title: Evaluation of temperature gradient capillary electrophoresis for detection of the Factor V Leiden mutation: coincident identification of a novel polymorphism in Factor V.
abstract: OBJECTIVE
  The Factor V Leiden mutation (G1691A) is a clinically important polymorphism that results in an increased risk of thrombosis. The goal of this study was to compare a temperature gradient capillary electrophoresis (TGCE) platform for the detection of Factor V gene mutations to a conventional restriction fragment length polymorphism (RFLP) assay.
  
  
  METHODS
  Three hundred and four samples were analyzed by both TGCE and a common clinical Mnl I/RFLP assay. Concordance of results between the two assays was observed for 302/304 (99.3%) of the samples.
  
  
  RESULTS
  All of the Leiden mutants (23/23, 100%) were identified by TGCE. Of the two discrepant results, one was caused by low peak heights in the TGCE output data and was easily rectified by the addition of a minimum peak height threshold. The second discrepancy resulted from the presence of a G-->A transition 95 bp downstream of the Leiden mutation site. This polymorphism represents a previously unreported alteration of the Factor V gene.
  
  
  CONCLUSIONS
  The TGCE assay is less labor-intensive and has a higher throughput capacity than the Mnl I/RFLP assay. TGCE is a less specific assay than the Mnl I/RFLP assay that allows for the detection of novel polymorphisms, but also creates the need for all positive TGCE results to be confirmed by an alternate method such as sequencing. Our results demonstrate that TGCE is a highly sensitive method for mutation detection and has utility for mutation discovery analysis.
authors.affiliation: Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Maryland 21287, USA. kmurphy4@jhmi.edu
authors.forename: Kathleen
authors.initials: K
authors.lastname: Murphy
authors.forename: Michael
authors.initials: M
authors.lastname: Hafez
authors.forename: Juliet
authors.initials: J
authors.lastname: Philips
authors.forename: Kellie
authors.initials: K
authors.lastname: Yarnell
authors.forename: Kevin
authors.initials: K
authors.lastname: Gutshall
authors.forename: Karin
authors.initials: K
authors.lastname: Berg
mesh_terms: D004252:DNA Mutational Analysis; D019075:Electrophoresis, Capillary; D005165:Factor V / Q000235:genetics*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length
chemical_list: C095381:factor V Leiden; D005165:Factor V