Browsing Doctoral Degrees (Genetics) by browse.metadata.advisor "Bardien, Soraya"
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- ItemInvestigation of the genetic aetiology of Parkinson's disease in South Africa(Stellenbosch : University of Stellenbosch, 2011-03) Keyser, Rowena J.; Bardien, Soraya; Carr, Jonathan; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Parkinson’s disease (PD), a neurodegenerative movement disorder characterized by resting tremors, bradykinesia, postural instability and rigidity, is due to a selective loss of dopaminergic neurons in the substantia nigra. Non-motoric symptoms include autonomic, cognitive and psychiatric problems. PD has been suggested to result from environmental factors, genetic factors or a combination of the two. Evidence has mounted over the last 13 years supporting the involvement of a significant genetic component. Mutations in the parkin, PINK1, DJ-1, ATP13A2, SNCA, and LRRK2 genes have been conclusively associated with PD. The aim of the present study was to establish the first study on the genetic etiology of PD in South African patients. Patients from the various South African ethnic groups with predominantly early-onset PD and/or a positive family history were recruited. Varying numbers of study participants (ranging from 88-205) were used for the different sections of this study depending on their availability at the time of the experiments and the specific clinical criteria applied. Mutation screening was conducted using High-resolution melt (HRM) analysis, DNA sequencing and multiplex ligation-dependent probe amplification (MLPA). HRM analysis and sequencing of the known PD genes identified the following mutations: parkin (T113fsX163), PINK1 (Y258X), and LRRK2 (G2019S and R1441C). Using haplotype analyses, the five South African LRRK2 G2019S-positive patients were found to share a common ancestor with other G2019S haplotype 1-associated families reported worldwide. Two commercially available MLPA kits, SALSA P051 and P052, were used to assess the study participants for exon dosage mutations. Exonic deletions and insertions in parkin were identified in five patients. In addition, a family with a whole-gene triplication mutation of SNCA was identified. This is the 4th family worldwide to have this specific mutation which leads to a severe phenotype with autonomic dysfunction and early-onset dementia. The CAESAR (CAndidatE Search And Rank) bioinformatic program was used to select novel candidate genes for PD. CAESAR produced a ranked list containing known PD causing genes as well as novel candidates. The MAPT and SNCAIP genes were selected from the list of ten highest scoring genes. HRM analysis identified novel sequence variants in both genes with unknown functional significance that warrants further study. A novel 16bp deletion (g.-6_+10del) in the promoter region of DJ-1 was identified in one PD patient. The functional significance of this variant was investigated using a Dual-Luciferase Reporter assay. The variant was found to significantly reduce luciferase activity in two separate cell lines, HEK293 and BE(2)-M17 neuroblastoma cells, both with and without oxidative stress (p<0.0001), and we proposed that the 16bp sequence might be important in transcriptional regulation of DJ-1. In addition, the activity of three transcription factors (AhR, ARNT and HIF- 1) with binding sites within the deletion sequence may be influenced by the variant. In conclusion, mutation screening resulted in the identification of mutations in six patients in parkin, six patients in LRRK2, one patient in PINK1 and one patient in SNCA. In addition, a number of novel sequence variants were identified with unknown functional significance. Investigating the genetic basis of PD in the unique South African ethnic groups has shown that the known PD associated genes play minor roles in causing the disease in this population which indicates the possible involvement of other as yet unidentified PD genes. Innovative bioinformatic and wet bench experimental strategies are therefore urgently needed to identify new candidate genes for PD.