The Association of CTLA-4 rs231775 and rs3087243 Polymorphisms with Latent Autoimmune Diabetes in Adults: A Meta-Analysis

Polymorphisms rs231775 and rs3087243 of cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) gene have been associated with risk of latent autoimmune diabetes in adults (LADA). However, the results were inconsistent. The purpose of this study was to quantitatively assess the relationship between polymorphisms rs231775 and rs3087243 of CTLA-4 and LADA in a larger pooled population by performing a meta-analysis. Systematic search for eligible studies was conducted in PubMed, Web of Science, and Embase. Case–control studies containing genotype frequencies of polymorphisms rs231775 or rs3087243 were selected, and pooled odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the associations between polymorphisms of CTLA-4 and LADA in allelic, dominant and recessive genetic model. A total of eleven studies, in which five studies reported rs231775, two studies reported rs3087342, and four studies reported both rs231775 and rs3087243, were identified. Among them, one study wasn’t included in the following meta-analysis because the distribution of genotypes in the control group didn’t comply with Hardy–Weinberg equilibrium. Significant associations with susceptibility to LADA were detected for rs231775 (785 cases and 3435 controls) and for rs3087243 (820 cases and 4824 controls) in overall population. Further subgroup analyses for ethnicity (Asian, Caucasian, and African) have also indicated the positive association between rs231775 and LADA. As for rs3087243, subgroup analyses detected the association between polymorphism and LADA in Caucasian population under recessive model. Polymorphisms rs231775 and rs3087243 of CTLA-4 gene are potential risk factors for LADA and may serve as novel genetic biomarkers of LADA.


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CTLA-4 protein, as a co-stimulatory molecule, can inhibit T cell activation, and therefore repress autoimmune response (Xie et al. 2014). Previous studies have indicated that the single nucleotide polymorphisms (SNPs) rs231775 and rs3087243 of CTLA-4 were potential risk factors for T1DM (Korolija et al. 2009; Sharma and B RA et al., 2021). These two polymorphisms may cause enhanced T-cell activation and resultant autoimmune responses due to decreased function of CTLA-4 (Ueda et al. 2003;Xie et al. 2014). In addition, many studies have evaluated the association between polymorphisms (rs231775 and rs3087243) and LADA in multiple populations (Alshareef et al. 2019;Caputo et al. 2005;Cosentino et al. 2002;Delitala et al. 2015;Douroudis et al. 2009;Haller et al. 2007;Jin et al. 2011Jin et al. , 2015Kisand and Uibo 2012;Pettersen et al. 2010;Qi et al. 2014). However, the results are inconclusive. Therefore, we performed this meta-analysis to further assess their associations based on existing relevant studies and aimed to improve the acknowledgement of pathogenesis of LADA.

Publication Search
This meta-analysis was complied with the PRISMA guideline (Moher D, Liberati A, Tetzlaff J, Altman DG and Group P 2009). Systematic literature searches (up to May 2021) were performed by using PubMed, Web of Science, and Embase. The searching strategy was as follows: ("LADA" OR "latent autoimmune diabetes in adults") and ("CTLA-4" OR "cytotoxic T-lymphocyte antigen-4") and ("polymorphism" OR "SNP" OR "allele" OR "variant" OR "genotype"). In addition, the reference list of original studies was manually screened to identify potentially relevant articles.

Inclusion and Exclusion Criteria
The included studies must meet the following criteria: (1) case-control study written in English; (2) report the associations between polymorphisms (rs231775 or rs3087243) of CTLA-4 and LADA; (3) provide odds ratios (ORs) and 95% confidential intervals (CIs) or detailed data to calculate ORs and 95% CIs. Family-based association studies, letters, conference abstracts, case reports, reviews, and commentary were excluded.

Data Extraction and Quality Assessment
The following data were collected from the included articles: (1) name of the first author; (2) publication year; (3) region and ethnicity of participants; (4) the genotyping methods (5) the number of cases and controls; (6) genotype frequencies of investigated polymorphisms of cases and controls; (7) OR value and 95% CI. The Hardy-Weinberg equilibrium (HWE) of control group was tested by online software (http:// ihg. gsf. de/ cgi-bin/ hw/ hwa1. pl). The Newcastle-Ottawa Scale (NOS) was applied to assess the quality of included studies from three aspects: the selection of case and control (0-4 points), comparability (0-2 points), and exposure assessment (0-3 points). Studies with at least 6 points were assumed to be of high quality.

Statistical Analysis
All statistical analyses were performed by the software STATA version 12.0 (STATA, College Station, TX). Higgins I-squared statistic and Cochran's Q test were used to evaluate the heterogeneity of included studies. If P heterogeneity > 0.1 and I 2 < 50%, the fixed-effects model was used to calculate the pooled ORs and 95% CIs, otherwise the random-effects model was applied. The subgroup analysis, sensitivity analysis, and meta-regression were used to seek the source of heterogeneity. Begg's test and Egger's test were used to assess the publication bias. P value less than 0.05 was considered as statistically significant.

Study Characteristics
Totally, eleven studies in which five studies reported rs231775, two studies reported rs3087342, and four studies reported both rs231775 and rs3087243, were identified (  Douroudis et al. 2009;Haller et al. 2007;Jin et al. 2011Jin et al. , 2015Kisand and Uibo 2012;Pettersen et al. 2010;Qi et al. 2014). Characteristics of identified articles were shown in Table 1. Among them, one study wasn't included in the following meta-analysis because the distribution of genotypes in the control group wasn't in accordance with HWE.

Associations Between Polymorphisms of CTLA-4 and LADA
The results of meta-analysis were shown in Table 2 To explore the potential source of heterogeneity among included studies, we performed sensitivity analysis to evaluate the influence of each study. No changes of results were detected for both rs231775 (Fig. 4)

Publication Bias
Begg's test and Egger's test were used to assess the publication bias. As shown in Table 3, Begg's and Egger's test showed statistically significant (P < 0.05) under dominant model for both rs231775 and rs3087243. Therefore, we performed a trim and fill method to estimate the number of missing studies and recalculate the pooled OR value after combining the missing hypothetical studies (rs231775: OR 1.797, 95% CI 1.188-2.719, random-effect model; rs3087243: OR 1.000, 95% CI 0.806-1.242, fixed-effect model). The funnel plots after addition of two missing hypothetical studies for rs231775 and three missing hypothetical studies for rs3087243 were basically symmetrical, respectively (Fig. 6).

Discussion
We performed a meta-analysis to determine the effects of polymorphisms rs231775 and rs3087243 of CTLA-4 on the LADA. In this meta-analysis, ten eligible studies including 785 cases and 3435 controls for rs231775 and 820 cases and 4828 controls for rs3087243 were analyzed. The present study revealed a significant association between polymorphism rs231775 and LADA under allelic, dominant, and recessive models. Similar positive associations were obtained in the following subgroup analysis by ethnicity except under recessive model in Caucasians. As for polymorphism rs3087243, significant association with LADA was identified in overall populations under allelic and recessive models. Subgroup analysis reported an association between rs3087243 and LADA in Caucasians under recessive model. Asian-derived populations indicated an unrelated result. Different genetic background and limited study may partially explain the difference.
The CTLA-4 gene is located on human chromosome 2q33.2. Its encoding product expressed on the cell surface of T lymphocytes and served as a negative regulator of T-cell activation (Ounissi-Benkalha and Polychronakos 2008). Therefore, dysfunction of CTLA-4 can increase self-reactivity of T lymphocyte and may  contribute to autoimmune response (Ueda et al. 2003;Atabani et al. 2005). Previous studies have also indicated associations between CTLA-4 and multiple autoimmune diseases, such as T1DM (Westra et al. 2018), systemic lupus erythematosus (Barreto et al. 2004), and rheumatoid arthritis (Lee et al. 2003). The rs231775 polymorphism located in exon 1 of CTLA-4 and led to substitution of alanine with threonine. The mutant protein caused the reduction of CTLA-4 cell surface expression (Xie et al. 2014). Polymorphism rs3087243 located in downstream of the poly (A) termination site and might play an important role in the mRNA stability of soluble CTLA-4 (sCTLA-4). Previous study has indicated that individuals with potential risk genotype G/G exhibited lower production of sCTLA-4 than those with A/A genotype (Ueda et al. 2003). Therefore, these two polymorphisms may lead to increased T-cell activation and higher probability of autoimmunity because of decreased function of CTLA-4. Latent autoimmune diabetes in adults, as an autoimmune disease, has also been associated with the polymorphisms of CTLA-4. However, the results are inconclusive. Our meta-analysis revealed significant associations between two investigated polymorphisms and susceptibility to LADA. This finding may contribute to early diagnosis and better prognosis of LADA patients, and identification of the underlying pathogenic mechanisms of LADA. There were some limitations in this meta-analysis. First, heterogeneity was detected for polymorphisms rs231775 and rs3087243 in the overall population analyses for LADA, thus the random-effects model was used. In subsequent subgroup analysis, reduced tendency of heterogeneity was observed in Asians, which indicated the ethnicity might partially explain the heterogeneity. However, other unidentified factors may still contribute to the heterogeneity of included studies. Second, the number of relevant studies was relatively limited, especially in Asians and Africans, therefore, more replication studies are warranted. Third, our results were drawn based on unadjusted data and failed to adjust baseline parameters of participants. Although the methodology quality of included studies was good, the confounding factors may influence the authenticity of our results. Finally, publication bias was detected in this meta-analysis. Although we performed trim and fill method to evaluate the potential missing studies, the results may still be influenced to some extent. Taken together, the present results should be interpreted with caution.

Conclusion
In conclusion, our results indicated the polymorphisms rs231775 and rs3087243 may serve as genetic biomarkers of LADA. Further studies, especially in Asians and Africans are needed to confirm these findings.