The novel FII c.*64_*66del prothrombin gene variant in women with pregnancy loss

Abstract

Normal pregnancy associated with complex changes of hemostasis, leading to hypercoagulability states. The presence of acquired or genetic prothrombotic risk factors might affect the proper maternal-fetal circulation and result in pregnancy loss. Hence, the screening for the novel prothrombotic variants associated with pregnancy loss would be beneficial. Our aim was to investigate the potential association of recently reported c.*64_*66del variant in prothrombin gene with the etiology of pregnancy loss. Study included 105 women with pregnancy loss and 155 controls. Analyses in patients? plasma samples, as well as in vitro analyses on transfected Cos-7 cell line were performed in order to investigate the mechanism by which this variant could perturb the coagulation and lead to pregnancy loss. Analyses in patients' DNA and plasma samples involved: DNA sequencing and PCR-RFLP assay for detection of FII c.*64_*66del variant, routine thrombophilia screening, thrombin generation assay and Western blot analysis of prothrombin plasma level. In vitro analyses included transient transfections of Cos-7 cell line with wild-type and c.*64_*66del mutated constructs of pCIneo?SV40 expression vector. Real-Time PCR and Western blot analysis were used to determine the effect of FII c.*64_*66del variant on mRNA and protein level in constructs. Three women in patients group (2.9%) were detected as heterozygous carriers of FII c.*64_*66del, while none was found among controls. The carriers routine thrombophilia parameters were in reference range and similar prothrombin plasma level in FII c.*64_*66del carriers and non-carriers were detected. The endogenous thrombin potential was slightly increased in FII c.*64_*66del carriers compared to control plasma, but this difference was not statistically significant. Results of in vitro analyses showed significantly decreased prothrombin mRNA and protein level for c.*64_*66del variant compared to wild-type. Results of our pilot study have shown a trend of higher prevalence of FII c.*64_*66del variant in women with pregnancy loss. However, further studies are needed to completely elucidate whether FII c.*64_*66del variant affects prothrombin expression during pregnancy and to account its potential role in etiology of pregnancy loss.