Mechanisms of Abnormal Diaphragm and Cardiac Development

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Project Summary Congenital diaphragmatic hernia (CDH) is a life-threatening birth defect that accounts for 8% of all major congenital anomalies. In cases where CDH co-occurs with cardiovascular malformations (CVMs), mortality rates increase from 30 to 60%, and long-term morbidity is common. Our goal is to identify genes that cause CDH and CDH/CVM, and to discover the mechanism by which they control diaphragm and heart development. Correctly identifying genes that contribute to specific phenotypes from the large list of candidate genes data generated in research and clinical studies is a major obstacle to progress in this and other research fields. In Specific Aim #1, we will address this challenge by generating ranked CDH and CDH/CVM pathogenicity scores for all RefSeq genes using a machine-learning algorithm that integrates data from large-scale genomic knowledge sources. We will use these scores to identify novel CDH and CDH/CVM genes from cytogenetically defined critical regions and form large next-generation sequencing databases as part of our multifaceted approach to novel gene discovery. We will also accelerate the pace at which human disease genes are discovered by making these scores and our machine-learning algorithm freely available. 8p23.1 microdeletions that encompass GATA4 and SOX7 are among the most frequently identified causes of CDH/CVM. In RNA-seq studies, we identified several CDH-associated genes that are dysregulated in the E15.5 diaphragms of Gata4flox/flox;Prx1-Cre embryos. These embryos are an ideal model of the sac hernias that comprise 20% of human CDH cases. In Specific Aim #2, we will combine data from RNA-seq and BioChIP-seq analyses with the priority scores generated in Aim #1 to identify primary GATA4 target genes whose dysregulation contribute to the development of sac CDH. We will then determine if alterations in the expression of these target genes can cause CDH, or can modify the CDH phenotypes of GATA4-deficient mice. We have shown that SOX7 deficiency causes septal defects by decreasing endothelial-to-mesenchymal transition (EMT) in the developing heart. In RNA-seq and in situ hybridization studies we have shown that the expression of Wnt4—a key regulator of EMT in the endocardium—is severely decreased in E9.5 Sox7-/- hearts. In Specific Aim #3 we will determine if Wnt4 is a primary target of SOX7, and whether modulation of WNT4 or its downstream effectors can rescue SOX7-related cardiac phenotypes. Several lines of evidence suggest that WNT4 is a novel CDH/CVM gene in humans. To confirm this association, and learn more about the role of WNT4 in diaphragm and heart development, we will determine if WNT4 deficiency causes CDH/CVM in mice, if Sox7 and Wnt4 interact genetically in the development of CDH/CVM and if SOX7 regulates Wnt4 transcription in the developing diaphragm. Through these studies we will identify novel CDH and CDH/CVM genes and pathways. The bioinformatic tools we develop in this grant will enhance gene discovery efforts across multiple research fields.