Identifying Thrombosis Modifier Genes in the Mouse

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Project 1" Identifying Thrombosis Modifier Genes in the Mouse Several highly prevalent genetic susceptibility factors for thrombosis have recently been identified, including the factor V Leiden mutation. However, most of the population attributable risk for both arterial and venous thrombotic disease remains unexplained. In addition, the genetic factors responsible for the highly variable clinical course of patients with factor V Leiden and other defined mutations are currently unknown. This project aims to identify a large subset of potential thrombosis modifier genes within the mammalian genome using a whole genome ENU mutagenesis strategy in the mouse. In preliminary studies, mice carrying the factor V Leiden mutation (R504Q) have been crossed with animals carrying targeted deletions in genes for other potential thrombosis risk factors. Mice homozygous for the factor V Leiden mutation (FvQ/Q) and heterozygous for Tfpi (Tfpi+/-) exhibit a uniformly lethal thrombotic phenotype in the immediate perinatal period. This observation serves as the basis for our proposed large-scale ENU mutagenesis screening strategy. Mutagenized FvQ/Q males will be bred with doubly heterozygote (FvQ/+ Tfpi+/-) females. The resulting G1 offspring should carry a large number of mutations with up to several hundred heterozygous gene inactivations per mouse. G1 mice will be genotyped to identify FvQ/Q Tfpi+/- animals who have survived the otherwise uniform perinatal lethality, presumably due to the suppressing effect of a mutated (haploinsufficient) modifier gene. In preliminary mutagenesis studies, 6 potential suppressor mutations have been identified. In addition, a spontaneous mutation that also rescues this lethal phenotype has recently been observed in our colony. These preliminary results demonstrate the feasibility of our approach for identifying modifier genes. Surviving FvQ/Q Tfpi+/- mice will be progeny tested and confirmed suppressor modifier genes mapped through an extended cross to homozygous FvQ/Q mice on the 129Sv genetic background. Candidate genes located within the mapped intervals will be tested by direct sequence analysis to identify the responsible mutation. Taken together, these studies should provide novel information about the total number of potential thrombosis modifier genes within the mammalian genome, provide new insight into the regulation of hemostasis in vivo, and identify a number of candidates as important modifier genes for inherited thrombotic and hemorrhagic diseases in humans.