Gestational Diabetes Mellitus (GDM) is defined as glucose intolerance that emerges for the first time during pregnancy or that begins during pregnancy [1,2]. Both women with GDM and their children are at increased risk of developing T2DM later in life [2]. Insulin resistance and destruction of pancreatic β-cells are seen as the most important factors in the pathophysiology of GDM. Although the etiology of GDM is not known exactly, it is thought to occur as a result of the interaction of genetic, epigenetic and environmental factors [3-5].

It is known that energy consumption increases during exercise and improves insulin sensitivity. Depending on physical activity and exercise activities, hormones known as myokines are secreted from muscle cells. These myokines are considered to be one of the factors in the development of insulin resistance underlying GDM. It has been determined that irisin, a myokine, is highly expressed during exercise and this molecule transforms white adipose tissue into brown adipose tissue. It is reported that this situation increases the metabolic rate and reduces insulin resistance [6,7]. In several studies, it was stated that serum irisin level is high in normal pregnancies, and serum irisin level is lower in patients with GDM compared to healthy pregnancies [8-11]. It has been reported that there is a significant decrease in serum irisin level in the postpartum three months period in both normal glucose tolerant pregnancies and pregnancies with GDM [9,11].

Irisin is a molecule formed by proteolytic cleavage of FNDC5 (fibronectin Type III Domain Containing 5), a plasma membrane protein consisting of 112 amino acids, weighing approximately 12kDa [7,12]. The FNDC5 gene contains 6 exons and 5 introns and is located at the p35.1 position of chromosome 1 [13,14]. It has been reported that FNDC5 gene variants affect circulating serum irisin levels and are associated with metabolic syndromes, insulin sensitivity, insulin resistance, T2DM and obesity [15,16]. Studies have reported that rs16835198 and rs726344 polymorphisms on the FNDC5 gene are associated with insulin sensitivity and the G allele of rs16835198 affects fasting insulin levels [17]. Likewise, in patients with Type 2 diabetes mellitus and diabetic nephropathy, the frequency of the TT genotype and T allele in the FNDC5 gene rs16835198 polymorphism was significantly lower than the control group, while the wild G allele had a desensitizing effect to insulin in patients with Type 2 diabetes mellitus [18]. The fact that the FNDC5 gene is expressed from the placenta, the serum irisin level rises during pregnancy, and irisin is associated with metabolic status, insulin resistance and diabetes, suggests the hypothesis that FNDC5 gene polymorphisms may affect the development of GDM. However, no study investigating the relationship between FNDC5 gene variants and GDM was found in the literature.

In this study, it was aimed to investigate whether common rs726344 and rs16835198 polymorphisms in the FNDC5 gene are associated with GDM.

This study adhered to the Declaration of Helsinki, and its protocols were approved by the Ethical Review Committee of Harran University number of 76244175-050.04.04. All participants provided written informed consent before taking part in the study.

The study included 233 pregnanat subjects from Southeastern Anatolian region. The study population was divided into two groups; the first one is 110 subjects of the GDM group and the second one is 123 subjects of the control (Non-GDM subjects) group. 110 subjects of the first group, who diagnosed with GDM at the 24-28 gestational period, were chosen as the patient group according to the criteria of ADA (American Diabetes Association).

The inclusion criterias for GDM subjects were; firstly diagnosed cases and no previous usage of oral hypoglycemic medications. The inclusion criterias for control group were; absence of family history for diabetes mellitus, clinical evidence of any major disease and usage of medications which may change glucose tolerance.

The exclusion criteria of all participants of the study were the presence of diabetes mellitus, pre-gestational, obesity and other known major diseases. All participants were subjected to; clinical assessment, a detailed survey and laboratory investigations. Two millilitres of peripheral venous blood samples were withdrawn from all participants after >10 h of fasting and transferred into EDTA (Ethylene Diamine Tetra Acetic Acid) tubes for DNA isolation.

Total genomic DNA was isolated from whole blood leucocytes using PureLink Genomic DNA Isolation Kit K1820-02 (Invitrogen, Inc. Carlsbad, CA).

Two of the most common FNDC5 SNPs that rs16835198 and rs726344, were selected from the International HapMap Project and they were genotyped by Real Time PCR (Rotor-Gene Q Series, Qiagen) using validated TaqMan Allelic Discrimination Assays (Assay ID: C_1838758_10, and C_3259700 _1_ Applied Biosystems, Foster City, CA were used for the two SNPs, respectively).

Real-time PCR amplification was carried out in a final volume of 20 lL reaction mixture, including 20 ng of genomic DNA, 5 mL of TaqManVR Universal PCR Master Mix, and 0.5 mL of 40X TaqManVR assay. Thermal cycling conditions were; initial denaturing at 94 C for 3 min, 40 cycles of 94 C for 15 s, and 60 C for 1 min. The Rotor-Gene Q Series Software Version Q 2.3.1 (Rotor-Gene Q Series, Qiagen) was used for allelic discrimination.

For this study, an independent t-test was used to compare the averages between the studying groups. X² test was used to analyze the distribution of alleles to the genotypes and to detect the compatibility of the distribution with the expected values (Hardy-Weinberg equilibrium). Statistical analysis of the data gained from the study was carried out by SPSS V20.0 package program. P < 0,05 was considered statistically significant.

110 patients with GDM and 123 healthy controls were enrolled in the study. General Characteristics of the study population are shown in Table 1. When evaluated in terms of age, it was observed that the mean age of the patient group was 28.89 (± 6.58), and the mean age of the control group was 29.03 (± 5.74). It was observed that there was no statistically significant difference between the patient and control groups in terms of age distribution and BMI (p> 0.05. When the glucose values of the patient and control groups were compared, it was seen that there was a statistically significant difference between them (p < 0. 0001).

Table 1: Demographic characteristics of Patient and Control groups.

*Unpaired t test applied

In this study, two polymorphisms of FNDC5 gene were studied in terms of genotype distributions and allele frequencies in the patient and control groups. The first of these polymorphisms is the G> A polymorphism of the FNDC5 gene (rs726344). The second one is the G> T polymorphism (rs16835198).

When the data in Table 2 is examined, it is seen that the homozygous GG genotype of FNDC5 gene G> A (rs726344) polymorphism has the same ratio in the patient and control groups, while the heterozygous GA geneotype has a very close

Percentage in the control and patient groups. Homozygous AA genotype was never seen in the patient group, and it was found only 1% in the control group. In terms of allele frequencies of mentioned polymorphism, in patients with GDM and control group are also shown in Table 2. Accordingly, the G allele was detected in 94% of the patient group and 93% in the control group. The A allele was found to be 6% in the patient group and 7% in the control group.

As a result of the analysis, no statistically significant difference was found in the genotype distribution and allele frequencies of the FNDC5 gene G> A (rs726344) polymorphism in the patient and control groups with GDM (p> 0.05).

Table 2: Genotype distribution and Allele frequencies of FNDC5 gene rs726344 G>A polymorphism in GDM patient and control group.

As can be understood from Table 3, it was observed that homozygous GG, heterozygous GT and homozygous TT genotypes were not statistically significant in terms of genotype distributions of FNDC5 gene rs16835198 G> T polymorphism when compared to patient and control groups (p> 0.05).

Also when the allele frequencies of the FNDC5 gene rs16835198 G>T polymorphism in the GDM patient and control group were examined in Table 3, it was observed that the G allele was 74% in both the patient group and the control group, and the T allele was 26% in both the patient group and the control group was seen. No statistically significant difference was found between the patient and control groups in terms of allele frequencies in the FNDC5 gene rs16835198 G> T polymorphism (p> 0.05).

Table 3: Genotype distribution and Allele frequencies of FNDC5 gene rs16835198 G> T polymorphism in patient and control groups with GDM.

(Pearson Chi-Square test was applied.)

From the discovery of irisin in 2012 by Bostrom et al. many scientists have tried to find its relationship with metabolic diseases and pregnancy [7]. Within the myokine group, irisin peptide, which is released from muscle cells and functions as a messenger between muscle and adipose tissue, has been shown to have an important role in insulin resistance and regulation of energy metabolism [19]. Irisin induces energy metabolism, the conversion of white adipose tissue to brown adipose tissue, and converts chemical energy into heat. In addition, irisin plays an important role in the regulation of metabolic homeostasis.

In the studies mentioned below, it was stated that the level of irisin in the circulation has significant effects on T2DM, insulin resistance and energy metabolism. Hee Park et al. demonstrate that irisin is associated with increased odds of having Metabolic syndrome (MetS) and with increased risk of insulin resistance and 10-year risk of Cardiovascular disease (CVD) and suggested that irisin may play a possible role in glucose metabolism (20). Choi et al. compared serum irisin levels of patients with newly diagnosed Type 2 DM and controls with normal glucose tolerance and found that serum irisin levels were significantly decreased in the group with newly diagnosed Type 2 DM (21). In their study, Kuzmicki et al. reported that serum irisin level significantly increased in normal pregnancies, but it was significantly lower in patients with GDM [22]. In some studies, serum irisin level was found to be positively related with HOMA-IR during pregnancy but in some studies, irisin level has been showed to be negatively related with HOMA-IR [20,23]. E.G. Khidr et al. found that the FNDC5 (rs16835198 G> T) gene polymorphism provides significant protection against T2DM without affecting nephropathy and serum irisin levels and they also reported that, this SNP’s wild G allele is associated with Dyslipidemia, high fasting insülin and HOMA-IR [23].

In addition to all studies mentioned above, irisin also plays a role in pregnancy and embryo development. In previous studies, it has been reported that the serum irisin level is high in normal pregnancies, the placenta contributes to the increase in the circulating irisin level in pregnancy, and the serum irisin level in patients with GDM is lower than in healthy individuals [8-11]. Irisin is a membrane protein formed by cleavage of the N-terminal part of FNDC5 [19]. Widely studied polymorphisms the precursor of irisin of the FNDC5 gene; are rs16835198, rs726344, rs3480, rs1746661, rs1298190 and rs1570569. Al-Daghri NM et al. reported that SNPs in the FNDC5 gene are associated with obesity and glucose lipid metabolism, probably because they modulate serum irisin levels [24]. In their study, Sanchis-Gomar F et al, with the luciferase reporter gene analysis method, they found that rs16835198 and rs726344 polymorphisms were functional and the minor alleles of both SNPs had more functional effects [25]. Abdu Allah AM et al. also found that rs16835198 and rs726344 polymorphisms were associated with serum irisin levels in their study on obesity. In their study, they observed that FNDC5, rs16835198 and rs726344 SNPs were associated with obesity in the Egyptian population. Also, they observed that rs726344 variant, GG genotype and G allele, and rs16835198 variant's TT genotype and T allele may increase the susceptibility to obesity, and that there is a genotype-phenotype correlation between these SNPs with circulating serum irisin level and various metabolic parameters [18]. E.G. Khidr et al. reported that the rs16835198 G >T polymorphism in the FNDC5 gene provides significant protection against T2DM in Egyptian patients, independent of nephropathy and serum irisin levels, and they have stated that it is related the wild G allele of this SNP is associated with high fasting insulin, HOMA-IR, and dyslipidemia [23]. In another study, it was stated that the 767 C>G polymorphism of the Fetuin-A gene can be seen as a risk factor for GDM, and the GG variant may have a protective effect for GDM [26].

Based on the above studies, it was aimed to investigate the effect of two polymorphisms in the FNDC5 gene, which is responsible for the synthesis of irisin, on GDM. The relationship between two polymorphisms that affect serum irisin level and function in FNDC5 gene and their relationship with GDM were investigated.

According to the obtained data, it was concluded that the studied rs726344 and rs16835198 polymorphisms were not associated with GDM. We assume that stronger statistical results regarding to these polymorphisms can be obtained by increasing the sample size of study group. These results do not seem to be compatible with studies in which these polymorphisms are associated with metabolic syndrome, insulin sensitivity, insulin resistance, T2DM and obesity [18,23]. In other words, these polymorphisms in the gene may not have an effect on serum irisin level, contrary to previous studies. As a matter of fact, Kumpei Tanisawa et al. reported in a study that there was no relationship between common FNDC5 SNPs and serum irisin level [27]. Since these polymorphisms are located in the 3' flanking region (rs16835198) and intron 5 (rs726344) of the gene, they are not expected to affect the amino acid sequence of the synthesized protein. However, alteration in the 3'UTR region of a gene (eg rs16835198) has the potential to affect the level, location and timing of gene expression. Intronic genomic variants (eg, rs726344) have the potential to affect gene expression by altering mRNA stability, generating alternative mRNA splicing, or acting on the binding of transcription factors. Indeed, Sanchis-Gomar F et al. With the luciferase reporter analysis method, it was determined that these two variants had a functional effect on the gene, but similar to the results obtained, they were not related to aging, which is the subject of the study [25]. In addition, although polymorphisms may have effects on gene expression and ultimately on serum irisin level, as noted in the above studies, they may not have an effect on the development of GDM. The limited sample size in the study population and the fact that the polymorphic variants in question were not compared with the expression of the FNDC5 gene and serum irisin levels are the main limitations of the study. We think that it would be useful to examine the irisin molecule and its precursor, the FNDC5 gene, which we think has an effect on human metabolism, with a larger sample volume and in detail.

With the findings obtained as a result of the study, it was concluded that the FNDC5 gene rs726344 and rs16835198 polymorphisms were not associated with Gestational Diabetes Mellitus. These results need to be confirmed by studies with higher sample volumes from different ethnic communities.

This study was supported by HUBAK (Scientific Researches Coordinating Council of Harran University) with project number of 19258.

The authors declare that they have no conflicts of interest.

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