Design and Validation of a Colorimetric Test for the Genetic Diagnosis of Hemochromatosis Using α-Phosphorothioate Nucleotides

Hereditary hemochromatosis is an autosomal recessive disease highly prevalent in Northern Europe. Here we describe the performance of a genetic test for two mutations of the HFE gene (C282Y and H63D). It is based on a solid-phase PCR coupled with an α-phosphorothioate-mediated primer extension, conferring resistance to hydrolysis by ExoIII. Next, Elisa-like detection allows a colorimetric reading of the genetic test. We performed 322 tests (212 on the C282Y mutation, 110 on the H63D mutation) and compared the results with the RFLP method. Using OD ranges giving the minimum of uncertainty, the tests lead to high specificity and sensitivity, and they address the detection of mutated or normal bases in the HFE gene or the deduced phenotype (safe or ill), with positive predictive values or negative ones greater than 0.96. This method is therefore proposed as a primary test or as a confirming test.