Implementation of Targeted Resequencing Strategies to Identify Pathogenic Mutations in Nigerian and South African Patients with Parkinson’S Disease
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Implementation of Targeted Resequencing Strategies to Identify Pathogenic Mutations in Nigerian and South African Patients with Parkinson’s Disease Oluwafemi Gabriel Oluwole Dissertation presented for the degree; Doctor of Philosophy in the Faculty of Medicine and Health Sciences at Stellenbosch University Supervisor: Professor Helena Kuivaniemi Faculty of Medicine and Health Sciences Department of Biomedical Sciences Co-supervisor: Professor Gerardus Tromp Faculty of Medicine and Health Sciences Centre for Bioinformatics and Computational Biology Department of Biomedical Sciences April 2019 Stellenbosch University https://scholar.sun.ac.za DECLARATION This dissertation includes one original paper published in a peer-reviewed journal, and one unpublished publication (submitted for publication). The development and writing of the published paper were the principal responsibility of myself and for each of the cases where this is not the case a declaration is included in the dissertation indicating the nature and extent of the contributions of co-authors. Declaration by the candidate: With regard to Chapter 2, the nature and scope of my contribution were as follows: I was the project lead, carried out literature searches, appraised the articles, summarized results, prepared the tables and figures and drafted and revised the manuscript, which has now been published on-line in Parkinsonism and Related Disorders. (DOI: https://doi.org/10.1016/j.parkreldis.2018.12.004). I have obtained a permission from the publisher (Elsevier) to use the accepted version of the manuscript as part of my thesis (Chapter 2). Declaration by co-authors: The undersigned hereby confirm that 1. the declaration above accurately reflects the nature and extent of the contributions of the candidate and the co-authors to Chapter 2, 2. no other authors contributed to Chapter 2 besides those specified below, and 3. potential conflicts of interest have been revealed to all interested parties and that the necessary arrangements have been made to use the material in Chapter 2 of this dissertation. ii Stellenbosch University https://scholar.sun.ac.za Name e-mail address Nature of Extent of contribution contribution (%) Helena [email protected] Carried out 15 Kuivaniemi literature searches, reviewed the articles and edited the manuscript Jonathan A. [email protected] Provided clinical 2 Carr expertise, critically reviewed and edited the manuscript Owen A. Ross [email protected] Provided clinical 2 expertise, critically reviewed and edited the manuscript Matthew O.B. [email protected] Provided clinical 1 Olaogun expertise, critically reviewed and edited the manuscript Soraya Bardien [email protected] Conceptualized the 10 idea for the research, obtained funding, supervised the project and wrote sections of the manuscript Morenikeji A. [email protected] Conceptualized the 5 Komolafe idea for the research, obtained funding, supervised the project and wrote sections of the manuscript iii Stellenbosch University https://scholar.sun.ac.za With regard to Chapter 4, the nature and scope of my contribution were as follows: I led the project, obtained ethics approvals for the study, did literature searches, carried out Sanger sequencing, high resolution melt experiments, performed data analyses using Ion Torrent software, prepared some of the tables and figures and drafted and revised the manuscript that has been submitted for publication. Declaration by co-authors: The undersigned hereby confirm that 1. the declaration above accurately reflects the nature and extent of the contributions of the candidate and the co-authors to Chapter 4, 2. no other authors contributed to Chapter 4 besides those specified below, and 3. potential conflicts of interest have been revealed to all interested parties and that the necessary arrangements have been made to use the material in Chapter 4 of this dissertation. Name e-mail address Nature of contribution Extent of contribut ion (%) Helena [email protected] Supervised the project, 10 Kuivaniemi critically appraised the results in this study and edited the manuscript Shameemah [email protected] Revised the manuscript and 1 Abrahams contributed to knowledge William L. [email protected] Revised the manuscript and 1 Haylett contributed to knowledge Alvera A. Vorster [email protected] Performed tNGS 1 Carel J. van [email protected] Performed tNGS 1 Heerden David L. Tabb [email protected] Revised the manuscript and 1 contributed to knowledge iv Stellenbosch University https://scholar.sun.ac.za Michael B. [email protected] Recruited patients in Nigeria, 1 Fawale provided clinical expertise and revised the manuscript Taofiki A. [email protected] Recruited patients in Nigeria, 1 Sunmonu m provided clinical expertise and revised the manuscript Abiodun Ajose abiodun_ajose57@yaho Revised the manuscript and 1 o.com contributed to knowledge Matthew O. [email protected]. Revised the manuscript and 1 Olaogun uk contributed to knowledge Anastasia C. [email protected] Recruited patients in South 1 Rossouw om; Africa, provided clinical expertise and revised the manuscript Ludo S. van [email protected] Recruited patients in South 1 Hillegondsberg Africa, provided clinical expertise and revised the manuscript Jonathan Carr [email protected] Led the clinical team in 2 South Africa, obtained ethics approvals, provided clinical expertise, contributed to knowledge, and revised the manuscript Owen A. Ross [email protected] Revised the manuscript and 1 contributed to knowledge Morenikeji A. [email protected] Conceptualised the idea for 5 Komolafe om this research, obtained ethics approvals, invited other collaborators, obtained funding, recruited patients in Nigeria, supervised part of the work, contributed to knowledge, and revised the manuscript Gerard Tromp [email protected] Co-supervised the project, 10 performed data analysis, wrote results, generated figures and tables, and contributed to knowledge Soraya Bardien [email protected] Conceptualised the idea for 7 this research, obtained ethics approvals, invited other collaborators, obtained funding, supervised part of the work, contributed to knowledge, and revised the manuscript v Stellenbosch University https://scholar.sun.ac.za vi Stellenbosch University https://scholar.sun.ac.za Copyright © 2019 Stellenbosch University, All rights reserved. vii Stellenbosch University https://scholar.sun.ac.za ABSTRACT Parkinson's disease (PD) is a debilitating neurodegenerative movement disorder. It is characterized by the progressive loss of specific neurons in the brain, the dopaminergic neurons of the substantia nigra pars compacta of the midbrain. Increasing age is considered to be one of the strongest risk factors for PD. The symptoms of PD profoundly affect the quality of life of patients and their family members. The aetiology of PD is not completely understood. Several genes harbour mutations that contribute to developing PD. Little, however is known about the genetics of PD in the sub-Saharan African patients. African populations have greatly admixed ancestry with the greatest genetic diversity in the world. This diversity has important ramifications for genetic research. Sporadic or idiopathic cases of PD are the most recurrent in South Africa and Nigeria. Familial PD cases are relatively rare. It is also possible that genetic mutations interact with environmental factors to cause PD onset and its progression. Several genes including PRKN, PINK1, PARK7, ATP13A2, SNCA, LRRK2, VPS35, EIF4G1, CHCHD2 and LRP10 have been associated with PD. The mutations in these genes provide new opportunities to understand the disease by suggesting biological pathways that could be involved in PD pathogenesis. The PD genes have been predominantly identified and studied in European, North American, North African and Asian populations. It is possible that the genetic information obtained from other populations may not be transferable to the sub-Saharan African populations. The present study focused on identifying pathogenic mutations in Black South African and Nigerian PD patients. This study is the first collaborative genetics study on PD between South Africa and Nigeria. This study hypothesized that novel disease-causing genes will be identified in Black South African and Nigerian PD patients. A total of 33 Black South African and 42 Nigerian PD patients were recruited and screened for genetic defects using various methods. The mean age at onset of PD in the South Africans and Nigerians was 48±8 and 65±10 years, respectively. Two South African patients had a family history of PD. viii Stellenbosch University https://scholar.sun.ac.za This study evaluated the presence of exonic rearrangements such as multiplications or deletions of exons in the PD patients. A total of 42 Nigerian and 15 South African PD patients were studied for exonic rearrangement using a commercially available assay kit. The results revealed three Nigerian patients with exonic rearrangements: (1) Heterozygous deletion in PARK7 exon 1; (2) Heterozygous duplications in PINK1 exon 5 and PRKN exon 1; and (3) Two heterozygous duplications in PRKN exon 1 and 2 gene. The study also implemented targeted next-generation sequencing (NGS) to screen the 751 genes known to harbour pathogenic mutations in patients with neurological diseases using the AmpliSeqTM Neurological Disease panel and the Ion Torrent sequencing platform. NGS is fast, scalable, reliable and could reveal putative pathogenic variants. Bcftools was used for assessing the data