A rapid automated SSCP multiplex capillary electrophoresis protocol that detects the two common mutations implicated in hereditary hemochromatosis (HH)

Currently two mutations in the HFE gene are known to be associated with the manifestation of the autosomal recessive disorder hereditary hemochromatos is (HH). A single-base mutation resulting in Cys282Tyr appears to have a ca usative role in the development of the disease, and a point mutation result ing in His63Asp may also be involved. Recent observations with a fully auto mated capillary electrophoresis (CE) system (ABI Prism 310)suggested that t his instrument could be used for the precise identification of known mutati ons based on single-strand conformation polymorphism (SSCP). Two DNA fragme nts, each specific for one of the HFE mutation sites and labeled with a dif ferent fluorophor, were coamplified and without further manipulation simult aneously analyzed by CE-SSCP. Wild-type samples showed a mobility pattern t hat was clearly distinguishable from homozygous Cys282Tyr, homozygous His63 Asp, or a compound heterozygous sample. To evaluate the reliability of this system for the detection of both mutations, 20 samples were analyzed blind . All genotypes, which were called automatically, were in concordance with those obtained by a previously validated restriction fragment length polymo rphism method. Thus, SSCP in combination with CE provides a fast and precis e research tool for the simultaneous identification of the two common mutat ions implicated in HH.