Development of a custom-designed targeted resequencing gene panel for Parkinson’s disease

Date
2020-03
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : Stellenbosch University
Abstract
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.
AFRIKAANSE OPSOMMING: Parkinson se siekte (PS) is ʼn komplekse neurodegeneratiewe siekte wat gekenmerk word deur die verlies van dopaminergiese neurone in die substantia nigra van die brein. Selektiewe verlies van díe neurone lei tot motoriese afwykings, ook bekend as die kardinale simptome van PS, onder andere bewerigheid, rigiditeit, traer bewegings en posturale instabiliteit. Daarbenewens, het PS ook nie-motoriese simptome soos bui versteurings, kognitiewe afname, slaapafwykings, verlies van reuk en laastens gastrointestinale- en outonomiese versteurings tot gevolg. Tot op hede betaan daar geen kuur vir PS nie en verder bly die onderliggende patobiologie van PS steeds onbekend. Omtrent 90% van PS gevalle is idiopaties en word voorgestel as die gevolg van ʼn komplekse interaksie tussen omgewings- en genetiese faktore. Die oorblywende 10% van PS gevalle word as monogenies gedefinieer en volg ‘n Mendeliese oorerwingspatroon. Sedert die ontdekking van die eerste monogeniese PS-veroorsakende geen, naamlik SNCA, is daar al talle gene ontdek en bestudeer in Europese en Asiatiese populasies. Dit sluit in, ATP13A2, CHCHD2, DJ-1, DNAJC13, DNAJC6, EIF4G1, FBXO7, GBA, GCH1, GIGYF2, HTRA2, LRRK2, PINK1, PLA2G6, PRKN, RAB39B, RIC3, SLC6A3, SNCA, SYNJ1, TMEM230, VPS13C en VPS35. Baie van die gene is egter nog nie intensief in die sub-Sahariese Afrika (SSA) populasies bestudeer nie. Verder, het studies wat wel al sommige van die gene bestudeer het van eerste generasie volgorde-bepaling tegnologieë, soos Sanger volgorde-bepaling, gebruik gemaak. Dit behels tegnieke wat die sifting van slegs ʼn enkele area of mutasie op ʼn slag kan handhaaf. In meer onlangse studies word daar van volgende generasie volgorde-bepaling (“NGS-sequencing”) geen panele gebruik gemaak, om al die reeds bekende PS gene in pasiënte met vroeë aanvang en positiewe familie geskiendenis, wat dui op genetiese oorerwing as oorsaak van PS, te ondersoek. Die eerste doelwit van ons studie was dus die ontwerp van ʼn gespesialiseerde volgende generasie volgorde-bepaling geen paneel vir die spoedige sifting van die reeds bekende PS-veroorsakende gene in die Suid-Afrikaanse populasie. Die geen paneel was ontwerp deur van Agilent SureSelect Target Enrichment tegnologie gebruik te maak en het al 23 van die bogenoemde PS gene bevat. Daarna was ʼn siftingstoets, op 32 vroeë aanvang PS pasiënte met ʼn familie geskiedenis van PS, in twee aparte volgorde-bepaling lopies gedoen. ʼn Dekking van >200x vir beide lopies was bereik na die ontleding van die volgorde-bepaling data. Die eerste lopie het die insluiting van positiewe kontroles met reeds bekende patogeniese enkel-nukleotied mutasies, ʼn 40bp delesie en kopie nommer mutasies behels. Al die mutasies van die positiewe kontroles, behalwe die kopie nommer mutasies, was geverifieer. ʼn Gemiddeld van 79 variante per pasiënt was opgespoor waarvan slegs 15 geprioritiseer was op grond van vorige assosiasie met PS, skaarsheid (MAF <0.01), nuutheid en of dit deur die meerderheid van die in-siliko gereedskap voorspel was om skadelik te wees. Hierdie variante was ook verder volgens die American College Medical Genetics and Genomics (ACMG) aanbevelings geklassifiseer om die kliniesie belang daarvan te evalueer. Slegs ses het aan die kriteria vir patogenies GBA (p.L483P [p.L444P]) of waarskynlik patogenies (GBA p.R170L [p.R131L], p.D179H [p.D140H], p.E365K [p.E326K], PINK1 p.P305A, en PRKN p.E310D) voldoen. Een van die hoof genetiese risiko faktore vir PS en ʼn reeds bekende oorsaak van Gaucher se siekte wat by die geen panel ingereken was, is GBA wat ook ʼn pseudogeen naamlik GBAP1 het, met ʼn volgorde oorkeenkoms van 96%. Gespesialiseerde oligonukleotied eksemplare wat lei tot die amplifikasie van slegs GBA was dus gebruik om sodoende die opspoor van variante in die nie-funksionele pseudogeen te vermy. Die tweede doelwit van ons studie was dus om ʼn metode vir die sifting en validasie van GBA mutasies in ons laboratorium te ontwerp. Na die suksesvolle optimalisering van die gemodifiseerde polimerasie kettingreaksie- en Sanger volgorde-bepaling metodes, was sifting op 30 van ons swart Afrika PS pasiënte toegepas. Tot ons wete was dit die eerste sifting van GBA mutasies in PS wat in die populasie uitgevoer is. Agt variante, waarvan vier deur die meerderheid van in-siliko gereedskap as skadelik voorspel was, is geïndentifiseer. Dit sluit in drie reeds bekende patogeniese Gaucher se siekte-geassosieerde mutasies (p.R150W [p.R120W], p.R170L [p.R131L], and p.T75del [p.T36del]), waarvan p.R120W bekend is as ʼn risiko faktor vir PS, asook een variant met onbekende beduidenis (p.Q536* [p.Q497*]). Boonop was twee nuwe variant bevindings (p.F255L [p.F216L] and p.G517R [p.G478R]) geïdentifiseer waarvan p.F216L gereeld (9.9%) in die kontroles voorgekom het. Verder was die gespesialiseerde GBA oligonukleotied eksemplare ook gebruik om al die geprioritiseerde GBA variante, wat deur die geen paneel geïdentifiseer is, te verifieer. Slegs vier van die vyf variante (GBA p.L483P, p.R170L, p.D179H, p.E365K) is bevestig om in die funksionele geen voor te kom. Ter afsluiting, het ons ʼn suksesvolle metode vir die spoedige sifting van die reeds bekende PS gene asook ʼn tegniek vir die sifting en verifikasie van GBA mutasies ontwerp. Hierdie metodes kan gebruik word vir spoedige en hoë-deursettingsvermoë sifting van die genetiese bydrae tot PS in ons plaaslike populasies asook ander populasies in SSA. Die metodes sal ons dus in staat stel om nuwe kandidate te identifiseer wat gebruik kan word vir verifikasie werk in toekomstige funskionele studies. Gevolglik kan die werk ook bydra tot die ontwikkeling van presisie-medikasie wat aangepas word tot elke PS pasiënt.
Description
Thesis (MSc)--Stellenbosch University, 2020.
Keywords
South African patients, Parkinson's disease, Gene targeting, Neurodegenerative disease
Citation