published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics))--Stellenbosch University, 2008. / A South African family of Mixed Ancestry presented with a rapidly progressive dementia and a movement disorder which affected a number of individuals across three generations. The initial symptoms included personality changes and tremors that escalated to severe dementia and eventually a completely bedridden state. It was determined that the mean age at onset was in the third decade of life and affected individuals died within 10-15 years after the onset of symptoms. The aim of the present study was to elucidate the genetic cause of the disorder in this family and to further investigate the patho-biology of the disease. Mutations that could possibly cause the observed phenotype in this family were screened for. These included loci implicated in Huntington’s disease, Parkinson’s disease, Dentatorubral-Pallidoluysian Atrophy, Spinocerebellar ataxias (types 1, 2, 3, 6, and 7), Huntington’s disease-like 2 (HDL2) and several mitochondrial disorders. Single-strand Conformation Polymorphism (SSCP) analysis and direct sequencing were used to detect possible mutations while genotyping on an ABI genetic analyser was used to detect disorders caused by repeat expansions. Haplogroup and Short Tandem Repeats (STRs) analyses of the Y-chromosome and mitochondrial DNA of one affected family member was used to determine the family’s genetic ancestry. Reverse transcriptase polymerase chain reaction (RT- PCR) and complementary DNA (cDNA) analyses of the Junctophlin-3 (JPH3) gene was performed to provide information on the expression profile of this gene. After the exclusion of several genetic loci it was shown that this family had HDL2. This is a rare disease caused by a CAG/CTG repeat expansion in an alternatively spliced version of the JPH3 gene. HDL2 occurs almost exclusively in individuals of Black African ancestry. The genetic ancestry data suggested that the family member was most likely of South African Mixed Ancestry making this the first reported family of South African Mixed Ancestry with HDL2. A pilot study investigated the repeat distribution amongst three South African sub-populations in order to determine whether there was a bias in the repeat distribution that possibly predisposes Black Africans to develop the disease. The results showed a statistically significant difference (P= 0.0014) in the distribution of the repeats between the Black African and Caucasian cohorts. However, no conclusions could be drawn as to whether Black Africans harboured larger repeats that predisposes them to developing HDL2. The expanded repeat is located in an alternatively spliced version of the JPH3 mRNA. Interestingly, this repeat is not present in the mouse homologue of the gene although the rest of the genomic sequence is highly conserved across the human, mouse and chimpanzee genomes. Using foetal brain cDNA and PCR primers designed to be specific for different JPH3 isoforms, independent confirmation of the presence of two JPH3 mRNA transcripts (the full length and a shorter alternatively spliced version) was provided. In the absence of brain tissue from an HDL2-affected individual, it was investigated whether both JPH3 mRNA transcripts could be detected in lymphocytes. Using RNA isolated from the transformed lymphocytes of two HDL2-affected family members, real-time PCR was attempted. These experiments produced inconclusive results and required further optimisation. Further RT-PCR experiments for JHP3 expression in different tissues (brain and other) obtained from HDL2-affected individuals would be of interest. The present study identified the first Mixed Ancestry family with HDL2. This family will now be able to request genetic counselling and pre-symptomatic testing for all at-risk family members. Aspects of this study provided independent confirmation of characteristics of the mutated gene. More research on HDL2 will be crucial in understanding the pathogenesis of this disease.

Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer's disease (AD) is the most common form of dementia defined by loss in memory and cognitive abilities severe enough to interfere significantly with daily life activities. Amnestic mild cognitive impairment (MCI) is a clinical condition in which an individual has memory deficits not normal for the individual's age, but not severe enough to interfere significantly with daily functioning. Every year, approximately 10-15% of individuals with MCI will progress to dementia. Currently, there is no treatment to slow or halt AD progression, but research studies are being conducted to identify causes that can lead to its earlier diagnosis and treatment. Genetic variation plays a key role in the development of AD, but not all genetic factors associated with the disease have been identified. Copy number variants (CNVs), a form of genetic variation, are DNA regions that have added genetic material (duplications) or loss of genetic material (deletions). The regions may overlap one or more genes possibly affecting their function. CNVs have been shown to play a role in certain diseases. At the start of this work, only one published study had examined CNVs in late-onset AD and none had examined MCI. In order to determine the possible involvement of CNVs in AD and MCI susceptibility, genome-wide CNV analyses were performed in participants from three cohorts: the ADNI cohort, the NIA-LOAD/NCRAD Family Study cohort, and a unique cohort of clinically characterized and neuropathologically verified individuals. Only participants with DNA samples extracted from blood/brain tissue were included in the analyses. CNV calls were generated using genome-wide array data available on these samples. After detailed quality review, case (AD and/or MCI)/control association analyses including candidate gene and genome-wide approaches were performed. Although no excess CNV burden was observed in cases compared to controls in the three cohorts, gene-based association analyses identified a number of genes including the AD candidate genes CHRFAM7A, RELN and DOPEY2. Thus, the present work highlights the possible role of CNVs in AD and MCI susceptibility warranting further investigation. Future work will include replication of the findings in independent samples and confirmation by molecular validation experiments.