Determination of IL-1B (rs16944) and IL-6 (rs1800796) genetic polymorphisms in IgA nephropathy in a northwest Chinese Han population

IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide, but etiology and pathogenesis continue to be poorly understood. Polymorphisms in the cytokine genes may play a role in the etiology and pathogenesis of IgAN. The incidence of different between diverse ethnic groups suggested important genetic influences on its pathogenesis. We genotype 10 single nucleotide polymorphisms (SNPs) in IL-1B and IL-6 gene using Sequenom Mass-ARRAY technology from 417 IgAN patients and 463 healthy controls of the Chinese Han population. We evaluated these SNPs associated with IgAN utilising the chi-square tests and genetic model analysis. We identified that the minor alleles of rs16944 (“A”), rs1800796 (“G”) in IL-1B, IL-6 were involved in an increasingly risk of IgAN in allelic model analysis, respectively. The rs16944 in IL-1B and rs1800796 in IL-6 were associated with 1.23-fold (95% CI, 1.02-1.48, P = 0.031) and 1.33-fold (95% CI, 1.11-1.66, P = 0.003) increases in the risk of developing IgAN, respectively. There was only rs1800796 still correlated with IgAN in the allelic model after adjustment by age and gender and the Bonferroni correction. In addition, Haplotype Grs1800796A rs2069837G rs2069840 (P = 0.037) and G rs1800796A rs2069837C rs2069840 (P = 0.042) in IL-6were considered to be associated with increased IgAN risk. This study verified the IL-6, IL-1B genetic variants polymorphisms contributed to IgAN susceptibility in a Chinese Han population. Although we identified SNPs susceptibility, however, replication studies and functional research are required to confirm the genetic contribution in IgAN.


INTRODUCTION
Berger and Hinglais firstly described IgA nephropathy (IgAN) in 1968, and it is a relatively newly recognized disease [1,2]. In the next few decades, IgAN was considered as a common glomerulonephritis. At the initial time of the diagnosis of long-term prognosis, predicting is still difficult in this disease. There were four processes are as follows induce the renal injury in IgAN: (1) aberrant glycosylation of IgA1; (2) synthesis of antibodies directed against galactose-deficient IgA1; (3) binding of the galactose-deficient IgA1 by the anti-glycan/glycopeptide antibodies to form immune complexes; (4) accumulation of these complexes in the glomerular mesangium to initiate renal injury. Others consensual factors [3][4][5] include the occurrence of arterial hypertension, presence of severe lesions on initial renal biopsy, such as focal and segmental hyalinosis, and tubulointerstitial fibrosis. There also exist some other factors, such as age [6], sex, obesity or overweight,  hyperuricemia or hypertriglyceridemia [7], and different immune genetic markers, all of the which are not extensively confirmed or controversial. In IgAN patients, a previous study reported that Interleukin-6 (IL-6) contributed to the proliferation of cells [8]. IL-6 encodes a cytokine that functions in inflammation and the maturation of B cells. And the encoded protein has turned out to be an endogenous pyrogen can induce fever in people who carried the infections or autoimmune diseases. The functioning of IL-6 is associated with a variety of disease states, such as diabetes mellitus and systemic juvenile rheumatoid arthritis.   A study involved to patients with a progressive clinical course has reported that Increased IL-6 glomerular expression as well as urinary excretion [9]. It has been suggested to be a prognostic marker for IgAN, but its role is still debated [10,11]. At tumor sites, inflammatory cytokines can modulate the growth and invasive properties of tumor cells, and IL-1 is part of the most potent pro-inflammatory cytokines [12]. IL-1 exists in two agonistic forms, IL-1alpha and IL-beta (IL-1B). IL-1B coding for protein IL1-β is located in the 2q14 chromosome, has 7,153 kb and seven encoding regions. IL-1B plays a key role in the regulation of the immune responses and in the production of a variety of inflammatory mediators, together with other molecules [13].
In this study, we conducted an extensive association analysis to assess the roles of IL-1B and IL-6 polymorphisms and haplotypes on susceptibility to IgAN in a Chinese Han population from a case-control study.
A total of 10 single nucleotide polymorphisms (SNPs) were studied in this association analysis in an attempt to identify markers that might guide intervention decisions and improve patient survival.

RESULTS
A total of 880 participants, including 417 IgAN patients and 463 controls, were analyzed in this study. Basic patient characteristics (gender and age) are listed in Table  1. 31.0% of the IgAN patients were men and 16.4% were women, while 30.1% of the controls were men and 22.5% were women. The mean ages were 33.22 ±12.15 years (mean ± SD) for IgAN patients and 50.65 ±11.79 years for controls.
10 SNPs in the IL-1B and IL-6 gene were genotyped in the IgAN patients and the controls. Table 2 listed the basic characteristics of these SNPs in the study population. All of the ten SNPs have been conformed to HWE in the control group (P > 0.05), and two-sided Pearson chi-square tests were used to identify differences in allele frequency distributions between IgAN patients and controls. The rs2853550 and rs16944 in IL-1B, and rs1800796 in IL-6 were associated with 1.37-fold (95% CI, 1.00-1.87, P = 0.050), 1.23-fold (95% CI, 1.02-1.48, P = 0.031), and 1.33-fold (95% CI, 1.11-1.66, P = 0.003) increases in the risk of developing IgAN, respectively. The SNP rs2853550 had a weak significant difference. Only one (rs1800796) of the three SNPs was still correlated with IgAN in the allelic model after Bonferroni correction.
And then, we determined the association between these 10 SNPs and IgAN risk using four genetic models (Table 3); P-values were calculated using the chi-square test. The "A/G-A/A" genotype at the rs2853550 SNP increased the risk of IgAN in the dominant model (OR = 1.46; 95% CI, 1.04-2.05, P = 0.030), and "A/G" in the overdominant model with a P = 0.023 (OR = 1.151; 95%CI, 1.06 -2.15). The "G" allele in rs16944 decreased the IgAN risk in the log-additive model (OR = 0.80; 95% CI, 0.66-0.97, P = 0.026). However, these results were no statistical significance after adjustments for age and gender and the Bonferroni correction.
IL-1B and IL-6 genetic polymorphisms were characterized utilizing the haplotype and linkage disequilibrium (LD) analyses. LD was determined pairwise among all these 10 SNPs, the haplotype structure of the two genes were analyzed, two blocks ( Figure 1) were detected in studied IL-1B SNPs and one block (Figure 2) was found in IL-6 SNPs by haplotype analyses.

DISCUSSION
The onset of IgAN may be associated with infections in the upper respiratory tract. Genetic analysis of familial IgAN is the most promising approach to the identification of IgA disease [14,15]. For example, a recently study reported that 30 IgAN families were analyzed utilizing the whole-genome scanning [16]. However, in IgAN it is still not understood how the composition of circulating immune complexes affects the disease presentation, severity, and/or progression. Five novel variants were correlated with a protective effect against IgAN susceptibility were identified in a Genomewide study [17]. It was reported that people carried protective alleles and had the lowest prevalence of IgAN in African populations. But in Asians, it was reported that people have fewer protective variants and have the highest prevalence. In a genome-wide linkage analysis study with 30 multiplex kindreds, had demonstrated the linkage of IgAN to 6q22-23 [16].
There is no doubt that there exist genetic components to the IgAN pathogenesis and IgAN clinical expression. In ourstudy, we systematically examined the impact of 10 SNPs in the IL-1B and IL-6 loci on susceptibility to IgAN. We identified the association between SNPs and the susceptibility of esophageal cancer in five genetic models (codominant, dominant, recessive, overdominant and additive), which equivalent of repeated multiple comparisons, increasing the chance of making type I errors. Meanwhile, we should perform the P correction with Bonferroni correction. We found that the rs2853550, rs16944 in IL-1B and rs1800796 in IL-6 genetic polymorphisms were correlated with an increased the IgAN risk in a northwestern Chinese Han patients population. To the best of our knowledge, this is the first case-control study to investigate this association. Persistent inflammation is linked with cancer development and progression [18]. IL1-B, a key cytokine released from glial cells, is critically involved in the pathogenesis of chronic pain [19], memory deficit [20], via activation of IL-1R1 [21]. It has revealed that treatment with IL1B can promote the invasiveness of breast cancer (BC) cells in vitro [22,23]. A study reported that IL1B induced osteoprotegerin secretion, independent of BC subtype and basal osteoprotegerin levels [24]. The polymorphism rs1143630 is located in intron four, a noncoding region

Ethics and consent
All subjects were informed of the purpose of the study and the experimental procedures involved. The study protocol was approved by the ethics committee of the First Affiliated Hospital of Xi'an Jiaotong University. We also obtained signed informed consent from all participants. The experimental protocol was implemented in accordance with the approved guidelines.

Study participants
From August 2012 to July 2016, 417 patients (273 males and 114 females, mean age of 33.22±12.15) were recruited to participates form Han population in the northwest China, all the cases were diagnosed as IgAN by renal biopsy and none-familialy IgAN cases. 463 healthy subjects (265 males and 198 females, mean age of 50.65 ±11.79) were recruited from routine healthy examinations in the same hospitals. All subjects were unrelated Chinese Han people living in Xi'an city or nearby. There are detail inclusion/exclusion criteria: patients with cancer, infection, secondary IgAN (Secondary IgAN is seen most commonly in patients with liver disease or mucosal inflammation, in particular affecting the gastrointestinal tract), other renal diseases and autoimmune diseases were excluded. The exclusion criteria for healthy subjects included the chronic disease, central nervous systemrelated disease, and conditions involving vital organs (liver, heart, lung, brain) and more aggressive metabolic and endocrinological disease.

SNP selection and genotyping
We examined 7 SNPs in IL-1B and 3 SNPs in IL-6, and all of the 10 SNPs had minor allele frequencies (MAF) greater than 5%. Samples were centrifuged and stored at -80° until analysis. We extracted genomic DNA from peripheral blood samples using the GoldMag-Mini Whole Blood Genomic DNA Purification Kit (GoldMag Ltd. Xi'an, China) according to the manufacturer's protocol and measured DNA concentrations using a NanoDrop 2000. Sequenom MassARRAY Assay Design 3.0 Software was used to design primers for amplification and extension reactions [33]. SNP genotyping was performed using a Sequenom MassARRAY RS1000 according to the manufacturer's standard protocol [33]. Finally, Sequenom Typer 4.0 Software was used for data management and analysis [33,34]. www.impactjournals.com/oncotarget

Statistical analysis
Departure from Hardy-Weinberg Equilibrium (HWE) was assessed for the frequency of each SNP in genotype frequencies between the esophageal cancer and control groups were evaluated using the Chi-square test [35]. Microsoft Excel and the SPSS 17.0 statistical package (SPSS, Chicago, IL) were used for statistical analyses. All P-values presented in this study are twosided; P ≤ 0.05 was considered statistically significant. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using unconditional logistic regression analyses [36]. The web-based software SNP Stats was used to identify associations between SNPs and the risk of esophageal cancer in four genetic models (Codominant, dominant, recessive, and additive) [37]. We used the Haploview software package (version 4.2) and the SHEsis software platform (http://analysis.bio-x.cn/myAnalysis. php) to analyze linkage disequilibrium, haplotype construction, and genetic associations at polymorphism loci [38,39].