Doctoral Degrees (Molecular Biology and Human Genetics)
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Browsing Doctoral Degrees (Molecular Biology and Human Genetics) by browse.metadata.advisor "Corfield, Valerie A."
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- ItemA candidate and novel gene search to identify the PFHBII-causative gene(Stellenbosch : University of Stellenbosch, 2004-12) Fernandez, Pedro (Pedro Wallace); Corfield, Valerie A.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biochemical Sciences.ENGLISH ABSTRACT: Heart failure due to cardiomyopathy or cardiac conduction disease is a major cause of mortality and morbidity in both developed and developing countries. Although defined as separate clinical entities, inherited forms of cardiomyopathies and cardiac conduction disorders have been identified that present with overlapping clinical features and/or have common molecular aetiologies. The objective of the present study was to identify the molecular cause of progressive familial heart block type II (PFHBII), an inherited cardiac conduction disorder that segregates in a South African Caucasian Afrikaner family (Brink and Torrington, 1977). The availability of family data tracing the segregation of PFHBII meant that linkage analysis could be employed to identify the chromosomal location of the disease-causative gene. Human Genome Project (HGP) databases have provided additional resources to facilitate the identification of positional candidate genes. Clinical examinations were performed on individuals of the PFHBII-affected family, and, where available, clinical records of subjects examined in a previous study by Brink and Torrington (1977) were re-assessed. Retrospective data suggested redefining the classification of PFHBII. Subsequently, linkage analysis was used to test described dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM) and cardiac conduction-causative loci on chromosomes 1, 2, 3, 6, 7, 9, 11, 14, 15 and 19 for their involvement in the development of PFHBII. Once a locus was mapped, bioinformatics tools were applied to identify and prioritise positional candidate genes for mutation screening. The retrospective and prospective clinical study redefined PFHBII as a cardiac conduction and DCM-associated disorder and simultaneously allowed more family members to be traced.Fortuitously, candidate loci linkage analysis mapped the PFHBII locus to chromosome 1q32, to a region that overlapped a previously described DCM-associated disorder (CMD1D), by the generation of a maximum pairwise lod score of 3.13 at D1S3753 (theta [θ]=0.0) and a maximum multipoint lod score of 3.7 between D1S3753 and D1S414. However, genetic fine mapping and haplotype analysis placed the PFHBII-causative locus distal to the CMD1D locus, within a 3.9 centimorgan (cM) interval on chromosome 1q32.2-q32.3, telomeric of D1S70 and centromeric of D1S505. Bioinformatics analyses prioritised seven candidate genes for mutation analysis, namely, a gene encoding a potassium channel (KCNH1), an extracellular matrix protein (LAMB3), a protein phosphatase (PPP2R5A), an adapter protein that interacts with a cytoskeletal protein (T3JAM), a putative acyltransferase (KIAA0205) and two genes encoding proteins possibly involved in energy homeostasis (RAMP and VWS59). The PFHBII-causative mutation was not identified, although single sequence variations were identified in four of the seven candidate genes that were screened. Although the molecular aetiology was not established, the present study defined the underlying involvement of DCM in the pathogenesis of PFHBII. The new clinical classification of PFHBII has been published (Fernandez et al., 2004) and should lead to tracing more affected individuals in South Africa or elsewhere. The identification of a novel disease-causative locus may point toward the future identification of a new DCM-associated aetiology, which, in turn, might provide insights towards understanding the associated molecular pathophysiologies of heart failure.
- ItemA candidate and novel gene search to identify the PFHBII-causative gene(Stellenbosch : Stellenbosch University, 2004-12) Fernandez, Pedro; Corfield, Valerie A.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences.ENGLISH ABSTRACT: Heart failure due to cardiomyopathy or cardiac conduction disease is a major cause of mortality and morbidity in both developed and developing countries. Although defined as separate clinical entities, inherited forms of cardiomyopathies and cardiac conduction disorders have been identified that present with overlapping clinical features and/or have common molecular aetiologies. The objective of the present study was to identify the molecular cause of progressive familial heart block type II (PFHBII), an inherited cardiac conduction disorder that segregates in a South African Caucasian Afrikaner family (Brink and Torrington, 1977). The availability of family data tracing the segregation of PFHBII meant that linkage analysis could be employed to identify the chromosomal location of the disease-causative gene. Human Genome Project (HGP) databases have provided additional resources to facilitate the identification of positional candidate genes. Clinical examinations were performed on individuals of the PFHBII-affected family, and, where available, clinical records of subjects examined in a previous study by Brink and Torrington (1977) were re-assessed. Retrospective data suggested redefining the classification of PFHBII. Subsequently, linkage analysis was used to test described dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM) and cardiac conduction-causative loci on chromosomes 1, 2, 3, 6, 7, 9, 11, 14, 15 and 19 for their involvement in the development of PFHBII. Once a locus was mapped, bioinformatics tools were applied to identify and prioritise positional candidate genes for mutation screening. The retrospective and prospective clinical study redefined PFHBII as a cardiac conduction and DCM-associated disorder and simultaneously allowed more family members to be traced. Fortuitously, candidate loci linkage analysis mapped the PFHBII locus to chromosome 1q32, to a region that overlapped a previously described DCM-associated disorder (CMD1D), by the generation of a maximum pairwise lod score of 3.13 at D1S3753 (theta [θ]=0.0) and a maximum multipoint lod score of 3.7 between D1S3753 and D1S414. However, genetic fine mapping and haplotype analysis placed the PFHBII-causative locus distal to the CMD1D locus, within a 3.9 centimorgan (cM) interval on chromosome 1q32.2-q32.3, telomeric of D1S70 and centromeric of D1S505. Bioinformatics analyses prioritised seven candidate genes for mutation analysis, namely, a gene encoding a potassium channel (KCNH1), an extracellular matrix protein (LAMB3), a protein phosphatase (PPP2R5A), an adapter protein that interacts with a cytoskeletal protein (T3JAM), a putative acyltransferase (KIAA0205) and two genes encoding proteins possibly involved in energy homeostasis (RAMP and VWS59). The PFHBII-causative mutation was not identified, although single sequence variations were identified in four of the seven candidate genes that were screened. Although the molecular aetiology was not established, the present study defined the underlying involvement of DCM in the pathogenesis of PFHBII. The new clinical classification of PFHBII has been published (Fernandez et al., 2004) and should lead to tracing more affected individuals in South Africa or elsewhere. The identification of a novel disease-causative locus may point toward the future identification of a new DCM-associated aetiology, which, in turn, might provide insights towards understanding the associated molecular pathophysiologies of heart failure.
- ItemAn investigation of myosin binding protein C mutations in South Africa and a search for ligands binding to myosin binding protein C(Stellenbosch : University of Stellenbosch, 2004-12) De Lange, W. J. (Willem Jacobus); Corfield, Valerie A.; Moolman-Smook, Johanna C.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences.ENGLISH ABSTRACT: Hypertrophic cardiomyopathy (HCM) is an autosomal dominantly inherited primary cardiac disease. The primary features of HCM are left ventricular hypertrophy, myocardial disarray, fibrosis and an increased risk of sudden cardiac death. To date, more than 264 HCM-causing mutations, occurring in thirteen genes, have been identified. As the vast majority of HCM-causing mutations occur in components of the cardiac sarcomere, HCM has been considered a disease of the cardiac sarcomere. Functional analyses of HCM-causing mutations in sarcomeric protein-encoding genes revealed that HCM-causing mutations have a vast array of effects on contractile function. The discovery of HCMcausing mutations in the gamma two subunit of adenosine monophosphate activated protein kinase highlighted the fact that mutations in non-sarcomeric proteins can also cause HCM and supports a hypothesis that HCM-causing mutations may result in energy wastage leading to energy depletion. Mutations in the cardiac myosin binding protein C (cMyBPC) gene (MYBPC3) are the second most prevalent cause of HCM. cMyBPC is a modular protein that forms an integral part of the sarcomeric thick filament, where it acts as a regulator of thick filament structure and cardiac contractility. Although cMyBPC has been studied extensively, the mechanisms through which it fulfill these functions have remained elusive, largely due to a lack of a comprehensive understanding of its interactions with other sarcomeric components and its quaternary structure. The aims of the present study were, firstly, to screen MYBPC3 for HCM-causing mutations in a panel of HCM-affected individuals and, secondly, to identify the ligands of domains of cMyBPC in which HCM-causing mutations were found.A panel of deoxyribonucleic acid (DNA) samples obtained from unrelated HCM-affected individuals was screened for HCM-causing mutations in MYBPC3, using polymerase chain reaction (PCR)- based single-strand conformation polymorphism method, as well as restriction enzyme digestion, DNA sequencing and reverse transcription PCR techniques. In order to identify the ligands of domains in which HCM-causing mutations were found, yeast two-hybrid (Y2H) candidate-ligandand library-assays were performed. Three novel and two previously described putative HCM-causing mutations were identified in MYBPC3. Data generated in this and other studies, however, suggest that two of these “mutations” are likely to be either polymorphisms, or disease-modifying factors, rather than main-locus HCMcausing mutations. Recent findings showed a specific interaction between domains C5 and C8 of cMyBPC. This finding identified domains C6 or C10 as candidate ligands of domain C7. Y2H-assays revealed a specific C7:C10 interaction. Additional Y2H assays also identified C-zone titin as a ligand of domain C7 and domain C10 as a ligand of domain C3. Several other Y2H assays, however, yielded no known sarcomeric ligands of the N-terminal region of cMyBPC. Identification of the ligands of specific domains of cMyBPC led to the development of detailed models of cMyBPC quaternary structure when cMyBPC is both unphosphorylated and fully phosphorylated. The integration of these models into an existing model of thick filament quaternary structure allows new insights into the functioning of cMyBPC as a regulator of both thick filament structure and cardiac contractility, as well as the pathophysiology of cMyBPC-associated HCM.
- ItemMolecular and functional characterisation of Long QT Syndrome causing genes(Stellenbosch : Stellenbosch University, 2014-04) Hedley, Paula Louise; Corfield, Valerie A.; Moolman-Smook, Johanna C.; Christiansen, Michael; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Ventricular arrhythmias are the most important cause of sudden cardiac death (SCD) among adults living in industrialised nations. Genetic factors have substantial effects in determining population-based risk for SCD and may also account for inter-individual variability in susceptibility. Great progress has been made in identifying genes underlying various Mendelian disorders associated with inherited arrhythmia susceptibility. The most well studied familial arrhythmia syndrome is the congenital long QT syndrome (LQTS) caused by mutations in genes encoding subunits of myocardial ion channels. Not all mutation carriers have equal risk for experiencing the clinical manifestations of disease (i.e. syncope, sudden death). This observation has raised the possibility that additional genetic factors may modify the risk of LQTS manifestations. This study establishes the genetic aetiology of LQTS in South Africa and Denmark through the identification and characterisation of LQTS-causative mutations in five previously identified genes, as well as examining possible novel genetic causes of LQTS in a cohort comprising Danish and British probands. We have functionally characterised several of the mutations identified in this study and examined other cardiac phenotypes that may be explained by variants causing repolarisation disorders.
- ItemMolecular genetic strategies to identify Obsessive-compulsive disorder (OCD) and schizophrenia candidate genes in a South African sub-population group(Stellenbosch : Stellenbosch University, 2007-12) Kinnear, C. J. (Craig John); Moolman-Smook, Johanna C.; Corfield, Valerie A.; Emsley, Robin A.; Stellenbosch University. Faculty of Health Sciences. Dept. of Biomedical Sciences.ENGLISH ABSTRACT: Obsessive-compulsive disorder is a severe, debilitating psychiatric disorder for which the underlying molecular aetiology still remains unclear. Evidence from family studies have suggested that OCD may be caused by a complex interplay of environmental and genetic factors. In order to identify the genetic factors that mediate OCD susceptibility, several genetic association studies have been undertaken, which have yielded inconsistent findings. Moreover, the majority of these studies have focused on a small number of candidate genes that encode components of the serotonin and dopamine neurotransmitter pathways. However, based on the complexity of clinical manifestations observed in OCD, it is likely that its pathogenesis is mediated by a broader complex of interrelated neurotransmitter systems and signal transduction pathways; consequently there is a need to identify and assess novel candidate genes. One method of identifying such novel OCD candidate genes is by utilising knowledge of diseases with phenomenological overlap with OCD, which lend themselves to better genetic dissection through linkage analysis and animal studies. Genetic loci for such disorders, identified though linkage analysis, could potentially harbour novel OCD candidate genes, while genes implicated through animal models may lead to the identification of additional susceptibility genes through delineation of pathways by, for instance, interactome analysis. One such disorder is schizophrenia, which manifests overlap in both symptoms and brain circuits with OCD. In schizophrenia, in addition to several case-control association studies having been performed, linkage data, studies of chromosomal aberrations and animal models have led to the identification of many chromosomal regions that may contain genes involved in its aetiology and thus may also contain OCD candidate genes. In the present investigation, this approach was employed using previously reported schizophrenia susceptibility loci to identify novel OCD candidate genes. All genes residing in each of these loci were catalogued and individually analysed using a battery of bioinformatic techniques in order to assess their potential candidature for OCD susceptibility. These analyses yielded 13 credible OCD candidate genes.Additional candidates were sought using information regarding a well-defined schizophrenia animal model, the heterozygous reeler mouse, that exhibits neurodevelopmental, neuroanatomical and behavioural abnormalities, similar to those displayed by patients with schizophrenia. The phenotype of these mice is caused by a mutation in Reln, which encodes reelin, a large extracellular matrix protein that plays a pivotal role in the ordered migration of neurons during the development of laminar brain structures. The fact that both reelin protein and mRNA levels have been shown to be reduced in post-mortem brain sections of schizophrenic patients, coupled with the observed behaviour and neurochemical similarities between the heterozygous reeler mouse and schizophrenic patients suggests that reelin may be involved in the pathogenesis of schizophrenia and hence also OCD. Furthermore, genes encoding proteins that interact with reelin may thus also be considered plausible candidate genes for both schizophrenia and OCD. For this reason, novel reelin-interacting proteins were sought using the N-terminal reeler-domain of reelin, a domain only found in proteins involved in neuronal migration, as “bait” in a yeast twohybrid screen of a foetal brain cDNA library. Putative reelin ligands were subsequently reevaluated using co-immunopreciptitation and mammalian two-hybrid analysis to corroborate the yeast two-hybrid findings. Results of these analyses showed that WDR47, a WD40-repeat domain protein, interacts with reelin via its reeler-domain; therefore, the gene encoding this ligand protein, as well as RELN itself, was also considered a credible OCD candidate gene. Each of the candidate genes identified using the afore-mentioned strategies were assessed for their potential role in the aetiology of OCD by case-control association studies of a cohort of Afrikaner OCD patients and control individuals. Statistically significant associations were detected for two genes, DLX6 and SYN3, with the disorder. These associations are exciting as they may point to novel mechanisms involved in OCD development. The identification of WDR47 as a novel reelin-interacting protein has significant implications for our understanding of reelin-dependant signalling. Using this protein as the starting point, further novel components of the reelin signalling pathway may be unravelled, an investigation which may lead to the identification of novel roles for reelin in neurodevelopment. Such novel components may, of course, also be considered OCD and schizophrenia candidate genes, which may, in turn, augment the existing knowledge of the pathophysiologies of OCD, schizophrenia and other neurodevelopmental disorders. Taken together, the current study yielded exciting results that warrants follow-up investigation in future. The identification of DLX6 and SYN3 as novel OCD susceptibility genes as well as the identification of WDR47 as a reelin-interacting protein may provide investigators with alternative avenues of research into potential pathological mechanisms involved both in OCD and schizophrenia, which may ultimately lead to alternative pharmacotherapy.