Genomic rearrangements (exon dosage) are common mutations reported in Parkinson's disease (PD) patients. In the present study, we aimed to investigate the prevalence of genomic rearrangements in 88 ...South African patients with predominantly early-onset PD (age-at-onset ≤50 years). The multiplex ligation-dependent probe amplification method was used to detect exon dosage changes. Two commercially available probe kits, SALSA P051 and P052, were used and together the kits consisted of probes for exons of
α-synuclein
,
parkin, PINK1, DJ-1, LRRK2, UCH-L1, ATP13A2, LPA, TNFRSF9, CAV2, CAV1, GCH1,
and two-point mutations. We identified exonic rearrangements in
parkin
and
α-synuclein
in 8% of South African patients from different ethnic groups. One patient had a whole-gene triplication of
α-synuclein
; representing only the fourth family with this mutation reported to date. We found six patients with
parkin
mutations who had either heterozygous duplications and deletions, or homozygous deletions. A false positive result of an exonic deletion detected in one patient turned out to be homozygous point mutation (Y258X) in
PINK1
. No exonic rearrangements were found in four of the PD genes;
LRRK2, PINK1, DJ-1,
and
ATP13A2
. Mutations in
parkin
were the predominant genetic cause; however, the frequency of exon dosage in our study group is low compared with previous studies. This indicates the possible involvement of other as yet unidentified PD genes in the development of the disease in the South African population.
► One known homozygous mutation (Y258X) was identified. ► Two heterozygous missense variants (P305A and E476K) was identified. ► Thirteen polymorphisms of which five were novel were identified. ► No ...homozygous exonic deletions were detected. ► PINK1 is not a common cause of Parkinson’s disease in the South African population.
Mutations in the PINK1 gene are the second most common cause after parkin of autosomal recessive early-onset Parkinson’s disease (PD). PINK1 is a protein kinase that is localized to the mitochondrion and is ubiquitously expressed in the human brain. Recent studies aimed at elucidating the function of PINK1, have found that it has neuroprotective properties against mitochondrial dysfunction and proteasomally-induced apoptosis. In the present study, we aimed to investigate the prevalence of PINK1 genetic variants in 154 South African PD patients from all ethnic groups. Mutation screening was performed using the High-Resolution Melt technique and direct sequencing. A total of 16 sequence variants were identified: one known homozygous mutation (Y258X), two heterozygous missense variants (P305A and E476K), and 13 polymorphisms of which five were novel. No homozygous exonic deletions were detected. The novel P305A variant was found in a female patient of Black Xhosa ethnicity who has a positive family history of the disease and an age at onset of 30years. This variant has the potential to modulate enzymatic activity due to its location in the kinase domain. This is the first report on mutation screening of PINK1 in the South African population. Results from the present study showed that point mutations and homozygous exonic deletions in PINK1 are not a common cause of PD in the South African population.
Assuming that a significant cause of Parkinson’s disease (PD) is genetic, genetic factors have been shown to account for <10% of all PD cases to date, and it is therefore necessary to identify novel ...genes. The aim of the present study was to identify PD candidate genes using a bioinformatic approach and to screen them for possible PD-causing mutations. The CAESAR (CAndidatE Search And Rank) program was used in the present study to identify and prioritize PD candidate genes. CAESAR ranks annotated human genes as candidates by using ontologies to semantically map natural language descriptions of the trait under investigation to gene-centric databases. Two of the candidates were selected and screened for mutations in 202 South African PD patients using the High-Resolution Melt (HRM) method. Samples exhibiting altered HRM profiles were sequenced. CAESAR generated a prioritized list of candidates including both known and novel PD genes. The
MAPT
and
SNCAIP
genes were selected for mutation screening from the list of ten highest scoring genes. Two novel missense (A91V and V635I), four synonymous and three intronic sequence variants were identified in
MAPT
. For
SNCAIP
, three novel missense (T383N, R606Q, N906H), one known (E709Q), four synonymous and one intronic sequence variant were found. A bioinformatic approach was used to aid in the identification and selection of PD candidate genes in a group of South African patients. Mutation screening of
MAPT
and
SNCAIP
identified novel sequence variants in both genes and further studies are necessary to determine their possible functional consequences.
Abstract The molecular basis of Parkinson's disease (PD) has been extensively studied in numerous population groups over the past decade. However, very little is known of the molecular etiology of PD ...in the South African population. We aimed to assess the genetic contribution of parkin mutations to PD pathology by determining the frequency of both point mutations and exon rearrangements in all 12 exons of the parkin gene in a group of 229 South African patients diagnosed with PD. This was done by performing high resolution melt (HRM) as well as multiplex ligation-dependent probe amplification (MLPA) analyses. In total, seven patients (3.1%; 7/229) had either compound heterozygous or homozygous mutations in parkin , and seven patients (3.1%) had heterozygous sequence variants. Two of the patients with parkin mutations are of Black African ancestry. Reverse-transcription PCR on lymphocytes obtained from two patients verified the presence of parkin mutations on both alleles. In conclusion, the present study reveals that mutations in the parkin gene are not a major contributor to PD in the South African population. Further investigations of the molecular etiology of PD in the unique South African population, particularly the Black African and mixed ancestry sub-populations, are warranted.
DJ-1 forms part of the neuronal cellular defence mechanism against oxidative insults, due to its ability to undergo self-oxidation. Oxidative stress has been implicated in the pathogenesis of central ...nervous system damage in different neurodegenerative disorders including Alzheimer's disease and Parkinson's disease (PD). Various mutations in the DJ-1 (PARK7) gene have been shown to cause the autosomal recessive form of PD. In the present study South African PD patients were screened for mutations in DJ-1 and we aimed to investigate the functional significance of a novel 16 bp deletion variant identified in one patient.
The possible effect of the deletion on promoter activity was investigated using a Dual-Luciferase Reporter assay. The DJ-1 5'-UTR region containing the sequence flanking the 16 bp deletion was cloned into a pGL4.10-Basic luciferase-reporter vector and transfected into HEK293 and BE(2)-M17 neuroblastoma cells. Promoter activity under hydrogen peroxide-induced oxidative stress conditions was also investigated. Computational (in silico) cis-regulatory analysis of DJ-1 promoter sequence was performed using the transcription factor-binding site database, TRANSFAC via the PATCH and rVISTA platforms.
A novel 16 bp deletion variant (g.-6_+10del) was identified in DJ-1 which spans the transcription start site and is situated 93 bp 3' from a Sp1 site. The deletion caused a reduction in luciferase activity of approximately 47% in HEK293 cells and 60% in BE(2)-M17 cells compared to the wild-type (P < 0.0001), indicating the importance of the 16 bp sequence in transcription regulation. The activity of both constructs was up-regulated during oxidative stress. Bioinformatic analysis revealed putative binding sites for three transcription factors AhR, ARNT, HIF-1 within the 16 bp sequence. The frequency of the g.-6_+10del variant was determined to be 0.7% in South African PD patients (2 heterozygotes in 148 individuals).
This is the first report of a functional DJ-1 promoter variant, which has the potential to influence transcript stability or translation efficiency. Further work is necessary to determine the extent to which the g.-6_+10del variant affects the normal function of the DJ-1 promoter and whether this variant confers a risk for PD.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics. Medical Biochemistry))--University of Stellenbosch, 2007.
Connexins (Cx) are major proteins of gap junctions, dynamic ...pores mediating the relay of ions and metabolites between cells. Cxs 40, 43 and 45 are the predominant cardiac isoforms and their distinct distribution raises questions about their functional differences. Their cytoplasmic (C)-terminal domains are involved in protein-protein interactions. Furthermore, mutations in the myotonic dystrophy protein kinase (DMPK)-causative gene are associated with disruptions in cardiac conduction similar to that described for Cx knock-out mice. DMPK is a Cx43 ligand, raising the possibility that defects in Cx40 ligands may be involved in the development of cardiac conduction disturbances. We hypothesised that delineation of the protein ligands of the C-termini of Cx40 and of Cx45 (parallel study conducted by N Nxumalo) would help elucidate their functional roles.;
Yeast-two-hybrid methodology was used to identify putative Cx40 ligands. Primers were designed to amplify the C-terminus-encoding domain of the human Cx40 gene (Cx40), the DNA product was cloned into the pGBKT7 vector which was used to screen a cardiac cDNA library in Saccharomyces cerevisiae. Successive selection stages reduced the number of putative Cx40 ligand-containing colonies (preys) from 324 to 33. The DNA sequences of the 33 ligands were subjected to BLAST-searches and internet database literature searches to assign identity and function and to exclude false positive ligands based on subcellular location and function. Eleven plausible ligands were identified: cysteine-rich protein 2 (CRP2), beta-actin (ACTB), creatine kinase, muscle type (CKM), myosin, heavy polypeptide 7 (MYH7), mucolipin1 (MCOLN1), voltage-dependent anion channel 2 (VDAC2), aldehyde dehydrogenase 2 (ALDH2), DEAH box polypeptide 30 (DHX30), NADH dehydrogenase, 6, (NDUFA6), prosaposin (PSAP) and filamin A (FLNA). Cxs 40 and 45 showed differences in the classes of proteins with which they interacted; the majority of putative Cx40 interactors were cytoplasmic proteins, while Cx45 interactors were mitochondrial proteins. These results suggest that Cxs 40 and 45 are not only functionally different, but may also have different cellular distributions. Further analyses of these protein interactions will shed light on the independent roles of Cxs 40 and 45.
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
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