Background/Purpose: Systemic Sclerosis (SSc) is characterized by fibrosis of the skin and internal organs associated with vasculopathy and autoantibodies and exhibiting wide clinical heterogeneity. Contributors to pathogenesis include genetic variants and currently unidentified external triggers. Several genome-wide association studies (GWASs) have shed light on the genetic component and estimated that it contributes 30% to the risk of developing SSc. We used Whole Exome Sequencing (WES) data to identify novel SSc susceptibility loci, and to identify putative causative pathways.

Methods: Exome sequencing (ES) was performed by Regeneron in an academic-industry partnership. ES data was provided to the study team and all analyses were performed by the authors independently of Regeneron. We first performed GWAS analysis of 2,865 Caucasian cases and 1,043 controls from samples in the SSc Registry and DNA Repository. Genotype imputation was performed with the 1000 Genome Project Phase 3 panel which yielded 5,522,961 variants. Case-control association analysis was performed using PLINK v1.9 logistic regression model of additive effects, including gender and the first two principal components (PCs) as covariates. Replication testing was performed in an independent case-control set of European ancestry (949 SSc patients, 998 controls from collaborators Kerick and Martin).

The evolutionary action method estimates the functional and clinical impact (mutational harm) of missense protein coding variations.

Results: We identified a novel susceptibility locus at MICB (rs2516497, P = 8.42 × 10⁻¹⁵) within the HLA region. MICB (MHC class I chain-related gene B) is a protein coding gene in the MHC region whose gene product is a stress-inducible cell surface molecule that labels malfunctioning cells for targeting by cytotoxic lymphocytes such as natural killer cells.

Additionally, we confirmed and firmly established the role of HLA-DQA1 (rs1048372, P = 2.95 × 10⁻²¹) and HLA-DQB1 (rs2647032, P = 1.32× 10⁻¹⁶) gene regions as SSc genetic risk factors. Replication testing in an independent case-control set of European ancestry confirmed association of this MICB SNP (rs2516497, P = 7.17 × 10⁻⁹) to SSc. In addition, the replication cohort also confirmed that the association of this MICB SNP is an independent event, i.e., it is not in linkage disequilibrium with other HLA class genes.

Next, we applied the evolutionary action method to investigate genes and pathways associated with SSc. Evolutionary action predicts the impact of missense variants on protein function. Using the de novo missense variants from the ES data, this method corroborated genes associated with SSc and identified additional genes in the interferon pathway among others. IFI44L and IFIT5 are interferon genes and are shown to be dysregulated in SSc blood and skin.

Conclusion: Using a GWAS analysis applied to WES data, we identified MICB as a novel SSc susceptibility locus independent of other HLA class genes. Additionally, by application of the evolutionary action analysis method to this dataset, we identified missense mutations in the interferon pathway that may contribute to SSc pathogenesis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/whole-exome-sequencing-and-evolutionary-action-missense-mutation-analysis-identifies-micb-as-a-new-ssc-susceptibility-locus-and-the-interferon-pathway-as-contributors-to-ssc-pathogenesis/