Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by a reduced platelet count and an increased risk of bleeding (1). The pathogenesis of ITP includes enhanced platelet clearance and impaired platelet production, which is related to T cell-mediated effects, B cell-mediated effects, tolerance checkpoint defects, and more (2, 3). Although immense research has improved our understanding of ITP, the etiology is still not completely clear. However, it has been widely accepted that both genetic and environmental factors play a significant role in the pathogenesis of ITP (4).

Autoimmune disease is characterized by self-antigen-induced chronic activation of immune system and eventually leads to tissue inflammation in genetically predisposed individuals (5). ITP is an autoimmune disease and closely related to inflammation (6, 7). Besides, inflammation can trigger ITP. For example, serum uric acid, an inflammatory mediator, played a pathophysiological role in the occurrence of ITP (8). However, ITP can also induce inflammation through platelet dysfunction. Recent research revealed that the acute phase of platelet destruction in severe ITP may trigger inflammation in the lung, which may induce interstitial lung disease (9). Furthermore, platelets mediate inflammation and immune-mediated disorders through multiple mechanisms, such as release of pro-inflammatory mediators, surface inflammation-related molecules, and interaction with leukocytes and endothelial cells (10, 11). Even after being phagocytosed by the mononuclear phagocyte system, platelets modulated survival surface molecules in monocytes (12). In others words, platelets regulated inflammation even after being eliminated from the circulation.

Single-nucleotide polymorphisms (SNPs), the most common pattern of genetic variants, have drawn attention because of their universal distribution and gradually uncovered biological functions (13). Many SNPs of inflammatory cytokine genes, including IL-17F, IL-10, TNFA, TNFB, TGF-beta 1, IL-6, INF-gamma, and IL-1A, influence patients’ susceptibility to ITP (14–18). Among them, IL-17F rs763780 G allele has been identified as a protective factor in ITP, while TNFB + 252G/A A allele was a risk factor. There were also some SNPs associated with the severity of ITP. IL-10 rs1800872 AA genotype presented a significant association with severity of ITP in a cohort of Egyptian population (p = 0.008) (14). The IL-10 gene polymorphism was also identified to be associated with the severity in Japanese population (p = 0.01) (19). When it comes to corticosteroid sensitivity of ITP patients, an earlier study reported that stromal cell-derived factor-1 (SDF-1) rs2297630 GA and AA genotypes significantly increased the corticosteroid treatment sensitivity compared with GG genotype (p = 0.007) (20). Interestingly, a recent study showed that this SDF-1 polymorphism was associated with corticosteroid dependence in pediatric ITP patients (21). Furthermore, researchers identified that ATP-binding cassette gene B1 gene (ANCB1) G2677T/A polymorphism was significantly associated with corticosteroid sensitivity (22). Besides, there were several researches on the association between SNPs and the refractoriness of ITP. For example, it was identified that TNFA-308G/A A allele was a risk factor for refractory ITP (17). In addition, genetic variants of a cluster of inflammation-related genes, including CD24, CD226, FCRL3, IL2, IRF5, ITGAM, NLRP3, CARD8, PTPN22, SH2B3, STAT4, TNFAIP3, and TRAF1, are associated with diverse autoimmune diseases. For example, the rs8734 polymorphism in CD24 was associated with susceptibility to diverse autoimmune diseases including rheumatoid arthritis (RA), autoimmune thyroid disease (AITD), systemic lupus erythematosus (SLE), multiple sclerosis (MS), and inflammatory bowel disease (IBD) (23–29). In addition, four SNP candidates in FCRL3, including rs11264799, rs7528684, rs945635, and rs3761959, have been identified as potential risk factors in multiple autoimmune diseases, including RA, AITD, SLE, MS, autoimmune Addison’s disease, and autoimmune pancreatitis (30–36). Finally, rs6822844, located between the IL2 gene and the nearby IL21 gene, was associated with RA, IBD, type 1 diabetes, and psoriasis (37–40). Therefore, there are multiple inflammation-related SNPs that are robustly associated with multiple autoimmune diseases. The above studies demonstrated that multiple genetic factors involved in the pathogenesis of ITP and other autoimmune diseases. However, most previous researches only verified the associations between SNPs and ITP susceptibility or ITP treatment. None of them associated the polymorphisms with complete clinical data including susceptibility, severity, corticosensitivity, and refractoriness.

Given that ITP is an inflammatory and autoimmune disease and given the above associations between inflammation-related SNPs and other autoimmune diseases, we hypothesized that these SNPs were also associated with primary ITP. The aim of our study was to investigate the association between inflammation-related gene polymorphisms and the pathogenesis of primary ITP in the Chinese Han population.

Immune thrombocytopenia is an autoimmune and inflammatory disease characterized by immune-mediated platelet destruction. Although ITP has been associated with inflammation, only a few studies have identified the association between inflammation-related SNPs and the pathogenesis of ITP (43). By contrast, inflammation-related gene polymorphisms have been associated with multiple autoimmune diseases.

In this study, we assessed the associations between 25 inflammation-related SNPs and the occurrence and treatment of ITP in the Chinese Han population. Of these SNPs, the frequencies of the homozygous minor allele of FCRL3 rs11264799 and TNFAIP3 rs10499194 were significantly decreased in ITP patients. The A allele of CD24 rs8734 was associated with a decreased risk of ITP. In addition, compared with the CC genotype, individuals carrying pooled TT/CT genotypes of TNFAIP3 rs10499194 had a decreased risk of ITP under the dominant model. These three SNPs were all protective factors.

Our findings contrast evidence found regarding other autoimmune diseases (26, 44–48). For example, in the Spanish population, no association was found between FCRL3 rs11264799 and MS (44). Interestingly, however, the C allele of FCRL3 rs7528684 was a protective factor for MS. This suggests that FCRL3 polymorphisms may protect individuals from autoimmune diseases, which is somewhat consistent with our findings. In the Israeli population, individuals with CD24 rs8734 polymorphisms had an increased risk of IBD, ulcerative colitis, and Crohn’s disease (26). In the Chinese population, a significant association was not found between TNFAIP3 rs10499194 and SLE (45); however, carriers of the CT genotype and combined TT/CT genotype had an increased risk for RA in the Chinese Han population (46). A recent meta-analysis revealed that there was no association between TNFAIP3 rs10499194 and RA in Europeans, but a significant association in Asians (OR = 1.254, 95% CI = 1.101–1.429, p = 6.7 × 10⁻⁴) (48). The discrepancies between our results and previous research may be attributed to differences in the populations and autoimmune diseases studied.

When it comes to TNFAIP3 rs10499194, there were several researches identified that it was significantly associated with the susceptibility of autoimmune diseases. Strong associations were observed between TNFAIP3 rs10499194 and juvenile idiopathic arthritis (OR = 0.74, 95% CI = 0.61–0.91, p < 0.004) (49). Prahalad et al demonstrated that TNFAIP3 rs10499194 had a significant protective effect against childhood onset RA (OR = 0.60, 95% CI = 0.44–0.83, p = 0.002) (50). Consistent with our findings, TNFAIP3 rs2230926 and rs5029939 were significantly different between chronic ITP and control groups (43), suggesting that TNFAIP3 polymorphisms may affect the susceptibility to ITP. We found that TNFAIP3 rs10499194 was significantly associated with the susceptibility of ITP even after Bonferroni multiple correction. Therefore, TNFAIP3 rs10499194 may be an important susceptibility-related SNP for ITP.

To investigate whether TNFAIP3 rs10499194 was a functional polymorphism, quantitative real-time RT-PCR was performed to evaluate TNFAIP3 expression. The data showed that individuals with CT genotype on TNFAIP3 rs10499194 locus showed higher levels of TNFAIP3 mRNA expression compared with the CC genotype, which might play a role in the susceptibility of ITP. It is widely accepted that TNFAIP3 is a deubiquitinating protein which can deregulate pro-inflammatory signal pathways including NF-κB- and IRF3-dependent gene expression by deubiquitinating specific signaling molecules (51, 52). Some researchers revealed that TNFAIP3 was a central gatekeeper in inflammation and immunity (53). A recent study demonstrated that lack of TNFAIP3 in B cells resulted in overexpression of pro-inflammatory cytokines, which caused inflammation and autoimmunity in aged mice (54). TNFAIP3-deficient B cells displayed enhanced proliferation and development of autoantibodies (55). In our functional analysis, individuals with CT genotype on TNFAIP3 rs10499194 locus showed higher levels of TNFAIP3 mRNA expression compared with the wild genotype CC, which is in agreement with our genotyping analysis that CT genotype is a protective factor for ITP. However, the mechanism of polymorphism on TNFAIP3 expression awaits further investigations.

Furthermore, we found that the AG genotype of TRAF1 rs10818488 was significantly associated with increased risk of severe ITP after adjusting for age and gender. These novel findings will be studied further in order to understand the molecular mechanism of these genetic polymorphisms.

In addition to the pathogenesis of ITP, the mechanism of corticosteroid resistance remains poorly understood. In acute lymphoblastic leukemia, the NLRP3-CASP1 inflammasome induced glucocorticoid resistance (56). Overexpression of CASP1 may promote cleavage of the glucocorticoid receptor, which decreased glucocorticoid sensitivity. We identified that NLRP3 rs4353135 was significantly associated with increased risk of corticosteroid resistance of ITP under the recessive model; however, this observation was not significant after Bonferroni multiple correction. Thus, we propose that the NLRP3 rs4353135 polymorphism may enhance expression of the NLRP3-CASP1 inflammasome and lead to corticosteroid resistance. Future studies will examine this hypothesis.

Importantly, TNFAIP3 rs10499194 was associated with corticosteroid-sensitivity after Bonferroni correction. Future work will examine the potential of TNFAIP3 rs10499194 as a biomarker for corticosteroid sensitivity.

There were some limitations to our study. On the one hand, the participants enrolled in our study were solely from Chinese Han population. Larger trials investigating multi-racial populations are needed to examine whether the associations between the inflammation-related SNPs and ITP exist in the general population. Second, as mentioned earlier, the biological functions and signaling pathways of these SNPs are not yet known and future research with global collaborations are required. Third, there was a potential selection bias in our study. Only inpatients were recruited in our study, as the corticosteroid-sensitivity and refractoriness of outpatients were difficult to follow-up in our hospital. The inpatients have more severe thrombocytopenia and bleeding symptoms. The bleeding symptoms may include gastrointestinal hemorrhage, extensive skin and mucosal hemorrhage, or intracranial hemorrhage. Besides, the 25 susceptibility loci, we tested were selected basing on the pervious reported loci associated with other autoimmune diseases. The candidate gene approach cannot cover all susceptible loci, which may lead to the selection bias.

Our investigation of SNPs and ITP provides interesting results. We found that inflammation-related SNPs, especially TNFAIP3 rs10499194, may be genetic risk factors associated with the development and treatment of ITP. Our findings may lead to clinicians screening for these SNPs in order to predict the prognosis and guide the treatment of ITP.

Part of this study was presented as a poster with a title of “Inflammation-related Gene Polymorphisms Associated with Susceptibility to Primary Immune Thrombocytopenia” (Abstract Code: 3737) and won ASH Abstract Achievement Award at the 58th ASH Annual Meeting and Exposition in San Diego, CA, USA, December 3–6, 2016.

This study was carried out in accordance with the recommendations of Medical Ethical Committee of Qilu Hospital, Shandong University with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Medical Ethical Committee of Qilu Hospital, Shandong University.

JP, MH, and X-hZ designed research, analyzed data, and wrote the paper; JL performed research, analyzed data, and wrote the paper; SM, LS, CM, and CG performed research and analyzed data.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.