Real-time polymerase chain reaction with fluorescent hybridization probes for the detection of prevalent mutations causing common thrombophilic and iron overload phenotypes.

We evaluated more than 450 patients with thrombophilia or iron overload for the presence of a factor V Leiden (R506Q), prothrombin G20210A, or HFE C282Y mutation using a standard method (polymerase chain reaction [PCR]–restriction fragment length polymorphism) and a comparative real-time PCR fluorescent resonance energy transfer (FRET) hybridization probe melting curve method. There was 100% concordance between the genotypes ascertained by the 2 methods (at each loci). In addition, phenotypic biochemical laboratory parameters measured on a subset of referred patients correlated with their respective genotypes. In the iron overload cohort, HFE C282Y homozygotes (n = 74) had significantly higher (P < .0001) transferrin saturation levels (74% – 25%) than did nonhomozygotes (n = 340; 51.4% – 28%), suggesting a genotype-dependent increase in body iron loads. In the thrombophilic cohort, the degree of activated protein C resistance (APCR), measured by a clotting time–based test, was associated significantly with the presence of 0 (n = 255; APCR = 2.59 – 0.26), 1 (n = 84; APCR = 1.61 – 0.13), or 2 (n = 5; APCR = 1.16 – 0.04) copies of the mutant factor V Leiden allele. As the fluorescent genotyping method required no postamplification manipulation, genotypes could be determined more quickly and with minimized risk of handling errors or amplicon contamination. In addition to these practical advantages, the FRET method is diagnostically accurate and clinically predictive of phenotypic, disease-associated manifestations. The recognition that prevalent, well-conserved single nucleotide mutations predispose to many common disease syndromes has created the demand for better methods for their direct detection. Accurate, fast, and inexpensive assays to detect pathogenic single nucleotide alterations will be required in the molecular medicine era for confirming disease diagnoses in affected patients and for detecting at-risk family members for whom effective interventions may be available. Perhaps the best examples of the clinical usefulness of single nucleotide mutation assays in the management of common diseases are the detection of the factor V Leiden (R506Q) and prothrombin G20210A mutations in thrombophilic patients and the detection of the HFE C282Y mutation in patients with hemochromatosis-induced iron overload. Venous thromboembolic events not only are very common (affecting 0.1% of the population per year) 1,2 but also are often the result of one or several specific genetic defects in coagulation-related proteins. The most common of these identifiable genetic defects is the factor V (R506Q) Leiden mutation causing resistance to activated protein C (APC) and thrombotic predisposition via the destruction of a protein C cleavage and inactivation site in the factor V procoagulant protein. The factor V Leiden mutation is extremely common; heterozygotes represent 3% to 7% of the general population, approximately 20% of all patients with any venous thrombosis, and approximately 50% of patients with recurrent venous thrombosis. 3,4 The second most common thrombophilic genetic defect is the prothrombin G20210A mutation, imparting a 2- to 5-fold increased risk for venous thromboembolism (in heterozygotes) and being present in heterozygous form in 15% to 20% of patients with thrombophilia. 5 Unlike most other “genetic” diseases, for which treatment often is unavailable, anticoagulant therapy of