Can J Diabetes xxx (2015) 1e6
Contents lists available at ScienceDirect
Canadian Journal of Diabetes journal homepage: www.canadianjournalofdiabetes.com
Original Research
ABCG5 and ABCG8 Gene Polymorphisms in Type 2 Diabetes Mellitus in the Turkish Population Ozlem Gok MSc a, b, Zeynep Ermis Karaali MD c, Leyla Acar BSc a, Ulkan Kilic PhD, PhD b, Arzu Ergen PhD a, * a b c
Department of Molecular Medicine, Institute for Experimental Medicine, Istanbul University, Istanbul, Turkey Department of Medical Biology and Regenerative and Restorative Medicine Research Center (REMER), Faculty of Medicine, Istanbul Medipol University, Istanbul, Turkey Department of Internal Medicine, Haseki Training and Research Hospital, Istanbul, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 August 2014 Received in revised form 23 March 2015 Accepted 9 April 2015 Available online xxx
Objective: The aim of the present study was to investigate the relationship between ABCG5 and ABCG8 gene polymorphisms and plasma lipid concentrations in Turkish patients with type 2 diabetes mellitus. Methods: Included in this study were 80 patients with type 2 diabetes and 73 healthy controls. Two selected single nucleotide polymorphisms in ABC transporter genes, ABCG5 (rs6720173) and ABCG8 (rs4148211), were genotyped by using the polymerase chain reaction-restriction fragment length polymorphism technique. Results: The rate of having the ABCG8 AA genotype (p¼0.001) was significantly higher in the patients than in the control subjects. Correspondingly, the rates of having the AG genotype (p¼0.001) and the G allele (p¼0.001) were significantly lower in the patients than in controls. Upon comparing the groups regarding ABCG5, the frequencies of occurrence of the GG genotype (p¼0.031) and G allele (p¼0.003) were considerably higher in patients than in control subjects. In the patients, the rates of having the CC genotype (p¼0.003) and the C allele (p¼0.031) were also significantly lower than those in control subjects. There was no significant difference between G5 and G8 polymorphism and lipid levels in the study groups. The ABCG8 AA genotype carriers had higher triglyceride (p¼0.045) and very low-density-cholesterol (p¼0.045) levels than the ABCG8 GG genotype carriers in all study populations. Conclusions: These results indicate that the AA genotype for ABCG8 and the GG genotype and G allele for ABCG5 are risk factors for diabetes. This study reveals the first data concerning the ABCG5 and ABCG8 gene polymorphisms in Turkish patients with diabetes. Ó 2015 Canadian Diabetes Association
Keywords: ABCG5 ABCG8 diabetes mellitus lipid metabolism polymorphism
r é s u m é Mots clés : ABCG5 ABCG8 diabète sucré métabolisme des lipides polymorphisme
Objectif : Le but de la présente étude était d’étudier la relation entre les polymorphismes des gènes ABCG5 et ABCG8 et les concentrations plasmatiques de lipides chez les patients turcs souffrant du diabète sucré de type 2. Méthodes : Cette étude comprenait 80 patients souffrant du diabète de type 2 et 73 témoins en santé. Deux polymorphismes mononucléotidiques sélectionnés des gènes codant pour les transporteurs ABC, l’ABCG5 (rs6720173) et l’ABCG8 (rs4148211), ont été génotypés par la technique de réaction en chaîne de la polyméraseepolymorphisme de longueur des fragments de restriction. Résultats : Le risque d’être porteur du génotype AA du ABCG8 (p¼0,001) était significativement plus élevé chez les sujets témoins. Parallèlement, les risques d’être porteur du génotype AG (p¼0,001 et de l’allèle G (p¼0,001) étaient significativement plus faibles chez les patients que chez les témoins. En comparant les groupes au sujet de l’ABCG5, la fréquence du génotype GG (p¼0,031) et de l’allèle G (p¼0,003) était considérablement plus élevée chez les patients que les sujets témoins. De plus, les risques que les patients soient porteurs du génotype CC (p¼0,003) et de l’allèle C (p¼0,031) étaient significativement plus faibles que ceux des sujets témoins. Il n’existait aucune différence significative entre les polymorphismes G5 et G8 et les concentrations de lipides dans les groupes étudiés. Dans toutes les populations faisant l’objet de l’étude, les porteurs du génotype AA du ABCG8 avaient des concentrations plus élevées de
* Address for correspondence: Arzu Ergen, Department of Molecular Medicine, The Institute of Experimental Medicine, Istanbul University, Vakif Gureba Cad., Sehremini-Fatih TR-34093 Istanbul, Turkey. E-mail address: [email protected] 1499-2671/$ e see front matter Ó 2015 Canadian Diabetes Association http://dx.doi.org/10.1016/j.jcjd.2015.04.004
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triglycérides (p¼0,045) et de cholestérol à lipoprotéines de très basse densité (p¼0,045) que les porteurs du génotype GG du ABCG8. Conclusions : Ces résultats indiquent que le génotype AA du ABCG8 et que le génotype GG et l’allèle G du ABCG5 sont des facteurs de risque de diabète. Cette étude révèle les premières données concernant les polymorphismes des gènes ABCG5 et ABCG8 chez les patients turcs souffrant du diabète. Ó 2015 Canadian Diabetes Association
Introduction Diabetes mellitus is a multisystemic disorder of carbohydrate, protein and fat metabolism that is affected by both genetics and environmental factors. Although there are several types of the disease, 2 main types, namely insulin dependent (type 1 diabetes) and non-insulin dependent diabetes (type 2 diabetes), are the most common types. Diabetes disturbs the life quality of patients by causing serious health problems and complications, such as retinopathy, neuropathy, fatigue, weight loss, micro- and macrovascular abnormalities, nephropathy and several kinds of cardiovascular disorders, such as hypertension and atherosclerosis (1e6). Type 2 diabetes also causes dyslipidemia, which is characterized by high levels of triglyceride (hypertriglyceridemia) and low-density lipoprotein (LDL) (7,8), and shows close interaction with the risk for coronary artery disease (9). However, the pathogenesis and genetic mechanisms of diabetes are still unclear (10,11). Adenotriphosphate (ATP)-binding cassette (ABC) transporters, the most common type of transmembrane proteins, coded by 49 genes, have been subdivided into 8 subgroups: 1) A-G, based on sequence similarity and domain organization; 2) ABCA, 12 members; 3) ABCB, 11 members; 4) ABCC 13 members; 5) ABCD, 4 members; 6) ABCE, 1 member; 7) ABCF, 3 members; 8) ABCG, 5 members). In particular, these transmembrane proteins cross the membrane in an ATP-dependent manner and transport specific substrates, such as lipids, sterols and metabolic products. ABC transporters are expressed in liver, kidney, testis, intestine, ovary, lung, tumour cell, blood-brain barrier and placenta. Eukaryotic ABC transporters have been subdivided into being either full or half transporters. Full transporters contain 2 nucleotide-binding domains (NBDs) and two 6-transmembrane helices, namely transmembrane domains (TMDs). Some full transporters have additional transmembrane helices at the amino terminus. However, half transporters contain 1 NBD and 1 TMD on a single polypeptide (12e15). Human ABCG subfamily proteins are half-type ABC proteins and contain cytosolic N-terminal NBD and C-terminal TMD. They contain 5 members: ABCG1, ABCG2, ABCG4, ABCG5 and ABCG8, which transport sterols and xenobiotics. ABCG1, ABCG2 and ABCG4 function as homodimers on the plasma membrane. Correspondingly, ABCG5 and ABCG8 function as heterodimers on the plasma membrane and homodimers in the endoplasmic reticulum (12e18). ABCG5 and ABCG8 are located in head-to-head orientation of exon 13 of chromosome 2p21 (PUBMED, gene) and are expressed in both the liver and the intestine (19e21). Previous studies have reported that mutations in either ABCG5 or ABCG8 cause sitosterolemia, a rare lipid disorder that is characterized by high intestinal absorption of all sterols (cholesterol, plant sterols and shellfish sterols) (15,19,22e25). There are limited number of studies of the roles of ABCG5 (Gln604Glu¼C1810G, rs6720173) and ABCG8 (Tyr54Cys¼A161G, rs4148211) gene polymorphisms on the plasma cholesterol levels and familial hypercholesterolemia (21). There is no investigation related to ABCG5 Gln604Glu and ABCG8 Tyr54Cys single nucleotide polymorphisms (SNPs) and lipid levels in patients with diabetes. According to this knowledge, we aimed to investigate the
association between ABCG5 and ABCG8 gene polymorphisms and lipid levels in Turkish patients with type 2 diabetes as compared to controls.
Methods Study groups The experimental group consisted of 80 patients (59 female and 21 male, mean age 57.6414.23) diagnosed with type 2 diabetes who were in the follow-up group studied by the Istanbul University’s Faculty of Medicine, Department of Endocrinology. The control group included 73 randomly selected healthy individuals (26 female, 47 male; mean age 53.7710.54) who had no signs of coronary artery disease, hypertension, lipid metabolic disorders or cardiovascular diseases. The controls enrolled in this study were selected from a group of people without family histories of diabetes. This study was approved by the ethical committee of Istanbul University, Faculty of Medicine. All participants, after giving written informed consent, completed a structured questionnaire in order to collect demographic data. The study was conducted in accordance with the ethical principles described by the Declaration of Helsinki.
Biochemical and demographic analysis After 12 hours of fasting, blood samples of the participants were taken into plain tubes and tubes with ethylenediaminetetraacetic acid (EDTA) (Vacuette; Greiner Labortechnick, Frickenhausen, Germany). The samples were centrifuged for 10 minutes at 1.500 g at þ4 C, followed by the removal of serum and plasma and then were stored at e20 C. Fasting glucose, total cholesterol, triglyceride, high-density lipoprotein (HDL) cholesterol, lowdensity lipoprotein (LDL) cholesterol and very low-density lipoprotein (VLDL) cholesterol were determined in the study groups by standard laboratory methods in the Istanbul University and Haseki Training and Research hospitals (26,27). In addition, body mass index (BMI; weight in kilograms divided by the square of the height in meters) values were calculated and categorized according to World Health Organization recommendations. The smoking status of an individual was assigned as yes if he or she was smoking currently or had given up smoking 0.05). However, the BMIs (p¼0.018) were significantly higher in the subjects with the ABCG5 CC genotype (26.53 2.81) than in the subjects with the ABCG5 GG genotype (23.36 3.30) in the control group (Table 4). In the total group, HDL cholesterol levels (p¼0.025) were significantly higher in the female subjects with the ABCG5 CC genotype than in the female subjects with the ABCG5 CG genotype. Triglyceride and VLDL cholesterol levels (p¼0.045) were considerably higher in the subjects with the ABCG8 AA genotype than in the subjects with the ABCG8 GG genotype in the total group (Table 5). Discussion Type 2 diabetes mellitus is associated with gene-environment interactions due to epigenetic factors, such as defects in lipid metabolism, hyperlipidemia, hypertension, obesity, smoking and
O. Gok et al. / Can J Diabetes xxx (2015) 1e6
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Table 5 Comparison of ABCG5 Gln604Glu and ABCG8 Tyr54Cys genotypes with lipid/anthropometric parameters in all study populations ABCG5 Gln604Glu genotype
Triglyceride (mg/dL) Total cholesterol (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) VLDL cholesterol (mg/dL) Total cholesterol /HDL cholesterol (mg/dL) BMI (kg/m2)
ABCG8 Tyr54Cys genotype
CC (n¼73)
CG (n¼25)
CG (n¼55)
p
AA (n¼60)
GG (n¼19)
AG (n¼74)
p
154.3866.74 209.8845.87 38.229.64 140.7842.62 30.8813.34 5.852.24 26.353.63
161.7664.64 223.3158.29 37.168.49 153.8053.32 32.3512.92 6.422.53 25.853.76
152.0268.73 210.3750.92 35.588.63 144.3847.22 30.4013.74 6.282.31 26.483.83
NS NS NS NS NS NS NS
169.3080.27* 216.0956.92 38.038.58 144.2052.66 33.8616.05* 6.062.48 26.493.90
134.1141.37 212.2442.15 37.7412.50 147.6845.52 26.828.27 6.343.05 25.244.28
148.2357.67 209.1345.63 36.188.59 143.3141.64 29.6511.53 6.061.96 26.453.38
0.045 NS NS NS 0.045 NS NS
HDL, High density lipoprotein; LDL, low density lipoprotein; n, number of individuals; NS, not significant; SD, standard deviation; VLDL, very low-density lipoprotein; BMI, body mass index. Statistical evaluation was made by using the Student t test and the 1-way ANOVA test. The results are shown as mean SD. * p0.05). ABCG5 and ABCG8 genes have important effects on the regulation of lipid metabolism. These genes are involved not only in cholesterol absorption but also in cholesterol excretion (16,21,26,31). Santosa et al (31) observed that weight loss and defects in cholesterol metabolism were related to ABCG5/8 polymorphisms. Hubajeck et al (21) reported that total cholesterol and LDL cholesterol levels were low for ABCG8 Tyr54Cys only in female subjects. In addition, they observed no association between lipid profiles and genotype distribution and allele frequencies for ABCG5 Gln604Glu polymorphism in both genders of the study population (131 male and 154 female). Similar results were demonstrated in the study by Jakulj et al (37), which was performed for ABCG5 and ABCG8 SNPs in 245 patients with hypercholesterolemia. Because of the failure to find any association between G5/G8 and plasma sterol levels, they pooled their data for a meta-analysis of 3364 individuals from 16 studies. However, they did not find any association between plasma lipid levels and ABCG5/8. In this study, we similarly observed that there was no significant association between ABCG5 and ABCG8 genotypes and lipid parameters. Garcia Rios et al (38) genotyped ABCG5 and ABCG8 SNPs in 500 individuals with genetic diagnoses of familial hypercholesterolemia. In particular, carriers of the G allele had considerably lower VLDL cholesterol (p¼0.011) and triglyceride (p¼0.017) levels than carriers of the CC genotype for ABCG5_Gln604GluC>G. In the present study, the ABCG8 AA genotype carriers had higher triglyceride (p¼0.045) and VLDL cholesterol (p¼0.045) levels than the ABCG8 GG genotype carriers in the total population. Junyent et al (39) genotyped ABCG5 Gln604Glu and ABCG8 Tyr54Cys genetic variants in 845 subjects and observed that minor alleles in these SNPs had a lowering effect on HDL cholesterol concentrations. According to a study performed by Li et al (40) of G5/G8 polymorphisms in 719 unrelated subjects of Mulao nationality and 782 participants of Han nationality, there was an association between serum lipid parameters and ABCG5 and ABCGG8 genotypes, especially in Han males. In another study performed by Li et al (41) ABCG8 rs4148217 gene polymorphism was analyzed in 634 subjects of Mulao nationality and 717 subjects of Han nationality. HDL cholesterol levels were lower in the subjects with the A allele than in the subjects with the C allele in the Han. Carriers of the A allele had also higher triglyceride and lower HDL cholesterol levels than carriers of the C allele in females in the Han. In the current study, we established that the ABCG5 CC genotype carriers had higher HDL cholesterol levels, if female but not if male, than the ABCG5 CG genotype carriers in the all-study population (p¼0.025).
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Conclusions This study reports the first data about ABCG5 Gln604Glu and ABCG8 Tyr54Cys gene polymorphisms and lipid parameters in diabetes. The present results show that there may be an ethnic association between lipid parameters and ABCG5 and ABCG8 polymorphisms. This study has some potential limitations. The small number of study groups is the most important one, resulting in statistically insignificant differences between groups. Therefore, future studies with larger sample sizes in differing races will help us to understand the relationship between ABCG5 Gln604Glu and ABCG8 Tyr54Cys polymorphisms and lipid profiles in diabetes mellitus. Acknowledgments The present work was supported by the Research Fund of Istanbul University (Project #6403). References 1. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2004;27:S5e10. 2. Rottiers R. New classification and new diagnostic criteria of diabetes mellitus. Acta Clin Belg 2003;58:269e76. 3. Grundy SM, Benjamin U, Burke GL, et al. Diabetes and cardiovascular disease: A statement for healthcare professionals from the American Heart Association. Circulation 1999;100:1134e46. 4. Ergen HA, Zeybek U, Gök O, et al. Investigation of ABCA1 C69T polymorphism in patients with type 2 diabetes mellitus. Biochem Med (Zagreb) 2012;22:114e20. 5. Föger B. Lipid lowering therapy in type 2 diabetes. Wien Med Wochenschr 2011;161:289e96. 6. Kota SK, Meher LK, Jammula S, et al. Genetics of type 2 diabetes mellitus and other specific types of diabetes: Its role in treatment modalities. Diabetes Metab Syndr 2012;6:54e8. 7. Mooradian AD. Dyslipidemia in type 2 diabetes mellitus. Nat Clin Pract Endocrinol Metab 2009;5:150e9. 8. Beckman JA, Creager MA, Libby P. Diabetes and atherosclerosis: Epidemiology, pathophysiology and management. JAMA 2002;287:2570e81. 9. Mazzone T, Chait A, Plutzky J. Cardiovascular disease risk in type 2 diabetes mellitus: Insights from mechanistic studies. Lancet 2008;371:1800e9. 10. Horikawa Y, Iwasaki N, Hara M, et al. Mutations in the hepatocyte nuclear factor-1a gene in maturity-onset diabetes of the young (MODY3). Nature 1996;384:455e8. 11. Clement K, Hercberg S, Passinge B, et al. The Pro115Gln and Pro12Ala PPAR gene mutations in obesity and type 2 diabetes. Int J Obes Relat Metab Disord 2000;24:391e3. 12. Higgins CF. ABC transporters: From microorganisms to man. Annu Rev Cell Biol 1992;8:67e113. 13. Dean M, Allikmets R. Evolution of ATP-binding cassette transporter genes. Curr Opin Genet Dev 1995;5:779e85. 14. Klein I, Sarkadi B, Varadi A. An inventory of the human ABC proteins. Biochim Biophys Acta 1999;1461:237e62. 15. Graf AG, Yu L, Li WP, et al. ABCG5 and ABCG8 are obligate heterodimers for protein trafficking and biliary cholesterol excretion. J Biol Chem 2003;278:48275e82. 16. Hirata T, Okabe M, Kobayashi A, et al. Molecular mechanisms of subcellular localization of ABCG5 and ABCG8. Biosci Biotechnol Biochem 2009;73:619e26. 17. Ozvegy C, Varadi A, Sarkadi B. Characterization of drug transport, ATP hydrolysis, and nucleotide trapping by the human ABCG2 multidrug transporter: Modulation of substrate specificity by a point mutation. J Biol Chem 2002;277: 47980e90.
18. Mitomo H, Kato R, Ito A, et al. A functional study on polymorphism of the ATP-binding cassette transporter ABCG2: Critical role of arginine-482 in methotrexate transport. Biochem J 2003;373:767e74. 19. Berge KE, Tian H, Graf GA, et al. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters. Science 2000; 292:1771e5. 20. Lee MH, Lu K, Hazard S, et al. Identification of a gene, ABCG5, important in the regulation of dietary cholesterol absorption. Nat Genet 2001; 27:79e83. 21. Hubacek JA, Berge KE, Stefkova J, et al. Polymorphisms in ABCG5 and ABCG8 transporters and plasma cholesterol levels. Physiol Res 2004;53: 395e401. 22. Stefkova J, Poledne R, Hubacek JA. ATP-Binding cassette (ABC) transporters in human metabolism and diseases. Physiol Res 2004;53:235e43. 23. Lu K, Lee MH, Hazard S, et al. Two genes that map to the STSL locus cause sitosterolemia: Genomic structure and spectrum of mutations involving sterolin-1 and sterolin-2, encoded by ABCG5 and ABCG8, respectively. Am J Hum Genet 2001;69:278e90. 24. Hubacek JA, Berge KE, Cohen JC. Mutations in ATP-cassette binding proteins G5 (ABCG5) and G8 (ABCG8) causing sitosterolemia. Hum Mutat 2001a;18: 359e60. 25. Heimer S, Langmann T, Moehle C, et al. Mutations in the human ATP-binding cassette transporters ABCG5 and ABCG8 in sitosterolemia. Hum Mutat 2002; 20:151. 26. Wang J, Mitsche MA, Lütjohann D, et al. Relative roles of ABCG5/ABCG8 in liver and intestine. J Lipid Res 2015;56:319e30. 27. Yu XH, Qian K, Jiang N, et al. ABCG5/ABCG8 in cholesterol excretion and atherosclerosis. Clin Chim Acta 2014;428:82e8. 28. Zhang LS, Xu M, Yang Q, et al. ABCG5/G8 deficiency in mice reduces dietary triacylglycerol and cholesterol transport into the lymph. Lipids 2015;50: 371e9. 29. Tomkin GH. Dyslipidaemia: Hepatic and intestinal cross-talk. Atheroscler Suppl 2010;11:5e9. 30. Li J, Wang X, Liu H, et al. Impaired hepatic and intestinal ATP-binding cassette transporter G5/8 was associated with high exposure of b-sitosterol and the potential risks to blood-brain barrier integrity in diabetic rats. J Pharm Pharmacol 2014;66:428e36. 31. Santosa S, Demonty I, Lichtenstein AH, et al. Single nucleotide polymorphisms in ABCG5 and ABCG8 are associated with changes in cholesterol metabolism during weight loss. J Lipid Res 2007;48:2607e13. 32. Liang Y, Hou D, Zhao X, et al. Childhood obesity affects adult metabolic syndrome and diabetes. Endocrine 2015. Epub ahead of print. 33. Vollenweider P, von Eckardstein A, Widmann C. HDLs, diabetes, and metabolic syndrome. Handb Exp Pharmacol 2015;224:405e21. 34. Kilic U, Gok O, Elibol-Can B, et al. SIRT1 gene variants are related to risk of childhood obesity. Eur J Pediatr 2015;174:473e9. 35. Kilic U, Gok O, Bacaksiz A, et al. SIRT1 gene polymorphisms affect the protein expression in cardiovascular diseases. PLOS ONE 2014;9:e90428. 36. Gylling H, Hallikainen M, Pihlajamaki J, et al. Polymorphisms in the ABCG5 and ABCG8 genes associate with cholesterol absorption and insulin sensitivity. J Lipid Res 2004;45:1660e5. 37. Jakulj L, Vissers MN, Tanck MW, et al. ABCG5/G8 polymorphisms and markers of cholesterol metabolism: Systematic review and meta-analysis. J Lipid Res 2010;51:3016e23. 38. Garcia-Rios A, Perez-Martinez P, Fuentes F, et al. Genetic variations at ABCG5/ G8 genes modulate plasma lipids concentrations in patients with familial hypercholesterolemia. Atherosclerosis 2010;210:486e92. 39. Junyent M, Tucker KL, Smith CE, et al. The effects of ABCG5/G8 polymorphisms on plasma HDL cholesterol concentrations depend on smoking habit in the Boston Puerto Rican Health Study. J Lipid Res 2009;50: 565e73. 40. Li Q, Yin RX, Wei XL, et al. ATP-binding cassette transporter G5 and G8 polymorphisms and several environmental factors with serum lipid levels. PLOS ONE 2012;7:e37972. 41. Li Q, Wei XL, Yin RX. Association of ATP binding cassette transporter G8 rs4148217 SNP and serum lipid levels in Mulao and Han nationalities. Lipids Health Dis 2012;11:46.
Contents lists available at ScienceDirect
Canadian Journal of Diabetes journal homepage: www.canadianjournalofdiabetes.com
Original Research
ABCG5 and ABCG8 Gene Polymorphisms in Type 2 Diabetes Mellitus in the Turkish Population Ozlem Gok MSc a, b, Zeynep Ermis Karaali MD c, Leyla Acar BSc a, Ulkan Kilic PhD, PhD b, Arzu Ergen PhD a, * a b c
Department of Molecular Medicine, Institute for Experimental Medicine, Istanbul University, Istanbul, Turkey Department of Medical Biology and Regenerative and Restorative Medicine Research Center (REMER), Faculty of Medicine, Istanbul Medipol University, Istanbul, Turkey Department of Internal Medicine, Haseki Training and Research Hospital, Istanbul, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 August 2014 Received in revised form 23 March 2015 Accepted 9 April 2015 Available online xxx
Objective: The aim of the present study was to investigate the relationship between ABCG5 and ABCG8 gene polymorphisms and plasma lipid concentrations in Turkish patients with type 2 diabetes mellitus. Methods: Included in this study were 80 patients with type 2 diabetes and 73 healthy controls. Two selected single nucleotide polymorphisms in ABC transporter genes, ABCG5 (rs6720173) and ABCG8 (rs4148211), were genotyped by using the polymerase chain reaction-restriction fragment length polymorphism technique. Results: The rate of having the ABCG8 AA genotype (p¼0.001) was significantly higher in the patients than in the control subjects. Correspondingly, the rates of having the AG genotype (p¼0.001) and the G allele (p¼0.001) were significantly lower in the patients than in controls. Upon comparing the groups regarding ABCG5, the frequencies of occurrence of the GG genotype (p¼0.031) and G allele (p¼0.003) were considerably higher in patients than in control subjects. In the patients, the rates of having the CC genotype (p¼0.003) and the C allele (p¼0.031) were also significantly lower than those in control subjects. There was no significant difference between G5 and G8 polymorphism and lipid levels in the study groups. The ABCG8 AA genotype carriers had higher triglyceride (p¼0.045) and very low-density-cholesterol (p¼0.045) levels than the ABCG8 GG genotype carriers in all study populations. Conclusions: These results indicate that the AA genotype for ABCG8 and the GG genotype and G allele for ABCG5 are risk factors for diabetes. This study reveals the first data concerning the ABCG5 and ABCG8 gene polymorphisms in Turkish patients with diabetes. Ó 2015 Canadian Diabetes Association
Keywords: ABCG5 ABCG8 diabetes mellitus lipid metabolism polymorphism
r é s u m é Mots clés : ABCG5 ABCG8 diabète sucré métabolisme des lipides polymorphisme
Objectif : Le but de la présente étude était d’étudier la relation entre les polymorphismes des gènes ABCG5 et ABCG8 et les concentrations plasmatiques de lipides chez les patients turcs souffrant du diabète sucré de type 2. Méthodes : Cette étude comprenait 80 patients souffrant du diabète de type 2 et 73 témoins en santé. Deux polymorphismes mononucléotidiques sélectionnés des gènes codant pour les transporteurs ABC, l’ABCG5 (rs6720173) et l’ABCG8 (rs4148211), ont été génotypés par la technique de réaction en chaîne de la polyméraseepolymorphisme de longueur des fragments de restriction. Résultats : Le risque d’être porteur du génotype AA du ABCG8 (p¼0,001) était significativement plus élevé chez les sujets témoins. Parallèlement, les risques d’être porteur du génotype AG (p¼0,001 et de l’allèle G (p¼0,001) étaient significativement plus faibles chez les patients que chez les témoins. En comparant les groupes au sujet de l’ABCG5, la fréquence du génotype GG (p¼0,031) et de l’allèle G (p¼0,003) était considérablement plus élevée chez les patients que les sujets témoins. De plus, les risques que les patients soient porteurs du génotype CC (p¼0,003) et de l’allèle C (p¼0,031) étaient significativement plus faibles que ceux des sujets témoins. Il n’existait aucune différence significative entre les polymorphismes G5 et G8 et les concentrations de lipides dans les groupes étudiés. Dans toutes les populations faisant l’objet de l’étude, les porteurs du génotype AA du ABCG8 avaient des concentrations plus élevées de
* Address for correspondence: Arzu Ergen, Department of Molecular Medicine, The Institute of Experimental Medicine, Istanbul University, Vakif Gureba Cad., Sehremini-Fatih TR-34093 Istanbul, Turkey. E-mail address: [email protected] 1499-2671/$ e see front matter Ó 2015 Canadian Diabetes Association http://dx.doi.org/10.1016/j.jcjd.2015.04.004
2
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triglycérides (p¼0,045) et de cholestérol à lipoprotéines de très basse densité (p¼0,045) que les porteurs du génotype GG du ABCG8. Conclusions : Ces résultats indiquent que le génotype AA du ABCG8 et que le génotype GG et l’allèle G du ABCG5 sont des facteurs de risque de diabète. Cette étude révèle les premières données concernant les polymorphismes des gènes ABCG5 et ABCG8 chez les patients turcs souffrant du diabète. Ó 2015 Canadian Diabetes Association
Introduction Diabetes mellitus is a multisystemic disorder of carbohydrate, protein and fat metabolism that is affected by both genetics and environmental factors. Although there are several types of the disease, 2 main types, namely insulin dependent (type 1 diabetes) and non-insulin dependent diabetes (type 2 diabetes), are the most common types. Diabetes disturbs the life quality of patients by causing serious health problems and complications, such as retinopathy, neuropathy, fatigue, weight loss, micro- and macrovascular abnormalities, nephropathy and several kinds of cardiovascular disorders, such as hypertension and atherosclerosis (1e6). Type 2 diabetes also causes dyslipidemia, which is characterized by high levels of triglyceride (hypertriglyceridemia) and low-density lipoprotein (LDL) (7,8), and shows close interaction with the risk for coronary artery disease (9). However, the pathogenesis and genetic mechanisms of diabetes are still unclear (10,11). Adenotriphosphate (ATP)-binding cassette (ABC) transporters, the most common type of transmembrane proteins, coded by 49 genes, have been subdivided into 8 subgroups: 1) A-G, based on sequence similarity and domain organization; 2) ABCA, 12 members; 3) ABCB, 11 members; 4) ABCC 13 members; 5) ABCD, 4 members; 6) ABCE, 1 member; 7) ABCF, 3 members; 8) ABCG, 5 members). In particular, these transmembrane proteins cross the membrane in an ATP-dependent manner and transport specific substrates, such as lipids, sterols and metabolic products. ABC transporters are expressed in liver, kidney, testis, intestine, ovary, lung, tumour cell, blood-brain barrier and placenta. Eukaryotic ABC transporters have been subdivided into being either full or half transporters. Full transporters contain 2 nucleotide-binding domains (NBDs) and two 6-transmembrane helices, namely transmembrane domains (TMDs). Some full transporters have additional transmembrane helices at the amino terminus. However, half transporters contain 1 NBD and 1 TMD on a single polypeptide (12e15). Human ABCG subfamily proteins are half-type ABC proteins and contain cytosolic N-terminal NBD and C-terminal TMD. They contain 5 members: ABCG1, ABCG2, ABCG4, ABCG5 and ABCG8, which transport sterols and xenobiotics. ABCG1, ABCG2 and ABCG4 function as homodimers on the plasma membrane. Correspondingly, ABCG5 and ABCG8 function as heterodimers on the plasma membrane and homodimers in the endoplasmic reticulum (12e18). ABCG5 and ABCG8 are located in head-to-head orientation of exon 13 of chromosome 2p21 (PUBMED, gene) and are expressed in both the liver and the intestine (19e21). Previous studies have reported that mutations in either ABCG5 or ABCG8 cause sitosterolemia, a rare lipid disorder that is characterized by high intestinal absorption of all sterols (cholesterol, plant sterols and shellfish sterols) (15,19,22e25). There are limited number of studies of the roles of ABCG5 (Gln604Glu¼C1810G, rs6720173) and ABCG8 (Tyr54Cys¼A161G, rs4148211) gene polymorphisms on the plasma cholesterol levels and familial hypercholesterolemia (21). There is no investigation related to ABCG5 Gln604Glu and ABCG8 Tyr54Cys single nucleotide polymorphisms (SNPs) and lipid levels in patients with diabetes. According to this knowledge, we aimed to investigate the
association between ABCG5 and ABCG8 gene polymorphisms and lipid levels in Turkish patients with type 2 diabetes as compared to controls.
Methods Study groups The experimental group consisted of 80 patients (59 female and 21 male, mean age 57.6414.23) diagnosed with type 2 diabetes who were in the follow-up group studied by the Istanbul University’s Faculty of Medicine, Department of Endocrinology. The control group included 73 randomly selected healthy individuals (26 female, 47 male; mean age 53.7710.54) who had no signs of coronary artery disease, hypertension, lipid metabolic disorders or cardiovascular diseases. The controls enrolled in this study were selected from a group of people without family histories of diabetes. This study was approved by the ethical committee of Istanbul University, Faculty of Medicine. All participants, after giving written informed consent, completed a structured questionnaire in order to collect demographic data. The study was conducted in accordance with the ethical principles described by the Declaration of Helsinki.
Biochemical and demographic analysis After 12 hours of fasting, blood samples of the participants were taken into plain tubes and tubes with ethylenediaminetetraacetic acid (EDTA) (Vacuette; Greiner Labortechnick, Frickenhausen, Germany). The samples were centrifuged for 10 minutes at 1.500 g at þ4 C, followed by the removal of serum and plasma and then were stored at e20 C. Fasting glucose, total cholesterol, triglyceride, high-density lipoprotein (HDL) cholesterol, lowdensity lipoprotein (LDL) cholesterol and very low-density lipoprotein (VLDL) cholesterol were determined in the study groups by standard laboratory methods in the Istanbul University and Haseki Training and Research hospitals (26,27). In addition, body mass index (BMI; weight in kilograms divided by the square of the height in meters) values were calculated and categorized according to World Health Organization recommendations. The smoking status of an individual was assigned as yes if he or she was smoking currently or had given up smoking 0.05). However, the BMIs (p¼0.018) were significantly higher in the subjects with the ABCG5 CC genotype (26.53 2.81) than in the subjects with the ABCG5 GG genotype (23.36 3.30) in the control group (Table 4). In the total group, HDL cholesterol levels (p¼0.025) were significantly higher in the female subjects with the ABCG5 CC genotype than in the female subjects with the ABCG5 CG genotype. Triglyceride and VLDL cholesterol levels (p¼0.045) were considerably higher in the subjects with the ABCG8 AA genotype than in the subjects with the ABCG8 GG genotype in the total group (Table 5). Discussion Type 2 diabetes mellitus is associated with gene-environment interactions due to epigenetic factors, such as defects in lipid metabolism, hyperlipidemia, hypertension, obesity, smoking and
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Table 5 Comparison of ABCG5 Gln604Glu and ABCG8 Tyr54Cys genotypes with lipid/anthropometric parameters in all study populations ABCG5 Gln604Glu genotype
Triglyceride (mg/dL) Total cholesterol (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) VLDL cholesterol (mg/dL) Total cholesterol /HDL cholesterol (mg/dL) BMI (kg/m2)
ABCG8 Tyr54Cys genotype
CC (n¼73)
CG (n¼25)
CG (n¼55)
p
AA (n¼60)
GG (n¼19)
AG (n¼74)
p
154.3866.74 209.8845.87 38.229.64 140.7842.62 30.8813.34 5.852.24 26.353.63
161.7664.64 223.3158.29 37.168.49 153.8053.32 32.3512.92 6.422.53 25.853.76
152.0268.73 210.3750.92 35.588.63 144.3847.22 30.4013.74 6.282.31 26.483.83
NS NS NS NS NS NS NS
169.3080.27* 216.0956.92 38.038.58 144.2052.66 33.8616.05* 6.062.48 26.493.90
134.1141.37 212.2442.15 37.7412.50 147.6845.52 26.828.27 6.343.05 25.244.28
148.2357.67 209.1345.63 36.188.59 143.3141.64 29.6511.53 6.061.96 26.453.38
0.045 NS NS NS 0.045 NS NS
HDL, High density lipoprotein; LDL, low density lipoprotein; n, number of individuals; NS, not significant; SD, standard deviation; VLDL, very low-density lipoprotein; BMI, body mass index. Statistical evaluation was made by using the Student t test and the 1-way ANOVA test. The results are shown as mean SD. * p0.05). ABCG5 and ABCG8 genes have important effects on the regulation of lipid metabolism. These genes are involved not only in cholesterol absorption but also in cholesterol excretion (16,21,26,31). Santosa et al (31) observed that weight loss and defects in cholesterol metabolism were related to ABCG5/8 polymorphisms. Hubajeck et al (21) reported that total cholesterol and LDL cholesterol levels were low for ABCG8 Tyr54Cys only in female subjects. In addition, they observed no association between lipid profiles and genotype distribution and allele frequencies for ABCG5 Gln604Glu polymorphism in both genders of the study population (131 male and 154 female). Similar results were demonstrated in the study by Jakulj et al (37), which was performed for ABCG5 and ABCG8 SNPs in 245 patients with hypercholesterolemia. Because of the failure to find any association between G5/G8 and plasma sterol levels, they pooled their data for a meta-analysis of 3364 individuals from 16 studies. However, they did not find any association between plasma lipid levels and ABCG5/8. In this study, we similarly observed that there was no significant association between ABCG5 and ABCG8 genotypes and lipid parameters. Garcia Rios et al (38) genotyped ABCG5 and ABCG8 SNPs in 500 individuals with genetic diagnoses of familial hypercholesterolemia. In particular, carriers of the G allele had considerably lower VLDL cholesterol (p¼0.011) and triglyceride (p¼0.017) levels than carriers of the CC genotype for ABCG5_Gln604GluC>G. In the present study, the ABCG8 AA genotype carriers had higher triglyceride (p¼0.045) and VLDL cholesterol (p¼0.045) levels than the ABCG8 GG genotype carriers in the total population. Junyent et al (39) genotyped ABCG5 Gln604Glu and ABCG8 Tyr54Cys genetic variants in 845 subjects and observed that minor alleles in these SNPs had a lowering effect on HDL cholesterol concentrations. According to a study performed by Li et al (40) of G5/G8 polymorphisms in 719 unrelated subjects of Mulao nationality and 782 participants of Han nationality, there was an association between serum lipid parameters and ABCG5 and ABCGG8 genotypes, especially in Han males. In another study performed by Li et al (41) ABCG8 rs4148217 gene polymorphism was analyzed in 634 subjects of Mulao nationality and 717 subjects of Han nationality. HDL cholesterol levels were lower in the subjects with the A allele than in the subjects with the C allele in the Han. Carriers of the A allele had also higher triglyceride and lower HDL cholesterol levels than carriers of the C allele in females in the Han. In the current study, we established that the ABCG5 CC genotype carriers had higher HDL cholesterol levels, if female but not if male, than the ABCG5 CG genotype carriers in the all-study population (p¼0.025).
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Conclusions This study reports the first data about ABCG5 Gln604Glu and ABCG8 Tyr54Cys gene polymorphisms and lipid parameters in diabetes. The present results show that there may be an ethnic association between lipid parameters and ABCG5 and ABCG8 polymorphisms. This study has some potential limitations. The small number of study groups is the most important one, resulting in statistically insignificant differences between groups. Therefore, future studies with larger sample sizes in differing races will help us to understand the relationship between ABCG5 Gln604Glu and ABCG8 Tyr54Cys polymorphisms and lipid profiles in diabetes mellitus. Acknowledgments The present work was supported by the Research Fund of Istanbul University (Project #6403). References 1. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2004;27:S5e10. 2. Rottiers R. New classification and new diagnostic criteria of diabetes mellitus. Acta Clin Belg 2003;58:269e76. 3. Grundy SM, Benjamin U, Burke GL, et al. Diabetes and cardiovascular disease: A statement for healthcare professionals from the American Heart Association. Circulation 1999;100:1134e46. 4. Ergen HA, Zeybek U, Gök O, et al. Investigation of ABCA1 C69T polymorphism in patients with type 2 diabetes mellitus. Biochem Med (Zagreb) 2012;22:114e20. 5. Föger B. Lipid lowering therapy in type 2 diabetes. Wien Med Wochenschr 2011;161:289e96. 6. Kota SK, Meher LK, Jammula S, et al. Genetics of type 2 diabetes mellitus and other specific types of diabetes: Its role in treatment modalities. Diabetes Metab Syndr 2012;6:54e8. 7. Mooradian AD. Dyslipidemia in type 2 diabetes mellitus. Nat Clin Pract Endocrinol Metab 2009;5:150e9. 8. Beckman JA, Creager MA, Libby P. Diabetes and atherosclerosis: Epidemiology, pathophysiology and management. JAMA 2002;287:2570e81. 9. Mazzone T, Chait A, Plutzky J. Cardiovascular disease risk in type 2 diabetes mellitus: Insights from mechanistic studies. Lancet 2008;371:1800e9. 10. Horikawa Y, Iwasaki N, Hara M, et al. Mutations in the hepatocyte nuclear factor-1a gene in maturity-onset diabetes of the young (MODY3). Nature 1996;384:455e8. 11. Clement K, Hercberg S, Passinge B, et al. The Pro115Gln and Pro12Ala PPAR gene mutations in obesity and type 2 diabetes. Int J Obes Relat Metab Disord 2000;24:391e3. 12. Higgins CF. ABC transporters: From microorganisms to man. Annu Rev Cell Biol 1992;8:67e113. 13. Dean M, Allikmets R. Evolution of ATP-binding cassette transporter genes. Curr Opin Genet Dev 1995;5:779e85. 14. Klein I, Sarkadi B, Varadi A. An inventory of the human ABC proteins. Biochim Biophys Acta 1999;1461:237e62. 15. Graf AG, Yu L, Li WP, et al. ABCG5 and ABCG8 are obligate heterodimers for protein trafficking and biliary cholesterol excretion. J Biol Chem 2003;278:48275e82. 16. Hirata T, Okabe M, Kobayashi A, et al. Molecular mechanisms of subcellular localization of ABCG5 and ABCG8. Biosci Biotechnol Biochem 2009;73:619e26. 17. Ozvegy C, Varadi A, Sarkadi B. Characterization of drug transport, ATP hydrolysis, and nucleotide trapping by the human ABCG2 multidrug transporter: Modulation of substrate specificity by a point mutation. J Biol Chem 2002;277: 47980e90.
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