Transcription Factor 7-like 2 (TCF7L2) rs7903146 Polymorphism, Association with Type 2 Diabetes Mellitus Susceptibility

Objectives: The aim of this study was to investigate the possible role of TCF7L2 rs 7903146 (C/T) variant on susceptibility of T2DM among Egyptian children and adolescents.

Patients and methods: 30 T2DM pediatric patients, 20 obese children and 20 control subjects were enrolled in the study and subjected to: anthropometric measurements, routine laboratory studies including lipid profile, fasting serum insulin level and homeostatic model assessment of insulin secretion and β cell function. The rs7903146 (C/T) polymorphism was genotyped using the PCR-RFLP method.

Results: T allele of TCF7L2 rs7 903146 (C/T) was associated with T2DM in the study (P<0.001; OR=5.96, 95% CI: (2.58-16.22). Haplotype analyses showed a higher distribution of haplotype TT in the T2DM patients than the control group [56.7% vs. 15.0%, P<0.002; χ²=11.66]. Association of TCF7L2 rs7903146 and clinical and metabolic measures in T2D patients revealed significantly lower levels of fasting insulin and Homa β among carriers of T allele. Also, no significant interaction was found between T2D risk and BMI (SDS) regarding rs7903146 SNP. Conclusion: Our data prove that rs7903146 (C/T) variant of the TCF7L2 gene is associated with T2DM in our study.

TCF7L2; Gene; Polymorphism; T2DM

Type 2 diabetes is associated with impaired insulin secretion. Both 1^st and 2^nd phase insulin secretions are reduced, but the effect is particularly pronounced for the 1st phase. Although both genetic and environmental factors are thought to play a role, the processes culminating in impaired insulin secretion are not completely understood, but both genetic and environmental factors are thought to play a role. Over the past 2 years, genome-wide association scans have transformed the genetic landscape of type 2 diabetes susceptibility [1]. TCF7L2, the susceptibility gene with the strongest effect on disease susceptibility discovered to date, was conferred pre genome-wide association by Grant et al. in 2006, with rapid replication of its consequence on diabetes susceptibility in multiple populations [2]. TCF7L2 was a positional candidate gene that mapped to a region spanning 215.9 Kb on human chromosome 10q25 with replicated linkage to T2DM.

The transcription factor 7-like 2 (TCF7L2) gene is a member of the T-cell factor (TCF)/lymphoid enhancing factor family of high mobility group (HMG) box-containing transcription factors that play a key role in the Wnt signaling pathway [3]. Wnt signaling is initiated by the binding of Wnts to their receptor complex, which results in the release of catenin from its degradation complex and translocation to the nucleus. In the nucleus, catenin heterodimerizes with the TCF/lymphoid-enhancing factor family of transcription factors to regulate the expression of Wnt/beta-Catenin signaling pathway which has been implicated in blood glucose homeostasis through the regulation of pro-glucagon gene expression [4]. There is also a provided indication that active Wnt signaling stimulates the multiplication of beta cells, thus offering physiological evidence of an important role of TCF7L2 in maintaining beta cell's mass and /or function [5]. Grant and colleagues have reported the association of (DG10S478); a common microsatellite with intronic region of TCF7L2 and T2DM [6]. There are at least four well-studied single nucleotide polymorphism (SNP) markers in the humanTCF7L2 gene, which are associated with T2DM, viz., rs7903146, rs7901695, rs12255372 and rs11196205. Previous studies have shown an association between T2DM and rs7903146 polymorphism of (TCF7L2) gene [6].

The aim of this study is to investigate the possible association of TCF7L2 rs7903146 (C/T) variant on susceptibility of T2DM.

Study subjects

Anthropometric assessment

Genotyping of TCF7L2rs7903146 polymorphism

Statistical analyses

This study was conducted on 30, T2DM patients, 20 obese children. 20 age and sex matched subjects were taken as control. Their mean ages were 12.69 ± 2.22, 12.95 ± 1.87 and 12.75 ± 2.12 respectively.

The results of our study were illustrated in the following tables and figure:

Table 1 shows the clinical and biochemical characteristics of diabetic, obese and control groups: Obese group has significantly higher BMI (SDS), waist circumference and serum cholesterol than both diabetic and control groups. Diabetic group has significantly higher fasting plasma glucose, glucose 120min, glycated haemoglobin, serum triglycerides and HDL cholesterol than both obese and control groups. Obese group has significantly higher fasting serum insulin and LDL cholesterol than diabetic group. Obese and diabetic groups have significantly higher LDL cholesterol than the control group.

Table 2 demonstrates frequency distribution of rs7903146 (IVS3C/T) polymorphism with T2D. There was a significant difference between T2D cases and obese in risk allele distribution; higher frequency of TT haplotype of 56.7 % with OR=11.49, 95% CI: (2.37-55.96) and P value=0.003, vs. 23.3 % frequency of the CT haplotype, OR=4.12 and 95%CI: 4.12(0.67-16.14). The T allele of rs7903146 variant is associated with T2DM risk (OR=5.96, 95 % CI: (2.58-16.22) and P value<0.001 in comparison to C-allele (reference allele) in obese group.

Table 3 shows association of TCF7L2 rs7903146 genotype and clinical and biochemical parameters in T2D patients and controls. As compared to carriers of C- allele (individuals with CC genotype), carriers of T-allele (those with CT,TT genotype) had higher FBG and 2hr post load glucose levels, in addition to significantly lower levels of FI and HOMA-β . P-value=0.001.

Table 4 shows comparison of TCF7 L2 rs7903146 polymorphism in the study groups according to BMI categorization.

Regarding interaction of rs7903146 variant of TCF7L2 in all studied participants according to BMI categorization, no interaction was found between T2D risk and obesity or high BMI status regarding rs7903146 SNP of TCF7L2 gene as shown in Table 4, where obese individuals had TT haplotype frequency of 20.0 % with OR=1.32, 95% CI: (0.95-1.82), χ ²=2.69 and p value=0.261. On the other hand, obese individuals showed higher significant association with C- allele (frequency of 67.5 %, χ ²=3.94 and P=0.047), in comparison to lean (non over weight) individuals in our study.

Figure 1 shows distribution of the studied groups regarding genotype.

Figure 2 shows distribution of the studied obese and average weight groups regarding genotype.

Figure 3 shows genotyping of RsaI digest product of a PCR- amplified 205 bp fragment of TCF7L2 gene.

The first lane (M) indicates100 bps ladder. The presence of rs7903146 restriction site was shown by the presence of bands at 151,53 bps and was assigned as homozygous TT genotype as in lane (4,5). Homozygous CC genotype was demonstrated by the presence of band at 205 bps as in lane (1,6), while the presence of corresponding bands at 205, 151, 53 bps indicate heterozygous CT genotype; lane (2,3,7).

Despite serious efforts to identify genetic variants that predispose to common forms of type 2 diabetes, till now only a few genes, such as KCNJ11, PPARG and HNF4A have been reproducibly associated with this complex disease in a variety of large-scale studies performed in the different populations [16,17]. Replication studies show that the TCF7L2 gene is an important candidate gene contributing to susceptibility to type 2 diabetes. Since the most strongly associated SNP is within an intron of the TCF7L2 gene, this SNP is not likely to be the true disease variant. However, the intronic variants may influence the risk of T2D through altering gene expression in human islets or possibly transcription regulation. TCF7L2 SNP rs7903146 is the strongest variant polymorphism associated with T2DM [18,19] and has been confirmed in numerous populations throughout the world [18,20,21]. In this study, we aimed to investigate the association between the TCF7L2 rs7903146 (C/T) variant and T2DM among study groups. Our case-control study, along with the previous reports from different region of the world prove that the rs7903146 (C/T) variant of the TCF7L2 gene is associated with T2DM. The T allele of TCF7L2 rs7903146 (C/T) was observed to be significantly associated with T2D risk in our studied diabetic patients (p<0.001, OR=6.74, 95% CI: (2.63-15.91).

This finding is consistent with the previous reports from other populations, except for Arab population of Persian Gulf [22,23]. This reflects the role of other genetic factors that influence the disease risk among these populations [22].

T allele frequency for rs7903146 was 32.5% in the control subjects and 68.5% in T2DM subjects of this study. The frequency of the minor T allele in the control subjects was consistent with that of rs7903146 T in the populations of Icelandic, Palestinian, Danish, Asian-Indian, Dutch, (UK-resident South Asians and Emirati populations (30.4, 29.3, 29, 26.1, 28, 29 and 37.2%, respectively) [18,21,23-25] but strikingly different from that reported in the Japanese (4.2%) and Chinese (~2%) populations [6,26].The frequency of the minor T allele in the diabetic subjects was consistent with that of rs7903146 T among broad ethnic backgrounds, including for example populations of UK [27], Dutch [25], Amish [28], Finnish [29], Swedish [30], French [31], and US [31,32], Indian [33], and Japanese [26] origin but strikingly different from that reported in Pima Indians and Chinese diabetics [6,34]. The variability in the allele frequency, which probably reflects different ethnic backgrounds, would partly account for the discrepancies found in the results among different studies. Other factors include small sample size, age of subjects [26] and effects of environmental factors, such as life-style.

Haplotype analysis involving rs7903146 (IVS3C/T) of TCF7L2 in our study participants revealed a significant association of the T-allele and T2DM, compared to obese (χ ²=16.07 with p value<0.001). Obese group showed no significant p-value for TT genotype.

Reinehr et al. 2004 found that reduction of overweight improves glucose metabolism in overweight children [35]. However, not all children reducing overweight demonstrated an improvement of glucose metabolism suggesting further influencing factors such as genetic markers [36]. The SNP rs7903146 in TCF7L2 is associated with an increased risk of type 2 diabetes mellitus [2,34,37-39]. The TT haplotype increases the risk of T2D in diabetic group. As well, we analyzed the association of metabolic measurements and genotype of the locus. For TCF7L2 rs7903146, mean FBG significantly increased with the number of T-allelels; CC 103.0 mg/dl ,C/T 108.41, TT 117.67 (p value=0.001). In this study, our results showed that over expression HOMA β significantly and inversely associated with T2D risk. Functional studies demonstrated that TCF7L2 specifically risk allele (T) of rs7903146 polymorphism was associated with impaired pancreatic β cell function through regulation of insulin production and secretion pathways [40]. However, the exact molecular genetic mechanisms need yet to be determined. The rs7903146 variant showed an association with T2D, but no statistically significant interactions was observed between the genotype and BMI, HDL, LDL or TG levels. Studies have reported modulating factors on the effect of TCF7L2 variant such as lipid profile, nutrition and obesity status [41]. Similarly, Hansson et al. found that allele- specific over expression of TCF7L2 in human pancreatic islets that may cause impaired insulin secretion was not altered by either insulin sensitivity or BMI [42].

Interestingly, the TCF7L2 rs7903146 variant has been associated with decreased BMI and this variant polymorphism is thought to exert a greater effect on T2D risk in lean individuals compared to obese as speculated by Van Vlit-Ostaptchouk [25]. Also, Hegalson et al. [24] reported that the rs7903146 T allele, probably an ancestral and not causative variant, tags an unidentified functional variant lying outside the studied locus. It is challenging to study gene-environmental interaction effect on β cell function and genetic variant risk for T2D. Moreover, our group will conduct knowledge to confirm the association of this SNP with T2D in Egypt.

We have reported the association of rs7903146 variant of the TCF7L2 gene with type2 diabetes susceptibility in our study group. Also, HOMA-β implies functional association with impaired β-cell secretory function. More insights into molecular mechanism of action of the TCF7L2 gene are recommended to clarify the pathogenesis of type 2 diabetes for prevention of the disease with attempts that could offer an early intervention health care program among at-risk children and adolescents.