Detecting the C282Y and H63D Mutations of the HFE Gene by Holliday Junction-Based Allele-Specific Genotyping Methods

Abstract

Hereditary hemochromatosis, one of the most common genetic diseases in Caucasians, is characterized by excessive iron deposition secondary to hyperabsorption of dietary iron and can potentially can lead to multiorgan failure if untreated. The C282Y and H63D mutations of the HFE gene are the most common mutations associated with symptomatic hemochromatosis. Recently, several genotyping methods have been used to identify hemochromatosis mutations and other single-nucleotide polymorphisms (SNPs) (1). There are advantages and limitations for each methodology in terms of cost and efficiency (2). We report here the application of a new SNP/point mutation genotyping platform developed by our group, the Holliday junction-based allele-specific genotyping (HAS) platform (3), to identify the C282Y and H63D mutations associated with hemochromatosis. For the HAS technology, we developed two detection modalities based on differences in the physical and biochemical properties between Holliday junctions (HJs) and duplex DNA or single-stranded DNA. The junctions that form in an allele-specific manner can be detected heterogeneously through gel electrophoresis (acrylamide or agarose; Fig. 1A⇓ ) or homogeneously through a fluorescence polarization (FP) competition assay (3). Using the HAS genotyping platform, we developed an assay for genotyping the C282Y and H63D mutations with both gel electrophoresis and FP for detection. Five primers (one forward, two reference, and two reverse primers) were designed for each of the two mutations (see Table 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue1/). PCR amplification was performed with a PTC-200 DNA Engine thermocycler (MJ Research, Inc.). For each point mutation locus, two PCR reactions in separate tubes were carried out in parallel for each DNA sample to amplify the target DNA with each of its two DNA references. Each PCR reaction contained four primers [one forward, one reference, reverse tail I, and reverse tail II (Table …