Complete scanning of the hereditary hemochromatosis gene (HFE) by use of denaturing HPLC

Background: Between 4% and 35% of hereditary hemochromatosis (HC) probands are C282Y or H63D heterozygotes or lack both of these two common HFE mutati ons, and 15 novel HFE mutations have been described recently. We evaluated denaturing HPLC (DHPLC) for screening of the whole HFE coding region and fu rther defined whether HC probands with an incomplete HFE genotype carry unc ommon mutations. Methods: Analytical conditions for each coding exon were determined by a co mbination of computer melting profile predictions and experimental melting curves. To test accuracy for scanning the complete HFE coding region and op timize DHPLC running conditions, each melting domain was investigated with at least one mutation or one polymorphism as reference. We tested 100 DNA s amples harboring the C282Y, H63D, or S65C mutations and 17 artificially cre ated positive controls that carried either 1 of the 14 other known HFE muta tions or 3 selected polymorphisms. Results: Investigations on each of the coding exons 1, 2, 4, 5, and 6 could be performed at one analysis temperature. Coding exon 3 displayed a more c omplex melting profile and required two analysis temperatures. DHPLC detect ed all known HFE mutations as well as the three selected polymorphisms. Conclusions: DHPLC can be used to scan the HFE gene in HC probands in whom at least one chromosome lacks an assigned mutation. (C) 2001 American Assoc iation for Clinical Chemistry.