A sensitized mutagenesis screen in Factor V Leiden mice identifies novel thrombosis suppressor loci

Abstract

AbstractFactor V Leiden (F5L) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized ENU mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous forF5L(F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/−).F8deficiency enhanced survival ofF5L/LTfpi+/−mice, demonstrating thatF5L/LTfpi+/−lethality is genetically suppressible. ENU-mutagenizedF5L/Lmales andF5L/+Tfpi+/−females were crossed to generate 6,729 progeny, with 98F5L/LTfpi+/−offspring surviving until weaning. Sixteen lines exhibited transmission of a putative thrombosuppressor to subsequent generations, with these lines referred to asMF5L(Modifier ofFactor5 Leiden) 1-16. Linkage analysis inMF5L6identified a chromosome 3 locus containing the tissue factor gene (F3). Though no ENU-inducedF3mutation was identified, haploinsufficiency forF3(F3+/−) suppressedF5L/LTfpi+/−lethality. Whole exome sequencing inMF5L12identified anActr2gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression ofF5L/LTfpi+/−lethality (p=1.7x10−6), suggesting thatActr2p.R258Gis thrombosuppressive. CRISPR/Cas9 experiments to generate an independentActr2knockin/knockout demonstrated thatActr2haploinsufficiency is lethal, supporting a hypomorphic or gain of function mechanism of action forActr2p.R258G. Our findings identifyF8and theTfpi/F3axis as key regulators in determining thrombosis balance in the setting ofF5Land also suggest a novel role forActr2in this process.Significance StatementVenous thromboembolism (VTE) is a common disease characterized by the formation of inappropriate blood clots. Inheritance of specific genetic variants, such as the Factor V Leiden polymorphism, increases VTE susceptibility. However, only ~10% of people inheriting Factor V Leiden develop VTE, suggesting the involvement of other genes that are currently unknown. By inducing random genetic mutations into mice with a genetic predisposition to VTE, we identified two genomic regions that reduce VTE susceptibility. The first includes the gene for blood coagulation Factor 3 and its role was confirmed by analyzing mice with an independent mutation in this gene. The second contains a mutation in the Actr2 gene. These findings identify critical genes for the regulation of blood clotting risk.