Implementation of a cost-effective unlabeled probe high-resolution melt assay for genotyping of Factor V Leiden.
The Factor V Leiden mutation (FVL; c.1601G>A, p.Arg534Gln), the most common aberration underlying activated Protein C resistance, results in disruption of a major anticoagulation pathway and is a leading cause of inherited thrombophilia. A high-throughput assay for FVL mutation detection was developed using a single unlabeled probe on a high-resolution platform, the 96-well Roche 480 LightCycler (LC480) instrument. This method replaced the U.S. Food and Drug Administration-approved Roche Factor V Leiden kit assay on the LightCycler PCR instrument, decreasing total cost by 48%. The analytical sensitivity and specificity of the LC480 high-resolution assay approached 100% for the FVL mutation. Factor V mutations in proximity to the FVL locus may influence probe binding efficiency and melt characteristics. One out of three very rare variants tested in a separate study, 1600delC, was not distinguishable from FVL using the described high-resolution assay. However, a c.1598G>A variant, which changes the amino acid sequence from arginine to lysine at position 533, was detected by this high-resolution assay and confirmed by bidirectional sequencing. In the labeled probe LightCycler assay, the c.1598G>A variant was indistinguishable from the heterozygous FVL control. The c.1598G>A variant has not been described previously and its clinical significance is uncertain. In conclusion, the LC480 FVL assay is cost effective in a high-throughput setting, with capability to detect both previously described and novel FV variants.
Department of Pathology, University of Utah, Salt Lake City, Utah, USA. asvensson@hsc.wvu.edu
Annika M
AM
Svensson
Lan-Szu
LS
Chou
Cindy
C
Meadows
Christine E
CE
Miller
Robert
R
Palais
Kelli
K
Sumner
Tyler C
TC
Wayman
Rong
R
Mao
Elaine
E
Lyon
D003362:Cost-Benefit Analysis; D015342:DNA Probes; D005165:Factor V / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods*; D005838:Genotype; D057166:High-Throughput Screening Assays; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000191:economics* / Q000295:instrumentation / Q000379:methods*; D019851:Thrombophilia / Q000235:genetics; D044366:Transition Temperature
D015342:DNA Probes; C095381:factor V Leiden; D005165:Factor V