Are single nucleotide polymorphisms rs7903146 and rs12255372 in transcription factor 7-like 2 gene associated with an increased risk for gestational diabetes mellitus in Egyptian women?

Background Genetic variants in the transcription factor 7-like 2 (TCF7L2) gene are related with type 2 diabetes (T2D) and gestational diabetes mellitus (GDM) in various populations, but there are not enough statistics regarding GDM among Egyptian women. We aimed by this study to evaluate the effect of two polymorphisms of rs7903146 and rs12255372 in the TCF7L2 gene with the development of GDM among Egyptian women. Results We enrolled 114 pregnant women with normal glucose tolerance and 114 with GDM according to the International Association of Diabetes and Pregnancy Study Groups (IADPSG) guidelines. We gathered records on blood pressure, body mass index (BMI), blood glucose level, hemoglobin A1C (HbA1c), and lipid profile. The genotyping of rs7903146 and rs12255372 polymorphisms was carried out using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). The statistical significance of prepregnancy BMI, fasting blood sugar (FBS), HbA1c, low-density lipoprotein (LDL), and total cholesterol (Tch) was higher, P < 0.001, in GDM women in comparison to pregnant women without GDM. CT and TT genotypes in rs7903146 SNP were 46.5% vs. 54%, P <0.04, OR; CI = 1.9 (1.0 to 3.78); TT carriers were 37.7% vs. 9.6%, P <0.001, OR (CI) = 8.9 (3.7–21.1), respectively. For the TCFL2 gene rs12255372 SNP, GT carriers were 48.2% vs. 39.5%, P= 0.004, OR (CI) = 2.3 (1.3–4.2), while TT carriers were 24.6% vs. 7.9%, P < 0.001, OR (CI) = 6 (2.5–14.3). Conclusion The study showed there is a significantly higher incidence of CT/TT genotypes in rs7903146 SNP and GT/TT genotypes in rs12255372 SNP in TCF7L2 gene among GDM women in comparison to healthy pregnant women (controls).


Background
Gestational diabetes mellitus (GDM) is a kind of diabetes that is acquired during the gestation period. It is important to distinguish it from overt diabetes. There is a continuous increase in the incidence of GDM [1]. In 2020, the Center of Disease Control (CDC) reported that the prevalence of GDM is around 10% [2]. However, incidence differs from one population to another. There are Page 2 of 8 Shalabi et al. J Genet Eng Biotechnol (2021) 19:169 only 2 existing studies regarding the prevalence of GDM among Egyptians. One took place in Upper Egypt where incidence was about 17.5% and the other in the Delta region (Menoufia governorate) with an incidence of 6%. Among the American population, the occurrence of GDM is around 7% [3,4]. In Gulf countries, the occurrence of GDM varies between 4.2 and 24.9% [5]. In 2010, many countries followed the guidelines of the International screening of Gestational Diabetes according to the Association of Diabetes and Pregnancy Study Groups (IADPSG) [6]. A couple of years later, after following IADPSG guidelines, the incidence of GDM has been assessed to be approximately 17% worldwide and in specific 10% among North Americans and 25% in Southeast Asia [7]. Due to the global widespread of obesity among young fertile women, the prevalence of GDM is in a constant rapid increase [8]. Hyperglycemic state during the gestation period is directly correlated with the increase in the number of feto-maternal co-morbidities [9]. Type 2 diabetes (T2D) is more common to evolve among pregnant women with a history of GDM as well as their offsprings are highly vulnerable to having diabetes and obesity during their grownup life [10]. Obesity and family history for diabetes both are important risk factors associated with GDM and T2D [11].
Due to the resemblance between GDM and T2D, it was documented that both might have some genetic factors in common such as single nucleotide polymorphisms (SNPs) which increase the risk for diabetes [12]. TCF7L2 is positioned at chromosome 10q25.3 and its amino acid controls hyperglycemic state in the blood [13]. The actual action of TCF7L2 in developing DM is still under debate though there are theories that DM might occur due to malfunction or decreased number in pancreatic islets [14]. It is assumed that the TCF7L2 gene controls proglucagon expression inside entero-endocrine cells [15]. TCF7L2 is important in the propagation of pancreatic B-cell as well as the production of several incretin hormones, glucose-dependent insulin-tropic peptide (GLP-1) which are released from entero-endocrine cells. GLP-1 also stimulates insulin secretion [16]. TCF7L2 regulates the metabolism of hepatic glucose mainly through inhibiting gluconeogenesis [17,18]. TCF7L2 gene has been related to T2D in various populations [16,19,20]. Intronic regions of the TCF7L2 gene have variants which are well known as strong risk genetic markers for T2D [21]. TCF7L2 polymorphisms have a dual effect on blood glucose level and insulin excretion [16]. TCF7L2 polymorphism rs7903146 has been related with GDM in Danish [22], Australian [20], Greek [23], and Swedish populations [19] as well as other populations [19,24,25]. The aim of this study is to evaluate the association of two polymorphisms of rs7903146 and rs12255372 in the TCF7L2 gene with the risk of development of GDM among Egyptian women.

Methods
A total of 228 Egyptian women were examined. Healthy pregnant women were classified as the control group (n = 114) and pregnant women with GDM as the GDM group (n = 114). Screening for GDM using oral glucose tolerance test (OGTT) was done for the participants according to the IADPSG guidelines [26]. Participants were given 75 g of glucose. Fasting and 2-h blood glucose levels were assessed after that. The diagnosis of GDM was made after which any one of the following plasma glucose values were exceeded: if the fasting plasma glucose was 92 mg/dL or more or when the 2-h value is 153 mg/dL or more. Values (fasting ≥ 126 mg/dL or 2 h postprandial ≥ 200 mg/dL) specify overt diabetes mellitus (DM), which was excluded from this study. The study was conducted from the period of 2019-2021. Participants were referred to our prenatal diagnosis and fetal medicine department. The study was done after approval was obtained from the ethical committee (Medical research ethical Committee). With approval reference number 19266, it conforms to the provisions of the Declaration of Helsinki. Informed written consent was obtained from all participants in the study. The control group was selected from low-risk pregnant women who came for regular follow-up at our clinic unit to do a 20-week ultrasound. Women who were appropriate to be in the control group in the study were called at 28 weeks to check a normal blood glucose level then accordingly were involved in the study protocol.

Inclusion criteria
All non-diabetic pregnant women, coming for antenatal care, were assessed. Fasting and 2h postprandial, HbA1c were done at 12 weeks for high-risk pregnancies for GDM (first degree relative with diabetes, history of GDM during previous pregnancies, history of delivering overweight babies > 4 kg or prepregnancy overweight; BMI > 25kg/m 2 , age 20-45 years, and fetal Doppler scan within the normal reference range) [27]. Then, reassessment for the high-risk group was taken at 24-28 weeks, and whoever tested positive for hyperglycemias was included in the case group.

Exclusion criteria
Pregnant women with diabetes history before pregnancy or overt diabetes mellitus (DM) (fasting ≥ 126 mg/dL or 2 h postprandial ≥ 200mg/dL) were excluded from the study. Women known to be hypertensive were also excluded from the study. Any woman who is a smoker or having any pathology other than GDM before or during pregnancy was excluded.

Clinical and laboratory data
Clinical and anthropometric records were collected from patient files containing fasting blood glucose, postprandial, and HA1c, and fat metabolism was evaluated (total cholesterol (TCh), low-density lipoprotein (LDL), highdensity lipoprotein cholesterol (HDL), serum triglycerides (TG)).

Genotyping
DNA was extracted from whole blood using the "salting out" method [28] and normalized to 20 ng/μL for the following analyses. Only samples with 280/260 absorbance ratios of 1.8 to 2.0 (NanoDrop, Thermo Scientific) were used in this study. TCF7L2 gene polymorphisms of rs12255372 and rs7903146 gene were carried out using PCR-RFLP analysis as described by Bodhini et al. [29] and Szepietowska et al. [30]. Two sets of primers were used to amplify the loci in the TCF7L2 gene containing the two polymorphisms. The primers used and the size of the PCR products and restriction enzyme used are listed in Table 1 [29]. PCR was carried out in a 50-μl total final volume containing 200 μM dNTPs (Finzyme, Finland), 10 pmole of each primer, 2U of Taq polymerase (Finzyme, Finland), and 500 ng DNA. Thermal cycling conditions were as follows: denaturation at 95°C for 10 min, followed by 30 cycles of denaturation at 95 °C for 50 s, annealing at 56 °C 50 s, and elongation at 72 °C for 50 s followed by a final elongation of 5 min. Ten microliters of a successfully amplified PCR fragment containing the rs7903146 polymorphism was digested with RsaI enzyme (Fermentas, Germany) and incubated at 37 °C for 3 h and the fragments were run on 3.5% agarose gel stained with ethidium bromide and analyzed under ultraviolet light. The RsaI enzyme cuts digested PCR product (113 bp) into two fragments (91 and 22 bp ) in the presence of the C allele as shown in Fig. 1.
On the other hand, ten microliters of PCR fragments containing the rs12255372 was digested with Tsp509I (Fermentas, Germany) and incubated at 65 °C for 3 h and the fragments were run in 3.5% agarose gel stained with ethidium bromide and analyzed under ultraviolet light. The Tsp509I enzyme cuts PCR product (346 bp) in three fragments (143, 104, 99 bp) in the presence of the G allele while the T allele creates an additional site for the enzyme resulting in five fragments (128, 104, 104, 99, 17 bp) as shown in Fig. 2.

Statistical analysis
Statistical analysis was done using IBM© SPSS© Statistics version 22 (IBM© Corp., Armonk, NY, USA). Normality was tested using the Kolmogorov-Smirnov test. Table 1 Sequence of primers, size of the PCR products, and restriction enzyme used in genotyping of the rs7903146 and rs12255372 in the TCF7L2 gene Comparisons of parameters with normal distribution were performed using the Student t-test for independent samples. Categorical variables were compared using the Fisher exact test (two-tailed) or the chi-square test, as appropriate. Allele frequencies and Hardy-Weinberg (HW) equilibrium were verified using the chi-square test. Probability lower than 5% (P < 0.05) was considered significant. The genotypic relative risk was assessed by comparing the GDM with the control group and calculating the odds ratio and 95% confidence interval.

Results
The anthropometric characteristics and laboratory data of all participants are demonstrated in Table 2. Both groups were comparable in age, P= 0.05. GDM patients were significantly weightier with a higher body mass index, P= 0.05. Blood pressure was within the normal range, P <0.001, but still significantly higher among the GDM group in comparison to healthy pregnant women in the control group. Family history of diabetes was more prominent among the GDM group, P=0.01. Among the GDM group, fasting glucose and glycated hemoglobin were lying in the normal reference range, signifying that those patients had a past good glycemic index. Postprandial hyperglycemia was observed among the GDM group, which was more than 153 mg/dL but less than 200 mg/dL indicating the presence of GDM and the absence of preexisting overt diabetes according to IADPSG guidelines [26]. Those results display the occurrence of a hyperglycemic surge in the GDM group, which is well-matched with the studied disease as shown in Table 1. Lipid profile was normal apart from total cholesterol (Tch) and LDL which were significantly higher among the GDM group in comparison to controls, P = 0.02 and 0.001, respectively.
All genotypes in both groups were in consistency with HW equilibrium (P N 0.05). Regarding rs7903146 SNP, CT and TT genotypes were more prominent among GDM in comparison to the control group; CT carriers among the GDM group were 46.5% vs. 54% and TT carriers were 37.7% vs. 9.6%, respectively. Consequently, dominant and recessive models were highly significant among the GDM group in comparison to the control group. As for the rs12255372 SNP, GT and TT genotypes were also more prominent among the GDM group in comparison   Table 3.

Discussion
GDM is broadly studied, yet its pathophysiology is not clearly understood. In the meantime, it is important to consider women either with a positive family history of T2D or had a previous history of GDM are at a high risk for developing GDM in upcoming future pregnancies. It is hypothesized that GDM might have similar risk factors and genetic liabilities with T2D [31]. The approximate incidence of acquiring diabetes is nearly 60% in the upcoming future among females with GDM. Associated factors predicting the development of T2D afterwards stay comparable between several cohorts [32]. We aimed by this study to evaluate the effect of two polymorphisms of rs7903146 and rs12255372 in the TCF7L2 gene with the development of GDM among Egyptian women.
Among different populations, TCF7L2 is known to be the most common risk gene for T2D. It is the most frequently examined gene among women with GDM [33,34]. The TCF7L2 gene polymorphisms were documented to be correlated with GDM in many populations [24,35,36]. Conferring to the literature, there is not a highlight on the specific geographical area for TCF7L2 SNP allele frequency [37]. In our study, clinical parameters including BMI and hypertension measurements among the GDM group were comparable with other published studies that stated that pregnant women with GDM have a higher BMI and more frequency of hypertension in comparison to healthy controls [38,39]. Throughout the normal gestation period, Tch and TG levels in the maternal blood are more prone to a sustained increase [40]. Those fluctuations in lipid profile are intensified with the existence of GDM [41]. This explains the difference found in the lipid profiles among the studied groups as shown in Table 1. Percentages of the other laboratory parameters were as predicted, and the results were comparable to other reported studies representing GDM patients with acceptable glycemic index as shown in Table 1 [42].
Women enrolled in this study were entirely Egyptians. CT and TT genotypes in rs7903146 SNP were more prominent among the GDM group in comparison to the control group. Women carrying the T allele have a nearly . Dan Ye and his colleagues studied 18 SNPs within TCF7L2 and found out that rs290487, rs6585194, and rs7094463 polymorphisms are significantly related with the existence of GDM [31]. A meta-analysis study done by Chang and his colleagues reported a direct relationship between TCF7L2 alleles and the presence of chronic pregnancy hyperglycemia [44]. The study reported that six polymorphisms in the TCF7L2 gene had a highly significant correlation with the increased risk of GDM among the entire population. Chang and his colleagues also highlighted the high significance found between diverse ethnic populations [44]. Reyes-Lopez and his colleagues reported that rs12255372 is more prominent among women increasing the liability for insulin glucose tolerance (IGT) status as well as increasing the frequency for developing T2D [42]. Potasso and his colleagues reported that T mutant allele in TCF7L2 rs7903146 is related to the loss of initial postprandial glycemic control and stressed on the necessity of insulin treatment among pregnant women with chronic hyperglycemia, even if other risk interfering factors like BMI had been regulated [41]. There is an increased possibility of having T2D in different cohorts especially among CT/TT genotype carriers of SNP rs7903146 [15]. In a long time study of around 22 years of follow-up, mutant T allele carriers had a poor insulin feedback mechanism to random OGTT as well as greater chance for developing T2D in comparison to CC genotype carriers [13]. Lin and his colleagues conducted a meta-analysis and they reported a significant correlation between mutant allele of rs7903146 with the high chances of acquiring GDM among all genomic models in the entire population of white and Hispanic/Latino as well as Asian subcategories. Lin and his colleagues also stated that homozygous TT allele carriers for the SNP rs7903146 among Asians were highly correlated with the risk of GDM (OR = 2.08) followed by Hispanics/Latinos (OR = 1.80) and whites (OR = 1.51) [45]. It is reported that TCF7L2 has a strong impact on the breakdown and absorption of glucose and lipids; therefore, it is hypothesized that TT carriers might have a greater chance for developing cardiovascular diseases [40]. Francaite et al. reported that CT/TT genotypes in rs7903146 SNP and GT/TT genotypes in rs12255372 SNP in TCF7L2 gene were more prominent among GDM Lithuanian women in comparison to women from the general population [46]. In contrast to our results, de Melo et al. reported that rs12255372 and rs7903146 were not associated with GDM in the Euro-Brazilian population [47]. Siudak et al. also found no relationship between rs7903146 polymorphism of the TCF7L2 gene and GDM in the Polish population [48]. A study was conducted in North India on 115 women with GDM, and TCF7L2 variants rs 7903146 and rs 12255372 showed no association with GDM among the Indian population [49]. Rizk et al. reported that from 114 Arab pregnant women and 45 non-Arab pregnant women with GDM, TCF7L2 rs 7903146 polymorphism showed no association with GDM but the T allele of rs 12255372 showed a significant association with GDM in comparison to healthy controls [50].

Conclusion
In conclusion, we detected a significantly high prevalence of CT/TT genotypes in rs7903146 and GT/TT genotypes in rs12255372 of TCF7L2 gene in women with GDM compared to healthy pregnant women. A good overview of genetic influences might assist in finding a clear path for early detection and efficient management plans regarding GDM. The results from this study will probably help to highlight the possible roles of those variants in our population in developing GDM; yet, wider base studies are needed to find other possible risk factors that increase the prevalence of GDM in the Egyptian population.