scholarly journals A sensitized mutagenesis screen in Factor V Leiden mice identifies novel thrombosis suppressor loci

2016 ◽  
Author(s):  
Randal J. Westrick ◽  
Kärt Tomberg ◽  
Amy E. Siebert ◽  
Guojing Zhu ◽  
Mary E. Winn ◽  
...  
Keyword(s):  
Venous Thromboembolism ◽  
Tissue Factor ◽  
Factor V Leiden ◽  
Coagulation Factor ◽  
Common Disease ◽  
Factor V ◽  
Mutagenesis Screen ◽  
Function Mechanism ◽  
Whole Exome ◽  
Genomic Regions

AbstractFactor V Leiden (F5L) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized ENU mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous forF5L(F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/−).F8deficiency enhanced survival ofF5L/LTfpi+/−mice, demonstrating thatF5L/LTfpi+/−lethality is genetically suppressible. ENU-mutagenizedF5L/Lmales andF5L/+Tfpi+/−females were crossed to generate 6,729 progeny, with 98F5L/LTfpi+/−offspring surviving until weaning. Sixteen lines exhibited transmission of a putative thrombosuppressor to subsequent generations, with these lines referred to asMF5L(Modifier ofFactor5 Leiden) 1-16. Linkage analysis inMF5L6identified a chromosome 3 locus containing the tissue factor gene (F3). Though no ENU-inducedF3mutation was identified, haploinsufficiency forF3(F3+/−) suppressedF5L/LTfpi+/−lethality. Whole exome sequencing inMF5L12identified anActr2gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression ofF5L/LTfpi+/−lethality (p=1.7x10−6), suggesting thatActr2p.R258Gis thrombosuppressive. CRISPR/Cas9 experiments to generate an independentActr2knockin/knockout demonstrated thatActr2haploinsufficiency is lethal, supporting a hypomorphic or gain of function mechanism of action forActr2p.R258G. Our findings identifyF8and theTfpi/F3axis as key regulators in determining thrombosis balance in the setting ofF5Land also suggest a novel role forActr2in this process.Significance StatementVenous thromboembolism (VTE) is a common disease characterized by the formation of inappropriate blood clots. Inheritance of specific genetic variants, such as the Factor V Leiden polymorphism, increases VTE susceptibility. However, only ~10% of people inheriting Factor V Leiden develop VTE, suggesting the involvement of other genes that are currently unknown. By inducing random genetic mutations into mice with a genetic predisposition to VTE, we identified two genomic regions that reduce VTE susceptibility. The first includes the gene for blood coagulation Factor 3 and its role was confirmed by analyzing mice with an independent mutation in this gene. The second contains a mutation in the Actr2 gene. These findings identify critical genes for the regulation of blood clotting risk.

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

2017 ◽  
Vol 114 (36) ◽  
pp. 9659-9664 ◽  
Author(s):  
Randal J. Westrick ◽  
Kärt Tomberg ◽  
Amy E. Siebert ◽  
Guojing Zhu ◽  
Mary E. Winn ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Genetic Risk ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor V Leiden ◽  
Factor V ◽  
Factor Gene ◽  
Mutagenesis Screen ◽  
Function Mechanism ◽  
Whole Exome ◽  
Tissue Factor Pathway

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.

A Sensitized Whole Genome ENU Mutagenesis Screen Identifies an Arp2 Missense Mutation As a Novel Suppressor of Lethal Thrombosis in the Factor V Leiden Mouse

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 493-493
Author(s):  
Randal Joseph Westrick ◽  
Guojing Zhu ◽  
Kart Tomberg ◽  
David R. Siemieniak ◽  
Sara A. Haynes ◽  
...  
Keyword(s):  
Factor V Leiden ◽  
Exome Capture ◽  
Volume Distribution ◽  
Factor V ◽  
Chromosome 11 ◽  
Enu Mutagenesis ◽  
Distribution Width ◽  
Mutagenesis Screen ◽  
Cell Counts ◽  
Whole Exome

Abstract Abstract 493 Only ∼10% of individuals carrying the common risk factor, Factor V Leiden (FVL), will develop venous thrombosis. In order to identify potential FVL modifier genes, we performed a sensitized dominant ENU mutagenesis screen based on the perinatal synthetic lethal thrombosis observed in mice homozygous for FVL (FVQ/Q) and hemizygous for tissue factor pathway inhibitor deficiency (Tfpi+/−). The screen was performed by crossing ENU-treated male FVQ/Q mice with FVQ/+ Tfpi+/− females. Surviving G1 offspring were analyzed to identify survivors with the lethal FVQ/Q Tfpi+/− genotype. Analysis of 7,128 G1 offspring (∼2X genome coverage) identified 98 FVQ/Q Tfpi+/− mice that survived to weaning. Fourteen FVQ/Q Tfpi+/− G1 mice exhibited successful transmission of a putative suppressor mutation to two or more FVQ/Q Tfpi+/− G2 offspring. Whole exome sequencing was performed on a progeny tested member of 8 of the 14 lines using the Agilent SureSelect mouse whole exome capture kit resulting in ∼100 fold coverage. Variant analysis revealed a small number of high confidence novel heterozygous (dominant) single nucleotide variants (SNVs) in each sample. Sanger re-sequencing of all 11 SNVs in a cohort of mice from line 1 confirmed that the G to C mutation (chromosome 11 base 19,977,300) in the Actr2 gene (present in 15/16 re-sequenced progeny p<0.0001) is the dominant FVL modifier in this line. This mutation resulted in an R286G substitution in a highly conserved amino acid in the Arp2 protein, which is essential for the function of the Arp2/3 complex. Arp2/3 is responsible for intracellular actin branching and polymerization, which is critical for the regulation of cell shape. Analysis of 31 progeny from an Arp2 R/G × Arp2 R/G cross revealed only one live Arp2 G/G mouse (p<0.05), suggesting that R286G is a loss of function mutation and that homozygous deficiency is lethal. Complete blood counts (Advia 2120) performed on 14 Actr2 heterozygous and 10 wildtype littermates revealed no significant differences in platelet count, red and white blood cell counts, hematocrit or hemoglobin. However, measurements of platelet size and size distribution including mean platelet dry mass, platelet volume distribution width and the platelet component distribution width were significantly altered in Actr2 heterozygous mutant mice (p<0.05 for each measure). Thus, partial deficiency of Arp2 appears to alter platelet structure/function resulting in a shift in hemostatic balance facilitating survival of the otherwise lethal FVQ/Q Tfpi+/− phenotype. These results suggest that variation in Arp2 or related genes could potentially modify thrombosis risk in humans, and might also identify novel therapeutic targets for the treatment of this class of disorders. Disclosures: Ginsburg: Shire Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children's Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

Whole-Exome Sequencing to Identify Mutations in Thrombosis Modifier Genes Isolated From a Factor V Leiden-Dependent Sensitized ENU Suppressor Screen in the Mouse

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1183-1183
Author(s):  
Randal Joseph Westrick ◽  
Guojing Zhu ◽  
Jishu Xu ◽  
Audrey C.A. Cleuren ◽  
Angela Yang ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Exome Sequencing ◽  
Board Of Directors ◽  
Whole Exome Sequencing ◽  
Factor V Leiden ◽  
Modifier Genes ◽  
Factor V ◽  
Advisory Committees ◽  
Mutagenesis Screen ◽  
Whole Exome

Abstract Abstract 1183 Only ∼10% of individuals carrying the common venous thrombosis risk factor, Factor V Leiden (FVL) will develop venous thrombosis in their lifetime. In order to identify potential FVL modifier genes, we performed a sensitized dominant ENU mutagenesis screen, based on the perinatal synthetic lethal thrombus previously observed in mice homozygous for FVL (FVQ/Q) and hemizygous for tissue factor pathway inhibitor deficiency (Tfpi+/−). The genome-wide ENU mutagenesis screen was performed by crossing ENU-treated male FVQ/Q mice with FVQ/+ Tfpi+/− females. Surviving G1 offspring were analyzed to identify survivors with the otherwise lethal FVQ/Q Tfpi+/− genotype. As proof of concept, we demonstrated that reduced tissue factor (Tf+/−) suppresses the lethal FVQ/Q Tfpi+/− phenotype, suggesting that mutations at Tf should be among the suppressor genes identified by our screen. Analysis of 7,128 G1 offspring (∼2X genome coverage) identified 98 FVQ/Q Tfpi+/− mice that survived to weaning. Fourteen FVQ/Q Tfpi+/− G1 mice exhibited successful transmission of a putative suppressor mutation to two or more FVQ/Q Tfpi+/− G2 offspring. Extensive genotyping of mice from an expanded genetic cross from one of these lines mapped a candidate suppressor locus to a chromosome 3 region encompassing the TF gene (LOD=4.93). With continued improvements in next generation sequencing technologies, we have now applied whole exome sequencing to analysis of 8 of the remaining 13 lines. The entire DNA coding region (the “exome”, totaling 49.6 Mb of DNA sequence) from a progeny-tested member of each line was captured using the Agilent SureSelect mouse exome capture system. Whole-exome sequencing using the Illumina HiSeq high-throughput sequencer yielded 12–15 gigabases of sequence data per sample, corresponding to an average of ∼200 fold sequencing coverage for each nucleotide position. Variant analysis using the Gene Analysis Toolkit revealed the presence of a small number of high confidence novel heterozygous (dominant) variants in each sample. Each of these heterozygous variants is a candidate suppressor mutation and these are presently being tested in remaining FVQ/Q Tfpi+/− mice from each respective line. Based on previous studies where ENU-induced mutations in mice have been identified, we anticipate the identification of putative exomic mutations in approximately 80% of tested suppressor lines. Identification of these mutations should provide candidate modifier genes for FVL and other human hemostatic disorders. Disclosures: Ginsburg: Portola Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Catalyst Biosciences: Membership on an entity's Board of Directors or advisory committees; Shire Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Coagulation Factor V Leiden Mutation Was Detected in the Patients with Activated Protein C Resistance in Thailand

1998 ◽  
Vol 80 (08) ◽  
pp. 344-345 ◽  
Author(s):  
Pasra Arnutti ◽  
Motofumi Hiyoshi ◽  
Wichai Prayoonwiwat ◽  
Oytip Nathalang ◽  
Chamaiporn Suwanasophon ◽  
...  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Coagulation Factor ◽  
Factor V ◽  
Activated Protein C Resistance ◽  
Factor V Leiden Mutation ◽  
Coagulation Factor V

Phenotyping and Genotyping of Coagulation Factor V Leiden

1996 ◽  
Vol 75 (02) ◽  
pp. 267-269 ◽  
Author(s):  
H Engel ◽  
L Zwang ◽  
H H D M van Vliet ◽  
J J Michiles ◽  
J Stibbe ◽  
...  
Keyword(s):  
Factor V Leiden ◽  
Coagulation Factor ◽  
Diagnostic Value ◽  
Resistance Test ◽  
Amplification Refractory Mutation System ◽  
Factor V ◽  
Chain Reactions ◽  
Apc Resistance ◽  
Specificity And Sensitivity ◽  
Coagulation Factor V

SummaryThe currently used activated Protein C resistance test demonstrated to be of limited diagnostic value for the detection of the mutant Factor V Leiden. Moreover, this assay is not useful for patients under anticoagulant therapy. A modification of the APC resistance test, applying Factor V deficient plasma is described which demonstrates a specificity and sensitivity of 1.0. The superiority of the modified APC resistance test over the existing APC resistance test was verified by genotyping.For that purpose, the Amplification Refractory Mutation System (ARMS) was applied to the detection of the G to A mutation at position 1691 in the gene encoding coagulation Factor V. The mutation at that position could be easily detected by using each of two allele-specific oligonucleotide primers concomitantly with one common primer in two separate polymerase chain reactions, thereby amplifying a fragment of 186 base-pairs of the Factor V gene.

Pregnancy-associated risk for venous thromboembolism and pregnancy outcome in women homozygous for factor V Leiden

2000 ◽  
Vol 1 (1) ◽  
pp. 37-41 ◽  
Author(s):  
I Pabinger ◽  
L Nemes ◽  
C Rintelen ◽  
S Koder ◽  
E Lechler ◽  
...  
Keyword(s):  
Venous Thromboembolism ◽  
Pregnancy Outcome ◽  
Factor V Leiden ◽  
Factor V

The procoagulant effects of factor V Leiden may be balanced against decreased levels of factor V and do not reflect in vivo thrombin formation in newborns

2006 ◽  
Vol 95 (03) ◽  
pp. 434-440 ◽  
Author(s):  
Satu Hyytiäinen ◽  
Ulla Wartiovaara-Kautto ◽  
Veli-Matti Ulander ◽  
Risto Kaaja ◽  
Markku Heikinheimo ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Platelet Rich Plasma ◽  
Venous Blood ◽  
Factor V Leiden ◽  
Factor V ◽  
Cord Plasma ◽  
Adult Plasma ◽  
Thrombin Formation

SummaryThrombin regulation in newborns remains incompletely understood.We studied tissue factor-initiated thrombin formation in cord plasma in vitro, and the effects of Factor VLeiden (FVL) heterozygosity on thrombin regulation both in vitro and in vivo in newborns. Pregnant women with known thrombophilia (n=27) were enrolled in the study. Cord blood and venous blood at the age of 14 days were collected from 11 FVL heterozygous newborns (FVL-positive) and from 16 FVL-negative newborns. Prothrombin fragment F1+2 and coagulation factors were measured. Tissue factor-initiated thrombin formation was studied in cord platelet-poor plasma (PPP) of FVL-negative and -positive newborns, and in both PPP and platelet-rich plasma (PRP) of healthy controls. The endogenous thrombin potential (ETP) in cord PPP or PRP was ∼60% of that in adult plasma, while thrombin formation started ∼55% and ∼40% earlier in cord PPP and PRP, respectively. Further, in FVL-positive newborns thrombin formation started significantly earlier than in FVL-negative newborns. Exogenous activated protein C (APC) decreased ETP significantly more in cord than in adult PRP. In FVL-negative cord plasma 5nM APC decreased ETP by 17.4±3.5% (mean±SEM) compared with only 3.5±3.8% in FVL-positive cord plasma (p=0.01). FVL-positive newborns showed similar levels of F1+2 but significantly decreased levels of factor V compared with FVL negative newborns both in cord plasma (FV 0.82±0.07 U/ml vs. 0.98±0.05 U/ml, p=0.03) and at the age of two weeks (FV 1.15±0.04 U/ml vs. 1.32±0.05 U/ml, p=0.03). In conclusion, newborn plasma showed more rapid thrombin formation and enhanced sensitivity to APC compared with adult plasma. FVL conveyed APC resistance and a procoagulant effect in newborn plasma. Lack of elevated F1+2 levels in FVL-positive infants, however, suggested the existence of balancing mechanisms; one could be the observed lower level of factor V in FVL heterozygous newborns.

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