Hereditary haemochromatosis (HHC) is a disease of iron metabolism characterised by systemic iron overload. The gene affected in this disorder, HFE, was cloned in August 1996 and lies at the telomeric end of the major histocompatibility complex distal to HLAA. The overall aims of this thesis were a) to conduct a linkage disequilibrium (LD) analysis of an extended haplotype covering a region between HLA-A and HFE in Australian HHC patients, and b) to characterise the role of HFE in iron metabolism. Since the identification of the gene, neither genotype-phenotype relationships nor HFE function have been clearly defined. Therefore, this study was carried out in order to further understand the genetics of haemochromatosis and to provide some insights into the function of HFE in iron metabolism.
The first results chapter (Chapter 3) describes analyses of an extended haplotype and linkage disequilibrium in Australian HHC patients between the markers HLA-A and D6S1281, a 5.5 Mb region of chromosome 6p21.3. These analyses were performed using DNA samples from HHC patients, HHC family members and unrelated controls. A common haplotype was found to be present in 23% of 56 affected chromosomes and LD analysis in this region showed two peaks, at the markers D6S105 (centromeric to HFE) and D6S2240 (close to HFE), which are separated by approximately 2 Mb. The association between these two peaks and HHC may relate to variations in the clinical phenotype of the disorder.
The relationship between alleles of the microsatellite D6S105 (genotype) and clinical expression (phenotype) in Australian HHC patients homozygous for the C282Y mutation in HFE has been examined. Statistical analysis shows a significant difference in hepatic iron index between HHC patients homozygous for D6S105 allele 8 and those heterozygous for this allele or without it. This study supports the concept that a modifying gene may exist in the D6S105 region.
Studies on the role of HFE in iron metabolism have been carried out through the investigation of its association with the transferrin receptor. This study used Chinese Hamster Ovary (CHO) cells deficient in endogenous transferrin receptor and the same cells transfected with human transferrin receptor. Plasmid constructs bearing either wild type HFE or mutant HFE (C282Y and H63D) were transfected into these CHO cells. These studies demonstrated that HFE can be transported to the cell surface in the absence of an association with the transferrin receptor.
In order to study the flinction of HFE using mouse models, polyclonal antibodies against mouse HFE were raised in rabbits. The animals were immunised with a 16 amino acid peptide from the carboxy terminal region of the mouse HFE molecule. Based on Westem blot analysis, these antibodies are specific for a 45-46 kD protein which is similar to the molecular weight of human HFE. Immunocytochemistry shows that antibodies to mouse HFE recognise a protein which is expressed on both the cell surface and intracellular vesicles in NIH 3T3 (mouse) cells and in Clone 9 (rat) cells. However, the level of HFE expression in mouse and rat cell lines is lower than that observed in mouse liver tissue. Immunohistochemical analysis demonstrated that the distribution of HFE in the mouse intestine is predominantly in the crypt cells. In the liver, HFE is located most probably in the Kupffer cells. Thus these antibodies appear to be specific for the mouse HFE and are suitable for use in further studies.
In conclusion, the LD analysis over an extended genetic region containing the HFE locus shows two peaks of association with HHC. Statistical analysis of the genotype-phenotype of HHC patients indicates that modifying gene(s) affecting HHC clinical expression may be located in the region of D6S105. Studies to characterise the role of HFE in iron metabolism suggest that HFE can be transported to the cell surface without associating with the transferrin receptor. However, these studies must be confirmed using other models. Polyclonal antibodies against mouse HFE have been raised and used to localised HFE both in mouse cell lines and tissues. These antibodies can be used for further studies on the role of HFE in iron metabolism using mouse models.