A Sensitized ENU Mutagenesis Screen for Thrombosis Modifiers Identifies Suppressor Variants in Non-mutagenized Parental Generations Due to Antithrombotic Selective Pressures

Thrombosis is a leading cause of morbidity and mortality. We used a mouse forward genetic ENU screen to identify genomic variants that suppress <i>F5</i> ^L/L <i>Tfpi</i> ^+/- lethal thrombosis. Surviving <i>F5</i> ^L/L <i>Tfpi</i> ^+/- mice from our <i>M</i> odifier of <i>F</i> actor <i>5 L</i> eiden 16 ( <i>MF5L16</i> ) ENU line were subjected to whole-genome sequencing analysis. This revealed that instead of an ENU-induced mutation, four mutations introduced from our <i>F5</i> ^L/L breeding stock were responsible for survival, which we named <i>sMF5L1-4</i> for <i>s</i> pontaneous <i>M</i> odifier of <i>F</i> actor <i>5 L</i> eiden. In our colony, <i>F5</i> ^L/L female breeders carrying all four <i>sMF5L</i> mutations produced more litters and offspring than breeders with three or fewer mutations (p<0.006). Genotyping of 13 additional <i>MF5L</i> lines demonstrated that the four <i>sMF5L</i> mutations were present in all lines and were consistently associated with survival. Of these four mutations, a single G to A intergenic variant on Chromosome 18 (Chr18 ^A , <i>sMF5L4</i> ), was most significantly associated with survival (p=0.003), with ∼15% penetrance for conferring the survival phenotype. Furthermore, platelet aggregation was significantly reduced in Chr18 ^A mice, suggesting an additional mechanism by which Chr18 ^A could suppress lethal thrombosis. Comparative transcriptomics analysis of livers from Chr18 ^A mice versus wildtype littermate controls revealed a small number of differentially expressed genes both known and unknown to affect thrombosis. In summary, we have identified four variants exerting a significant selective breeding advantage along with antithrombotic effects. Superimposing our mutagenesis screen on a selective background illustrates the interplay of natural strain background variants and <i>de novo</i> ENU mutations in suppressing <i>F5</i> ^L/L <i>Tfpi</i> ^+/- lethal thrombosis.