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 F5 L/L Tfpi +/- lethal thrombosis. Surviving F5 L/L Tfpi +/- mice from our M odifier of F actor 5 L eiden 16 ( MF5L16 ) ENU line were subjected to whole-genome sequencing analysis. This revealed that instead of an ENU-induced mutation, four mutations introduced from our F5 L/L breeding stock were responsible for survival, which we named sMF5L1-4 for s pontaneous M odifier of F actor 5 L eiden. In our colony, F5 L/L female breeders carrying all four sMF5L mutations produced more litters and offspring than breeders with three or fewer mutations (p<0.006). Genotyping of 13 additional MF5L lines demonstrated that the four sMF5L 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 , sMF5L4 ), 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 de novo ENU mutations in suppressing F5 L/L Tfpi +/- lethal thrombosis.