A Sensitized Whole Genome ENU Mutagenesis Screen Identifies an Arp2 Missense Mutation As a Novel Suppressor of Lethal Thrombosis in the Factor V Leiden Mouse

Abstract 493

Only ∼10% of individuals carrying the common risk factor, Factor V Leiden (FVL), will develop venous thrombosis. In order to identify potential FVL modifier genes, we performed a sensitized dominant ENU mutagenesis screen based on the perinatal synthetic lethal thrombosis observed in mice homozygous for FVL (FVQ/Q) and hemizygous for tissue factor pathway inhibitor deficiency (Tfpi+/−). The screen was performed by crossing ENU-treated male FVQ/Q mice with FVQ/+ Tfpi+/− females. Surviving G1 offspring were analyzed to identify survivors with the lethal FVQ/Q Tfpi+/− genotype. Analysis of 7,128 G1 offspring (∼2X genome coverage) identified 98 FVQ/Q Tfpi+/− mice that survived to weaning. Fourteen FVQ/Q Tfpi+/− G1 mice exhibited successful transmission of a putative suppressor mutation to two or more FVQ/Q Tfpi+/− G2 offspring. Whole exome sequencing was performed on a progeny tested member of 8 of the 14 lines using the Agilent SureSelect mouse whole exome capture kit resulting in ∼100 fold coverage. Variant analysis revealed a small number of high confidence novel heterozygous (dominant) single nucleotide variants (SNVs) in each sample. Sanger re-sequencing of all 11 SNVs in a cohort of mice from line 1 confirmed that the G to C mutation (chromosome 11 base 19,977,300) in the Actr2 gene (present in 15/16 re-sequenced progeny p<0.0001) is the dominant FVL modifier in this line. This mutation resulted in an R286G substitution in a highly conserved amino acid in the Arp2 protein, which is essential for the function of the Arp2/3 complex. Arp2/3 is responsible for intracellular actin branching and polymerization, which is critical for the regulation of cell shape. Analysis of 31 progeny from an Arp2 R/G × Arp2 R/G cross revealed only one live Arp2 G/G mouse (p<0.05), suggesting that R286G is a loss of function mutation and that homozygous deficiency is lethal. Complete blood counts (Advia 2120) performed on 14 Actr2 heterozygous and 10 wildtype littermates revealed no significant differences in platelet count, red and white blood cell counts, hematocrit or hemoglobin. However, measurements of platelet size and size distribution including mean platelet dry mass, platelet volume distribution width and the platelet component distribution width were significantly altered in Actr2 heterozygous mutant mice (p<0.05 for each measure). Thus, partial deficiency of Arp2 appears to alter platelet structure/function resulting in a shift in hemostatic balance facilitating survival of the otherwise lethal FVQ/Q Tfpi+/− phenotype. These results suggest that variation in Arp2 or related genes could potentially modify thrombosis risk in humans, and might also identify novel therapeutic targets for the treatment of this class of disorders.