LOF variants identifying candidate genes of laterality defects patients with congenital heart disease

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.