Ensuring Accurate Molecular Genetic Testing

Abstract

In this issue of Clinical Chemistry , Lutz et al. (1) report on a multicenter evaluation of polymerase chain reaction (PCR) methods for the detection of Factor V Leiden genotypes. Mutations in the Factor V Leiden gene have been shown to result in activated protein C (APC) resistance, which is the most common cause of inherited venous thrombosis in Caucasians (2)(3)(4). In particular, a specific mutation in the Factor V Leiden gene, a guanine- to-adenine substitution at nucleotide 1651 that results in a glutamine-to-arginine substitution at position 506 (R506Q), has been shown to be associated with APC resistance (2)(3). Clinically, individuals who are heterozygous for this mutant allele have a 5- to 10-fold increased risk for thrombotic events, and individuals who are homozygous have a 50- to 100-fold increase (2)(5). In the past, patients with APC resistance could be identified only by coagulation-based assays that are limited by decreased reliability of results in a number of conditions (e.g., pregnancy) and in patients receiving certain pharmacologic agents (e.g., anticoagulant therapy) (6). In contrast, molecular testing for the identification of Factor V Leiden gene mutation heterozygotes and homozygotes is not subject to these limitations. This advantage over coagulation-based assays has resulted in the rapid integration of this testing into clinical practice and its widespread availability. For example, in a recent survey of molecular genetic testing laboratory directors, Factor V Leiden mutation detection was available in 29% of 245 responding laboratories (7). In the study by Lutz et al., six laboratories that offer Factor V Leiden mutation analysis simultaneously tested a set of 62 blinded patient samples to determine the Factor V Leiden genotype. Although each of the participating laboratories had developed PCR-based assays, the exact testing system conditions varied substantially (e.g., PCR primer …