Two rare confounding polymorphisms proximal to the factor V Leiden mutation

Comprehensive assessment of thrombophilia risk includes characterization of the R506Q (Leiden) mutation in factor V in many patients. Although activated protein C resistance is often assessed by means of a coagulation test, molecular interrogation of the G1691A mutation provides confirmation and interpretive utility in patients undergoing anticoagulation. Many molecular methods are available to provide genotyping at this locus. Among these, PCR-restriction fragment length polymorphism (RFLP) is widely used. Unfortunately, because this common mutation is 11 bp from the 3' end of exon 10, one PCR primer often anneals within intron 10. As a consequence, polymorphism can confound test results.

In the course of a clinical diagnostic test of 15,301 patients, two samples repeatedly showed two different unusual electrophoretic mobilities after PCR and restriction enzyme digestion. After stripping patient identifiers and entering a research protocol, the amplicons from these DNAs were sequenced in parallel with normal and heterozygous G1691A control genomic DNA samples. This sequencing showed two novel polymorphisms, each mapping to intron 10.

PCR-RFLP-based methods rely on sequence conservation in the interrogated region. Amplification of mutated loci adjacent to introns present a special risk for confounding restriction patterns. Sequencing amplicons with reproducibly unusual restriction patterns resolved the paradoxical restriction pattern in this case.