Genetic Relationship Between Interleukın-6 rs1800796 Variants, Interleukın-6 Plasma Levels and Susceptibility to Type 2 Diabetes Mellitus and Diabetic Nephropathy

Type 2 diabetes mellitus (T2DM) is very common worldwide and genetically heterogeneous. One of the microvascular complications is diabetic nephropathy (DN). In recent years, T2DM has been described as a disease caused by chronic inammation. The imbalance between pro- and anti-inammatory cytokines causes inammation. One of the candidate genes associated with T2DM and DN is the Interleukin-6 (IL-6) gene, one of the pro-inammatory cytokines. This study was conducted to determine the polymorphism frequencies of the IL-6 gene rs1800796 and investigate the role of this polymorphism in the development of T2DM and DN. Genomic DNA that was obtained from 261 people was used in the study. IL-6 gene rs1800796 polymorphism was determined using the PCR, restriction fragment length polymorphism (RFLP) and electrophoresis. IL-6 gene PCR products were discontinued by treatment with restriction enzyme BsrBI and were analyzed in 2% agarose gel electrophoresis. IL-6 (Bioassay technology laboratory, Shangai, China) level was examined by enzyme-linked immunosorbent assay (ELISA) using a commercial kit. The results were statistically analyzed. The frequencies of rs1800796 genotypes were found to be GG 70.7%, GC 28.5%, CC 0.8% in the control group and GG 87.8%, GC 9.9 %, CC 2.3% in T2DM patients. Although there was a statistically signicant difference between the control group and the T2DM patient group in genotype and allele frequencies, there was no signicant difference in DN. The G allele frequency was also signicantly higher in the T2DM group (p=0.000). IL-6 levels were determinated increased in patients with Type-2 diabetes compared to the control group. However; there was no signicant statistically. We can say that IL-6 rs1800796 polymorphism is related to T2DM and G allele can be used as a useful genetic marker; this


Introduction
In ammation caused by an imbalance between pro-and anti-in ammatory cytokines causes T2DM and its complications [1]. Recently, T2DM has been described as a metabolic pro-in ammatory disease which is characterized by chronic hyperglycemia and increased circulatory cytokine levels [2]. In chronic low-grade in ammation, which is de ned as a risk factor for the development of T2DM, increases circulating amounts of pro-in ammatory cytokines such as tumor necrosis factor-α (TNF-α), IL-6, and C-reactive protein (CRP).
These cytokines are stated to cause insulin resistance development, as well as β cell death, and ultimately T2DM by weakening insulin signaling, preventing insulin sensitivity and effect [3,4]. Especially in abdominal obesity (from abdominal adipose tissue), large amounts of in ammatory cytokines such as TNF-α and IL-6 are secreted, and these cytokines by stimulating the production of CRP in the liver, trigger chronic in ammation [5]. IL-6 levels increase in obese individuals and patients with T2DM. IL-6 level decreases with the reduction of fat mass in obesity [6]. IL-6 plays an important role in regulating the energy balance with its effects on glucose metabolism by regulating the effects of insulin [7]. IL-6 is secreted by both immune cells and adipose tissue [8]. IL-6 is also produced from glomerular mesangial cells [9].
One of the most important complications of T2DM is DN. In ammatory cytokines such as IL-6 have been recognized as having an important role in the pathogenesis and progression of DN [10]. Studies have shown that the − 572 G/C polymorphism (rs1800796) found in the IL6 gene promoter region may affect IL6 gene transcription and serum levels [8,9]. Recently, several epidemiological studies have also been conducted to evaluate the relationship between IL6 gene rs1800796 polymorphism and T2DM and DN risk in various populations [8,9,11,12].
Based on the relationship between in ammation and T2DM, we thought that genetic variations in the IL-6 gene, one of the pro-in ammatory cytokines, may cause susceptibility to the disease by altering the gene's function or expression, and in our study, we aimed to determine the frequency of variants of the IL-6 gene in patients with T2DM and DN and to investigate the relationship with T2DM and DN.

Materials And Methods
Peripheral blood samples were obtained from 131 patients [80 patients without DN (DN − ) and 51 with DN (DN + )] who applied to the Artvin State Hospital internal medicine department. The control group consisted of volunteers who came for a routine health screening with no evidence or family history of T2DM (130 volunteers). T2DM was diagnosed by quali ed clinicians based on fasting blood glucose (FBG) ≥ 7.0mmol/l levels and normoalbuminuria and HbA1c (6.5%) for two consecutive routine screening readings. This study was approved by the local ethics committee of Karadeniz Technical University (Protokol number: 2019/164), Turkey. An informed consent was obtained from all patients prior to inclusion in this study in accordance with the Declaration of Helsinki.
Genomic DNA was extracted using DNA isolation Kit (EZ-10 Spin Colon Blood Genomic DNA Minipreps Kit, Biotechnology Department Bio Basic Inc., Markham, Ontario, Canada). The isolated DNA samples were ampli ed with the PCR conditions and primers for the IL-6 gene indicated in Table 1. After ampli cation, all PCR products were stored at 4°C till the next procedure. After the ampli ed PCR products were checked by using agarose gel electrophoresis, a 164-bp fragment was cleaved with 1U BsrBI restriction endonuclease (NEB, R0102S) according to the manufacturer's instructions. All PCR products obtained from the digestion reaction were separated using 2% agarose gel electrophoresis. The results were valued via gel analysis software (LabWorks, Cambridge, UK). Following cleavage with BsrBI restriction endonuclease, three different genotypes were determined including CC (164-bp), GC (164 − 101, and 63-bp) and GG (101 and 63-bp).

Statistical Analysis
Pearson Chi Square test, Independent Two Sample t test, Mann-Whitney U test, and one-way ANOVA p value were used to compare the categorical variables (allele, genotype, biochemical parameters, etc.) between groups. Statistical Package for Social Sciences software (SPSS v.19 package program) was used to analyze the data. The Shapiro-Wilk test was used for the normality. The p values < 0.05 were considered to be statistically signi cant. The odds ratio (OR) were estimated with 95% con dence interval (95% CI) and the probability value (p value) of less than 5% was considered to be statistically signi cant. Sampling number was determined by power analysis.

Results
In our study; There were statistically signi cant differences between the patient and control groups in terms of weight (p = 0.000), height (p = 0.001), BMI (p = 0.000), Fasting Glucose (p = 0.000), Postprandial Glucose  (Table 2).  Different models of gene inheritance were evaluated to check any predisposition to elevated risk or protection against T2DM and DN by comparing the two groups (Table 4 and Table 5). According to the inheritance model, GC-CC (OR: 0.33, 95% CI = 0.17-0.64; p = 0.000) genotype was signi cantly associated with T2 DM.    (Table 6). In recent years, evidence has revealed that T2DM may be due to the disorder of the natural immune system and is associated with chronic in ammation. In some studies, T2DM has been described as a disease caused by chronic in ammation, and this pathogenic role of in ammation in diabetes has been proven by many studies [14][15][16]. In ammation biomarkers such as TNF-alpha, IL-6 and C-reactive protein have been reported to predict the development of T2DM [16,17]. It has also been stated that proin ammatory cytokines can cause insulin resistance by inhibiting the transmission of insulin signal in skeletal muscle, liver and adipose tissue [18]. Another study has stated that such cytokines increase the risk of T2DM by increasing insulin resistance in fat and other tissues [19]. Three single nucleotide polymorphisms of the IL-6 promoter at positions − 174 (rs 1800795), -572 (rs 1800796) and − 597 (rs 1800797) can cause an interpersonal change in the transcription and expression of IL-6 [13,20].
In this study, we found IL-6 gene rs 1800796 polymorphism to be signi cantly different between the control group and the T2DM patient group. The CC genotype was rare, but there was no statistically signi cant difference between DN + and DN- (Table 3).
In accordance with our results, in the study conducted by Wang et al., the prevalence of IL-6 gene rs 1800796 GG genotype was 4.7% in T2DM patient group and 1.84% in the control group. There was a signi cant difference between the two groups; based on these data, they reported that the risk of T2DM might be high in the IL-6 gene rs 1800796 GG genotype [21]. Yin [24].
In our study, no signi cant difference was found between the patients with and without DN in terms of IL6 gene rs1800796 polymorphism genotype numbers and percentages and allele numbers and percentage values (Table 3).
There are also studies that yielded different results from ours. In their study, Chen et al. reported that IL-6 gene promoter rs1800796 polymorphism is associated with type 2 DN, and the G allele may be a genetic risk factor for type 2 DN. Also, they stated that the G allele may increase the risk by increasing IL-6 expression in the pathogenesis of type 2 DN [25]. Another study conducted by Chang et al. showed that in general, rs1800796 GG and rs1524107 CC homozygous genotypes may pose a higher risk for the development of nephropathy in T2DM [11].
Kitamura et al. conducted a study with Japanese patients with T2DM and stated that IL-6 rs1800796 polymorphism may be associated with and responsible for the progression of DN. They also reported that GG genotype requires con rmation of the effect of DN on the development/progression with a large-scale prospective study [9].
Pradhan et al. In their studies; reported that IL-6 levels in female patients with type 2 diabetes were statistically different compared to controls [15]. Unlike our results, in other studies were conducted with patients with type 2 diabetes, IL-6 levels were found to be statistically different in the control and patient groups [12,14].

Conclusion
Early diagnosis of T2DM, which has a wide prevalence and economic burden throughout the world, is important for both increasing the quality of life of patients and protecting them from diabetes-related complications. Therefore, we think that it would be useful to investigate T2DM, which is known to have a genetic predisposition to early diagnosis in molecular detail and may be an auxiliary parameter for doctors.
No studies have been conducted between T2DM and DN and IL-6 rs1800796 polymorphism in Turkey. In this respect; although there are studies reporting a signi cant relationship between IL-6 rs1800796 polymorphism in various populations and T2DM and DN susceptibility, our ndings show that IL-6 rs1800796 polymorphism is associated with T2DM but not DN susceptibility in Turkish population. We can say that the G allele frequency is high in T2DM patients and can be used as a useful genetic marker. Declarations