PT  - JOURNAL ARTICLE
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 C. Cleuren
AU  - Linzi M. Hobbs
AU  - Lena M. Mishack
AU  - Alexander Johnston
AU  - Emilee Kotnik
AU  - David R. Siemieniak
AU  - Jishu Xu
AU  - Jun Z. Li
AU  - Thomas L. Saunders
AU  - David Ginsburg
TI  - A sensitized mutagenesis screen in Factor V Leiden mice identifies novel thrombosis suppressor loci
AID  - 10.1101/080432
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 080432
4099  - http://biorxiv.org/content/early/2017/04/06/080432.short
4100  - http://biorxiv.org/content/early/2017/04/06/080432.full
AB  - 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. Sixteen lines exhibited transmission of a putative thrombosuppressor to subsequent generations, with these lines 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=1.7×10−6), 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.Significance Statement Venous thromboembolism (VTE) is a common disease characterized by the formation of abnormal 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 will 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 tissue factor gene and its role was confirmed by analyzing mice with an independent tissue factor gene mutation. The second contains a mutation in the Actr2 gene. These findings identify critical genes for the regulation of blood clotting risk.