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Table of Contents    
Year : 2019  |  Volume : 67  |  Issue : 2  |  Page : 582-588

Genetic analysis of a family from India with Machado–Joseph disease

1 Molecular Biology and Genetics Unit, Human Molecular Genetics Laboratory, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, India
2 Department of Psychiatry, Molecular Genetics Laboratory, National Institute of Mental Health and Neurosciences, Bengaluru, India
3 Neuro Specialities Centre, Belgaum, Karnataka, India

Date of Web Publication13-May-2019

Correspondence Address:
Dr. Meera Purushottam
Department of Psychiatry, Molecular Genetics Laboratory, National Institute of Mental Health and Neurosciences, Bengaluru, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0028-3886.258030

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How to cite this article:
Anjanappa RM, Jain S, Wali GM, Purushottam M. Genetic analysis of a family from India with Machado–Joseph disease. Neurol India 2019;67:582-8

How to cite this URL:
Anjanappa RM, Jain S, Wali GM, Purushottam M. Genetic analysis of a family from India with Machado–Joseph disease. Neurol India [serial online] 2019 [cited 2021 Jan 27];67:582-8. Available from:


Machado–Joseph disease (MJD), also known as spinocerebellar ataxia Type 3 (SCA3), is a dominantly inherited late-onset neurodegenerative disorder.[1] It is clinically characterized by cerebellar ataxia and progressive external ophthalmoplegia, along with the presence of other symptoms to a variable degree, such as pyramidal signs, dystonia, rigidity, amyotrophies, and peripheral neuropathy.[2],[3],[4] The mean age at onset (AAO) is approximately 40 years (range 5–73 years)[5] and the mean survival time is 21 years (range 7–29 years).[6]

MJD is associated with the CAG repeat expansion located in exon 10 of the ATXN3 on chromosome 14q32.1.[7] The CAG repeat ranges from 12 to 44 triplets in normal individuals, and ~60 to 87 in MJD patients.[8],[9] Intermediate size alleles are rare, but there are a few reports of smaller disease associated alleles,[10],[11],[12],[13] and the smallest pathogenic repeat allele being 45 in a family from India.[14] In an earlier study, we have reported a pathogenic allele range of 69–78 in Indian MJD families.[15] Like other polyglutamine disorders, there is an inverse correlation between the age of onset, and the number of CAG repeats. However, the CAG repeat number accounts for only 50–80% of the variability.[16],[17],[18]

Haplotype analyses involving three intragenic single nucleotide polymorphisms (SNP s) (A669 TG/G669 TG, C987 GG/G987 GG, and TAA1118/TAC1118) from 249 MJD families across the world showed four haplotypes, of which two (A-C-A and G-G-C) were more common.[19] Region-specific population analysis showed the presence of the A-C-A haplotype in the island of Flores, and G-G-C haplotype in the island of São Miguel, whereas mainland Portugal showed both the haplotypes. Worldwide, a large proportion (72%) of families with MJD has the A-C-A haplotype, and the distribution of the A-C-A haplotype suggested a founder effect for the MJD mutation. In our previous study, all (CAG)n expanded alleles were associated with A-C-A haplotype in nine Indian origin MJD families.[20]

The objective of this study was to analyze the CAG repeat length in ATXN3 and study the haplotype using three intragenic SNPs in a large family with multiple individuals with MJD from southwestern part of India. This would also help to illustrate the phenotypic diversity within a shared familial and environmental context.

A large family [Figure 1] with multiple members clinically diagnosed with MJD was recruited from the Neuro Specialities Centre, Belgaum, India. This family has a southwestern geographical origin and most of the family members are living in the same geographical area through generations. The family could be traced back to 7 generations and data were collected for 150 members from the family. A detailed neurological examination was performed for 38 family members, of whom 18 were clinically diagnosed to be having MJD. The mean AAO was 29.17 ± 16.95 years. All the participants were interviewed in person, and each provided information on their relatives, following a structured questionnaire. By personal examination, and by compiling data from the informants, 52 members could be assigned a provisional diagnosis of MJD. The study was approved by the Institute Ethics Committee. All 38 participants gave written informed consent for blood sampling, after being provided a complete description of the study.
Figure 1: Pedigree structure of the family with MJD under study. Squares represent males and circles represent females. Filled symbols indicate affected members and unfilled symbols indicate apparently unaffected members. Diagonal line across the symbol indicates deceased members. *Indicates the members available for genotyping

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The CAG repeat fragment of ATXN3 was amplified by polymerase chain reaction followed by sizing on a ABI 3500XL DNA analyzer (ThermoFisher Scientific Inc., Waltham, MA, USA). Analysis of intragenic SNP-based haplotype was done for all samples based on SNPs A669 TG/G669 TG, C987 GG/G987 GG, and TAA1118/TAC1118.[7],[19]

A detailed clinical assessment of the affected members examined from the family is presented in [Table 1]. Based on the clinical examination, the affected members were grouped into subgroups of MJD. Type I consisted of one individual with AAO of 13 years and with pronounced pyramidal and extrapyramidal signs besides the common features of cerebellar ataxia and ophthalmoplegia, and severe generalized dystonia. Type II consisted of nine members with cerebellar and pyramidal deficits without extrapyramidal signs with a mean AAO of 16.44 ± 8.01 years. Type III included eight members with pronounced peripheral signs in addition to ataxia with a mean AAO of 45.5 ± 8.58 years.
Table 1: Detailed clinical description of the affected family members where genetic data were available

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An expanded allele was found in all 18 individuals considered to be affected clinically [Table 2]. These 18 individuals had one expanded allele, other being normal, and the size of the expanded allele varied from 57 and 70 repeats. The mean size of the expanded allele was 63 ± 4.72 repeats. We analyzed the data to test if the repeat size influences the age of onset in patients with the expanded allele, by Pearson's correlation analysis. A significant negative correlation was observed for the AAO with the expanded repeat length, r (16)= −0.880 (P < 0.001) [Figure 2]. The mean repeat size of the expanded allele was 66.45 ± 2.13 (range 63–70) and 58.25 ± 1.04 (range 57–59) for members with MJD Types II and III, respectively. The type I individual with a very early onset had the highest repeat length of 70, as expected. The means of repeat sizes were significantly different between the Type II and III, with the Type III being smaller by almost 10 repeats (P < 0.0001; Student's t-test). The size of the normal chromosome in the affected members ranged from 8 to 22 repeats. An allele range of 8–26 repeats was found in the unaffected members of the family.
Table 2: Details of the family members genotyped in the study with the CAG repeats and haplotype is shown

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Figure 2: Correlation of CAG repeats length in the expanded chromosomes of affected individuals with age at onset of disease. A squared linear correlation coefficient of r2 = -0.775 was obtained for Pearson's correlation using R statistical software package version 3.3.2

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Instability of the CAG repeat number during transmission from parent to offspring has been observed in MJD. Within the family, there were four affected parent-to-offspring transmission, of the expanded alleles. We observed increase in the repeat length in two father-to-son transmissions. In the transmission from V: 13 to VI: 12, there was an increase from 59 repeats to 66 repeats, and from V: 16 to VI: 16, repeat size increased from 63 to 68. There were three affected sibpairs and one sibship of three members in the family, where we could assess the repeat length. The repeat size varied among the siblings for V: 16 and V: 23 (63 and 70 repeats), VI: 9 and VI: 11 (64 and 67 repeats), and VII: 1 and VII: 2 (68 and 70 repeats). All these three affected sibpairs, which show variations in the repeat size, had paternal lineage. There was a tendency for an increase in the expanded allele with increasing paternal age, with younger of the sibpair showing an increased repeat size.

Two mother-to-son transmissions were stable, with no increase in the repeat length (V: 34 to VI: 46, 57 repeats; and V: 26 to VI: 32; 66 repeats). In the sibship, which has a maternal transmission, consisting of VI: 39, VI: 41, and VI: 42, there was no variation observed in the repeat length.

Analysis[21] of intragenic SNP-based haplotype revealed the presence of six combinations of haplotype in the family, namely, A-C-A, G-G-C, G-G-A, A-C-A, G-C-A, A-C-C, and A-G-A. All the individuals with the expanded (CAG), had A-C-A halpotype. The haplotype analysis in the unexpanded normal chromosomes (n = 58) is shown in [Figure 3].
Figure 3: Haplotype frequency distribution in the non-expanded chromosomes from the family

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Previous studies have found an inverse correlation of the age of onset of MJD with the size of the expanded CAG repeat size.[16] Overall, our data also show similar trend; however, variation in the age of onset was often seen in members with same repeat size. The individuals V: 26, VI: 32, and VI: 12 had repeat size of 66, but the AAO was 30, 15, and 7 years, respectively. This observation suggests that other factors may also be involved in determining the age of onset in MJD. The influence of environmental factors, in addition to other genetic factors, such as modifier genes, for this variation in the AAO may need to be explored.

Earlier reports have suggested that the larger CAG repeats result in a more severe phenotype.[16],[22],[23] We also observed significantly increased repeat sizes with increased severity of the disease. There was an increase in the mean repeat size of ~8 repeats in Type II cases when compared to the Type III cases. The larger repeats were mostly associated with pyramidal signs, whereas the smaller repeat size showed an increased ataxia symptom severity [Table 1]. Similar observations were also made in a previous study from Japan with an earlier onset and larger expansions. This study showed marked extrapyramidal features. The studies with late onset and smaller expansions, however, have shown cerebellar ataxia with neuropathy.[22]

A decrease in the AAO (anticipation by 28 years) and increased disease severity (Type III–II) was seen in V: 13 and VI: 12 transmission. Other transmission – V: 16–VI: 16 – also showed a decreased age of onset (-19 years), and no change in severity of the disease, which remained as Type II in the parent and off-spring, although the follow-up showed mild worsening of the symptoms in VI: 16. The results of our analysis of the intragenic SNPs confirm our previous observations found in nine MJD families from India.[20] All the expanded alleles from these families were exclusively associated with the A-C-A haplotype. The A-C-A and G-G-C haplotypes, which were shown to be present in more than 80% of the population in our previous study,[20] were 65% in the present study. Based on haplotype analyses, the world-wide presence of MJD is explained by two founder lineages. The first lineage seems to have an Asian origin with an estimated age of around 7000 years (TTACAC; Joseph lineage) and the second seems to have a Portuguese/Azorean origin, with a more recent one with around 1500 years (GTGGCA; Machado lineage).[24],[25] The Joseph lineage is found in a vast majority of the population of non-Portuguese families, and the Machado lineage is seen in some regions of Portugal. We were also able to detect the presence of the Joseph lineage haplotype in the family, with the expanded chromosomes.

This genetic study reveals significant clinical correlations. It provides genetic support for the presence of MJD (SCA3) of Asian origin in this geographical region, although previous epidemiological studies have revealed that SCA2 and SCA1 are most commonly seen in the neighboring eastern and southern regions, respectively. It lends genetic confirmation to the clinical recognition of various subphenotypes (Types I–III). Apart from confirming most of the previously described genotype-phenotype correlations, the study hints at the possible role of epigenetics and environmental factors in the pathogenesis of MJD. The reported family is described from a geographical region where consanguineous marriages are very common. As the social and financial burden of the disease on the family can be devastating, there may be advantages in educating the population on the demerits of consanguineous marriages.

In conclusion, our study shows CAG repeat expansions in all the affected members of the family at the ATXN3. The haplotype analysis suggests an Asian origin hypothesis for the MJD mutation in India.

Financial support and sponsorship

The study was funded by Indian Council of Medical Research (ICMR), New Delhi, India.

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2]


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