LOF variants identifying candidate genes of laterality defects patients with congenital heart disease.
LOF variants identifying candidate genes of laterality defects patients with congenital heart disease.
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DOI：
10.1371/journal.pgen.1010530
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发表时间：
2022-12
期刊：
影响因子：
4.5
通讯作者：
中科院分区：
文献类型：
--
作者：
偏侧畸形可导致胚胎发育早期内脏器官位置异常，表现为异位综合征和内翻位，而偏侧畸形占所有先天性心脏病的3~7%。然而，大多数偏侧缺陷的致病机制仍不清楚。在这项研究中，我们招募了70名患有先天性心脏病的偏侧缺陷患者，通过外显子组测序确定候选疾病基因。然后，我们评估了罕见的功能丧失(LOF)变体，通过参考以前的文献确定了候选基因。我们从符合初步筛选标准的776个LOF变异体中选择了TRIP11、DNHD1、CFAP74和EGR4作为候选者。在变异到基因定位之后，我们对这些候选基因进行了功能研究。通过整体原位杂交、基因敲除和基因拯救等方法研究了这4个候选基因在斑马鱼模型中的表达模式和功能。在这4个基因中，Trip11、dnhd1和cfap74变异斑马鱼在心环和早期信号分子的表达模式上都表现出异常，这表明这些基因在建立偏侧性模式中发挥了重要作用。此外，我们还进行了免疫染色和高速纤毛视频显微镜研究斑马鱼的库普弗氏囊泡器官发生和纤毛发生。在Trip11、dnhd1和cfap74变异斑马鱼中发现Kupffer囊泡器官发生或纤毛发生的损害，这揭示了它们的LOF变异体在偏侧缺陷中的可能致病机制。这些结果强调了罕见的LOF变异在识别疾病相关基因和确定TRIP11、DNHD1和CFAP74在左右模式中的新角色方面的重要性。此外，这些发现与偏侧缺陷的复杂遗传学是一致的。在胚胎发育的早期阶段，偏侧模式的缺陷会导致内部器官的位置异常。合并偏侧畸形的冠心病患者的病死率高于无偏侧异常的冠心病患者。然而，大多数偏侧缺陷的致病机制仍不清楚。在这项研究中，我们招募了70名患有偏侧缺陷的冠心病患者，通过外显子组测序来识别候选疾病基因。然后我们评估了罕见的功能丧失变体，确定TRIP11、DNHD1、CFAP74和EGR4为候选。在斑马鱼模型中研究了这4个候选基因的表达模式和功能。在这四个基因中，Trip11、dnhd1和cfap74变异斑马鱼在心脏环路和早期信号通路的表达模式上都显示出异常。此外，在trip11、dnhd1和cfap74变异斑马鱼中发现库普弗氏囊泡器官发生或纤毛发生障碍。这些结果强调了罕见的功能丧失变异在识别疾病相关基因方面的重要性，并展示了TRIP11、DNHD1和CFAP74在左右模式中的新角色。首次在偏侧缺陷患者中发现TRIP11和CFAP74的变异。
Defects in laterality pattern can result in abnormal positioning of the internal organs during the early stages of embryogenesis, as manifested in heterotaxy syndrome and situs inversus, while laterality defects account for 3~7% of all congenital heart defects (CHDs). However, the pathogenic mechanism underlying most laterality defects remains unknown. In this study, we recruited 70 laterality defect patients with CHDs to identify candidate disease genes by exome sequencing. We then evaluated rare, loss-of-function (LOF) variants, identifying candidates by referring to previous literature. We chose TRIP11, DNHD1, CFAP74, and EGR4 as candidates from 776 LOF variants that met the initial screening criteria. After the variants-to-gene mapping, we performed function research on these candidate genes. The expression patterns and functions of these four candidate genes were studied by whole-mount in situ hybridization, gene knockdown, and gene rescue methods in zebrafish models. Among the four genes, trip11, dnhd1, and cfap74 morphant zebrafish displayed abnormalities in both cardiac looping and expression patterns of early signaling molecules, suggesting that these genes play important roles in the establishment of laterality patterns. Furthermore, we performed immunostaining and high-speed cilia video microscopy to investigate Kupffer’s vesicle organogenesis and ciliogenesis of morphant zebrafish. Impairments of Kupffer’s vesicle organogenesis or ciliogenesis were found in trip11, dnhd1, and cfap74 morphant zebrafish, which revealed the possible pathogenic mechanism of their LOF variants in laterality defects. These results highlight the importance of rare, LOF variants in identifying disease-related genes and identifying new roles for TRIP11, DNHD1, and CFAP74 in left-right patterning. Additionally, these findings are consistent with the complex genetics of laterality defects. Defects in laterality pattern can result in abnormal positioning of the internal organs during the early stages of embryogenesis. Patients with laterality anomalies complicated by CHD have higher mortality as compared to their CHD peers without laterality anomalies. However, the pathogenic mechanism underlying most laterality defects remains unknown. In this study, we recruited 70 laterality defects patients with CHD to identify candidate disease genes by exome sequencing. We then evaluated rare, loss-of-function variants, identifying TRIP11, DNHD1, CFAP74, and EGR4 as candidates. The expression patterns and functions of these four candidate genes were studied in zebrafish models. Among the four genes, trip11, dnhd1, and cfap74 morphant zebrafish displayed abnormalities in both cardiac looping and expression patterns of early signaling pathways. In addition, impairments of Kupffer’s vesicle organogenesis or ciliogenesis were found in trip11, dnhd1, and cfap74 morphant zebrafish. These results highlight the importance of rare, loss-of-function variants in identifying disease-related genes and display the new roles for TRIP11, DNHD1, and CFAP74 in left-right patterning. Variants in TRIP11 and CFAP74 are identified in laterality defect patients for the first time.
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