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 multi plex detection of the two single nucleotide mutations commonly found in her editary hemochromatosis (HH). HH is associated with a major G to A transiti on at position 845 (mutation Cys282Tyr) acid a minor C to G transition at p osition 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 followin g multiplex PCR amplification. RFLP genotypes the two sites as dsDNA fragme nts of different lengths generated by restriction enzymes Rsa I/Bcl I. SNE extends primers 5 ' -adjacent to the sites of interest with a dideoxynucleo tide triphosphate (ddNTP) to generate extended ssDNA. The identity of the a dded ddNTP reveals the identity of the original possible mutation site(s). Application of these methods with HPLC analysis provides simple and reliabl e genotyping for HH and can be applied to other single nucleotide polymorph ism studies. (C) 2001 Elsevier Science B.V. All rights reserved.