Spontaneous 8bp Deletion in Nbeal2 Recapitulates the Gray Platelet Syndrome in Mice

Tomberg, Kärt; Khoriaty, Rami; Westrick, Randal J.; Fairfield, Heather; Reinholdt, Laura G.; Brodsky, Gary; Davizon‐Castillo, Pavel; Ginsburg, David; Paola, Jorge Di

doi:10.1371/journal.pone.0150852

Cited by 13 publications

(9 citation statements)

References 47 publications

(56 reference statements)

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“…Relative expression was estimated at SNP sites by dividing the area under the Sanger sequencing peak of one allele to another (52,53). Next, the relative expression of each SNP was compared between the B6 and 129S1 allele carrying progeny.…”

Section: Estimation Of F3 Allelic Expressionmentioning

confidence: 99%

“…Heterozygous variants within exonic regions with >6X coverage unique for only one mouse in the cohort were regarded as potential ENU induced variants. A total of 125 heterozygous variants were identified as candidate suppressor mutations, using an in-house filtering pipeline (52), with 79 variants occurring within coding sequence. The number of ENU variants identified in each exome sequenced mouse varied by genealogical distance from the G1 MF5L founder.…”

Section: Mouse Whole-exome Sequencingmentioning

confidence: 99%

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

Westrick

Tomberg²,

Siebert³

et al. 2016

Preprint

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Factor V Leiden (F5 L ) 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 for F5 L (F5 L/L ) and haploinsufficient for tissue factor pathway inhibitor (Tfpi +/-). F8 deficiency enhanced survival of F5 L/L Tfpi +/mice, demonstrating that F5 L/L Tfpi +/lethality is genetically suppressible. ENU-mutagenized F5 L/L males and F5 L/+ Tfpi +/females were crossed to generate 6,729 progeny, with 98 F5 L/L Tfpi +/offspring surviving until weaning. Sixteen lines exhibited transmission of a putative thrombosuppressor to subsequent generations, with these lines referred to as MF5L (Modifier of Factor 5 Leiden) 1-16. Linkage analysis in MF5L6 identified a chromosome 3 locus containing the tissue factor gene (F3). Though no ENU-induced F3 mutation was identified, haploinsufficiency for F3 (F3 +/-) suppressed F5 L/L Tfpi +/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 F5 L/L Tfpi +/lethality (p=1.7x10 -6 ), suggesting that Actr2 p.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 Actr2 p.R258G . Our findings identify F8 and the Tfpi/F3 axis as key regulators in determining thrombosis balance in the setting of F5 L and also suggest a novel role for Actr2 in this process.Significance Statement (120 words max):Venous 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.

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Section: Estimation Of F3 Allelic Expressionmentioning

confidence: 99%

Section: Mouse Whole-exome Sequencingmentioning

confidence: 99%

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

Westrick

Tomberg²,

Siebert³

et al. 2016

Preprint

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“…The lack of macrothrombocytopenia in our family could be the result of a modifier gene(s) difference in these disparate genetic backgrounds. Strain differences in mice have been demonstrated to contribute to variations in platelet size in Gray Platelet Syndrome (Tomberg et al , ), analogous to this observation in humans.…”

Section: Resultsmentioning

confidence: 70%

Genome‐wide linkage analysis and whole‐exome sequencing identifies an ITGA2B mutation in a family with thrombocytopenia

et al. 2019

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Summary Hereditary thrombocytopenias can be subclassified based on mode of inheritance and platelet size. Here we report a family with autosomal dominant (AD) thrombocytopenia with normal platelet size. Linkage analysis and whole exome sequencing identified the R1026W substitution in ITGA2B as the causative defect. The same mutation has been previously reported in 7 Japanese families/patients with AD thrombocytopenia, but all of these patients had macrothrombocytopenia. This is the first report of a family with AD thrombocytopenia with normal platelet size resulting from mutation in ITGA2B. ITGA2B mutations should therefore be included in the differential diagnosis of this latter disorder.

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“…Failure to map the causal loci in any of these pedigrees was likely due to insufficient marker coverage. However, in these analyses, we could not exclude the contribution from a non-ENU-induced variant [ 18 ] or an unexpectedly high phenocopy rate. While WES has been successfully applied to identify causal ENU variants within inbred lines [ 19 ] and in mixed background lines [ 20 , 21 ], whole genome sequencing (WGS) provides much denser and more even coverage of the entire genome (~3,000 ENU variants/genome expected) and outperforms WES for mapping [ 15 ].…”

Section: Resultsmentioning

confidence: 99%

Whole exome sequencing of ENU-induced thrombosis modifier mutations in the mouse

et al. 2018

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Although the Factor V Leiden (FVL) gene variant is the most prevalent genetic risk factor for venous thrombosis, only 10% of FVL carriers will experience such an event in their lifetime. To identify potential FVL modifier genes contributing to this incomplete penetrance, we took advantage of a perinatal synthetic lethal thrombosis phenotype in mice homozygous for FVL (F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/-) to perform a sensitized dominant ENU mutagenesis screen. Linkage analysis conducted in the 3 largest pedigrees generated from the surviving F5L/L Tfpi+/- mice (‘rescues’) using ENU-induced coding variants as genetic markers was unsuccessful in identifying major suppressor loci. Whole exome sequencing was applied to DNA from 107 rescue mice to identify candidate genes enriched for ENU mutations. A total of 3,481 potentially deleterious candidate ENU variants were identified in 2,984 genes. After correcting for gene size and multiple testing, Arl6ip5 was identified as the most enriched gene, though not reaching genome-wide significance. Evaluation of CRISPR/Cas9 induced loss of function in the top 6 genes failed to demonstrate a clear rescue phenotype. However, a maternally inherited (not ENU-induced) de novo mutation (Plcb4R335Q) exhibited significant co-segregation with the rescue phenotype (p = 0.003) in the corresponding pedigree. Thrombosis suppression by heterozygous Plcb4 loss of function was confirmed through analysis of an independent, CRISPR/Cas9-induced Plcb4 mutation (p = 0.01).

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Spontaneous 8bp Deletion in Nbeal2 Recapitulates the Gray Platelet Syndrome in Mice

Cited by 13 publications

References 47 publications

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

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

Genome‐wide linkage analysis and whole‐exome sequencing identifies an ITGA2B mutation in a family with thrombocytopenia

Whole exome sequencing of ENU-induced thrombosis modifier mutations in the mouse

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