Maternal genetic contribution to pre-pregnancy obesity, gestational weight gain, and gestational diabetes mellitus

Introduction Pre-pregnancy obesity, gestational diabetes mellitus (GDM), and gestational weight gain (GWG) are associated with each other. This is the first study to investigate whether genetic variants were associated with having GDM, and whether genetic variants-related GDM were associated with adiposity including pre-pregnancy obesity and excessive GWG in Turkish women. Patients and methods Women with GDM (n = 160) and without GDM (n = 145) were included in case-controlled study. Genotyping of the HNF1A gene (p.I27L rs1169288, p.98V rs1800574, p.S487N rs2464196), the VDR gene (p.BsmI rs1544410, p.ApaI rs7975232, p.TaqI rs731236, p.FokI rs2228570), and FTO gene (rs9939609) SNPs were performed by using RT-PCR. Results The FTO AA genotype was associated with an increased risk of having GDM (AA vs. AT + TT, 24.4% vs. 12.4%, OR = 2.27, 95% CI [1.23–4.19], p = 0.007). The HNF1A p.I27L GT/TT genotype was associated with increased GDM risk (GT + TT vs. GG-wild, 79.4% vs. 65.5%, OR = 2.02, 95% CI 1.21–3.38], p = 0.007). However, all VDR gene SNPs and the HNF1A p.A98V, p.S487N were not associated with having GDM (p > 0.05). The FTO AA genotype was associated with an increased risk for pre-pregnancy overweight/obesity (OR = 1.43, 95% CI [1.25–3.4], p = 0.035), but not associated with excessive GWG after adjusting for pre-pregnancy weight (p > 0.05). Pre-pregnancy weight, weight at delivery, and GWG did not differ in both VDR and HNF1A gene carriers (p > 0.05). HOMA-IR and HbA1c were increased in both p.I27L TT and FTO AA genotype carriers (p < 0.05). Conclusion The adiposity-related gene FTO is associated with GDM by the effect of FTO on pre-pregnancy obesity. The diabetes-related p.I27L gene is associated with GDM by increasing insulin resistance.


Introduction
Maternal obesity and gestational diabetes mellitus (GDM) is a growing public health problem worldwide [1]. The Institute of Medicine (IOM) developed guidelines for gestational weight gain (GWG) during pregnancy; however, no specific recommendations could be made for GDM and multiethnic differences [2,3]. Both pre-pregnancy obesity and excessive GWG are related to increased risk of maternal obesity and GDM [3]. Becoming pregnant or gaining too much weight during pregnancy are the risk factors for adverse perinatal complications and increased risk for future metabolic disease in overweight/obese women, both for the mothers and their offspring [1,4]. Pre-pregnancy obesity and excessive GWG may have additive negative impact on maternal and neonatal outcomes in women with GDM [5,6]. Pre-pregnancy obesity, gestational diabetes, and excessive GWG are associated with multiple factors such as the environment, behavior, and genetics; however,

Open Access
Diabetology & Metabolic Syndrome understanding these associations is complex [1,3]. Diabetes-related or maternal and/or fetal adiposity-related genetic variants have been associated with GDM, prepregnancy weight, and GWG during pregnancy [7][8][9]. Kawai et al. reported that common type 2 diabetes risk variants were associated with increased risk of GDM [8]. Genetic variants were associated with GDM and progression to pre-diabetes and type 2 diabetes mellitus in women with prior GDM [9]. Evidence has been presented for a genetic predisposition to GDM risk and also a change in GWG during pregnancy [7,[10][11][12][13], and gene-environment interactions could explain the variation in GWG and GDM.
The fat mass and obesity-associated gene (FTO) rs9939609 single nucleotide polymorphism (SNP) was associated with increased risk of obesity and type 2 diabetes, as well as GDM [10]. The FTO SNPs have been reported to be associated with pre-pregnancy obesity [8] and excessive GWG [11]. The FTO variants related to type 2 diabetes are mediated by the effect of the FTO gene on body mass index (BMI); however, the exact mechanisms of this relation have not been identified [10,11]. Vitamin D shows its cellular activity by binding to vitamin D receptors (VDR). VDR, as a transcription factor, has a role in the regulation of insulin secretion from pancreatic beta cells [14]. VDR has effect on proliferation, differentiation, and activation of immune cells and cytokine production, and subsequently type 2 diabetes occurs [15,16]. Hepatocyte nuclear factor 1A (HNF1A), as a transcription factor, has a role in the function of pancreas beta cells [17]. Endocrine and exocrine pancreatic cells express HNF1A in the developmental stage. HNF1A is necessary for the glucose response to insulin secretion and glucose metabolism [18]. Women with HNF1A mutation are diagnosed as having monogenic form of diabetes type 3 (MODY3), and these women usually present with GDM, and diabetes persisting after delivery [17][18][19].
This is the first study to investigate the effect of HNF1A gene, VDR gene, and FTO gene variants on having GDM, pre-pregnancy obesity, and excessive GWG in Turkey. We aimed to examine whether these genetic variants would associate with having GDM, and then, whether the genetic variants that associated with GDM would associate with adiposity including pre-pregnancy obesity and excessive GWG. The VDR gene (encoding as SNPs p.BsmI, p.ApaI, p.TaqI, and p.FokI), and, HNF1A gene (encoding as SNPs p.I27L, p.A98V, and p.S487N) were chosen because these genetic variants have been reported to be associated with type 2 diabetes, as well as GDM risk [12,[14][15][16][17][18][19][20]. We also investigated the obesityrelated FTO gene rs9939609 SNP because it is associated with both GDM and gestational body weight during pregnancy [10,13,20]. Genetic variants are implicated in the pathogenesis of GDM. Evidence suggests the genetic alterations in genes responsible for metabolic changes during pregnancy predispose to GDM [7]. We also hypothesized that these diabetes and adiposity-related genetic variants would likely be associated with GDM risk and gestational body weight during pregnancy.

Study population
Pregnant women referred to tertiary hospital, Obstetrics and Gynecology Clinic, Ankara, from 2015 to 2016, were included in this case-control study. Women with GDM (n = 160) and age-and gestational age-matched women without GDM as controls (n = 145) were included in the study. Gestational age was assessed from the date of the last menstrual period and clinical assessment. A 2-h, 75-g oral glucose tolerance test at 24 to 28 weeks gestation age was performed for all pregnant women, irrespective of family history of DM or any other risk factors for GDM. Glucose concentrations after fasting, and 1 and 2 h after glucose administration < 92 mg/dl, < 180 mg/ dl, and < 153 mg/dl, respectively, were considered normal. When the pregnant women's glucose concentration was higher than any of these values, the women were diagnosed as having GDM [12]. Women whose GDM was diagnosed according to these criteria, aged 22-38 years, and whose pregnancy age was 24-48 weeks were included in the study. Women with GDM who had pre-existing type 2 diabetes, GDM observed in prior pregnancy, GDM with chronic disease such as hypertension, thyroid disorders, cardiac, hepatic or renal dysfunction were excluded. Women aged 22-38 years and with pregnancy age 24-28 weeks, with no GDM, type 2 diabetes, hypertension, thyroid disorders, cardiac, hepatic or renal dysfunction were accepted as controls and included in the study. Treatment of diet with or without insulin therapy was recorded. Weight, height, and systolic (SBP) and diastolic blood pressure (DBP) were measured in all participants. Body mass index (BMI, kg/m 2 ) was calculated as weight (kg)/height 2 (m 2 ). Women were categorized as underweight (BMI < 18.5 kg/m 2 ), normal weight (BMI = 18.5-24.9 kg/m 2 ), overweight (BMI = 25-29.9 kg/ m 2 ), and obese (BMI ≥ 30 kg/m 2 ). Maternal weight before pregnancy, pre-pregnancy weight, was obtained through a questionnaire. Maternal weight was measured at delivery. Gestational weight gain (GWG) was calculated as the difference between the maternal weight at delivery and pre-pregnancy weight. The recommended GWG was calculated based on IOM guidelines related with prepregnancy BMI: underweight, a gain of 12.5-18 kg; normal weight, a gain of 11.5-16 kg; overweight, a gain of 7-11.5 kg; and obese, a gain of 5-9 kg. After this, GWG was divided into three categories: low, if the weight was

Statistical analysis
Statistical analysis was performed using the SPSS 18.0 (SPSS, Inc) software. Variables are presented as mean ± standard deviation (SD) or median (min-max), percentages (%), odds ratios (OR), 95% confidence intervals (CI). Normality was tested using the Kolmogorov-Smirnov and Shapiro-Wilk W test. SNPs are expressed as allelic frequency (q) or prevalence of genotypes (%). Categorical variables were analyzed using the Chi-square test or Fisher's exact test, where appropriate. Student's t-test was used for normally distributed continuous variables or log-transformed variables between two groups. The Hardy-Weinberg equilibrium (HWE) at individual loci was assessed using the Chi-square test. Multiple logistic regression analysis and the Chi-square test or Fisher's exact test was tested using models and ORs were calculated: dominant (major allele homozygotes vs. heterozygotes + minor allele homozygotes), recessive (major allele homozygotes + heterozygotes vs. minor allele homozygotes) and codominant (major allele homozygotes vs. heterozygote and minor allele homozygotes vs. major allele homozygotes). Pair-wise linkage disequilibrium (LD) and correlation coefficients (r 2 ) were analyzed using the HAPLOVIEW program. We made a variable reflecting all possible combinations of genotypes for each SNP. Power analysis was performed using web-based software http://osse.bii.a-star.edu.sg/calcu latio n2.php. The power of study was 65%. Statistical significance was defined as p < 0.05.

Results
The mean age, gestational age, and height were similar between the women with GDM and controls (p > 0.05). Pre-pregnancy overweight/obesity were increased in women with GDM compared with controls (p < 0.05). Weight at delivery and excessive GWG were increased in women with GDM compared with the controls (p < 0.05). Serum glucose, insulin, HOMA-IR, and HbA1c were increased in women with GDM compared with the controls (p < 0.05, each). The clinical features of the subjects are shown in Table 1. Minor allele frequency of the HNF1A, VDR, and FTO genes is shown in Table 2. These frequencies were in HWE except p.A98V. Haploview analysis showed that the HNF1A, VDR, and FTO genes were not in LD. The risk alleles of the HNF1A gene (p.S487N, and p.A98V) and, VDR gene (p.ApaI, p.TaqI, p.BsmI and p.FokI) were similar between women with GDM and the controls (p > 0.05, each). Genotype analysis is shown in Table 3.
The FTO gene rs9939609 distribution was TT-wild, heterozygote AT, and homozygote AA at 50.3%, 37.2%, and 12.4% in the controls, and 36.9%, 38.8%, and 24.4% in women with GDM (p = 0.011). The FTO gene AA genotype was associated with an increased risk of GDM more than the TT/AT genotype in co-dominant, dominant, and recessive models (dominant: AT + AA vs.  Table 3). The FTO AA/AT genotype had a greater association with pre-pregnancy overweight/obesity than TT-wild genotype (p < 0.05) ( Table 4). Pre-pregnancy weight (p < 0.05) and weight at delivery (p < 0.05) progressively increased from the AA genotype to the TT genotype. GWG was increased in AT/AA genotype compared with the TT genotype (p < 0.05). Serum glucose, insulin, HOMA-IR, and HbA1c were higher in the AA genotype compared with the TT genotype (p < 0.05). The FTO AA genotype was associated with a greater risk of pre-pregnancy overweight/obesity compared with AT/TT genotypes ( Table 3). Pre-pregnancy weight, weight at delivery, and GWG were similar between p.I27L genotypes (p > 0.05) ( Table 5). Glucose, HOMA-IR, and HbA1c were increased in the p.I27L TT genotype compared with the GG-wild type (p < 0.05). Pre-pregnancy weight, weight at delivery, and GWG did not differ between the VDR and HNF1A gene carriers (p > 0.05).     but not associated with excessive GWG after adjusting for pre-pregnancy weight. The association of the adiposity-related gene FTO with GDM might be mediated by the effect of FTO on pre-pregnancy obesity. The diabetes-related p.I27L gene was associated with GDM by increasing insulin resistance. Our results demonstrated that the VDR gene p.ApaI, p.TaqI, p.BsmI, and p.FokI genotypes were not associated with having GDM in Turkish women. The VDR gene and HNF1A gene SNPs were not associated with pre-pregnancy weight, weight at delivery, and GWG during pregnancy. The associations of the VDR gene and HNF1A gene with pre-pregnancy weight, weight at delivery, and GWG have not been investigated in previous studies. El-Beshbishy et al. reported that p.BsmI and p.FokI were not associated with GDM in Saudi women [22]. Incompatible to our results, p.FokI [23], p.ApaI, and p.TaqI [22] were associated with an increased risk of GDM in Iranian women [24]. We found that the HNF1A gene p.A98V and p.S487N were not associated with GDM in Turkish women. Zurawek et al. reported that p.I27L, p.A98V, and p.S487N were not associated with GDM in Polish women [25]. No relationship was reported between p.A98V and GDM in Danish women [12]; however, insulin secretion was decreased in p.A98V carriers without GDM [26], which is compensated by increasing insulin sensitivity [27]. Our data show that the HNF1A gene p.I27L GT/ GG genotype was associated with an increased risk of GDM (OR = 2.02, 95% CI [1.21-3.38], p = 0.007). Prepregnancy weight, weight at delivery, and GWG were not associated with p.I27L genotypes. Insulin resistance and impaired glucose metabolism was observed in p.I27L TT carriers. We suggest that the diabetes-related p.I27L gene was associated with the increased risk of GDM by impairing glucose metabolism and increasing insulin resistance. Similarly, p.I27L was associated with an increased GDM risk in Scandinavian women by the effect of p.I27L on pancreas beta cell function [28] and insulin resistance [29]. Decreased beta cell function/transcriptional activity, decreased glucose-stimulated insulin secretion, increased insulin resistance, and increased type2 diabetes risk have been found in p.I27L + p.S487N carriers (if also including p.A98V) [27,30,31]. HNF1A controls beta cell function by regulating target genes such as glucose transporter 2, liver pyruvate kinase, collectrin, hepatocyte growth factor activator, and HNF4A. Decreased HNF1A activity causes decreased beta cell mass and expression of these target genes, which lead to impaired insulin secretion [17,18]. Beta-cell dysfunction is more prone to developing impaired glucose tolerance during pregnancy [28]. Insulin resistance and impaired glucose metabolism were observed in FTO AA genotype carriers. We suggest that the adiposity-related gene FTO was associated with increased risk of GDM by increasing pre-pregnancy obesity. Similarly, previous studies have shown that the FTO rs9939609 AA genotype was associated with higher prepregnancy weight [10,13,32]. Lawlor et al. reported that maternal fat or fetal fat adiposity-related variants were not associated with excessive GWG, but the FTO gene was associated with pre-pregnancy overweight [33]. The FTO gene has a role in the regulation of adiposity-related phenotypes through the effect of FTO on weight gain during younger ages [34] and continues throughout life [10]. FTO is expressed in the hypothalamic region, which regulates appetite [35], and this would contribute to energy intake and body fat mass [36]. Our data demonstrated that FTO gene AA genotype carriers were heavier before pregnancy, but AA carriers did not have significant weight gain during pregnancy. Chiou et al. reported that the FTO gene was associated with pre-pregnancy obesity and a tendency to gain less weight throughout pregnancy [5]. Consistent with our data, the FTO gene was not associated with greater GWG after adjusting for pre-pregnancy BMI in Caucasian and African-American populations [37]. The FTO gene was not associated with GWG according to the period of pregnancy in British [33] and Brazilian women [10]. Moreover, GWG comprises other factors such as the fetus, amniotic fluid, and placenta [10]. Pregnant women have biologic, behavioral, and hormonal changes throughout pregnancy [11].
Pre-pregnancy body weight shows maternal nutritional changes before conception, whereas GWG represents fetal-maternal physiologic conditions associated with genetic and nutrition factors [1]. This could modify the genetic contributions of the maternal FTO, HNF1A, and VDR gene variants on pre-gestational weight and GWG, as well as GDM [13,33]; however it is not fully known which of these conditions is more associated with these disorders.
There are some limitations in our study that should be considered. We did not report the GWG according to gestational weeks. The small sample size resulted in a lower power for investigating a significant effect of any of the HNF1A, VDR, and FTO gene SNPs on weight changes during pregnancy. Also, we did not control our data for confounding variables such as nutrition, education, smoking and parity.

Conclusion
Both the FTO AA genotype and HNF1A p.I27L GT/ TT genotype were associated with increased GDM risk in Turkish pregnant women. However, the VDR gene p.ApaI, p.TaqI, p.FokI, p.BsmI and the HNF1A gene p.A98V, p.S487N genotypes were not associated with having GDM. The diabetes-related p.I27L gene was associated with GDM by increasing insulin resistance. The diabetes-related HNF1A p.I27L gene was associated with insulin resistance, which might contribute to developing GDM. The FTO AA genotype was associated with prepregnancy overweight/obesity, but did not contribute to significant weight gain during pregnancy. The adiposity-related gene FTO was associated with GDM by the effect of FTO on pre-pregnancy obesity. The FTO gene was associated with pre-pregnancy obesity, which might contribute to developing GDM. Genetic factors involved in GDM, pre-pregnancy weight, and GWG should be identified for the prevention of adverse complications of GDM and obesity during pregnancy. Further studies with multiethnic and larger populations are needed to find genetic variants related to GDM, pre-pregnancy obesity, and GWG during pregnancy.