Evaluation of the Relationship Between Cytochrome P450 Polymorphisms and T2DM Risk

Background: Recent studies have identied some genetic polymorphisms of CYP2C8 and CYP2D6 related to disease susceptibility. However, it has not been reported whether polymorphisms in CYP2C8 and CYP2D6 are associated with the risk of type 2 diabetes mellitus (T2DM). We designed a case-control study to evaluate the relationship between those CYP polymorphisms and T2DM risk. Methods: Four single nucleotide polymorphisms (SNPs) of CYP2C8 and CYP2D6 were genotyped from 512 patients and 515 healthy controls using Agena MassARRAY. The chi-square test was used to compare the differences in allele and genotype frequencies between the two groups. Odds ratios (ORs) and 95% condence intervals (CIs) were estimated by logistic regression analysis to evaluate the relationship between polymorphism and T2DM risk. Results: The results found that the rs1065852 in CYP2D6 was correlated with the T2DM risk in overall (A vs. G: OR = 1.22, 95% CI: 1.03–1.45, P = 0.024; AA vs.GG: OR = 1.46, 95% CI: 1.04–2.06, P = 0.031; AA-AG vs. GG: OR = 1.36, 95% CI: 1.04– 1.79, P = 0.026; additive: OR = 1.21, 95% CI: 1.02–1.44, P = 0.027). Gender stratication analysis results demonstrated that the rs1065852 in CYP2D6 was related with an increased the risk of T2DM in male and age < 59 years old. However, no statistical signicance relation was found between CYP2C8 SNPs and T2DM risk. Conclusions: This study revealed that CYP2D6 (rs1065852) could be potential genetic markers of susceptibility to T2DM. Further studies are required to conrm our ndings. P = 0.031). Signicant associations were also found under the dominant model (AA-AG vs. GG: OR = 1.36, 95% CI: 1.04–1.79, P = 0.026) and additive model (OR = 1.21, 95% CI: 1.02– 1.44, P = 0.027) before and after adjusting with age and gender. However, no statistical signicance relation was found between CYP2C8 SNPs and the risk of T2DM.


Background
Diabetes mellitus (DM) is one of the most common chronic disease, characterized by impaired glucose metabolism due to defective insulin secretion or action [1]. The global prevalence of DM has increased signi cantly over the past two decades, primarily due to the obesity epidemic. It has been estimated that approximately 451 million people worldwide suffering from DM in 2017 and it is expected to reach more than 693 million in 2045 [2]. Type 2 diabetes mellitus (T2DM) is the most common form of DM, which accounts for more than 90% of the all of DM patients. According to reports, the prevalence of T2DM among Chinese adults was approximately 11.6% in 2010 [3]. T2DM is a non-autoimmune, complex, heterogeneous, and polygenic metabolic disease. Environmental risk factors and genetic components have been reported to play important roles in the etiology and pathogenesis of T2DM [4]. This genetic factor for T2DM is likely to be due to single-nucleotide polymorphisms (SNPs) [5]. Studies have identi ed that several genes polymorphisms were associated with susceptibility to T2DM, such as CYP3A4 [6], CYP2C9 [7], and CYP2D6 [8].
Cytochrome P450 (CYP) enzymes play a central role in the oxidative metabolisms of a diverse number of drugs and chemicals [9]. CYP2C8 enzyme encoded by the CYP2C8 gene is a member of the human CYP2C enzyme family. CYP2C8 comprises 7% of the total hepatic CYP content and converts endogenous compounds such as arachidonic acid to biologically active epoxide metabolites [10,11]. Recently, studies have reported that CYP2C8 polymorphisms are associated with susceptibility to coronary artery disease [12], colorectal cancer [13], ischemic stroke [14]. CYP2D6 enzyme encoded by the CYP2D6 gene is a highly polymorphic enzyme. CYP2D6 comprises a small percentage of the total hepatic CYP content and catalyzes the biotransformation of about 20-25% of the clinically used drugs [15]. Some polymorphisms of CYP2D6 have been reported to be associated with risk of pemphigoid [16], Parkinson's disease [17], autoimmune Diseases [18].
However, there are very few reports of the relationship between CYP2C8 and CYP2D6 polymorphisms and T2DM risk in the Chinese Han population. Therefore, to exported whether the ve polymorphisms in CYP2C8 (rs1934953, rs1934951, rs2275620, and rs17110453) and CYP2D6 (rs1065852) are associated with the risk of T2DM, we designed this study with 512 T2DM patients and 515 healthy controls. Understanding SNPs related with T2DM susceptibility may be helpful for providing personalized diagnosis and prevention.

Study Subjects
This case-control study was based on 1027 individuals of Chinese Han population. The case group included 512 patients who had been recently diagnosed with T2DM in the First A liated Hospital of Xi'an Jiaotong University according to the WHO guidelines, fasting plasma venous glucose more than 126 mg/dl, or plasma glucose more than 200 mg/dl after 2 h of oral glucose, or glycated hemoglobin level more than 6.5%. Subjects who had been diagnosed with cancer, acute infections, myocardial infarction, or kidney disease were excluded from this study. A total of 515 healthy controls were recruited from the medical center of the First A liated Hospital of Xi'an Jiaotong University. We collected the clinical and laboratory ndings of each T2DM patient from the clinical charts, including fasting blood glucose, glycated hemoglobin, total cholesterol, high-density lipoproteins (HDL) cholesterol, low-density lipoproteins (LDL) cholesterol, triglyceride, urea, creatinine, cystatin C and glomerular ltration rate (GFR).

DNA extraction
We collected 5 mL of peripheral blood from each participant using Ethylene diamine tetraacetic acid (EDTA) tube, then stored at − 20 °C until analysis. Genomic DNA was isolated from whole blood samples using GoldMag-Mini Whole Blood Genomic DNA Puri cation Kit (GoldMag. Co. Ltd., Xi'an, China). We used a Nanodrop 2000 spectrophotometer (Thermo Fisher Scienti c, Waltham, MA, USA) to detect the concentration and purityof extracted DNA. The DNA yield at optical density (OD) 260 nm/OD280nm between 1.8 and 2.0 was considered as pure. The isolated DNA was stored at − 20 °C for further experiment use.

SNP genotyping
We selected the ve SNPs in CYP2C8 (rs1934953, rs1934951, rs2275620, and rs17110453) and CYP2D6 (rs1065852) based on previously published articles [19][20][21][22][23][24]. The HapMap database showed that the minor allele frequency (MAF) of all SNPs is more than 0.05 in global population. The polymerase chain reaction (PCR) primers were designed by the online software Agena Bioscience Assay Design Suite Version 2.0 (https://agenacx.com/online-tools/). We used the Agena MassARRAY platform (Agena Bioscience, San Diego, CA, USA) to genotype ve CYP2C8 and CYP2D6 SNPs following the manufacturer's instructions. The Agena Bioscience TYPER software (version 4.0) was used to manage and analyze the genotyping results.

Statistical analysis
The data were analyzed using the SPSS 20.0 statistical software (SPSS, Chicago, IL, USA) and PLINK 1.07. The chi-square test was used to determine whether the genotype frequencies distributions of CYP2C8 and CYP2D6 genes SNPs in control group were in Hardy-Weinberg Equilibrium (HWE). The distributions of the alleles and genotypes frequencies of polymorphisms were compared between the case and control groups using chi-square test. The continuous variables were provided as mean ± standard deviation (SD). We used Student's t test to compare the numerical values of the continuous variables. The odds ratio (OR) and 95% con dence interval (CI) were calculated to evaluate the association between the polymorphisms CYP2C8 and CYP2D6 and T2DM risk by logistic regression model. The threshold for signi cance was set at P < 0.05.

Results
In this study, we recruited a total of 512 T2DM patients and 515 healthy controls. The demographics and clinical parameters of all participants are shown in Table 1. The case group comprised 281(55%) male T2DM patients and 231(45%) female T2DM patients. The control group had 283 (55%) males and 232 (45%) females. The chi-square test result showed that no signi cant difference in gender distribution was observed between the case group and the control group (P = 0.982). The average age of the case group and control group was 59.23 ± 9.592 and 59.27 ± 11.97, respectively.
T test results showed no signi cant difference in age distribution between the two groups (P = 0.962). The clinical indicators including fasting blood glucose, glycated hemoglobin, total cholesterol, triglyceride, urea, creatinine and GFR were signi cantly different in cases compared with the controls (P < 0.05). However, no signi cant difference was observed between the two groups concerning LDL, HDL and Cystatin C. The detailed information and allelic frequencies of the CYP2C8 and CYP2D6 genes SNPs in case and control groups are shown in Table 2. Chi-square test results showed that the genotype frequencies of the ve CYP2C8 and CYP2D6 SNPs in control group conformed to HWE (P > 0.05). As presented, there is no statistically signi cant difference in the allelic frequencies of the CYP2C8 gene SNPs between the T2DM patients and healthy controls (P > 0.05). However, the rs1065852 in CYP2D6 was found to be related with an increased the risk of T2DM (A vs. G: OR = 1.22, 95% CI: 1.03-1.45, P = 0.024).   No relationship between genotypes of the rs1065852 and clinical characteristics (fasting blood glucose, glycated hemoglobin, total cholesterol, HDL, LDL, triglyceride, urea, creatinine, Cystatin C and GFR) ( Table 5).

Discussion
In this study, we recruited 512 T2DM patients and 515 healthy controls to investigate the relationship between polymorphisms of CYP2C8 and CYP2D6 and the risk of T2DM. The analysis results demonstrated that the rs1065852 in CYP2D6 was related with an increased the risk of T2DM in overall, male and age < 59 years old subgroup. However, no association was found between the four SNPs in CYP2C8 and T2DM risk in the Chinese Han population.
CYP2C8 mapped in chromosome 10q23 encodes a signi cant enzyme in metabolism of numerous drugs and chemicals [11]. No association between the four SNPs in CYP2C8 (rs1934953, rs1934951, rs2275620, and rs17110453) and T2DM risk was found in this study. However, studies on the correlation of these loci with other diseases have been reported.
Previous study has reported no signi cant correlation between rs1934953 in CYP2C8 and risk of coronary heart disease in Russian population [19]. However, the SNP rs1934953 showed signi cantly association with essential hypertension risk in Russians [20]. A genome-wide association study previously identi ed that rs1934951 and rs17110453 in CYP2C8 were risk factors for the development of osteonecrosis of the jaw (ONJ) in multiple myeloma patients under bisphosphonate therapy [25]. English et.al [26] found that rs1934951 was not associated with the development of bisphosphonate-related ONJ in men with castrate-resistant prostate cancer. Esperanza et.al [21] veri ed the frequency of SNP rs193451 in 79 patients with multiple myeloma and found that there was no signi cant correlation between this polymorphism and the risk of ONJ. A meta-analysis indicated that AA and AG genotypes of rs1934951 might be predictors for multiple myeloma patients at high risk to develop bisphosphonate-related ONJ [27]. The two-locus interaction (rs17110453 and rs751141) [14] and three-locus interaction (rs17110453, rs751141, and rs9333025) [28] confer signi cantly higher risk for ischemic stroke. Therefore, further well-designed studies are needed to con rm this nding.
CYP2D6 genetic polymorphisms because may in uence on the enzymatic activity of CYP2D6, which is responsible for the metabolism of numerous drug, including tricyclic antidepressants, antipsychotics, beta-blockers, anti-arrhythmics, antidiabetics [29]. In present study, the results rstly indicated that rs1065852 in CYP2D6 was correlated with an increased the risk of T2DM in overall, male and age < 59 years old subgroup. Several studies about the effect of rs1065852 on drug metabolism and disease susceptibility have been reported. Previous study suggested that rs1065852 was a favorable factor in escitalopram treatment for major depressive disorder [30]. The mutation rs1065852 affects the pharmacokinetics of iloperidone and its metabolites in Chinese schizophrenia patients [31]. The SNP rs1065852 was found to be associated with increased risk of lung cancer in the Northwestern Chinese Han population [24]. Our results suggest that rs1065852 may be a good genetic marker for susceptibility to T2DM.

Conclusion
In conclusion, this is the rst study performed to investigate the relationship between polymorphisms in CYP2C8 and CYP2D6 and T2DM risk in the Chinese Han population. Our data have demonstrated that rs1065852 in CYP2D6 could be potential genetic markers of susceptibility to T2DM. Further studies are required to con rm our ndings and better understand the pathogenesis of T2DM.

Availability of data and materials
The data used to support the ndings of this study are available from the corresponding author upon request.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
Qingbin Zhao contributed to the study design and drafted the manuscript. Huiyi Wei contributed to the information collection and critically revised the manuscript. Zhiying Li and Haoyang Wei contributed to the data analysis and interpretation. All authors gave nal approval and are accountable for all aspects of the work and its integrity and accuracy.