Homogeneous multiplex genotyping of hemochromatosis mutations with fluorescent hybridization probes

Multiplex polymerase chain reaction amplification and genotyping by fluorescent probe melting temperature (Tm) was used to simultaneously detect multiple variants in the hereditary hemochromatosis gene. Homogenous real-time analysis by fluorescent melting curves has previously been used to genotype single base mismatches; however, the current method introduces a new probe design for fluorescence resonance energy transfer and demonstrates allele multiplexing by Tm for the first time. The new probe design uses a 3'-fluorescein-labeled probe and a 5'-Cy5-labeled probe that are in fluorescence energy transfer when hybridized to the same strand internal to an unlabeled primer set. Two hundred and fifty samples were genotyped for the C282Y and H63D hemochromatosis causing mutations by fluorescent melting curves. Multiplexing was performed by including two primer sets and two probe sets in a single tube. In clinically defined groups of 117 patients and 56 controls, the C282Y mutation was found in 87% (204/234) of patient chromosomes, and the relative penetrance of the H63D mutation was 2.4% of the homozygous C282Y mutation. Results were confirmed by restriction enzyme digestion and agarose gel electrophoresis. In addition, the probe covering the H63D mutation unexpectedly identified the A193T polymorphism in some samples. This method is amenable to multiplexing and has promise for scanning unknown mutations.