Multiplex Allele-Specific PCR for Simultaneous Detection of H63D and C282Y HFE Mutations in Hereditary Hemochromatosis

BACKGROUND: Hereditary hemochromatosis (HH) is characterized by excessive iron absorption in the intestine, which can lead to failure of vital organs such as the heart, liver, and pancreas. Among northern Europeans, HH is most often associated with the C282Y and H63D mutations of the HFE gene. We developed a test that allows screening for both mutations in a single reaction. METHODS: A multiplex allele-specific PCR was developed for simultaneous genotyping of the H63D and C282Y HFE mutations. PCR fragments were designed such that the resulting PCR product can be analyzed in a single polyacrylamide gel lane. RESULTS: Test results from our multiplex assay were concordant with genotypes of 55 Canadian patients with suspected hemochromatosis, which had previously been established by allele-specific PCRs that targeted H63D and C282Y in separate reactions. CONCLUSIONS: Molecular diagnostic detection of H63D and C282Y mutations can be achieved by a variety of methods, but these are not necessarily time-efficient or economical. Multiplex allele-specific PCR is an excellent tool for molecular diagnostic screening for H63D and C282Y mutations in patients with suspected hemochromatosis. This method is inexpensive, accurate, and highly efficient in terms of labor, throughput, and turnaround time.