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Proc Natl Acad Sci U S A. 2017 Sep 5;114(36):9659-9664. doi: 10.1073/pnas.1705762114. Epub 2017 Aug 21.

Sensitized mutagenesis screen in Factor V Leiden mice identifies thrombosis suppressor loci.

Author information

1
Department of Biological Sciences, Oakland University, Rochester, MI 48309; ginsburg@umich.edu rjwestrick@oakland.edu.
2
Center for Data Science and Big Data Analysis, Oakland University, Rochester, MI 48309.
3
Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109.
4
Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109.
5
Department of Biological Sciences, Oakland University, Rochester, MI 48309.
6
Bioinformatics and Biostatistics Core, Van Andel Research Institute, Grand Rapids, MI 49503.
7
Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI 48109.
8
Transgenic Animal Model Core, University of Michigan, Ann Arbor, MI 48109.
9
Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109; ginsburg@umich.edu rjwestrick@oakland.edu.
10
Department of Internal Medicine, Ann Arbor, MI 48109.
11
Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109.

Abstract

Factor V Leiden (F5L ) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized N-ethyl-N-nitrosourea (ENU) mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for F5L (F5L/L ) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/- ). F8 deficiency enhanced the survival of F5L/LTfpi+/- mice, demonstrating that F5L/LTfpi+/- lethality is genetically suppressible. ENU-mutagenized F5L/L males and F5L/+Tfpi+/- females were crossed to generate 6,729 progeny, with 98 F5L/LTfpi+/- offspring surviving until weaning. Sixteen lines, referred to as "modifier of Factor 5 Leiden (MF5L1-16)," exhibited transmission of a putative thrombosuppressor to subsequent generations. Linkage analysis in MF5L6 identified a chromosome 3 locus containing the tissue factor gene (F3). Although no ENU-induced F3 mutation was identified, haploinsufficiency for F3 (F3+/- ) suppressed F5L/LTfpi+/- lethality. Whole-exome sequencing in MF5L12 identified an Actr2 gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression of F5L/LTfpi+/- lethality (P = 1.7 × 10-6), suggesting that Actr2p.R258G is thrombosuppressive. CRISPR/Cas9 experiments to generate an independent Actr2 knockin/knockout demonstrated that Actr2 haploinsufficiency is lethal, supporting a hypomorphic or gain-of-function mechanism of action for Actr2p.R258G Our findings identify F8 and the Tfpi/F3 axis as key regulators in determining thrombosis balance in the setting of F5L and also suggest a role for Actr2 in this process.

KEYWORDS:

ENU mutagenesis; Factor V Leiden; genetic screen; tissue factor pathway inhibitor; venous thromboembolism

PMID:
28827327
PMCID:
PMC5594664
DOI:
10.1073/pnas.1705762114
[Indexed for MEDLINE]
Free PMC Article

Conflict of interest statement

The authors declare no conflict of interest.

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