Background:Human heterotaxy is a group of congenital disorders characterized by misplacement of one or more organs according to the left-right axis. The genetic causes of human heterotaxy are highly heterogeneous.Methods:We performed exome sequencing in a cohort of 26 probands with heterotaxy followed by gene burden analysis for the enrichment of novel rare damaging mutations. Transcription activator-like effector nuclease was used to generate somatic loss-of-function mutants in a zebrafish model. Ciliary defects were examined by whole-mount immunostaining of acetylated α-tubulin.Results:We identified a significant enrichment of novel rare damaging mutations in the CC2D1A gene. Seven occurrences of CC2D1A mutations were found to affect 4 highly conserved amino acid residues of the protein. Functional analyses in the transcription activator-like effector nuclease–mediated zebrafish knockout models were performed, and heterotaxy phenotypes of the cardiovascular and gastrointestinal systems in both somatic and germline mutants were observed. Defective cilia were demonstrated by whole-mount immunostaining of acetylated α-tubulin. These abnormalities were rescued by wild-type cc2d1a mRNA but not cc2d1a mutant mRNA, strongly suggesting a loss-of-function mechanism. On the other hand, overexpression of cc2d1a orthologous mutations cc2d1a P559L and cc2d1a G808V (orthologous to human CC2D1A P532L and CC2D1A G781V) did not affect embryonic development.Conclusions:Using a zebrafish model, we were able to establish a novel association of CC2D1A with heterotaxy and ciliary dysfunction in the F2 generation via a loss-of-function mechanism. Future mechanistic studies are needed for a better understanding of the role of CC2D1A in left-right patterning and ciliary dysfunction.

中文翻译：

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CC2D1A 中的单等位基因突变表明在人类异质性和纤毛功能障碍中的新作用

背景：人类异位症是一组以一个或多个器官沿左右轴错位为特征的先天性疾病。人类异位性的遗传原因具有高度异质性。方法：我们对 26 名具有异位性的先证者进行外显子组测序，然后进行基因负荷分析，以富集新的罕见破坏性突变。转录激活剂样效应核酸酶用于在斑马鱼模型中产生体细胞功能丧失突变体。通过乙酰化α-微管蛋白的整体免疫染色检查纤毛缺陷。结果：我们在CC2D1A基因中发现了新的罕见破坏性突变的显着富集。CC2D1A出现七次发现突变影响蛋白质的 4 个高度保守的氨基酸残基。在转录激活因子样效应核酸酶介导的斑马鱼敲除模型中进行功能分析，并观察到体细胞和种系突变体中心血管和胃肠系统的异位表型。有缺陷的纤毛通过乙酰化 α-微管蛋白的整体免疫染色证明。这些异常被野生型cc2d1a mRNA 而不是cc2d1a突变体 mRNA拯救，强烈表明功能丧失机制。另一方面，cc2d1a直系同源突变cc2d1a P559L 和cc2d1a G808V（与人类CC2D1A直系同源）的过度表达P532L 和CC2D1A G781V) 不影响胚胎发育。结论：使用斑马鱼模型，我们能够通过功能丧失机制建立CC2D1A与 F2 代异位性和纤毛功能障碍的新关联。需要进一步的机制研究来更好地了解CC2D1A在左右模式和纤毛功能障碍中的作用。