Polymorphisms in FTO and TCF7L2 genes of Euro-Brazilian women with gestational diabetes

CLB-09055; No. of pages: 4; 4C: Clinical Biochemistry xxx (2015) xxx–xxx Contents lists available at ScienceDirect Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem Polymorphisms in FTO and TCF7L2 genes of Euro-Brazilian women with gestational diabetes Sandra Fabrico de Melo a, Henrique Ravanhol Frigeri a,b, Izabella Castilhos Ribeiro dos Santos-Weiss a, Rosângela Roginski Réa c, Emanuel Maltempi de Souza d, Dayane Alberton a, Fabiane Gomes de Moraes Rego a, Geraldo Picheth a,⁎ a Post-Graduate Program in Pharmaceutical Science, Federal University of Parana, Brazil Health and Biosciences School, Pontifical Catholic University of Parana, Curitiba, Parana, Brazil c Endocrinology and Metabolism Service (SEMPR), Clinical Hospital, Federal University of Parana, Brazil d Department of Biochemistry and Molecular Biology, Federal University of Parana, Brazil b a r t i c l e i n f o Article history: Received 27 February 2015 Received in revised form 14 May 2015 Accepted 15 June 2015 Available online xxxx Keywords: Gestational diabetes mellitus FTO TCF7L2 Polymorphisms Case-controlled study SNP a b s t r a c t Objective: To investigate the association between fat mass and obesity-associated (FTO) gene polymorphisms rs8050136C N A and rs9939609T N A, and transcription factor 7-like 2 (TCF7L2) gene polymorphisms rs12255372G N T and rs7903146C N T, in a sample group of pregnant Euro-Brazilian women with or without gestational diabetes mellitus (GDM). Methods: Subjects were classified as either healthy pregnant control (n = 200) or GDM (n = 200) according to the 2010 criteria of the American Diabetes Association. The polymorphisms were genotyped using fluorescent probes (TaqMan®). Results: All groups were in the Hardy–Weinberg equilibrium. The genotype and allele frequencies of the examined polymorphisms did not exhibit significant difference (P N 0.05) between the groups. In the healthy and GDM pregnant women groups, the A-allele frequencies (95% CI) of FTO polymorphisms rs8050136 and rs9939609 were 39% (34–44%); 38% (33–43%) and 40% (35–45%); 41% (36–46%), respectively; and the T-allele frequencies of TCF7L2 polymorphisms rs12255372 and rs7903146 were 30% (26–35%), 32% (27–37%) and 29% (25–34%), 36% (31–41%), respectively. Conclusion: The examined polymorphisms were not associated with GDM in the Euro-Brazilian population studied. © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Introduction Gestational diabetes mellitus (GDM) is a type of diabetes diagnosed during pregnancy, which is not considered to be overt diabetes; GDM affects 1–14% of all pregnancies worldwide, with rising incidence level [1]. GDM occurs in about 7% of pregnancies in Brazil, a prevalence that is rapidly increasing in association with the obesity epidemic [2]. Chronic hyperglycemia in GDM is associated with an increase in the morbimortality of both the mother and fetus [3]. Patients with GDM have increased risk of developing type 2 diabetes (T2D), and the children of GDM mothers are more susceptible to diabetes and obesity in adult life [1]. Obesity and family history of diabetes are two major risk factors common to GDM and T2D [4]. These similarities suggest that both ⁎ Corresponding author at: Clinical Analysis Department, Federal University of Paraná, Curitiba, Parana, Brazil Rua Prefeito Lothário Meissner, 632, 80210-170 Curitiba, PR, Brazil. E-mail address: gpicheth@ufpr.br (G. Picheth). these diseases could share genetic factors such as single nucleotide polymorphisms (SNPs) involved in disease predisposition [5]. Fat mass and obesity-associated (FTO) is a protein-coding gene located in chromosome region 16q12.2 (NCBI GeneID: 79068). It was initially described in mice and encodes for a 502 amino acid protein [6]. The FTO is associated with the control of food intake and energy balance. However, the function of FTO remains to be elucidated [7]. Polymorphisms (SNPs) in this gene have been associated with obesity in European subjects, as well as with the increase of fat mass in other populations [7–9]. FTO SNPs rs8050136C N A and rs9939609T N A were shown to be associated with fat body mass accumulation and obesity in several studies, primarily among Asians and Europeans [6,10,11]. The transcription factor 7-like 2 (TCF7L2) gene is located in chromosome region 10q25.3 (NCBI GeneID: 6934), and encodes for a protein of 596 amino acids [12]. The TCF7L2 protein is involved in the Wnt signaling pathway. The Wnt glycoprotein enhances the formation of heterodimers of beta-catenin with TCF7L2, which induces the expression of several genes such as glucagon-like peptide-1 (GLP-1), insulin, and http://dx.doi.org/10.1016/j.clinbiochem.2015.06.013 0009-9120/© 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Please cite this article as: de Melo SF, et al, Polymorphisms in FTO and TCF7L2 genes of Euro-Brazilian women with gestational diabetes, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.06.013 2 S.F. de Melo et al. / Clinical Biochemistry xxx (2015) xxx–xxx those encoding proteins associated with exocytosis of insulin granules in pancreatic beta cells [13,14]. Polymorphisms in the TCF7L2 have been associated with increased susceptibility to T2D [15]. The best studied SNPs of the TCF7L2 (rs12255372G N T and rs7903146C N T) have been correlated with higher body mass index (BMI) and T2D susceptibility in related studies [16–18]. Few studies have examined the association between polymorphisms of the FTO and TCF7L2 and gestational diabetes, particularly in the Brazilian population. In this study, we examined the association of the FTO polymorphisms rs8050136 and rs9939609, and the TCF7L2 polymorphisms rs12255372 and rs7903146 in pregnant Euro-Brazilian women with or without gestational diabetes. Materials and methods Subjects A total of 400 unrelated Euro-Brazilian women were examined. Healthy pregnant women were classified as control (n = 200), and pregnant women with GDM as GDM group (n = 200), according to the 2010 criteria of the American Diabetes Association [19]. The studied groups were unmatched by age or BMI. The control group was recruited from the Municipal Laboratory of Curitiba, and the GDM patients from the Clinical Hospital of the Federal University of Parana. Both institutions are public, and are located in Curitiba, State of Parana, Brazil. This research was approved by the Federal University of Parana's Ethics Committee (CEP/SD: 1045.170.10.11 and CAAE: 5876.0.000.09110). Clinical and laboratory data Clinical and anthropometric data were collected from patient files or from electronic registries. Routine biochemical parameters were determined using an Architect® automated system. Reagents, calibrators, and controls were provided by the manufacturer (Architect, Abbott Diagnostics). 1,5-Anhydroglucitol levels were measured by an enzymatic method (GlycoMark, Inc.). Glycated hemoglobin (HbA1C) was quantified using HPLC (Varian II, BioRad Diagnostics). Genotyping DNA was extracted from whole blood using the “salting out” method [20] and normalized to 20 ng/μL for subsequent assays. Only samples with 280/260 absorbance ratios of 1.8 to 2.0 (NanoDrop, ThermoScientific) were used in this study. Polymorphisms (TaqMan codes) rs8050136 (C_20311259_10), rs9939609 (C_30090620_10), rs12255372 (C_291484_20), and rs7903146 (C_29347861_10) were genotyped using fluorescent probes (TaqMan®, Life Technologies) and the real time PCR StepOnePlus™ System (Life Technologies). All reagents were supplied by Life Technologies. The reaction mixtures contained 3.0 μL of Master Mix, 0.3 μL of SNP Genotyping Assay (40 ×), 1.7 μL ultra-pure water and 1.0 μL of DNA (20 ng/μL). The PCR cycle used was as follows: 10 min at 95 °C (1 cycle) followed by 40 cycles of 15 s at 92 °C and 60 s at 60 °C. All genotypes were analyzed using the StepOnePlus software, and genotypes obtained exhibited a minimal quality threshold of 95%. Statistical analysis Normality was tested using the Kolmogorov–Smirnov test. Comparisons of parameters with normal distribution were performed using the Student t-test for independent samples, or the Mann–Whitney U test for non-normal distribution variables. 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 (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl). Statistical analyses were performed with the Statistica for Windows version 8.0 software (StatSoft Inc., Tulsa, OK, USA). A probability lower than 5% (P b 0.05) was considered significant. Results The anthropometric and laboratory characteristics of the study's subjects are detailed in Table 1. GDM patients were significantly older, heavier (high body mass index), and more hypertensive than healthy pregnant women (control). GDM patients also had a high prevalence (approximately 70%) of diabetes in their family history. Fasting glucose (b5.6 mmol/L) and glycated hemoglobin (HbA1c b 6.5%; 47.5 mmol/mol) levels of the GDM group were within the reference range, suggesting that these patients showed good glycemic control. The low levels of 1,5-anhydroglucitol (b60.9 μmol/L; b10 μg/mL), a marker for post-prandial hyperglycemia, in the GDM group indicated the presence of hyperglycemic excursions in this group, which is compatible with the disease. Uric acid, total cholesterol, HDL-cholesterol, and triglycerides levels were significantly higher (P b 0.05) in GDM patients. Only LDLcholesterol levels were not significantly different between the groups. None of the patients had clinical symptoms of kidney disease or serum creatinine above 97.2 μmol/L (1.1 mg/dL). All genotypes in both groups were in HW equilibrium (P N 0.05). The genotype and allele frequencies of the studied polymorphisms were not significantly different (P N 0.05) between the groups (Table 2). The sample size and power calculation for the results in Table 2 are low and adequate for a prospective study. For an alpha of 0.05 and a power of 0.80 a sample size larger than 1000 subjects per group should be used. Discussion Several studies indicate that pregnant women with GDM are often heavier and more hypertensive than healthy pregnant women [21–23]. The characteristics of the GDM patients in our study are similar to others reported. Table 1 Anthropometric and laboratory characteristics of the study groups. Characteristics Control n = 200 GDM n = 200 P Age, years Body mass index, kg/m2 Hypertension, % Family history of diabetes, % Fasting glucose, mmol/L 2-h 75 g glucose, mmol/L HbA1c, %c 1,5 anhydroglucitol, μmol/L Creatinine, μmol/L Urea, mmol/L Uric acid, μmol/L 24.0 (20.0–28.0) 24.9 ± 4.0 0.5 – 4.59 (4.29–4.80) – – 90.8 (63.9–121.8) 70.7 (62.8–75.1) 3.32 (2.82–4.15) 202.2 (178.4–226.0) 4.92 ± 1.32 1,20 ± 0.28 3.15 ± 1.05 1.14 (0.87–1.49) 32.0 (28.0–36.0) 33.0 ± 6.4 6.5 69.0 4.90 (4.50–5.55) 9.09 (8.29–10.18) 5.7 (5.4–6.1) [39] 57.8 (36.6–77.9) 53.0 (50.4–63.6) 2.66 (2.16–3.24) 261.7 (226.0–309.3) 5.94 ± 1.27 1.44 ± 0.34 3.20 ± 1.06 2.53 (2.08–3.23) b0.001a b0.001 b0.001b – b0.001a – – b0.001a b0.001a b0.001a b0.001a Total cholesterol, mmol/L HDL-cholesterol, mmol/L LDL-cholesterol, mmol/L Triglycerides, mmol/L b0.001 b0.001 0.612 b0.001a Values are presented as mean ± SD, median (interquartile range) or %; and – , no information available. Control, healthy pregnant women; and GDM, gestational diabetes. P values, t-test independent variables. a Mann–Whitney U test or. b Chi-square test. c HbA1c values in mmol/mol 39 (36–43). Please cite this article as: de Melo SF, et al, Polymorphisms in FTO and TCF7L2 genes of Euro-Brazilian women with gestational diabetes, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.06.013 3 S.F. de Melo et al. / Clinical Biochemistry xxx (2015) xxx–xxx Table 2 Genotype and allele frequencies of the FTO and TCF7L2 polymorphisms in the absence (control) or presence of gestational diabetes (GDM). Polymorphisms Genotype allele Control (n = 200) GDM (n = 200) C/C C/A A/A A 74 (37.0) 96 (48.0) 30 (15.0) 39.0 [34–44] 73 (35.5) 102 (51.0) 25 (12.5) 38.0 [33–43] T/T T/A A/A A 71 (35.5) 97 (48.5) 32 (16.0) 40.3 [35–45] 68 (34.0) 100 (50.2) 32 (15.8) 41.0 [36–46] G/G G/T T/T T 102 (51.0) 75 (37.5) 23 (11.5) 30.3 [26–35] 92 (46.0) 88 (44.0) 20 (10.0) 32.0 [27–37] C/C C/T T/T T 98 (49.0) 86 (43.0) 16 (8.0) 29.5 [25–34] 76 (38.0) 104 (52.0) 20 (10.0) 36.0 [31–41] 0.725 FTO rs8050136 Allele frequency (%) FTO rs9939609 Allele frequency (%) TCF7L2 rs12255372 Allele frequency (%) TCF7L2 rs7903146 Allele frequency (%) P (χ2) 0.771 0.946 (χ2) 0.829 0.205 (χ2) 0.593 0.085 (χ2) 0.050 Control, healthy pregnant women; and GDM, gestational diabetes. Values of genotypes are presented as n (%); allele frequencies as % [95% Confidence Interval]. All genotypes were in Hardy–Weinberg equilibrium. P-value, Fisher exact test two-tailed (genotype) or allele Chi-square test (χ2). During normal pregnancy, the levels of total cholesterol and triglycerides in the plasma change atherogenically [24,25]. These changes are aggravated in the presence of GDM [26]. This could explain the differences in the lipid profile between the study groups (Table 1). The levels of the other laboratory parameters (Table 1) examined were as expected, and the results were similar to those of several reports demonstrating GDM patients with good glycemic control [21–25]. FTO polymorphisms have been associated with obesity, which is a major risk factor for diabetes [11,27]. In our study, polymorphisms rs9939609 and rs8050136, found in the FTO, were not associated with GDM (Table 2). The A-allele of rs8050136 was found to correlate with diabetes in a Korean test-population, which exhibited increased levels of insulin secretion (indicating a possible protective effect of this polymorphism in relation to the development of diabetes) [28]. The product of the TCF7L2 is involved in glycemic regulation and has been associated with type 2 diabetes in different studies [13,29,30]. The two intronic polymorphisms of TCF7L2 (rs7903146 and rs12255372) studied did not correlate (P N 0.05) with gestational diabetes in our test population (Table 2). Our results differed from those of a metaanalysis of four studies that examined rs12255372, and nine studies evaluating rs7903146, which demonstrated an association among the T-alleles and the risk for GDM [18]. The systematic review conducted by Zhang et al. [18] showed a compelling evidence for association of rs7903146T with GDM (Odds ratio 1.44; 95% CI 1.29–1.60, P b 0.001). These authors also showed a significative heterogeneity among Caucasians (I2 68.4%; P = 0.007). We hypothesized that the high miscegenation of the Brazilian population added to the small sample size of this study could be the major factors for the divergence of our results and the described systematic review. The difference between the rs7903146 T-allele frequencies of the groups was not significant (P = 0.050), but suggestive to more investigation, especially with a large sample size. The T-allele was associated with impaired insulin secretion, enhanced rate of hepatic glucose production [31,32], and with a high risk for GDM [32,33]. The association of this polymorphism with T2D in a Euro-Brazilian population revealed that the T-allele frequencies for healthy and T2D subjects were 27.0% and 35.8%, respectively [29]. These results were similar to those obtained in the present study for GDM. Marquezine et al. [34] evaluated two cohorts of Brazilian patients and found an association between type 2 diabetes and rs7903146 (P = 0.003; OR = 1.57 [1.16–2.11]) in one of the cohorts. Table 3 compares the studied risk allele frequencies in healthy subjects from different populations. Overall, the risk allele frequencies for these polymorphisms among Brazilians were comparable to most other populations, but significantly higher than Asian populations. Since Brazilians are an admixed population, differences in genetic background in the various regions could explain the disparity between the results of our study and others regarding the rs7903146 polymorphisms. Additional studies with larger sample sizes are required to clarify the association between these polymorphisms and GDM. To the best of our knowledge, this is the first study to examine polymorphisms in a Brazilian population with GDM. In conclusion, polymorphisms rs7903146 and rs12255372 of TCF7L2 and polymorphisms rs9939609 and rs8050136 of the FTO were not associated with GDM in the Euro-Brazilian population sample examined in this study. Acknowledgments The CNPq and Araucaria Foundation supported this project. No potential conflicts of interest relevant to this article have been reported. Table 3 Comparison between the allele frequencies of healthy pregnant women with those obtained from other studies. Gene polymorphism Population Allele frequencies (%) References FTO rs8050136 (A-allele) Euro-Brazilian 39 [34–44] This study European African American Asian Chinese Han Euro-Brazilian 44.4 48.0 13.5 12.0 40 [35–45] (31) (35) (28) (9) This study Danish Caucasians Europeans African Americans Spanish British white Chinese Han Euro-Brazilian 46.2 44.4 48.0 39.3 40.0 12.0 30 [26–35] (36) (31) (35) (37) (38) (9) This study Mexican American Southern Sweden Czech Caucasian British Indian Mexican Asian Euro-Brazilian 39.4 30.9 30.6 36.0 29.0 18.0 11.4 0.2 29 [25–34] (32) (39) (40) (41) (42) (43) (44) (28) This study Greek women European Czech Southern Sweden Brazilian Danish Caucasian Scandinavian British Caucasian Mexican Sweden Euro-Brazilian Asian 39.5 38.4/32.2 33.8 32.9/39.1 33.5 34.6 31.9 30.7 34.0 32.0 28.0 27.0 3.9/6.0 (28) (31) (45) (40) (46) (34) (36) (47) (42) (48) (49) (49) (29) (45) (28) FTO rs9939609 (A-allele) TCF7L2 rs12255372 (T-allele) TCF7L2 rs7903146 (T-allele) The frequencies are presented as % [95% confidence interval]. Please cite this article as: de Melo SF, et al, Polymorphisms in FTO and TCF7L2 genes of Euro-Brazilian women with gestational diabetes, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.06.013 4 S.F. de Melo et al. / Clinical Biochemistry xxx (2015) xxx–xxx References [1] American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2013;36:S67–74. [2] Sociedade-Brasileira-de-Diabetes. Diretrizes da Sociedade Brasileira de Diabetes 2012–2013. Barueri, São Paulo: Guanabara Koogan; 2013. p. 388. [3] Metzger BE, Gabbe SG, Persson B, et al. International association of diabetes and pregnancy study groups recommendations on the diagnosis and classification of hyperglycemia in pregnancy. Diabetes Care 2010;33:676–82. [4] Buchanan TA, Xiang A, Kjos SL, Watanabe R. What is gestational diabetes? Diabetes Care 2007;30(Suppl. 2):S105–11. [5] Castorino K, Jovanovic L. Pregnancy and diabetes management: advances and controversies. Clin Chem 2011;57:221–30. [6] Lima WA, Glaner MF, Taylor AP. Fenótipo da gordura, fatores associados e o polimorfismo rs9939609 do gene FTO. Rev Bras Cineantropom Desempenho Hum 2010;12:164–72. [7] Labayen I, Ruiz JR, Ortega FB, et al. Association between the FTO rs9939609 polymorphism and leptin in European adolescents: a possible link with energy balance control. The HELENA study. Int J Obes (Lond) 2011;35:66–71. [8] Hotta K, Nakata Y, Matsuo T, et al. Variations in the FTO gene are associated with severe obesity in the Japanese. J Hum Genet 2008;53:546–53. [9] Li H, Wu Y, Loos RJ, et al. Variants in the fat mass- and obesity-associated (FTO) gene are not associated with obesity in a Chinese Han population. Diabetes 2008;57:264–8. [10] Ahmad T, Chasman DI, Mora S, et al. The fat-mass and obesity-associated (FTO) gene, physical activity, and risk of incident cardiovascular events in white women. Am Heart J 2010;160:1163–9. [11] Liu Y, Liu Z, Song Y, et al. Meta-analysis added power to identify variants in FTO associated with type 2 diabetes and obesity in the Asian population. Obesity (Silver Spring) 2010;18:1619–24. [12] Tong Y, Lin Y, Zhang Y, Yang J, Liu H, Zhang B. Association between TCF7L2 gene polymorphisms and susceptibility to type 2 diabetes mellitus: a large Human Genome Epidemiology (HuGE) review and meta-analysis. BMC Med Genet 2009;10:15. [13] Prestwich TC, Macdougald OA. Wnt/beta-catenin signaling in adipogenesis and metabolism. Curr Opin Cell Biol 2007;19:612–7. [14] Raitakari OT, Ronnemaa T, Huupponen R, et al. Variation of the transcription factor 7-like 2 (TCF7L2) gene predicts impaired fasting glucose in healthy young adults: the cardiovascular risk in young Finns study. Diabetes Care 2007;30:2299–301. [15] Yi F, Brubaker PL, Jin T. TCF-4 mediates cell type-specific regulation of proglucagon gene expression by beta-catenin and glycogen synthase kinase-3beta. J Biol Chem 2005;280:1457–64. [16] Ciccacci C, Di Fusco D, Cacciotti L, et al. TCF7L2 gene polymorphisms and type 2 diabetes: association with diabetic retinopathy and cardiovascular autonomic neuropathy. Acta Diabetol 2013;50:789–99. [17] Klein K, Haslinger P, Bancher-Todesca D, et al. Transcription factor 7-like 2 gene polymorphisms and gestational diabetes mellitus. J Matern Fetal Neonatal Med 2012;25:1783–6. [18] Zhang C, Bao W, Rong Y, et al. Genetic variants and the risk of gestational diabetes mellitus: a systematic review. Hum Reprod Update 2013;19:376–90. [19] American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2010;33(Suppl. 1):S62–9. [20] Lahiri DK, Nurnberger Jr JI. A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Res 1991;19:5444. [21] Bryson CL, Ioannou GN, Rulyak SJ, Critchlow C. Association between gestational diabetes and pregnancy-induced hypertension. Am J Epidemiol 2003;158: 1148–53. [22] Langer O, Yogev Y, Xenakis EM, Brustman L. Overweight and obese in gestational diabetes: the impact on pregnancy outcome. Am J Obstet Gynecol 2005;192:1768–76. [23] Lindheimer MD, Taler SJ, Cunningham FG. Hypertension in pregnancy. J Am Soc Hypertens 2008;2:484–94. [24] Chiang AN, Yang ML, Hung JH, Chou P, Shyn SK, Ng HT. Alterations of serum lipid levels and their biological relevances during and after pregnancy. Life Sci 1995;56: 2367–75. [25] Toescu V, Nuttall SL, Martin U, et al. Changes in plasma lipids and markers of oxidative stress in normal pregnancy and pregnancies complicated by diabetes. Clin Sci (Lond) 2004;106:93–8. [26] McGrowder D, Grant K, Irving R, et al. Lipid profile and clinical characteristics of women with gestational diabetes mellitus and preeclampsia. J Med Biochim 2009; 28:72–81. [27] Ramya K, Radha V, Ghosh S, Majumder PP, Mohan V. Genetic variations in the FTO gene are associated with type 2 diabetes and obesity in south Indians (CURES-79). Diabetes Technol Ther 2011;13:33–42. [28] Cho YM, Kim TH, Lim S, et al. Type 2 diabetes-associated genetic variants discovered in the recent genome-wide association studies are related to gestational diabetes mellitus in the Korean population. Diabetologia 2009;52:253–61. [29] Barra GB, Dutra LA, Watanabe SC, et al. Association of the rs7903146 single nucleotide polymorphism at the transcription factor 7-like 2 (TCF7L2) locus with type 2 diabetes in Brazilian subjects. Arq Bras Endocrinol Metabol 2012;56:479–84. [30] Scott LJ, Bonnycastle LL, Willer CJ, et al. Association of transcription factor 7-like 2 (TCF7L2) variants with type 2 diabetes in a Finnish sample. Diabetes 2006;55: 2649–53. [31] Ekelund M, Shaat N, Almgren P, et al. Genetic prediction of postpartum diabetes in women with gestational diabetes mellitus. Diabetes Res Clin Pract 2012;97:394–8. [32] Watanabe RM, Black MH, Xiang AH, Allayee H, Lawrence JM, Buchanan TA. Genetics of gestational diabetes mellitus and type 2 diabetes. Diabetes Care 2007;30(Suppl. 2):S134–40. [33] Shaat N, Lernmark A, Karlsson E, et al. A variant in the transcription factor 7-like 2 (TCF7L2) gene is associated with an increased risk of gestational diabetes mellitus. Diabetologia 2007;50:972–9. [34] Marquezine GF, Pereira AC, Sousa AG, Mill JG, Hueb WA, Krieger JE. TCF7L2 variant genotypes and type 2 diabetes risk in Brazil: significant association, but not a significant tool for risk stratification in the general population. BMC Med Genet 2008;9: 106. Please cite this article as: de Melo SF, et al, Polymorphisms in FTO and TCF7L2 genes of Euro-Brazilian women with gestational diabetes, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.06.013