PROJECT SUMMARY: Our lab recently discovered COPA syndrome, an autoimmune disease affecting the lungs and joints caused by dominant mutations in the coatomer subunit alpha (COPA) gene. COPA syndrome typically presents in childhood with either inflammatory arthritis or lung disease that manifests as pulmonary capillaritis or interstitial lung disease. Although genetic testing is used to confirm the diagnosis, uncertainty can arise when sequencing uncovers COPA variants of unknown significance (VUS). This occurs relatively frequently because verified mutations lie within a ~50 base pair (bp) region that spans less than 1% of the full coding sequence of the gene. COPA is a subunit of coat protein complex I (COPI) that mediates retrograde movement of proteins from the Golgi apparatus to the endoplasmic reticulum (ER). Established COPA mutations alter the protein’s WD40 domain and impair binding of COPA to cargo proteins marked for ER retrieval by a C-terminal dilysine motif. In this grant, we will employ molecular dynamics simulations and eigenvector centrality analysis to resolve how mutations alter the allosteric binding site of COPA and to predict novel mutations. We will functionally validate mutants through complementary experimental approaches based on our recent discovery that mutant COPA impairs retrieval of STING from the Golgi and leads to type I interferon signaling. We have identified several COPA variants of unknown significance in patients that require further functional analysis and have been contacted by clinicians throughout the world with VUS for us to study. We will search for novel COPA variants by performing whole exome sequencing in subjects enrolled in the PedVas initiative registry, the largest cohort of pediatric vasculitis patients in the world. Subjects will be identified for sequencing using screening criteria of common pulmonary and extra-pulmonary features delineated in a recently published international cohort of COPA syndrome patients. In subjects without COPA variants, we will search for novel genes suspected to cause COPA syndrome based on the cellular and molecular pathways implicated in disease. Our grant will use computational modeling and functional studies to provide insight into the basic mechanisms of COPA function by analyzing how pathogenic mutations impair binding of COPA to the C-terminal dilysine motif of cargo. This work will allow us to accurately predict whether a mutation causes COPA syndrome and has the potential to expand the molecular definition of the disease. We will perform whole exome sequencing in patients suspected to have COPA syndrome to identify new disease-causing COPA variants or identify novel genes that lead to inflammatory lung and joint disease.