In: Trends in Birth Defects Research ISBN 1-60021-398-7 Editor: Pia R. Klausen, pp. 83-94 © 2006 Nova Science Publishers, Inc.

Chapter III

Genetic Testing for Late-Onset Disorders: The Experience

of Machado-Joseph Disease in the Azores Islands (Portugal)

Manuela Lima1, Cristina Santos1, Conceição Bettencourt1 and

Patrícia Maciel2

1 Center of Research in Natural Resources (CIRN) and Department of Biology, University of the Azores, Ponta Delgada, Portugal

2 Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal

Machado-Joseph Disease: Clinical and Genetic Features

Machado-Joseph disease (MJD) (MIM 109150), also named spino-cerebellar ataxia type

III (SCA3), is an autosomal dominant late-onset neurodegenerative disorder which involves the cerebellar, ocular motor, pyramidal, extrapyramidal and peripheral motor systems. Mean age at onset is 40.5 years, with extremes of 7 and 70 years; the estimated mean survival time is of 20 years (Coutinho, 1992). Detailed clinical investigation of the variability in MJD led to its classification into three phenotypes, which can be occasionally present in a single family, and constitute three clinical types, cerebellar ataxia and ophthalmoplegia being common to all of them. Type 1 has an early symptom onset (around 25 yr), and is additionally characterised by pyramidal signs and dystonic postures. Type 2 is the most common type and occurs around 40 yr, being dominated by ataxia and ophtalmoplegia, with or without pyramidal signs. Type 3 is later in onset (mean 47 yr) and progresses slowly with prominent amyotrophy (Coutinho and Andrade, 1978).

Pathologically, the disease is characterized by degeneration of the spinocerebellar, dentate, pontine and vestibular nuclei as well as extra pyramidal structures. In addition, there

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is neuronal loss in motor cranial nerve nuclei, anterior horn cells, and posterior root ganglia (Coutinho and Andrade, 1978). Presently there is no effective treatment to delay or to halt the progression of the disease, although a few studies have reported the effect of specific drugs in the progression of the disease. Improvement of several symptoms (namely ataxia) has been reported following the administration of tandospirone citrate, a 5-HT1A agonist (Takei et al., 2004, 2005). Furthermore, clinical trials that evaluated the response to lamotrigine (LTG) on a small number of affected individuals concluded that LTG might bring benefits for the patients, mainly by relieving gait disturbance (Liu et al., 2005).

MJD was first described in North American patients, who had emigrated from the Portuguese islands of the Azores (Nakano et al., 1972; Woods and Schaumburg, 1972). For this reason the disease's origin was initially believed to be Azorean and the entity referred to in the literature as "Azorean disease of the nervous system" (Romanul et al., 1977). Notwithstanding the fact that molecular epidemiology studies have latter demonstrated a wide epidemiological distribution of the disease, revealing MJD as one of the most common dominant spinocerebellar ataxias, it is in the Azores Archipelago, specifically in Flores Island, that the disease reaches its worldwide highest value of prevalence (Lima et al., 1997a).

The MJD locus was first mapped to the long arm of chromosome 14 (14q24.3-q32) in Japanese pedigrees (Takiyama et al., 1993). By 1994 Kawaguchi and collaborators isolated and characterized a cDNA clone designated as MJD1a, mapping the MJD gene to 14q32.1 and identifying the causative mutation of the disease as being an expansion of the trinucleotide CAG, located in the coding region of the gene (Kawaguchi et al., 1994). The complete structure of the MJD gene, however, was not described until 2001 (Ichikawa et al., 2001). The gene consists of 11 exons, spanning a genomic region of about 48 kilobases (kb), the CAG repeat being located in exon 10. Northern blot analysis showed that the MJD mRNA was ubiquitously expressed in human tissues, in at least four different transcript sizes, that probably result from alternative splicing (Ichikawa et al, 2001).

In the MJD locus healthy individuals present 12 to 44 CAG units, whereas in MJD patients the repeat copy number in the expanded allele is between 61 and 87 (Maciel et al., 2001). The expanded allele displays intergenerational instability, particularly in male meiosis, which has been associated with the clinical phenomenon of anticipation. The mild instability of the expanded CAG repeat provided a molecular basis to explain the disease’s considerable variability, both in age at onset as well as in clinical presentation. A significant correlation between the expanded repeat size and age at onset accounts from 50 to nearly 80% of the variation in age of the appearance of first symptoms (e.g. Maciel et al., 1995; Maruyama et al., 1995). The remaining variance in age at onset should be the consequence of other factors (genetic, environmental or both), unstudied so far. In MJD the size of the expanded allele was not only inversely correlated with the age at onset of the disease, but was also associated with the frequency of other clinical features, such as pseudoexophthalmos and pyramidal signs, that are more frequent in subjects with larger repeats (Takiyama et al., 1995). Moreover, the disease phenotype was significantly more severe and had an early age of onset in subjects carrying the expanded allele in both chromosomes. Reports of homozygous individuals can be found, for example, in Lerer et al. (1996) and Sobue et al. (1996).

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The instability of the expanded polyglutamine-coding (CAG)n tracts during cell division can lead to changes in repeat length, either contractions or more frequently expansions, resulting in certain populations of cells carrying different repeat lengths. For MJD expanded alleles, both somatic (Cancel et al., 1995; Lopes-Cendes et al., 1996; Maciel et al., 1997) and gonadal mosaicism (Cancel et al., 1995) have been described.

Although the mechanisms of trinucleotide repeat instability are still not well understood, a variety of factors have been implicated as modulators of intergenerational repeat instability in MJD, namely the sex of the transmitting parent (Maciel et al., 1995) and the intragenic environment. Igarashi et al. (1996) and Maciel et al. (1999) investigated the association of intergenerational instability of the expanded CAG repeat in MJD with a CAG/CAA polymorphism in the CAG motif and a CGG/GGG polymorphism at the 3-prime end of the CAG array. Their results strongly suggested that an interallelic interaction is involved in the intergenerational instability of the expanded CAG repeat, through yet unclear mechanisms.

Population Genetics of Wild-Type CAG Repeats

and Origins of the MJD Mutation It has been postulated that a mutational bias in favour of expansions exists in

trinucleotide repeat loci, suggesting that the large majority of new mutations at these loci originated from the upper end of the normal allele distribution that would provide a reservoir from which expanded alleles would be generated (Rubinsztein et al., 1994). In Portugal, despite the relatively high prevalence of MJD, with the exception of a healthy individual described by Maciel et al. (2001), there are no reports of intermediate alleles in normal individuals. In a study of a large representative sample of the 20 Portuguese districts (~1000 individuals) no intermediate alleles were detected (Lima et al., 2005). In accordance with this absence, haplotype studies have shown that most of the mutant chromosomes worldwide share a common haplotype. A second haplotype is present in a subgroup of Portuguese patients on the Azorean Island of S. Miguel and on the mainland of Portugal, as well as in a few Japanese, North American, Brazilian and Spanish families. Two additional haplotypes have been identified, corresponding to a very small proportion of families, which may actually derive from the main haplotypes as a result of mutation of microsatellites (Gaspar et al., 2001).

The Portuguese population is extensively characterized at the epidemiological level, resulting from a systematic nationwide survey of MJD (Silva et al., 1997); furthermore, extensive genealogical information concerning the affected families (Lima et al., 1998; Abade et al., 2000; Santos et al., 2000) is available. This population provided an adequate background to test several aspects related with the relationship between the behaviour of the (CAG)n tract of the MJD gene in the normal population and the prevalence of the disease. We have studied the polymorphism of wild-type MJD alleles in a large representative sample of the Portuguese population and detected a negative skew for the allele size distribution of the (CAG)n tract in the MJD gene (Lima et al., 2005). This finding contrasts with what was reported by Takano et al. (1998), who described a positive skew (implying an excess of normal alleles with longer repeats) for the allele size distribution of the MJD alleles in the

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Japanese population. Moreover, no difference in allele distribution was detected between Portuguese districts presenting distinct values of epidemiological representation of MJD. Overall, the analyses conducted by Lima and co-authors (2005) failed to detect the existence of an association between the epidemiological representation of MJD and the frequency of large alleles within the normal range.

Survival Mechanisms of the MJD Gene: The Azorean Scenario

The Azores are a group of 9 Portuguese islands (Santa Maria, S. Miguel, Terceira,

Graciosa, Pico, S. Jorge, Faial, Flores and Corvo), located in the North Atlantic Ocean, 1500 km from the European mainland. To date, thirty-four extended affected families have been identified in this population, where the current estimated prevalence of the disease is 1/2402. On the island of Flores, where 1 in 106 individuals is affected (1 in 35 is a pre-symptomatic carrier and 1 in 21 is at 50% risk) (Lima et al., 1997a), MJD constitutes a public health problem. The mechanisms that underlay the survival and the high representation of a degenerative, highly disabling disease in a small population such as the Azores Islands have been the target of a comprehensive research program. An important part of such program had the purpose of documenting the historical genetics of the disease (Lima et al., 1997a, 1998).

To analyse the patterns of diffusion of the MJD mutation a study of the geographical distribution of the birthplace of the patients was undertaken. This distribution was contrasted with that of the founders. The founders, identified after the reconstruction of extensive ascending genealogies, represented individuals born between 1525 and 1846. On the basis of the geographical distribution of patients and founders, it was possible to identify specific populations that were clusters for both, and that were crucial sites for the introduction of the disease in the Azorean Islands (Lima, 1996). The kinship coefficient calculated between proponents of the Azorean MJD genealogies revealed that some of the families were isolated, without any link to another MJD family. This fact was only in part attributed to the many limitations of genealogical reconstruction using parish records, that refer principally to the lack or incompleteness of records, irregular surname transmission, exogamy and illegitimacy (Lima et al., 1998). No link could be established between the affected families originating from S. Miguel and families from Flores, the two Azorean Islands that present the large majority of MJD families. Emerging as one of the main conclusions of the historical genetics research was the fact that no unique set of founders for all Azorean MJD families could be established (Lima et al., 1998). Thus, before molecular data was available to provide insights into the historical genetics of MJD, genealogical data did not support a founder effect for MJD in the Azores. The chronological and geographical distribution of the founders of the MJD genealogies indicated that more than one mutation was introduced in the Azores, probably by settlers coming from the mainland of Portugal (Lima et al., 1998). Later work by Gaspar et al. (2001) that typed 5 microsatellite markers surrounding the MJD1 locus and 3 intragenic single base-pair polimorphisms in 249 families of different geographical origin, corroborated this finding by demonstration the presence of 2 distinct haplotypes in the Azores, one in the families originating from S. Miguel and the other in the families from

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Flores Island. In the MJD families from the mainland of Portugal both haplotypes were found (Gaspar et al., 2001). The genealogical investigation of the MJD families allowed the framing of a model for the disease’s origin (Lima, 1996). According to this model there were at least two different introductions of the MJD gene, probably by Portuguese settlers before the 17th century. Based on genealogical evidence, the centre region of Portugal appears to be the most likely centre of diffusion of the mutation segregating in the MJD families from S. Miguel. Genealogical data did not provide clues as to which region in the Mainland of Portugal served as the center of diffusion of the second mutational event.

The reproduction patterns of the MJD families have also been the target of genealogical based studies, aiming to determine the Darwinian fitness of the MJD phenotype. Work by Boutte (1987, 1992) reported that in Flores Island the MJD families were occasionally seen as “more prolific” than the average, and that this fact would have helped the “dissemination” of the disease. A case-control survey of MJD families found no differences in the reproductive output of MJD couples, compared to unaffected families (Lima et al, 1997b). However, this survey was limited, by the study design, to the fertility of married MJD patients and could not consider whether affected individuals survived and married at the same rate as unaffected ones. A latter paper by Lima and collaborators (2001) investigated whether there was selection against the MJD gene, acting through differential survival, nuptiality and fertility, associated with clinical type and age at onset (Lima et al., 2001a). The authors found no reduction of fertility among married patients in whom MJD onset below the age of fifty. Moreover, among married patients there was no dependence of fertility on clinical type or age at onset. Lima et al. (2001a) further described an association between clinical type and onset, since in the sample of patients analysed, there was a clear impairment both in nuptiality and fertility in the type 1 patients, as compared with the remaining clinical types. Because longer repeats are associated with clinical type 1,it was considered that the high-repeat CAG haplotypes associated with early age at onset and clinical Type 1 were selected against through reduced survival and fertility. The fertility component of selection was mediated by nuptiality rather than marital fertility (Lima et al., 2001a). These results have significance for the debate about the regulation of the number of CAG repeats, supporting a mechanism of selective elimination of the largest CAG repeat numbers via natural selection against early onset forms of MJD (Lima et al., 2005).

Genetic Testing for Machado-Joseph Disease:

Molecular Aspects In several disorders that affect preferentially certain ethnic groups, the ascertainment of

the geographical or ethnic background of the patients plays an important role in the clinical diagnosis. Although the detection of the MJD mutation in many patients from non-Portuguese origins showed that MJD was not confined to the Portuguese population, as it was initially thought (Romanul et al., 1977; Sachdev et al., 1980), the belief that this was a disease “of the Portuguese” contributed to a biased epidemiological profile, since a Portuguese background was, at a certain stage of the MJD investigation, a major element in the MJD diagnosis (Spinella and Sheridan, 1992).

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MJD is a well-defined clinical entity; however, in the early stages of the disease, if minor but specific signs are missing, if it appears isolated or in small family units or if the ethnic or geographical origin is thought to be unusual for this disease, a diagnosis of MJD may not be evident. The molecular screening of the MJD gene allowed performing worldwide molecular studies in this disease, and has lead to a distribution of molecularly confirmed cases that was clearly different from the initial scenario obtained exclusively by clinical criteria (Lopes-Cendes et al., 1996).

The initial location of the MJD locus to chromosome 14 (Takiyama et al., 1993) made possible the search of the mutation by indirect genetic testing. However, the genetic markers used in linkage analysis were not sufficiently close to the locus, allowing the occurrence of recombination events, and being of no practical utility as a genetic test. The gene cloning and the identification of the MJD mutation by Kawaguchi et al. (1994) permitted the analysis of the CAG expansion in the MJD gene and the direct detection of the mutation, thus validating a molecular diagnosis for this disease (Maciel et al., 1995). This achievement also made possible the development of the Predictive Test (PT), which gives an accurate confirmation of the carrier/non carrier status in asymptomatic individuals. A programme for Predictive testing and Genetic Counselling (GC) in MJD (Sequeiros, 1996), including the direct molecular testing, genetic counselling and psychological evaluation and support, was delineated in Portugal by the end of 1995, following the guidelines previously established for Huntington’s disease (Craufurd and Tyler, 1992). The general protocol of the Portuguese Program of GC and PT for MJD aims to provide to adult individuals at risk the access to the genetic information that can reduce the uncertainty about their genetic status, as well as to provide the necessary support to allow a healthy adaptation to the test results, so that psychological harm can be avoided (Sequeiros, 1996). Candidates for MJD Predictive Test are all the individuals that: a) are at 50% risk and wish to receive genetic information; b) are over 18 years old and are capable to provide informed consent; c) have a molecularly confirmed familiar history of MJD; and d) are asymptomatic for the disease. With the direct molecular testing available, the number of individuals requesting PT has increased. However, new ethical, individual, familial, as well as laboratorial complexities emerged from this technical possibility (Sequeiros, 1996).

Improvements in the diagnosis of MJD were made in order to resolve the main technical difficulties of presymptomatic testing. The first problem was the occurrence of intermediate alleles, for which it is not yet possible to determine whether a phenotype is associated (Maciel et al., 2001). The clinical and molecular analysis (including the determination of CAG repeat length and the establishment of intragenic haplotypes) of large pedigrees of the affected families, as well as the study of the healthy population from the same region, to assess the distribution of the normal (CAG)n length in that specific population (Lima et al., 2005) can contribute to minimize this issue. The second problem was homoallelism, ie, homozygosity for two normal alleles with exactly the same (CAG)n length, which is reported to occur in about 10% of all test results. This was solved by studying intragenic polymorphisms, which allowed the distinction of both normal chromosomes. Furthermore, using a new Southern blot based method, the possibility of existence of an expanded allele in the presumed homoallelic individuals can also be excluded (Maciel et al., 2001).

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Precision on sizing the MJD CAG repeat motif has its limitations, given the existence of somatic mosaicism, that originates differences in (CAG)n length among subpopulations of lymphocytes as well as between lymphocytes (where length is usually measured) and central nervous system cells. Thus an error of ±1 CAG repeat is considered as acceptable (Maciel et al. 2001). This somatic mosaicism contributes to the incompleteness of the existent inverse correlation between repeat size measured in the lymphocytes and the clinical features; (Maciel et al, 1995; DeStefano et al, 1995, 1996).

Sequeiros et al. (1998) described the first requests of Prenatal Diagnosis (PND) in MJD and showed that PND by direct mutation analysis of the MJD gene is technically feasible. However, since a positive result for the MJD mutation should imply the termination of the pregnancy, several psychological and ethical questions have emerged. As an alternative for PND, the Preimplantation Genetic Diagnosis is presently also feasible (Drusedau et al., 2004).

Predictive Testing for MJD: Social and Ethical

Issues in “Face-to-Face” Communities The discovery of the MJD mutation opened new prospects for pre-symptomatic testing,

forcing health professionals to review their approach to the patients and to their relatives (Lima et al., 2001b). In the Azores Islands, the involvement of the MJD families in the early description of MJD resulted to some extent in the stigmatisation of the families, specially in the small Island of Flores, where the reduced population size and the social structure (“every-one-knows-everyone” or “face-to-face” community) favours the occurrence of this phenomenon (Boutté, 1987, 1992). Therefore, the implementation of a Genetic Counselling Program for this disorder, in this particular population, had to be tailored to accommodate such specificities.

Before the PT was offered to the Azorean individuals at risk, a survey of the level of information of the MJD families concerning the disease was undertaken (Lima et al., 2001b). Results from a questionnaire applied in the study by Lima and colleagues (2001b) highlighted the fact that a large percentage of individuals could not comprehend the notion of “pre-symptomatic carrier” and therefore could not quantify the objective risk of inheriting/transmitting the disease. This reinforced the responsibility that the GC team has in providing the families with adequate information concerning the genetics of MJD. Furthermore, it drew the attention to the importance of encouraging the affected families to share their experience with MJD, since this could lead to a better understanding of the clinical and genetic aspects of the disease.

The study by Lima et al (2001b) had revealed high levels of intention (83.3%) of the individuals at risk to undertake the genetic test. However, and in accordance with the experience of other populations with similar disorders, namely Huntington disease (HD), the level of participation in the PT in the Azores Islands, ascertained nearly 5 years after the test became available in Portugal, was of 20.7% (Gonzalez et al., 2004), a value that although being high comparatively with other similar disorders, represents less than one fourth of the level of request estimated from the intentions of at-risk individuals.

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Levels of adherence to the PT in the Island of Flores (35.8% of all individuals at risk that were eligible to enter the PT Program undertook the test) are very peculiar and were extensively debated in the work by Gonzalez and colleagues (2004). As previously refered, according to the work of Boutté (1987, 1992) in the particular context of Flores Island MJD represents a source of stigmatization to the families, by providing a definitive mark that brings into discredit the individual’s life project. However, the high levels of adherence to the PT in Flores seem contradictory relatively to this interpretation. Gonzalez et al. (2004) discussed the levels of adherence to the PT in Flores Islands by considering that, prior to the availability of the PT, the stigma was perceived not only by the patients themselves, but also by their descendants, thus constituting a cultural reality. Individuals at risk, because of their high probability of developing the disease saw themselves necessarily as a menace to others (Gonzalez et al., 2004). With the availability of the PT, the individuals at risk had the possibility of confronting (and resolving) the dubious aspects inherent to their at-risk status (to resolve the threat that the disease represents). If being a carrier meant the confirmation of a menace, the proof of absence of the MJD mutation offers the individuals at risk from Flores Island the possibility of being liberated from the stigma, and hence, from exclusion by others. The study by Gonzalez and colleagues highlights the fact that, in communities such as Flores Island, characterized by a small population size, high isolation and a very high prevalence of a genetic disorder, a careful intervention regarding genetic tests is mandatory, since in similar contexts, genetic diseases can represent a source of stigmatization to the affected families, and thus become cultural forces (Gonzalez et al., 2004).

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