Idiopathic membranous nephropathy (iMN), one of the most common cause of nephrotic syndrome in the adult population, is a kidney-specific, autoimmune glomerular disease that presents with increased protein in the urine. It is now recognized that iMN is characterized by subepithelial immune deposits that consist immunoglobulin (Ig)G and complement.[1,2] Many studies demonstrated that the major target antigen of autoantibodies in iMN is an M-type phospholipase A2 receptor (PLA2R).[3,4] However, little is known about the therapeutic strategies of iMN. In recent years, numerous studies have indicated that certain genetic polymorphisms associated with susceptibility to this disease.[5,6] And this discovery has opened a new horizon in the therapy of iMN.
PLA2R, a major target antigen in iMN, are detectable in about 70% of patients from various ethnic groups. Epitopes of PLA2R must be presented by the human leucocyte antigen (HLA)-encoded major histocompatibility complex (MHC) class II molecules to stimulate autoantibody production. Therefore, HLA plays a critical role in the pathogenesis of iMN. The HLA region resides on chromosome 6p21.3, and this region is one of the most polymorphic regions of the genome in humans. human leucocyte antigen -DQ a-chain 1 (HLA-DQA1), belonging to the group of HLA Class II regions, is part of the heterodimer anchored in the membrane, forming the antigen-binding groove. HLA-DQA1 plays a central role in the immune system by presenting peptides derived from extracellular proteins to immunocompetent cells. Genome-wide association study (GWAS) identified risk alleles at HLA and PLA2R loci in European populations, with the top variant rs2187668 within HLA-DQA1 showing higher risk effect compared with the top variant rs4664308 within PLA2R1. Recent years, more and more published papers have revealed that the rs2187668 G > A polymorphism of HLA-DQA1 was associated with iMN.[9–15]
Considering the relatively small sample size in most studies, it is possible to perform a quantitative synthesis of the evidence with rigorous methods. Meta-analysis has been proven to be an effective statistical method combining available studies to produce a precise conclusion. Therefore, we performed a meta-analysis on 11 published case and control studies to identify the precise association between HLA-DQA1 rs2187668 G/A polymorphism and iMN risk.
2 Materials and methods
2.1 Selection of studies
Medical Subject Headings (MeSH) terms (“HLA-DQ alpha-chains” or “HLA-DQ a-chain 1” or “HLA-DQA1” or “rs2187668”) and (“membranous nephropathy” or “membranous glomerulonephritis” or “idiopathic membranous nephropathy” or “MN” “iMN” or “IMN”) were used in PubMed. These keywords retrieval strategies were also used in other databases (Google Scholar, Embase, Cochrane Library) for entries until April 2018. References of the retrieved publications were also reviewed. All eligible studies were retrieved, and their bibliographies were checked for any additional relevant and potential eligible studies.
2.2 Inclusion and exclusion criteria
Literatures fulfilled the following criteria:
- published in English;
- experimental subjects were diagnosed as iMN by renal biopsy;
- evaluated HLA-DQA1 gene polymorphism rs2187668 and risk of iMN;
- were cohort-based or case-control;
- included sufficient data for calculating an odds ratio (OR) with 95% confidence interval (CI);
- case control groups genotype conformed to the Hardy–Weinberg (H–W) balance. If such data were unavailable, we attempted to contact the corresponding author to provide the missing data before the study was excluded. The major exclusion criteria included: review articles, meeting abstract, case reports, editorials, treatment outcome studies,meta-analysis were excluded; lack of sufficient data for calculation of ORs with 95% CIs; and when there were multiple publications from the same study, only the largest population study was adopted, others were excluded.
2.3 Data extraction and synthesis
We performed this meta-analysis based on published studies. So there is no need to conduct special ethic review, and the ethical approval is not necessary. To exclude irrelevant and overlapping studies, two independent investigators (Liping Bao and Jushuang Li) examined the articles by using a standardized data extraction form. If genotype distributions were not given in the study, we calculated them from allele frequencies and number of cases and controls if the reported study was in accordance with Hardy–Weinberg equilibrium (HWE). Disagreements were resolved by discussion and consensus. If discussion and consensus were not achieved, the third reviewer (Shuang Hu) would make an ultimate decision. We extracted the following information from each study: first author, year of publication, ethnicity, and the number of cases and controls for each genotype, gene detection method, source of control groups, and statement of HWE.
2.4 Statistical analysis
The OR with 95% CI was used to assess the strength of association between HLA-DQA1 rs2187668 G/A polymorphisms and iMN risk in 5 genetic models (A vs G, AA vs GA + GG, GG vs GA + AA, GG vs AA and GA vs GG). The HLA-DQA1 rs2187668 polymorphism distribution in the control group was tested for HWE using the Pearson chi-square test. Newcastle–Ottawa Scale (NOS) was used to access the quality of the inclusive studies. Cochran's chi-square-based Q-test and I test were performed to assess the between-study heterogeneity of studies. If the heterogeneity was not significant (P >.1, I2 < 50.0%), the fixed-effect model can be used to pool ORs; otherwise, the random-effect model was used. Subgroup analyses were performed based on variables, such as ethnicities and control tipies. Sensitivity analysis was conducted by deletion of a single study, in turn to, identify the influence of the individual data on pooled results and test the reliability of results. Begg funnel plots and Egger tests were used to access the potential publication bias. A 2-sided P value of less than .05 was considered statistical significant. All statistical analyses were conducted by Stata version 12.0 (StataCorp LP, College Station, TX).
3.1 Study characteristics
A total of 36 potentially relevant citations were identified from databases. After we screened the titles and abstracts, 26 citations were removed due to irrelevant topics (not about iMN and HLA-DQA1 rs2187668 polymorphism). Then, the full-text of the rest of 10 citations were downloaded for reading carefully; we removed 3 citations due to insufficient genotype data for extraction. All 11 case and control studies from 7 articles were included in our meta-analysis, incorporating 3209 iMN cases and 7358 controls. The populations were from Europe (France, Britain, Netherlands and Spain), Asia (Japan, India and China), Caucasus and America. The characteristics of these studies and the quality scores were presented in Table 1.
3.2 Main results
The evaluation of association between HLA-DQA1 rs2187668 G/A polymorphism and iMN risk was presented in Table 2.
Overall, there was correlation between the susceptibility of HLA-DQA1 gene and iMN, and the difference has statistical significance (A vs G: OR = 3.34, 95% CI = 2.70-4.13; AA vs GA + GG: OR = 8.69, 95%CI = 6.64–11.36; GG vs GA + AA: OR = 0.25, 95% CI = 0.19–0.33;AA vs GG: OR = 12.61, 95% CI = 8.02–19.81; GA vs GG: OR = 3.45, 95% CI = 2.79–4.25, respectively). In order to identify potential differences based on ethnicity, subgroup analysis was performed. Since only one study was performed in an American population, however, it should be noted that the ethnicity-based analysis may not be reliable in regard to the American subgroup. The results suggested the HLA-DQA1 rs2187668 G/A gene has a certain correlation with iMN susceptibility among Asian, European and Caucasian subjects. As shown in Figures 1–5.
3.3 Sensitivity analysis
Sensitivity analysis was performed by omitting each study in turn to estimate the influence of individual study on the pooled OR (Fig. 6). The result of sensitivity analysis showed that the corresponding pooled OR and principal results did not change appreciably, suggesting stability of the meta-analyses.
3.4 Publication bias
Begg funnel plots and Egger test were generated to detect the potential publication bias in the literature used. The result did not show any evidence of publication bias based on Begg funnel plot (PBegg = 1, A vs G, Fig. 7) or Egger regression test (PEgger = 0.523, A versus G). Similarly, no publication bias for the association between HLA-DQA1 rs2187668 polymorphism and iMN susceptibility under the other genetic models.
iMN, is an autoimmune disease that represents approximately 80% of MN cases, in which circulating antibodies to a conformation-dependent epitope on the target antigen. The autoimmune response is regulated by genes at the HLA-DQA1 locus. Therapies for iMN that include the use of immunosuppressive drugs and nonspecific antiproteinuric measures have led to disappointing results, which prompted increased interest in the discovery of new therapeutic targets. A variety of studies have focused on the association between the HLA-DQA1 gene rs2187668 polymorphism and iMN.[9–15] And recent studies indicated that HLA-DQA1 gene rs2187668 may confer susceptibility to iMN by presenting T cell epitopes on PLA2R. However, the results obtained from such investigations have been inconclusive. To derive a more precise estimation of the relationship, we performed this meta-analysis, combining data from similar studies to increase sample size and statistical power and achieve a more robust result.
In this meta-analysis, a total of 7 articles including 11 case and control studies were used to evaluate the association between HLA-DQA1 gene rs2187668 polymorphism and iMN risk. In order to eliminate heterogeneity, we established strict inclusion and exclusion criteria, and only high-quality research which aim is the same can be included considering the consistency of the research object, exposure factors, etc. However, heterogeneities were still observed in the models except recessive model in our meta-analysis. The fixed-effect model was used in recessive model, and the result showed a significant association between rs2187668 genetic polymorphisms and iMN (Fig. 2). Because of heterogeneity, the random-effects model was adopted in other genetic models, which make the results relatively conservative, and the results also showed significant statistical differences (Fig. 1, Figs. 3–5). In addition, subgroup analysis was conducted to explore potential sources of heterogeneity. Considering that the polymorphism frequencies might differ among ethnic groups and control types, we performed a subgroup analysis by ethnicity and control source (Table 2). In the ethnicity-specific meta-analysis, a separate analysis was performed in Asian, European, Caucasian and American populations. The results demonstrated that the HLA-DQA1 rs2187668 G/A polymorphism was associated with iMN risk in Asian, European and Caucasian. However, the result of American subgroup may not be reliable, since only one study was performed in American population. Moreover, the control source-specific meta-analysis was performed in hospital-based, population-based and not reported control sources. The results also showed that the HLA-DQA1 rs2187668 G/A polymorphism was associated with iMN risk. After subgroup analysis by ethnicity and control source, heterogeneity still existed in the different genetic models, therefore, ethnic differences and control sources are not the cause of heterogeneity. iMN is a multifactorial disease, which interactions between many factors including age, gender, and so on. But data for people of different age and gender were limited, and data were not available to perform subgroup analyses based on age or gender. Anyway, our result indicated that HLA-DQA1 gene polymorphism was associated with iMN risk. And rs2187668 A allele was a risk factor for iMN. No publication bias was observed in our meta-analysis.
Some potential limitations of the present meta-analysis should be considered. First, all of the 11 studies included in this meta-analysis were based only on papers published in English, so studies in other languages did not attend this meta-analysis, which may cause selection bias. Second, there were only 2 studies with Caucasian populations and 1study with American populations, the exploration of moderator variables was limited by the low number of studies. Third, there was heterogeneity between studies of HLA-DQA1 rs2187668 polymorphisms. Therefore, other risk factors are not well considered into analysis, such as age and gender which may affect the risk of iMN.
In conclusion, the results of our study indicate that HLA-DQA1 rs2187668 polymorphism is associated with the susceptibility to iMN in Asians, Europeans, and Caucasians. Accordingly, the rs2187668 mutation was important in the iMN, and the intervention of this mutation might bring new therapeutic strategy for iMN. In the future, it is critical that further investigations with larger sample size, more ethnic groups and strict protocols to further validate the results of the current meta-analysis.
Conceptualization: Liping Bao, Xiaoyan Wu.
Data curation: Liping Bao, Jushuang Li.
Formal analysis: Jushuang Li.
Funding acquisition: Xiaoyan Wu.
Investigation: Liping Bao, Jushuang Li.
Methodology: Liping Bao.
Resources: Shuang Hu, Xiaoyan Wu.
Software: Shuang Hu.
Supervision: Xiaoyan Wu.
Validation: Shuang Hu.
Visualization: Shuang Hu.
Writing – original draft: Liping Bao.
Writing – review & editing: Xiaoyan Wu.
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Keywords:Copyright © 2018 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.
gene polymorphism; HLA-DQA1; idiopathic membranous nephropathy; meta-analysis; rs2187668