Presence of the Hemochromatosis S65C Mutation Leads to Failure of Amplification in a Multiplex C282Y/H63D PCR

Hemochromatosis is an autosomal recessive disorder of iron metabolism affecting 0.2%–0.5% of white populations. Approximately 90% of affected individuals are homozygous for the C282Y mutation, but the H63D and S65C mutations are also of interest. Various molecular diagnostic methods for detection of these mutations have been described, including a multiplex PCR from Stott et al. (1) that simultaneously detects the C282Y and H63D mutations. In this method, PCR-mediated site-directed mutagenesis is used to create a Bbr PI restriction site in the wild-type C282Y and H63D products. To detect the C282Y mutation and the H63D mutation, respectively, 1 specific mismatch in the forward …