Thrombosis Research 92 (1998) 251–259 REGULAR ARTICLE New DNA Diagnostic System for Detection of Factor V Leiden Lev I. Patrushev 1 , Elena S. Zykova 1,2 , Alexei L. Kayushin 1 , Maria D. Korosteleva 1 , Anatoly I. Miroshnikov 1 , Igor N. Bokarew 2 , Stanislav G. Leont’ev 3 , Valery M. Koshkin 3 and Evgeny S. Severin 2 1 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences; 2 Medical Sechenov Academy, 3 Russian Medical University, Moscow, Russia. (Received 3 April 1997 by Editor B. Bouma; revised/accepted 24 June 1998) G enomic mutations often cause hereditary Abstract diseases and very often reveal predisposi- tion to disease [1]. As shown recently, the Use was made of allele-specific PCR to develop a single point mutation in exon 10 of the human highly effective DNA diagnostic system for detec- factor V gene is the main reason for hereditary tion of the factor V Leiden mutation in exon 10 of human factor V gene. The allele-specific primers resistance of factor Va to the activated protein C contain a 3 ' -OH end nucleotide, which matches a [2–4]. As a result of the mutation designated factor mutant or wild-type nucleotide of the template V Leiden, the nucleotide G is substituted by A in DNA (A-allele and G-allele, respectively) and also position 1691, which in turn induces the substitu- one mismatched nucleotide near the 3 ' -end. The tion of Arg-506 by Gln in the factor V polypeptide. universal primers have an internal mismatch with The factor V polypeptide loses a processing site for the mutant nucleotide of the template DNA and APC, thus producing the APC resistance phenotype. another mismatched nucleotide at 3 ' -OH end. The To identify known mutations in genomic DNA, A-allele-specific primer enables the preferential of use are allele-specific oligonucleotide hybridiza- amplification of both the homozygous and het- tion (ASO) [5], restriction fragment length poly- erozygous mutant alleles. The extension of the morphism (RFLP) [6], ligase chain reaction (LCR) G-allele-specific primer or the universal one is in- [7] as well as allele-specific PCR [8]. Most of these hibited in the presence of the homozygous factor approaches and some others were recently em- V Leiden. The developed assay system allowed us ployed for detection of the factor V Leiden. [9–27]. to detect five patients, who are heterozygous for Factor V is encoded by a 80-kb gene consisting of factor V Leiden among the 48 patients with deep 25 exons of different length: from 72 to 2820 bp venous thrombosis and pulmonary thromboembo- (see Figure 1). The factor V polypeptide precursor lism.  1998 Elsevier Science Ltd. consists of 2224 amino acids containing the leader peptide of 24 amino acids [28]. The factor V Leiden Key Words: Allele-specific PCR; factor V Leiden; Throm- destroys the site for restriction endonuclease Mnl bophilia; DNA diagnostics I rendering it uncapable of cutting the mutant DNA at this site (see Figure 1). In accordance with this Abbreviations: ASO, allele-specific oligonucleotide hybridization; fact the factor V Leiden was for the first time de- RFLP, restriction fragment length polymorphism; LCR, ligase tected due to resistance of the corresponding PCR chain reaction. Corresponding author: Lev I. Patrushev, Research Group of Ge- product to restriction enzyme Mnl I [2,3]. But al- nome Analysis and Correction, Shemyakin-Ovchinnikov Institute lele-specific PCR makes it possible to detect point of Bioorganic Chemistry, Russian Academy of Sciences, Mik- mutations much easier without employment of the lukho-Maklaya, 16/10, 117871 Moscow, GSP-7 V-437, Russia. E-mail: patrush@ibch.siobc.ras.ru. restriction enzymes. We designed a simple and ef- 0049-3848/98 $–see front matter  1998 Elsevier Science Ltd. Printed in the USA. All rights reserved. PII S0049-3848(98)00133-9