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. Westrick 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 Cleuren AU - Lena M. Mishack AU - Linzi M. Hobbs AU - Alexander Johnston AU - David R. Siemieniak AU - Jishu Xu AU - Jun Z. Li AU - David Ginsburg Y1 - 2016/01/01 UR - http://biorxiv.org/content/early/2016/10/16/080432.abstract N2 - Factor V Leiden (F5L) is a common genetic risk factor for venous thromboembolism (VTE), though it exhibits only 10% penetrance. 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+/−). The observation that F8 deficiency enhanced survival of F5L/L Tfpi+/− mice demonstrated the potential for genetic suppression of F5L/L Tfpi+/− lethality. G0 ENU-mutagenized F5L/L males and F5L/+ Tfpi+/− females were next crossed to generate 6,739 G1 progeny, with 98 F5L/L Tfpi+/− offspring surviving until weaning and 16 exhibiting transmission of a putative thrombosuppressor to subsequent generations. The resulting 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, heterozygous F3 deficient mice (F3+/-) suppressed F5L/L Tfpi+/− lethality. Thus, like F8 deficiency, reduced F3 activity suppresses F5L/L Tfpi+/− thrombosis. Whole exome sequencing in MF5L12 identified an Actr2 gene point mutation (p.R258G) as the sole candidate. Attempts to generate an independent Actr2 knockin/knockout via CRISPR/Cas9 failed for any mouse beyond the 60-cell stage. Our findings identify F8 and the TFPI/F3 axis as key regulators of thrombosis balance in the setting of F5L and demonstrate the utility of this sensitized ENU mutagenesis approach for the identification of dominant thrombosis suppressor loci. ER -