PROJECT SUMMARY Clubfoot is one of the most common structural birth defects with a prevalence of 1 in 1000 live births world- wide. Therapeutic options for children born with clubfoot are limited to supportive care including skeletal bracing and invasive surgical interventions. If left untreated, which is common in low- to middle- income countries that lack specialized institutional management, clubfoot can incur lifelong economic, social, and physical burdens. The genetic causes of clubfoot are not confined to a single locus and pathogenic variants have been identified in B3GAT3, CHST14, CHST3, COG4, MYBPC1, MYH3, PITX1, and TPM2. Large-scale sequencing initiatives have identified countless variants in clubfoot-associated loci, but a vast majority of these variants remain uncharacterized. From a developmental standpoint, the genes associated with clubfoot control the morphogenesis of multiple systems including bone, muscle, and connective tissues, and the disease mechanisms contributing to clubfoot likely involve incomplete or inappropriate interactions among multiple organs. Our overarching hypothesis is that understanding and ultimately treating disorders involving clubfoot will require in vivo models that can validate variant pathogenicity and uncover the affected developmental programs that promote disease. By leveraging the speed and versatility of zebrafish genetics, we will screen variants of unknown significance (VUS) identified in patients with clubfoot-associated disorders and use pathogenic variants to further model disorders. Distal Arthrogryposes (DA) are a group of related disorders characterized by contractures, which are defined as a permanent shortening of muscles and joints that inhibit limb movement and function. Clubfoot is one diagnostic feature of DA, but affected patients often show involvement outside the extremities including craniofacial defects and scoliosis. Pathogenic Tropomyosin 2 (TPM2) variants are causative of DA, but a large number of uncharacterized TPM2 variants have been identified in DA patients. As an entry point to modeling DA, we will (1) use zebrafish embryos as a screening platform to characterize fourteen VUS in TPM2, and (2) perform comparative longitudinal studies of three pathogenic TPM2 variants. We expect that these proof-of- principle studies will show that defects in muscle development lead to dysfunctional assembly of the musculoskeletal system, and in turn to the contractures associated with clubfoot and structural birth defects. Our long-term goals are to use the methods developed in this application to study variants in an array of loci associated with DA, and to identify small molecules that ameliorate variant pathogenicity.