published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

 Genome-wide mapping of susceptibility genes was conducted in two complex disorders of hypertension and epilepsy, allowing the dissection of the genetic architecture of these common diseases and related quantitative traits. The study performed comprehensive genetic analyses in a genome-wide scale, using different structure of data – sib-pairs and case-control samples. To identify genes influencing hypertension and blood pressure, a combined linkage and association study was conducted using over half a million SNPs genotyped in 328 siblings. Regions of significant linkage were identified for blood pressure traits on chromosomes 2q22.3 and 5p13.2, respectively. Further family-based association analysis of the linkage peak on chromosome 5 yielded a significant association (rs1605685, P < 7  10-5) for hypertension. One candidate gene, PDC, was replicated in the family-based association tests. A two-stage genome-wide association study (GWAS) was performed in a total of 1,087 cases and 3,444 controls, to identify common susceptibility variants of epilepsy in Chinese. The combined analysis identified two association signals in CAMSAP1L1, rs2292096 [G] (P=1.0×10-8, OR =0.63) and rs6660197 [T] (P=9.9×10-7, OR=0.69), which are highly correlated, achieving genome-wide significance. One SNP (rs9390754, P = 1.7 × 10-5) in GRIK2 was refined as a previously-implicated association. In addition to SNPs, the assessment of CNVs in GWAS was performed, which could provide valuable clues to discover genes contributing to the heritability of epilepsy. A genome-wide scan for epilepsy through the use of DNA pooling also provides an alternative approach to reducing the substantial cost and thus increase efficiency in large-scale genetic association studies. The genome-wide mapping studies in families and unrelated individuals are complementary and together offer a comprehensive catalog of common variations and structural variants implicated for both quantitative and qualitative traits. / published_or_final_version / Psychiatry / Doctoral / Doctor of Philosophy

Complex diseases, unlike Mendialian diseases, are often characterized by genetic heterogeneity and multifactorial inheritance, involving defects in genes from the same or multiple alternative pathways. Many congenital diseases and psychiatric disorders are complex diseases, and incur heavy health care burden on the society. With the advancement in high-throughput genotyping technologies and the availability of the human single nucleotide polymorphism (SNP) catalogue, genome-wide association study (GWAS) has been widely used to investigate the genetic component of complex diseases. Copy number variations (CNV) can also be identified using the data from the same SNP array. Aiming to identify more disease susceptibility loci for complex diseases, separate GWAS using a case-control design were conducted on anorectal malformations (ARMs) and schizophrenia. ARMs are rare congenital diseases with heterogeneous phenotypes which could probably be explained by the genetic heterogeneity among patients, while schizophrenia is a common psychiatric disorder that is well known for its multigenic inheritance. The GWAS studies on ARM and schizophrenia included 4,369 (patients: N=363; controls: N=4,006) and 1,231 Han Chinese (patients: N=381; controls: N=850) respectively. The two studies were mainly focused on investigating the contribution of rare CNVs to the diseases, involving analyses on global CNV burden, rare CNV association, protein-protein interaction (PPI) network, pathway and chromosomal aberrations. The associations of SNPs with ARMs were also examined. Apart from elucidating the genetic components in these two diseases, a systematic analysis on four CNV detection programs (CNV partition, PennCNV, QuantiSNP and iPattern) was also undertaken. In the study of schizophrenia, a new approach in CNV filtering which was based on latent class analysis was adopted to gather information from multiple CNV prediction programs. The study of ARMs revealed 79 genes which were disrupted by CNVs in patients only. In particular, a de novo duplication of DKK4 (an antagonist of WNT signaling) was identified, and addition of Dkk4 protein was demonstrated to cause ARMs in mice. Another 10 genes uniquely disrupted in ARMs patients are also related to WNT signaling. Interestingly, this pathway was also significantly inferred by CNV in patients with schizophrenia. A different set of genes related to WNT signaling was disrupted in ARMs patients and patients with schizophrenia. WNT signaling is crucial for the development of multiple parts in the embryo. The contribution of different WNT signaling pathways at different development stages may vary. Apart from the WNT signaling pathway, other genes with biological relevance were also implicated in the two studies through gene-network and pathway analyses. The results from these two GWAS studies support our existing understanding of complex diseases that defects in various interacting genes could contribute to the same disease. In summary, the CNV results from the two studies have demonstrated the genetic heterogeneity nature of these two complex diseases. The findings also uncovered a set of putative disease candidate genes, which can be used as reference materials for future genetic research for ARMs and schizophrenia. / published_or_final_version / Psychiatry / Doctoral / Doctor of Philosophy

published_or_final_version / Molecular Biology / Doctoral / Doctor of Philosophy

No description available.

Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Variegate porphyria (VP, MIM 176200) is a low penetrance autosomal dominant disorder that stems from mutations in the protoporphyrinogen oxidase (PPOX) gene. VP is found in most populations, but has a high prevalence in the South African Afrikaner population with most patients inheriting the same PPOX mutation (R59W) from a common ancestor. The clinical manifestations of the disease include acute neurovisceral attacks and/or cutaneous photosensitivity. Great variation in the clinical presentation of VP is observed; even in members of the same family that share a common genetic background and that have been exposed to similar environmental factors. Candidate genes that may have an influence on phenotypic variation due to the regulatory function in the haem biosynthetic pathway include the two deltaaminolevulinic acid synthase (ALAS) genes and the porphobilinogen deaminase (PBGD) gene. Sequence homology searches between different species indicated that the ALAS-1, ALAS-2 and PBGD genes are highly conserved, indicating that these genes have an important function to fulfill in the haem biosynthetic pathway. The study population of 25 R59W individuals were divided in four categories according to their clinical presentation. The distribution of clinical symptoms observed in this study corresponds with results from previous studies. Conformation sensitive gel electrophoresis (CSGE), conventional single stranded conformation polymorphism analysis (SSCP) and two buffer SSCP analysis were implemented to screen for possible sequence variants. The exons of all three genes as well as the adjacent intronic sequences were investigated. A total of six sequence variation sites were identified of which five had previously been described single nucleotide polymorphisms (ALAS-1: 4713 T>C; PBGD: -64 C>T, 3581 A>G, 6479 G>T, 7064 C>A)] and a novel 8bp deletion (PBGD: 4582_ 4589del). No sequence variant was identified in the ALAS-2 gene. The CSGE method proved to have the highest sensitivity (83%), identifying five of six sequence variant sites. The conventional SSCP method identified only three (50%) sequence variant sites, while the two buffer system detected two (33%) of the sequence variants. The 4713 T>C SNP in exon 4 of the ALAS-1 gene and the -64 C>T SNP in the PBGD gene were selected for further investigation due to their location in the respective genes. These sequence variants were typed in 50 patients and 50 control subjects matched for ethnic background. The relationship between variation at these loci and clinical features was investigated. No statistical significant association was observed for either of the 4713 T>C SNP (P= 0.717) or the -64 C>T SNP (P= 0.931). Genetic modifying factors make a variable contribution to the total clinical picture and are difficult to identify in small populations. Due to the fact that we only had a limited number of VP samples, association cannot be ruled out. This study does, however, provide insight into investigational approaches that should be undertaken in future research concerning the ALAS and PBGD genes. Further knowledge concerning the haem biosynthetic pathway could ultimately lead to the understanding and assessment of the clinical expression observed in individuals with VP. / AFRIKAANSE OPSOMMING: Variegate porfirie (VP, MIM 176200) is 'n lae penetrasie outosomaal dominante siekte wat veroorsaak word deur mutasies in die protoporfirienogeen oksidase (PPOX) geen. VP word gevind in die meeste populasies, maar het 'n hoë voorkoms in die Suid- Afrikaanse populasie waar meeste pasiente dieselfde PPOX mutasie (R59W) van 'n gemeenskaplike voorouer oorgeërf het. VP word gekenmerk deur akute neuroviserale aanvalle en/of fotosensitiewe vel. Groot variasie word egter waargeneem in die kliniese uitdrukking van VP, selfs in lede van dieselfde familie wat 'n gemeenskaplike genetiese agtergrond deel en wat blootgestel is aan dieselfde omgewingsfaktore. Kandidaat gene wat as gevolg van hulle regulatoriese funksie in die heem biosintetiese padweg 'n effek op die ekspressie van VP mag hê, sluit in die twee deltaaminolevuliniese suur sintase (ALAS) en die porfobilinogeen deaminase (PBGD) gene. Homologie ondersoeke van die ALAS-1, ALAS-2 en PBGD gene in verskillende spesies dui daarop dat die gene hoogs gekonserveerd is en dus gevolglik 'n belangrike funksie in die heem biosintetiese padweg vertolk. Die studie populasie van 25 R59W individue is verdeel in vier kategorieë op grond van hulle kliniese simptome. Die verspreiding van die kliniese simptome wat waargeneem is tydens hierdie studie stem ooreen met die resultate van vorige studies. Konformasie sensitiewe gel elektroforese (CSGE), konvensionele enkelstring konformasie polimorfisme analise (SSCP) en twee buffer SSCP analise is gebruik vir die identifisering van genetiese variasie. Die eksons van al drie gene, sowel as die aangrensende intron volgordes, is ondersoek. 'n Totaal van ses areas van genetiese variasie is geïdentifiseer, waarvan vyf reeds beskryfde polimorfismes is (ALAS-1: 4713 T>C; PBGD: -64 C>T, 3581 A>G, 6479 G>T, 7064 C>A) en 'n nuwe 8bp delesie (PBGD: 4582_ 4589del). Geen genetiese volgorde variasie is gevind in die ALAS-2 geen nie. Die CSGE metode het die hoogste sensitiwiteit getoon (83%) en het vyf van die ses volgorde variasies geïdentifiseer. Die konvensionele SSCP metode het slegs drie volgorde variasies geïdentifiseer (50%), terwyl die twee buffer deteksie-sisteem twee variasies geïdentifiseer (33%) het. Die 4713 T>C polimorfisme in ekson 4 van die ALAS-1 geen en die -64 C>T polimorfisme in die PBGD geen, is geselekteer vir verdere ondersoek as gevolg van hulle posisie in die respektiewe gene. Die volgorde variasies is getipeer in 50 R59W pasiënte sowel as in 'n kontrole groep van 50 individue met dieselfde etniese agtergrond. Die verband tussen die variasie by die lokusse en die kliniese kenmerke is ondersoek. Geen statisties beduidende assosiasie is waargeneem vir hetsy die 4713 T>C SNP (P= 0.717) of die -64 C>T SNP (P= 0.931). Genetiese modifiserende faktore word moeilik geïdentifiseer in klein populasies omdat hulle afsonderlike bydra tot die geheelbeeld van die kliniese simptome so varieerbaar is. 'n Relatiewe klein groep van VP pasiënte was tydens die studie beskikbaar en dus kan assosiasie nie uitgesluit word nie. Die studie verskaf egter insig in verband met toekomstige benaderings wat volg kan word in verdere ondersoeke van die ALAS en PBGD gene. Verdere kennis in verband met die heem biosihtetiese padweg kan uiteiHdelik lei tot die verduideliking en assesering van die kliHiese uitdrukking in vI=' individue.

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Psychiatry / Master / Master of Medical Sciences

A human isolate of hepatitis A virus (HAV) strain HAS-15 was adapted to rapid growth FRhK-4 cells and a one-step growth curve was determined. Detectable virion production was absent for approximately 20 h post-infection (p.i.) and was followed by a 4-day logarithmic phase of virus production. A maximum intracellular virus titer of 10⁹ radioimmunofocus-forming units (RFU) per milliliter was achieved and remained essentially constant for a period of up to 14 days p.i. An adsorption study with HAV HAS-15 using FRhK-4 cells demonstrated greater than 99.9% of infectious virus adsorbed at 25 C in less than 20 min. Milligram amounts of purified HAV HAS-15 were obtained from persistently-infected RFhK-4 cells. The HAV polypeptides were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and transferred to nitocellulose for detection by an enzyme-linked immunotransfer blot (EITB) procedure. HAV nucleotide-derived amino acid sequence was subjected to computer analysis to identify potential immunogenic regions within the HAV capsid polypeptides. Synthetic peptides corresponding to selected regions of each of the larger putative capsid polypeptides were coupled to keyhole limpet hemocyanin and used to immunize rabbits. EITB reactivities of HAV specific anti-peptide sera have allowed the identification of the gene order for the larger HAV P1 gene products and the determination of the following molecular weights: HAV VP2 or 1B (MW 27,000), HAV VP3 or 1C (MW 29,000), and HAV VP1 or 1D (MW 33,000). The disposition of the HAV capsid polypeptides with respect to the virion external surface was evaluated by EITB reactivity of HAV polypeptides with specific antisera. Hyperimmune rabbit anti-157S HAV and human IgM reacted with VP1, VP2, and VP3, while IgG reacted predominantly with VP1 and VP2. Further evaluation of the HAV virion structure was attempted by examining the relative accessibility of the virion polypeptides to various labeling reagents. Reaction of intact virions with Iodogen resulted in the predominant labeling of VP1 while labeling of VP2 and 3 was barely detectable. Selective labeling of VP1 under controlled conditions, combined with the anti-HAV IgG immunologic reactivity against VP1 and VP2, suggests that these two capsid components are more exposed on the virion surface and may play an important role in the generation of neutralizing antibodies.