Potential Misdiagnosis of Factor V Leiden: A Comparison of Two Genotyping Techniques

Resistance to activated protein C (APCR) (1) is, in 95 percent or more of cases, due to the presence of a mutation in the factor V gene, a G to A substitution at nucleotide 1691 (2). This mutation gives rise to a variant form of factor V, FV Leiden, which has normal procoagulant properties but is inactivated only slowly by activated protein C (APC). APCR is the most common of the known inherited risk factors for venous thrombosis, with a prevalence of 20 to 50 percent in subjects with a history of deep vein thrombosis (3,4), depending on patient population selection criteria. The most commonly used method to screen for the FV Leiden mutation involves polymerase chain reaction (PCR) amplification and restriction enzyme digestion of the PCR product. The FV gene nucleotide substitution associated with FV Leiden (G1691A) results in the loss of a Mi11 1 restriction site which can be revealed by gel electrophoresis. In theory, however, this assay may not be wholly specific for the FV Leiden mutation. Alternative substitutions within the 4-base-pair Mnl I recognition site (GAGG) will also abolish M n l l digestion giving rise to an electrophoresis pattern which is identical to that associated with the FV Leiden mutation. This will be so even if the alternative substitution is silent and the amino acid at position 506 in the FV molecule remains an arginine, thus preserving the APC cleavage site. Screening for the FV Leiden mutation is an important part of thrombophilia investigations, and it is essential that detection strategies are reliable. Recently, a case was described where genotyping by PCR and Mnl I restriction enzyme digestion produced a false positive finding of heterozygosity for the FV Leiden mutation (5). The patient concerned had a normal coagulation assay for APCR and subsequent sequencing of the FV Leiden-related Mnl 1 restriction site revealed a silent A to C transition at nucleotide position 1692. This resulted in the loss of the M n l l restriction site, but produced no change in the amino acid structure of the FV molecule. This finding potentially has important implications for the use of Mnl I digestion to screen for the FV Leiden mutation. We have utilised 2 detection strategies to investigate the prevalence of the reported FV gene nucleotide 1692 polymorphism and its potential for causing misdiagnosis of FV Leiden. The first strategy relied upon PCR followed by Mnl 1 restriction enzyme digestion and agarose gel electrophoresis (2), whilst the second utilised the Amplification Refractory Mutation System (ARMS) (6). In the latter method, 2 PCR reactions were performed on each DNA sample, using oligonucleotide primers designed specifically to amplify the F V gene wild-type allele or the mutant FV gene Leiden allele respectively. A false positive result would be obtained using the Mnl I digestion method if disruption of the restriction enzyme site occurred at nucleotide 1692. Using ARMS, however, the FV Leiden allele specific primer, whose 3' base matched only the A transition at position 1691, would fail to amplify if there was no such base change. This would indicate that the subject was in fact negative for the FV Leiden mutation. We used the ARMS genotyping method to screen 273 patients found by Mnl 1 digestion to be FV Leiden heterozygotes, and 7 patients similarly found to be homozygotes. Complete concordance was observed between the results obtained by M n l l digestion or by ARMS. We have also screened by PCR and Mnl I digestion 1,258 individuals who were found to have normal Mnl 1 digestion patterns, indicating that the Mnl 1 recognition site was intact. Overall, therefore, we have found the FV gene nucleotide 1692 polymorphism to be absent in a total of 1,538 individuals (3,076 alleles). In conclusion, the silent base change at the FV gene nucleotide 1692 is at most a rare polymorphism in our patient population, indicating that PCRlMnl I restriction enzyme detection of the FV Leiden mutation is reliable. Theoretically, however, there remains a slight risk of obtaining a false positive result when using this method.