W175 - Functional Assessment of Genetic Variants of CARD11 and BCL10 with Saturation Genome Editing

Abstract Text: The effective use of genomic medicine is often hindered by variants of uncertain significance - genetic variants in disease-associated genes whose impact on disease development remains unknown. The CARD11-BCL10-MALT1 signalosome plays a critical role in B and T cell function, with each gene implicated in various immune-related diseases. Loss-of-function in these genes are linked to immunodeficiency and, in some cases, primary immune regulatory disorders, while gain-of-function is associated with lymphoma. In this study, we used cloning-free saturation genome editing to introduce all possible single nucleotide variants of the coiled-coil domain of CARD11, and the CARD domain of BCL10 into human primary T cells and a B cell line. Using a proliferation based assay we were able to functionally score 3,022 genomic variants spanning 321 amino acids of the coiled-coil domain of CARD11 and 2,178 variants covering 234 amino acids of the coding region of BCL10. We were able to show that loss-of-function variants that clinically present as primary immune regulatory disorders, such as those in CADINS Disease (C223T and R235P) or atopic dermatitis (L194P), were scored as loss-of-function in the T-cell assay. Known variants associated with gain-of-function diseases such as lymphoma (L245P, S250P) were not distinguishable in the T-cell assay, but were scored as gain-of-function in B cells treated with inhibitors of B cell receptor signaling. Collectively, these data demonstrate how saturation genome editing can be used to score and classify variants of uncertain significance in genes associated with immune disease.