B17-07 Functional Validation of Variants of Unknown Significance in COPA Syndrome

Abstract Rationale COPA syndrome is an autosomal dominant disorder causing childhood onset of systemic autoimmunity and interstitial lung disease (ILD) characterized by pulmonary cysts, recurrent alveolar hemorrhage, and fibrosis. Mutations causing disease cluster in the WD40 domain of coatomer subunit α (COPA), impacting the ability of COPA to bind proteins with C-terminal dilysine motifs and impairing their Golgi to Endoplasmic Reticulum retrograde trafficking. Disease is specifically caused by Golgi accumulation and activation of the innate immune sensor STING (stimulator of interferon genes), with patients demonstrating activation of type I interferons, secretion of nuclear factor kappa B (NFkB) dependent cytokines, and increased ER stress. Clinical sequencing has identified variants of unknown significance (VUS) in COPA in individuals with a wide range of phenotypes, with some VUS unlikely to impair COPA function or cause COPA syndrome. We optimized quantifiable assays to functionally evaluate COPA VUS. Methods Plasmids encoding COPA VUS were generated via site directed mutagenesis and co-transfected with STING into naturally STING-deficient HEK293T human cells. Cell lysates were probed with beads coated with the C-terminus of DDOST, a known dilysine peptide recognized by both COPA and its paralog COPB’, prior to quantification of STING by flow cytometry. COPA-dependent activation of STING-dependent pathways was determined for each VUS using luciferase assays measuring induction of interferon β (IFNβ), NFkB, Activating Transcription Factor 6 (ATF6) as a readout of ER stress, and ATF4 as a readout of the related integrated stress pathway. Results Transfection conditions and read out assays were optimized using WT and E241K COPA, a well-validated pathogenic variant. Pathogenic ranges of STING pulldown were established using COPA variants previously validated to cause COPA syndrome. Pathogenic ranges of COPA-dependent activation of IFNβ, NFkB, ATF6, and ATF4 were similarly established. COPA VUS results were compared to these ranges, allowing classification as pathogenic (poor STING pulldown with increases of all 3 reporters), likely pathogenic (poor STING pulldown with increase of 2 reporters), non-COPA syndrome disease (intact STING pulldown with increase of 1 or more reporters), and likely benign (intact STING pulldown without increases in reporters). Conclusions We created and validated functional assays for COPA VUS with which we are testing over 40 variants found in individuals whose phenotypes demonstrate variable overlap with COPA syndrome. Some variants tested, including three WD-40 domain variants, are well tolerated. VUS classification will inform patient care by clarifying which of the identified COPA mutations are expected to cause disease. This abstract is funded by: NIH, ATS/chILD Foundation