Partridge, Jason; (2003) Haemochromatosis: Genetic and functional studies. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Genetic haemochromatosis (GH) is an autosomal recessive condition common in Northern Europeans. It causes excess absorption of iron, resulting in tissue damage. Two approaches were used to study GH: Positional cloning: P1 derived artificial chromosomes (PACs), containing large inserts of human genomic DNA, were isolated from around D6S1260, at that time the observed peak of linkage disequilibrium with GH. These clones were used to screen a human small intestine cDNA library. Four of the cDNAs isolated from this library contained C2H2 zinc finger motifs, comprising a single locus. The cDNA and genomic sequence and expression pattern of this locus were determined. The locus has characteristics similar to that of an expressed processed pseudogene, although it retains an open reading frame of 1.2kb. No evidence for a parent locus at another chromosomal site was detected using sequence database screening, somatic cell hybrid analysis and fluorescent in situ hybridisation. The sequence conservation displayed by this zinc finger pseudogene makes it an excellent tool for the identification of zinc finger genes in model organisms. Assessment of a cellular phenotype; a cellular phenotype for GH would allow functional cloning of the gene, as well as investigation of GH in vitro. The enzyme ferric reductase was previously shown to have increased activity in duodenal biopsies from GH patients compared to controls. This increase paralleled increased uptake of ⁵⁹Fe in GH. GH patients and controls for ferric reductase studies were characterised by investigation of both haplotype and HFE mutations. No significant difference in lymphocyte, monocyte or macrophage ferric reductase activity was observed when GH and control preparations were compared. However, a significant (p<0.05) ten-fold increase in ferric reductase activity accompanied the differentiation of monocytes to macrophages. This increase most likely reflects the co-ordinate upregulation of proteins of iron metabolism during the differentiation of monocyte to macrophage.

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