Thesis (MScMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Parkinson's disease (PD) is a severely debilitating neurodegenerative disorder that results in
motor circuit dysregulation and ultimately, causes impairment of movement. This condition
is due to the selective degradation of the dopaminergic neurons in the substantia nigra pars
compacta in the midbrain, which subsequently results in the pathological symptoms namely
bradykinesia, resting tremor, postural instability and rigidity. It was initially hypothesized
that individuals who develop PD were exposed to an environmental trigger(s) that caused the
onset of the disease, but more recently, a significant genetic component, coupled to
environmental factors have been implicated in disease pathogenesis. Currently, there are
eight genes (Parkin, PINK1, LRRK2, SNCA, DJ-1, ATP13A2, EIF4G1 and VPS35) that have
been directly implicated in PD.
Worldwide, the prevalence of neurodegenerative disorders is increasing as populations are
living longer. In Europe, Canada and USA, it has been projected that the prevalence of PD
may increase by a factor of two between 2010 and 2050; approximately a 92% increase. In
Tanzania (the only study done in sub-Saharan Africa) an even larger increase of 184%
between 2005 and 2025 is predicted, due to the fact that the speed of populations ageing in
developing countries, will exceed that of developed countries. Research into the causes and
risk factors underlying neurodegenerative disorders such as PD is therefore urgently needed
for policy makers and governments in developing nations to take appropriate action to deal
with this impending health care problem.
The aim of the present study was to investigate the molecular aetiology of a group of South
African PD patients. A total of 262 patients from various ethnic backgrounds were recruited
for the study, and 35% had a positive family history of PD with the average age at onset
(AAO) being 54.3 years of age (SD = 12.5 years). Mutation screening of the known PD
genes (Parkin, PINK1, LRRK2, SNCA and DJ-1) was performed using high resolution melt
and Sanger sequencing. Genotyping was done using fluorescently-labelled PCR primers
followed by electrophoresis on an ABI 3130xl genetic analyser (for CTG repeats in JPH3)
and with a KASP™ Genotyping Assay (for a 16bp indel in DJ-1). In order to identify a
novel PD-causing gene, whole exome sequencing (WES) was conducted on three Afrikaner
probands with an Illumina Genome Hiseq 2000TM and the sequences were aligned using the NCBI Human Reference Genome 37.2. The BORG (Bio-Ontological Relationship Graph)
semantic database, which models the relationship of human and model organism genes to
functions, pathways and phenotypes, was used to filter and prioritise genetic variants shared
between the three PD exomes.
It was determined that the known PD genes do not play a significant role in disease
pathogenesis in the South African patients as only 15/262 (5.7%) of the patients harboured
mutations: seven in Parkin, one in PINK1, six in LRRK2 and one in SNCA. Only one of the
patients harboured a 16bp indel variant at the transcription start site of DJ-1. None of the
Black PD patients had pathogenic repeat expansions in JPH3 thereby excluding Huntington
disease-like 2 as a cause of the disease phenotype.
Genealogical analysis revealed that six of the apparently unrelated Afrikaner PD probands
were related to a founder couple that immigrated to South Africa in the 1600s which suggests
that there is a possible founder effect for the disease. Bioinformatics analysis of WES data
on three of the probands identified 21 variants in 12 genes that were present in all three PD
exomes and fulfilled various criteria. Sanger sequencing was used for verification of five
variants and of these, two (in CDC27 and NEDD4) were found to be artefacts. The
remaining three (in HECDT1, TBCC and RNF40) were excluded based on the lack of cosegregation
with disease and the high frequency of the allele in controls. Further work is
necessary to verify the presence of the remaining sixteen variants and to characterise each of
them for their possible pathogenicity.
The discovery of novel PD-causing genes is important as this may shed light on the pathways
or processes that are involved. A current hypothesis implicates the lysosome-dependent
pathway as a unifying biochemical pathway that can account for the phenotypic spectrum
within PD. Notably, although Mendelian forms are thought to account for only about 10-
15% of cases, the study of Mendelian inherited variants is likely to provide insight into the
pathophysiology of the more common sporadic form of this condition. Dissecting the key
molecular mechanisms underlying PD will provide critical information for improved
treatment strategies and drug interventions that will ultimately prevent or halt neuronal cell
loss in susceptible individuals. / AFRIKAANSE OPSOMMING: Parkinson se siekte (PS) is 'n erge neurodegeneratiewe bewegings-siekte, wat motorstroombaan
disregulasie veroorsaak. Dit lei uiteindelik tot beperkte bewegings vermoëns. Hierdie toestand
word veroorsaak weens die selektiewe agteruitgang van die dopaminergeniese neurone in die
substantia nigra pars compacta in die midbrein, wat later lei tot die patologiese simptome
naamlik: bradykinesia, rustende spiersametrekkings, posturale onstabiliteit en rigiditeit. Daar is
aanvanklik vermoed dat individue wat PS ontwikkel, aan 'n omgewingsfaktor(e) blootgestel is
wat die aanvang van die siekte veroorsaak het, terwyl meer onlangs is daar 'n aansienlike
genetiese komponent tesame met omgewingsfaktore geïdentifiseer, wat betrokke is by die
patogenese van die siekte. Tans is daar agt gene (Parkin, PINK1, LRRK2, SNCA, DJ-1,
ATP13A2, EIF4G1 en VPS35) wat direk by PS geïmpliseer is.
Wêreldwyd is daar ‗n toenemende voorkoms van neurodegeneratiewe siektes aangesien
bevolkings langer leef. In Europa, Kanada en die VSA, is daar geprojekteer dat die voorkoms
van PS tussen 2010 en 2050 met 'n faktor van twee verhoog kan word. Dit is ongeveer 'n 92%-
verhoging. In Tanzanië (die enigste studie wat tot dusver in sub-Sahara Afrika gedoen is) word
daar selfs ‗n groter toename, van 184% tussen 2005 en 2025 voorspel. Dit is te danke aan die feit
dat die bevolkings- veroudering in ontwikkelende lande die van ontwikkelde lande sal oorskry.
Ondersoeke na die oorsake en risiko-faktore onderliggend aan neurodegeneratiewe siektes,
byvoorbeeld PS, word dus dringend benodig deur beleidmakers en regerings in ontwikkelende
lande, sodat hulle die nodige stappe kan neem om hierdie dreigende gesondheidsorg-probleem op
te los.
Die doel van die huidige studie was om ondersoek in te stel na die molekulêre etiologie van 'n
groep Suid-Afrikaanse PS pasiënte. 'n Totaal van 262 pasiënte van verskillende etniese
agtergronde, is gewerf vir die studie. Hiervan het 35% 'n positiewe familiegeskiedenis van PS en
die gemiddelde aanvangs ouderdom (AAO) was 54,3 jaar (SD = 12,5 jaar). Mutasie-analise van
die bekende PS gene is uitgevoer met behulp van hoë resolusie smelt en Sanger
volgordebepaling. Genotipering is gedoen met behulp van fluoresserend geëtiketteerde PKR
inleiers met elektroforese, op 'n ABI 3130xl genetiese analiseerder (CTG herhalings in JPH3), en
met 'n KASP ™ Genotipering toets (vir 'n 16bp indel in DJ-1). Ten einde, om 'n nuwe PSveroorsakende
geen te identifiseer was heel eksoom volgordebepaling (WES) uitgevoer op drie
Afrikaner PS positiewe pasiënte met 'n Illumina Genome Hiseq 2000™ en die volgorders is gerangskik met behulp van die NCBI Menslike Verwysings Genoom 37.2. Die BORG (Bio-
Ontologiese Verhoudings Grafiek) semantiese databasis, wat gebaseer is op die verhouding van
die mens en model organisme gene funksies, paaie en fenotipes, en is gebruik om genetiese
variante, wat gedeel word tussen die drie PS exome te filtreer en te prioritiseer.
Daar is vasgestel dat die bekende PS gene nie 'n belangrike rol in die patogenese van die siekte
in die Suid-Afrikaanse pasiënte speel nie. Dit is aangesien slegs 15/262 (5.7%) van die pasiënte
bekende mutasies dra: sewe in Parkin, een in PINK1, ses in LRRK2 en een in SNCA. Slegs een
van die pasiënte het 'n 16bp delesie variant in die transkripsie promotor area van DJ-1 gedra.
Geen van die Swart PS pasiënte het patogeniese herhalings in JPH3 vertoon nie. Gevolglik is
Huntington siekte-agtige 2 uitgesluit as 'n oorsaak van die siekte fenotipe.
Genealogiese analise het getoon dat ses van die skynbaar onverwante Afrikaner PS pasiënte
verwant is aan 'n stigter paartjie wat in die 1600's na Suid-Afrika geïmigreer het, wat daarop dui
dat daar 'n moontlike stigter effek vir die siekte is. Bioinformatiese analise van WES data vir drie
van die pasiënte, het 21 variante in 12 gene geïdentifiseer, wat in al drie PS exome teenwoordig
was en verskeie kriteria vervul het. Sanger volgordebepaling is gebruik vir die bevestiging van
vyf variante en van hierdie, is twee (in CDC27 en NEDD4) bevind om artefakte te wees. Die
oorblywende drie (in HECDT1, TBCC en RNF40) is uitgesluit gebaseer op die gebrek aan
gesamentlike-segregasie met die siekte en die hoë frekwensie van die allele in die kontrole groep.
Verdere werk is nodig om die teenwoordigheid van die oorblywende variante te verifieer en om
elkeen van hulle te karakteriseer vir hulle moontlike patogenisiteit.
Die ontdekking van die nuwe PS-veroorsakende gene is belangrik aangesien dit lig kan werp op
die stelsels of prosesse wat betrokke is. 'n Huidige hipotese impliseer die lisosoom-afhanklike pad
as 'n verenigende biochemiese padweg, wat verantwoordelik is vir die fenotipiese spektrum binne
PS. Alhoewel Mendeliese vorms vermoedelik verantwoordelik is vir slegs omgeveer 10-15% van
die gevalle, is die studie van Mendelse gene geneig om insig te verkry in die patofisiologie van
die meer algemene sporadiese vorm van hierdie toestand. Ontleding van die kern molekulêre
meganismes onderliggend aan PS sal kritiese inligting vir beter strategieë vir behandeling en
geneesmiddel-intervensies voorsien, wat gevolglik neuronale sel verlies in vatbare individue sal
voorkom of beëindig. / Medical Research Council / National Research Foundation / Harry Crossley Foundation
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/79831 |
| Date | 03 1900 |
| Creators | Glanzmann, Brigitte |
| Contributors | Bardien, Soraya, Carr, Jonathan, Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Molecular Biology and Human Genetics. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xix, 202 p. : ill., (some col. ) |
| Rights | Stellenbosch University |
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