A Common Intron 3 Mutation (IVS3 −48c→g) Leads to Misdiagnosis of the c.845G→A (C282Y) HFE Gene Mutation

Amplification of the region of the HFE gene that contains the c.845G→A (C282Y) mutation is usually performed using one amplimer that binds to a sequence in intron 3 and another that binds to a sequence in intron 4. Previously, a mutation that interferes with efficient binding to the intron 4 site has been described. We now find that another common mutation in intron 3, IVS3 −48c→g, prevents binding of the amplimer to that site. This polymorphism occurs at a gene frequency of 0.128 in the African–American population and at a frequency of only 0.006 in the European population. DNA samples heterozygous for the IVS3 −48c→g polymorphism and C282Y were undistinguishable from samples homozygous for the C282Y mutation when they were examined by allele-specific oligonucleotide hybridization (ASOH) and showed only a weak normal band when examined by electrophoresis following restriction endonuclease digestion. Although the polymorphism occurs in a DNA sequence almost identical to the intron 3 splice donor site, we found no evidence of alternative splice forms. Moreover, serum iron, transferrin saturation, and serum ferritin levels were normal in subjects heterozygous for the polymorphism. It appears, therefore, that the main importance of this polymorphism is that it may lead to misdiagnosis of heterozygotes for the C282Y mutation as homozygotes. We therefore recommend exonic amplimers that avoid sites that contain polymorphisms and that can be multiplexed for detection of the c.187C→G (H63D) and c.845G→A (C282Y) mutations.