Single nucleotide polymorphism (SNP) genotyping methods are widely used for the detection of hereditary thrombophilias caused by genetic defects in the coagulation system. The hereditary thrombophilias are frequently associated with higher incidences of point mutations in hemostasis (F2 20210G>A, F5 1691G>A) and folate metabolism (MTHFR 677C>Т, MTHFR 1298A>C) genes. Moreover, the combination of gene abnormalities in F2 or/and MTHFR with F5 Leiden mutation leads to increased risk of developing thrombosis. Thus, simultaneous detection of the multiple gene mutations in a sample has important clinical relevance. The microchip-based multiplex real time PCR for estimation of allele specific polymorphism in hemostatic and folate metabolism genes presented here has a high efficiency and may be used for laboratory diagnosis. The optimized protocol for estimation of 4 different types of genetic polymorphisms allowed PCR to be performed with minimal quantity of DNA template and PCR reagents including Taq polymerase and a short-term thermocycling.
Gennaia diagnostika shiroko ispol'zuetsia dlia vyiavleniia faktorov riska razvitiia nasledstvennoĭ trombofilii, voznikaiushcheĭ vsledstvie molekuliarnykh defektov v sisteme svertyvaniia krovi. Chastym geneticheskim izmeneniem, obuslavlivaiushchim razvitie nasledstvennoĭ trombofilii, iavliaetsia polimorfizm genov gemostaza (F2 20210G>A, F5 1691G>A) i folatnogo tsikla (MTHFR 677C>T, MTHFR 1298A>C). Risk razvitiia trombozov znachitel'no vozrastaet, esli mutatsii v genakh F2 i/ili MTHFR vstrechaiutsia v sochetanii s mutatsieĭ Leĭdena (F5 1691G>A). Takim obrazom, vypolnenie analiza s tsel'iu opredeleniia allel'nogo polimorfizma odnovremenno vsekh nazvannykh genov imeet vazhnoe klinicheskoe znachenie. V nastoiashchem issledovanii pokazana vysokaia éffektivnost' metoda dlia otsenki allel'nogo polimorfizma genov gemostaza i folatnogo tsikla s ispol'zovaniem mikrochipovoĭ allel'-spetsifichnoĭ PTsR. Étot test pozvoliaet odnovremenno proanalizirovat' polimorfizm po trem genam (chetyrem tochkam mutatsii) za korotkoe vremia s ispol'zovaniem minimal'nogo kolichestva DNK-matritsy i reaktivov PTsR, vkliuchaia DNK polimerazu, i mozhet byt' rekomendovan dlia klinicheskoĭ laboratornoĭ diagnostiki.
Keywords: F2; F5; MTHFR; PCR; hereditary thrombophilias; single nucleotide polymorphism.