Novel mutagenesis screen used to identify genetic suppressors of thrombosis

Researchers using a novel sensitized ENU (N-ethyl-N-nitrosourea, a carcinogen-induced animal tumor model established to grow malignant and benign tumors) mutagenesis (induction of mutation) screen in laboratory mice have developed a promising approach to genetic screening that can increase the likelihood of identifying dominant suppressors of thrombosis.

Using this novel genetic screening process, the researchers sought to identify potential modifier genes contributing to penetrance of Factor V Leiden, an inherited disorder of blood clotting in which the clotting process is unopposed, increasing the chance of developing abnormal morbid and life-threatening blood clots. The results of the study were presented at the 46th Annual Meeting of the American Society of Hematology.

Factor V Leiden (FVL) thrombophilia is an inherited disorder of blood clotting. It is the most commonly known inherited thrombotic risk factor and is present in about five percent of most Western populations. People who have the factor V Leiden mutation are at somewhat higher than average risk for a type of clot that forms in large veins in the legs (deep venous thrombosis) or a clot that travels through the bloodstream and lodges in the lungs (pulmonary embolism). FVL is incompletely penetrant with only about 10 percent of FVL carriers developing thrombosis in their lifetime.

A team of scientists from the University of Michigan and the Howard Hughes Medical Institute set out to identify the genetic factors responsible for the incomplete penetrance of FVL. They hypothesized that dominant mutations in key components of the coagulation system will improve hemostatic balance and allow survival in mice carrying both the FVL mutation and a partial deficiency of a key coagulation component, tissue factor pathway inhibitor (TFPI). TFPI, a natural anti-coagulant, is a key mechanism for the body's control of coagulation. Complete TFPI deficiency in mice is lethal in the embryo stage of development, whereas heterozygosity (having two genes at corresponding alleles on homologous chromosomes different for one or more alleles) is compatible with normal survival. Homozygosity (having the two genes at corresponding alleles on homologous chromosomes identical for one or more alleles) for FVL (FvQ/Q), however, in the context of heterozygosity for TFPI (Tfpi+/-), is uniformly lethal due to disseminated perinatal thrombosis.

In order to identify candidate modifier genes, the investigators performed a whole genome mutagenesis screen. In this screen, male FvQ/Q mice were mutagenized with ENU and bred to FvQ/+ Tfpi+/- double heterozygous females. DNAs from surviving offspring were PCR assayed to identify rescued mice with the FvQ/Q Tfpi+/- genotype.

Utilizing this lethal genetic interaction as a phenotyping tool, they analyzed 2,250 mouse offspring, corresponding to approximately half genome coverage, and identified 15 mice that survived to weaning. Heritability was demonstrated for the five mutant lines subjected to progeny testing to date. Genetic crosses are still in progress to map the mutant genes in three of the five progeny tested lines. Based on these data, the research team estimates that there are likely 10 to 20 mammalian genes for which a less than fifty percent reduction in expression could result in a major shift in hemostatic balance sufficient to rescue the lethal thrombosis associated with the heterozygous mice.

According to the authors of the study, each of the gene loci they have identified represents a candidate for a human modifier gene in patients with FVL and other thrombophylic mutations. The pathways uncovered in these studies should provide new insights into the overall regulation of hemostatic balance and identify potential new targets for therapeutic interventions.