An Intergenic Noncoding Chromosome 18 Variant Suppresses Lethal Thrombosis in Mice By Normalizing Blood Coagulation and Reducing Platelet Reactivity
Abstract Background: Factor V Leiden (FVL) is a common thrombosis susceptibility variant in humans. It is incompletely penetrant; this indicates that there are modifiers of FVL that alter thrombosis susceptibility. We used mouse models of FVL (F5L) and heterozygous tissue factor pathway inhibitor deficiency (Tfpi+/-), to identify a perinatal lethal genetic interaction when mice inherited F5L/L Tfpi+/-. This phenotype was used as the basis for a sensitized genome wide ENU mutagenesis screen to identify mutations suppressing lethal thrombosis in F5L/L Tfpi+/- mice. From this screen, we generated multiple independent lines of thrombosuppressed mice, called MF5L, for Modifier of F5L. MF5L16 was a large, highly penetrant (77.2%), multigenerational pedigree containing 136 viable F5L/L Tfpi+/- mice. Aims: In the present study, we aimed to identify and functionally characterize the thrombosuppressor mutation present in MF5L16. Methods: Genomic analyses: We performed whole genome sequencing (WGS) on four MF5L16 F5L/L Tfpi+/- mice. We used comparative bioinformatic analyses to identify variants inherited by all four mice and compiled these variants into candidate variant list. PCR and Sanger sequencing were used to analyze the 136 F5L/L Tfpi+/- mice for inheritance of each of the candidate variants. Functional analyses: We performed biochemical blood coagulation and platelet assays of blood from the Chr18 A mice . Complete blood counts were measured using the Advia 2120 with settings optimized for C57BL/6 mouse blood. Platelet aggregation studies were performed using the Roche Multiplate Aggregometer with ADP and type 1 collagen as the aggregating agents. Results: We analyzed four MF5L16 mice by WGS and identified seven spontaneous mutations that arose in our F5L/L breeding colony that were introduced into MF5L16. Importantly, no coding variants were linked to these variants. Analysis of these seven mutations in all 136 MF5L16 F5L/L Tfpi+/- mice revealed a significant association between a Chromosome 18 intergenic variant (Chr18 G to A, Chr18 A) and F5L/L Tfpi+/- mouse survival (p=0.003). To re-create the suppression of the lethal F5L/L Tfpi+/- phenotype, we bred F5+/L Tfpi+/- Chr18 +/A triple heterozygous mice to F5L/L Chr18 A/A mice to observe the effects of Chr18 A on F5L/L Tfpi+/- mouse survival. Out of 109 mice from this cross, two F5L/L Tfpi+/- Chr18 +/A mice were produced (expected ratio ~1:8). This suggests that the Chr18 A variant suppresses F5L/L Tfpi+/- lethal thrombosis at ~15% penetrance. Complete blood count analysis on Chr18 +/+,Chr18 +/A, and Chr18 A/A mice determined that Chr18 A/A mice had reduced platelet count and distribution width and increased variability in red blood cell (RBC) mean corpuscular volume (n≥4; p<0.05). The Chr18 A/A mice did not display differences in PT or aPTT assays, but had significantly reduced platelet aggregation velocity when stimulated by both ADP and collagen agonists (n≥4; p=0.0002). Additionally, blood smears revealed the presence of poikilocytic RBCs in the Chr18 A/A mice. Conclusions and future directions: Our results establish that a noncoding intergenic Chr18 variant at nucleotide position 62,970,011 (G>A, Chr18 A) contributes to thrombosuppression by reducing platelet reactivity. The observed platelet and RBC phenotypes suggest that a major mechanism of Chr18 A thrombosuppression could be through regulation of gene expression in cells of the myeloid lineage. We are performing additional platelet and blood coagulation analyses to refine the phenotypic differences due to the Chr18 A variant. Comparative transcriptomic analyses are also being performed to identify the genetic pathways involved. Understanding the mechanism in which this intergenic mutation suppresses thrombosis could provide insights into human thrombosis regulation. Disclosures No relevant conflicts of interest to declare.
