PROJECT SUMMARY In this proposal, we will apply robust analytical approaches to whole genome sequencing (WGS) data [obtained from our X01 Gabriella Miller Kids First (GMKF)-funded dataset] from 20 large multigenerational families of Hispanic and nonHispanic white ethnicities to identify variants individually and in aggregate contributing to nonsyndromic cleft lip/palate (NSCLP). NSCLP is a common birth defect annually affecting approximately 135,000 newborns/year worldwide. Despite improvement in treatments, NSCLP imposes significant medical, psychosocial and financial burdens to affected individuals and their families. NSCLP is complex, caused by genetic and environmental factors, and their interactions. It is estimated that ~25% of the genetic liability for NSCLP has been uncovered; however, most of the identified variants reflect common, modest risk-variants often located in noncoding regions of the genome. More recently, it has been suggested that part of the missing heritability for NSCLP lies in rare, high-risk variants, independently or via interactions with common variants. These observations highlight the complexity of NSCLP and difficulty in unraveling risk alleles, and may explain the lack of consistent findings among studies. In this proposal, we will select 20 large and informative families sequenced (WGS) through the X01 mechanism and conduct genome-wide linkage and association, and PRS- informed analyses to assess the contribution of rare and common genetic variation to familial NSCLP. In support of our proposed approach, we have exciting and robust preliminary data showing the identification of a rare variant segregating with NSCLP in a large multiplex family that was modified by common risk variants likely impacting penetrance of the gene. Moreover, we showed that this variant was a null allele and resulted in craniofacial notching mimicking NSCLP. These findings demonstrate the strengths of our approach and the high potential for discovery of novel NSCLP genes/variants. The results of this study will provide novel and important insights about the genetic architecture of NSCLP and lay the foundation for more extensive analysis of WGS data from NSCLP families. The results will also inform putatively causal variants to be used in future functional characterization studies in vitro and in vivo. Ultimately, this study will provide the groundwork for better risk assessment for NSCLP individuals and their families that can be translated into more precise recurrence risk counseling in clinical practice.