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.

Thesis (MSc (Genetics))--University of Stellenbosch, 2008. / The porphyrias are a group of genetic diseases resulting from the accumulation of haem precursors due to defective enzyme activity in either one of the last seven enzymes in the haem biosynthesis pathway. One of the common hepatic porphyrias, porphyria cutanea tarda (PCT), arises from the inhibition of uroporphyrinogen decarboxylase (UROD) activity. It is characterised by excessive urinary and hepatic excretion of uroporphyrinogens and manifests cutaneously in the form of dermatitis. Two main forms of PCT have been described, namely familial PCT (fPCT) and sporadic PCT (sPCT). PCT is a complex disease and a few genetic (including modifier loci) and environmental precipitating factors have been implicated in the aetiology of PCT. An important exacerbating factor, iron overload, is observed in the majority of PCT patients. The aim of this study was to determine whether DNA sequence variation in the 5' untranslated regulatory region of four genes involved in iron metabolism i.e. CP, CYBRD1, HAMP and SLC40A1 may in any way be associated with PCT. The study cohort consisted of 74 patients from three diverse South African populations including 15 Black (eight males and seven females), 30 Caucasian (13 male and 17 females) and 29 Coloured (18 males and 11 females) individuals as well as 132 population-matched controls. The promoter region of the selected genes were screened for variation utilising the techniques of polymerase chain reaction (PCR) amplification, heteroduplex single-stranded conformational polymorphism (HEX-SCCP) analysis, restriction fragment length polymorphism (RFLP) analysis and bi-directional semi-automated DNA sequencing. Twenty three previously described and eleven novel variants were identified. The novel variants comprised CYBRD1: -1540G/A, -1474G/A, -1452T/C, -1346T/C, -1272T/C, -645T/C; G(T)8G(T)nG(T)nG(T)9; HAMP: -429G/T and SLC40A1: -1461T/C, -1399G/A, -524C/T. Statistically significant associations were observed at a number of loci. In silico analysis revealed several putative transcription factor binding sites (TFBSs) spanning the regions of variation. The disruption of an existing (or creation of a novel) TFBS is thought to occur in the presence of a variant in a number of instances. This may lead to the manipulation of transcription rates, thereby depicting a possible mechanism for gene dysregulation. The study presented here was undertaken as a preliminary investigation to determine the contribution (if any) of variants in the regulatory regions of candidate genes in iron metabolism in South African PCT patients. Considering the increasing incidence of PCT, in particular the Black South African population, it is necessary to elucidate the underlying mechanisms of iron overload in PCT patients. The propitious findings signified in the study, in conjunction with phenotypegenotype correlations, will assist in clarifying the association between iron overload and PCT. / jfl2010 / Imported from http://etd.sun.ac.za April 2010.