PCR screening method to detect factor v leiden mutation in tia and stroke

D. De Lucia, G. A. Puca, A. M. Molman, S. Pezzella, C. Di Mauro, M. Conte, V. Rems, A. Belli, M. B. Donati, Iacoviello Land, V. Nigro

Research output: Contribution to journalArticlepeer-review


Inherited resistance to APC is caused by a single factor V gene mutation changing arginine 506 to a glutamine (FV Leiden) in exon 10. We studied APC-resistance in a kindred of 5 subjects with a familial history of transient ischemic attacks (TIA) or strokes. Two siblings had suffered from TIAs. and one of them had an ischemic stroke. We assayed APC-resistance according to the method of Dahlback Two out of five subjects showed APC-resistance but no deficiencies in the natural anticoagulants (protein C, protein S, antithrombin III) The family history demonstrated a distribution of APC-resistance compatible with dominant autosomal inheritance. None of the subjects was positive for antiphospholipid antibodies. Fibrinolytic protein deficiencies were not found. After biochemical analyses we set-up a PCR screening method (without sequencing or restriction enzyme digestion) to detect factor V (FV) Leiden mutation. Genomic DNA (200 ng) was amplified in two separate reactions, one containing the primers for the wild-type allele (forward/5′-gatgaacccacagaaaatga and reverse/5′-aaaagtacctgtattcctc) and the other containing the primers for the mutant allele (forward/5′-cagatccctggacaggca and reverse/5′-tgttatcacactggtgctaa) The fragments were generated from the genomic DNA sequence spanning the 3′ end of exon 10 (including nucleotide 1,691) and 146 bp of the flanking intron 10 region. PCR reaction for the wild-type allele yielded a 149 bp product, while a separate PCR reaction for the mutant allele yielded a 174 bp product. In normal subjects only the 149 bp product is observed, while in homozygous resistant subjects only the 174 bp product as well. Both products are amplified from the DNA of heterozygous resistant subjects. An heterozygous carrier status for this mutant gene was found and associated with symptoms of TIAs and relative angiographic abnormalities in one subject with an ischemic stroke. Therefore, we propose this simple PCR test to study young patients with an episode of cerebral thrombosis and a positive family history for TIAs in transectional and epidemiological studies.

Original languageEnglish
Pages (from-to)99
Number of pages1
Issue numberSUPPL. 3
Publication statusPublished - 1996

ASJC Scopus subject areas

  • Hematology


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