Polymorphism of Transcription Factor 7-Like 2 in Obese Patients with Type 2 Diabetes Mellitus

Objective: The article is to investigate the relationship between the polymorphism of transcription factor 7-like 2 (TCF7L2) gene rs7903146 and type 2 diabetes mellitus with obesity in Chinese Han population. Methods: 216 patients with type 2 diabetes mellitus (T2DM), 92 males, 124 females, 194 normal controls, 89 males and 105 females. The polymorphism of TCF7L2 gene rs7903146 was measured by PCR-RFLP. PCR amplifier was used for amplification reaction, followed by polymorphism analysis of TCF7L2 gene: the reaction system was 20 μl, among which restriction endonuclease was 0.5 μl and PCR product was 7.5 μl. Restriction enzyme SspI (Fermentas) was used for PCR product and was bathed at 37˚C for 16 h. The enzyme digestion products were detected and genotypes were identified by 2% agarose gel electrophoresis. The genotyping was repeated in 10% of the samples, and the genotyping rate was 100%. Height and weight were measured and BMI calculated. According to BMI, the experimental group was further divided into the obese group (n = 137, BMI > 25 kg/m 2 ) and the normal body mass group (n = 79, BMI < 25 kgm 2 ). Normal control group: BMI < 25 kgm 2 : FBG < 6.1 mmol/L tween the T2DM group and the control group (χ 2 = 78.154, P = 0.006) and the T2DM obesity group and the control group (χ 2 = 7.247, P = 0.009), but there was no significant difference between the T2DM normal body mass group and the control group (P > 0.05). In addition, there was no significant difference between TC type + TT type and CC type between the T2DM group and the control group and the T2DM obesity group and the control group (P > 0.05). Conclusion: There were significant differences in the genotype frequency and recessive inheritance pattern (TT/CC + TC) of rs7903146, suggesting that this SNP may be associated with the incidence of T2DM in obesity and insulin resistance.


Introduction
China is the second largest number of diabetics in the world with nearly 30 million now [1]. Previous study shows rs7903146 of transcription factor 7-like 2 (TCF7L2) is closely related to the occurrence of type 2 diabetes mellitus (T2DM) in different populations and nationalities, which plays a very important role [2]. Other studies have shown that this gene was associated with increasing body mass index (BMI), increasing LDL cholesterol (LDLC), decreasing triglycerides (RG), decreasing insulin secretion and obesity [3]. In this study, PCR-RFLP was used to investigate the relationship between rs7903146 polymorphism and type 2 diabetes with obesity in Chinese Han population [4].

Research Objects
There were 216 cases of Type 2 diabetes in Han nationality, including 92 males and 124 females. There was no blood relationship among the patients and the diagnostic criteria of diabetes were in accordance with the standards stipulated by WHO in 1999. There were 194 healthy subjects in the normal control group, including 89 males and 105 females. There were Han ethnic groups with no history of diabetes, which were impaired glucose tolerance or family history of diabetes and excluded diabetes and impaired glucose tolerance.

Polymorphism Determination rs7903146 of TCF7L2 Gene: PCR-RFLP
Each restriction enzyme genotype was determined by sequencing. 1) PCR primers are shown in Table 1 for 25 s, and after 32 cycles, it was extended at the end at 72˚C for 5 min. PCR amplification was performed with a PCR amplification instrument (German company Eppendorf), and PCR amplification products were detected by 1% agarose gel electrophoresis. 4) Polygenicity analysis of TCF7L2 gene: the reaction system was set as 20 L, and the restriction enzyme was 0.5 L and PCR product was 7.5 L. PCR products were incubated with restriction enzyme SspI (Fermentas) for 16 h at 37˚C. Enzyme digestion products were detected and genotypes were identified by 2% agarose gel electrophoresis. PCR products and enzyme digestion products after gel electrophoresis were analyzed by gel imager from Amersham Biosciences, USA. In addition, genotyping was repeated in 10% of the samples, with a 100% incidence. They then measured height, weight and calculated BMI.

Statistical Methods
SPSS18.0 Software Package was used for Statistical Analysis. The frequency of alleles and genes in each group was in accordance with Hardy-Weinberg equilibrium. The measurement data were expressed as M±SD, and t-test was performed. Comparison of gene and allele frequency distribution was performed by χ 2 test. Logistic regression was used to analyze the correlation between genes and alleles and obesity in T2DM patients and P < 0.05 was considered statistically significant.

The Clinical Data of FBG
The clinical data of FBG, 2 h postpranational BG, FIN and LgHOMA-IR were significantly different between the control group and the T2DM groups (P < 0.01), while the other parameters showed no statistical difference (P > 0.05).

TCF7L2 Allele rs7903146 and Its Genotype Frequency Distribution
Allele frequency and genotype frequency accord with Hardy-Weinberg equilibrium. The results showed that there was a significant difference in genotype frequency between the experimental group and the control group (χ 2 = 17.382, P < 0.001), while there was no significant difference in allele frequency distribution between the control group and the T2DM group (P > 0.05). The genotype frequency of T2DM patients was significantly different between the control group and the obesity group (χ 2 = 20.427, P = 0.001). However, there was a significant difference in allele frequency distribution between the two groups (P > 0.05) ( Table 2).

Group Comparison of TT Type and TC + CC Type
There was significant difference between T2DM normal body quality group and control group (χ 2 = 78.154, P = 0.006) and T2DM obesity group and control group (χ 2 = 7.247, P = 0.009), but there was no significant difference between T2DM normal body quality group and control group (P > 0.05). In addition, the group comparison of TT type and TC + CC type showed that there was no significant difference between T2DM and the control group and T2DM obesity group and control group (P > 0.05, Table 2), but allele T was associated with

Discussion
In recent years, there has been a global explosion of molecular genetics research aiming at understanding the TCF7L2 polymorphism that predicts the incidence and impact of T2DM in individuals. In this study, the correlation between the onset of type 2 diabetes mellitus and this gene was studied by using case-control method to observe the effect of this gene on genetic factors.  [11] showed that the T allele frequency, CT and TT genotype frequency of RS7903146 were significantly lower in the normal control group than in the T2DM group.
Generally, diabetes is caused by insulin resistance and insufficient insulin secretion [12]. Non-obese patients with T2DM most caused by a lack of insulin secretion and most obese T2DM patients with insulin resistance. This research was further divided into normal body mass group (BMl < 25 kg/m 2 ) and obese group (BMI > 25 kgm 2 ) of the group of T2DM. BMI is a clinical index of T2DM population stratification. This study showed that the experimental index based on BMI level was consistent with the clinical classification. TCF7L2 gene polymorphism locus rs7903146 recessive genetic pattern (TT/CC+TC) and its genotype frequency were significantly different suggesting that this gene polymorphism locus may be related to the incidence of T2DM in obesity and insulin resistance. However, the area of introns rs7903146 polymorphism loci located in gene, which is directly involved in the regulation of gene function and expression change. There maybe just a real pathogenic polymorphisms of polymorphism loci in linkage disequilibrium logo and need further studies to prove gene function.