LightCycler PCR Assay for Simultaneous Detection of the H63D and S65C Mutations in the HFE Hemochromatosis Gene Based on Opposite Melting Temperature Shifts

Abstract

Hereditary hemochromatosis is a common autosomal recessive disorder of iron metabolism, which in its homozygous form occurs in Caucasian populations with a prevalence of 0.2–0.5% (1)(2). Characteristic of the disease is the excessive accumulation of dietary iron and a progressive rise in iron stores. This may lead to serious clinical consequences, including cirrhosis, cardiac failure, diabetes, arthritis, and hepatocellular carcinoma. Treatment involves removal of the iron burden by regular venesection and leads to a normal life expectancy if implemented before the development of cirrhosis (3). Thus, early detection and treatment are critically important. The recent identification of a hemochromatosis gene ( HFE , initially termed HLA-H) by Feder et al. (4) allows early genetic diagnosis and greatly simplifies the screening of a family once affected individuals have been identified. The HFE gene protein product is structurally similar to MHC class I-type molecules and interacts with β2-microglobulin and the transferrin receptor to limit iron absorption (5)(6). Three disease-associated mutations have been detected in the HFE gene. Most individuals with hemochromatosis (80–100%, depending on the population studied) are homozygous for the mutation C282Y. In addition, a small number of compound heterozygotes for C282Y and a second mutation, H63D, may develop clinical iron overload (7). Recently, we demonstrated that a third mutation, S65C, is enriched in hereditary hemochromatosis patients who have a mild form of the disease and who have no mutations at C282 or H63 (8). Although mutations in the HFE gene thus account for most cases of hereditary hemochromatosis, it is clinically important that a minority of hereditary iron overload syndromes are not associated with mutations in the HFE gene. Absence of such mutations thus should not be interpreted to mean that the patient in question does not have hereditary iron overload (1)(2). …