A sensitized mutagenesis screen in Factor V Leiden mice identifies novel thrombosis suppressor loci

Abstract

Factor V Leiden (F5 L ) 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 F5 L (F5 L/L ) and haploinsufficient for tissue factor pathway inhibitor (Tfpi +/-). F8 deficiency enhanced survival of F5 L/L Tfpi +/mice, demonstrating that F5 L/L Tfpi +/lethality is genetically suppressible. ENU-mutagenized F5 L/L males and F5 L/+ Tfpi +/females were crossed to generate 6,729 progeny, with 98 F5 L/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 F5 L/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 F5 L/L Tfpi +/lethality (p=1.7x10 -6 ), suggesting that Actr2 p.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 Actr2 p.R258G . Our findings identify F8 and the Tfpi/F3 axis as key regulators in determining thrombosis balance in the setting of F5 L and also suggest a novel role for Actr2 in this process.Significance Statement (120 words max):Venous thromboembolism (VTE) is a common disease characterized by the formation of inappropriate 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 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 gene for blood coagulation Factor 3 and its role was confirmed by analyzing mice with an independent mutation in this gene. The second contains a mutation in the Actr2 gene. These findings identify critical genes for the regulation of blood clotting risk.

Whole exome sequencing of ENU-induced thrombosis modifier mutations in the mouse

“…F5 L/L Tfpi +/rescues were on average 25-30% smaller than their littermates at 2-3 weeks of age (p=7x10 -13 , Fig 1C-D). The proportion of identified rescues among G1 offspring, their smaller weight compared to littermates, and fewer overall females among rescues, were all consistent with our previous report [5].…”

“…F5 L/L Tfpi +/mice exhibit reduced survival and smaller size A previously described [5] sensitized ENU-mutagenesis for thrombosis modifiers was extended to screen for dominant suppressors of the perinatal lethal F5 L/L Tfpi +/genotype ( Fig 1A). 2834 G1 offspring were generated from mutagenized C57BL/6J F5 L/L males crossed to unmutagenized C57BL/6J F5 L/+ Tfpi +/females, with a total of 76 viable F5 L/L Tfpi +/rescue progeny identified at weaning.…”

“…In order to identify all/most exonic ENU mutations, a total of 107 G1 rescues (57 from the current ENU screen and an additional 50 rescues from the previous screen [5]) were subjected to WES (S1 Table). From ~1.5 million initially called variants, 6735 SNVs and 36 insertions-deletions (INDELs) within exonic regions were identified as potential ENUinduced mutations, using an in-house filtering pipeline (see Materials and methods).…”

“…To identify genetic variants potentially modifying the FVL, we recently employed a dominant ENU screen [5] in mice sensitized for thrombosis. Mice homozygous for the FVL mutation (F5 L/L ) and haploinsufficient for tissue factor pathway inhibitor (Tfpi +/-) die of perinatal thrombosis [6].…”

“…After ENU mutagenesis, 98 G1 F5 L/L Tfpi +/progeny survived to weaning ('rescues') and 16 of them exhibited successful transmission of the ENUinduced suppressor mutation. However, subsequent efforts to genetically map the corresponding suppressor loci were confounded by complex strain-specific differences introduced by the required genetic outcross [5]. Similar genetic background effects have complicated previous mapping efforts [7] and have been noted to significantly alter other phenotypes of interest [8,9].…”