Submitted in fulfillment of the requirements for the degree of Master of Technology: Health Sciences, Durban University of Technology, Durban, South Africa, 2013. / Several genes are associated with an increased susceptibility to respiratory diseases, including asthma, which may be exacerbated by ambient air pollution. These genes include the Tumour Necrosis Factor alpha (TNFα) gene and the Cluster of Differentiation 14 (CD14) gene A total of 104 schoolchildren from seven primary schools in a heavily industrialized region of south Durban participated in the study. For the purpose of this study, DNA was extracted from whole blood using the GENTRA Puregene kit. Genotyping for the TNF-308α G/A polymorphism was conducted using restriction fragment length polymorphism (RFLP) polymerase chain reaction (PCR). The CD14 (-159) C/T genotype was determined using real time PCR and Taqman probes (Applied Biosystems). Multiple logistic models and Pearson’s chi-squared tests were used to evaluate the association between any asthma, BHR, atopy, persistent asthma and genotype. Covariate-adjusted generalised estimating equations (Martin et al.) with lags of 1-5 days were used to evaluate genotype effect modification of exposure-response. The TNF-308α variant A allele was quite common in the population, it was detected in more than forty percent of the population and with an allelic frequency of 0.24. Similarly almost 38% of the population carried the variant CD14 (-159) T allele, with an allelic frequency of 0.24. TNF-308α G/A and CD14 (-159) C/T polymorphisms were not associated significantly with asthma, and its related respiratory phenotypes. In addition there was no association detected between any of the gene polymorphisms and the levels of their respective cytokine proteins. Increased TNFα levels were associated with persistent asthma. On the other hand lower sCD14 levels were associated with atopy in children. There was a significant relationship between TNF- α levels and acute asthma (p=0.03) and sCD14 levels and atopy (p=0.04) GEE models showed that the TNF- 308-α A allele carriers had a greater deterioration of lung function post pollution exposure to SO2 (intraday variability FEV1 readings lag 2) β= 2.62, CI (0.51, 4.71) p= 0.02 and p (interaction=0.03). There was a statistically significant gene environment interaction with NO in individuals who were carriers of the TNF- A allele (Nadir of PF readings lag 2: β= -12.3, CI (-22.09, -2.51), p=0.01 p (interaction) =0.03.and 5 day average β= - 42.83, CI (-70.11,-15.55), p≤0.005 and p (interaction) =0.01). With analysis of the CD14 gene polymorphism gene environment interaction, adverse effects of SO2 were limited to individuals carrying the C allele of this polymorphism, β= - 1.50, CI (-0.36, 3.37), p=0.01, p (interaction) =0.01. Carriers of the T allele seemed to have a protective effect with NO2 and NO exposure. Intraday variability of FEV1 improved 5 days post exposure to NO2 β= -4.02, CI (-6.52,- 1.53), p=0, p (interact) =0.05. There was also improvement five days post exposure to NO β= -9.42, CI (-12.45, -6.03), p= p≤0.005, p (interact) ≤0.005 There was no association of co-inheritance of the 2 gene polymorphisms, CD14 (-159) C/T and TNF-308α G/A, and protein expression or respiratory phenotype. The GEE model results were consistent with modification of air pollutant-pulmonary function relationships by proinflammatory cytokine associated genotypes. Results indicate that genetic susceptibility combined with exposure to pollutants causes adverse respiratory effects. This study supports the importance of further investigation on these and other genotype variants involved in inflammation and respiratory linked phenotypes in larger cohorts. / M

HLA (Human Leukocyte Antigen) molecules provide a framework for T-cell recognition of antigenic peptides and thus play an important role in the immune system and defence against pathogens. HLA molecules are encoded for by genes on the short arm of chromosome six in the human genome, a region of over 4000 kilo bases (kb) known as the human major histocompatibility complex (MHC) or HLA complex. According to structural and functional characteristics, the genes of this region have been classified into three families, namely classes ƒ¹, ƒ¹ƒ¹ and ƒ¹ƒ¹ƒ¹. The HLA genes are highly polymorphic and because of this characteristic, it increases the functional range of recognition of different antigens and contributes to immunological specificity. Previous studies on population groups such as Indians and Cambodians, have identified an association between HLA polymorphisms and susceptibility to TB. A large number of different population groups with diverse gene pools reside in South Africa, of which the Cape Coloured population is one. This anthropologically distinct population group¡¦s diverse gene pool originated from founder individuals of the colonizing population, which came from various nations and cultural backgrounds, including Europe, Africa (such as Khoi, San Xhosa, Sotho and East African populations), Madagascar and the Far East. Unusual MHC allele frequencies and haplotypes have been identified in the Cape Coloured population. The Cape Coloureds are highly susceptible to TB and reside in the Western Cape, which has a TB incidence rate that is higher than that of any other province in South Africa. The recently admixed Coloured population is a valuable candidate population for the identification of genes/mutations underlying complex diseases. This study focussed on polymorphisms of class ƒ¹ƒ¹ genes, specifically HLA-DRB, and their possible contribution to disease susceptibility in populations of South Africa. Two questions have been formulated: 1) What knowledge can we gain from the current literature on HLA variants in the different population groups of South Africa and disease susceptibility? 2) Is there an association between alleles of the most polymorphic class ƒ¹ƒ¹ MHC gene, HLA-DRB, and susceptibility or resistance to TB in the Cape Coloured population? These two primary questions have been addressed by: 1) Reviewing the literature concerning HLA alleles in diverse population groups in South Africa and their contribution to disease susceptibility. Chapter 2 addresses this objective and is written in the format of a review article to facilitate its future publication, 2) Conducting a TB case-control study, typing HLA-DRB in Cape Coloured individuals residing in the Western Cape to investigate the possible association between TB susceptibility or resistance and specific DRB alleles. The HLA-DRB1, DRB3 and DRB4 typing by means of PCR-SSP (polymerase chain reaction ¡V sequence specific primers) was done on DNA isolated from 106 TB patients and 107 controls from the Cape Coloured population. The results obtained for this experimental investigation is presented (also in publication format) in Chapter 3. Summary of main findings: 1) The literature overview of publications describing HLA related disease association in the different population groups in South Africa (Chapter 2), revealed that unique alleles contributing to susceptibility of various diseases have been identified in some South African populations. The large number of ethnic groups in South Africa and unique populations such as the Cape Coloureds provide a genetic resource, which has the potential to be utilized for candidate gene hunting. 2) A weak association between susceptibility for TB and HLA-DRB1*0301-0302 (DR3) and HLA-DRB3*0101-0301 (DR52) exists in the Cape Coloured population (Chapter 3). Since the South African population consists of a large number of different population groups with diverse gene pools, a unique opportunity to study disease predispositions among specific population groups with diverse genetic make-up is presented. With these different populations, often residing in a common environment, the interplay of environmental and genetic factors in disease development could be studied. For example, HLA typing of these populations could clarify the extent to which inter-population HLA variation contributes towards disease susceptibility. The identification of certain HLA-DRB alleles potentially contributing to TB susceptibility, could lead to an understanding of the differential factors involved in disease susceptibility and in turn lead to the understanding of the fundamental mechanisms involved. This could result in therapeutic approaches and treatments that will be advantageous to the population concerned. / Prof. L. Bornman

Indiana University-Purdue University Indianapolis (IUPUI) / Vincristine is metabolized by the cytochrome P450 3A subfamily of enzymes possibly including CYP3A5, a genetically polymorphic enzyme. The contribution of CYP3A5 to the metabolism of vincristine was quantified by various in vitro models: cDNA-expressed enzymes, human liver microsomes, and human hepatocytes. With these models, the major CYP metabolite of vincristine, M1, was identified and extensively characterized. The rates of M1 formation in the cDNA-expressed enzyme models were at least 7-fold higher with CYP3A5 than CYP3A4; approximately 90% of the hepatic metabolism was predicted to be CYP3A5-mediated. For human liver microsomes with high CYP3A5 expression, the CYP3A5 contribution was substantial, approximately 80%. Human hepatocytes with at least one CYP3A5*1 allele also metabolized vincristine, albeit at a slower rate (10-fold) than human liver microsomes. The CYP3A5 low-expressing hepatocytes did not metabolize vincristine. We conclude that for high CYP3A5 expressers, the majority of the CYP metabolism is mediated by CYP3A5. By in vitro/in vivo scaling with microsomes, the hepatic clearances of high CYP3A5 expressers are predicted to have a 5-fold higher hepatic clearance than low expressers. However, the role of metabolism in the systemic clearance of vincristine is unknown. To study the disposition of vincristine in vivo, a sensitive and selective LC/MS/MS assay was validated for the quantification of vincristine and M1 quantification in human plasma. Vincristine and M1 were identified and quantified in select pediatric plasma and urine samples. For future large-scale clinical studies, the vincristine and M1 concentrations in plasma will be quantified to understand the role of CYP3A5 genotype in vincristine pharmacokinetics. For patients that are CYP3A5 high expressers, the systemic clearance of vincristine may be higher than that of low CYP3A5 expressers. Thus, CYP3A5 genotype may be an important determinant of inter-individual variability in clinical outcomes.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. Johannesburg, 17 May 1994. / Two black South African subjects presenting with hereditary elliptocytosis were investigated and the defect defined as Spol/74, a previously described spectrin variant leading to defective heterodimer self-association and instability of the erythrocyte membrane. [Abbreviated Abstract. Open document to view full version]

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published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Master / Master of Philosophy