TGF-beta1 plays an important role in the pathogenesis of experimental and clinical glomerulosclerosis and tubufointerstitial fibrosis. Associations have been described between polymorphisms of cytokine and growth factor genes and susceptibility to, or progression of, an increasing number of diseases. In this study, single nucleotide polymorphisms (SNPs) in the TGFbeta1 gene were investigated as possible markers for the progression of chronic renal failure (CRF). One hundred and forty two Caucasian patients with CRF were screened for four TGFB1 SNPs: T-509C in the promoter region; Arg25Pro and Leu10Pro in exon 1 and Thr263Ile in exon 5. There were significant differences between CRF patients and controls in allele frequencies of two of the SNPs (Leu10Pro and C-509T), indicating an association with susceptibility to CRF, We also observed a significant association between rate of progression of CRF (the slope of the reciprocal of serum creatinine v time) and genotype, both at codon 25 (odds ratio 3.77, 95% confidence interval, 2.2 - 6, p < 0.001) and at the -509 promoter site (odds ratio 1.67, 95% confidence interval 1.1-2.5), p < 0.005) in patients with primary nephropathy (excluding PKD). Genotype at codon 25 was also associated with severity of proteinuria (p= 0.038), plasma TGF-B1 protein levels (p = 0,01), and the severity of glomerulosclerosis (p < 0.05). Genotype at C-509T was associated with the level of renal tubular TGF-B1 immunostmning (p = 0.0006) and with renal interstitial inflammatory cellular infiltration (p=0,015). There was a highly significant correlation between the degree of cellular infiltration in renal tissues and tubular TGF-beta1 immunostaining.

As knowledge of the human genome has accelerated, various diseases and individuals’ responses to drugs have been pinpointed to specific DNA variations in one’s genome. Among many different types of variants, the most common and simplest is the single nucleotide polymorphism (SNP) in which a single base substitution occurs. Although there have been considerable improvements in technologies that can reveal a single base difference in a DNA strand, simple and affordable methods that have high detection sensitivity and require small sample volume are expected to facilitate widespread adoption of routine SNP analysis in clinical settings. One such method that meets these requirements is to use nanopore as a single molecule detector, an emerging analytic system that detects changes in current related to molecules occupying a nanometer aperture. This dissertation thus chronicles our endeavors in developing a nanopore-based SNP assay using polymer tagged dideoxynucleotides (ddNTPs). The fundamental principles of this method rely on single base extension (SBE) of a primer by DNA polymerase using polymer tagged ddNTP analogs for allele discrimination and simple electronic readout of an alpha hemolysin (αHL) nanopore current for allele detection at the single molecule level. Using four uniquely tagged ddNTPs, a characteristic current level that is specific to each base is produced, thus identifying the SNP alleles present and the genotype at the site. To demonstrate the feasibility of this approach, four polymer attached ddNTPs, each with a different tag that generates a characteristic current blockade level in the αHL nanopore, were designed and synthesized. To search for a DNA polymerase that can accept these tagged ddNTP analogs as substrates, several candidate DNA polymerases were surveyed and their relative efficiencies for incorporation of the analogs were compared (Chapter 2). To generate a steady and stable blockade event for accurate SNP analysis, two different means of positioning a tag molecule in the αHL nanopore after the SBE reaction have been explored: covalent conjugation of DNA primer to the pore and immobilization of biotinylated primer within the pore by streptavidin. To find a suitable position for primer attachment on the pore, three αHL mutants, each with a different single conjugation point, were constructed. Using these mutants, different DNA-pore conjugates were produced and purified via various chromatography systems (Chapter 3). In the nanopore system, charged molecules such as DNA are electrophoretically driven through the pore under an applied voltage, thereby modulating the ionic current through the nanopore. This current reveals useful information about the structure and dynamic motion of the molecule at the single molecule level. Before performing SNP analysis, we first studied single molecule behaviors of oligonucleotides of different lengths and structures in the αHL pore and their ensuing current signatures in the system (Chapter 4). Finally, harnessed with tools and insights from the nanopore single molecule studies, actual SNP assays were performed in our nanopore system using the polymer tagged ddNTPs and SBE. Chapter 5 discusses the integrated approach where SBE is achieved on a primer-conjugated αHL nanopore and Chapter 6 presents the results using a biotin-streptavidin complex for immobilization of tag molecules in the pore. Overall, this thesis validates adaptation of the nanopore detection system for SNP analysis using the polymer tagged ddNTPs.

The SLC30A2 zinc transporter has been investigated due to its important role in zinc secretion into human milk. SLC30A2 is expressed in mammary epithelial cells, and the presence of genetic variations in this transporter could cause low zinc transport into the milk, leading to Transient Neonatal Zinc Deficiency (TNZD) in newborns. Through bioinformatics analysis 22 SNPs were identified. Therefore, we aim to identify the functional changes caused by 4 SNPs. By subcloning the SLC30A2 open reading frames into the Gateway expression plasmid tagged with red and green fluorescent proteins (mCherry, tGFP). Four SNPs were introduced by mutagenesis and tagged with mCherry. We transfected individual plasmids into mammary epithelial cells (HC11) and observed cellular targeting using epifluorescent imaging. The most common variants located to secreting endosomes and membrane in HC11 cells. Incorrect targeting of SLC30A2 causes mislocalization. It may be possible to identify mothers carrying risk genotypes for infant zinc deficiency.

>Magister Scientiae - MSc / Autism or autism spectrum disorder (ASD) is a very diverse neurological disorder that manifests specifically in children and infants between the ages of two to three years of age. An individual suffering is deemed as autistic and individuals suffering would be classed under the banner of ASD. It is observed that sufferers have impairment in their social and interactive skills. It has both genetic and environmental factors that contribute to its diversity and although the primary cause of autism is still unclear, scientist are investigating both factors. In this study we aimed to investigate the molecular genetics of autism in the South African (SA) population. This was done in two parts, a genetic association study and afunctional genomics (proteomic study). An association study of the 2 single nucleotide polymorphisms (SNPs) of the Cadherin 10 type II gene (CDH10) (rs4307059 and rs4327572) was investigated in the SA healthy and autistic population. The proteomic approach was used to determine the differential expression of genes of the healthy population and compared to the autistic population of African descent. In both parts of the project, objectives were achieved. The SNPs were successfully genotyped however no association was determined for autism in the SA population. The urine protein profiles with 1 dimensional (1D) and 2dimensional (2D) Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis (SDSPAGE)generated in this study has revealed the following proteins, Uromodulin, Vitelline membrane outer layer protein homologue, kinninogen-1, Alpha-1-Antitrypsin, Ig Kappa chain region C, and CD59 glycoprotein that require further investigation. The results indicated that six of the identified proteins were expressed in both groups but were found to be either quantitatively or statistically significant. However, a statistically significant difference was observed in the expression of one protein (Uromodulin) which was observed to be expressed in the healthy group but absent in the experimental group. However further investigation is required validation of these findings.

Thesis (MMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The aetiology of multiple sclerosis (MS) remains largely unknown due to the multifactorial nature of disease susceptibility determined by both environmental and genetic factors. Progress has been made in identifying the genetic component of MS , as well as the possible interactions with the environment. In this study single nucleotide polymorphisms (SNPs) in the FTO (rs9939609, Intron 1 T>A), MTR (rs1805087, 2756 A>G), MTRR (rs1801394, 66 A>G), MTHFR (rs1801133, 677 C>T and rs1801131, 1298 A>C) and COMT (rs4680, 472 G>A) genes involved in the methylation metabolic pathway were studied in the context of MS. The overall objective of this study was to elucidate the mechanism underlying raised homocysteine levels in MS patients. The specific aims were 1) to analytically validate high throughput real-time polymerase chain reaction (RT-PCR) genotyping assays for the 6 selected SNPs against direct sequencing as the gold standard for 2) possible integration into a pathology-supported genetic testing strategy aimed at improved clinical management of MS. The study population included a total of 114 unrelated Caucasian MS patients (98 females and 16 males) and 195 unrelated Caucasian control individuals without a diagnosis of neurological disease (128 females and 67 males). A novel finding of this study was that the risk-associated FTO rs9939609 A-allele was associated with raised homocysteine levels (p=0.003) in patients diagnosed with MS, but not in controls. Furthermore, homocysteine levels correlated significantly with bo dy mass index (BMI) (p=0.046) and total cholesterol levels (p=0.048). Both homocysteine (p=0.011) and BMI (p=0.017) were significantly reduced with increasing intake of folate in the diet, while high saturated/trans fat intake correlated significantly with increased BMI (p<0.001). High physical activity correlated with reduced BMI (p<0.006) in the study population, adjusted for age, gender and disease status. Daily intake of at least five fruit and vegetable portions and the COMT rs4680 (472 G>A) AA genotype had a favourable lowering effect on MS disability as assessed by the expanded disability status scale (EDSS) (p=0.035), while smoking increased MS disability significantly (p<0.001). All SNPs studied were found to be in Hardy-Weinberg equilibrium (HWE), with no significant differences detected between patients and control individuals in genotype distribution or allele frequencies. This study has shown for the first time that the underlying disease process of MS moderates the effect of the FTO rs9939609 polymorphism on homocysteine levels , which is consistent with the role of FTO in demethylation and epigenetic changes. Identification of FTO rs9939609 reinforces the importance of adequate folate intake in the diet that can be assessed accurately with use of the Medical History and Lifestyle Questionnaire applied in this study. Finally, the finding that raised homocysteine levels and BMI are significantly influenced by lifestyle factors such as diet and physical activity in our study cohort , offers a solution to counteract the detrimental effects of genetic risk factors contributing to the development of these established vascular risk factors for MS. Combining this information with FTO rs9939609 and COMT rs4680 genotyping may in future translate into a comprehensive pathology supported genetic testing strategy aimed at improved risk management and quality of life in MS patients. / AFRIKAANSE OPSOMMING: Die etiologie van meervoudige sklerose (MS) is grootliks onbekend as gevolg van die multifaktoriale aard van siekte vatbaarheid wat bepaal word deur beide genetiese en omgewingsfaktore. Vordering is reeds gemaak in die identifisering van die genetiese component van MS, asook moontlike interaksie met die omgewing. In hierdie studie is enkel nukleotied polimorfismes (SNPs) in die FTO (rs9939609, Intron 1 T > A), MTR (rs1805087, 2756 A> G), MTRR (rs1801394, 66 A> G), MTHFR (rs1801133, 677 C > T en rs1801131, 1298 A> C) en COMT (rs4680, 472 G > A) gene, wat betrokke is in die metilering metaboliese padweg, in die konteks van MS bestudeer. Die oorhoofse doel van hierdie studie was om die onderliggende meganisme betrokke by verhoogde homosisteïen vlakke in MS pasiënte uit te lig. Die spesifieke doelwitte was 1) om die analitiese geldigheid van die hoë deurvoer riëeltyd polymerase kettingreaksie (RT-PCR) genotipering metode soos toegepas vir die 6 geselekteerde SNPs te bevestig teen direkte DNA volgorde bepaling as die goue standaard, vir 2) moontlike integrasie in 'n patologie-gesteunde genetiese toetsing (PSGT) stategie wat gemik is op verbeterde kliniese hantering van MS. Die studiepopulasie bestaan uit 'n totaal van 114 nie-verwante Kaukasiese MS pasiënte (98 vroue en 16 mans) en 195 nie-verwante Kaukasiese kontroles sonder ‘n diagnose van neurologiese siektes (128 vroue en 67 mans). 'n Nuwe bevinding van hierdie studie was dat die risiko-verwante FTO rs9939609 A- alleel geassosieer was met verhoogde homosisteïen vlakke (p = 0,003) in pasiënte gediagnoseer met MS, maar nie in kontroles nie. Homosisteïen vlakke was verder beduidend geassosieer met liggaamsmassa-indeks (BMI) (p=0,046) en totale cholesterol vlakke (p=0.048). Beide homosisteïen (p=0,011) en BMI (p=0,017) het aansienlik verminder met 'n hoër inname van folaat in die dieet, terwyl 'n hoë versadigde/trans vet en koolhidrate inname beduidend gekorreleer het met 'n verhoogde BMI (p <0.001). Hoë fisiese aktiwiteit was gekorreleer met 'n verminderde BMI (p< 0.006) in die gekombineerde groep, aangepas vir die ouderdom, geslag en MS diagnose. Daaglikse inname van ten minste vyf vrugte en groente porsies en die COMT rs4680 (472 G>A) AA genotipe het 'n gunstige uitwerking op vermindering van gestremdheid gehad, soos bepaal deur die uitgebreide gestremdheid status skaal (EDSS) (p=0,035), terwyl rook MS gestremdheid beduidend verhoog het (p <0.001). Alle SNPs bestudeer was in Hardy-Weinberg ewewig (HWE), met geen beduidende verskille waargeneem in genotipe verspreiding of alleelfrekwensies tussen pasiënte en kontroles nie. Hierdie studie het vir die eeste keer aangetoon dat ‘n diagnose van MS die effek van die FTO rs9939609 polimorfisme op homosisteïen vlakke modereer, wat ooreenstem met die rol van FTO in demetilering en epigenetiese veranderinge. Identifikasie van FTO rs9939609 versterk die belangrikheid van genoegsame folaat inname in die dieet wat akkuraat gemeet kon word deur gebruik te maak van die Mediese Geskiedenis en Leefstyl Vraelys soos toegepas in hierdie studie. Ten slotte, die bevinding dat verhoogde homosisteïen vlakke en BMI statisties betekenisvol beïnvloed word deur leefstylfaktore soos dieet en fisiese aktiwiteit in ons studie populasie, verskaf 'n oplossing om die genetiese bydrae tot hierdie gevestigde vaskulêre risikofaktore vir MS teen te werk. Kombinasie van hierdie inligting met FTO rs9939609 en COMT rs4680 genotipering kan moontlik in die toekoms benut word as deel van 'n omvattende patologie- gesteunende genetiese toetsing strategie wat daarop gemik is om die risikobestuur en kwaliteit van lewe te verbeter in MS pasiënte.

Asthma is a common complex disease that affects millions of people around the world. Studies indicate the increase in prevalence of asthma worldwide during the past century and report asthma as an important cause of morbidity and mortality. Asthma can be considered as an important health condition in the UK that ranks amongst the countries with the highest rate of asthma prevalence, hospital admissions and mortality due to asthma. Asthma is caused by a combination of genetic and environmental factors. Genetics has an important role in development of asthma with the heritability of around 70% in most studies. To date, more than 100 asthma . associated genes have been identified but they account for only a small proportion of the heritability of asthma. The centerpiece of this thesis is the investigation of genetic association of cystatin and cathepsin genes with asthma and associated phenotypes including atopy and IgE levels. Cathepsinsl cystatins, as proteases and the related antiproteases have been suggested to have a role in airway remodeling. The investigation included three phases; initial association study, replication study in two independent samples sets and complementary analyses. Three sample panels were used in the studies; AUS1/UK1, MRC- AlMRC-E and DLM-4264. The results of this work identified CSTL 1 (cystatin like-1) associated with asthma and IgE levels.

Background: Essential hypertension is regarded as a complex disease, the phenotype of which results from interactions between numerous genes and environmental factors. Genome-wide association studies of blood pressure (BP) and hypertension have been developed to explore the potential genes involved in blood pressure and identified a number of trait-associated variants. Among those variants, single nucleotide polymorphisms (SNPs) rs1173771 (G/A) and rs1421811 (G/C) are located at the natriuretic peptide receptor C (NPR3) gene locus. Their major alleles are related with blood pressure elevation. Studies have implicated NPR-C in mediating some of the cardio-protective actions of natriuretic peptides and its direct involvement in the pathogenesis of hypertension. However, the precise role of these association between genetic variants at NPR3 and blood pressure control has not been elucidated. Objective: To functionally characterise the effect of BP-associated SNPs at the NPR3 gene locus in the context of BP regulatory pathways. Methods: Primary human umbilical artery smooth muscle (HUASMCs) and vein endothelial (HUVECs) cells were genotyped for BP-associated NPR3 variants. Endogenous mRNA and protein expression levels were assessed by qRT-PCR, allelic expression imbalance assay and western blotting. Open chromatin regions were assayed using formaldehyde-assisted isolation of regulatory elements (FAIRE). Interaction between variants flanking region with nuclear protein was detected by electrophoretic mobility shift assay (EMSA). Cell proliferation and migration were 4 determined by cell counting and scratch assays. Angiotensin II (Ang II)-induced calcium flux was evaluated using the intracellular fluorescent probe. Results: The BP-elevating allele of the NPR3 variants in rs1173771 linkage disequilibrium (LD) block was associated with lower endogenous mRNA and protein levels in HUASMCs. This is consistent with the finding that BP-elevating allele is less located within open chromatin. The decreased NPR3 expression in HUASMCs carrying the BP-elevating allele is associated with increased cell proliferation and intracellular calcium flux in response to Ang II stimulation. No differences in migration rates were detected. No genotype-dependent characteristics were observed in HUVECs NPR3 expression and cell proliferation. Moreover, RT-PCR showed a linkage between of the BP-elevating allele of the NPR3 variants in rs1421811 LD block and lower endogenous mRNA in HUASMCs. Intracellular calcium flux detection also revealed a trend of higher response to Ang II stimulation in BP-elevating allele homozygous HUASMCs. However, No genetic differences were detected in proliferation and migration rates of HUASMCs, and HUVECs NPR3 expression and cell proliferation studies did not present any significant genotype-dependent association. Conclusions: This study has identified a potential mechanism for BP-associated SNPs at NPR3 locus to influence BP predominantly via an effect on vascular smooth muscle cell behaviours.

No description available.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and subsequent destruction of synovial joints. Although its causes remain largely unknown, a substantial genetic contribution is known to exist. During the last decades the benefits of early aggressive treatment have become evident, and more potent therapeutic options have become available. These advances have increased the demands for rapid accurate diagnosis and prognostic markers of disease course and therapy response. The ‘rheumatoid factor’ (RF) has long been used as a diagnostic and prognostic marker of RA. In this thesis, the utility of measuring antibodies to cyclic citrullinated peptides (CCP) was investigated. In a population-based arthritis incidence study, 69 very early arthritis patients (symptom duration &lt; 3 months) were identified. The anti-CCP test, performed at baseline and related to diagnosis at the 2-year follow-up, had a diagnostic specificity for RA of 96% and a sensitivity of 44%, both of which were superior to RF. In a prospective cohort of 242 incident cases of RA (symptom duration &lt; 1 year), 64% of the patients tested positive for anti-CCP at baseline (equal to RF). Despite receiving more active anti-rheumatic therapy, the anti-CCP-positive patients had a more aggressive disease course during 3 years as compared to those testing negative. The 158VV genotype of Fcγ Receptor type IIIA (FcγRIIIA), which binds IgG with higher affinity than 158FF, was associated with an increased susceptibility to RA in men, but not in women. Previous studies report conflicting results, and none stratified according to gender. The 158V/F polymorphism of FcγRIIIA was not found to influence outcome of anti-tumour necrosis factor therapy in 282 RA patients, contradicting hints from previous studies. Genetic variation in proteins of the inflammasome, an interleukin-1 (IL-1) regulating intracellular protein complex, is associated with rare autoinflammatory conditions and possibly with Crohn’s disease. In this first study on genetic variation of the inflammasome in RA, we describe a compound polymorphism of the genes CIAS1 and TUCAN that associates both with susceptibility to RA and to the severity of the disease. Hypothetically, these genes may identify a subgroup of RA patients that would benefit from anti-IL-1 therapy. This thesis work emphasizes the benefits of testing for anti-CCP in the diagnosis and outcome prediction in early arthritis. FcγRIIIA genotype is likely to affect RA susceptibility and further work should apply a gender perspective. Inflammasome genetics may influence the risk of developing RA. Additional studies are warranted to settle whether it also identifies a subgroup of RA patients benefiting from IL-1 targeted therapy.

No description available.