Molecular characteristics of Machado-Joseph disease mutation in 25 newly described Brazilian families

Iscia Lopes-Cendes¹*, Hélio G.A. Teive ², Francisco Cardoso³, Erika M. Viana ³,
Maria E. Calcagnotto ⁴, Jaderson C. da Costa ⁴, Paulo C. Trevisol-Bittencourt ⁵,
Jayme A. Maciel ⁶, Marylene Rousseau¹, André S. Santos ⁷, Abelardo Q.C. Araújo⁸
and G.A. Rouleau^1
1Centre for Research in Neuroscience and the Montreal General Hospital Research Institute, McGill University, Montreal, Quebec, Canada. E-mail: iscia@turing.unicamp.br. Send correspondence to I.L.-C. *Present address: Departamento de Genética Médica, Faculdade de Ciências Médicas (FCM), Universidade Estadual de Campinas (UNICAMP), Cidade Universitária Zeferino Vaz, Caixa Postal 6111, Distrito de Barão Geraldo, 13083-970 Campinas, SP, Brasil. Tel. (019) 788-8210, Fax (019) 239-3114.
²Serviço de Neurologia, Universidade Federal do Paraná, Curitiba, PR, Brasil.
³Departamento de Neurologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil.
⁴Departamento de Neurologia, Hospital Universitário São Lucas, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brasil.
⁵Serviço de Neurologia, Hospital Universitário da Universidade Federal de Santa Catarina, Florianópolis, SC, Brasil.
⁶Departamento de Neurologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, Brasil.
⁷Hospital de Caridade, Florianópolis, SC, Brasil.
⁸Departamento de Neurologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.

ABSTRACT
Machado-Joseph disease (MJD) is a form of autosomal dominant spinocerebellar ataxia first described in North-American patients originating from the Portuguese islands of the Azores. Clinically this disorder is characterized by late onset progressive ataxia with associated features, such as: ophthalmoplegia, pyramidal and extrapyramidal signs and distal muscular atrophies. The causative mutation is an expansion of a CAG repeat in the coding region of the MJD1 gene.
We have identified 25 unrelated families segregating the MJD mutation during a large collaborative study of spinocerebellar ataxias in Brazil. In the present study a total of 62 family members were genotyped for the CAG repeat in the MJD1 gene, as well as 63 non-MJD individuals (126 normal chromosomes), used as normal controls. We observed a wide gap between the size range of the normal and expanded CAG repeats: the normal allele had from 12 to 33 CAGs (mean = 23 CAGs), whereas the expanded alleles ranged from 66 to 78 CAGs (mean = 71.5 CAGs). There were no differences in CAG tract length according to gender of affected individuals or transmitting parent. We observed a significant negative correlation between age at onset of the disease and length of the CAG tract in the expended allele (r = -0.6, P = 0.00006); however, the size of the expanded CAG repeat could explain only about 40% of the variability in age at onset (r² = 0.4).
There was instability of the expanded CAG tract during transmission from parent to offspring, both expansions and contractions were observed; however, there was an overall tendency for expansion, with a mean increase of +2.4 CAGs. The tendency for expansion appeared to the greater in paternal (mean increase of +3.5 CAGs) than in maternal transmissions (mean increase of +1.3 CAGs). Anticipation was observed in all transmissions in which ages at onset for parent and offspring were known; however, anticipation was not always associated with an increase in the expanded CAG repeat length. Our results indicate that the molecular diagnosis of MJD can be confirmed or excluded in all suspected individuals, since alleles of intermediary size were not observed.

INTRODUCTION

Machado-Joseph disease (MJD) is an adultonset neurodegenerative disorder with autosomal dominant inheritance. It was first reported in North-American families of Portuguese-Azorean descent (Nakano et al., 1972; Rosenberg et al., 1976; Romanul et al., 1977; Coutinho and Andrade, 1978), but it is now shown to occur in patients from many different ethnic backgrounds (Cancel et al., 1995; Ranum et al., 1995; Schöls et al., 1995; Brunt et al., 1996; Burt et al., 1996; Lopes-Cendes et al., 1996a; Silveira et al., 1996; Zhou et al., 1997). Clinically, MJD is characterized by progressive cerebellar ataxia, with associated features such as external ophthalmoplegia, pyramidal signs, dystonic postures, distal muscular atrophies, eyelid retraction, faciolingual fasciculations and no mental impairment (Coutinho, 1994). The different clinical expressions of MJD resulted in its classification into three clinical subtypes (Lima and Coutinho, 1980): type 1 shows an early onset, a rapid course, and patients have marked pyramidal and extrapyramidal signs in addition to the common features of ataxia and ophthalmoplegia; type 2, the most common subtype, is a more benign form with clinical manifestations limited to cerebellar ataxia, pyramidal signs and ophthalmoplegia, and type 3 shows a late onset and a very slow progression of the disease, which is characterized mainly by peripheral signs in addition to the features of type 2.

The MJD locus, also known as SCA3, was first mapped to chromosome (ch) 14q in Japanese pedigrees (Takiyama et al., 1993) and subsequently found to assign to the same chromosomal region in Portuguese (Sequeiros et al., 1994), Brazilian (Twist et al., 1995) and French families (Stevanin et al., 1994). Recently, the expansion of a trinucleotide CAG repeat located in the coding region of the MJD1 gene was identified as the causative mutation (Kawaguchi et al., 1994). Expansions of CAG repeats have been associated with several neurodegenerative disorders, including Huntington disease, Kennedy disease, and five types of autosomal dominant spinocerebellar ataxia (SCA), including MJD (Sutherland and Richards, 1995; Zoghbi, 1996; Zhuchenko et al., 1997). In all of these disorders affected individuals carry abnormally expanded and unstable CAG tracts in the coding region of the disease genes.

We have studied 62 individuals belonging to 25 unrelated Brazilian families segregating the MJD mutation in order to: a) compare the size range of the CAG repeat in the MJD and control populations, b) investigate the relationship between the size of the expanded CAG allele and age at onset of the disease, and c) analyze the instability of the repeat size in its transmission from parent to progeny, in an attempt to determine the molecular basis for the previously described effect of anticipation in this disease (Sequeiros and Coutinho, 1981).

SUBJECTS

This study was performed using DNA samples from 62 family members, including 49 clinically affected individuals, belonging to 25 unrelated families. Families were ascertained in six states from the southern and southeastern regions of Brazil: Rio Grande do Sul, Paraná, Santa Catarina, São Paulo, Minas Gerais and Bahia. The clinical features of one of these families were reported previously (Teive et al., 1991). The 25 families were identified as segregating the MJD mutation during a large collaborative project involving the screening for mutations in Brazilian families with different types of SCA ( Lopes-Cendes et al., in press). Of the 25 families, 22 were of Portuguese background, two were of Italian origin and one had a mixed Portuguese and African ancestry. None of the families could trace their ancestors back to the Portuguese Islands of the Azores. Ages at onset were based on information provided by the patient and/or a close relative. For determination of the frequency of the normal alleles we used 37 normal chs identified in the MJD families, as well as 126 normal chs identified in 63 non-MJD Brazilian individuals.

METHODS

Genomic DNA was isolated from peripheral lymphocytes by standard methods (Sambrook et al., 1989). The CAG-containing fragment of the MJD gene was amplified by PCR using primers MJD52 and MJD25 (Kawaguchi et al., 1994). PCR was performed in a final volume of 12.5 ml, containing 100 ng of genomic DNA, 10 mM Tris-HCl (pH 8.8), 1.5 mM MgCl₂, 50 mM KCl, 2% formamide, 250 mM each dCTP, dGTP and dTTP, 25 mM ATP, 1.5 mCi ³⁵S a-dATP, 100 ng of each primer, 1 unit of Taq polymerase (Perkin Elmer). The DNA was denatured at 94^oC for 5 min, then 32 cycles at 94^oC for 1 min, 60^oC for 1 min, and 72^oC for 1 min were performed, followed by a final extension at 72^oC for 5 min.

For determination of allele sizes (AS), PCR products were analyzed on 6% polyacrylamide gels in parallel with an M13 sequencing ladder (-40 primer) and were visualized by autoradiography. Allele sizes were determined by comparison with the M13 sequence and were converted into CAG unit number (N), using the formula N = (AS-121)/3, assuming that the variation in size of the PCR product occurred within the repetitive CAG stretch (Maciel et al., 1995).

Linear regression techniques were used to determine the association between repeat number in the MJD or normal alleles and age at onset. Differences in size of the CAG repeat among groups were assessed by analysis of variance and post hoc tests (Tukey HSD).

RESULTS

We found expanded alleles in 49 patients, as well as in five individuals at risk of developing the disease. The size of the expanded alleles varied from 66 to 78 CAGs (mean = 71.5 CAGs) (Figure 1B). There were no significant differences in the size of the expanded alelles inherited paternally vs. maternally (73 CAGs for paternal and 71 CAGs for maternal transmission, P = 0.3), as well as between affected males and females (71 CAGs for male and 73 CAGs for female patients, P = 0.3).

Age at onset was known for 34 of the 49 affected individuals studied. It varied from 15 to 53 years (mean = 34 years). There were no differences between mean age at onset due to gender of transmitting parent (30 years for paternal and 35 years for maternal transmission, P = 0.6) or between affected males and females (35 years for males and 34 years for females, P = 0.9).

The relationship between the CAG repeat length in the MJD allele of affected individuals and age at onset of the disease is shown in Figure 2A. A correlation coefficient of -0.6 was obtained (r² = 0.4, P = 0.00006), assuming a linear relationship between age at onset and repeat length in MJD chs. This indicates a tendency for age at onset to decrease as the CAG repeat length increases. No significant correlation was found between  the  normal   CAG   allele  and  age  at  onset (r = -0.05, r² = 0.003, P = 0.8) as shown in Figure 2B.

We observed instability of the expanded CAG repeat during transmission from parent to offspring. Expansions as well as contractions of the expanded CAG repeat length occurred in our families (Figure 3). We documented eight transmissions of the expanded CAG allele, four maternal and four paternal. There was an overall tendency for increase in the expanded CAG repeat length, with a mean increase of +2.4 CAGs. This tendency was greater for paternal vs. maternal transmissions (mean increase of +3.5 CAGs in paternal and +1.3 CAGs in maternal transmissions); however, this difference was not statistically significant (P = 0.5). Expansions of the CAG tract occurred 3/4 times in paternal and 1/2 times in maternal transmissions. In four transmissions, ages at onset for parent and offspring were known (two paternal and two maternal), and anticipation occurred in all four instances; however, an increase in expanded CAG repeat length was observed in only three of these transmissions (Table I). Normal CAG alleles were always stable during transmission (Figure 3).

Table I - Relationship between instability of the expanded CAG repeat in the MJD1 gene and anticipation during transmission from parent to offspring.

Normal and expanded alleles had a different appearance in the autoradiographs. The normal alleles had a single strong band distinctively seen in the X-ray films, whereas the expanded alleles showed several bands, suggesting the presence of cells with different lengths of the expanded CAG tract (Figure 3).

DISCUSSION

Over the past six years a new class of mutations, dynamic mutations, has been identified (Richards and Sutherland, 1992; Sutherland and Richards, 1995). These mutations are characterized by expansions of different types of unstable trinucleotide repeats. They have been divided into three main classes according to the type of trinucleotide involved, their location within the disease gene and their possible mechanism of action in determining disease (Strachan and Read, 1996): a) large scale expansions of very unstable (CGG)n which are associated with fragile sites, as in the fragile X syndromes (FRAXA and FRAXE). In these syndromes it is likely that the massive expansion of the CGG repeat causes loss of function by disrupting nearby genes (Richards and Sutherland, 1992); b) large expansions of CTG repeats located in the 3’untranslated region of a protein kinase gene (DMPK), causing myotonic dystrophy (MD). The molecular pathology of MD is not known; however, no other type of mutation has been found in MD patients, suggesting a specific gain of function caused by the expansion of the CTG repeat (Caskey et al., 1992) and c) moderately expanded CAG repeats within coding regions, encoding expanded polyglutamine tracts at the protein level. The exact mechanism by which expansions of CAG repeats cause disease is not understood; however, evidence points to a mechanism involving gain of function (Ross, 1995). To date, expansions of trinucleotide CAG repeats have been found in neurodegenerative disorders such as Huntington disease (Huntington’s Disease Collaborative Research Group, 1993), Kennedy disease (LaSpada et al., 1991) and five types of autosomal dominant SCA: spinocerebellar ataxia type 1 (SCA1) (Orr et al., 1993), spinocerebellar ataxia type 2 (SCA2) (Imbert et al., 1996; Pulst et al., 1996; Sampei et al., 1996), Machado-Joseph disease or spinocerebellar ataxia type 3 (MJD/SCA3) (Kawaguchi et al., 1994), spinocerebellar ataxia type 6 (SCA6) (Zhuchenko et al., 1997) and a related disorder dentatorubropallidoluysian atrophy (DRPLA) that can be present as SCA in late onset cases (Koide et al., 1994; Nagafuchi et al., 1994). More recently, a GAA expansion in the first intron of a novel gene (c25) was identified as the causative mutation in 94% of Friedreich ataxia (FA) patients (Campuzano et al., 1996). Point mutations have also been identified in a few FA patients, suggesting that the disease is probably caused by loss of function.

We (Silveira et al., 1996) and others (Ranum et al., 1995; Schöls et al., 1995) have established that the MJD mutation is the most frequent cause of autosomal dominant SCA worldwide, including the Brazilian SCA patients (Lopes-Cendes et al., in press). In Brazilian patients, as in other ethnic groups (Cancel et al., 1995; Maciel et al., 1995; Schöls et al., 1995) there is a wide gap between the normal and expanded allele sizes, thus allowing a clear differentiation between normal and disease alleles, making it possible to confirm or exclude the molecular diagnosis of MJD in small families or in single affected individuals. In addition, the high heterozygosity observed for the normal CAG allele in the Brazilian population (91.3%) reduces the probability of finding individuals that are homozygous for the normal CAG allele. Individuals homozygous for the CAG allele in the normal range may be difficult to differentiate from failure to amplify an expanded allele during the PCR testing.

We have shown that there is a significant inverse correlation between the length of the expanded CAG allele and age at onset of the disease; however, only part of the variability in age at onset can be explained by the length of the expanded CAG tract (about 40%). Thus, the majority of the variation in age at onset is determined by other, still unidentified, factors. Therefore, a prediction of age at onset of the disease based on the length of the expanded CAG allele is not accurate. In our series, individuals with an expanded allele of 72 CAGs (the average size found in our study) had ages at onset varying from 29 to 38 years.

Anticipation is defined as a progressive earlier onset of a disease in successive generations, usually associated with increased severity (Bell, 1942; Penrose, 1948). Anticipation has been observed in most disorders associated with dynamic mutations (Caskey et al., 1992; Richards and Sutherland, 1992). However, in the CAG repeat disorders the effect of anticipation is less marked, possibly due to the mild instability of the expanded allele observed in these disorders (Ross, 1995; Zoghbi, 1996). Anticipation occurred in all transmissions that were documented in this study; nonetheless, this was associated with both increases and decreases in the size of the expanded CAG repeat. Many factors, such as an increased awareness of the disease in the family, and difficulty in recalling age at onset in the older generation may introduce bias in the evaluation of anticipation (Penrose, 1948). We observed an overall tendency for further increase in size of the expanded CAG repeat over successive generations (+2.4 CAGs); however, anticipation did not always correlate with expansions in individual transmissions. The greater overall increase in size of the expanded repeat in males (+3.5 CAGs) than in females (+1.3 CAGs) was observed previously in MJD (Maciel et al., 1995) and in other CAG related disorders such as Huntington disease (Andrew et al., 1993) and SCA1 (Chung et al., 1993).

The multiple bands shown by the expanded alleles in the autoradiographs (Figure 3) suggest the presence of somatic mosaicism, with different cells containing different lengths of the expanded CAG allele. Somatic mosaicism of the expanded CAG repeat has been described in MJD, but no relationship was found between the size of the expanded CAG repeat in different parts of the central nervous system and site of neurodegeneration (Lopes-Cendes et al., 1996b).

In conclusion, although some of the parameters examined here have been previously studied in other populations (Cancel et al., 1995; Maciel et al., 1995; Schöls et al., 1995), and molecular studies were conducted in Brazilian MJD patients (Stevanin et al., 1995; Iughetti et al., 1996), we believe that the present study provides further information that can be used to guide the molecular testing of Brazilian MJD patients, as well as the genetic counseling of patients and family members. The molecular diagnosis, based on the determination of the length of the CAG repeat in the MJD1 gene, is highly sensitive and specific. However, the length of the expanded CAG allele should not be used to predict age at onset or severity of clinical presentation, since genetic and/or environmental factors, yet to be identified, are responsible for most of the clinical variability seen in these patients.

The authors would like to thank the family members who participated in this study. This work was supported by: the joint Program FRSQ-ACAF (Fonds de la Recherche en Santé du Québec and Association Canadienne de l’Ataxie de Friedreich), the NIH (grant NS 31687) and the Network of Centres of Excellence (Canadian Genetic Disease Network). G.A.R. is supported by the Medical Research Council of Canada and the Fonds de la Recherche en Santé du Québec.

RESUMO
A doença de Machado-Joseph (MJD) é uma forma de ataxia espinocerebelar (AEC) de herança autossômica domi nante, que foi descrita inicialmente em pacientes norte-americanos provenientes das ilhas portuguesas dos Açores. Clinicamente essa doença é caracterizada por uma ataxia cerebelar progressiva, de início tardio e com algumas características associadas tais como: oftalmoplegia, sinais piramidais e extrapiramidais e amiotrofias. A mutação responsável é uma expansão de trinucleotídeos CAG localizada na região codificadora do gene MJD1.
Como parte de um estudo colaborativo sobre AEC no Brasil, nós identificamos 25 famílias, não aparentadas, segregando a mutação MJD. Nesse artigo nós relatamos as características moleculares do trinucleotídeo CAG presente no gene MJD1 em 62 indivíduos dessas famílias com MJD e em 63 indivíduos que não apresentam a mutação MJD (126 cromossomos normais) e que foram considerados como controles. Nós observamos uma grande diferença entre o tamanho dos alelos CAG normais e expandidos. Os alelos normais variaram entre 12 e 33 CAGs (média de 23 CAGs), enquanto que os alelos expandidos tiveram de 66 a 78 CAGs (média de 71.5 CAGs). Não encontramos nenhuma diferença entre o tamanho dos alelos expandidos em pacientes masculinos e femininos ou entre alelos transmitidos via paterna ou materna. Uma correlação negativa significante foi observada entre a idade de início da doença e o tamanho do segmento de CAG expandido (r = -0,6, P = 0,00006); no entanto o tamanho do segmento de CAG expandido foi responsável por somente 40% da variabilidade na idade de início da doença (r² = 0.4).
Nós observamos também instabilidade do segmento expandido de CAG durante a transmissão de pais para filhos. Expansões e contrações foram observadas; contudo, houve uma tendência geral para expansão, com um aumento médio de +2,4 CAGs. Essa tendência para expansão pareceu ser maior nas transmissões paternas (aumento médio de +3,5 CAGs) que nas maternas (aumento médio de +1,3 CAGs). Antecipação foi observada em todas as transmissões nas quais as idades de início para pais e filhos eram conhecidas, porém, nem todas as antecipações foram acompanhadas de aumento no tamanho do segmento de CAG expandido.
Em conclusão, os nossos resultados mostram que o diagnóstico molecular da mutação responsável pela MJD pode ser estabelecido em todos os indivíduos que apresentam a doença, bem como excluído naqueles portadores de outras formas de AEC, já que não encontramos alelos de tamanho intermediário que poderiam dificultar a interpretação do teste molecular. Porém, o tamanho do segmento de CAG expandido não deve ser usado para predizer a idade de início da doença, já que apenas 40% da variabilidade na idade de início pode ser explicada pelo tamanho do segmento de CAG expandido.

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Figure 1 - Frequency distribution of CAG repeat sizes in controls and MJD individuals. A) Distribution of normal alleles; black bars show the distribution for 37 normal alleles found in 25 unrelated MJD families, hatched bars show the distribution for 126 normal alleles found in 63 unrelated non-MJD individuals. There was no significant difference in the distribution of the normal alleles in the two groups (P = 0.2). B) Overall distribution of 163 normal CAG alleles and 54 expanded alleles found in 25 unrelated Brazilian MJD families.

Figure 2 - A) Correlation of CAG repeat length in the MJD chromosomes of 34 affected individuals with age at onset of the disease (Pearson correlation coefficient (r) = -0.6, r² = 0.4, P = 0.00006). B) Absence of correlation between age at onset and CAG repeat length in the normal chromosomes of 34 affected individuals (r = -0.05, r² = 0.003 and P = 0.8).

Figure 3 - Pattern of segregation for the normal and expanded CAG alleles in two MJD sibships. Top panel (a and b): Pedigrees of two small MJD sibships. Black symbols represent clinically affected individuals and diagonal lines indicate deceased individuals. Bottom panel: Analysis of PCR products containing normal and expanded CAG repeats at the MJD1 gene. Genomic DNA, extracted from peripheral blood leukocytes, was amplified using primers MJD52 and MJD25 (Kawaguchi et al., 1994). PCR products were analyzed on 6% polyacrylamide gels. Normal alleles had sizes varying from 21 to 31 CAG units and expanded alleles varied from 68 to 78 CAGs. In pedigree a, variability in size of the expanded CAG allele can be seen. The same expanded allele, which was transmitted from the deceased mother, had different lengths in the offspring (lane 1: 77 CAGs, lane 2: 68 CAGs, lane 3: 78 CAGs and lane 5: 72 CAGs); whereas Mendelian segregation can be observed for the normal allele, the offspring inherited the normal allele with 23 CAGs from the unaffected father (lane 4: normal alleles = 23 and 27 CAGs). In pedigree b, differences in the length of the expanded allele in the same sibship are also seen (lane 6: 71 CAGs, lane 7: 68 CAGs and lane 9: 68 CAGs). A maternal transmission showing a contraction of the expanded allele can be observed (lane 6: 71 CAGs to lane 10: 70 CAGs). By contrast, there is a stable segregation of the normal allele (23 CAGs) from the unaffected female in lane 8 (normal alleles = 21 and 23 CAGs) to her unaffected daughter in lane 11 (normal alleles = 23 and 31 CAGs).