Spelling suggestions: "subject:"dissertations -- human genetics"" "subject:"dissertations -- suman genetics""
1 |
A molecular investigation of a mixed ancestry family displaying dementia and movement disordersAbrahams-Salaam, Fatima 12 1900 (has links)
Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics))--Stellenbosch University, 2008. / A South African family of Mixed Ancestry presented with a rapidly progressive dementia and a
movement disorder which affected a number of individuals across three generations. The initial
symptoms included personality changes and tremors that escalated to severe dementia and
eventually a completely bedridden state. It was determined that the mean age at onset was in the
third decade of life and affected individuals died within 10-15 years after the onset of symptoms.
The aim of the present study was to elucidate the genetic cause of the disorder in this family and to
further investigate the patho-biology of the disease.
Mutations that could possibly cause the observed phenotype in this family were screened for. These
included loci implicated in Huntington’s disease, Parkinson’s disease, Dentatorubral-Pallidoluysian
Atrophy, Spinocerebellar ataxias (types 1, 2, 3, 6, and 7), Huntington’s disease-like 2 (HDL2) and
several mitochondrial disorders. Single-strand Conformation Polymorphism (SSCP) analysis and
direct sequencing were used to detect possible mutations while genotyping on an ABI genetic
analyser was used to detect disorders caused by repeat expansions. Haplogroup and Short Tandem
Repeats (STRs) analyses of the Y-chromosome and mitochondrial DNA of one affected family
member was used to determine the family’s genetic ancestry. Reverse transcriptase polymerase
chain reaction (RT- PCR) and complementary DNA (cDNA) analyses of the Junctophlin-3 (JPH3)
gene was performed to provide information on the expression profile of this gene.
After the exclusion of several genetic loci it was shown that this family had HDL2. This is a rare
disease caused by a CAG/CTG repeat expansion in an alternatively spliced version of the JPH3
gene. HDL2 occurs almost exclusively in individuals of Black African ancestry. The genetic ancestry
data suggested that the family member was most likely of South African Mixed Ancestry making this
the first reported family of South African Mixed Ancestry with HDL2. A pilot study investigated the
repeat distribution amongst three South African sub-populations in order to determine whether there
was a bias in the repeat distribution that possibly predisposes Black Africans to develop the disease.
The results showed a statistically significant difference (P= 0.0014) in the distribution of the repeats
between the Black African and Caucasian cohorts. However, no conclusions could be drawn as to
whether Black Africans harboured larger repeats that predisposes them to developing HDL2.
The expanded repeat is located in an alternatively spliced version of the JPH3 mRNA. Interestingly,
this repeat is not present in the mouse homologue of the gene although the rest of the genomic
sequence is highly conserved across the human, mouse and chimpanzee genomes. Using foetal
brain cDNA and PCR primers designed to be specific for different JPH3 isoforms, independent
confirmation of the presence of two JPH3 mRNA transcripts (the full length and a shorter alternatively
spliced version) was provided. In the absence of brain tissue from an HDL2-affected individual, it was
investigated whether both JPH3 mRNA transcripts could be detected in lymphocytes. Using RNA
isolated from the transformed lymphocytes of two HDL2-affected family members, real-time PCR
was attempted. These experiments produced inconclusive results and required further optimisation.
Further RT-PCR experiments for JHP3 expression in different tissues (brain and other) obtained
from HDL2-affected individuals would be of interest.
The present study identified the first Mixed Ancestry family with HDL2. This family will now be able
to request genetic counselling and pre-symptomatic testing for all at-risk family members. Aspects of
this study provided independent confirmation of characteristics of the mutated gene. More research
on HDL2 will be crucial in understanding the pathogenesis of this disease.
|
2 |
Differential expression of genes in clinical strains of mycobacterium tuberculosis in response to isoniazidSeepe, Prudy Mashika 03 1900 (has links)
Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Isoniazid forms part of the first line anti-tuberculosis therapy and it is generally used to treat latent Mycobacterium tuberculosis infection. Isoniazid inhibits synthesis of long chain mycolic acids found in cell wall of Mycobacterium tuberculosis, which have proven vital for the survival of the bacterium. Mycolic acids are primarily synthesized by the fatty acid synthase enzyme (FAS) system found in mycobacteria as the FAS-I and FAS-II complex. Isoniazid kills the bacteria by blocking the FAS-II complex, required for extension of mycolates. It does this by entering the tubercle bacilli as a prodrug where isoniazid becomes activated by catalase peroxidase encoded by katG gene. The activated isoniazid then forms a complex with NAD+ which targets InhA (NADH-dependent enoyl-acyl carrier protein reductase) located in the FAS-II complex. Loss of catalase peroxidase, due to gene mutations or a complete katG gene deletion is one of the primary mechanisms conferring resistance to INH in Mycobacterium tuberculosis. In addition, four other genes (inhA, KasA, ndh and ahpC) are also associated with INH resistance. Nonetheless, mutations in these five genes are present in only 70-80% of INH resistant clinical isolates, implying that other mechanisms are involved in resistance of Mycobacterium tuberculosis to isoniazid.
This study aims to quantify the expression level of genes induced by isoniazid in the mycolic acid pathway and drug transport in two closely related Mycobacterium tuberculosis Beijing cluster 208 isolates. These are the fully susceptible (K636) and isoniazid mono-resistance strains (R55), with minimum inhibitory concentrations of 0.1 and 4 µg/ml, respectively. Both these isolate had no isoniazid gene associated mutations. The isolates were cultured in the presence and absence of 0.1µg/ml isoniazid for 24 hours after which RNA was extracted followed by QRT-PCR analysis to identify differentially expressed genes. This result has shown that various genes were differentially expressed in response to low level INH exposure. The most significant up-regulation was observed in genes (acpM, fabD, Accd6 and fbpC) encoding the FAS-II complex and genes (efpA, iniA, iniB, and mmpl7) involved in drug transport. In addition, two genes (ndh and fbpC) were significantly down-regulated in the isoniazid mono-resistant isolate. Based on these findings, we propose a model whereby isoniazid exposure in the susceptible isolate inhibits FAS-II complex and with its associated accumulation in mycolates kills the bacterium. In contrast, we propose that in the resistance isolate the bacterium acquires additional resistance by the activation of efflux pumps in combination with disruption in INH-NAD+ complex formation that protect inhibition of InhA located in FAS-II complex. / AFRIKAANSE OPSOMMING: Isoniasied vorm deel van die eerste linie van behandeling teen tuberkulose en word algemeen gebruik om latente Mycobacterium tuberculosis infeksie te behandel. Isoniasied inhibeer die sintese van langketting mikolitiese sure wat in die selwand van Mycobacterium tuberculosis voorkom. Dit is bewys dat hierdie sure essensieel is vir die oorlewing van die bakterie. Mikolitiese sure word hoofsaaklik gesintetiseer deur die vetsuur sintase ensiem (FAS) sisteem wat in mikobakteriee voorkom as die FAS-I en FAS-II komplekse. Isoniasied dood die bakteriee deur die FAS-II kompleks, wat nodig is om die verlenging van mikoliete, te blokkeer. Dit word bewerkstellig deurdat 'n pro-vorm van die middel die tuberkulose bacilli binnedring, waarna isoniasied geaktiveer word deur katalase peroksidase, wat deur die katG geen geenkodeer word. Die geaktiveerde isoniasied vorm 'n kompleks met NAD+, wat InhA (NADH-afhanklike eno.asiel draer prote.enreduktase), gelee in die FAS-II kompleks teiken. Een van die primere meganismes wat weerstandigheid teen isoniasied bewerkstellig, is die verlies van katalase peroksidase weens geenmutasies of algehele delesie van die katG geen. 'n verdere vier gene (inhA, kasA, ndh en ahpC) word ook verbind met isoniasied weerstandigheid. Nietemin is mutasies in hierdie vyf gene teenwoordig in slegs 70-80% van isoniasied weerstandige kliniese isolate, wat impliseer dat ander meganismes ook betrokke is in die weerstandigheid van Mycobacterium tuberculosis teen isoniasied.
Die doel van hierdie studie is om die vlak van uitdrukking van gene wat deur isoniasied in die mikolitiese suur biochemiese pad geïnduseer word, asook middel transport te kwantifiseer in twee naby verwante Mycobacterium tuberculosis isolate van Beijing groep 208. Die twee isolate is die volledig sensitiewe (K636) en isoniasied monoweerstandige (R55), met minimum inhiberende konsentrasies van onderskeidelik 0.1 en 4µg/ml. Mutasies wat geassosieer word met isoniasied weerstandigheid was afwesig in beide die isolate. Kulture is van die isolate gemaak met en sonder 0.1µg/ml isoniasied vir 24 uur, waarna RNA geekstraeer is deur middel van QRT-PCR analise om gene te identifiseer wat verskillend uitgedruk word.
Die resultate toon dat verskeie gene verskillend uitgedruk is in reaksie op laevlak isoniasied blootstelling. Die mees prominente opregulering is waargeneem in die gene (acpM, fabD, accd6 en fbpC) wat die FAS-II kompleks enkodeer, asook die gene (efpA, iniA, iniB en mmpl7) wat betrokke is in middel transport. Beduidende afregulering van 'n verdere twee gene in die isoniasied monoweerstandige isolate, naamlik ndh en fbpC is waargeneem. Op grond van hierdie waarnemings, stel ons 'n model voor waarvolgens isoniasied blootstelling in die sensitiewe isolaat die FAS-II kompleks inhibeer, en met die gevolglike akkumulasie van mikoliete, dood dit die bakterium. In teenstelling stel ons voor dat addisionele weerstandigheid bekom word in die weerstandige isolaat deur die aktivering van uitvloeipompe, in kombinasie met die ontwrigting van die INH-NAD+ kompleksvorming wat die inhibisie van InhA binne die FAS-II kompleks beskerm.
|
3 |
Long QT syndrome : the identification and verification of putative KCNE2-interacting proteinsNeethling, Annika 12 1900 (has links)
Thesis (MScMedSc)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Long QT syndrome (LQTS) is a cardiac repolarization disorder affecting every 1:2000-1:3000 individuals. This disease is characterized by a prolonged QT interval on the surface electrocardiogram (ECG) of patients. Symptoms of LQTS range from dizziness and syncope to more severe symptoms such as seizures and sudden cardiac death (SCD). Clinical features of LQTS are a result of the precipitations of Torsades de Pointes, which is a polymorphic form of ventricular tachycardia. A number of genetic forms of LQTS have been identified with more than 700 mutations in 12 different genes leading to disease pathogenesis. However it has been estimated that approximately 25% of patients with compelling LQTS have no mutations within the known LQT genes. This proves to be problematic since treatment regimens depend on the genetic diagnosis of affected individuals. Of the known mutated genes, KCNE2 is associated with LQT6. KCNE2 encodes the beta-subunit of potassium ion channel proteins. These proteins contain cytoplasmic C-terminal domains in which many mutations have been identified.
We hypothesize that genes encoding KCNE2-interacting proteins might be identified as disease-causing or modifying genes. The present study aimed to use yeast two-hybrid (Y2H) methodology to screen a pre-transformed cardiac cDNA library in order to identify putative interactors of the C-terminal of KCNE2. Through specific selection methods the number of KCNE2 ligands was reduced from 296 to 83. These interactors were sequenced and 14 were identified as putative interacting proteins. False positive ligands were excluded based on their function and subcellular location. Ultimately three strong candidate ligands were selected for further analysis: Alpha-B crystallin (CRYAB), Filamin C (FLNC) and voltage-dependent anion-selective channel protein 1 (VDAC1). Three-dimensional (3D) co-localization and co-immunoprecipitation were used to verify these proposed interactions and succeeded in doing so.
The genes encoding verified interactors will be screened in our SA panel of LQT patients, to potentially identify novel LQT causative or modifying genes. Furthermore, the interactions verified in the present study may shed some light on the mechanism of pathogenesis of LQT causative mutations in KCNE2. / AFRIKAANSE OPSOMMING: Lang QT-sindroom (LQTS) is 'n hart her-polariserende siekte wat elke 1:2000-1:3000 individue affekteer. Hierdie siekte word gekenmerk deur 'n lang QT-interval op die oppervlak elektrokardiogram (EKG) van pasiënte. Simptome van LQTS wissel van duiseligheid en floutes tot meer ernstige simptome soos stuiptrekkings of aanvalle en skielike kardiale dood (SKD). Kliniese kenmerke van LQTS is 'n gevolg van die neerslag van Torsades de Pointes; 'n polimorfiese vorm van ventrikulêre tagikardie. Verskeie genetiese vorms van LQTS is geïdentifiseer met meer as 700 mutasies in 12 verskillende gene wat lei tot siekte patogenese. Dit is ergter beraam dat ongeveer 25% van pasiënte met dwingende LQTS geen mutasies in die bekend LQT gene besit nie. Dit is problematies aangesien siekte behandeling af hang van die genetiese diagnose van geaffekteerde individue. Een van die bekende gemuteerde gene is KCNE2 wat verband hou met LQT6. KCNE2 kodeer die beta-subeenheid van kalium ioonkanaal proteïene. Hierdie proteïene bevat sitoplasmiese C-terminale waarin baie mutasies alreeds geïdentifiseer is.
Ons veronderstel dat gene wat proteïene kodeer wat met KCNE2 interaksie toon, geïdentifiseer kan word as siekte veroorsaakende of wysigings gene. Die huidige studie het die gis twee-hibried metode gebruik om 'n vooraf-getransformeerde hart cDNS biblioteek te sif om vermeende protein interaksies van die C-terminaal van KCNE2 te identifiseer. Deur middel van seleksie metodes is die aantal KCNE2 ligande verminder van 296 tot 83. Die identiteit van die proteïene is bekend gemaak deur volgorderbepaling waarna 14 geïdentifiseer is as proteïene wat moontlik interaksie kan toon met KCNE2. Vals positiewe ligande is uitgesluit op grond van hul funksie en subsellulêre lokasering. Drie kandidaat ligande is gekies vir verdere analise: Alfa-B crystallin (CRYAB), Filamin C (FLNC) en spanning-afhanklike anioon-selektiewe kanaal proteïen 1 (VDAC1). Drie-dimensionele (3D) mede-lokalisering en mede-immunopresipitasie tegnieke is gebruik om hierdie voorgestelde interaksies te verifieer en het geslaag om dit te doen.
Die gene wat geverifieerde proteïene kodeer, sal gekeur word in ons Suid-Afrikaanse paneel van LQT pasiënte om sodoende potensieel nuwe LQT veroorsakende of wysigings gene te identifiseer. Verder kan die geverifieer interaksies in die huidige studie lig werp op die meganisme van die ontstaan van LQT veroorsakende mutasies in KCNE2. / Harry Crossley Foundation (South Africa) / Stellenbosch University / South African Council for Scientific and Industrial Research / Stella and Paul Loewenstein Charitable and Educational Trust
|
4 |
Investigation of the genetic aetiology of Parkinson's disease in South AfricaKeyser, Rowena J. 03 1900 (has links)
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
|
5 |
Identification of ligands interacting with the Wolframin protein (WFS1), a candidate in the pathophysiology of posttraumatic stress disorder (PTSD)Honing, Candice 03 1900 (has links)
Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Posttraumatic stress disorder (PTSD) is a multifactorial disorder, with substantial evidence for a genetic
contribution. Although genetic association studies have been conducted to identify vulnerability factors
in PTSD, the results remain largely inconsistent. Identifying ligands of proteins that are involved in the
aetiology of PTSD represents a means of delineating the network of interactions that may play a role in
the development of the disorder. Numerous animal studies have identified the Wolframin protein
(WFS1) as a putative biomarker for the development of PTSD. However, the function of WFS1 has not
yet been fully elucidated. The aim of the present investigation was to identify proteins that interact with
the N-terminal domain of WFS1, in order to possibly elucidate the function of the protein, and to
subsequently hypothesise on the role that WFS1 may play in the development of PTSD.
Yeast two-hybrid (Y2H) methodology was used to identify putative ligands of the N-terminal domain
of WFS1 (amino acids 1-300) by screening a human adult brain complementary DNA (cDNA) library.
Successive selection stages reduced the number of putative WFS1 N-terminal ligand-containing
colonies (preys) from 878 to three. Putative ligands were sequenced and indentified by BLAST-search.
Four preys were excluded because they were either out of frame with the vector or the protein they
encoded occurred in a subcellular location that was not compatible with the location of the N-terminal
domain of WFS1. An interesting putative ligand was identified as carboxypeptidase E (CPE).
Colocalisation analyses verified that CPE colocalises with WFS1 in rat hypothalamic GT1-7 cells. Coimmunoprecipitation
(Co-IP) further verified a direct interaction between WFS1 and CPE in rat
hypothalamic GT1-7 cells, providing conclusive evidence that WFS1 and CPE interact.
Both WFS1 and CPE are upregulated in response to fear and both are localised to the secretory
granules of the regulated secretory pathway. WFS1 has been detected in both the ER and secretory
granules it seems to play an important role in protein biosynthesis, modification, folding, trafficking
and the regulation of calcium homeostasis. CPE is involved in neuropeptide processing and trafficking
of secreted proteins. The interaction between CPE and WFS1 may thus serve to facilitate an optimal
environment in which neuropeptides can be processed and secreted. / AFRIKAANSE OPSOMMING: Posttraumatiese stresversteuring (PTSV) is 'n multifaktoriese siekte, met aansienlike bewyse vir 'n
genetiese bydrae. Hoewel genetiese assosiasie-studies uitgevoer word om kwesbaarheidsfaktore in
PTSV te identifiseer, is die resultate grootliks teenstrydig. Identifiseering van ligande van proteїene wat
betrokke is in die etiologie van PTSV dien as middel om die netwerk van interaksies wat ń moontlike
rol in die ontwikkeling van die versteuring kan speel, te oudersoek talle diere studies het die Wolframin
proteien (WFS1) geїdentifiseer as 'n moontlike biomerker vir die ontwikkeling van PTSV. Die funksie
van WFS1 is egter nog nie ten volle beskryf nie. Die doel van die huidige studie was om proteїene wat
interaksie met die N-terminale domein van WFS1 her te identifiseer, om sodoende die funksie van die
proteїen uit te lig, en daardeur die rol wat WFS1 kan speel in die ontwikkeling van PTSV te bepaal.
Die gis twee-hibried metodologie is gebruik om moontlike ligande van die N-terminale domein van
WFS1 te identifiseer, deur die sifting van 'n mens volwasse brein komplementêre DNS
biblioteek. Opeenvolgende seleksie stappe het die aantal moontlike WFS1 N-terminale ligand wat
moontlike prooi kolonies bevat van 878 tot en met ses verminder. Die DNS volgorde van die moontlike
prooi-plasmiede is bepaal en geїdentifiseer deur die BLAST soek-engin. Vier prooi-plasmiede is
uitgesluit omdat hulle of nie in die korrekte lees-raam in die vektor was nie of die subsellulêre ligging
van die proteїen wat uitgedrukword is nie versoenbaar met die N-terminale domein van WFS1. 'n
Interessante moontlike ligand is geїdentifiseer as Karboxypeptidase E (CPE). Ko-lokalisering ontleding
bevestig dat CPE ko-lokaliseer met WFS1 in rot hipotalamiese selle (GT1-7). Ko-immunopresipitasie
(Ko-IP) toon verder 'n direkte interaksie tussen WFS1 and CPE in rot GT1-7 selle. Wat dus bewys dat
WFS1 en CPE wel met mekaar 'n interaksie het.
Beide WFS1 en CPE toon 'n verhoogde uitdrukking in respons tot ń vrees-situasie. Beide van hierdie
proteїene kom voor in die sekretoriese korrels van die gereguleerde sekretoriese pad. Die WFS1
proteien word bevind in die endoplasmiese retikulum (ER) van die sel, waar dit verantwoordelik is vir
proteien biosintese, modifikasie, vouing, vervoer en die reguleering van kalsium homeostase. Die CPE
proteїen is verantwoordelik vir die proseseering van neuropeptiede en die vervoer van uitgeskiede
proteїene. Dus kan die interaksie tussen CPE en WFS1 dien om 'n optimale omgewing te skep waarin
neuropeptiede geproseseer en uitgeskei kan word. / The National Research Foundation (NRF), the Harry Crossley Foundation and the Medical Research Council
(MRC)
|
6 |
Investigation of the genetic aetiology of aminoglycoside-induced hearing loss in South African populationsHuman, Hannique 12 1900 (has links)
Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: South Africa is currently facing a major multidrug-resistant tuberculosis (MDR-TB) epidemic and
has one of the highest incidences in the world. Aminoglycoside antibiotics are commonly used in
this country as a treatment against MDR-TB. A well known side-effect of aminoglycosides is
permanent hearing loss and this is thought to have a significant genetic component. To date, at least
six mutations in the mitochondrial genome are known to confer susceptibility to aminoglycosideinduced
hearing loss. It is imperative that we investigate the frequency of these mutations in our
populations and determine whether certain sub-groups are at increased risk. The aim of the present
study was therefore to investigate the genetic aetiology of aminoglycoside-induced hearing loss in
the South African population.
A multiplex method using the ABI Prism® SNaPshotTM Multiplex system was optimised to screen
for six mutations in the MT-RNR1: A1555G, C1494T, T1095C, 961delT+C(n), A827G and T1291C.
A total of 115 MDR-TB patients from the Brooklyn Chest Hospital in Cape Town who were
receiving high doses of either streptomycin, kanamycin or capreomycin were recruited for this
study. Furthermore, 439 control samples, comprising of 93 Afrikaner, 104 Caucasian, 112 Black
and 130 Mixed Ancestry individuals were recruited and screened for the presence of the six
mutations. Identification of novel variants in the MT-RNR1 and the entire mitochondrial genome
was performed using High Resolution Melt analysis (HRM) and whole mitochondrial DNA
sequencing, respectively. A total of 97 family members from a South African family known to
harbour the A1555G mutation were recruited and genotyped using SNaPshot analysis. In addition,
mitochondrial functioning in the presence of different streptomycin drug concentrations, in
transformed lymphoblasts of an individual harbouring the A1555G, was assessed by means of the
MTT colorimetric assay. Detection of heteroplasmic mutations was performed using PCRRestriction
Fragment Length Polymorphism (RFLP) analysis and UN-SCAN-IT software.
We successfully developed a robust and cost-effective method that detects the presence of all six
mutations simultaneously. The method worked equally well on both blood (from adults) and buccal
swabs (from children). The C1494T, T1095C and T1291C mutations were not detected in any of
the MDR-TB or control groups. Alarmingly, the A1555G mutation was detected in 0.9% of the
Black control samples and in 1.1% of the Afrikaner controls (in one sample in the heteroplasmic
state 25%). The A827G mutation was present at a frequency of 0.9% in the MDR-TB patients and
in 1.1% of the Afrikaner controls. The 961delT + insC(n) mutation was found in relatively high
frequencies in both the MDR-TB patients (3.5%) and control groups (1.1% of the Afrikaner, 1.5%
of the Mixed Ancestry and 7.1% of the Black samples). Similarly, the T961G mutation was
III
detected at high frequencies in the Caucasian (2.9%) and Afrikaner (3.2%) controls. Screening for
novel variants in MT-RNR1 in MDR-TB patients experiencing ototoxicity revealed two novel
variants (G719A and T1040C). However, G719A and T1040C are not likely to be pathogenic since
they were detected in ethnic-matched controls: Mixed Ancestry (20.7%) and Black (1.8%) controls.
Furthermore, a total of 50 novel variants were identified within the mitochondrial genome of eight
MDR-TB patients with ototoxicity. Only five of the 50 variants (one in the MT-TH, ND3, COX3
and two in the CYTB gene) were shown to reside at positions that are evolutionarily conserved
across five species from human to frog, and the four variants in the protein coding genes resulted in
missense changes. A total of 76 of the 97 family members recruited were found to be A1555Gpositive
(on mitochondrial haplogroup L0d) and are therefore at risk of developing irreversible
hearing loss. Genes and variants known to act as genetic modifiers: tRNASer(UCN), homozygous
A10S in TRMU and 35delG in GJB2 were not present in this family. For the MTT assay, decreased
mitochondrial functioning of cells harbouring the A1555G mutation in the presence of streptomycin
were (compared to wild type) observed but this was not statistically significant (p-value: 0.615-
0.999).
The high frequency of the A1555G mutation (0.9%) in the Black population in South Africa is of
concern given the high incidence of MDR-TB in this particular ethnic group. However, future
studies with larger numbers of samples are warranted to determine the true frequencies of the
aminoglycoside deafness mutations in the general South African population. Our data suggests that
the 961delT + insC(n) and T961G variants are common non-pathogenic polymorphisms due to the
high frequencies observed in controls (>1%). The identification of the first novel variants within
protein coding genes that could possibly be associated with aminoglycoside-induced hearing loss
holds great possibilities with regards to the identification of a second gene involved in drug induced
hearing loss. Future studies where the possible effect of these variants on the normal functioning of
these genes could be assessed would contribute greatly to this field of research. All 76 A1555Gpositive
members of the family were given genetic reports and counseled about their risk and that of
their children for developing hearing loss due to aminoglycoside use.
The development of a rapid and cost-effective genetic method facilitates the identification of
individuals at high risk of developing hearing loss prior to the start of aminoglycoside therapy. This
is of critical important in a low-resource country like South Africa where, despite their adverse sideeffects,
aminoglycosides will be continue to be used routinely and are accompanied with very
limited or no audiological monitoring. Future studies and greater public awareness is therefore
needed to address this serious problem. / AFRIKAANSE OPSOMMING: Suid Afrika beleef tans „n grootskaalse tuberculose epidemie (veral weerstandige vorme van
tuberculose) (MDR-TB), met een van die hoogste voorkomssyfers in die wêreld. Aminoglikosied
antibiotikums word baie algemeen gebruik in Suid Afrika vir die behandeling van MDR-TB. ‟n
Bekende newe effek van die middels is permanente gehoor verlies en dit is van mening dat dit
gekoppel is aan „n genetiese component. Daar is tans ses mutasies in die mitochondriale genoom
wat vatbaarheid tot aminoglikosied-geinduseerde gehoor verlies veroorsaak. Daarom is dit van
uiterse belang dat die frekwensie van die mutasies in ons populasies bepaal word sodat daar
vasgestel kan word watter groepe „n hoë risiko het om gehoor verlies te kan ontwikkel.
Die ABI Prism® SNaPshotTM Multipleks sisteem is gebruik en geoptimiseer om te toets vir die ses
mutasies in die MT-RNR1: C1494T, T1095C, 961delT+C(n), A827G and T1291C. „n Totaal van 115
MDR-TB pasiente van die Brooklyn Chest Hospital in Kaap Stad is gewerf vir die studie. Hierdie
pasiente ontvang daaglikse hoë dosese van een van die volgende aminoglikosiede: streptomycin,
kanamycin of capreomycin. Verder is „n totaal van 439 kontrole DNA monsters gewerf vanuit die
volgende etniese groepe: 93 Afrikaner, 104 Blank, 112 Swart and 130 Kleurling. Hierdie monsters
is ook getoets vir die ses mutatsies. Hoë Resolusie Smelt analise (HRS) is gebruik om nuwe DNS
volgorde veranderinge in die MT-RNR geen te identifiseer. Die hele mitochondriale genoom is
blootgestel aan DNA volgorde bepaling in „n poging om nuwe DNS volgorde verandering in die
genoom te identifiseer wat moontlik betrokke kan wees by aminoglikosied-geinduseerde gehoor
verlies. „n Total van 97 lede van „n Suid Afrikaanse familie waar die A1555G mutasie teenwoordig
is, is deur middle van die SNaPshot metode gegenotipeer. Verder is die normale funcitoneering van
die mitochondrion in getransformeerde witbloed selle, getoets in die teenwoordigheid van
verskillende konsentrasies streptomycin met behulp van die MTT kleurmetrie toets. Deteksie van
heteroplasmiese mutasies is gedoen deur middle van die PCR-RFLP tegniek en alle analises is
gedoen op die UN-SCAN-IT program.
Ons was suksesvol in die ontwikkeling van „n vinnige, koste effektiewe en kragtige tegniek wat al
ses die mutasies in MT-RNR1 in een reaksie kan optel. Hierdie tegniek het goed gewerk met DNA
monsters van bloed en van selle verkry vanuit die wangholte (geneem van kinders jonger as 12 jaar).
Die C1494T, T1095C en T1291C mutasies is glad nie waargeneem in enige van ons MDR-TB
patiente of kontroles nie. Skrikwekkend is die hoë frekwensie (0.9%) waarby die A1555G mutasie
in die Swart kontrole groep waargeneem is. Hierdie mutasie is ook in 1.1% van die Afrikaner
kontrole groep opgemerk in heteroplasmie van 25%. Die A827G mutasie was teenwoordig in 0.9%
en 1.1% van die MDR-TB patiente en Afrikaner kontrole monsters, onerskeidelik. Die 961delT +
insC(n) mutasie is opgemerk in baie hoë frekwensies in beide die MDR-TB (3.5%) en kontrole
groepe (1.1% van die Afrikaner, 1.5% van die Kleurling en 7.1% van die Swart monsters). Die
T961G mutasie is ook in hoë frekwensies in slegs die Blanke (2.9%) en die Afrikaner (3.2%)
kontrole groepe waargeneem. Nuwe DNS volgorde veranderinge in MT-RNR1 is gesoek in „n groep
MDR-TB patiente wat gehoor verlies ondervind. Slegs twee nuwe verandering is ontdek (G719A en
T1040C). Dit is onwaarskynlik dat hierdie veranderinge patogenies is siende dat hulle teen
frekwensies van 20.7% en 1.8% waargeneem is in die Kleurling en Swart kontrole groepe
onderskeidelik. Tydens die soeke na nuwe DNS volgorde veranderinge wat moontlik geassosieer is
met aminoglikosied-geinduseerde gehoor verlies in die mitochondriale genoom is 50 onbekende
veranderinge ontdek (een in die MT-TH, ND3, COX3 en twee in die CYTB gene). Die veranderinge
is verder ondersoek vir evolusionêre konservasie op beide die nukliotied en amino suur vlak van
mens to padda. Dit is bevind dat 76 uit die 97 familie lede positief is vir die A1555G mutasie en het
dus „n hoë risiko om aminoglikosied-geinduseerde gehoor verlies te ontwikkel as hul bloot gestel
word aan hierdie antibiotikums. Verder is gevind dat hierdie familie op die L0d mitochondriale
haplogroep lê. Geen van die sogenaamde genetiese modifiseerde gene of DNS volgorde
veranderinge in hierdie gene (tRNASer(UCN), A10S in TRMU in homosigotiese vorm en die 35delG in
GJB2) is gevind in die familie nie. Die MTT toets het „n afname in die mitochondriale
funksioneering van selle waar die A1555G mutasie teenwoordig was getoon, alhoewel die verskil
tussen selle wat nie die A1555G mutasie het nie, nie statisties betekenisvol was nie (p-waarde:
0.615-0.999).
Die hoë frekwensie van die A1555G mutasie (0.9%) in die Swart populasie van Suid Afrika is
skrikwekkend siende dat die voorkomssyfer van MDR-TB in hierdie groep baie hoog is.
Toekomstige studies met grooter getalle is nodig om die ware frekwensie van die mutasies
geassosieer met aminoglikosied-geinduseerde gehoor verlies in die algemende Suid Afrikaanse
populasie te bepaal. Ons data dui aan dat die 961delT + insC(n) en die T961G mutasies slegs
algemene nie-patogeniese polimorphismis is siende dat dit in sulke hoë frekwensies (>1%) in
kontroles opgemerk is. Die identifiseering van die eerste DNS volgorde veranderinge in proteïen
kodeerende gene wat moontlik geassosieer is met aminoglikosied-geinduseerde gehoor verlies hou
groot en belowende moontlikehede in, interme van die identifiseering van „n tweede geen.
Toekomstige studies waarin die effek van hierdie veranderinge op die normale funktioneering van
hierdie gene ondersoek word sal „n besondere groot bydrae lewer tot hierdie veld van navorsing. Al
76 van die A1555G positiewe familie lede is voorsien van genetiese verslae en het berading ontvang
in verband met hul risiko en die risiko van hul kinders om aminoglikosied-geinduseerde gehoor
verlies te ontwikkel.
Die ontwikkeling van „n kragtige, vinnige en koste-effektiewe genetiese metode vergemaklik die
vinnige identifiseering van hoë risiko individue vir die ontwikkeling van gehoor verlies voordat
hulle met hul aminoglikosiede behandeling begin. Dit is veral noodsaaklik in „n derde wêreld land
soos Suid Afrika waar, ten spyte van hul gevaarlike newe effekte, aminoglikosied antibiotikums
steeds gebruik sal word. Daarom is grooter publieke bewusmaking nodig om hierdie problem te
probeer oplos en te verhoed.
|
7 |
Improving methods for genotypic drug resistance testing in Mycobacterium tuberculosisMlamla, Zandile Cleopatra 03 1900 (has links)
Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: An important next step to Tuberculosis control relies on the translation of basic science and modern diagnostic techniques into primary health care clinics. These assays must be rapid, inexpensive, interpretation of results must be easy and they must be simple so that a healthcare worker with limited training can perform the tests under safe conditions. This study consists of four aims. The first aim was to develop a methodology to sterilize sputum specimens for rapid TB diagnosis and drug resistance testing. Candidate bactericides were identified from the literature, and tested for their bactericidal activity in Mycobacterium tuberculosis. We identified ultraseptin®aktiv as a powerful bactericidal agent which sterilizes sputum specimens for subsequent safe handling prior to light emitting diode microscopy and it also provides a DNA template for PCR-based tests. An algorithm has been proposed for the processing of specimens and rapid diagnosis of TB and drug resistant TB while patients wait for results.
Recently, the World Health Organization has endorsed the MTBDRplus test for diagnosis of TB and drug resistant TB. However genotypic tests may have more problems than anticipated. With the HIV pandemic, an increase of non-tuberculous mycobacteria has been reported. The sensitivity of genotypic tests in specimens with underlying non-tuberculous mycobacterial species therefore requires further evaluation. This study therefore also aimed at determining the reliability of the MTBDRplus assay for detection of drug resistant TB where non-tuberculous bacterial load is high. Clinically relevant non-tuberculous mycobacterium DNA and DNA from a multi-drug resistant TB isolate were obtained. Ratios of the different NTM with the MDR-TB DNA were made and subjected to the MTBDRplus assay. Known mix NTM and TB infected clinical isolates and sputum sediments were also evaluated for TB and drug resistance detection on the MTBDRplus assay. Under these conditions, this study provides evidence that the MTBDRplus test cannot reliably detect TB and drug resistance TB in specimens with underlying non-tuberculous mycobacteria.
Thirdly, to evaluate the sensitivity of the MTBDRplus assay for detecting drug resistance in hetero-resistant isolates, ratios were made using purified DNA from an MDR and pan-susceptible TB isolate. The MTBDRplus assay was then performed on the different ratios. We report that the MTBDRplus assay can efficiently detect wild type DNA in genes associated with resistance during the early evolution of drug resistance. However, in the later stage during treatment when both the wild type and mutants are present, the detection limit for the mutant DNA was 1:55. Due to these results, the MTBDRplus assay should still be further improved or other tests should be developed to address these limitations.
And finally to combat cross amplicon contamination during the final steps of genotypic detection with the MTBDRplus assay, a proof of concept for a patentable closed tube line probe device was proposed on the 4th aim. This device can be improved to enable automated drug resistance genotyping of multiple specimens.
The results of this study highlight the need for a sensitive inexpensive point of care drug resistance test that does not require intensive training. / AFRIKAANSE OPSOMMING: 'n Belangrike volgende stap om Tuberkulose te beheer is om basiese wetenskap resultate te gebruik sodat moderne diagnose tegnieke ontwikkel kan word wat in primêre gesondheidsorg klinieke toegepas kan word. Hierdie toetse moet vinnig, goedkoop, en die interpretasie van resultate moet maklik wees. Die toetse moet eenvoudig wees sodat 'n gesondheidswerker met beperkte opleiding die toetse onder veilige omstandighede kan uitvoer. Hierdie studie bestaan uit vier doelwitte, waarvan die eerste was om 'n metode te ontwikkel vir die sterilisasie van sputum monsters vir vinnige TB diagnose en die toesting van middelweerstandigheid. Kandidaat kiemdodende middels was geïdentifiseer vanaf die literatuur en die middels se kiekdodende aktiviteit was getoets op Mycobacterium tuberculosis. Ons het ultraseptin®aktiv geïdentifiseer as 'n kragtige kiemdodende middel wat bakteria in sputum monsters steriliseer vir veilige hantering voordat diagnose met 'n lig uitstralende diode mikroskopie gedoen kan word. Hierdie behandeling met ultraseptin®aktiv bied ook 'n DNA templaat vir PCR-gebaseerde toetse. 'n Algoritme is voorgestel vir die hantering van monsters en die vinnige diagnose van sensitiewe- en middel weerstandige Tuberkulose terwyl die pasiënte by die kliniek wag vir die resultate.
Onlangs het die Wêreld Gesondheid Organisasie die genotipiese MTBDRplus toets vir die diagnose van Tuberkulose en middel-weerstandige Tuberkulose onderskryf. Hierdie toets word tans op groot skaal in Suid Afrika gebruik. Dit kan egter wees dat genotipiese toetse baie meer probleme kan he as wat aanvanklik verwag is. Die HIV pandemie gaan toenemend gepaard met n toename van nie-tuberkulose mycobacteria. Die sensitiwiteit van genotipiese toetse op monsters met onderliggende nie-tuberkulose mikobakteriese spesies vereis dus verdere evaluasie. Die doel van hierdie studie was ook om die betroubaarheid van die MTBDRplus-toets te bepaal vir die opsporing van middelweerstandige TB waar die nie-tuberkulose bakteriële lading hoog is. DNA van kliniese relevante nie-tuberkulose mikobakteria en multi-middelweerstige TB isolate was bekom. Verskillende verdunnigs van die spesifieke NTM DNA te same met die van MDR-TB DNA is gemaak en onderwerp aan die MTBDRplus toets. Bekende gemengde NTM- en TB geïnfekteerde kliniese isolate en sputum sedimente was ook geevalueer vir die opsporing van TB en middel weerstandigheid met die MTBDRplus toets. Hierdie studie verskaf bewyse dat die
MTBDRplus toets nie betroubaar is met die diagnose van sensitiewe- en middel weerstandige Tuberkulose in monsters met onderliggende nie-tuberkulose mycobacteria nie.
Verskillende verdunnings van gesuiwerde DNA van MDR en pan-sensitiewe TB isolate is gemaak om die sensitiwiteit van die MTBDRplus toets vir die opsporing van middelweerstandigheid te bepaal. Die MDRDRplus toets is gebruik met hierdie verdunnings. Resultate in hierdie studie toon dat die MTBDRplus toets effektief is met die identifisering van wilde-tipe DNA (dit beteken middel sensitief) in gene wat geassosieer word met middel weerstandigheid gedurende die vroeë ontwikkeling van weerstandigheid. Hier teenoor toon die resultate dat in die later stadium tydens behandeling, wanneer beide die wilde-tipe (sensitief) en mutante DNA (weerstandig) teenwoordig is, is die opsporingslimiet vir die mutante DNA maar 1:55. As gevolg van hierdie resultate raai ons aan dat die MTBDRplus toets nog verder verbeter moet word of dat ander toetse ontwikkel moet word om hierdie beperkinge aan te spreek.
Amplikon kruiskontaminasie kan n groot impak hê op die betroubaarheid van enige genotipiese diagnostiese toets. Die finale stappe van MTBDRplus toets behels die gebruik van 'n oop sisteem sodat kontaminasie maklik kan plaasvind. In die 4de doewit 'n konsep vir 'n patenteerbare geslotebuis toestel ontwikkel en die resultate het getoon dat kontaminasie suksesvol uitgeskakel kan word. Hierdie toestel kan verbeter na 'n outomatiese apparaat verbeter word sodat die module genotipering van verskeie monsters moontlik kan maak.
Die resultate van hierdie studie beklemtoon die noodsaaklikheid van 'n sensitiewe goedkoop “point of care” diagnostiese toets wat nie intensiewe opleiding benodig nie. / Medical Research Council of South Africa / University of Stellenbosch, Dept. of Molecular Biology and Human Genetics
|
8 |
Investigation into genotypic diagnostics for mycobacterium tuberculosisHoek, Kim Gilberte Pauline 12 1900 (has links)
Thesis (PhD )--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Diagnostic delay is regarded as a major contributor to the continuous rise in tuberculosis (TB)
cases and the emergence and transmission of multidrug-resistant tuberculosis (MDR-TB) and
extensively drug resistant tuberculosis (XDR-TB). It is therefore essential that more rapid
diagnostic methods are developed. Molecular-based assays have the potential for the rapid
species-specific diagnosis of TB and associated drug-resistances directly from clinical
specimens. We investigated whether high resolution melting analysis (HRM) could enable
the rapid diagnosis of TB and associated drug resistance, since the HRM apparatus and
reagents are relatively inexpensive and the methodology can easily be implemented in high incidence,
low income regions.
Application of this methodology allowed for the rapid identification of mycobacterial lymphadenitis
from fine-needle aspiration biopsy (FNAB) samples in 2 studies. This was done by targeting the
region of deletion 9 (RD9), present in M. tuberculosis and M. canettii, but absent from all other
members of the complex. However, the sensitivity of the method was low (51.9% and 46.3%,
respectively) when compared to the reference standard (positive cytology and/or positive
culture). Despite this limitation our method was able to provide a rapid diagnosis in more than
half of the infected patients with a relatively high specificity (94.0% and 83.3%, respectively). We
therefore proposed a diagnostic algorithm allowing the early treatment of patients with both HRM
and cytology results indicative of mycobacterial disease.
We developed the Fluorometric Assay for Susceptibility Testing of Rifampicin (FAST-Rif) which
allowed the rapid diagnosis of MDR-TB by detecting rifampicin (RIF) resistance mutations in the
rpoB gene with a sensitivity and specificity of 98% and 100%, respectively. The FAST-Rif method
was easily adapted to detect ethambutol (EMB) resistance due to mutations in the embB gene
with a sensitivity and specificity of 94.4% and 98.4% respectively, as compared to DNA
sequencing. The FAST-EMB method was a significant improvement over the inaccurate culture
based method. We identified a strong association between EMB resistance (and pyrazinamide
resistance) and MDR-TB and subsequently advised modifications to the current (2008) South
African National TB Control Programme draft policy guidelines.
Due to the potential for amplicon release, we adapted the FAST-Rif and FAST-EMB methods to
a closed-tube one-step method using the detection of inhA promoter mutations conferring
isoniazid (INH) resistance as a model. The method (FASTest-inhA) was able to identify inhA
promoter mutations with a sensitivity and specificity of 100% and 83.3%. These mutations are of
particular interest as they confer low level INH resistance and cross-resistance to ethionamide
(Eto). Since inhA promoter mutations are strongly associated with XDR-TB in the Western and
Eastern Cape Provinces of South Africa, data generated by the recently implemented
GenoType® MDRTBPlus assay may allow individualised treatment regimens to be designed for a
patient depending on their INH mutation profile. Our proposed treatment algorithm may be
particularly useful in XDR-TB cases, for which only few active drugs remain available.
Since current diagnostic methods all carry advantages and disadvantages, a combination of
phenotypic and genotypic-based methodologies may be the best scenario while awaiting
superior methods. / AFRIKAANSE OPSOMMING: Die onvermoë om tuberkulose (TB), multi-weerstandige tuberkulose (MDR-TB) en uiters
weerstandige tuberkulose (XDR-TB) vinnig te diagnoseer, is ‘n belangrike oorsaak vir die
volgehoue toename en verspreiding daarvan. Dit is noodsaaklik dat diagnostiese toetse wat
vinniger resultate oplewer, ontwikkel word. Molukulêre toetsing het die potensiaal om vinnig
spesie-spesifieke diagnoses van TB en die weerstandigheid teen TB-medikasie te lewer. Hierdie
studie wil vasstel of hoë-resolusie smeltingsanalise (HRS) ‘n vinnige diagnose van TB en die
weerstandigheid teen TB-medikasie kan oplewer aangesien die relatiewe lae koste van reagense
en apparaat, asook die minimale infrastruktuur en vaardighede wat vir dié toets benodig word, dit
uiters geskik maak vir pasiënte in gebiede met ‘n hoë TB-insidensie en lae inkomste.
Die toepassing van die HRS-metode op fynnaald-aspiraatbiopsies in twee afsonderlike studies,
het gelei tot die vinnige identifisering van mikrobakteriële-limfadenitis. Dit is bemiddel deur die
gebied van delesie 9 (RD9) teenwoordig in Mycobacterium tuberculosis en M. canettii, maar
afwesig in al die ander lede van die kompleks, te teiken. Die sensitiwiteit van die metode was
(51.9% en 46.3%, vir die twee studies onderskeidelik) in vergelyking met die verwysingstandaard
(positiewe sitologie en/of positiewe kultuur). Ten spyte van dié beperking was ‘n vinnige
diagnose in meer as die helfte van geïnfekteerde pasiënte met ‘n redelike hoë spesifisiteit
(94.0% en 83.3%, onderskeidelik) moontlik. ‘n Diagnostiese algoritme wat gebaseer is op die
resultate van die HRS en sitologie-toetse, is voorgestel om pasiënte vroeër te behandel.
‘n Fluorometriese toets (FAST-Rif) is ontwikkel vir die vinnige diagnose van MDR-TB deur
mutasies in die rpoB-geen op te spoor met ‘n hoë sensitiwiteit en spesifisiteit (98% en 100%,
onderskeidelik). Hierdie mutasies is verantwoordelik vir weerstandigheid teen die antibiotikum
rifampicin (FAST-Rif) en word beskou as ‘n vinnige diagnose vir MDR-TB. Die FAST-Rif metode
kon maklik aangepas word om mutasies in die embB-gene, verantwoordelik vir weerstandigheid
teen die antibiotikum ethambutol (EMB), op te spoor. Die FAST-EMB-metode het ‘n sensitiwiteit
en spesifisiteit van 94.4% en 98.4% onderskeidelik getoon in vergelyking met DNS volgordebepaling.
Die FAST-EMB-metode was ‘n betekenisvolle verbetering op die onakkurate
kultuurgebaseerde metodes. ‘n Sterk korrelasie tussen EMB-weerstandigheid (en
weerstandigheid teen pyrazinamide) en MDR-TB is geïdentifiseer. Vervolgens is veranderinge
aan die Suid-Afrikaanse Nasionale TB-beheerprogram se Konsepbeleidsgids (2008) voorgestel.
Om die potensiële vrylating van amplikone te verhoed, is die FAST-Rif en FAST-EMB aangepas
tot ‘n enkelstap geslote buissisteem deur gebruik te maak van die opsporing van inhA promotormutasies
wat weerstandigheid teen isoniazid (INH) veroorsaak. Die metode het ‘n
sensitiwiteit en spesifisiteit van 100% en 83.3% onderskeidelik, getoon. Hierdie mutasies
veroorsaak laevlak weerstandigheid teen INH, maar ook kruisweerstandigheid teen ethionamide
(Eto). Aangesien daar ‘n sterk verbintenis tussen inhA-promotormutasies en XDR-TB in die Oos en
Wes-Kaapprovinsies van Suid-Afrika is, kan data van die GenoType® MDRTBPlus-toets
moontlik gebruik word om ‘n meer geïndividualiseerde behandeling te ontwerp afhangende van
die pasiënt se INH-mutasieprofiel. Ons behandelingsalgoritme is veral geskik vir XDR-TB pasiënte
vir wie daar weinig aktiewe antibiotika beskikbaar is.
Huidige diagnostiese metodes het almal voor- en nadele, dus bied ‘n kombinasie van fenotipiese
en genotipiese metodes moontlik die beste oplossing totdat beter metodes ontwikkel word.
|
9 |
Gene expression and cytokine pattern of pulmonary tuberculosis patients and their contacts in EthiopiaBekele, Adane Mihret 03 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The immune response against M. tuberculosis is multifactorial, involving a network of innate
and adaptive immune responses. Characterization of the immune response, a clear
understanding of the dynamics and interplay of different arms of the immune response and
the identification of infection-stage specific biomarkers are critical to allow the
development of better tools for combating tuberculosis. In an attempt to identify such
biomarkers, we studied pulmonary tuberculosis patients and their contacts in Addis Ababa,
Ethiopia as part of EDCTP and BMGF funded tuberculosis projects by using multiplex
techniques. We analysed 45 genes using the Multiplex Ligation Dependent Probe
Amplification (MLPA) technique and the expression of IL-4δ2, BLR1, MARCO, CCL-19, IL7R, Bcl2, FcyR1A, MMP9, and LTF genes discriminate TB cases from their healthy contacts.
FoxP3, TGFß1 and CCL-19 discriminate latently infected from uninfected contacts. Single
genes predict with an area under the Receiver Operating Characteristic (ROC) curve of 0.68
to 0.85 while a combination of genes identified up to 95% of the different groups. Similarly,
the multiplex analysis of cytokines and chemokines also showed that single or combinations
of plasma cytokines and chemokines discriminate between different clinical groups
accurately. The median plasma level of EGF, fractalkine, IFN-y, IL-4, MCP-3 and IP-10 is
significantly different (p<0.05) in active tuberculosis and non active tuberculosis infection
and the median plasma levels of IFN-y, IL-4, MCP-3, MIP-1ß and IP-10 were significantly
different (p<0.05) before and after treatment. We also found a significant difference
(p<0.05) in plasma levels of cytokines of patients infected with the different lineages and
different families of the modern lineage. The plasma level of IL-4 was significantly higher in
patients infected with lineage 3 (p<0.05) as compared to lineage 4 and the CAS familyinfected
patients had a higher plasma level of IL-4 (P<0.05) as compared to patients infected
with H and T families but there was no difference between H and T families.
We identified genes and cytokines which had been reported from other studies in different
settings and we believe that these molecules are very promising biomarkers for classifying
active tuberculosis, latent infection, absence of infection and treated infection. These
markers may be suitable for the development of clinically useful tools but require further
validation and qualification in different populations and in larger studies. / AFRIKAANSE OPSOMMING: Die immuunrespons teen M. tuberculosis is multifaktoriaal en betrek ‘n netwerk van niespesifieke
and spesifieke immuunresponse. Karakterisering van die immuunrespons, ‘n
duidelike insig in die dinamika en tussenspel deur die verskillende arms van die
immuunrespons en die identifikasie van spesifieke biomerkers is krities belangrik om die
ontwikkeling van nuwe hulpmiddels teen tuberkulose te bevorder. In ‘n poging om sulke
biomerkers te identifiseer het ons pulmonale tuberkulose pasiënte en hulle kontakte in
Addis Ababa, Etiopië, as deel van die EDCTP en BMGF befondste tuberkulose projekte
bestudeer met multipleks tegnieke. Ons het 45 gene analiseer met ‘Multiplex Ligation
Dependent Probe Amplification (MLPA)’ en gevind dat die geenuitdrukking van IL-4•2, BLR1,
MARCO, CCL-19, IL7R, Bcl2, Fc•R1A, MMP9, en LTF TB pasiënte van hulle kontakte
onderskei. FoxP3, TGF•1 en CCL-19 onderskei tussen latent infekteerde en ongeïnfekteerde
kontakte. Enkele gene voorspel met ‘n area onder die ‘Receiver Operating Characteristic
(ROC)’ kurwe van 0.68 tot 0.85 terwyl die kombinasie van gene 95% van die verskillende
groepe identifiseer. Soortgelyk het multipleks analise van sitokiene en chemokiene
verskillende kliniese groepe akkuraat van mekaar onderskei. Die mediane plasmavlakke van
EGF, fractalkine, IFN-•, IL-4, MCP-3 en IP-10 is beduidend verskillend (p<0.05) in aktiewe
tuberkulose en nie-aktiewe tuberkulose infeksie en die mediane plasmavlak van IFN-•, IL-4,
MCP-3, MIP-1• en IP-10 was beduidend verskillend voor en na behandeling. Ons het ook
beduidende verskille (p<0.05) in plasmavlakke van sitokiene in pasiënte gevind wat
infekteer is met verskillende stamme and verskillende families van die moderne stamme.
Die plasmavlak van IL-4 was beduidend hoër in pasiënte wat infekteer is met stam 3
(p<0.05) teenoor stam 4 en die CAS familie-infekteerde pasiënte het ‘n hoër plasmavlak van
IL-4 (p<0.05) teenoor pasiënte met H en T familie infeksie hoewel daar geen versikke was
tussen die H en T families nie. Ons het gene en sitokiene identifiseer wat deur ander werkers onder verskillende
omstandighede ook beskryf is en ons glo dat hierdie molekules baie belowende biomerkers
is om aktiewe tuberkulose, latent tuberkulose, die afwesigheid van infeksie en behandelde
infeksie van mekaar te onderskei. Hierdie merkers mag toepaslik wees vir die ontwikkeling
van bruikbare kliniese hulpmiddele maar benodig verdere validasie en kwalifikasie in
verskillende populasiegroepe en in groter studies. / Bill and Melinda Gates Foundation (BMGF) / European and Developing Countries Clinical Trials Partnership (EDCTP) / African European Tuberculosis Consortium (AE TBC).
|
10 |
Investigating the Human-M. tuberculosis interactome to identify the host targets of ESAT-6 and other mycobacterial antigensBruiners, Natalie 12 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The causative agent of human tuberculosis, Mycobacterium tuberculosis, is an intracellular
pathogen that secretes virulence factors, namely ESAT-6 and CFP-10, as substrates of the
ESX-1 secretion system. It is hypothesised that these substrates interact with host proteins in a
targeted manner in order to elicit a required immune response, and they have been shown to
be involved in processes related to pro-inflammatory responses, necrosis, apoptosis,
membrane lysis and cytolysis. However, the biological function of ESX-1 substrates during
host-pathogen interactions remains poorly and incompletely understood. Therefore, the
present study was designed to gain insight into the role of the ESX-1 secretion system
substrates in host-pathogen interactions and to identify how M. tuberculosis mediates the
response of the human host.
In this study, a cDNA yeast two-hybrid library was constructed from human lung mRNA, to
identify mycobacterial-host protein-protein interactions that occur within the lung alveoli. The
ESX-1 secretion system substrates, ESAT-6 and CFP-10, were cloned in-frame into the
pGBKT7 vector, which was used in the yeast two-hybrid system to screen the lung cDNA
library in Saccharomyces cerevisiae. The ESAT-6 and CFP-10 screens identified 79 and 19
positive colonies, respectively. Of the total number of clones characterised, only two in-frame
inserts were identified with the ESAT-6 screen, corresponding to the human proteins filamin
A and complement component 1, q subcomponent, A chain (C1QA). In addition, the screen
with CFP-10 also identified C1QA as binding partner.
Subsequent in vitro and in vivo experiments were unable to confirm the putative interactions
of C1QA with ESAT-6 and CFP-10. However, the interaction between filamin A and ESAT-6
was demonstrated and confirmed by both in vivo co-localisation and co-immunoprecipitation.
Furthermore, the degradation of filamin A in the presence of ESAT-6 was shown to be
reflective of cytoskeleton remodelling and the induction of cell death. The work presented
here suggests that as ESAT-6 gains access to the cytosol, it initiates cell death by inducing
destabilisation of the cytoskeleton cell structure. This may possibly be driven by the
interaction of ESAT-6 and filamin A.
Finally, we also initiated an investigation of the identified putative binding partners (filamin A and C1QA) as possible genetic markers for genetic susceptibility studies to tuberculosis. A case-control analysis was performed involving 604 cases, of which 109 were Tuberculous
Meningitis (TBM), and 486 were controls from the South African Coloured (SAC) population
within the Ravensmead-Uitsig catchment area. The results of this analysis demonstrated a
novel association of a regulatory variant (rs587585) located upstream of the C1QA gene and
demonstrated an increasing trend towards increased values in tuberculosis patients with the
associated genotype.
This study has contributed significantly to our understanding of human-mycobacterial hostpathogen
protein-protein interactions and has opened the way for future studies further
exploring the consequences and function of the identified ESAT-6-filamin A interaction. It
has also led to the identification of a novel genetic association with tuberculosis. Finally, it
demonstrates the usefulness of the yeast two-hybrid system to identify potential proteinprotein
(host-pathogen) interactions that can lead to additional important and exciting research. / AFRIKAANSE OPSOMMING: Die organisme wat tuberkulose veroorsaak, Mycobacterium tuberculosis, is `n intrasellulȇre
patogeen wat virulensie faktore afskei, naamlik ESAT-6 en CFP-10, as substrate van die
ESX-1 sekresiesisteem. Daar word vermoed dat hierdie substrate met gasheerproteïene in „n
teiken wyse interaksie het om `n vereiste immuunreaksie voort te bring. Hierdie substrate is
betrokke by prosesse soos pro-inflammatoriese reaksies, nekrose, apoptose, membraanlise en
sitolise. Die biologiese funksie van die ESX-1 substrate tydens gasheer-patogeen interaksies
word egter tans swak en onvolledig verstaan. Daarom was die huidige studie ontwerp om
insig te bekom oor die rol hiervan in gasheer-patogeen interaksies en om te identifiseer hoe M.
tuberculosis die reaksie teenoor die gasheer bemiddel.
In hierdie studie was `n komplementȇre deoksiribonukleïensuur (kDNS) gis twee-hibried
biblioteek gemaak vanaf long boodskapper ribonukleïensuur (bRNS) om proteïen-proteïen
interaksies wat in die long plaasvind, te identifiseer. Die substrate van die ESX-1
sekresiesisteem, ESAT-6 en CFP-10, is in volgorde gekloneer in die pGBKT7 vektor en is
gebruik om die long kDNS biblioteek in Saccharomyces cerevisiae te ondersoek. In die soeke
na interaksies met ESAT-6 and CFP-10, was 79 en 19 positiewe kolonies onderskeidelik
geïdentifiseer. Van die aantal klone, was slegs twee volgordes in-leesraam geïdentifiseer met
ESAT-6. Hierdie proteïene het ooreengestem met filamin A en “complement component 1, q
subcomponent, A chain” (C1QA). Bykomend hiertoe, is C1QA ook geïdentifiseer as „n
bindende vennoot met CFP-10.
Daaropvolgende in vitro and in vivo eksperimente kon nie die vermeende interaksie van
C1QA met ESAT-6 en CFP-10 bevestig nie. Maar die interaksie tussen filamin A en ESAT-6
kon wel gedemonstreer word deur die gebruik van mede-lokalisering en medeimunopresipitasie.
Die afbreek van filamin A in die teenwoordigheid van ESAT-6 is ook
aangetoon en blyk „n weerspieëling te wees van sitoskelet hermodellering en die induksie van
seldood. Die werk wat hier aangebied word, dui daarop dat soos ESAT-6 toegang kry tot die
sitosol, inisieër dit seldood deur die destabilisaisie van die sitoskelet selstruktuur. Dit word
moontlik aangedryf deur die interaksie van ESAT-6 met filamin A. Laastens het ons `n ondersoek van die geïdentifiseerde bindingsvennote (filamin A and
C1QA) as moontlike genetiese merkers vir genetiese vatbaarheidsstudies vir tuberkulose
uitgevoer. `n Pasiënt-kontrole studie is gedoen waarby 604 individue ingesluit is, waarvan 109
gediagnoseer is met Tuberculosis Meningitis (TBM), en die ander 486 kontrole individue was
van die Suid Afrikaanse Kleurling (SAC) bevolking binne die Ravenmead-Uitsig
opvanggebied. Die resultate het „n nuwe assosiasie van „n regulerende variant (rs587585) wat
stroomop van die C1QA geen gelokaliseer is, getoon. Hierdie variant het `n verhoogde
neiging in tuberkulose pasiënte met die geassosieërde genotipe getoon.
Hierdie studie het `n beduidende bydrae gemaak tot ons begrip van menslike-mikobakteriese
gasheer-patogeen proteïen-proteïen interaksies. Hierdie resultate het die weg oopgemaak om
die gevolge en funksie van die geïdentifiseerde ESAT-6-filamin A interaksie verder te
ondersoek. Dit het ook aanleiding gegee tot die identifikasie van `n genetiese assosiasie met
tuberkulose. Om saam te vat, hierdie werk bewys die bruikbaarheid van die gis twee-hibriede
sisteem, om potensiële proteïen-proteïen interaksies te ontdek wat die moontlikheid het om
aanleiding te gee tot addisionele navorsingsvrae. / The National Research Foundation, / Harry Crossley Foundation / Medical Research Council of South Africa / Stellenbosch University Postgraduate bursary / Prof. Paul van Helden
|
Page generated in 0.1403 seconds