- Email: [email protected]
comorbidity profile in type 2 diabetes patients
diabetes research and clinical practice 93 (2011) 390–395
Contents lists available at ScienceDirect
Diabetes Research and Clinical Practice jou rnal hom ep ag e: w ww.e l s e v i er . c om/ loca te / d i ab r es
Transcription factor 7-like 2 (TCF7L2) gene polymorphism and complication/comorbidity profile in type 2 diabetes patients Monika Buraczynska *, Andrzej Swatowski, Dorota Markowska-Gosik, Agata Kuczmaszewska, Andrzej Ksiazek Laboratory for DNA Analysis and Molecular Diagnostics, Department of Nephrology, Medical University of Lublin, Dr K. Jaczewskiego 8, 20-954 Lublin, Poland
article info
abstract
Article history:
Transcription factor 7-like 2 gene (TCF7L2) has been associated with type 2 diabetes. We
Received 17 January 2011
investigated the association of the rs7903146 SNP in this gene with clinical profile of type 2
Received in revised form
diabetes (T2DM) patients.
11 April 2011
The study involved 980 patients with diabetic nephropathy (44%), diabetic retinopathy
Accepted 9 May 2011
(42%), CVD (65%) and early onset of diabetes (45%) and 924 healthy controls. Subjects were
Published on line 8 June 2011
genotyped for rs7903146 by PCR-RFLP.
Keywords:
( p < 0.01, odds ratio (OR) for TT genotype 2.49 (95% confidence interval (CI) 1.84-3.39). An
Genotype frequencies significantly differed between T2DM patients and controls Diabetic nephropathy
association was observed between rs7903146 and nephropathy ( p < 0.001), with 22% of TT
Gene polymorphism
homozygotes in this subgroup vs. 11% in patients without nephropathy ( p = 0.006, OR for TT
Risk alleles
2.83, 95% CI 1.94–4.13). Association was stronger in patients with early onset of diabetes (34%
TCF7L2
of TT vs. 12% in the late onset, p < 0.001). In DN group 71% of TT homozygotes had an early
Type 2 diabetes
onset (OR 7.64, 95% CI 4.98–11.73 vs. controls). Our results confirm association of rs7903146 in the TCF7L2 gene with increased risk of type 2 diabetes. The T allele is strongly associated with nephropathy, especially in early onset of diabetes. # 2011 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
A combination of multiple genetic and environmental factors contributes to the pathogenesis of type 2 diabetes (T2DM) [1]. The identification of causative genes predisposing to T2DM could provide means to better understanding the pathogenesis of the disease and result in better prevention, diagnosis and treatment. With the advent of genome-wide association scans, numerous risk variants have been identified as candidates for conferring susceptibility to type 2 diabetes but most of them with only modest effects [2].
Transcription factor 7-like 2 (TCF7L2), a protein belonging to a family of TCF/lymphoid enhancer factor (LEF) transcription factors, is a key component of the Wnt signaling pathway involved in the regulation of pancreatic beta-cell proliferation, differentiation and insulin secretion [3,4]. The gene encoding TCF7L2 spans a 215,863 bp region on chromosome 10q25.3 [5]. TCF7L2 is the most significant and consistent genetic risk factor of diabetes identified to date. It was first identified as a diabetes risk conferring gene in 2006 [6]. Later, the common polymorphisms in the TCF7L2 have been associated with type 2 diabetes in numerous studies in different populations. The
* Corresponding author. Tel.: +48 81 7244 716; fax: +48 81 7244 357. E-mail addresses: [email protected], [email protected] (M. Buraczynska). 0168-8227/$ – see front matter # 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2011.05.017
diabetes research and clinical practice 93 (2011) 390–395
most significant genetic association was detected for two intronic single nucleotide polymorphisms (SNPs), rs7903146 (intron 3) and rs12255372 (intron 4) located 50 kb from each other [6–14]. The primary site of action for the TCF7L2 gene product in type 2 diabetes is unknown. It was suggested that TCF7L2 might be associated with beta-cell dysfunction and decreased insulin secretion but not with insulin resistance [15]. In this study we investigated the potential association of the rs7903146 (C/T) polymorphism in the intron 3 of the TCF7L2 gene with type 2 diabetes. We also analyzed the effect of this SNP on different clinical phenotypes in type 2 diabetes patients.
2.
Methods
2.1.
Subjects
The study population consisted of 980 unrelated type 2 diabetes mellitus (T2DM) patients, consecutively enrolled between January 2004 and September 2008. All subjects were Caucasians of Polish origin. The patients were stratified into subgroups with different clinical phenotypes: diabetic nephropathy (DN) (n = 432), diabetic retinopathy (DR) (n = 408), cardiovascular disease (CVD) (n = 635), early onset of diabetes (45 years) (n = 438), and obesity (n = 189). In the diabetic nephropathy subgroup 199 patients (46%) had also diabetic retinopathy. Similarly, in the diabetic retinopathy subgroup, 216 patients (53%) had also nephropathy. Diabetes was diagnosed according to American Diabetes Association criteria. One or more of the following conditions were met: the classic symptoms of hyperglycemia (polyuria, polydipsia, and weight loss), fasting plasma glucose 126 mg/dl or random plasma glucose 200 mg/dl, the use of insulin or oral hypoglycemic agents. The mean duration of diabetes was 12.6 years (range 6–27). It was estimated from time of the first symptoms attributable to the disease or time of first detection of glycosuria. Diabetic nephropathy was diagnosed clinically when the patient had persistent albuminuria 300 mg/24 h in at least two consecutive determinations in the absence of hematuria or infection. The patients without diabetic nephropathy were normoalbuminuric, those with microalbuminuria were excluded. Diabetic retinopathy was diagnosed by independent ophthalmologists. All patients underwent a complete ophthalmological examination, including corrected visual acuity, fundoscopic examination and fundus photography (three 458 fields per eye) at least every year. Fundoscopic findings were determined by retinal specialists. Retinopathy was diagnosed according to the Early Treatment Diabetic Retinopathy Study (ETDRS) criteria: the presence of microaneurysms, hemorrhages, cotton wool spots, intraretinal microvascular abnormalities, hard exudates, venous beading and new vessels. Cardiovascular disease was diagnosed and documented as one or the combination of several pathological states: congestive heart failure, left ventricular hypertrophy, angina pectoris, ischemic heart disease, myocardial infarction, ischemic cerebral stroke, vascular calcifications or atheromatous lesions. Each clinical manifestation of CVD was confirmed by appropriate biochemical, radiographic,
391
echocardiographic and vascular diagnostic criteria. There was a substantial overlap between categories. No gender differences between the clinical phenotypes of CVD were observed. Arrhythmia was more prevalent in male patients but the difference did not reach statistical significance. At the beginning of the study 642 individuals (65.5%) in the patient group were hypertensives. Hypertension was defined according to WHO criteria (World Health Organization, 1999). All patients had persistent systolic blood pressure > 140 mm Hg and diastolic blood pressure > 90 mm Hg and/or were receiving anti-hypertensive treatment. Secondary hypertension was excluded by clinical and laboratory examination. Obesity was defined as BMI 30 kg/m2. A positive family history of diabetes in first-degree relatives was reported by 224 patients (23%). Glycemic control was evaluated by measuring glycated HbA1c levels by turbidimetric inhibition immunoassay TINIA using Tina-quant hemoglobin A1cII (Roche-Hitachi 747). All other biochemical parameters were measured by standard laboratory procedures. Control subjects (n = 924) were healthy normotensive volunteers (mostly blood donors and hospital staff members), with no history of diabetes or hypertension. Written informed consent was obtained from all subjects in accordance with principles of the Declaration of Helsinki. The study protocol was approved by the institutional ethics committee.
2.2.
Genotyping
Genomic DNA was extracted from peripheral blood leukocytes (obtained from EDTA anticoagulated blood) using a standard technique. All subjects were genotyped for the rs7903146 single nucleotide polymorphism (SNP) by polymerase chain reaction (PCR) and subsequent cleavage of amplified fragment with Rsa I restriction endonuclease (Fermentas GmbH, St Leon-Rot, Germany). DNA fragments were visualized on 3% agarose gels. The quality of genotyping was controlled by using blind DNA duplicates for some samples (96). Also, 30 samples were randomly selected for each genotype (CC, CT and TT) and the PCR products were sequenced by automated sequencing in CEQ 8000 Genetic Analysis System (Beckman Coulter, High Wycombe, England). Observed concordance between genotyping assays was 100%.
2.3.
Statistical analysis
Statistical calculations were performed using SPSS 11.0 for Windows (SPSS, Inc., Chicago, IL, USA). For baseline characteristics the normally distributed continuous variables are presented as means SD. The Hardy–Weinberg equilibrium was tested with the x2 test. Genotype distribution and allele frequencies were compared between groups using a x2 test of independence with 2 2 contingency and z statistics. Student’s t-test and ANOVA were used for statistical significance between variables/groups. Where appropriate, the odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were calculated. A Bonferroni correction was applied for multiple testing (4 comparisons: subgroups/nephropathy, retinopathy, CVD, early onset of diabetes). Multivariate logistic regression was performed for analysis of independent risk factor for
392
diabetes research and clinical practice 93 (2011) 390–395
Table 1 – Demographic and clinical profile of studied subjects. Variable
T2DM patients
Controls
N Male/female Age at study (years) Diabetes duration (years) Diabetic retinopathy (%) Diabetic nephropathy (%) Hypertension (%) HbA1c (%) Total cholesterol (mmol/l) HDL cholesterol (mmol/l) Triglycerides (mmol/l) BMI (kg/m2)
980 512/468 57 18 12.6 8a 408 (42) 432 (44) 642 (65.5) 8.2 3.8 5.1 1.51 1.2 0.65 2.3 1.78 27.4 3.6
924 492/432 54 17 NA 0 0 0 ND 3.9 1.71 1.82 0.86 1.17 0.93 26.2 3.4
p value NS <0.001
<0.001 <0.001 <0.001 <0.001
T2DM: type 2 diabetes mellitus. Values are presented as mean SD or numbers (%). NA: not applicable and ND: not determined. Mean diabetes duration was 14.0 9.2 for patients with DN and 11.2 6.8 for patients without DN.
a
diabetic nephropathy. A two-tailed type I error rate of 5% was considered statistically significant. Power calculations were done using on-line available power calculator (http://calculators.stat.ucla.edu).
3.
Results
The genotype of the rs7903146 polymorphism in the TCF7L2 gene was determined in 980 patients with type 2 diabetes and 924 healthy individuals. The demographic and clinical profile of studied subjects has been presented in Table 1. The frequencies of the genotypes and alleles in the control group were similar to those reported for other European populations [16,17]. The genotype distribution among the controls was in Hardy–Weinberg equilibrium. Table 2 shows the genotypes of rs7903146 SNP in diabetes patients and controls. The genotype distribution and allele frequencies were significantly different between the entire diabetes group and healthy individuals ( p < 0.01). The odds ratio (OR) for the TT genotype vs. CC was 2.49 (95% CI 1.84–3.39). Genotype and allele frequencies were compared in subgroups of diabetes patients with diabetic nephropathy, diabetic retinopathy, CVD, early onset of diabetes and obesity. The results are presented in Table 3. The frequency of the TT genotype was higher in patients with CVD than in those without cardiovascular comorbidity although the difference did not reach a statistical significance ( p = 0.061). The same tendency was observed for diabetic retinopathy and early onset of diabetes, with no statistical significance after adjusting for confounding factors. The T allele was associated with an earlier age at diagnosis (54.3 years for CC, 54.1 years for
CT and 52.7 years for TT genotype, p < 0.01). There were no statistically significant differences in the allele/genotype distribution between obese and non-obese subjects (data not shown). After the Bonferroni correction for multiple comparisons a strong association was observed between the rs7903146 SNP and diabetic nephropathy (Table 4). The frequency of the TT homozygous genotype in the diabetic nephropathy subgroup was 22% compared to 9% in patients with no microvascular complications after 10 years of T2DM duration. The OR for TT genotype in DN patients, after adjusting for multiple testing, was 3.71 (95% CI 1.99–6.93) vs. no complications subgroup. After adjusting for age, sex, blood pressure, BMI and diabetes duration, the odds ratio was 2.98 (95% CI 1.16–6.87). The observed association of the rs7903146 SNP with diabetic nephropathy was even stronger in patients with an early onset of diabetes. The minor allele frequency was 0.52 vs. 0.28 in patients with no complications. The frequency of the TT homozygotes in T2DM patients with early onset of diabetes was 34% compared to 12% in patients with nephropathy and late onset of diabetes ( p < 0.001). Of all TT homozygotes in diabetic nephropathy group 71% were among patients with an early onset of diabetes. After adjusting for multiple testing, OR for TT genotype was 6.60 (95% CI 3.38–12.87) vs. no complications subgroup. Logistic regression analysis revealed that the association of minor allele of rs7903146 polymorphism with type 2 diabetes and diabetic nephropathy remained significant after adjusting for other covariates potentially associated with T2DM risk, age, gender, hypertension, BMI, HbA1c and also diabetes duration (OR 2.36, 95% CI 2.11–3.66 for T2DM and OR 2.73, 95% CI 2.29–3.42 for diabetic nephropathy).
Table 2 – Genotype and allele distribution of rs7903146 SNP in the TCF7L2 gene in type 2 diabetes patients and controls. Subjects
T2 DM (n = 980) Controls (n = 924)
CC
416 (42) 490 (53)
CT
407 (42) 360 (39)
TT
157 (16) 74 (8)
MAF
0.37 0.27
OR (95% CI) For T allele
For TT genotype
1.53 (1.33–1.76) 1.0 (ref.)
2.49 (1.84–3.39) 1.0 (ref.)
T2DM: type 2 diabetes mellitus and MAF: minor allele frequency. Data are n (%). HWE test: T2DM x2 = 11.22, p = 0.000809; Controls x2 = 0.48, p = 0.488422. Statistical power for T2DM vs. controls = 99.1%.
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diabetes research and clinical practice 93 (2011) 390–395
Table 3 – The rs7903146 SNP and different clinical profiles in T2DM patient group. Subjects CVD (n = 635) No CVD (n = 345) DR (n = 408) No DR (n = 572) Early onset of T2DM (n = 438) Late onset of T2DM (n = 542)
CC 261 155 162 254 178 238
CT
(41) (45) (40) (44) (41) (44)
254 153 167 240 172 235
TT
(40) (44) (41) (42) (39) (43)
120 37 79 78 88 69
(19) (11) (19) (14) (20) (13)
MAF 0.39 0.33 0.40 0.35 0.40 0.34
T allele carriers 374 190 246 318 260 304
(59) (55) (60) (56) (59) (56)
OR (95% CI)a for T allele 1.29 (1.06–1.57) 1.0 (ref.) 1.25 (1.03–1.50) 1.0 (ref.) 1.25 (1.04–1.51) 1.0 (ref.)
T2DM: type 2 diabetes mellitus; CVD: cardiovascular disease; DR: diabetic retinopathy; and MAF: minor allele frequency. Odds ratios were calculated: CVD vs. no CVD, DR vs. no DR, early onset vs. late onset, and adjusted for age, sex, blood pressure, BMI, HbA1c and diabetes duration. a
4.
for nephropathy. However, this study did not distinguish whether observed associations were with type 2 diabetes or diabetic nephropathy [23]. Wu et al. investigated association of diabetes related genes with diabetic nephropathy in Taiwanese population. In a single locus analysis they found the marginally significant association of rs7903146 variant with DN [24]. In the study of French population there was no evidence of association of the rs7903146 variant with type 2 diabetes complications such as CHD, severe nephropathy or retinopathy (exact data were not reported) [18]. In our study there was a trend towards association of TT genotype with CVD in diabetes patients ( p = 0.061). Similar trend was observed for CHD in the ARIC study [25]. In contrast, Sousa et al. found a significant association between TCF7L2 rs7903146 genotypes and coronary artery disease in non-diabetic subjects but were not able to demonstrate this association in diabetic patients [26]. We found that the T allele of rs7903146 was associated with an earlier age at diagnosis. It is in agreement with earlier studies [18,27,28]. A gene dosage effect has been observed. The risk of type 2 diabetes in TT homozygotes was increased by 2.49, similar to the result obtained in another study performed in a group of 290 Polish patients with T2DM [29]. It is not clear how rs7903146 variant, located approximately 9 kb downstream of exon 4 of TCF7L2 gene, might affect its expression or the function of the protein product. Functional studies are required to elucidate this. Our results should be viewed with caution since there is a deviation from HWE estimates in cases. The genotyping errors can be excluded since the quality of genotyping in our study
Discussion
Several recent studies have investigated the effect of SNPs in TCF7L2 on type 2 diabetes development. So far TCF7L2 is the most reproducible susceptibility gene for type 2 diabetes in various ethnic groups [4,6,12,13,15,18,19]. Recently, in the study of African American population, rs7903146 SNP in the TCF7L2 gene was implicated as a causal variant in type 2 diabetes [20]. Functional studies also point to a functional role of this SNP. Gaulton et al. [21] found that rs7903146 is located in islet-selective open chromatin and shows allele-specific islet enhancer activity. In the present study we investigated the potential association between type 2 diabetes and rs7903146 SNP in the intron 3 of TCF7L2 gene in a large group of Polish patients with type 2 diabetes. The results successfully replicated the previously found association between this SNP and type 2 diabetes. In addition, after stratification it was apparent that there is a strong association of this SNP with diabetic nephropathy, especially in patients with an early onset of diabetes. This could indicate to the higher risk of microvascular complications of T2DM for the T allele carriers. Previously published papers suggested a possible association of the rs7903146 variant with renal dysfunction in diabetic patients [22–24]. In the older population of Italian subjects the T allele carriers were more likely to have the key microvascular complication of poor renal function [22]. Another study investigated association of the TCF7L2 rs7903146 variant with type 2 diabetes in an African-American population enriched
Table 4 – Genotype and allele distribution of rs7903146 SNP in diabetes patients with and without diabetic nephropathy. Subjects DN (n = 432) No DN (n = 548) No complicationsb (n = 154) Early onset with DN (n = 198) Late onset with DN (n = 234)
CC 146 270 80 58 88
(34) (50) (52) (29) (38)
CT 191 216 60 73 118
(44) (39) (39) (37) (50)
TT 95 62 14 67 28
(22) (11) (9) (34) (12)
MAF
OR (95% CI)a for T allele
0.44 0.31 0.28 0.52 0.37
1.97 (1.49–2.61) – 1.0 (ref.) 2.74 (1.99–3.75) 1.47 (1.08–2.01)
DN: diabetic nephropathy and MAF: minor allele frequency. After adjusting for multiple testing. Data are n (%). The OR for TT genotype: DN patients vs. no complications 3.71 (1.99–6.93) and adjusted for age, sex, blood pressure, BMI and diabetes duration 2.98 (1.16–6.87), early onset with DN vs. no complications 6.60 (3.38–12.87) and adjusted 5.93 (2.96–11.04). Statistical power for DN vs. no complications = 97.4%. b No microvascular complications after 10 years of T2DM duration. a
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diabetes research and clinical practice 93 (2011) 390–395
was carefully controlled with using blind sample duplicates and direct sequencing. The likely reason for deviation from HWE may be selection bias. As most of the association studies, ours has some limitations. Since it is a retrospective case-control study, a selection bias cannot be excluded. To limit this bias we included consecutive patients in the study and tried to adjust for known confounding risk factors. All normoglycemic individuals with traits related to type 2 diabetes, like obesity, hypertension or hyperlipidemia, were excluded from the study. The strength of our study is that all patients and controls are of the same ethnic origin. Furthermore all subjects were examined employing well defined diagnostic criteria and genotyping was performed blind with respect to case-control status. The sample size in our study had 97.4% power to detect the genetic effect of TCF7L2 allele at a p value of 0.05. In conclusion, the findings of our study confirm that the risk allele of the rs7903146 SNP in the TCF7L2 gene is strongly associated with type 2 diabetes, even after correcting for traditional risk factors. The T allele of this polymorphism also conferred the risk of developing diabetic nephropathy.
Acknowledgements This study was supported by the grants DS 379/09 and DS 383/ 09 from Medical University of Lublin.
Conflict of interest The authors declare no conflict of interest.
references
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Contents lists available at ScienceDirect
Diabetes Research and Clinical Practice jou rnal hom ep ag e: w ww.e l s e v i er . c om/ loca te / d i ab r es
Transcription factor 7-like 2 (TCF7L2) gene polymorphism and complication/comorbidity profile in type 2 diabetes patients Monika Buraczynska *, Andrzej Swatowski, Dorota Markowska-Gosik, Agata Kuczmaszewska, Andrzej Ksiazek Laboratory for DNA Analysis and Molecular Diagnostics, Department of Nephrology, Medical University of Lublin, Dr K. Jaczewskiego 8, 20-954 Lublin, Poland
article info
abstract
Article history:
Transcription factor 7-like 2 gene (TCF7L2) has been associated with type 2 diabetes. We
Received 17 January 2011
investigated the association of the rs7903146 SNP in this gene with clinical profile of type 2
Received in revised form
diabetes (T2DM) patients.
11 April 2011
The study involved 980 patients with diabetic nephropathy (44%), diabetic retinopathy
Accepted 9 May 2011
(42%), CVD (65%) and early onset of diabetes (45%) and 924 healthy controls. Subjects were
Published on line 8 June 2011
genotyped for rs7903146 by PCR-RFLP.
Keywords:
( p < 0.01, odds ratio (OR) for TT genotype 2.49 (95% confidence interval (CI) 1.84-3.39). An
Genotype frequencies significantly differed between T2DM patients and controls Diabetic nephropathy
association was observed between rs7903146 and nephropathy ( p < 0.001), with 22% of TT
Gene polymorphism
homozygotes in this subgroup vs. 11% in patients without nephropathy ( p = 0.006, OR for TT
Risk alleles
2.83, 95% CI 1.94–4.13). Association was stronger in patients with early onset of diabetes (34%
TCF7L2
of TT vs. 12% in the late onset, p < 0.001). In DN group 71% of TT homozygotes had an early
Type 2 diabetes
onset (OR 7.64, 95% CI 4.98–11.73 vs. controls). Our results confirm association of rs7903146 in the TCF7L2 gene with increased risk of type 2 diabetes. The T allele is strongly associated with nephropathy, especially in early onset of diabetes. # 2011 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
A combination of multiple genetic and environmental factors contributes to the pathogenesis of type 2 diabetes (T2DM) [1]. The identification of causative genes predisposing to T2DM could provide means to better understanding the pathogenesis of the disease and result in better prevention, diagnosis and treatment. With the advent of genome-wide association scans, numerous risk variants have been identified as candidates for conferring susceptibility to type 2 diabetes but most of them with only modest effects [2].
Transcription factor 7-like 2 (TCF7L2), a protein belonging to a family of TCF/lymphoid enhancer factor (LEF) transcription factors, is a key component of the Wnt signaling pathway involved in the regulation of pancreatic beta-cell proliferation, differentiation and insulin secretion [3,4]. The gene encoding TCF7L2 spans a 215,863 bp region on chromosome 10q25.3 [5]. TCF7L2 is the most significant and consistent genetic risk factor of diabetes identified to date. It was first identified as a diabetes risk conferring gene in 2006 [6]. Later, the common polymorphisms in the TCF7L2 have been associated with type 2 diabetes in numerous studies in different populations. The
* Corresponding author. Tel.: +48 81 7244 716; fax: +48 81 7244 357. E-mail addresses: [email protected], [email protected] (M. Buraczynska). 0168-8227/$ – see front matter # 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2011.05.017
diabetes research and clinical practice 93 (2011) 390–395
most significant genetic association was detected for two intronic single nucleotide polymorphisms (SNPs), rs7903146 (intron 3) and rs12255372 (intron 4) located 50 kb from each other [6–14]. The primary site of action for the TCF7L2 gene product in type 2 diabetes is unknown. It was suggested that TCF7L2 might be associated with beta-cell dysfunction and decreased insulin secretion but not with insulin resistance [15]. In this study we investigated the potential association of the rs7903146 (C/T) polymorphism in the intron 3 of the TCF7L2 gene with type 2 diabetes. We also analyzed the effect of this SNP on different clinical phenotypes in type 2 diabetes patients.
2.
Methods
2.1.
Subjects
The study population consisted of 980 unrelated type 2 diabetes mellitus (T2DM) patients, consecutively enrolled between January 2004 and September 2008. All subjects were Caucasians of Polish origin. The patients were stratified into subgroups with different clinical phenotypes: diabetic nephropathy (DN) (n = 432), diabetic retinopathy (DR) (n = 408), cardiovascular disease (CVD) (n = 635), early onset of diabetes (45 years) (n = 438), and obesity (n = 189). In the diabetic nephropathy subgroup 199 patients (46%) had also diabetic retinopathy. Similarly, in the diabetic retinopathy subgroup, 216 patients (53%) had also nephropathy. Diabetes was diagnosed according to American Diabetes Association criteria. One or more of the following conditions were met: the classic symptoms of hyperglycemia (polyuria, polydipsia, and weight loss), fasting plasma glucose 126 mg/dl or random plasma glucose 200 mg/dl, the use of insulin or oral hypoglycemic agents. The mean duration of diabetes was 12.6 years (range 6–27). It was estimated from time of the first symptoms attributable to the disease or time of first detection of glycosuria. Diabetic nephropathy was diagnosed clinically when the patient had persistent albuminuria 300 mg/24 h in at least two consecutive determinations in the absence of hematuria or infection. The patients without diabetic nephropathy were normoalbuminuric, those with microalbuminuria were excluded. Diabetic retinopathy was diagnosed by independent ophthalmologists. All patients underwent a complete ophthalmological examination, including corrected visual acuity, fundoscopic examination and fundus photography (three 458 fields per eye) at least every year. Fundoscopic findings were determined by retinal specialists. Retinopathy was diagnosed according to the Early Treatment Diabetic Retinopathy Study (ETDRS) criteria: the presence of microaneurysms, hemorrhages, cotton wool spots, intraretinal microvascular abnormalities, hard exudates, venous beading and new vessels. Cardiovascular disease was diagnosed and documented as one or the combination of several pathological states: congestive heart failure, left ventricular hypertrophy, angina pectoris, ischemic heart disease, myocardial infarction, ischemic cerebral stroke, vascular calcifications or atheromatous lesions. Each clinical manifestation of CVD was confirmed by appropriate biochemical, radiographic,
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echocardiographic and vascular diagnostic criteria. There was a substantial overlap between categories. No gender differences between the clinical phenotypes of CVD were observed. Arrhythmia was more prevalent in male patients but the difference did not reach statistical significance. At the beginning of the study 642 individuals (65.5%) in the patient group were hypertensives. Hypertension was defined according to WHO criteria (World Health Organization, 1999). All patients had persistent systolic blood pressure > 140 mm Hg and diastolic blood pressure > 90 mm Hg and/or were receiving anti-hypertensive treatment. Secondary hypertension was excluded by clinical and laboratory examination. Obesity was defined as BMI 30 kg/m2. A positive family history of diabetes in first-degree relatives was reported by 224 patients (23%). Glycemic control was evaluated by measuring glycated HbA1c levels by turbidimetric inhibition immunoassay TINIA using Tina-quant hemoglobin A1cII (Roche-Hitachi 747). All other biochemical parameters were measured by standard laboratory procedures. Control subjects (n = 924) were healthy normotensive volunteers (mostly blood donors and hospital staff members), with no history of diabetes or hypertension. Written informed consent was obtained from all subjects in accordance with principles of the Declaration of Helsinki. The study protocol was approved by the institutional ethics committee.
2.2.
Genotyping
Genomic DNA was extracted from peripheral blood leukocytes (obtained from EDTA anticoagulated blood) using a standard technique. All subjects were genotyped for the rs7903146 single nucleotide polymorphism (SNP) by polymerase chain reaction (PCR) and subsequent cleavage of amplified fragment with Rsa I restriction endonuclease (Fermentas GmbH, St Leon-Rot, Germany). DNA fragments were visualized on 3% agarose gels. The quality of genotyping was controlled by using blind DNA duplicates for some samples (96). Also, 30 samples were randomly selected for each genotype (CC, CT and TT) and the PCR products were sequenced by automated sequencing in CEQ 8000 Genetic Analysis System (Beckman Coulter, High Wycombe, England). Observed concordance between genotyping assays was 100%.
2.3.
Statistical analysis
Statistical calculations were performed using SPSS 11.0 for Windows (SPSS, Inc., Chicago, IL, USA). For baseline characteristics the normally distributed continuous variables are presented as means SD. The Hardy–Weinberg equilibrium was tested with the x2 test. Genotype distribution and allele frequencies were compared between groups using a x2 test of independence with 2 2 contingency and z statistics. Student’s t-test and ANOVA were used for statistical significance between variables/groups. Where appropriate, the odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were calculated. A Bonferroni correction was applied for multiple testing (4 comparisons: subgroups/nephropathy, retinopathy, CVD, early onset of diabetes). Multivariate logistic regression was performed for analysis of independent risk factor for
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Table 1 – Demographic and clinical profile of studied subjects. Variable
T2DM patients
Controls
N Male/female Age at study (years) Diabetes duration (years) Diabetic retinopathy (%) Diabetic nephropathy (%) Hypertension (%) HbA1c (%) Total cholesterol (mmol/l) HDL cholesterol (mmol/l) Triglycerides (mmol/l) BMI (kg/m2)
980 512/468 57 18 12.6 8a 408 (42) 432 (44) 642 (65.5) 8.2 3.8 5.1 1.51 1.2 0.65 2.3 1.78 27.4 3.6
924 492/432 54 17 NA 0 0 0 ND 3.9 1.71 1.82 0.86 1.17 0.93 26.2 3.4
p value NS <0.001
<0.001 <0.001 <0.001 <0.001
T2DM: type 2 diabetes mellitus. Values are presented as mean SD or numbers (%). NA: not applicable and ND: not determined. Mean diabetes duration was 14.0 9.2 for patients with DN and 11.2 6.8 for patients without DN.
a
diabetic nephropathy. A two-tailed type I error rate of 5% was considered statistically significant. Power calculations were done using on-line available power calculator (http://calculators.stat.ucla.edu).
3.
Results
The genotype of the rs7903146 polymorphism in the TCF7L2 gene was determined in 980 patients with type 2 diabetes and 924 healthy individuals. The demographic and clinical profile of studied subjects has been presented in Table 1. The frequencies of the genotypes and alleles in the control group were similar to those reported for other European populations [16,17]. The genotype distribution among the controls was in Hardy–Weinberg equilibrium. Table 2 shows the genotypes of rs7903146 SNP in diabetes patients and controls. The genotype distribution and allele frequencies were significantly different between the entire diabetes group and healthy individuals ( p < 0.01). The odds ratio (OR) for the TT genotype vs. CC was 2.49 (95% CI 1.84–3.39). Genotype and allele frequencies were compared in subgroups of diabetes patients with diabetic nephropathy, diabetic retinopathy, CVD, early onset of diabetes and obesity. The results are presented in Table 3. The frequency of the TT genotype was higher in patients with CVD than in those without cardiovascular comorbidity although the difference did not reach a statistical significance ( p = 0.061). The same tendency was observed for diabetic retinopathy and early onset of diabetes, with no statistical significance after adjusting for confounding factors. The T allele was associated with an earlier age at diagnosis (54.3 years for CC, 54.1 years for
CT and 52.7 years for TT genotype, p < 0.01). There were no statistically significant differences in the allele/genotype distribution between obese and non-obese subjects (data not shown). After the Bonferroni correction for multiple comparisons a strong association was observed between the rs7903146 SNP and diabetic nephropathy (Table 4). The frequency of the TT homozygous genotype in the diabetic nephropathy subgroup was 22% compared to 9% in patients with no microvascular complications after 10 years of T2DM duration. The OR for TT genotype in DN patients, after adjusting for multiple testing, was 3.71 (95% CI 1.99–6.93) vs. no complications subgroup. After adjusting for age, sex, blood pressure, BMI and diabetes duration, the odds ratio was 2.98 (95% CI 1.16–6.87). The observed association of the rs7903146 SNP with diabetic nephropathy was even stronger in patients with an early onset of diabetes. The minor allele frequency was 0.52 vs. 0.28 in patients with no complications. The frequency of the TT homozygotes in T2DM patients with early onset of diabetes was 34% compared to 12% in patients with nephropathy and late onset of diabetes ( p < 0.001). Of all TT homozygotes in diabetic nephropathy group 71% were among patients with an early onset of diabetes. After adjusting for multiple testing, OR for TT genotype was 6.60 (95% CI 3.38–12.87) vs. no complications subgroup. Logistic regression analysis revealed that the association of minor allele of rs7903146 polymorphism with type 2 diabetes and diabetic nephropathy remained significant after adjusting for other covariates potentially associated with T2DM risk, age, gender, hypertension, BMI, HbA1c and also diabetes duration (OR 2.36, 95% CI 2.11–3.66 for T2DM and OR 2.73, 95% CI 2.29–3.42 for diabetic nephropathy).
Table 2 – Genotype and allele distribution of rs7903146 SNP in the TCF7L2 gene in type 2 diabetes patients and controls. Subjects
T2 DM (n = 980) Controls (n = 924)
CC
416 (42) 490 (53)
CT
407 (42) 360 (39)
TT
157 (16) 74 (8)
MAF
0.37 0.27
OR (95% CI) For T allele
For TT genotype
1.53 (1.33–1.76) 1.0 (ref.)
2.49 (1.84–3.39) 1.0 (ref.)
T2DM: type 2 diabetes mellitus and MAF: minor allele frequency. Data are n (%). HWE test: T2DM x2 = 11.22, p = 0.000809; Controls x2 = 0.48, p = 0.488422. Statistical power for T2DM vs. controls = 99.1%.
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Table 3 – The rs7903146 SNP and different clinical profiles in T2DM patient group. Subjects CVD (n = 635) No CVD (n = 345) DR (n = 408) No DR (n = 572) Early onset of T2DM (n = 438) Late onset of T2DM (n = 542)
CC 261 155 162 254 178 238
CT
(41) (45) (40) (44) (41) (44)
254 153 167 240 172 235
TT
(40) (44) (41) (42) (39) (43)
120 37 79 78 88 69
(19) (11) (19) (14) (20) (13)
MAF 0.39 0.33 0.40 0.35 0.40 0.34
T allele carriers 374 190 246 318 260 304
(59) (55) (60) (56) (59) (56)
OR (95% CI)a for T allele 1.29 (1.06–1.57) 1.0 (ref.) 1.25 (1.03–1.50) 1.0 (ref.) 1.25 (1.04–1.51) 1.0 (ref.)
T2DM: type 2 diabetes mellitus; CVD: cardiovascular disease; DR: diabetic retinopathy; and MAF: minor allele frequency. Odds ratios were calculated: CVD vs. no CVD, DR vs. no DR, early onset vs. late onset, and adjusted for age, sex, blood pressure, BMI, HbA1c and diabetes duration. a
4.
for nephropathy. However, this study did not distinguish whether observed associations were with type 2 diabetes or diabetic nephropathy [23]. Wu et al. investigated association of diabetes related genes with diabetic nephropathy in Taiwanese population. In a single locus analysis they found the marginally significant association of rs7903146 variant with DN [24]. In the study of French population there was no evidence of association of the rs7903146 variant with type 2 diabetes complications such as CHD, severe nephropathy or retinopathy (exact data were not reported) [18]. In our study there was a trend towards association of TT genotype with CVD in diabetes patients ( p = 0.061). Similar trend was observed for CHD in the ARIC study [25]. In contrast, Sousa et al. found a significant association between TCF7L2 rs7903146 genotypes and coronary artery disease in non-diabetic subjects but were not able to demonstrate this association in diabetic patients [26]. We found that the T allele of rs7903146 was associated with an earlier age at diagnosis. It is in agreement with earlier studies [18,27,28]. A gene dosage effect has been observed. The risk of type 2 diabetes in TT homozygotes was increased by 2.49, similar to the result obtained in another study performed in a group of 290 Polish patients with T2DM [29]. It is not clear how rs7903146 variant, located approximately 9 kb downstream of exon 4 of TCF7L2 gene, might affect its expression or the function of the protein product. Functional studies are required to elucidate this. Our results should be viewed with caution since there is a deviation from HWE estimates in cases. The genotyping errors can be excluded since the quality of genotyping in our study
Discussion
Several recent studies have investigated the effect of SNPs in TCF7L2 on type 2 diabetes development. So far TCF7L2 is the most reproducible susceptibility gene for type 2 diabetes in various ethnic groups [4,6,12,13,15,18,19]. Recently, in the study of African American population, rs7903146 SNP in the TCF7L2 gene was implicated as a causal variant in type 2 diabetes [20]. Functional studies also point to a functional role of this SNP. Gaulton et al. [21] found that rs7903146 is located in islet-selective open chromatin and shows allele-specific islet enhancer activity. In the present study we investigated the potential association between type 2 diabetes and rs7903146 SNP in the intron 3 of TCF7L2 gene in a large group of Polish patients with type 2 diabetes. The results successfully replicated the previously found association between this SNP and type 2 diabetes. In addition, after stratification it was apparent that there is a strong association of this SNP with diabetic nephropathy, especially in patients with an early onset of diabetes. This could indicate to the higher risk of microvascular complications of T2DM for the T allele carriers. Previously published papers suggested a possible association of the rs7903146 variant with renal dysfunction in diabetic patients [22–24]. In the older population of Italian subjects the T allele carriers were more likely to have the key microvascular complication of poor renal function [22]. Another study investigated association of the TCF7L2 rs7903146 variant with type 2 diabetes in an African-American population enriched
Table 4 – Genotype and allele distribution of rs7903146 SNP in diabetes patients with and without diabetic nephropathy. Subjects DN (n = 432) No DN (n = 548) No complicationsb (n = 154) Early onset with DN (n = 198) Late onset with DN (n = 234)
CC 146 270 80 58 88
(34) (50) (52) (29) (38)
CT 191 216 60 73 118
(44) (39) (39) (37) (50)
TT 95 62 14 67 28
(22) (11) (9) (34) (12)
MAF
OR (95% CI)a for T allele
0.44 0.31 0.28 0.52 0.37
1.97 (1.49–2.61) – 1.0 (ref.) 2.74 (1.99–3.75) 1.47 (1.08–2.01)
DN: diabetic nephropathy and MAF: minor allele frequency. After adjusting for multiple testing. Data are n (%). The OR for TT genotype: DN patients vs. no complications 3.71 (1.99–6.93) and adjusted for age, sex, blood pressure, BMI and diabetes duration 2.98 (1.16–6.87), early onset with DN vs. no complications 6.60 (3.38–12.87) and adjusted 5.93 (2.96–11.04). Statistical power for DN vs. no complications = 97.4%. b No microvascular complications after 10 years of T2DM duration. a
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was carefully controlled with using blind sample duplicates and direct sequencing. The likely reason for deviation from HWE may be selection bias. As most of the association studies, ours has some limitations. Since it is a retrospective case-control study, a selection bias cannot be excluded. To limit this bias we included consecutive patients in the study and tried to adjust for known confounding risk factors. All normoglycemic individuals with traits related to type 2 diabetes, like obesity, hypertension or hyperlipidemia, were excluded from the study. The strength of our study is that all patients and controls are of the same ethnic origin. Furthermore all subjects were examined employing well defined diagnostic criteria and genotyping was performed blind with respect to case-control status. The sample size in our study had 97.4% power to detect the genetic effect of TCF7L2 allele at a p value of 0.05. In conclusion, the findings of our study confirm that the risk allele of the rs7903146 SNP in the TCF7L2 gene is strongly associated with type 2 diabetes, even after correcting for traditional risk factors. The T allele of this polymorphism also conferred the risk of developing diabetic nephropathy.
Acknowledgements This study was supported by the grants DS 379/09 and DS 383/ 09 from Medical University of Lublin.
Conflict of interest The authors declare no conflict of interest.
references
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