Identification of a suitable SNP for allele-specific silencing of the disease-causing gene in SCA1 patients in South Africa

Includes bibliographical references (leaves 118-132).Spinocerebellar ataxia 1 (SCA1) is part of a broader group of dominant neurodegenerative disorders caused by an unstable CAG trinucleotide repeat. There is no known cure for this disease and symptoms worsen progressively culminating in death. The disease-causing mutation in SCA1 occurs in the ATXN1 gene. The function of the gene product (the ataxin-1 protein) is unknown, however; the protein has been linked to RNA processing in the cell. The first part of this study followed on a 1997 report of two founder haplotypes in the Mixed Ancestry SCA1 families in the Western Cape of South Africa, using microsatellites. The aim was to narrow the region investigated in the previous study, and confirm the existence of founder haplotypes using a SNP-based haplotype. The SNPbased haplotype was constructed using 4 SNPs in individuals from 5 different families of Mixed Ancestry origin from the Western Cape and the two founder events were confirmed. The SNP-based haplotype also shows the existence of a minimum common interval and indicates regions of possible break-points which may be useful in determining the extent and origins of the two haplotypes. The second aim of the study was to preferentially silence the mutant transcript of the ATXN1 gene by targeting a single nucleotide difference. Two of the SNPs genotyped for the SNP-based haplotype were found to be heterozygous in over half of the patient cohort. Eight shRNA effector molecules were screened against short target sequences incorporating one of these SNPs. Results are promising, with significant discrimination achieved between the wild-type and mutant alleles by targeting this SNP. This study has shown that RNAi may be developed as a beneficial therapeutic technique for a subset of SCA1 patients in South Africa

A probable cis-acting genetic modifier of Huntington disease frequent in individuals with African ancestry

Huntington disease (HD)is a dominantly inherited neurodegenerative disorder caused by the expansion of a polyglutamine encoding CAG repeat in the huntingtin gene. Recently, it has been established that disease severity in HD is best predicted by the number of pure CAG repeats rather than total glutamines encoded. Along with uncovering DNA repair gene variants as trans-acting modifiers of HD severity, these data reveal somatic expansion of the CAG repeat as a key driver of HD onset. Using high-throughput DNA sequencing, we have determined the precise sequence and somatic expansion profiles of the HTT repeat tract of 68 HD-affected and 158 HD-unaffected African ancestry individuals. A high level of HTT repeat sequence diversity was observed, with three likely African-specific alleles identified. In the most common disease allele (30 out of 68), the typical proline-encoding CCGCCA sequence was absent. This CCGCCA-loss disease allele was associated with an earlier age of diagnosis of approximately 7.1 years and occurred exclusively on haplotype B2. Although somatic expansion was associated with an earlier age of diagnosis in the study overall, the CCGCCA-loss disease allele displayed reduced somatic expansion relative to the typical HTT expansions in blood DNA. We propose that the CCGCCA loss occurring on haplotype B2 is an African cis-acting modifier that appears to alter disease diagnosis of HD through a mechanism that is not driven by somatic expansion. The assessment of a group of individuals from an understudied population has highlighted population-specific differences that emphasize the importance of studying genetically diverse populations in the context of disease

Implications of direct-to-consumer whole-exome sequencing in South Africa

This editorial examines a number of vitally important ethical, legal and scientific concerns that have to be addressed to ensure proper and ethical implementation of direct-to-consumer whole-exome sequencing in South Africa. Individuals taking part in this endeavour must be fully informed of the positive and negative sequelae

A molecular investigation of Huntington disease; origins of the mutation and current prevalence in South Africa

Huntington disease (HD) is a devastating neurodegenerative condition characterised by a triad of symptoms: behavioural/psychiatric changes, cognitive decline and movement disorder. The dominantly inherited disease-causing mutation is an expanded trinucleotide (CAG) repeat in the Huntingtin(HTT) gene. Clinical symptoms are believed to be the result of degeneration of specific neuronal populations that are susceptible to the presence of a toxic expanded protein product. The disease is incurable and following the onset of symptoms, is progressively debilitating over 10-20 years and eventually fatal. Although typical epidemiological studies of prevalence are challenging for a genetic disorder such as HD, family studies and various other methods of ascertainment have been used to estimate its occurrence in different populations. Prevalence is therefore known to vary geographically; population-specific haplotypes have been hypothesised to be the basis of this variation between ethnic groups. High prevalence estimates for populations with European ancestry led to the supposition that the HD mutation was introduced to different regions by Europeans. In South Africa, a survey in the 1970s estimated that the prevalence of HD in the white and coloured subpopulations was similar at 2 per 100 000 individuals; while that in the black subpopulation was significantly lower, at less than 0.01 per 100 000 individuals. Molecular genetic analyses have since revealed links between the white and coloured subpopulations which would explain the similarity in prevalence; however, our knowledge of the genetics of HD in the black subpopulation, has been sorely lacking. This study provides, for the first time, a comprehensive analysis of the HTT gene in an African population. An evaluation of the normal distribution of CAG-tract sizes highlighted significant differences between the subpopulations. Haplotype analysis identified population-specific disease-associated haplotypes, confirming distinct origins of the HD mutation in the different subpopulations. In a coloured family with the rare juvenile form of the disease, DNA sequencing revealed no novel variants within the immediate vicinity of the CAG-tract that could be associated with the observed instability. This indicates that genome-wide analyses may be more useful in identifying factors related to repeat instability and future investigations are planned for a cohort of South African patients affected by juvenile onset HD

A CIS-acting Modifier of Age at Diagnosis of Huntington Disease in Black South African Patients

Background: Huntington disease (HD) is a dominantly inherited neurodegenerative disorder, presenting with a movement disorder, cognitive decline and psychiatric complications. HD is caused by the expansion of a CAG repeat tract in exon 1 of the huntingtin gene (HTT). In other repeat expansion disorders, variants within the associated repeat tracts may affect stability, which in turn modifies disease severity. Although sequence variation has been reported within the sequence encoding the HTT polyglutamine and polyproline tract, little is known about its effect on disease. Aim: To determine the sequence diversity precisely within the HTT exon 1 trinucleotide repeat in black South African individuals and assess the effect of sequence variation in modifying disease. Methods/techniques: A sample of black South African individuals (59 patients and 153 controls) was analysed using a specialised high-throughput DNA sequencing assay and data analysis pipeline, specific for the HTT exon 1 repeat. Results/outcomes: Significant diversity was observed in comparison to a European sample of patients, with novel African alleles defined by the description of allele sub-structures. The most common disease allele structure in our patients was associated with the absence of the CCGCCA cassette that precedes the polyproline CCG repeat in typical HD-causing alleles. Regression analysis determined that age at diagnosis was approximately 6 to 13 years earlier in patients carrying HD-causing alleles lacking the CCGCCA cassette. Conclusion: This is the first study to describe allele sequence diversity within the HTT exon 1 repeat in an African population. The absence of the CCGCCA cassette, which appears to be African-specific and associated with an earlier age at diagnosis in African HD patients, is a potential cis-acting modifier of HD onset

A CIS-acting Modifier of Age at Diagnosis of Huntington Disease in Black South African Patients

Background: Huntington disease (HD) is a dominantly inherited neurodegenerative disorder, presenting with a movement disorder, cognitive decline and psychiatric complications. HD is caused by the expansion of a CAG repeat tract in exon 1 of the huntingtin gene (HTT). In other repeat expansion disorders, variants within the associated repeat tracts may affect stability, which in turn modifies disease severity. Although sequence variation has been reported within the sequence encoding the HTT polyglutamine and polyproline tract, little is known about its effect on disease. Aim: To determine the sequence diversity precisely within the HTT exon 1 trinucleotide repeat in black South African individuals and assess the effect of sequence variation in modifying disease. Methods/techniques: A sample of black South African individuals (59 patients and 153 controls) was analysed using a specialised high-throughput DNA sequencing assay and data analysis pipeline, specific for the HTT exon 1 repeat. Results/outcomes: Significant diversity was observed in comparison to a European sample of patients, with novel African alleles defined by the description of allele sub-structures. The most common disease allele structure in our patients was associated with the absence of the CCGCCA cassette that precedes the polyproline CCG repeat in typical HD-causing alleles. Regression analysis determined that age at diagnosis was approximately 6 to 13 years earlier in patients carrying HD-causing alleles lacking the CCGCCA cassette. Conclusion: This is the first study to describe allele sequence diversity within the HTT exon 1 repeat in an African population. The absence of the CCGCCA cassette, which appears to be African-specific and associated with an earlier age at diagnosis in African HD patients, is a potential cis-acting modifier of HD onset

Huntington disease in the South African population occurs on diverse and ethnically distinct genetic haplotypes

<p>Huntington disease (HD) is a neurodegenerative disorder resulting from the expansion of a CAG trinucleotide repeat in the huntingtin (HTT) gene. Worldwide prevalence varies geographically with the highest figures reported in populations of European ancestry. HD in South Africa has been reported in Caucasian, black and mixed subpopulations, with similar estimated prevalence in the Caucasian and mixed groups and a lower estimate in the black subpopulation. Recent studies have associated specific HTT haplotypes with HD in distinct populations. Expanded HD alleles in Europe occur predominantly on haplogroup A (specifically high-risk variants A1/A2), whereas in East Asian populations, HD alleles are associated with haplogroup C. Whether specific HTT haplotypes associate with HD in black Africans and how these compare with haplotypes found in European and East Asian populations remains unknown. The current study genotyped the HTT region in unaffected individuals and HD patients from each of the South African subpopulations, and haplotypes were constructed. CAG repeat sizes were determined and phased to haplotype. Results indicate that HD alleles from Caucasian and mixed patients are predominantly associated with haplogroup A, signifying a similar European origin for HD. However, in black patients, HD occurs predominantly on haplogroup B, suggesting several distinct origins of the mutation in South Africa. The absence of high-risk variants (A1/A2) in the black subpopulation may also explain the reported low prevalence of HD. Identification of haplotypes associated with HD-expanded alleles is particularly relevant to the development of population-specific therapeutic targets for selective suppression of the expanded HTT transcript.</p>