Whole exome sequencing of ENU-induced thrombosis modifier mutations in the mouse.
Whole exome sequencing of ENU-induced thrombosis modifier mutations in the mouse.
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DOI：
10.1371/journal.pgen.1007658
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发表时间：
2018-09
期刊：
影响因子：
4.5
通讯作者：
Ginsburg D
中科院分区：
文献类型：
--
作者：
Tomberg K;Westrick RJ;Kotnik EN;Cleuren AC;Siemieniak DR;Zhu G;Saunders TL;Ginsburg D
尽管因子V Leiden(FVL)基因变异是静脉血栓形成最常见的遗传危险因素，但只有10%的FVL携带者会在他们的一生中经历这样的事件。为了确定可能导致这种不完全外显的FVL修饰基因，我们利用FVL纯合子(F5L/L)和组织因子途径抑制物(TFP I+/-)单倍体不足的围产期合成致命性血栓形成表型进行了敏化显性ENU突变筛选。用ENU诱导的编码变异作为遗传标记，对存活的F5L/L TFPI+/-小鼠产生的3个最大家系(‘拯救’)进行连锁分析，未能成功识别主要抑制基因座。对107只拯救小鼠的DNA进行了全外显子组测序，以确定ENU突变的候选基因。在2984个基因中共鉴定出3481个潜在有害的候选ENU变体。在校正了基因大小和多次测试后，Arl6ip5被确定为最丰富的基因，尽管还没有达到全基因组的意义。对CRISPR/Cas9导致的前6个基因功能丧失的评估未能显示出明显的救援表型。然而，在相应的家系中，母系遗传(不是ENU诱导的)新生突变(Plcb4R335Q)与救援表型显示出显著的共分离(p=0.003)。通过对CRISPR/Cas9诱导的一个独立的Plcb4突变的分析，证实了杂合性Plcb4功能丧失对血栓形成的抑制(p=0.01)。静脉或动脉中的异常血液凝结(静脉血栓形成)或动脉血栓形成(动脉血栓形成)是主要的健康问题，在美国每年每千人中约有1人受到静脉血栓的影响。静脉血栓形成的易感性由基因和环境共同决定，大约60%的风险归因于遗传影响。尽管已知几种遗传风险因素，但仍有50%的遗传风险无法解释。大约5%的人携带最常见的已知风险因素--因子V莱顿。然而，这些人中只有10%的人会在他们的一生中发展成血栓。携带两个F V莱顿突变和第二个基因突变的小鼠在出生后不久就会发生致命的血栓形成。为了确定防止这种致命血栓形成的基因，我们研究了一大组携带随机分布在基因组中的失活基因变化的小鼠。我们确定了几个基因作为潜在的候选基因来改变易发生血栓形成的小鼠和人类的凝血平衡，并证实了其中一个基因(Plcb4)的这种DNA变化的保护功能。
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). Abnormal blood clotting in veins (venous thrombosis) or arteries (arterial thrombosis) are major health problems, with venous thrombosis affecting approximately 1 in every thousand individuals annually in the United States. Susceptibility to venous thrombosis is governed by both genes and environment, with approximately 60% of the risk attributed to genetic influences. Though several genetic risk factors are known, >50% of genetic risk remains unexplained. Approximately 5% of people carry the most common known risk factor, Factor V Leiden. However, only 10% of these individuals will develop a blood clot in their lifetime. Mice carrying two copies of the Factor V Leiden mutation together with a mutation in a second gene called tissue factor pathway inhibitor develop fatal thrombosis shortly after birth. To identify genes that prevent this fatal thrombosis, we studied a large panel of mice carrying inactivating gene changes randomly distributed throughout the genome. We identified several genes as potential candidates to alter blood clotting balance in mice and humans with predisposition to thrombosis, and confirmed this protective function for DNA changes in one of these genes (Plcb4).
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