Thesis (MSc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Progressive familial heart block type I (PFHBI) is a cardiac ventricular conduction disorder of unknown cause associated with risk of sudden death, which has been described in several South African families. Clinically, PFHBI is characterised by right bundle branch block on ECG, which may progress to complete heart block, necessitating pacemaker implantation. The disease shows an autosomal dominant pattern of inheritance with evidence of genetic anticipation. Using genetic linkage analysis, the PFHBI-causative gene was mapped to a 10 eentimorgan (cM) gene-rich area of chromosome (C) 19q13.3, which has, subsequently, been reduced to 7cM by fine mapping with polymorphic dinucleotide (CA)n short tandem repeat (STR) markers. Several attractive candidate genes, including muscle glycogen synthase (GSY 1) and histidine-rich calcium binding protein (HRC), lie within this region. The aim of the present study was two-fold: 1) to identify and characterise tetranucleotide (AAAT)n STRs within the PFHBI critical region that could be developed as polymorphic markers for use in genetic fine mapping and 2) to screen selected regions of GSY 1and HRC, positional candidate genes, for the presence ofPFHBI-causing mutation(s). Cosmids harbouring CI9q13.3 insert DNA were screened for the presence of (AAAT)n STRs by dot blot and Southern blot hybridisation using a radiolabelled (AAAT)lO oligonucleotide probe. To characterise the harboured (AAAT)n STRs, the positively hybridising fragments identified by Southern blot were sub-cloned, sequenced and primers designed from the unique repeat-flanking sequences. These primers were used to genotype the (AAAT)n repeat locus to assess its polymorphic nature in a panel of unrelated individuals. Alternatively, vectorette PCR, a rapid method of identifying repeat sequences and obtaining the flanking sequences in large inserts, was employed to develop polymorphic markers from the positively hybridising clones. Selected exons of GSY1 and HRC were screened for the presence of potentially disease-causing mutations by PCR-SSCP analysis and direct sequencing, respectively, in PFHBI-affected and unaffected family members. Of the available cosmid clones that gave strong signals on dot blot and Southern blot hybridisation, three, 29395, 24493 and 20381, were located within the critical PFHBI area and were used for marker development. An interrupted (AAAT)n repeat motif (n less than 5) was identified in cosmid 29395, however, the repeat locus was not polymorphic in the tested population. No (AAAT)n motif, single or repeated was observed in the partial sequence of the sub-cloned fragment of cosmid 24493. Using vectorette peR, no repeated (AAAT)n motif was identified on sequencing the generated products in either cosmid 24493 or 2038l. However, diffuse single AAAT motifs were detected in both cosmids. Exons 4, 5, 11, 12 and 16 of GSY 1, containing domains that are conserved across species, and the conserved eterminus- encoding exons 2-6 of HRC were selected for screening for potential PFHBI-causing mutation(s). However, no sequence variations were detected. The interrupted (AAAT)n repeat identified in cosmid 29395 was not polymorphic, which confirmed reports that complex repeats, especially those containing AAAT motifs of less than 6 repeats, are not polymorphic. One possible explanation for the absence of a repeated AAAT motif in cosmids 24493 and 20381, which both gave positive hybridisation signals, is that the low annealing temperature of the AfT -rich repeat-anchored primers used in vectorette peR may have resulted in transient annealing to the diffuse single AAAT motifs detected on sequencing. The screened regions of candidate genes GSYI and HRC were excluded from carrying the disease-causing mutation(s). The availability of new sequence data generated by the Human Genome Project will influence future strategies to identify the PFHBI gene. Electronic searches will allow identification of STR sequences for development of polymorphic markers and gene annotation will allow selection of new candidate genes for mutation screening. / AFRIKAANSE OPSOMMING: Sien volteks vir opsomming

Doctor of Philosophy(PhD) / Atherosclerosis manifests a state of increased oxidative stress characterized by comparable lipid and protein oxidation in the affected arterial wall. While oxidative modification of low density lipoprotein (LDL) has been extensively studied, increasing attention has been focused recently on oxidation of high-density lipoproteins (HDL) and its functional consequences in relation to atherosclerosis. Oxidative modification is thought to generate “dysfunctional” HDL that has lost anti-atherosclerotic activities, including the ability to remove cholesterol from lipid-laden cells. Therefore, there has been much interest in the detection of oxidized HDL. Unfortunately, available methods to detect oxidized HDL are limited at present, in part because oxidative modification of HDL is a complex process and ‘oxidized HDL’ is not a chemically defined entity. What is known however is that conversion of methionine (Met) residues of apolipoprotein (apo) A-I to methionine sulfoxide (MetO) is a process that occurs commonly as HDL undergoes oxidative modification. For example, human apoA-I+16 (containing MetO86 or MetO112) and apoA-I+32 (MetO86 plus MetO112) are generated when apoA-I reacts with lipid hydroperoxides formed as a consequence of the lipoprotein being exposed to 1e−oxidants. The formation of MetO in apoA−I induced by 2e−oxidants (i.e., hydrogen peroxide, hypochlorous acid or myeloperoxidase/hydrogen peroxide/chloride system) is associated with an impaired ability of the apolipoprotein to facilitate reactions relevant to reverse cholesterol transport. In addition, a previous study has suggested the plasma content of apoA-I+32 to be increased in certain subjects that have an increased risk to develop cardiovascular disease (CVD). Moreover, the MetO content in circulating, HDL−associated apoA−I is elevated in type 1 diabetes, a disorder commonly associated with increased oxidative stress and a risk factor for atherosclerosis. Therefore, in the present study, an existing HPLC method was applied to HDL samples from the Fletcher−Challenge study, a nested case control study, to test the potential usefulness of MetO-containing apoA-I as a marker of oxidative stress and/or CVD in a general population. Plasma samples whose HDL contained detectable apoA-I+16 and/or apoA-I+32 had significantly elevated levels of F2-isoprostanes, a marker of in vivo lipid oxidation, consistent with MetO-containing apoA-I being a useful marker of in vivo protein oxidation. Despite this however, there was no significant difference between controls and cases in their concentrations of HDL apoA-I+16 and apoA-I+32 or F2-isoprostanes, suggesting that markers of protein and lipid oxidation are not associated with the risk of coronary heart disease (CHD) in this general population. A limitation of the Fletcher−Challenge study was that only 22% of the 534 HDL samples analyzed contained apoA-I+16 and/or apoA-I+32. In addition, the HPLC−based method used is expensive and time−consuming and may lack the sensitivity needed for apolipoproteins to clinical studies. Thus, a mouse monoclonal anti-human apoA-I+32 antibody (MOA−1) was raised using HPLC−purified apoA-I+32 as immunogen. A sensitive ELISA was then developed using a commercial anti-human apoA-I monoclonal antibody as capture and biotinylated MOA−1 as detection antibody, respectively. The assay detected lipid−free HPLC−purified human apoA-I+32 in a concentration-dependent manner and with a significantly lower limit of detection (i.e., 3 ng/mL) than the HPLC method (1 μg/mL). The ELISA also detected lipid-free apoA-I modified by 2e-oxidants (hydrogen peroxide, hypochlorous acid, peroxynitrite), and HDL oxidized by 1e- or 2e-oxidants and present in buffer or human plasma. Moreover, the extent of recognition of MetO by MOA−1 increased with increasing numbers of MetO in apoA−I, as assessed by the experiments with H2O2−oxidized forms of apoA−I mutants, in which one, two or three Met residues were replaced with Leu. Their detection was concentration-dependent, reproducible, and exhibited a linear response over a physiologically plausible range of concentrations of oxidized HDL. In contrast, MOA-I failed to recognize native apoA-I, native apoA-II, apoA-I modified by hydroxyl radicals or metal ions, or LDL modified by 2e-oxidants. Furthermore, MOA−1 did not detect other Met−containing proteins oxidized by either hypochlorous acid or hydrogen peroxide. Taken together, the results showed that recognition of oxidized proteins by MOA−1 is limited to MetO contained in apoA−I. Finally, in a pilot study, plasma samples obtained from subjects with coronary artery disease (CAD) proven by angiography, and samples from CAD patients undergoing percutaneous coronary intervention (PCI) were analyzed by the ELISA. The preliminary data obtained showed elevated levels of MetO-containing apoA-I in plasma samples of CAD patients compared to those of corresponding control subjects. Unexpectedly, levels of MetOcontaining apoA-I decreased PCI compared to before PCI. A possible explanation for these results is that HDL−associated apoA−I become displaced by acute phase proteins, such as serum amyloid A, in response to PCI. In summary, the ELISA developed here specifically detects apoA-I containing MetO in HDL and human plasma. As such it may provide a useful tool for investigating the relationship between oxidized HDL and CAD.

Doctor of Philosophy(PhD) / Atherosclerosis manifests a state of increased oxidative stress characterized by comparable lipid and protein oxidation in the affected arterial wall. While oxidative modification of low density lipoprotein (LDL) has been extensively studied, increasing attention has been focused recently on oxidation of high-density lipoproteins (HDL) and its functional consequences in relation to atherosclerosis. Oxidative modification is thought to generate “dysfunctional” HDL that has lost anti-atherosclerotic activities, including the ability to remove cholesterol from lipid-laden cells. Therefore, there has been much interest in the detection of oxidized HDL. Unfortunately, available methods to detect oxidized HDL are limited at present, in part because oxidative modification of HDL is a complex process and ‘oxidized HDL’ is not a chemically defined entity. What is known however is that conversion of methionine (Met) residues of apolipoprotein (apo) A-I to methionine sulfoxide (MetO) is a process that occurs commonly as HDL undergoes oxidative modification. For example, human apoA-I+16 (containing MetO86 or MetO112) and apoA-I+32 (MetO86 plus MetO112) are generated when apoA-I reacts with lipid hydroperoxides formed as a consequence of the lipoprotein being exposed to 1e−oxidants. The formation of MetO in apoA−I induced by 2e−oxidants (i.e., hydrogen peroxide, hypochlorous acid or myeloperoxidase/hydrogen peroxide/chloride system) is associated with an impaired ability of the apolipoprotein to facilitate reactions relevant to reverse cholesterol transport. In addition, a previous study has suggested the plasma content of apoA-I+32 to be increased in certain subjects that have an increased risk to develop cardiovascular disease (CVD). Moreover, the MetO content in circulating, HDL−associated apoA−I is elevated in type 1 diabetes, a disorder commonly associated with increased oxidative stress and a risk factor for atherosclerosis. Therefore, in the present study, an existing HPLC method was applied to HDL samples from the Fletcher−Challenge study, a nested case control study, to test the potential usefulness of MetO-containing apoA-I as a marker of oxidative stress and/or CVD in a general population. Plasma samples whose HDL contained detectable apoA-I+16 and/or apoA-I+32 had significantly elevated levels of F2-isoprostanes, a marker of in vivo lipid oxidation, consistent with MetO-containing apoA-I being a useful marker of in vivo protein oxidation. Despite this however, there was no significant difference between controls and cases in their concentrations of HDL apoA-I+16 and apoA-I+32 or F2-isoprostanes, suggesting that markers of protein and lipid oxidation are not associated with the risk of coronary heart disease (CHD) in this general population. A limitation of the Fletcher−Challenge study was that only 22% of the 534 HDL samples analyzed contained apoA-I+16 and/or apoA-I+32. In addition, the HPLC−based method used is expensive and time−consuming and may lack the sensitivity needed for apolipoproteins to clinical studies. Thus, a mouse monoclonal anti-human apoA-I+32 antibody (MOA−1) was raised using HPLC−purified apoA-I+32 as immunogen. A sensitive ELISA was then developed using a commercial anti-human apoA-I monoclonal antibody as capture and biotinylated MOA−1 as detection antibody, respectively. The assay detected lipid−free HPLC−purified human apoA-I+32 in a concentration-dependent manner and with a significantly lower limit of detection (i.e., 3 ng/mL) than the HPLC method (1 μg/mL). The ELISA also detected lipid-free apoA-I modified by 2e-oxidants (hydrogen peroxide, hypochlorous acid, peroxynitrite), and HDL oxidized by 1e- or 2e-oxidants and present in buffer or human plasma. Moreover, the extent of recognition of MetO by MOA−1 increased with increasing numbers of MetO in apoA−I, as assessed by the experiments with H2O2−oxidized forms of apoA−I mutants, in which one, two or three Met residues were replaced with Leu. Their detection was concentration-dependent, reproducible, and exhibited a linear response over a physiologically plausible range of concentrations of oxidized HDL. In contrast, MOA-I failed to recognize native apoA-I, native apoA-II, apoA-I modified by hydroxyl radicals or metal ions, or LDL modified by 2e-oxidants. Furthermore, MOA−1 did not detect other Met−containing proteins oxidized by either hypochlorous acid or hydrogen peroxide. Taken together, the results showed that recognition of oxidized proteins by MOA−1 is limited to MetO contained in apoA−I. Finally, in a pilot study, plasma samples obtained from subjects with coronary artery disease (CAD) proven by angiography, and samples from CAD patients undergoing percutaneous coronary intervention (PCI) were analyzed by the ELISA. The preliminary data obtained showed elevated levels of MetO-containing apoA-I in plasma samples of CAD patients compared to those of corresponding control subjects. Unexpectedly, levels of MetOcontaining apoA-I decreased PCI compared to before PCI. A possible explanation for these results is that HDL−associated apoA−I become displaced by acute phase proteins, such as serum amyloid A, in response to PCI. In summary, the ELISA developed here specifically detects apoA-I containing MetO in HDL and human plasma. As such it may provide a useful tool for investigating the relationship between oxidized HDL and CAD.

Ce travail a pour objectifs de déterminer la prévalence des maladies coronariennes (MC) au Liban, de décrire le profil des personnes qui en souffrent, d'explorer la tendance de l'hypertension artérielle (HTA), du diabète et de la dyslipidémie à se regrouper chez les mêmes individus, et enfin d'évaluer le contrôle de l'HTA. Une étude transversale fut menée au niveau national. Un échantillon de 1515 individus fut choisi. Des données sur les caractéristiques sociodémographiques, sur les facteurs de risque (FDR) les plus importants de la MC et l'histoire médicale des participants furent collectées. Le clustering des FDR biologiques (FDRbio) fut examiné par la méthode du ratio de cas observés sur les cas attendus. Notre travail a révélé une MC prématurée avec une prévalence de 13.4%, a confirmé la pertinence des FDR classiques dans la population libanaise, a montré que les FDRbio se regroupent plus fréquemment qu'attendu et a révélé un contrôle insuffisant de l'HTA. / This thesis aimed to determine the prevalence of coronary heart diseases (CHD) in Lebanon, to describe the profile of people who suffer from it, to explore the tendency of hypertension, diabetes, and dyslipidemia to cluster in the same individuals beyond what could be attributed to chance, and finally to evaluate the control of high blood pressure. A national cross-sectional study was conducted by choosing a sample of 1515 individuals. We collected data on sociodemographic characteristics of the participants, their medical history and the most important risk factors (RFs) for CHD. The clustering of biological RFs (bioRFs) was assessed using the observed to expected ratio method. Our work revealed premature CHD with a prevalence of 13.4%, confirmed the relevance of classic RFs of CHD and their applicability to the Lebanese population, showed that bioRFs cluster more often than expected, and revealed an insufficient control of hypertension.

The purpose of this study was to describe the prevalence of increasing coronary heart diseases (CHD) among the Indian population in Chatsworth, Kwa Zulu-Natal. A quantitative, descriptive survey was conducted using non-probability purposive sampling. Respondents were sampled from four medical wards at the RK Khan Hospital. The results of this study indicate that the Indian population is at risk for the development of CHD. Although the increasing rate could not be attributed to a single risk factor, 72% of the respondents had a familial history of CHD and 52% had both parents at risk of CHD. Implementation of the primary prevention programmes recommended by the American Heart Association might reduce the prevalence among the Indian population. / Health Studies / M.A. (Health Studies)

The purpose of this study was to describe the prevalence of increasing coronary heart diseases (CHD) among the Indian population in Chatsworth, Kwa Zulu-Natal. A quantitative, descriptive survey was conducted using non-probability purposive sampling. Respondents were sampled from four medical wards at the RK Khan Hospital. The results of this study indicate that the Indian population is at risk for the development of CHD. Although the increasing rate could not be attributed to a single risk factor, 72% of the respondents had a familial history of CHD and 52% had both parents at risk of CHD. Implementation of the primary prevention programmes recommended by the American Heart Association might reduce the prevalence among the Indian population. / Health Studies / M.A. (Health Studies)