TY - JOUR T1 - A sensitized mutagenesis screen in Factor V Leiden mice identifies novel thrombosis suppressor loci JF - bioRxiv DO - 10.1101/080432 SP - 080432 AU - Randal J. Westricka AU - Kärt Tomberg AU - Amy E. Siebert AU - Guojing Zhu AU - Mary E. Winn AU - Sarah L. Dobies AU - Sara L. Manning AU - Marisa A. Brake AU - Audrey C. Cleuren AU - Linzi M. Hobbs AU - Lena M. Mishack AU - Alexander Johnston AU - Emilee N. Kotnik AU - David R. Siemieniak AU - Jishu Xu AU - Jun Z. Li AU - Thomas L. Saunders AU - David Ginsburg Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/02/12/080432.abstract N2 - Factor 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 for F5L (F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/−). F8 deficiency enhanced survival of F5L/L Tfpi+/− mice, demonstrating that F5L/L Tfpi+/− lethality is genetically suppressible. ENU-mutagenized F5L/L males and F5L/+ Tfpi+/− females were crossed to generate 6,729 progeny, with 98 F5L/L Tfpi+/− offspring surviving until weaning and 16 lines exhibiting transmission of a putative thrombosuppressor to subsequent generations. These lines are referred to as MF5L (Modifier of Factor 5 Leiden) 1-16. Linkage analysis in MF5L6 identified a chromosome 3 locus containing the tissue factor gene (F3). Though no ENU-induced F3 mutation was identified, haploinsufficiency for F3 (F3+/−) suppressed F5L/L Tfpi+/− lethality. Whole exome sequencing in MF5L12 identified an Actr2 gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression of F5L/L Tfpi+/− lethality (p=1.7x10−6), suggesting that Actr2p.R258G is thrombosuppressive. CRISPR/Cas9 experiments to generate an independent Actr2 knockin/knockout demonstrated that Actr2 haploinsufficiency is lethal, supporting a hypomorphic or gain of function mechanism of action for Actr2p.R258G. Our findings identify F8 and the Tfpi/F3 axis as key regulators in determining thrombosis balance in the setting of F5L and also suggest a novel role for Actr2 in this process. ER -