IADR Abstract Archives
A Novel TRIM21(E38G) Mutation Affects IFN and Cytokine Driving a SjöGren’s-Like Phenotype
Objectives: Sjögren's Disease (SjD) is an insidious systemic autoimmune disorder targeting exocrine glands leading to dry mouth and eyes. SjD has a multifactorial etiology whereby genetic and environmental factors contribute to disease evolution; exact molecular mechanisms of SjD remain mercurial. Type I interferons (IFN) are pivotal to the systemic and organ-specific damage in many autoimmune disorders including SjD. Genome-wide association studies have identified ~20 risk loci for SjD, primarily in non-coding regions of the genome. We hypothesized the existence of rare coding variants recapitulating the cardinal features of SjD. Using whole-exome sequencing (WES) and functional genomics we identify a single nucleotide polymorphism (T>C) mutation in the RING domain of TRIM21, an E3 ubiquitin ligase, in a SjD patient (pE38G) without SSA autoantibodies.
Methods: Consented SjD or healthy subjects had clinical evaluations including oral, rheumatologic, ophthalmologic, lower lip biopsies, and blood draws. WES was performed using the NIH Clinical Center Genomics Opportunity. Primary salivary gland epithelial cell cultures (pSGEC) were established from E38G and controls. Identified variants were tested using bespoke in vitro assays (ubiquitylation, NF-kB/AP1 activity, qPCR, immunoblot), flow cytometry, and confocal immunofluorescence (IF) were employed.
Results: TRIM21 wild-type and mutant constructs exhibited reduced ubiquitylation processivity. pSGEC and PBMCs treated with adenovirus or antibody-opsonized adenovirus, showed 4- and 2-folds higher NF-kB and AP1 activity, respectively, and higher IFN and cytokine (TNFa, IL6) expression in pE38G. IF showed enhanced nuclear phosphoIRF3 present post-activation in pE38G. pE38G salivary gland biopsies showed increased TRIM21, IL8, MX1, ISG15 protein.
Conclusions: We show strong biological evidence of a novel mutation affecting TRIM21 RING domain function altering control of innate immune signaling systemically and in target organs. Altered TRIM21 function and the resultant dysregulation of IFN and cytokine expressions in both epithelial cells and PBMC disrupt proper response to pathogens (e.g., viruses) and contribute to the patient’s Sjögren-like phenotype.
Methods: Consented SjD or healthy subjects had clinical evaluations including oral, rheumatologic, ophthalmologic, lower lip biopsies, and blood draws. WES was performed using the NIH Clinical Center Genomics Opportunity. Primary salivary gland epithelial cell cultures (pSGEC) were established from E38G and controls. Identified variants were tested using bespoke in vitro assays (ubiquitylation, NF-kB/AP1 activity, qPCR, immunoblot), flow cytometry, and confocal immunofluorescence (IF) were employed.
Results: TRIM21 wild-type and mutant constructs exhibited reduced ubiquitylation processivity. pSGEC and PBMCs treated with adenovirus or antibody-opsonized adenovirus, showed 4- and 2-folds higher NF-kB and AP1 activity, respectively, and higher IFN and cytokine (TNFa, IL6) expression in pE38G. IF showed enhanced nuclear phosphoIRF3 present post-activation in pE38G. pE38G salivary gland biopsies showed increased TRIM21, IL8, MX1, ISG15 protein.
Conclusions: We show strong biological evidence of a novel mutation affecting TRIM21 RING domain function altering control of innate immune signaling systemically and in target organs. Altered TRIM21 function and the resultant dysregulation of IFN and cytokine expressions in both epithelial cells and PBMC disrupt proper response to pathogens (e.g., viruses) and contribute to the patient’s Sjögren-like phenotype.