Leveraging whole genome sequencing and functional genomic characterization to improve NSCLP gene discovery

NSCLP is a common birth defect affecting ~135,000 newborns/year worldwide. The etiology is complex with genetic and environmental factors, and their interactions all playing roles. It is estimated that only ~25% of the genetic liability for NSCLP has been uncovered with most of the identified variants reflecting common, modest risk-variants often located in noncoding regions of the genome. Despite advances in NSCLP gene discovery, the specific effects on biological signaling networks for most variants are largely unknown and a challenge remains on recognizing which of these variations have functional effects on the phenotype. Recently, we and others have shown that part of the missing heritability for NSCLP lies in rare, high-risk variants potentially interacting with common variants. Further, our work confirmed polygenic inheritance for NSCLP which may explain part of the missing heritability and the variable phenotypes observed within families. These observations highlight the complexity of NSCLP and difficulty in unraveling risk alleles and may explain the lack of consistent findings among studies. Through an X01DE031445 from the Gabriella Miller Kids First (GMKF) Program, we have obtained whole genome sequencing (WGS) data for 172 large and well-characterized multigenerational families of Hispanic and nonHispanic white ethnicities. In this proposal, we leverage detailed phenotypic data and high-quality WGS data from our multigenerational NSCLP families, additional GMKF NSCLP datasets, and the All of Us program to systematically evaluate and validate the contributions of common and rare variants to NSCLP, and functionally characterize prioritized variants in vitro and in vivo. Specifically, we will (1) integrate gene- and variant-based linkage and case-control association analyses, as well as polygenic risk score (PRS)- informed analyses to determine the genetic architecture underlying familial NSCLP, (2) perform functionally- oriented analysis for screening prioritized variants using cell-based assays, and (3) determine the impact of prioritized variants on phenotypic changes in zebrafish embryos. This proposal overcomes the limitations of previous studies by providing a paradigm for advancing knowledge of the underlying biological mechanisms leading to NSCLP through the use of large and well-characterized NSCLP and control datasets and rigorous methodologies for gene discovery with validation, and functional genomic characterization in vitro and in vivo. The results will allow us to identify complex inheritance patterns contributing to the genetic etiology of NSCLP and inform additional mechanistic studies that will provide the groundwork for improved risk assessment and translation into more precise genetic counseling in clinical practice. Our proposal meets the goals of PAR-21- 229 as it addresses a challenging gap between identifying sequence variations of potential interest and recognizing which of those variations have functional effects on NSCLP. Project Number: 5R01DE033908-02 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Dental and Craniofacial Research (NIDCR) | Principal Investigator: Ariadne Letra (+2 co-PIs) | Institution: UNIVERSITY OF PITTSBURGH AT PITTSBURGH, PITTSBURGH, PA | Award Amount: $598,122 | Activity Code: R01 | Study Section: Special Emphasis Panel[ZRG1-MGG-K(02)M] View on NIH RePORTER: https://reporter.nih.gov/project-details/11322004