DIAB-6297; No. of Pages 6 diabetes research and clinical practice xxx (2015) xxx–xxx
Contents available at ScienceDirect
Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Association of thioredoxin reductase 2 (TXNRD2) gene polymorphisms with myocardial infarction in Slovene patients with type 2 diabetes mellitus Stojan Karizˇ a,1, Sara Mankocˇ b,1, Daniel Petrovicˇ b,* a
Internal Department, General Hospital Izola, Polje 35, 6310 Izola, Slovenia Institute of Histology and Embryology, Faculty of Medicine, University Ljubljana, Korytkova 2, 1105 Ljubljana, Slovenia b
article info
abstract
Article history:
Objectives: The aim of our study was to investigate the relationship between genetic
Received 29 July 2014
polymorphisms in the mitochondrial thioredoxin reductase 2 (TrxR2) and myocardial
Received in revised form
infarction (MI) in subjects with type 2 diabetes mellitus (T2DM) of Slovenian origin.
28 December 2014
Methods: The study population consisted of 972 Caucasian subjects with T2DM of more than
Accepted 30 January 2015
10 years’ duration: 161 patients with MI and 811 patients with no history of coronary artery
Available online xxx
disease. Polymorphisms in thioredoxin reductase 2 (TXNRD2) gene, rs1548357, rs4485648,
Keywords:
Results: Individuals carrying CC + CT genotypes of rs1548357 TXNRD2 polymorphism had
Oxidative stress
lower prevalence of MI compared with TT genotype group (41.6% vs 52.8%, OR = 0.589, 95%
and rs5748469, were studied.
Thioredoxin reductase 2
CI = 0.368–0.942, P = 0.027).
Genetic polymorphisms
Conclusions: The TXNRD2 rs 1548357 polymorphism might be a genetic risk factor for MI in
Type 2 diabetes mellitus
subjects with T2DM of Slovenian origin. # 2015 Elsevier Ireland Ltd. All rights reserved.
Myocardial infarction
1.
Introduction
Type 2 diabetes mellitus (T2DM) is a complex metabolic disease with a strong genetic component and familial inheritance. According to the latest estimates in 2013, it affected 320 million people worldwide, with a rapidly increasing incidence and prevalence [1]. T2DM is associated with accelerated atherosclerosis, the major cause of vascular complications leading to increased morbidity and mortality [2]. The risk of myocardial
infarction (MI) has increased 2- to 4-fold, and over 50% of all patients with T2DM die of coronary artery disease (CAD) [3]. Oxidative stress has been proposed as a major contributor in the development and progression of T2DM and its complications [4]. The metabolic abnormalities of T2DM cause mitochondrial overproduction of reactive oxygen species (ROS), which is central to the pathogenesis of diabetic vascular complications [5]. Vulnerability to oxidative stress is likely modulated by gene polymorphisms of the key enzymes involved in the regulation of redox status [6].
* Corresponding author. Tel.: +386 1 543 7367; fax: +386 1 543 7361. E-mail addresses: [email protected], [email protected] (D. Petrovicˇ). 1 The authors contributed equally to the manuscript. http://dx.doi.org/10.1016/j.diabres.2015.01.038 0168-8227/# 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Karizˇ S, et al. Association of thioredoxin reductase 2 (TXNRD2) gene polymorphisms with myocardial infarction in Slovene patients with type 2 diabetes mellitus. Diabetes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.diabres.2015.01.038
DIAB-6297; No. of Pages 6
2
diabetes research and clinical practice xxx (2015) xxx–xxx
Mitochondria produce 85–90% of cellular ROS and are increasingly appreciated as a central source of ROS under stressful energetic and redox conditions [7]. The thioredoxin (Trx) system is the major H2O2 scavenger in mitochondria and provides a primary line of defense against ROS produced by the mitochondrial respiratory chain [7]. Trx reduces ROS via reversible oxidation at two cysteine residues (Cys-32 and Cys-35) and is subsequently reduced by Trx reductase (TrxR) and NADPH [8]. In the mitochondrial matrix, the Trx system is composed of Trx2, Trx reductase 2 (TrxR2), and peroxiredoxin 3 [7]. Trx2 plays a critical role in maintenance of vascular endothelial cell function and prevention of atherosclerosis development, in part by reducing oxidative stress and increasing NO bioavailability [9]. Moreover, mitochondrial Trx2-system has an important protective role in cardiomyocyte ischemic/reperfusion injury. Cardiospecific deletion of Txnrd2 in the adult mouse heart resulted in excessive vulnerability during early postischemic reperfusion, evidenced by increased infarct size and cardiomyocyte apoptosis, as well as impaired postischemic functional recovery of the heart [10]. Thioredoxin reductase 2 (TXNRD2) gene is localized on chromosome 22q11.21. TXNRD2 consists of 18 exons of relatively small size with intron sizes ranking between 200 and 10 000 bp [11]. Thus far, genetic variation in the TXNRD2 was found to be associated with prostate cancer [12], gastric cancer [13], colon and rectal cancer [14], and the development of subcutaneous fibrosis in breast cancer patients treated with radiation therapy [15]. More importantly, rare mutations in human TXNRD2 were found in patients with dilated cardiomyopathy [16]. Interestingly, in contrast to the Txnrd2-knockout mouse model, where embryonic lethality as a consequence of hematopoietic and cardiac defects is described, absence of TXNRD2 in humans leads to glucocorticoid deficiency. This recent finding was the first report of a homozygous mutation in any component of the Trx antioxidant system leading to inherited disease in humans [17]. Functional polymorphisms of several antioxidant enzymes have been reported to be involved in the pathogenesis of T2DM [18], as well as with diabetic complications, such as MI [19,20]. Likewise, genetically determined variances in mitochondrial Trx2 system may be partially responsible for predisposition to macrovascular complications in T2DM subjects. To our knowledge no previous study has assessed the connection between the genetic variability of TXNRD2 gene and MI in subjects with T2DM. Accordingly, we tested the hypothesis that polymorphisms rs1548357, rs4485648, and rs5748469 of the TXNRD2 gene are associated with MI in Slovenian population with T2DM.
2.
Patients and methods
The study population of this cross-sectional analysis consisted of 972 subjects with T2DM lasting more than 10 years: 161 patients with MI and 811 patients with no history of CAD, no signs of ischemic changes on electrocardiogram and no ischemic changes during submaximal stress testing. Patients
were classified as having T2DM according to the American Diabetes Association (ADA) criteria for the diagnosis and classification of diabetes [21]. The diagnosis of MI was made according to the accepted criteria [22]. Patients with MI were included in the study 1 to 9 months after the acute event. All the subjects enrolled in the study were Slovenes of Slavic origin (Caucasians). The patients and control subjects came from independent families. Body mass index (BMI) was calculated as weight in kilograms divided by the height in square meters. Glucose, total cholesterol, low density lipoproteins (LDL), high density lipoproteins (HDL), and triglycerides were determined by standard biochemical methods. Patients were asked whether they were actively smoking at the time of recruitment (current smokers). The national medical ethic committee approved the research protocol. All patients participating in the study gave written informed consent.
3.
Genotyping
Genomic DNA was extracted from 100 ml of whole blood using a Qiagen isolation kit. The rs1548357, rs4485648, and rs5748469 polymorphism of the TXNRD2 gene were genotyped by KBioscience Ltd using their novel fluorescence-based competitive allele-specific PCR (KASPar) assay. Details of the method used can be found at http://www.kbioscience.co.uk/.
4.
Statistical analysis
Continuous variables were expressed as means standard deviations. Continuous clinical data were compared using an unpaired Student’s t test. The Pearson chi-squared test (x2) was used to compare discrete variables and to test whether the genotypes distribution is in Hardy–Weinberg equilibrium. Further, all variables that showed significant differences by univariate analysis were put into a stepwise multiple logistic regression. A P-value of T) TT (Ser/Ser) GT (Ala/Ser) GG (Ala/Ala) G allele (Ala) T allele (Ser) PHWEy
Cases (161)
Controls (811)
Unadjusted P*
13 (8.1%) 54 (33.5%) 94 (58.4%) 80 (24.9%) 242 (75.1%) 0.196
81 (10%) 347 (42.8%) 383 (47.2%) 509 (31.4%) 1113 (68.6%) 0.853
0.035
2 (1.3%) 55 (34%) 104 (64.7%) 59 (18.3%) 263 (82.7%) 0.07
31 (3.8%) 233 (28.7%) 547 (67.5%) 295 (18.2%) 1327 (81.8%) 0.325
0.127
16 (10%) 72 (44.4%) 73 (45.6%) 218 (67.7%) 104 (32.3%) 0.774
120 (14.8%) 369 (45.5%) 322 (39.7%) 1013 (62.5%) 609 (37.5%) 0.396
0.189
0.024
1.00
0.085
Data are presented as number of carriers (%) or proportion of sample. z We have given the dbSNP rs number for each SNP which uniquely identifies it. The nucleotide position in intronic variants or codon number in coding SNPs is referred related to the start codon. The major allele appears before the nucleotide position and the minor allele after it. * P values were computed using chi-square tests to compare case and control groups. y PHWE values were computed using Pearson’s goodness-of-fit chisquare (1 df).
Controls
P Value
811 62.8 9.1 416 (51.3) 14.16 7.73 462 (56.9) 151 20 85 11 30.39 4.35 665 (82) 11.02 9.0 73 (9.0) 4.8 1.12 2.77 0.92 1.28 0.36 1.89 1.42
Contents available at ScienceDirect
Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Association of thioredoxin reductase 2 (TXNRD2) gene polymorphisms with myocardial infarction in Slovene patients with type 2 diabetes mellitus Stojan Karizˇ a,1, Sara Mankocˇ b,1, Daniel Petrovicˇ b,* a
Internal Department, General Hospital Izola, Polje 35, 6310 Izola, Slovenia Institute of Histology and Embryology, Faculty of Medicine, University Ljubljana, Korytkova 2, 1105 Ljubljana, Slovenia b
article info
abstract
Article history:
Objectives: The aim of our study was to investigate the relationship between genetic
Received 29 July 2014
polymorphisms in the mitochondrial thioredoxin reductase 2 (TrxR2) and myocardial
Received in revised form
infarction (MI) in subjects with type 2 diabetes mellitus (T2DM) of Slovenian origin.
28 December 2014
Methods: The study population consisted of 972 Caucasian subjects with T2DM of more than
Accepted 30 January 2015
10 years’ duration: 161 patients with MI and 811 patients with no history of coronary artery
Available online xxx
disease. Polymorphisms in thioredoxin reductase 2 (TXNRD2) gene, rs1548357, rs4485648,
Keywords:
Results: Individuals carrying CC + CT genotypes of rs1548357 TXNRD2 polymorphism had
Oxidative stress
lower prevalence of MI compared with TT genotype group (41.6% vs 52.8%, OR = 0.589, 95%
and rs5748469, were studied.
Thioredoxin reductase 2
CI = 0.368–0.942, P = 0.027).
Genetic polymorphisms
Conclusions: The TXNRD2 rs 1548357 polymorphism might be a genetic risk factor for MI in
Type 2 diabetes mellitus
subjects with T2DM of Slovenian origin. # 2015 Elsevier Ireland Ltd. All rights reserved.
Myocardial infarction
1.
Introduction
Type 2 diabetes mellitus (T2DM) is a complex metabolic disease with a strong genetic component and familial inheritance. According to the latest estimates in 2013, it affected 320 million people worldwide, with a rapidly increasing incidence and prevalence [1]. T2DM is associated with accelerated atherosclerosis, the major cause of vascular complications leading to increased morbidity and mortality [2]. The risk of myocardial
infarction (MI) has increased 2- to 4-fold, and over 50% of all patients with T2DM die of coronary artery disease (CAD) [3]. Oxidative stress has been proposed as a major contributor in the development and progression of T2DM and its complications [4]. The metabolic abnormalities of T2DM cause mitochondrial overproduction of reactive oxygen species (ROS), which is central to the pathogenesis of diabetic vascular complications [5]. Vulnerability to oxidative stress is likely modulated by gene polymorphisms of the key enzymes involved in the regulation of redox status [6].
* Corresponding author. Tel.: +386 1 543 7367; fax: +386 1 543 7361. E-mail addresses: [email protected], [email protected] (D. Petrovicˇ). 1 The authors contributed equally to the manuscript. http://dx.doi.org/10.1016/j.diabres.2015.01.038 0168-8227/# 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Karizˇ S, et al. Association of thioredoxin reductase 2 (TXNRD2) gene polymorphisms with myocardial infarction in Slovene patients with type 2 diabetes mellitus. Diabetes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.diabres.2015.01.038
DIAB-6297; No. of Pages 6
2
diabetes research and clinical practice xxx (2015) xxx–xxx
Mitochondria produce 85–90% of cellular ROS and are increasingly appreciated as a central source of ROS under stressful energetic and redox conditions [7]. The thioredoxin (Trx) system is the major H2O2 scavenger in mitochondria and provides a primary line of defense against ROS produced by the mitochondrial respiratory chain [7]. Trx reduces ROS via reversible oxidation at two cysteine residues (Cys-32 and Cys-35) and is subsequently reduced by Trx reductase (TrxR) and NADPH [8]. In the mitochondrial matrix, the Trx system is composed of Trx2, Trx reductase 2 (TrxR2), and peroxiredoxin 3 [7]. Trx2 plays a critical role in maintenance of vascular endothelial cell function and prevention of atherosclerosis development, in part by reducing oxidative stress and increasing NO bioavailability [9]. Moreover, mitochondrial Trx2-system has an important protective role in cardiomyocyte ischemic/reperfusion injury. Cardiospecific deletion of Txnrd2 in the adult mouse heart resulted in excessive vulnerability during early postischemic reperfusion, evidenced by increased infarct size and cardiomyocyte apoptosis, as well as impaired postischemic functional recovery of the heart [10]. Thioredoxin reductase 2 (TXNRD2) gene is localized on chromosome 22q11.21. TXNRD2 consists of 18 exons of relatively small size with intron sizes ranking between 200 and 10 000 bp [11]. Thus far, genetic variation in the TXNRD2 was found to be associated with prostate cancer [12], gastric cancer [13], colon and rectal cancer [14], and the development of subcutaneous fibrosis in breast cancer patients treated with radiation therapy [15]. More importantly, rare mutations in human TXNRD2 were found in patients with dilated cardiomyopathy [16]. Interestingly, in contrast to the Txnrd2-knockout mouse model, where embryonic lethality as a consequence of hematopoietic and cardiac defects is described, absence of TXNRD2 in humans leads to glucocorticoid deficiency. This recent finding was the first report of a homozygous mutation in any component of the Trx antioxidant system leading to inherited disease in humans [17]. Functional polymorphisms of several antioxidant enzymes have been reported to be involved in the pathogenesis of T2DM [18], as well as with diabetic complications, such as MI [19,20]. Likewise, genetically determined variances in mitochondrial Trx2 system may be partially responsible for predisposition to macrovascular complications in T2DM subjects. To our knowledge no previous study has assessed the connection between the genetic variability of TXNRD2 gene and MI in subjects with T2DM. Accordingly, we tested the hypothesis that polymorphisms rs1548357, rs4485648, and rs5748469 of the TXNRD2 gene are associated with MI in Slovenian population with T2DM.
2.
Patients and methods
The study population of this cross-sectional analysis consisted of 972 subjects with T2DM lasting more than 10 years: 161 patients with MI and 811 patients with no history of CAD, no signs of ischemic changes on electrocardiogram and no ischemic changes during submaximal stress testing. Patients
were classified as having T2DM according to the American Diabetes Association (ADA) criteria for the diagnosis and classification of diabetes [21]. The diagnosis of MI was made according to the accepted criteria [22]. Patients with MI were included in the study 1 to 9 months after the acute event. All the subjects enrolled in the study were Slovenes of Slavic origin (Caucasians). The patients and control subjects came from independent families. Body mass index (BMI) was calculated as weight in kilograms divided by the height in square meters. Glucose, total cholesterol, low density lipoproteins (LDL), high density lipoproteins (HDL), and triglycerides were determined by standard biochemical methods. Patients were asked whether they were actively smoking at the time of recruitment (current smokers). The national medical ethic committee approved the research protocol. All patients participating in the study gave written informed consent.
3.
Genotyping
Genomic DNA was extracted from 100 ml of whole blood using a Qiagen isolation kit. The rs1548357, rs4485648, and rs5748469 polymorphism of the TXNRD2 gene were genotyped by KBioscience Ltd using their novel fluorescence-based competitive allele-specific PCR (KASPar) assay. Details of the method used can be found at http://www.kbioscience.co.uk/.
4.
Statistical analysis
Continuous variables were expressed as means standard deviations. Continuous clinical data were compared using an unpaired Student’s t test. The Pearson chi-squared test (x2) was used to compare discrete variables and to test whether the genotypes distribution is in Hardy–Weinberg equilibrium. Further, all variables that showed significant differences by univariate analysis were put into a stepwise multiple logistic regression. A P-value of T) TT (Ser/Ser) GT (Ala/Ser) GG (Ala/Ala) G allele (Ala) T allele (Ser) PHWEy
Cases (161)
Controls (811)
Unadjusted P*
13 (8.1%) 54 (33.5%) 94 (58.4%) 80 (24.9%) 242 (75.1%) 0.196
81 (10%) 347 (42.8%) 383 (47.2%) 509 (31.4%) 1113 (68.6%) 0.853
0.035
2 (1.3%) 55 (34%) 104 (64.7%) 59 (18.3%) 263 (82.7%) 0.07
31 (3.8%) 233 (28.7%) 547 (67.5%) 295 (18.2%) 1327 (81.8%) 0.325
0.127
16 (10%) 72 (44.4%) 73 (45.6%) 218 (67.7%) 104 (32.3%) 0.774
120 (14.8%) 369 (45.5%) 322 (39.7%) 1013 (62.5%) 609 (37.5%) 0.396
0.189
0.024
1.00
0.085
Data are presented as number of carriers (%) or proportion of sample. z We have given the dbSNP rs number for each SNP which uniquely identifies it. The nucleotide position in intronic variants or codon number in coding SNPs is referred related to the start codon. The major allele appears before the nucleotide position and the minor allele after it. * P values were computed using chi-square tests to compare case and control groups. y PHWE values were computed using Pearson’s goodness-of-fit chisquare (1 df).
Controls
P Value
811 62.8 9.1 416 (51.3) 14.16 7.73 462 (56.9) 151 20 85 11 30.39 4.35 665 (82) 11.02 9.0 73 (9.0) 4.8 1.12 2.77 0.92 1.28 0.36 1.89 1.42
