Mutation analysis of four genes implicated in iron homeostasis in porphyria cutanea tarda (PCT) patients

Panton, Nicola (2008-03)

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.

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