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

Abstract

Factor V Leiden (<i>F5</i>^<i>L</i> ) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized <i>N</i>-ethyl-<i>N</i>-nitrosourea (ENU) mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for <i>F5</i>^<i>L</i> (<i>F5</i>^<i>L/L</i> ) and haploinsufficient for tissue factor pathway inhibitor (<i>Tfpi</i>^<i>+/-</i> ). <i>F8</i> deficiency enhanced the survival of <i>F5</i>^<i>L/L</i><i>Tfpi</i>^<i>+/-</i> mice, demonstrating that <i>F5</i>^<i>L/L</i><i>Tfpi</i>^<i>+/-</i> lethality is genetically suppressible. ENU-mutagenized <i>F5</i>^<i>L/L</i> males and <i>F5</i>^<i>L/+</i><i>Tfpi</i>^<i>+/-</i> females were crossed to generate 6,729 progeny, with 98 <i>F5</i>^<i>L/L</i><i>Tfpi</i>^<i>+/-</i> offspring surviving until weaning. Sixteen lines, referred to as "modifier of Factor 5 Leiden (<i>MF5L1-16</i>)," exhibited transmission of a putative thrombosuppressor to subsequent generations. Linkage analysis in <i>MF5L6</i> identified a chromosome 3 locus containing the tissue factor gene (<i>F3</i>). Although no ENU-induced <i>F3</i> mutation was identified, haploinsufficiency for <i>F3</i> (<i>F3</i>^<i>+/-</i> ) suppressed <i>F5</i>^<i>L/L</i><i>Tfpi</i>^<i>+/-</i> lethality. Whole-exome sequencing in <i>MF5L12</i> identified an <i>Actr2</i> gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression of <i>F5</i>^<i>L/L</i><i>Tfpi</i>^<i>+/-</i> lethality (<i>P</i> = 1.7 × 10^-6), suggesting that <i>Actr2</i>^p.R258G is thrombosuppressive. CRISPR/Cas9 experiments to generate an independent <i>Actr2</i> knockin/knockout demonstrated that <i>Actr2</i> haploinsufficiency is lethal, supporting a hypomorphic or gain-of-function mechanism of action for <i>Actr2</i>^p.R258G Our findings identify <i>F8</i> and the <i>Tfpi/F3</i> axis as key regulators in determining thrombosis balance in the setting of <i>F5</i>^<i>L</i> and also suggest a role for <i>Actr2</i> in this process.