Molecular Diagnostics in Hereditary Hemochromatosis (HFE)

The HFE gene mutation, nucleotide 845 G>C (resulting in the codon change C282Y) is found in most patients affected with clinical hemochromatosis, an autosomal recessive disorder of iron metabolism (see recent clinical review by Dooley et al. (1997) Lancet 349:1688-1693; original citation Feder et al. (1996) Nature Genetics 13:399-408). Several studies indicate that the carrier frequency of hemochromatosis is about 10 % in the Caucasian population and in individuals of certain ethnic groups, may be lower or higher. Although there is controversy whether C282Y is unequivocally a pathogenic mutation in hemochromatosis, about 82 - 100% of hemochromatosis patients (depending on the study) are homozygous for this mutation or, less commonly, are heterozygous (about 5% of cases) with a second change, H63D. The relative risk for hemochromatosis is decreased when H63D is present as a compound heterozygote with C282Y or, even less, when H63D is present in the homozygous form (Risch (1997) Nature Genetics 17:375-376). The C282Y mutation has been found at increased frequencies in other disorders with inappropriate iron absorption such as porphyia cutanea tarda (Roberts et al. (1997) Lancet 349:321-323; Santos et al. (1997) NEJM 336:1327-1328) and the mutation may be a modifying factor in such disorders.

The acquisition of patent rights to detect this natural human DNA polymorphism by SmithKline Beecham, Collegeville, PA prevents CompGene from performing this simple assay on a clinical basis.

The figure below illustrates an HFE assay for the nucleotide 845 G>C mutation. Lane 1 shows DNA size markers, lane 2 shows a negative DNA control and lane 3 shows the uncut HFE PCR product of 387 bp. Lanes 4 and 7 are results for a normal individual, lanes 5 and 8 are the results from an HFE carrier, while lanes 6 and 9 are the results from a query hemochromatosis patient. This patient did not carry the mutation despite a clinical diagnosis of hemochromatosis. This result illustrates the fact that approximately 10 - 15% of patients diagnosed with clinical hemochromatosis will not carry the nucleotide 845 G>C mutation. The yellow arrows indicate the positions of normal restriction fragments while the red arrows indicate restriction fragments obtained by the gain of either a Rsa I site (lane 5) or a Sna BI site (lane8) in a carrier of the nucleotide 845 G>C mutation.



The figure below illustrates an HFE assay for the C >G mutation which causes the H63D mutation. Lane 1 shows DNA size markers, lane 2 shows a negative DNA control and lane 3 shows the uncut exon 2 HFE PCR product of 208 bp. Lanes 4 - 6 are results for normal individuals while lane 7 is the result from a query hemochromatosis patient. This patient did not carry the H63D or as shown above, the C282Ymutation despite a clinical diagnosis of hemochromatosis. This result illustrates the fact that approximately 10 - 15% of patients diagnosed with clinical hemochromatosis will not carry either mutation. In addition, this figure demonstrates the high frequency of the H63D mutation in the "normal" population. Various studies have shown a carrier frequency of about 15 - 20% for H63D. The yellow arrows indicate the positions of normal restriction fragments. The H63D mutation results in the loss of a Bcl I site (lanes 5 and 6).