goto et al.

376 Table 1 Haplotype Family Ok En Ii Id YE Ya On Sa Na Yo MJD-4 MJD-5

ID

analysis

~Yeuroscience Researcll 28 (1997) .17.7-377

of MJD families Ethnic

origin

Japanese Japanese Japanese Japanese Japanese Japanese Japanese Japanese Japanese Japanese Caucasian Caucasian

Haplotype

of disease chromosome

A A A A A A A A A A C C

This study This study This study This study Takiyama Takiyama Takiyama Takiyama Takiyama Takiyama Maciel et Maciel et

both alleles might be non-functional or unstable. Alternatively, the carboxyl terminal domain of the product might be processed posttranslationally and so the polymorphism might have no effect on the function of the mature protein, or the additional carboxy terminal amino acid residues in the pMJD2-1 protein may not affect the function of the protein. Resolving this puzzle may yield a clue to MJDl function, which remains unknown. The alternative stop codon usage and single amino acid substitutions produce at least four isoforms of the MJDl product, which we have named josephin. Recently a polymorphic stop codon was reported to have been identified in the familial breast cancer susceptibility gene BRCAZ (Mazoyer et al., 1996). To our knowledge, this is the second report of a polymorphic stop codon in a gene. We examined the relationship between the stop codon polymorphism and the CAG expansion in MJDl. To determine haplotypes we analyzed the genotypes of this polymorphism and the CAG repeats of MJD families of which multiple members were available for molecular analysis and suitable for haplotyping. They included four families from Tokyo, six from Niigata and two from Montreal, The families from Niigata and Montreal were previously described (Takiyama et al., 1995; Endo et al., 1996; Maciel et al., 1995). Expansions of the CAG repeat in MJDl were confirmed by the previouly described method (Kawaguchi et al., 1994). The results of Table 2 Genotype frequencies of the stop codon control and MJD populations

Reference

polymorphism

in Japanese

Genotype

Control

MJD

A/A A;‘C C/C Total

7 32 23 62

11 12 0 23

Test for Hardy-Weinberg df= I, P>O.400; MJD:

Equilibrium: Control: Chi Square = 0.695. Chi Square = 28.59, df= I, P
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1996 1996 1996 1996 1996 1996

haplotyping are shown in Table 1. In all 10 Japanese MJD families the expansions were associated with allele A. In contrast, in the two Caucasian families they were associated with allele C. For further investigation of association between allele A and the MJD mutations in the Japanese population we analyzed the genotypes of the stop codon polymorphism of 23 Japanese MJD families of which single affected members were available for molecular analysis. None of the Japanese MJD patients was found to be homozygous for allele C. It was demonstrated that allele A is significantly associated with expansion of the MJDl CAG repeat in the Japanese population (Table 2). These findings suggest that ancestors of Japanese MJD patients had a common haplotype. MJD was originally identified in families of Portuguese-Azorean descent (Nakano et al., 1972; Woods and Schaumburg, 1972; Rosenberg et al., 1976). One could speculate that the MJD mutations spread worldwide during the 16th century and were introduced into Japan by the Portuguese. It was reported that Japanese and Caucasian MJD patients share haplotypes at markers flanking the disease gene, suggesting the existence of common founders (Takiyama et al., 1995). An alternative explanation for the sharing of haplotypes was also proposed (Takiyama et al., 1995; Endo et al., 1996) according to which precursor MJD alleles are prone to be amplified pathologically. Linkage disequilibrium has been reported to exist between an expansion of the CAG repeat and another intragenic polymorphism in MJDl (Igarashi et al., 1996). For the ‘*‘CGG/ GGG polymorphism 3’ adjacent to the CAG repeat in MJDl the 987CGG allele is associated exclusively with the expansions of the CAG repeat in Japanese MJD patients. Therefore, these previous observations and ours suggest that Japanese MJD mutations are strongly associated with the haplotype of 3’XArg(9X7CGG)at the intra‘h’Stop(TA “‘XA). Analysis of haplotypes genie polymorphisms in additional populations could provide a clue to uuderstanding the mechanism of triplet repeat expansion.

J. Goto et al. ~Neuroscience Research 28 (1997) 373-377

Acknowledgements We wish to thank Dr Yoshio Misumi of Fukuoka University for constructing a cDNA library, the members of the MJD families for participating in this study, and MS Kiyoko Matsuba, MS Misae Arai and MS Noriko Tsuji for technical assistance. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Culture and Sports of Japan, a Grant from the Research Committee of Ataxic Diseases of the Ministry of Health and Welfare of Japan, and Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of Japan.

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expansions in a novel gene for MachadooJoseph disease at chromosome 14q32.1. Nat. Genet. 8, 221-228. Maciel, P., Gaspar, C., DeStefdno, A.L., Silveira, I., Coutinho, P., Radvany, J., Dawson, D.M., Sudarsky, L., Guimaraes, J., Loureiro. J.E.L., Nezarati, M.M., Corwin, L.I., Lopes-Cendes, I., Rooke, K., Rosenberg, R.. MacLeod, P., Farrer, L.A., Sequeriros, J., Rouleau, G.A., 1995. Correlation between CAG repeat length and clinical features in MachadooJoseph disease. Am. J. Hum. Genet. 57, 54-61. Mazoyer, S.. Dunning, A.M., Serova, O., Dearden, J., Puget, N., Healey, C.S., Gayther, S.A., Mangion. J., Stratton, M.R., Lynch, H.T., Goldgar, D.E., Ponder, B.A.J., Lenoir, G.M., 1996. A polymorphic stop codon in BRCAZ. Nat. Genet. 14. 2533254. Nakano, K.K., Dawson, D.M., Spence, A., 1972. Machado disease: a hereditary ataxia in Portuguese emigrants to Massachusetts. Neurology 22, 49955. Rosenberg, R.N., Nyhan, W.L., Bay, C., Shore. P., 1976. Autosomdl dominant striatonigral degeneration: a clinical, pathologic, and biochemical study of a new genetic disorder. Neurology 26, 703-714. St George-Hyslop, P., Rogaeva, E., Huterer, J., Tsuda, T., Santos, J., Haines, J.L., Schlumpf, K., Rogaev, E.I., Laing, Y.. McLachlan, D.R.C., Kennedy, J., Weissenbach, J., Billingsley, G.D., Cox, D.W., Lang, A.E., Wherrett, J.R., 1994. MachadooJoseph disease in pedigrees of Azorean decent is linked to chromosome 14. Am. J. Hum. Genet. 55, 120-125. Takiyama, Y., Nishizawa, M., Tanaka, H., Kawashima, S., Sakamoto, H., Karube, Y., Shimazaki, H., Soutome, M., Endo. K., Ohta, S., Kagawa, Y., Kanazawa, I., Mizuno, Y., Yoshida, M., Yuasa, T., Horikawa, Y., Oyanagi, K., Nagai, H., Kondo, T., Inuzuka, T‘.. Onodera, 0.. Tsuji. S., 1993. The gene for Machado-Joseph disease maps to human chromosome 14. Nat. Genet. 4. 300-304. 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., Nishizawa, M., Kidd. K.K., Tsuji, S., St George-Hyslop, P.H., 1995. Evidence for inter-generational instability in the CAG repeat in the MJDl gene and for conserved haplotypes at flanking markers amongst Japanese and Caucasian subjects with Machado-Joseph disease. Hum. Mol. Genet. 4, 1137~1146. Woods, B.T., Schaumburg. H.H., 1972. Nigro-spino-dental degeneration with nuclear ophthalmoplegia: a unique and partially treatable clinico- pathological entity. J. Neurol. Sci. 17, 1499166.