Identifying Thrombosis Modifier Genes in the Mouse The genetic factors responsible for the highly variable clinical course of patients with factor V Leiden and other forms of thrombophilia are unknown.
Aim 1 of this project will identify a large subset of potential thrombosis modifier genes within the mammalian genome using a whole genome ENU mutagenesis strategy in the mouse. We previously reported a uniformly lethal, perinatal thrombosis in mice homozygous for the factor V Leiden mutation (FvQ/Q) and heterozygous for Tfpi (Tfpi+/-). In preliminary studies, we used this phenotype as the basis for a large scale genetic suppressor screen. Mutagenized FvQ/Q males were bred with doubly heterozygote (FvQ/+Tfpi+/-) females and >6300 surviving G1 offspring genotyped to identify rare FvQ/QTfpi+/- animals who have survived the otherwise uniform perinatal lethality, presumably due to the suppressing effect of a mutated (haploinsufficient) modifier gene. As proof of principal, a genetic cross with TF knockout mice demonstrates that mutation at the TF locus will rescue the lethal FvQ/Q Tfpi+/- genotype. To date, 82 candidate suppressor mutants have been identified. Though most of these mutants have been lost due to decreased survival and/or infertility, genetic mapping for 7 of these lines is in progress and an additional 4 lines are undergoing progeny testing. We will expand this screen to achieve 4-6 fold genome coverage and identify the corresponding genes by positional cloning.
Aim 2 will further characterize a strain specific modifier of the FvQ/QTfpi+/- lethal phenotype identified in the DBA/2J strain, by a similar positional cloning strategy.
Aim 3 will take advantage of natural strain variation in the mouse to identify genetic factors critical for susceptibility to thrombotic thrombocytopenic purpura (TTP). Previous work by us and others suggests that deficiency of the metalloprotease ADAMTS13 is necessary, but not sufficient for the development of TTP in humans and in mice. In preliminary studies, we have shown that the development of TTP in ADAMTS13 null mice is dependent on genetic factors differing among mouse strains. Genetic crosses between susceptible and resistant strains will be performed to identify the underlying gene(s), which should provide critical insight into the pathogenesis of TTP as well as offering candidates for useful diagnostic and prognostic markers in humans. Relevance: These studies should provide new insight into the regulation of blood clotting, and identify a number of genes that may be important predictors of severity for many common inherited blood clotting diseases. This information may also suggest new approaches to therapy for these conditions.
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