Genes Influencing the Development and Severity of Chronic ITP Identified through Whole Exome Sequencing

Background: Chronic immune thrombocytopenia (ITP) is a complex autoimmune disease characterized by antibody mediated platelet destruction and impaired production. Sustained autoimmunity in chronic ITP appears to be due to generalized immune dysregulation including altered T cell balance with a shift toward immune activation (increased T_h1/T_h2 ratio) as well as decreased number and impaired function of regulatory T cells (T_reg). The cause of these abnormalities has not been fully elucidated and is likely multifactorial, but genetic factors may be involved in ITP pathogenesis. Improved understanding of genetic influences could lead to novel therapeutic approaches.

Aim: To identify genetic variants that may be involved in chronic ITP susceptibility and severity.

Methods: Whole exome sequencing (WES) was performed on 262 samples with robust phenotype data on children with chronic ITP from the North American Chronic ITP Registry (NACIR, n= 173) and the Platelet Disorders Center at the Weill-Cornell Medical Center (n=89). All but three patients were ≤19 years old at diagnosis; 83% had primary ITP, 10% had Evans syndrome, 7% had other autoimmune disorders. Sequencing data for ITP cases of European American (EA) ancestry were compared to EA controls with platelets >150 x 10⁹/L sequenced in the Atherosclerosis Risk in Communities (ARIC) Study (N=5664) to identify candidate genes associated with ITP susceptibility. Analyses filtered variants on a minor allele frequency (MAF) <0.01 as well as functionality of nonsynonymous, stop gain, splicing, stop loss, and indel variants. Both Fisher-Exact tests of single variants and Firth logistic regression for gene-based tests, accounting for an unequal proportion of cases compared to controls, were used. A Bonferroni corrected threshold based on 16,532 genes was calculated at 3.0x10^-6. In a separate analysis, phenotype data for ITP cases were reviewed and cases stratified by disease severity according to second line treatment needed (Yes =139, No=113) and compared to ARIC EA controls with platelet count >150 x 10⁹/L (N=5664).

Results: Several damaging variants identified in genes involved in cellular immunity had a significantly increased frequency in the EA ITP cohort (Table). The most significant associations were detected in the IFNA17 gene, which is involved in TGF-β secretion and could affect number and function of the T_reg compartment. IFNA17 rs9298814 (9:21227622 A>C) was identified in 26% of cases in the EA ITP cohort compared to <0.01% of EA controls, and other low frequency but presumed deleterious variants were also identified in IFNA17. IFNA17 gene variants remained significant in the most severely affected patients, specifically those requiring second line therapy, providing further evidence for this gene's functional relevance in the pathogenesis and pathophysiology of ITP. Other genes with known impact on T cell number or function, including DGCR14, SMAD2 and CD83 also contained variants with increased frequency in the EA ITP cohort. IFNLR1 and REL genes were also significantly associated with need for second line ITP therapy. Analysis of this large cohort did not validate any of over 20 variants that have been previously published as candidates for ITP susceptibility or evolution to chronic ITP.

Conclusion: Damaging variants in genes associated with cellular immunity have an increased frequency in children with chronic ITP compared to controls, providing further evidence for the role of T cell abnormalities in the pathophysiology of ITP. The IFNA17 and IFNLR1 genes maintained significance when the ITP cohort was stratified according to disease severity, and may be important candidate genes involved in immune regulation and sustained autoimmunity associated with chronic ITP.