evaluation of tcf7l2 and ppar-γ gene polymorphisms and insulin
Transkript
evaluation of tcf7l2 and ppar-γ gene polymorphisms and insulin
Acta Medica Mediterranea, 2014, 30: 1203 EVALUATION OF TCF7L2 AND PPAR-Γ GENE POLYMORPHISMS AND INSULIN SECRETION ABILITIES IN PATIENTS WITH TYPE II DIABETES AND THEIR UNAFFECTED SIBLINGS KEMAL TÜRKER1, MEHMET NUMAN TAMER2, OĞUZHAN AKSU2, BANU KALE KÖROĞLU2, ISMAIL HAKKI ERSOY2, RAMAZAN YILMAZ3, EMEL BARAN4, RECEP SÜTCÜ5, SITKI ÖZTAS6 1 Faculty of Medicine, Suleyman Demirel University, Department of Internal Medicine Isparta - 2Faculty of Medicine, Suleyman Demirel University, Department of Internal Medicine, Division of Endocrinology and Metabolism, Isparta - 3Faculty of Medicine, Mevlana University, Medical Biology, Konya - 4Faculty of Medicine, Suleyman Demirel University, Division of Endocrinology and Metabolism, Diabetes nurse, Isparta - 5Faculty of Medicine, Katip Çelebi University, Department of Biochemistry, Izmir - 6Faculty of Medicine, Suleyman Demirel University, Department of Genetics, Isparta, Turkey ABSTRACT Peroxisome proliferator activated receptor-γ (PPAR-γ) and transcription factor 7-like 2 (TCF7L2) gene polymorphisms are some of the genetic variants that are commonly held responsible for the genetic pathogenesis of type 2 Diabetes Mellitus (T2DM ).In this study the genetic polymorphisms of TCF7L2 and PPAR-γ genes in affected siblings of families with rich diabetic history and their unaffected siblings who are thought to be resistant to diabetes development were evaluated. 28 T2DM patient group (Group 1), 27 siblings who still have not developed T2DM 10 years after the earliest T2DM diagnosis age in the family (Group 2) and 20 healthy individuals who do not have T2DM and no history in the first degree relatives (Group 3). PPAR-γ Pro12Ala and TCF7L2 gene polymorphisms (rs12255372 and rs7903146) were searched for in these groups. Moreover, oral glucose tests and insulin secreting abilities of individuals in group 2 were investigated. PPAR-γ Pro12Ala gene polymorphism was not detected in any of the patients in our study group. There were no significant differences between group 1 and 2 regarding TCF7L2 gene polymorphism genotyping. TT genotype and T allele ratio was less in group 3 when compared to other groups but this was not statistically significant.As a result, it was observed that PPAR-γ Pro12Ala polymorphisms do not have an affect on T2DM development. However, TCF7L2 gene polymorphisms, although not a strong determinant, may play a role in diabetes along with other genetic and environmental factors. Key words:Type 2 Diabetes Mellitus, PPAR-γ, TCF7L2, polymorphism Received February 18, 2014; Accepted June 19, 2014 Introduction Diabetes mellitus (DM) is a metabolic disease that results from insulin resistance and / or insulin deficiency that is characterized by hyperglycemia(1). Insulin resistance in peripheric tissues (muscle, liver, adipose) and disorder of insulin secretion from pancreatic beta cells play a role in the pathogenesis of T2DM(2). These disorders have a role in DM pathogenesis and DM development occurs with the interaction of genetic and environmetal factors(3) (fig. 1). Obesity, hypertension, dyslipidemia, insulin resistance, sedantary life style and family history increase the risk of DM development(3). In individuals who are genetically vulnerable to insulin secretion disorder and insulin resistance, DM slowly develops in existence of environmental factors(4). Apart from rare monogenic DM forms, T2DM is mostly polygenic(4). The mutations and polymorphisms in the genes encoding the proteins that play a role in events such as insulin resistance, insulin secretion and glucose transport that are crucial in the pathogenesis of DM pathogenesis are the genet- 1204 ic basis of DM(4). The functional candidate genes in T2DM are genes that are contributing to insulin’s effects(insulin receptor, insulin receptor substrates (IRS), PI-3 Kinase, glucose transport and metabolism), genes that are participating to insulin secretion (GLUT2, glucokinase, ion channels, preproinsülin, β cell transcription factors), and obesity genes (2-4). Proline-alanine substitution in the 12th codon of PPAR-γ gene (Pro12Ala) is one of the first genetic polymorphisms shown to be linked to T2DM(5-7). In this study, the aim was to investigate the TCF7L2 and PPAR-γ gene polymorphisms of T2DM patients with rich DM family history, their unaffected siblings and healthy control group individuals and also to evaluate the relation between insulin resistance, insulin secretion, pancreatic capacity and these polymorphisms. Kemal Türker,Mehmet Numan Tamer et Al the vertical line that goes to iliac crista from the lateral side of the costal arch, was measured during expiration(10). DNA Isolation from Peripheral Blood QIAGEN DNA isolation kit was used for isolating DNA. TCF7L2 (rs12255372 and rs7903146) ve PPAR-γ (Pro12Ala) genes were amplified by PCR by Bioer ProGen device using the obtained genomic DNA. DNA segments amplified by PCR were compared with restriction fragment lenghts and mutations were identified (for example figure 1). Following PCR, 295 bp fragments belonging to PPAR-γ and 119 and 113 bp fragments belonging to TCF7L2 were identified. Methods and methods Selection of Patient and Control Groups Surveys were done on 295 T2DM patients and their family and diabetes histories were recorded. 3 study groups were constructed. Group 1: Diabetic index patients with three or more first degree relative with T2DM (diabetes rich family) and the first degree relatives of these patients that could be summoned. Group 2: The siblings of the index patients who have not developed T2DM although they are 10 years older than the age in which the individual who got the earliest T2DM diagnosis was. Group 3: Healthy individuals who do not have diabetes and no history in the first degree relatives. DM in individuals in group 3 were excluded by oral glucose tolerance test (OGTT). 0, 30 and 120 minute blood sugar(8) and insulin levels (for measurement of beta cell function and insulin resistance) were recorded during OGTT. In those thought to be resistant to diabetes development, HOMA-IR, HOMA-B cell and insulionogenic index (30th minute) levels were calculated(9). The diagnosis of diabetic patients were ascertained by fasting blood sugar (FBS) and HbA1c. All relatives, regardless of T2DM, were checked for serum lipid profiles. Control group individuals who did not have DM history in the family as well were analyzed by FBS and DM was excluded. Anthropometric measurements and BMI parameters of DM and non DM siblings in the study were calculated by body mass kg/height² formula. Waist circumference, the diameter that crosses the middle of Fig. 1: ????????? Quantitatıve measurements Insulin releasing abilities of individuals in group 2 were assessed according to HOMA B-cell (HOMA-B-cell: 20 x fasting plasma insulin level (µU/ml) / (fasting plasma glucose level (mmol/L)3,5)) and insulinogenic index (30th minute: (Insulin 30-Insulin 0) / (glucose 30-glucose 0)) measurements and HOMA-IR (HOMA–IR=[fasting insulin(lU/mL) x fasting glucose (mmol/L)] / 22.5) measurements were used to assess insulin resistanc(11). The statistical analysis of the study was done using SPSS version 15.0. Constant variables were expressed in arithmetrical average ± standart deviation, while categorical variables were expressed as %’s. Mann Whitney U test was used in group comparsion of constant variables. Chi square test was used in the group comparison of categorical comparison. In the presence of more than two groups Kruskal - Wallis test was used. Results Demographic Characteristics of Patients 28 patients from group 1, 27 from group 2 and 20 from group 3 were included in this study. There were no statistically significant differences between Evaluation of TCF7L2 and PPAR-γ Gene Polymorphisms and insulin secretion abilities in patients with type II... the first and second groups regarding age and gender (p>0,05). TCF7L2 gene’s rs12255372 polymorphism genotype distribution between the groups were shown in Table 1. When all three groups were compared in regard to their TCF7L2 gene rs12255372 genotype distributions, it was observed that there were no significant differences between the groups regarding GG, GT and TT genotypes (p > 0,05). Moreover, when the groups were compared two by two there were no significant differences as well (p>0,05) (Table 2). Group 1 Group 2 Group 3 n % N % N % C allele 36 64,2 36 66,6 29 T allele 20 35,7 18 33,3 11 1205 P1 P2 P3 P4 72,5 0,729 0,309 0,501 0,594 27,5 0,729 0,309 0,501 0,594 Table 4: TCF7L2 gene’s rs7903146 polymorphism allele frequencies. P1 stands for the relevance values between group 1 and 2 . P2 is for group 1 and 3. P3 is for group 2 and 3. P4 is for all groups. pared to others, there were no statistically significant differences in G and T allele frequency among all three groups (p>0,05). TCF7L2 gene’s Group 1 Group 2 Group 3 P1 P2 P3 P4 rs7903146 polymorphism genotype distribution n % N % N % assesment between the three groups were shown Rs in table 4. When all three groups were compared 12255372 12 42,9 10 37 9 45 0,660 0,883 0,582 0,842 GG genotype according to their TCF7L2 gene rs7903146 genoRs type distributions, there were no significant differ12255372 12 42,9 13 48,1 10 50 0,694 0,624 0,900 0,871 GT genotype ences observed in their CC, CT and TT genotype Rs frequencies (p>0,05). 12255372 4 14,3 4 14,8 1 5 0,956 0,299 0,281 0,530 TT genotype Moreover, there were no significant differences observed when all three groups were comTable 1: TCF7L2 gene’s rs12255372 polymorphism genotype pared two by two (p>0,05), (Table 3). frequencies. When TCF7L2 gene’s rs7903146 polymorP1 stands for the relevance values between group 1 and 2. P2 is for group 1 and 3. P3 is for group 2 and 3. P4 is for all groups. phism was evaluated for alleles, there was a intergroup allele frequency distribution was obtained Group 1 Group 2 Group 3 which is shown in table 4. Although T allele freP1 P2 P3 P4 quency was less in group 3, there were no signifin % N % N % cant differences between the three groups in G allele 36 64,2 33 61,1 28 70 0,713 0,644 0,404 0,715 regard to C and T allele frequency (p>0,05) (Table T allele 20 35,7 21 38,8 12 30 0,713 0,644 0,404 0,715 4). When TCF7L2 gene’s rs12255372 and Table 2: TCF7L2 gene’s rs12255372 polymorphism’s allele rs7903146 polymorphisms were evaluated in non diabetic siblings for insulin secretion abilities, frequencies. P1 stands for the relevance values between group 1 and 2. P2 is for there were no significant differences between the group 1 and 3. P3 is for group 2 and 3. P4 is for all groups. two groups in parameters (age, gender, BMI, HOMA - β cell, insulin index). Group 1 Group 2 Group 3 P1 P2 P3 P4 45 0,703 0,364 0,582 0,662 55 0,701 0,517 0,770 0,807 N % N % N % rs7903146CC genotype 9 32,1 10 37 9 rs7903146CT genotype 18 64,3 16 59,3 11 Discussion T2DM is a metabolic disease that develops due to insulin resistance and / or insulin deficiency by interaction of genetic and environmental factors. It rs7903146TT 1 3,6 1 3,7 0 0 0,979 0,393 0,384 0,688 genotype was reported that parameters such as BMI, HOMATable 3: TCF7L2 rs7903146 polymorphism’s genotype fre- IR, LDL, triglyceride are inclined tro be higher in quencies. individuals with T2DM family history compared to P1 stands for the relevance values between group 1 and 2. P2 is for those who do not have any family history (12) . group 1 and 3. P3 is for group 2 and 3. P4 is for all groups. Although there were no significant differences in BMI of individuals with diabetes and a rich family When TCF7L2 gene’s rs12255372 polymorhistory and their unaffected siblings, total cholestrol phism was assessed in regard to its alleles, an allele and LDL levels of non diabetic siblings have been frequency distribution was reached which is shown found to be significantly higher when compared to in table 2. Although G allele was more frequent and the diabetic group. The fact that same family memT allele less frequent in the third group when combers carry the same genetic information may be a 1206 reason for this finding. The fact that lipid levels in the diabetic group was lower was attributed to the fact that this group was under control and using hyperlipidemic drugs (56%). Moreover, lipid profile is a parameter that can be affected by smoking, diet, exercise and hyperlipidemia drugs. These parameters were not taken into account in our study, and this is a weak spot for lipid profile asssesment. Unaffected siblings with family history had average HOMA-IR levels of 2.5 as expected. HOMA-IR levels of healthy individuals with no family history of T2DM were not checked for comparison and this may seem as incomplete, however since the aim of the study was to ascertain any relationship between genetic polymorphism and IR, this kind of a road map was chosen. The effects of PPAR- γ gene’s Pro12Ala polymorphism on IR, lipid metabolism and BMI are under the control of other factors as well and pretty complicated. PPAR- γ gene’s Pro12Ala polymorphism has been found to be associated with T2DM in many studies(13-15). On the contrary, in a study by Hasstedt S.J. et al in which relatives of type II diabetes patients took part Pro12Ala polymorphism was shown to have no effect on diabetes development(16). In a study conducted by Bendlová B. et al, there were no differences in glucose, lipid profile parameters and Pro12 Ala polymorphisms between Czech individuals with and without T2DM family history when corrected according to their BMIs(17). In the metaanalysis assessement by Tönjes et al which consisted of 57 studies, there were no relations between Pro12Ala polymorphism and BMI, insulin, HOMA-IR and glucose in the nondiabetic population(18). Pro12Ala polymorphism frequency differs by population and race. There is no extensive study that investigates Pro12Ala polymorphism rate and effects in the Turkish population. In the study of Tok et al, Pro12Ala polymorphism was found in 19.4% of mothers with gestational diabetes and in 16% of non diabetic pregnancies(19). In Zengi et al’s study in which carotis intima media thickness, metabolic parameters and PPARγ gene Pro12Ala polymorphism rate was compared in the non diabetic first degree relatives of type 2 diabetic patients, Pro12Ala polymorphism rate was found to be 9.9% in the non diabetic group with negative family history while this rate was 14.6 % in the non diabetic group with positive family history(20). Also, there were no significant correlations between Pro12Ala polymorphisms and metabolic parameters. Kemal Türker,Mehmet Numan Tamer et Al The fact that there were no significant correlations between Pro12Ala polymorphisms and metabolic parameters in our study is consistent with the previous studies. We have not observed any Pro12Ala polymorphisms in all our groups and this may be due to our limited study group. Lately, it is postulated that beta cell secretion (insulin secretion) defect is an important genetic pathogenic factor in the T2DM ethiopathogenesis in addition to ID(21-23). In a study in which genotype scoring was performed on 2377 individuals, 18 SNPs including rs7903146 was checked. rs7903146 polymorphism was found to be associated with T2DM development(3). In another study which comprised 17,284 Swedish and 2770 Finnish individuals that had an average follow up of 23,5 years, presence of T2DM was found to be correlated with family history, increased BMI, smoking, elevated blood pressure and triglyceride levels. 11 gene variants including TCF7L2 and PPAR - γ were examined. All 11 genes were found to have correlation with T2DM development, 8 gene variants, including TCF7L2, were observed to be related to decreased insulin secretion(24). In all the groups of our study, rs7903146 and rs12255372polymorphism and genotyping in TCF7L2 gene, which was found to be influential in insulin secretion in all previous studies, was performed. There were no significant differences between groups 1 and 2 regarding genotype. In the third group, genotype frequencies of TT and CT / GT were found to be lower than the other groups but this decrease was not statistically significant. When the allele distribution was viewed from the perspective of rs12255372 and rs7903146,no differences were observed in groups 1 and 2, but T allele rate was found to be lower in the third group compared to other groups however this was not statistically significant. This may be becaus of the limited number of individuals in the study group. The fact that T allele is decreased in non diabetic individuals with negative family histories when compared to diabetic and non diabetic individuals with positive family history underlines the importance ofTCF7L2 gene polymorphisms in the development of type II diabetes, and this finding is consistent with previous studies(25-27). However, similar T allele distribution in the diabetic relatives and the non diabetic individuals with positive family history shows that TCF7L2 gene polymorphisms are not solely adequate for the Evaluation of TCF7L2 and PPAR-γ Gene Polymorphisms and insulin secretion abilities in patients with type II... development of diabetes and that it may evolve with the interaction of genetic and environmental factors. As a result, there were no differences in PPAR-γ gene Pro12Ala and TCF7L2 geners7903146 and rs12255372polymorphisms between diabetic individuals from rich diabetes families and their unaffected siblings. The authors state that more extensive studies should be conducted in this field. References 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) Bennett PH, Knowler W.C. In: Joslin E.P. KCR Joslin’s diabetes mellitus. 14th ed. Philadelphia, Pa: Lippincott Williams & Willkins, 2005: 331. Barroso I, Luan J, Middelberg RP, Harding AH, Franks PW et al. Candidate gene association study in type 2 diabetes indicates a role for genes involved in beta-cell function as well as insulin action. PLoS Biol (2003); 1: 20. Meigs JB, Shrader P, Sullivan LM, McAteer JB, Fox CS et al. Genotype score in addition to common risk factors for prediction of type 2 diabetes. N Engl J Med (2008); 359: 2208-2219. Owen KR, McCarthy MI. Genetics of type 2 diabetes. Curr Opin Genet Dev (2007); 17: 239-244. Malecki MT. Genetics of type 2 diabetes mellitus. Diabetes Res Clin Pract (2005); 68: 10-21. Fujimoto WY, Bergstrom RW, Boyko EJ et al. Diabetes and diabetes risk factors in second- and third-generation Japanese Americans in Seattle, Washington. Diabetes Res Clin Pract (1994); 24: 43-52. Grant RW, Moore AF, Florez JC. Genetic architecture of type 2 diabetes: recent progress and clinical implications. Diabetes Care (2009); 32: 1107-1114. Bianchi C, Miccoli R, Bonadonna RC, Giorgino F, Frontoni S et al. 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Metabolism (2003); 52: 443-450. Ringel J, Engeli S, Distler A, Sharma AM. Pro12Ala missense mutation of the peroxisome proliferator activated receptor gamma and diabetes mellitus. Biochem Biophys Res Commun (1999); 254: 450-453. 14) 1207 Nolan JJ, Ludvik B, Beerdsen P, Joyce M, Olefsky J. Improvement in glucose tolerance and insulin resistance in obese subjects treated with troglitazone. N Engl J Med, (1994); 331: 1188-1193. 15) Stumvoll M, Haring H. The peroxisome proliferatoractivated receptor-gamma2 Pro12Ala polymorphism. Diabetes (2002); 51: 2341-2347. 16) Hasstedt SJ, Ren QF, Teng K, Elbein SC. Effect of the peroxisome proliferator-activated receptor-gamma 2 pro(12)ala variant on obesity, glucose homeostasis, and blood pressure in members of familial type 2 diabetic kindreds. J Clin Endocrinol Metab (2001), 86: 536-541. 17) Bendlova B, Vejrazkova D, Vcelak J et al. PPARgamma2 Pro12Ala polymorphism in relation to free fatty acids concentration and composition in lean healthy Czech individuals with and without family history of diabetes type 2. Physiol Res (2008); 57: 77-90. 18) Tonjes A, Scholz M, Loeffler M, Stumvoll M. Association of Pro12Ala polymorphism in peroxisome proliferator-activated receptor gamma with Pre-diabetic phenotypes: meta-analysis of 57 studies on nondiabetic individuals. Diabetes Care (2006); 29: 2489-2497. 19) Tok EC, Ertunc D, Bilgin O, Erdal EM, Kaplanoglu M, Dilek S. PPAR-gamma2 Pro12Ala polymorphism is associated with weight gain in women with gestational diabetes mellitus. Eur J Obstet Gynecol Reprod Biol (2006), 129: 25-30. 20) Zengi A, Saygılı, F. Non-diabeticfirst degree relatives of type 2 diabetics Pro12Ala PPAR-γ gene polymorphisms and carotid intima-media thickness with the metabolic parameters investigated 2009 2) İmamoğlu Ş, Ersoy C.Ö Diabetes Mellitus, Multidisciplinary Approaches toDiagnosis, Treatment, and Follow-up. Istanbul:DeomedPublishing (2009): 54-68. 22) Gloyn AL, McCarthy MI The genetics of type 2 diabetes. Best Pract Res Clin Endocrinol Metab (2001), 15: 293-308. 23) Stumvoll M, Fritsche A, Haring H. The OGTT as test for beta cell function? Eur J Clin Invest (2001), 31: 380-381. 24) Lyssenko V, Jonsson A, Almgren P, Pulizzi N, Isomaa B et al. Clinical risk factors, DNA variants, and the development of type 2 diabetes. N Engl J Med (2008); 359: 2220-2232. 25) Florez JC, Jablonski KA, Bayley N, Pollin TI, de Bakker PI et al. TCF7L2 polymorphisms and progression to diabetes in the Diabetes Prevention Program. N Engl J Med (2006); 355: 241-250. 26) Hu C, Zhang R, Wang C, Wang J, Ma X et al. Variants from GIPR, TCF7L2, DGKB, MADD, CRY2, GLIS3, PROX1, SLC30A8 and IGF1 are associated with glucose metabolism in the Chinese. PLOS One (2010), 5: 15542. 27) Lyssenko V, Lupi R, Marchetti P, Del Guerra S, OrhoMelander M et al. Mechanisms by which common variants in the TCF7L2 gene increase risk of type 2 diabetes. J Clin Invest (2007), 117: 2155-2163. _________ Corresponding Author Dr. OĞUZHAN AKSU Department of Endocrinology and Metabolism, Suleyman Demirel University, School of Medicine 3200, Isparta (Turkey)
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gene polymorphisms (rs12255372 and rs7903146) were searched for in these groups. Moreover, oral glucose tests and insulin secreting abilities of individuals in group 2 were investigated. PPAR-γ Pro...
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The effects of PPAR- γ gene’s Pro12Ala polymorphism on IR, lipid metabolism and BMI are
under the control of other factors as well and pretty
complicated. PPAR- γ gene’s Pro12Ala polymorphism has b...
031402 Prevalence of Impaired Glucose Tolerance
Group 1: Diabetic index patients with three or
more first degree relative with T2DM (diabetes rich
family) and the first degree relatives of these
patients that could be summoned.
Group 2: The sibl...