5 years ago

Sensitized mutagenesis screen in Factor V Leiden mice identifies thrombosis suppressor loci [Genetics]

Sensitized mutagenesis screen in Factor V Leiden mice identifies thrombosis suppressor loci [Genetics]
Linzi M. Hobbs, Guoȷing Zhu, Alexander J. Johnston, Lena M. Mishack, Audrey C. Cleuren, Marisa A. Brake, Sara L. Manning, Sarah L. Dobies, Mary E. Winn, Thomas L. Saunders, Kart Tomberg, David Ginsburg, David R. Siemieniak, Emilee Kotnik, Jun Z. Li, Jishu Xu, Randal J. Westrick, Amy E. Siebert

Factor V Leiden (F5L) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized N-ethyl-N-nitrosourea (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 the 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, referred to as “modifier of Factor 5 Leiden (MF5L1–16),” exhibited transmission of a putative thrombosuppressor to subsequent generations. Linkage analysis in MF5L6 identified a chromosome 3 locus containing the tissue factor gene (F3). Although 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 role for Actr2 in this process.

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