A Simple RFLP-Based Method for Gene Multiplex Amplification and Determination of Hereditary Hemochromatosis-Causing Mutation C282Y and H63D Variant with Highly Sensitive Determination of Contamination.

Hereditary hemochromatosis is an autosomal recessive disorder with incomplete penetrance that results from excess iron absorption and can lead to chronic liver disease, fibrosis, cirrhosis, and hepatocellular carcinoma. The most common form of hereditary hemochromatosis in Western Europe is due to a homozygous mutation (p.(Cys282Tyr) or C282Y), in the gene which encodes hereditary haemochromatosis protein. In the general European population, the frequency of the homozygous genotype is 0.4%, and this mutation explains up to 95% of hereditary hemochromatosis in France. We report here an improved PCR and restriction endonuclease assay based on multiplex amplification of exon 4 (for C282Y detection), exon 2 (for H63D detection), gene (for digestion controls), and two Short Tandem Repeats (SE33 and FGA) for identity monitoring and contamination tracking. Fluorescent primers allow capillary electrophoresis, accurate allele tagging, and sensitive contamination detection.