Mouse embryonic heart-expressed gene list.

Congenital heart defects (CHDs) occur in about 1% of live births and are the leading cause of infant death due to birth defects. While there have been remarkable efforts to pursue large-scale whole-exome and genome sequencing studies on CHD patient cohorts, it is estimated that these approaches have thus far accounted for only about 50% of the genetic contribution to CHDs. We sought to take a new approach to identify genetic causes of CHDs. By combining analyses of genes that are under strong selective constraint along with published embryonic heart transcriptomes, we identified over 200 new candidate genes for CHDs. We utilized protein-protein interaction (PPI) network analysis to identify a functionally-related subnetwork consisting of known CHD genes as well as genes encoding proteasome factors, in particular POMP, PSMA6, PSMA7, PSMD3, and PSMD6. We used CRISPR targeting in zebrafish embryos to preliminarily identify roles for the PPI subnetwork genes in heart development. We then used CRISPR to create new mutant zebrafish strains for two of the proteasome genes in the subnetwork: pomp and psmd6. We show that loss of proteasome gene functions leads to defects in zebrafish heart development, including dysmorphic hearts, myocardial cell blebbing, and reduced outflow tracts. We also identified deficits in cardiac function in pomp and psmd6 mutants. These heart defects resemble those seen in zebrafish mutants for known CHD genes and other critical heart development genes. Our study provides a novel systems genetics approach to further our understanding of the genetic causes of human CHDs.先天性心脏缺陷（Congenital Heart Defects, CHDs）的发生率约为活产婴儿的1%，是出生缺陷导致婴儿死亡的首要原因。尽管学界已针对CHDs患者队列开展了大规模全外显子组与基因组测序研究并付出诸多卓有成效的努力，但据估算，目前这类手段仅能解释约50%的CHDs遗传诱因。本研究旨在采用全新的研究方法鉴定CHDs的遗传致病因素。通过结合对受强选择约束的基因的分析，以及已发表的胚胎心脏转录组数据，本研究共鉴定出200余个CHDs潜在候选基因。本研究采用蛋白质相互作用（Protein-Protein Interaction, PPI）网络分析方法，鉴定出一个功能相关的子网，该子网包含已明确的CHDs致病基因以及编码蛋白酶体因子的基因，其中尤为关键的包括POMP、PSMA6、PSMA7、PSMD3及PSMD6。本研究在斑马鱼胚胎中采用CRISPR（Clustered Regularly Interspaced Short Palindromic Repeats）靶向技术，初步明确了该PPI子网基因在心脏发育过程中的作用。随后，本研究针对该子网中的2个蛋白酶体基因——pomp与psmd6，利用CRISPR技术构建了新型斑马鱼突变品系。研究证实，蛋白酶体基因功能缺失会导致斑马鱼心脏发育异常，具体表现为心脏形态异常、心肌细胞膜起泡以及流出道发育不全。本研究同时还检测到pomp与psmd6突变体存在心脏功能缺陷。上述心脏缺陷表型与已明确CHDs致病基因及其他关键心脏发育基因的斑马鱼突变体所呈现的表型高度相似。本研究提供了一种全新的系统遗传学研究方法，有助于进一步解析人类CHDs的遗传致病机制。