Use of Denaturing HPLC and a Heteroduplex Generator to Detect the HFE C282Y Mutation Associated with Genetic Hemochromatosis

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron metabolism. Most individuals with HH are homozygous for a 845G→A missense mutation (C282Y) in the HFE gene that encodes a MHC class I-like protein (1)(2)(3)(4)(5). One in 300 adults of Caucasian descent has this genotype (3)(6). The disease is characterized by increased absorption of dietary iron by the gastrointestinal tract and the progressive deposition of iron in the parenchymal cells. It produces a wide range of clinical complications, including hepatic fibrosis, cirrhosis, hepatocellular carcinoma, diabetes mellitus, hypopituitarism, hypogonadism, arthritis, and cardiomyopathy (7). These complications can be entirely prevented with regular iron removal by phlebotomy. The availability of this effective treatment highlights the value of screening appropriate populations for the C282Y mutation to detect HH before irreversible complications occur (8). Another frequent mutation in the HFE gene, H63D, has been described (1), but the relationship between this mutation and iron overload is less clear. Although compound heterozygotes C282Y/H63D (1) and H63D homozygotes (9) may show some degree of iron overload, the penetrance associated with these genotypes is very low, and clinical features of hemochromatosis, especially liver disease, are rare. In opposition to C282Y, screening the population for H63D is therefore not indicated.An important issue is the development of a cost-effective methodology suitable for genetically testing large numbers of samples for the C282Y mutation. Many methods have been used to detect C282Y, including oligonucleotide ligation assays (1), fragment length polymorphism analyses of PCR products after restriction enzyme digestion (5), allele-specific oligonucleotide hybridization assays (2), mutagenically separated PCR assays (10), primer extension assays (11)(12), and allele refractory mutation systems (13)(14). All of these assays are time-consuming. In addition, they rely on complicated multistep procedures involving radioisotopes or …