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Background:Thrombosis is initiated by tissue factor (TF, gene nameF3) binding to coagulationFVII, with tissue factor pathway inhibitor (TFPI) inhibiting this complex. Alterations in TF orTFPI expression significantly affect thrombosis. Reducing TFPI expression by 50% (Tfpi+/-) inmice results in a perinatal lethal phenotype on the Factor V Leiden homozygous(F5^L/L)prothrombotic background. We used theF5^L/LTfpi+/- lethal phenotype to conduct a dominantsensitized whole genome ENU mutagenesis screen to suppress theF5^L/LTfpi+/- lethality. Weidentified a Modifier of Factor 5 Leiden 6 (MF5L6) line with 72% penetrance and 85F5^L/LTfpi+/- offspring. A significant linkage peak (LOD=4.35),explaining half the suppressing effect andcontainingF3(Chromosome 3) was identified. Goals/Hypothesis:To identify the genomic variant controlling F3 expression in the MF5L6 line. Methods:To quantifyF3expression in the surviving mice from MF5L6, quantitative PCR onliver, lung, and heart tissues from MF5L6 was performed. We used Sanger DNA and highthroughput sequencing to identify candidate TF regulatory variants in the F3 locus. Theprothrombin time assay was used to test the effects of reduced TF expression on in vitro bloodcoagulation. Results:Two distinct expression profiles in the lung and liver of the MF5L6 mice wereobserved, those that had a 50% reduction inF3mRNA and those that did not. Heart tissuesexhibited one expression profile, suggesting that the variant regulates F3 expressiontissue-specifically. Sanger sequencing of theF3coding region revealed no coding mutations inMF5L6 mice. Whole genome sequencing identified two novel candidate variants (in unknownF3 regulatory elements) in the 200 kilobase upstream region ofF3. The 50% reduction inF3resulted in significant changes in coagulation by the prothrombin assay (n=18,p<0.0009). Conclusion:We identified novel candidate variants for regulatingF3gene expression and aredetermining their mechanism of action. Investigation of these variants will provide new insightsinto the regulation ofF3and enable us to identify the variant(s) responsible for the remainder ofthe thrombosis suppressing effect in MF5L6. Our findings provide new insights into the geneticregulation of thrombosis. 

Abstract U12-type or minor introns are found in most multicellular eukaryotes and constitute ∼0.5% of all introns in species with a minor spliceosome. Although the biological significance for the evolutionary conservation of U12-type introns is debated, mutations disrupting U12 splicing cause developmental defects in both plants and animals. In human hematopoietic stem cells, U12 splicing defects disrupt proper differentiation of myeloid lineages and are associated with myelodysplastic syndrome, predisposing individuals to acute myeloid leukemia. Mutants in the maize ortholog of RNA binding motif protein 48 (RBM48) have aberrant U12-type intron splicing. Human RBM48 was recently purified biochemically as part of the minor spliceosome and shown to recognize the 5′ end of the U6atac snRNA. In this report, we use CRISPR/Cas9-mediated ablation of RBM48 in human K-562 cells to show the genetic function of RBM48. RNA-seq analysis comparing wild-type and mutant K-562 genotypes found that 48% of minor intron-containing genes have significant U12-type intron retention in RBM48 mutants. Comparing these results to maize rbm48 mutants defined a subset of minor intron-containing genes disrupted in both species. Mutations in the majority of these orthologous minor intron-containing genes have been reported to cause developmental defects in both plants and animals. Our results provide genetic evidence that the primary defect of human RBM48 mutants is aberrant U12-type intron splicing, while a comparison of human and maize RNA-seq data identifies candidate genes likely to mediate mutant phenotypes of U12-type splicing defects.