Rapid Genotyping of Hemochromatosis Gene Mutations on the LightCycler with Fluorescent Hybridization Probes

摘要

Two point mutations in the hemochromatosis gene (HFE) are considered responsible for the development of hereditary hemochromatosis (HH), an autosomal recessive iron overload disease. One of these mutations produces a cysteine-to-tyrosine amino acid substitution at position 282 of the HFE protein (Cys282Tyr), caused by a G-to-A transition at nucleotide position 845. A homozygous Cys282Tyr mutation is present in 80–100% of hemochromatosis patients (1)(2). This mutation prevents interaction of HFE protein with β2-microglobulin, leading to an inability to bind to the transferrin receptor, a process that is necessary for iron resorption (3)(4).A second mutation, which changes histidine at position 63 to aspartic acid (His63Asp), caused by a C187G transversion does not affect this binding but possibly blocks the interaction with other ligands (3)(5). In contrast to the Cys282Tyr mutation, the role of the His63Asp mutation in the pathogenesis of the disease is uncertain. A heterozygous His63Asp mutation is often observed in Cys282Tyr heterozygous patients. This genotype combination is called compound heterozygosity and is observed in 4–5% of HH cases. Therefore, it must be considered, like the homozygous Cys282Tyr mutation, as a genetic disposition for HH. However, it seems to play a subordinate role and seems to be important only in combination with the Cys282Tyr mutation (6). Approximately 3% of the population is homozygous for the His63Asp mutation, and thus is not considered in the risk group for HH (6)(7). In rare HH cases, none of these known HFE mutations are present. Other mutations, possibly not associated with HFE , might contribute to the development of hemochromatosis (2).There are many current methods available for genotyping the two known hemochromatosis-causing mutations, including oligonucleotide ligation assay (8), single-strand conformation polymorphism (9), allele-specific PCR (10)(11), and PCR-restriction fragment length …