Identification of Thrombosis Modifier Genes Using ENU Mutagenesis in the Mouse.

Abstract

Abnormal formation of a blood clot in veins, also called venous thromboembolism (VTE), is a major health problem in Western societies that affects 1 in every 1,000 individuals per year. Susceptibility to VTE is governed by both genetic and environmental factors, with approximately 60% of the risk attributed to genetic influences. The most prevalent genetic risk factor among VTE patients is a variant in coagulation factor V, called Factor V Leiden (FVL). While 20-25% of VTE patients carry the FVL variant, only ~10% of FVL carriers develop a VTE in their lifetime, indicating that interactions between FVL and other genetic and/or environmental factors influence the incidence and severity of thrombosis. The goal of this thesis was to identify modifier genes that help understand the differences in VTE phenotype among FVL carriers and more generally the complex genetic factors regulating hemostasis balance. The work described here took advantage of the synthetic lethal thrombosis phenotype observed in mice carrying two copies of the orthologous FVL (F5L/L) mutation together with haploinsufficiency for tissue factor pathway inhibitor (Tfpi+/-). F8 deficiency was found to ‘rescue’ F5L/L Tfpi+/- lethality, and an initial ENU mutagenesis screen for dominant thrombosis modifier genes additionally identified F3 and Actr2 as suppressors for this lethal phenotype (Chapter II). During the genetic analysis of the ENU-induced mutations, we additionally identified a de novo deletion in Nbeal2 which originated from a non-ENU treated parent, highlighting the potentially confounding effect of spontaneous mutation events in well-characterized mouse strains. Though initially considered a plausible thrombosis modifier, this mutation failed to rescue the synthetic lethal thrombosis (Chapter III). A complementary burden test that highlights genes enriched for mutations applied to >100 independent F5L/L Tfpi+/- rescues identified 12 novel candidate thrombosis modifiers. Preliminary validation data using independent null alleles suggest successful rescue for mice haploinsufficient for Sntg1 (Chapter IV).