Thesis (PhD )--University of Stellenbosch, 2011. / 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. / AFRIKAANSE OPSOMMING: Parkinson se siekte (PS), ‘n neurodegeneratiewe bewegings-siekte, gekarakteriseer deur rustende
spiersametrekkings, bradykinesia, posturale onstabiliteit en rigiditeit, onstaan as gevolg van
geselekteerde verlies van dopaminergiese neurone in die substantia nigra. Nie-motoriese
simptome sluit in outonome, kognitiewe en psigiatriese afwykings. Dit is voorgestel dat PS
ontwikkel as gevolg van omgewings- en genetiese faktore of ‘n kombinasie van die twee. Daar
was ‘n toename in bewyse vir die verantwoordelikheid van die genetiese komponent oor die
afgelope 13 jaar. Mutasies in die parkin, PINK1, DJ-1, ATP13A2, SNCA, en LRRK2 gene word
met PS geassosieer.
Die doel van hierdie studie was om vir die eerste keer die genetiese etiologie van PS in Suid-
Afrikaanse pasiënte te ondersoek. Pasiënte van die verskillende Suid-Afrikaanse etniese groepe,
met hoofsaaklik vroeë-aanvang PS en/of ‘n positiewe familie-geskiedenis, was gebruik.
Wisselende getalle van studie-deelnemers (van 88-205) was gebruik vir die verskillende dele van
die studie, afhangende van hul beskikbaarheid op die tyd van die eksperimente en die spesifieke
kliniese kriteria wat van toepassing was. Mutasie-analiese was uitgevoer deur middel van Hoëresolusie
smelting (HRS)-analiese, DNS volgorde-bepaling en multipleks ligasie-afhanklike
‘probe’ amplifikasie (MLPA).
HRS-analiese en DNS volgorde-bepaling van die bekende PS gene het die volgende mutasies
deïdentifiseer: parkin (T113fsX163), PINK1 (Y258X), en LRRK2 (G2019S en R1441C).
Haplotiepe-analiese het gevind dat vyf Suid-Afrikaanse LRRK2 G2019S patiente ‘n
gemeenskaplike voorvader deel met ander wêreldwyd gerapporteerde LRRK2 haplotiepe 1-
geassosieerde families.
Twee kommersieel beskikbare MLPA ‘kits’, SALSA P051 en P052, was gebruik om die
deelnemers te toets vir exon-dosis mutaties. Exon-delesies en invoegings in parkin was gevind in
vyf patiente. ‘n Familie met ‘n volle geen triplikasie van SNCA was gevind. Dit is die 4de familie
wêreldwyd wat die spesifieke mutasie het en dit lei tot ‘n erge fenotiepe met outonomiese
afwykings en vroeë-aanvang dementia.
Die ‘CAESAR (CAndidatE Search And Rank)’ bioinformatiese program was gebruik om nuwe
kandidaat PS gene te selekteer. Die program het ‘n lys kandidaat gene, wat beide bekende geassosieerde en nuwe bevat, opgelewer. Die MAPT en SNCAIP gene was gekies uit tien gene
met die hoogste tellings. HRS analiese het nuwe DNS volgorde variante in beide gene gevind.
Die funksies van die variante is tans onbekend en moet verder ondersoek word.
‘n Onbekende 16bp delesie (g.-6_+10del) in die promotor area van DJ-1 was gevind in een PS
patient. ‘n Dubbel-lusiferase rapporteerder eksperiment was uitgevoer om die funksie van die
variant te ondersoek. Die variant het die lusiferase-aktiwiteit aansienlik verlaag in twee
afsonderlike sel lyne, HEK293 en BE(2)-M17 neuroblastoma selle, met en sonder oksidatiewe
spanning (p<0.0001). Dit was voorgestel dat die 16bp volgorde dalk belangrik kan wees vir
transkripsionele regulasie van DJ-1. Die variant mag dalk ook die aktiwiteit van drie transkripsie
faktore (AhR, ARNT and HIF-1) met bindings plekke in die delesie- volgorde, beïnvloed.
Ter afsluiting, mutasie analiese het gelei tot die identifikasie van mutasies in ses patiente in
parkin, ses patiente in LRRK2, een patient in PINK1 en een patient in SNCA. ‘n Aantal nuwe
variante was gevind met ombekende funksies. Ondersoek van die genetiese basis van PS in die
uniek Suid-Afrikaanse etniese groepe het gevind dat die bekende PS gene nie ‘n groot rol speel
in die ontwikkeling van die siekte in die populasie nie. Dit is moontlik dat ander onbekende PS
gene hier verantwoordelik is vir die siekte. Dit is dus belangrik om innoverende bioinformatiese
en eksperimentele strategieë toe te pas om nuwe kandidaat-gene, vir PS, te identifiseer. / University of Stellenbosch / Agence Nationale de la Recherche, France / South African Medical Research Council / Doris Crossley Foundation
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/6563 |
| Date | 03 1900 |
| Creators | Keyser, Rowena J. |
| Contributors | Bardien, S., Carr, J., University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xii, 174 p. : ill., mostly col. |
| Rights | University of Stellenbosch |
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