Functional Validation of Novel Variants Associated with Congenital Diaphragmatic Hernia

PROJECT SUMMARY/ABSTRACT Congenital Diaphragmatic Hernia (CDH) is a common and severe structural birth defect arising in 1 out of 3000 live births, accounting for 8% of all congenital anomalies and 1-2% of infant mortality. The high mortality rates are due to the abnormal lung hypoplasia and pulmonary hypertension that accompanies the herniation of the

diaphragm muscle. The genetic etiology of CDH is highly heterogeneous and our understanding of the anomaly is incomplete. Although whole exome and whole genome sequencing has identified more than 100 candidate genes, only ~25 have been validated with reproducibility, and we estimate that many additional CDH genes and

alleles are yet to be discovered. Animal model validation of these discoveries are often lacking, which is due, in part, to the fact that the diaphragm is a mammalian-specific structure. The generation and characterization of germline mouse models is costly and time-consuming, and therefore typically incompatible with the need to

screen many candidate genes and variants. The high efficiency of CRISPR/Cas9 genome editing, and the appearance of phenotypes in founder animals has allowed us to develop a platform that can reduce the time for validation from more than one year, to 2-3 months. The overarching goal of this proposal is to identify novel

genes and variants that are associated with human CDH and to validate these discoveries in the mouse. We will use genome data from our large, well-characterized and longitudinally studied CDH cohort to identify novel genes and variants in patients with CDH, and prioritize these genes and variants for validation in mouse models. We

will screen prioritized variants using our high-throughput mouse F0 platform and select a subset of hits for germline modeling and more extensive characterization. Together, these studies will advance our understanding of the genetics and mechanisms underlying CDH, improve diagnostic tools for patients, and pave the way for

the future development of therapeutics aimed at improving lung maturation and function.