High-performance liquid chromatography multiplex detection of two single nucleotide mutations associated with hereditary hemochromatosis

High-performance liquid chromatography (HPLC) has been applied to the multiplex detection of the two single nucleotide mutations commonly found in hereditary hemochromatosis (HH). HH is associated with a major G to A transition at position 845 (mutation Cys282Tyr) and a minor C to G transition at position 187 (mutation His63Asp) in the cDNA of the HFE gene. Two detection assays were developed based on HPLC analysis of restriction fragment length polymorphism (RFLP) or single nucleotide extension (SNE) products following multiplex PCR amplification. RFLP genotypes the two sites as dsDNA fragments of different lengths generated by restriction enzymes Rsa I/Bcl I. SNE extends primers 5′-adjacent to the sites of interest with a dideoxynucleotide triphosphate (ddNTP) to generate extended ssDNA. The identity of the added ddNTP reveals the identity of the original possible mutation site(s). Application of these methods with HPLC analysis provides simple and reliable genotyping for HH and can be applied to other single nucleotide polymorphism studies.