Implementation of targeted resequencing strategies to identify pathogenic mutations in Nigerian and South African patients with Parkinson’s Disease

Oluwole, Oluwafemi Gabriel (2019-04)

Thesis (PhD)--Stellenbosch University, 2019.

Thesis

ENGLISH 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. 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 quality and to filter the variants. Sequence variants with mapping quality score > 100 and a minimum read depth > 40 were selected for follow-up. We used annovar utility software for the annotation. We removed variants with minor allele frequency > 0.01 in any of the frequency databases. We also assessed how often each variant was found in common among samples. Variants predicted to be deleterious were selected based on the prediction scores by MetaLR and MetaSVM as these two showed the best performance on curated data sets. We generated radar plots of these selected variants predicted to be deleterious to illustrate samples overall pathogenicity prediction. All 47 samples passed the quality control after sequencing. In summary, a total of 14,655 rare variants with a minor allele frequency of ≤0.01 were identified. Of which, 7,934 were intronic and 5,695 exonic. They included, 198 UTR5, 341 UTR3, 32 frameshift, 3,175 synonymous and 2,448 missense variants. Altogether, 14,057 were single and 598 multiple variants comprising of 261 insertions, 600 deletions and 13,794 substitutions. Altogether 60 variants linked to 44 genes were considered pathogenic based on MetaLR and MetaSVM scores. Altogether 41 of these genes were not previously associated with PD. Seven novel mutations were identified in three known PD genes ATP13A2 (S960R), PRKN (P153R and D245E) and PINK1 (S73L, S228F, S284Y and P305A). Finally, four novel variants were selected for Sanger sequencing, which confirmed the NGS results. In conclusion, the implementation of tNGS and the advanced bioinformatics tools led to identification of novel pathogenic mutations in the Black South African and Nigerian PD patients. Mutations were found in known PD genes and in genes not previously known to harbour mutations in PD patients. Further studies are required to ascertain the biological functions of these mutations. The results could also advance the discovery and the development of improved treatment approaches and drug interventions for PD.

AFRIKAANSE OPSOMMING: Verskeie gene, insluitend PRKN, PINK1, PARK7, ATP13A2, SNCA, LRRK2, VPS35, EIF4G1, CHCHD2 en LRP10 is geassosieer met PS. Die mutasies in hierdie gene bied nuwe geleenthede om die siekte beter te verstaan, deur relevante biologiese roetes uit te lig wat kan bydra tot PS. Die PS gene is hoofsaaklik geïdentifiseer en bestudeer in Europeese, Noord-Amerikaanse, Noord-Afrika en Asiatiese populasies. Dit is moontlik dat die genetiese inligting wat verkry is van hierdie populasies nie van toepassing in bevolkingsgroepe van Afrika suid van die Sahara nie. Die huidige studie het beoog om patogeniese mutaties in Swart Suid-Afrikaanse en Nigeriese PS-pasiënte te identifiseer. Hierdie studie is die eerste gesamentlike PS-genetika studie tussen Suid-Afrika en Nigerië. Daar word voorgestel dat nuwe siekte-veroorsakende gene in hierdie pasiënte gevind sal word. ‘n Somtotaal van 33 Swart Suid-Afrikaanse en 42 Nigeriese PS pasiënte is bestudeer vir genetiese defekte deur middel van verskeie metodes. Die gemiddelde aanvangsouderdom van PS was onderskydelik 48±8 en 65±10 jaar in die Suid-Afrikaners en Nigeriërs. Twee Suid-Afrikaanse pasiënte het ‘n familie geskiedenis van PS. Hierdie studie het eers gesoek vir die teenwoordigheid van eksoniese herrangskikkings soos multiplikasies of delesies in PS-gene. A total van 42 Nigeriese en 12 Suid-Afrikaanse PS pasiënte is bestudeer vir eksoniese herrangskikkings met behulp van ‘n kommmersiële toetsstel. Die resultate dui op drie Nigeriese pasiënte met eksoniese herrangskikkings: (1) heterosigotiese delesie in PARK7 ekson 1; (2) heterosigotiese duplikasies in PINK1 ekson 5 en PRKN ekson 1; en (3) twee heterosigotiese duplikasies in PRKN eksone 1 en 2. Die studie het ook ‘n geteikende volgende-generasie volgordebepaling (VGV) benadering gebruik om te soek vir mutasies in 751 gene wat voorheen geassosieer is met neurologiese siektes, deur middel van die AmpliSeq™ Neurological Disease Panel as ook die Ion Torrent volgordebepalings-platform. VGV is vinnig, opskaalbaar en betroubaar, en kan patogeniese variante ontdek. Vir die VGV deel van die studie is daar DNS monsters van 33 Swart Suid-Afrikaanse en 14 Nigeriëse PS pasiënte gebruik. Bcftools is gebruik om die gehalte van die data te asseseer en om deur die variante te sif. Volgordevariante wat ‘n karteringsgehalte-telling > 100 en ‘n minimum leesdiepte > 40 het was geselekteer vir verdere studie. Ons het annovar sagteware gebruik vir die annotasie. Ons het variante met ‘n allel-frekwensie > 0.01 in enige van die databasisse bewyder. Ons het gekyk na hoe gereeld elke variant in meer as een DNS monster voorgekom het. Patogeniese variante is gekies op grond van MetaLR en MetaSVM tellings, aangesien hierdie twee die beste gedoen het op saamgestelde datastelle. Ons het radarplotte genereer van die geselekteerde patogeniese variante om die algehele patogenesiteit-voorspelling uit te beeld. Al 47 DNS monsters wat gebruik is vir VGV het voldoen aan gehaltebeheer toetse na volgordebepaling. Ter opsomming, 14,655 rare variante is geïdentifiseer met n alleel-frekwensie van ≤0.01. Dit sluit in 32 raamverskuiwing-, 3,175 sinonieme en 2,448 nie-sinonieme variante. Altesaam was daar 14,057 enkelbasis variante en 598 multibasis variante, insluitende 261 insersies, 600 delesies en 13,794 substitusies. ‘n Totaal van 60 variante in 44 verskillende gene was as patogenies aanskou na aanleiding van MetaLR en MetaSVM tellings. Sewe nuwe mutasies is geïdenfiseer in drie bekende PS gene, AP13A2 (S960R), PRKN (P153R en D245E) en PINK1 (S73L, S228F, S284Y en P305A). Vier van die nuwe gene was gekies vir Sanger volgordebepaling, wat die VGV resultate bevestig het. Ter slotte, the soektog vir genetiese herrangskikkings en die implementering van VGV tesame met gevorderde bioinformatika hulpmiddels het gelei tot die identifisering van nuwe patogeniese mutasies in Swart Suid-Afrikaanse en Nigeriese PS pasiënte. Mutasies is gevind in beide bekende PS gene en in gene waarin PS mutasies nog nie voorheen beskryf is. Verdere studies word benodig om die biologiese funksies van hierdie mutasies vas te stel. Die resultate kan ook bydra tot die ontdekking en ontwikkeling van beter behandeling-benaderinge en medikasies vir PS.

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