RESUMO
BACKGROUND: The diagnosis of hereditary hemochromatosis (HH) before the onset of iron-overload has been difficult in the past. However, a convincing candidate gene for HH: HFE has been described recently. The aims of this study were: 1) To determine the prevalence of the hemochromatosis associated mutations C282Y and H63D of the HFE gene in patients from Southern Germany with hemochromatosis phenotype; and 2) to test two new, time- and cost-saving methods: automated SSCP-based capillary electrophoresis (SSCP-CE) and a PCR-ELISA technique for the analysis of HFE mutations. METHODS: HFE genotype was studied in 36 unrelated HH patients and 126 controls from Southern Germany. In addition, family screening was performed in 76 relatives. The C282Y and H63D mutations were detected using SSCP-CE and restriction length polymorphism (RFLP). The C282Y mutation was additionally analysed by a PCR-ELISA. RESULTS: Twenty-six (72%) HH patients were homozygous for mutation C282Y, and three compound heterozygous for C282Y and H63D. One patient was homozygous for H63D. By performing family screening, six additional patients with the +/+ C282Y mutation were identified. The results of the SSCP-CE and the PCR-ELISA analysis agreed completely with data obtained by RFLP. CONCLUSIONS: SSCP-CE and PCR-ELISA analysis proved to be reliable methods for HFE genotyping and therefore represent cost- and time-effective alternative methods to the widely used restriction analysis allowing large populations to be screened for HH associated with HFE mutations. Surprisingly, only 72% of our HH patients had the C282Y +/+ genotype. This indicates that hemochromatosis in Southern Germany is genetically more heterogeneous than in other regions. A challenge for the future will be to define the genetic or environmental factors responsible for iron-overload in HH patients who do not show typical alterations of the HFE gene.
