HFE genotyping using multiplex allele-specific polymerase chain reaction and capillary electrophoresis

Context.--Hereditary hemochromatosis is recognized as one of the most common autosomal recessive disorders, with a prevalence of 1 in 200 to 400 in the white population. Early detection and treatment are completely effective in preventing pathology. It is anticipated that testing for hereditary hemochromatosis will increase, as will the need for a technology that can handle the demand. Objective.--To describe a high-throughput, single-tube, allele-specific multiplex polymerase chain reaction assay for identifying the 2 mutations in the HFE gene associated with hereditary hemochromatosis. Design.--Fluorescence-labeled polymerase chain reaction products from a multiplex polymerase chain reaction are analyzed by automated capillary electrophoresis. Data Analysis.--The assay was validated by analysis of 25 blinded samples, and results were concordant with an established laboratory assay. Conclusion.--The assay described offers a significant improvement over manual laboratory assays in throughput, reduced technologist time, and cost.