Browsing by Author "Mahungu, Amokelani Clementine"
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- ItemDevelopment of a custom-designed targeted resequencing gene panel for Parkinson’s disease(Stellenbosch : Stellenbosch University, 2020-03) Mahungu, Amokelani Clementine; Bardien, Soraya; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Parkinson’s disease (PD) is a complex neurodegenerative disease characterized by the loss of dopaminergic neurons in a part of the brain known as the substantia nigra. The selective loss of these neurons results in motor impairments also classified as cardinal PD symptoms which are tremor, rigidity, bradykinesia, and postural instability. Additionally, non-motor symptoms also occur namely loss of smell, mood disorders, cognitive decline, sleep disturbances, gastrointestinal and autonomic dysfunction. To date, no cure for PD exists and the underlying pathobiology of the disease is not fully understood. Approximately 90% of PD cases are idiopathic which is proposed to result from a complex interaction of environmental and genetic factors. The remaining 10% of PD cases are defined as monogenic and caused by genes that follow a Mendelian type of inheritance. Since the discovery of the first monogenic PD-causing gene SNCA, numerous genes have been identified and extensively studied in European and Asian populations. These include ATP13A2, CHCHD2, DJ-1, DNAJC13, DNAJC6, EIF4G1, FBXO7, GBA, GCH1, GIGYF2, HTRA2, LRRK2, PINK1, PLA2G6, PRKN, RAB39B, RIC3, SLC6A3, SNCA, SYNJ1, TMEM230, VPS13C and VPS35. However, many of these genes have not been widely studied in sub-Saharan African (SSA) populations. Furthermore, for the few that have been investigated, the studies used first-generation sequencing methods namely Sanger sequencing, that only allows screening of a single region or mutation at a time. More recently, next-generation sequencing (NGS) gene panels have been used to examine all the known PD genes in patients whose early disease onset and positive family history suggest a possible inherited genetic cause. Thus, the first aim of our study was to design a custom NGS gene panel for rapid screening of the known PD genes in South African patients. The gene panel was developed using Agilent SureSelect Target Enrichment technology and it included all of the above-mentioned 23 PD genes. Subsequently, 32 PD patients with early disease onset and family history were screened in two separate sequencing runs. Following analysis of the sequence data, we achieved a coverage of >200x for both runs. For our first run, we included positive controls with known pathogenic single nucleotide mutations, a 40bp deletion, and copy number mutations. All the mutations from the positive controls were validated except for the copy number mutations. On average, 79 variants were detected per patient of which only 15 were prioritized based on whether they were previously associated with PD, were rare (MAF <0.01), novel, and were predicted to be deleterious by the majority of the in-silico tools. These variants were further classified according to the American College Medical Genetics and Genomics (ACMG) recommendations to assess their clinical significance. Only six were found to meet the pathogenic (GBA p.L483P [p.L444P]) or likely pathogenic (GBA p.R170L [p.R131L], p.D179H [p.D140H], p.E365K [p.E326K], PINK1 p.P305A, and PRKN p.E310D) criteria. One of the major genetic risk factors for PD and a known cause of Gaucher’s disease included on the gene panel was GBA, which has a pseudogene (GBAP1) whose sequence is about 96% similar. Specialized primers that allow the amplification of only GBA are used to avoid the detection of variants in the non-functional pseudogene. Thus, the second aim of our study was to develop a method for screening and validating GBA mutations in our laboratory. Once the nested Polymerase Chain Reaction (PCR) and Sanger sequencing method was successfully optimized, 30 of our African Black PD patients were screened. To our knowledge, this is the first GBA mutation screening in PD performed within this population. Eight variants were identified of which four were predicted to be deleterious by the majority of the in-silico tools. These included three known pathogenic Gaucher’s disease-associated mutations (p.R150W [p.R120W], p.R170L [p.R131L], and p.T75del [p.T36del]) of which p.R120W is a known risk factor for PD, and one variant of uncertain significance (p.Q536* [p.Q497*]). In addition, two novel variants (p.F255L [p.F216L] and p.G517R [p.G478R]) were identified of which p.F216L was found to be common (9.9%) in controls. Furthermore, the specialized GBA primers were also used to validate all of the prioritized GBA variants identified using the gene panel. Only four of the five variants (GBA p.L483P, p.R170L, p.D179H, p.E365K) were confirmed to be in the functional gene. In conclusion, we successfully developed a method for rapid screening of the known PD genes and a technique for screening and validating GBA mutations. These methods can be used for rapid and high- throughput screening of the genetic contribution to PD in our local populations and other populations within SSA. Subsequently, these methods will enable us to identify novel candidates for validation in future functional studies. Consequently, this work will also contribute to the development of precision medicine tailored to each PD patient.