Association Between the g.14461A>G Genetic Polymorphism of the TLR4 Gene and Type 2 Diabetes Mellitus Risk in a Chinese Population

Abstract

Objective:

Toll-like receptor 4 (TLR4) is an important candidate gene for mediating the susceptibility to type 2 diabetes mellitus (T2DM). The purpose of this study was to investigate the association between the TLR4 gene polymorphisms and T2DM susceptibility.

Methods:

A total of 671 T2DM patients and 677 healthy controls were recruited in this study. The created restriction site-polymerase chain reaction and DNA sequencing methods have been used to analyze the TLR4 gene polymorphisms.

Results:

One novel genetic polymorphism (g.14461A>G) was found. Our data indicated that the g.14461A>G genetic polymorphism was significantly associated with the increased susceptibility to T2DM in a homozygote comparison (GG vs. AA: odds ratio [OR]=2.09, 95% confidence interval [CI] 1.44-3.04, p<0.001), dominant model (GG/AG vs. AA: OR=1.27, 95% CI 1.03-1.57, p=0.028), recessive model (GG vs. AG/AA: OR=1.98, 95% CI 1.39-2.83, p<0.001), and allele contrast (G vs. A: OR=1.33, 95% CI 1.13-1.57, p=0.001). The allele-G might be the risk allele for enhancing the susceptibility to T2DM.

Conclusion:

These preliminary findings suggest that the g.14461A>G genetic polymorphism of the TLR4 gene is potentially related to the susceptibility to T2DM in the studied population.

Introduction

Type 2 diabetes mellitus (T2DM) is a chronic and complex disease and represents a significant global health problem. The prevalence of T2DM is increasing rapidly worldwide. A large number of epidemical studies have reported that China has a very high T2DM incidence, and diabetes has become a major public health problem in China, with ∼92.4 million adults with the disease and 148.2 million with prediabetes (Yang et al., 2010; Jiang et al., 2012; Cai et al., 2013; Fu et al., 2013). Previous studies revealed that the human toll-like receptor 4 (TLR4) is one of the most important genes for influencing the risk of T2DM (Illig et al., 2003; Rudofsky et al., 2004; Creely et al., 2007; Kim et al., 2008; Buraczynska et al., 2009; Arora et al., 2011; Maldonado-Bernal et al., 2011; Jiang et al., 2012; Cai et al., 2013; Fu et al., 2013). TLR4 is a polygenetic gene, and many genetic variants have been reported and identified that influence the risk of T2DM (Raby et al., 2002; Kolz et al., 2008; Arora et al., 2011; Jiang et al., 2012; Cai et al., 2013; Fu et al., 2013), such as aspartic acid (Asp)299 glycine (Gly) (Rudofsky et al., 2004; Kim et al., 2008; Buraczynska et al., 2009; Arora et al., 2011; Maldonado-Bernal et al., 2011; Jiang et al., 2012) and threonine (Thr)399 isoleucine (Ile) (Rudofsky et al., 2004; Kim et al., 2008; Kolz et al., 2008; Buraczynska et al., 2009; Maldonado-Bernal et al., 2011; Jiang et al., 2012). Several studies indicated that these genetic variants of the TLR4 gene were statistically significantly associated with the risk of T2DM (Rudofsky et al., 2004; Kim et al., 2008; Kolz et al., 2008; Buraczynska et al., 2009; Arora et al., 2011; Maldonado-Bernal et al., 2011; Jiang et al., 2012; Cai et al., 2013; Fu et al., 2013). However, up to date, the association between the g.14461A>G genetic variant of the TLR4 gene and T2DM risk has not been analyzed. Considering the importance of TLR4 genetic variants for T2DM risk, the purpose of this study was to evaluate the potential influence of the g.14461A>G genetic polymorphism of the TLR4 gene on T2DM risk.

Materials and Methods

Study population

This study consisted of 671 T2DM patients and 677 healthy controls. All subjects were consecutively recruited from January 2009 and December 2012 in the Tongji Hospital (Tongji University School of Medicine, Shanghai, China). The cases were diagnosed by doctors according to the 2003 American Diabetes Association criteria (Genuth et al., 2003). The controls were matched with the cases in terms of age, sex, and residence area. All individuals were of unrelated Chinese Han ethnic. According to the previous studies (Jing et al., 2011; Fu et al., 2013), demographic and clinical characteristics of subjects enrolled in this study are summarized in Table 1, including the gender, age, body mass index, diabetes duration, diastolic blood pressure, low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol, systolic blood pressure, fasting plasma glucose, triglycerides, and total cholesterol. The protocol of this study was approved by the ethics committee of the Tongji Hospital, and a written informed consent form was obtained from each subject.

Table 1.

The Demographic and General Characteristics of Type 2 Diabetes Mellitus Cases and Healthy Controls

Characteristics	T2DM cases (n=671)	Healthy controls (n=677)	p-Value
Gender (male/female)	360/311	392/285	0.1161
Age (years)	54.67±11.18	55.46±9.72	0.4521
BMI (kg/m²)	27.6±4.5	24.62±3.3	<0.001
Diabetes duration (years)	8.8±6.5	NA	ND
SBP (mmHg)	125.3±15.7	1143±11.6	<0.001
DBP (mmHg)	78.9±8.4	75.6±6.1	<0.001
FPG (mM)	9.47±2.35	5.27±0.39	<0.001
TC (mM)	4.85±1.46	4.33±1.27	<0.001
TG (mM)	1.97±1.35	1.39±0.36	<0.001
HDLC (mM)	1.10±0.11	1.39±0.19	<0.001
LDLC (mM)	2.96±0.22	2.06±0.42	<0.001

Data are presented as mean±SD or numbers (%).

T2DM, type 2 diabetes mellitus; NA, not applicable; ND, not determined; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; FPG, fasting plasma glucose; TG, triglycerides; TC, total cholesterol; LDLC, low-density lipoprotein-cholesterol; HDLC, high-density lipoprotein-cholesterol.

Polymerase chain reaction amplification

Peripheral blood was collected from each enrolled subject. Genomic DNA was isolated using the standard extraction method and then stored at −80°C (Daly et al., 1996). The specific polymerase chain reaction (PCR) primers were designed by the Primer Premier 5.0 software. The primer sequences, annealing temperature, and fragment region and sizes are given in Table 2. The PCR was performed in a total volume of 20 μL solution, including 50 ng template DNA, 1× buffer (Tris-HCl 100 mM, pH 8.3; KCl 500 mM), 0.25 μM primers, 2.0 mM MgCl₂, 0.25 mM dNTPs, and 0.5U Taq DNA polymerase (Promega, Madison, WI). The PCR amplification conditions were as follows: an initial denaturation at 94°C for 5 min, followed by 32 cycles at 94°C for 30 s, 57.0°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 8 min.

Table 2.

Primers and CRS-PCR Analysis for g.14461A>G Genetic Polymorphism of the TLR4 Gene

Primer sequences	Annealing temperature (°C)	Amplification fragment (bp)	Region	Restriction enzyme	Genotype (bp)
5′-ATCACTCAGAAACCTCATTTACCT-3′	57.0	205	Exon3	Tsp4CI	AA: 184,21
5′-AGACAACTGCTCCAGTTGACA G -3′					AG: 205,184,21
					GG: 205

Underlined nucleotides mark nucleotide mismatches enabling the use of the selected restriction enzymes for discriminating sequence variations.

CRS-PCR, created restriction site-polymerase chain reaction; TLR4, toll-like receptor 4.

Genotyping

The genotyping for the g.14461A>G genetic polymorphism of the TLR4 gene was analyzed by the created restriction site-PCR (CRS-PCR) method with one of the primers containing a nucleotide mismatch, which enables the use of restriction enzymes for discriminating sequence variations (Haliassos et al., 1989; Zhao et al., 2003; Yuan et al., 2012; Yuan et al., 2013a, 2013b). According to the manufacturer protocol, the PCR-amplified products were digested with 5 units of Tsp4CI restriction enzymes (MBI Fermentas, St. Leon-Rot, Germany; Table 2) at 37°C for 10 h. The digested products were separated by agarose gel electrophoresis containing 0.5 μg/mL ethidium bromide and visualized under UV light. To confirm the concordance of the CRS-PCR method, about 10% of the random samples were verified by DNA sequencing analysis (ABI3730xl DNA Analyzer; Applied Biosystems, Foster City, CA).

Statistical analysis

The Hardy-Weinberg equilibrium in controls and the differences of demographic and general characteristics between T2DM patients and controls were evaluated by the chi-square (χ²) test. The odds ratios (ORs) with their 95% confidence intervals (CIs) from multivariate logistic regression models were used to evaluate the potential associations between allelic and genotypic frequencies of the TLR4 gene and the risk of T2DM. Statistical analyses were analyzed by the Statistical Package for Social Sciences software (SPSS, Windows version, release 15.0; SPSS, Inc., Chicago, IL). p-Values less than 0.05 were defined as statistically significant.

Results

Demographic and clinical characteristics of subjects

In this case-control study, a total of 1348 subjects were enrolled, including 671 T2DM cases and 677 healthy controls. Table 1 shows the demographic and clinical characteristics of studied subjects. There was no significant difference in gender and age between the T2DM patients and controls (p=0.1161 and p=0.4521, respectively). The clinical characteristics and anthropometric data were significantly different between the T2DM patients and controls (all ps<0.001; Table 1).

Genotyping of TLR4 genetic polymorphism

In this study, the g.14461A>G genetic polymorphism of the TLR4 gene was established through CRS-PCR and confirmed by DNA sequencing methods. Based on the sequence analyses, we detected that the g.14461A>G genetic variant was an A to G mutation in exon3 of the TLR4 gene and that this caused a glutanine (Gln) to arginine (Arg) amino acid replacement (p.Gln505Arg, GenBank IDs: NG_011475.1, NM_138554.4, and NP_612564.1). The PCR-amplified products were digested with Tsp4CI restriction enzyme and divided into three different genotypes: AA (184 and 21 bp), AG (205,184 and 21 bp), and GG (205 bp, Table 2). The genotype and allele frequencies in both T2DM patients and healthy subjects are summarized in Table 3. The frequencies of allele-A and genotype-AA were predominant in the T2DM patients and controls. The frequencies of alleles in T2DM patients (A, 65.05%; G, 34.95%) were significantly different from those in controls (A, 71.27%; G, 28.73%, χ²=12.0110, p=0.0005). The frequencies of genotypes in T2DM patients (AA, 44.26%; AG, 41.58%, GG, 14.16%) were not consistent with those controls (AA, 50.22%; AG, 42.10%, GG, 7.68%, χ²=15.5183, p=0.0004, Table 3). The genetic variants genotyped were in Hardy-Weinberg equilibrium (for T2DM patients, χ²=4.9086, p=0.0859; for controls, χ²=0.5302, p=0.7671).

Table 3.

The Genotype and Allele Frequencies of g.14461A>G Genetic Polymorphism of TLR4 Gene

	Genotype frequencies		Allele frequencies
Groups	AA	AG	GG	A	G	χ²-Value	p-Value
Cases (n=671)	297 (44.26)	279 (41.58)	95 (14.16)	873 (65.05)	469 (34.95)	4.9086	0.0859
Controls (n=677)	340 (50.22)	285 (42.10)	52 (7.68)	965 (71.27)	389 (28.73)	0.5302	0.7671
Total (n=1348)	637 (47.25)	564 (41.84)	147 (10.91)	1838 (68.18)	858 (31.82)	1.7279	0.4215
	χ²=15.5183, p=0.0004			χ²=12.0110, p=0.0005

Association between TLR4 genetic variants and T2DM risk

The association between the risk of T2DM and g.14461A>G genetic polymorphism of TLR4 gene is summarized in Table 4. We detected that the alleles and genotypes from this genetic variant were statistically associated with the risk of T2DM. The g.14461A>G genetic variant had significantly increased susceptibility to T2DM in the homozygote comparison (GG vs. AA: OR=2.09, 95% CI 1.44-3.04, χ²=15.46, p<0.001), dominant model (GG/AG vs. AA: OR=1.27, 95% CI 1.03-1.57, χ²=4.80, p=0.028), recessive model (GG vs. AG/AA: OR=1.98, 95% CI 1.39-2.83, χ²=14.54, p<0.001), and allele contrast (G vs. A: OR=1.33, 95% CI 1.13-1.57, χ²=12.01, p=0.001, Table 4). There was no significant difference in susceptibility to T2DM in heterozygote comparison (AG vs. AA: OR=1.12, 95% CI 0.89-1.41, χ²=0.97, p=0.325, Table 4).

Table 4.

The Association Between the Risk of Type 2 Diabetes Mellitus and g.14461A>G Genetic Polymorphism of TLR4 Gene

	Test of association
Comparisons	OR (95% CI)	χ²-Value	p-Value
Homozygote comparison (GG vs. AA)	2.09 (1.44-3.04)	15.46	<0.001
Heterozygote comparison (AG vs. AA)	1.12 (0.89-1.41)	0.97	0.325
Dominant model (GG/AG vs. AA)	1.27 (1.03-1.57)	4.80	0.028
Recessive model (GG vs. AG/AA)	1.98 (1.39-2.83)	14.54	<0.001
Allele contrast (G vs. A)	1.33 (1.13-1.57)	12.01	0.001

CI, confidence interval; OR, odds ratio.

Discussion

Many studies demonstrated that the T2DM is a polygenic disease that results from complex interactions between multiple factors, including environmental factors, genetic factors, and so on (Permutt et al., 2005; Freeman and Cox, 2006; Liu et al., 2011; Al-Daghri et al., 2012; Gan and Yang, 2012; Gonul et al., 2012; Cai et al., 2013). It is well known that the genetic factors play key roles for the development of T2DM. However, the exact mechanism remains poorly understood. It has been suggested that TLR4 is an important candidate gene for T2DM susceptibility, and TLR4 genetic variants have been reported to be significantly associated with the susceptibility to T2DM (Illig et al., 2003; Rudofsky et al., 2004; Creely et al., 2007; Kim et al., 2008; Buraczynska et al., 2009; Arora et al., 2011; Maldonado-Bernal et al., 2011; Jiang et al., 2012; Cai et al., 2013; Fu et al., 2013). However, the findings from these observations are inconclusive. In this case-control study, we investigated the role of the g.14461A>G genetic polymorphism of TLR4 gene in the susceptibility to T2DM in the Chinese Han population by association analyses. Significant differences were detected in the distribution of allele and genotype between the T2DM patients and controls (p<0.01, Table 3). Our data indicate that the g.14461A>G genetic polymorphism of the TLR4 gene might increase the susceptibility to T2DM in the Chinese Han population. The allele-G and genotype-GG could contribute to the occurrence of T2DM.

In conclusion, to the best of our knowledge, this is the first study for investigating the role of the g.14461A>G genetic polymorphism of the TLR4 gene on the susceptibility to T2DM. Our findings support that the g.14461A>G genetic polymorphism of the TLR4 gene has a potential influence on the susceptibility to T2DM. These results may provide support for using this genetic variant as a therapeutic target and a prognostic molecular marker to evaluate the susceptibility to T2DM. Epidemiological studies with a larger number of populations in different ethnicities would be necessary to confirm our findings.

Footnotes

Author Disclosure Statement

No conflicts of interest exist.

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