Polymorphism in microRNA-196a2 contributes to the risk of cardiovascular disease in type 2 diabetes patients Monika Buraczynska, Pawel Zukowski, Piotr Wacinski, Katarzyna Ksiazek, Wojciech Zaluska PII: DOI: Reference:
S1056-8727(14)00134-2 doi: 10.1016/j.jdiacomp.2014.05.006 JDC 6270
To appear in:
Journal of Diabetes and Its Complications
Received date: Revised date: Accepted date:
18 January 2014 3 May 2014 17 May 2014
Please cite this article as: Buraczynska, M., Zukowski, P., Wacinski, P., Ksiazek, K. & Zaluska, W., Polymorphism in microRNA-196a2 contributes to the risk of cardiovascular disease in type 2 diabetes patients, Journal of Diabetes and Its Complications (2014), doi: 10.1016/j.jdiacomp.2014.05.006
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Polymorphism in microRNA-196a2 contributes to the risk of
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cardiovascular disease in type 2 diabetes patients
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Monika Buraczynska a*, Pawel Zukowski a, Piotr Wacinski b, Katarzyna Ksiazek a,
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Wojciech Zaluska a
Department of Nephrology, Medical University of Lublin, Lublin, Poland
b
Department of Cardiology, Medical University of Lublin, Lublin, Poland
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a
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Running title :
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miRNA-196a2 gene polymorphism in type 2 diabetes patients
*Corresponding author : Monika Buraczynska, Department of Nephrology, Medical University of Lublin, Dr K. Jaczewskiego 8, 20-954 Lublin, Poland, tel: +48 (81) 7244 716, fax: +48 (81) 7244 357, e-mail: [email protected]
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ACCEPTED MANUSCRIPT ABSTRACT
Aims: To investigate the effect of the microRNA-196a2 gene polymorphism
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(rs11614913) on risk of cardiovascular disease in type 2 diabetes patients.
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Methods: We examined 920 patients with diabetes and 834 healthy controls. All subjects were genotyped for the miRNA-196a2 SNP by polymerase chain reaction (PCR) and restriction analysis.
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Results: The genotype distribution among controls and patients was in HardyWeinberg equilibrium (p = 0.227 and 0.308, respectively). The frequency of the T
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allele was lower in patients than in controls (p = 0.044). The odds ratio 0.66 (95 % CI 0.54-0.79) suggests an association of the T allele with decreased risk of T2DM. For the main purpose of the study, T2DM patients were stratified into patients with CVD and those without it. The T allele and TT genotype were significantly more frequent in patients with CVD compared to those without CVD
(p = 0.013, p < 0.001,
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respectively). The odds ratio for the T allele in the CVD+ subgroup vs. CVD- was 1.76 (1.35-2.30), p < 0.0001, mostly due to the overrepresentation of TT
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homozygotes. The highest risk of development of CVD was observed in the additive model for TT homozygotes (OR 3.33, 95 % CI 2.05-5.42, p < 0.0001).
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Conclusion: Our findings suggest that miRNA-196a2 T/C polymorphism (rs11614913) is associated with an increased risk of CVD
in type 2 diabetes patients. This
provides further insights on pathogenesis of cardiovascular disease in type 2
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diabetes patients.
Keywords Type 2 diabetes, miRNAs, Single nucleotide polymorphism, Genetics, Cardiovascular disease
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1. Introduction
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Diabetes mellitus (DM), a complex multifactorial disease, increases the risk for
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many serious vascular complications, such as retinopathy, nephropathy and neuropathy. The macrovascular complications are associated with cardiovascular diseases, mainly coronary artery disease, atherosclerosis, hypertension and stroke
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(Beckman, Creager & Libby, 2002; He & King, 2004; Villeneuve, Reddy & Natarajan, 2011) . The increased evidence of these complications in diabetic population has attributed
to
several
pathological
factors
such
as
hyperglycemia,
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been
hyperlipidemia, growth factors, inflammatory cytokines and chemokines, and advanced glycation end products (Winer & Sowers, 2004; Brownlee, 2005). Recently, advances in genomics and proteomics have generated many new candidate gene markers of cardiovascular disease (May & Wang, 2008; Gianfagna,
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Cugino, Santimone & Jacoviello, 2012). Recent studies have unveiled a novel class of gene regulators called microRNAs (miRNAs) that play important roles in diverse
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aspects of cardiac development and functions (Bartel, 2004; Barringhaus & Zamore, 2009; Small & Olson). MicroRNAs are small non-protein-coding RNAs, 18-25
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nucleotides in length. They have been shown to play an important role in regulating mammalian gene expression and therefore involved in several key cellular functions (Bartel, 2004; Ambros, 2004; He & Hannon, 2004; Bentwich et al., 2005). MicroRNAs
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have made an enormous impact on our understanding of gene regulation at the posttranscriptional level, and numerous studies have documented their role in cell differentiation, growth, proliferation and apoptosis (Filipowicz, Bhattaharya & Sonenberg, 2008). Dysregulation of miRNAs can lead to a variety of human diseases. MicroRNAs are expressed in the cardiovascular system and could have a crucial role in normal development and physiology, as well as in disease development. They have been shown
to
participate
in
cardiovascular
disease
pathogenesis
including
atherosclerosis, coronary artery disease, myocardial infarction, heart failure and cardiac arrhythmias (Kartha & Subramanian, 2010; Papageorgiou et al., 2012; Quiat & Olson, 2013). Single nucleotide polymorphisms (SNPs) or mutations may alter miRNA expression and/or maturation and make a significant contribution to
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ACCEPTED MANUSCRIPT phenotypic variation, including the susceptibility to cardiovascular diseases (Georges, Coppieters & Charlier, 2007). According to recent data, miR-196a2 T/C polymorphism (rs11614913) contributes
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to pathogenesis of variety of diseases and is possible genetic predisposing factor. It
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is closely associated with regulation of annexin A1 (ANXA1) that is connected to decreased TNF-α levels. This miRNA target is related to thrombosis and inflammation pathway in the circulation system (Luthra et al., 2008). Recently, a pre-
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miRNA SNP in his-mir196a2 was found to be associated with occurrence and prognosis in coronary artery disease (Zhi et al., 2012), and with increased risk of
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cardiovascular disease, contributing to susceptibility and/or unfavorable prognosis (Xu et al., 2009; Zhou et al., 2010).
Since cardiovascular complications are very common in diabetes, the aim of our study was to investigate the potential effect of the rs11614913 SNP in miRNA-196a2 on the risk of cardiovascular disease in type 2 diabetes patients. We have performed
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a case-control association study in a cohort of 920 T2DM patients and 834 healthy
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controls.
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2. Materials and methods
2.1 Study subjects
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The study was designed to analyze the miRNA-196a2 gene polymorphism in type 2 diabetes (T2DM) patients . To compare the prevalence of polymorphism in patients with a healthy population, we included a control group of healthy individuals. The study population consisted of 920 unrelated T2DM patients, consecutively enrolled between 2007 and 2011 from the Departments of Nephrology and Cardiology of Medical University of Lublin and outpatient diabetes clinic.
All subjects were
Caucasians of Polish origin. Diabetes was diagnosed according to American Diabetes Association criteria. One or more of the following conditions were met for diagnosis : the presence of classic symptoms of hyperglycemia (polyuria, polydipsia, weight loss), fasting plasma glucose ≥ 7 mmol/L or random plasma glucose ≥ 11 mmol/L, the use of insulin or oral hypoglycemic agents. Age at diagnosis of type 2 diabetes was > 35 years in all subjects. The mean duration of diabetes, estimated from time of the first symptoms attributable to the disease or from time of first 4
ACCEPTED MANUSCRIPT detection of glycosuria, was 13.2 years (range 7-31). Glycemic control was evaluated by measuring glycated HbA1c levels by turbidimetric inhibition immunoassay (TINIA) using Tina-quant hemoglobin A1cII (Roche-Hitachi 747). consecutive determinations in the
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albuminuria ≥ 300 mg/24 hrs in at least two
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Diabetic nephropathy was diagnosed clinically in the presence of persistent
absence of hematuria or infection. Diabetic retinopathy was diagnosed according to the Early Treatment Diabetic Retinopathy Study (ETDRS) criteria : the presence of
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microaneurysms, hemorrhages, cotton wool spots, intraretinal microvascular abnormalities, hard exudates, venous beading and new vessels.
as one or the combination of several
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disease was diagnosed and documented
Cardiovascular
pathological states: congestive heart failure (with left ventricular rejection fraction < 45 %) (mainly from atherosclerotic heart disease or dilated cardiomyopathy), left ventricular hypertrophy, angina pectoris, ischemic heart disease, myocardial infarction, ischemic cerebral stroke, vascular calcifications or atheromatous lesions. of CVD were confirmed
by appropriate biochemical,
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Clinical manifestations
radiographic, echocardiographic and vascular diagnostic criteria. There was a
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substantial overlap between categories. All cases of congenital heart disease were excluded from the study.
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Among diabetic patients 635 individuals (69 %) were hypertensive, as defined according to World Health Organization criteria. Hypertensive patients had persistent systolic blood pressure > 140 mm Hg and diastolic blood pressure > 90 mm Hg
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and/or were currently receiving anti-hypertensive treatment. Healthy control subjects of Polish origin (n = 834) were unrelated volunteers (mostly blood donors and hospital staff members) with no history of diabetes or cardiovascular events and no signs of disease or dysfunction of cardiovascular system upon examination.
Subjects with a positive family history of diabetes or
cardiovascular disease in first degree relatives were excluded from the study. A written informed consent was obtained from all subjects in accordance with principles of the Declaration of Helsinki. The protocol of the study was approved by the institutional ethics committee.
2.2 Genotype determination
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ACCEPTED MANUSCRIPT Genomic DNA was extracted from peripheral blood leukocytes (obtained from EDTA anticoagulated blood) using standard procedures. All subjects were genotyped by PCR-RFLP method for the miRNA-196a2 T/C polymorphism (rs11614913). The
-
3’
and
antisense
primer
5’
-
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CCCCTTCCCTTCTCCTCCAGATA
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following primers were used for amplification reaction : sense primer 5’ CGAAAACCGACTGATGTAACTCCG - 3’. Genomic DNA (300 ng) was amplified in a final volume of 30 μl , using the following conditions : initial denaturation at 96 oC for
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6 min, followed by 35 cycles of 94oC for 30 sec, annealing at 58oC for 30 sec and extension at 72oC for 45 sec. A final extension step was at 72oC for 7 min. Ten μl of
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the PCR product were digested with Msp I restriction endonuclease (Fermentas, Vilnius, Lithuania) and digestion products were separated on 3 % agarose gel. The T allele was detected as a 149 bp fragment and the C allele as 125 bp and 24 bp fragments. The quality of genotyping was controlled by using blind DNA duplicates for some samples). In addition, 20
samples were randomly selected for each
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genotype of studied SNP and the PCR products were sequenced in CEQ 8000 Genetic Analysis System (Beckman Coulter, High Wycombe, England). Observed
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concordance between genotyping assays was 100 %.
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2.3 Statistical analysis
Statistical calculations were performed using SPSS version 11.0 for Windows
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(SPSS, Inc., Chicago, IL, USA). For baseline characteristics the normally distributed continuous variables are presented as means ± SD. The Hardy-Weinberg equilibrium was evaluated with the Χ2 test. Genotype distribution and allele frequencies were compared between groups using a Pearson Χ2 test of independence with 2x2 contingency and z statistics. ANOVA was used to compare average values of biochemical parameters. Student’s t-test and Mann-Whitney test were used for comparing discrete and continuous variables. For significant allelic and genotyping associations the adjusted odds ratios (OR) with corresponding 95 % confidence intervals (CI) were calculated. Power calculations were performed with the program of
Purcell
et
al.
(Purcell,
Cherny
&
Sham,
2003)
(available
at
http://pngu.mgh.harvard.edu/~purcell/gpc/ ). The prevalence of CVD was 58 % and the frequency of the miRNA-196a2 T allele was 0.33. The study had 97 % power (α = 0.05) to detect an association (OR vs CVD- patients 1.76 , 95 % CI 1.35-2.30). An 6
ACCEPTED MANUSCRIPT interaction of the polymorphisms with diabetic complications and various risk factors was examined with multiple logistic regression analysis. The Bonferroni correction was applied for multiple comparisons to control type 1 error. Statistical significance
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was set at P < 0.05.
3. Results
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The genotypes of the miRNA-196a2 T/C polymorphism were determined in 920 type 2 diabetes patients and 834 healthy individuals. The baseline clinical and laboratory
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parameters of studied T2DM patients are summarized in Table 1. The CVD+ patients presented an average age 4 years older than patients without CVD and longer diabetes duration. Both diabetic retinopathy and nephropathy were more frequent in CVD+ subgroup (p = 0.043 and 0.007, respectively). Patients with CVD had also higher levels of total cholesterol and triglycerides and a lower level of HDL
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cholesterol. BMI in CVD+ subgroup was higher than in CVD- (p = 0.01). The frequencies of the genotypes and alleles in the control group were similar to
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those reported in other studies of European populations (Catucci et al., 2010; Hezova et al., 2012). The genotype distribution among the controls and T2DM patients was in
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Hardy-Weinberg equilibrium (p = 0.227 and p = 0.308, respectively). Table 2 presents the genotypes of the miRNA-196a2 polymorphism in patients and controls.
The
frequency of the T allele was lower in T2DM patients than in controls (for T allele
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carriers p = 0.044). The odds ratio 0.66 (95 % CI 0.54-0.79) might suggest an association of the T allele with decreased risk of type 2 diabetes. For the main purpose of the study T2DM patients were divided into subgroups with and without CVD (n = 534 and n = 386, respectively). The comparison between these subgroups showed that theT allele and TT genotype were significantly more frequent in patients with CVD compared to those without CVD (p = 0.013, p < 0.001, respectively). A multiple logistic regression analysis was performed to evaluate the association of the miRNA-196a2 polymorphism adjusted for variables such as age, sex and hypertension. The odds ratio for the T allele in the CVD+ subgroup of patients was 1.76 (1.35-2.30), p < 0.0001 vs. CVD- subgroup. The difference in allele distribution was mostly due to the overrepresentation of TT homozygotes. The CT heterozygote was present at the similar levels (p = 0.307) in CVD+ and CVD-
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ACCEPTED MANUSCRIPT subgroup. The highest risk of development of CVD was observed in the additive
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model for TT homozygotes (OR 3.33, 95 % CI 2.05-5.42, p < 0.0001).
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4. Discussion
MicroRNAs are involved in a wide range of biological processes such as
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development, differentiation, cell growth and proliferation, metabolism, immunity and diseases such as cancer, diabetes and cardiovascular diseases (Catalucci, Gallo &
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Condorelli, 2009; Roberts & Steer, 2010). In the cardiovascular field numerous studies have documented the implication of miRNAs in nearly every pathological process
of
cardiovascular
system,
including
ischemia,
heart
failure
and
atherosclerosis (Kartha & Subramanian, 2010; Catalucci, Gallo & Condorelli, 2009). Genetic polymorphisms of miRNAs (SNPs) can potentially influence the processing
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or selection of miRNA targets.
To our knowledge, this is the first study to provide evidence that common SNP in
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miRNA might play a role in the risk of cardiovascular disease in type 2 diabetes patients.
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The T allele and TT genotype of the miRNA-196a2 T/C polymorphism (rs11614913) were significantly more frequent in type 2 diabetes patients with CVD compared to those without CVD (p = 0.013, p < 0.001, respectively). The T allele
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was associated with a 1.76-fold increased risk of CVD in type 2 diabetes patients. At α = 0.05 the statistical power for T2DM CVD+ vs. T2DM CVD- was 97 %. There are few reports available that demonstrated the effect of the miRNA-196a2 gene polymorphism on cardiovascular disease. Xu et al. (Xu et al., 2009) found that miR-196a2 polymorphism plays a role in sporadic congenital heart disease in Chinese population. It was associated with significantly increased expression of mature mir-196a in cardiac tissue specimens from congenital heart disease. In another Chinese study, the T allele of miRNA-196a2 polymorphism was associated with significantly increased risk of dilated cardiomyopathy (Zhou et al., 2010). In the study of Zhi et al. (Zhi et al., 2012) the authors did not find any main effect of the hsa-mir-196a2 polymorphism on the risk of coronary artery disease. They demonstrated, however, that CC and CC/CT genotypes of this polymorphism were significantly associated with the unfavorable prognostic factors compared with TT 8
ACCEPTED MANUSCRIPT genotype. The discrepancies between obtained results might be explained by the population-specific factors such as different genetic background or other causative variants in linkage. There are also some recent studies evaluating a role of the
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miRNA-196a2 gene polymorphism in cerebrovascular events. Liu at al. (Liu et al., on risk of
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2013) investigated the effect of three miRNA gene polymorphisms
ischemic stroke in a Chinese population. The hsa-mir-196a2 polymorphism was not associated with increased risk of stroke in studied subjects. In contrast, in a Korean
196a2
polymorphism
among
them)
associated
with
ischemic
stroke
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pathogenesis (Jeon et al., 2013).
was
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study, the combination of alleles of four miRNA gene polymorphisms (with miRNA-
The mechanism of effect of miRNA gene polymorphism on cardiovascular disease is unknown. The miR-196a2 is operative in several biological pathways. It was found to be associated with regulation of annexin A1 (ANXA1) that in turn is connected to decreased TNF-α levels. This mRNA target is involved in thrombosis and
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inflammation pathway in the circulation system (Luthra et al., 2008). Yekta et al. (Yekta et al., 2004) reported that miR-196a is expressed from HOX gene clusters loci
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in mammals and that HOX genes in turn are targets of miR-196a. Xu and colleagues (Xu et al., 2009) showed that
a SNP (rs11614913) in miR-196a2 affects its
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expression and consequently, expression of the downstream gene HOXB8. They hypothesized that rs11614913 might influence miR-196a-HOXB8-Shh signaling and thus be associated with congenital heart disease susceptibility. The rs11614913
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variant of miRNA-196a2 has been shown to alter the expression of mature miRNA196a2 and binding activity of target mRNA (Hoffman et al., 2009). It might modulate the expression level of miRNA-196a2 that in turn might affect the risk and/or progression of the disease (Saunders, Liang & Li, 2007). Cardiovascular diseases are conditions that often develop over many years of life and even small effects on gene expression patterns can play a role in the regulation processes occurring during CVD development. As in most of the association studies, one has to consider potential limitations. Although our study has a considerable statistical power, some differences might be due to a chance. Also some weak associations may have been missed. A survival and selection bias cannot be ruled out in this retrospective case-control study. We included consecutive patients and tried to adjust for known confounding risk factors. The comorbidities, however, might still represent a confounding factor. The strengths 9
ACCEPTED MANUSCRIPT of the study are the relatively large size of studied groups and the same ethnic origin of all patients and controls. Furthermore, all subjects were examined in a standardized manner with well defined diagnostic criteria. The genotyping was
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performed blind with respect to case-control status. The occurrence of diabetes and
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comorbidities such as CVD depends on interaction between different risk alleles, environmental factors and the lifestyle. Since the influence of single polymorphisms is rather small, an interactive effect of several factors may lead to an underestimation or
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overestimation of a role of polymorphism in determining the phenotype. These results might not therefore apply to populations with different genetic or environmental
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background. Additional studies in other populations should be performed, since the potential contribution of the polymorphism to cardiovascular disease might differ between ethnic populations.
In conclusion, the results of this study suggest that miRNA-196a2 T/C polymorphism (rs11614913) is associated with an increased risk of CVD in type 2
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diabetes patients. Our findings provide further insights on pathogenesis of
Acknowledgments
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cardiovascular disease in type 2 diabetes patients.
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This study was supported by grants DS 379/11 (MB) and DS 379/13 (MB) from Medical University of Lublin. The authors thank Ms. Bozenna Jarzabkowska for
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skillful technical help. Conflict of interest
The authors declare no conflict of interest.
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Table 1 Demographic and clinical profile of T2DM subjects with and without cardiovascular disease
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T2DM CVD+
T2DM CVD-
(534)
(386)
P valuea
262 / 272 65 ± 16
Diabetes duration (years)
14.6 ± 7.5
Diabetic retinopathy (%)
191 (36)
Diabetic nephropathy (%)
214 (40)
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Age at study (years)
Diabetic neuropathy (%)
139 (26)
Cardiovascular disease (%)
534 (100)
Hypertension (%)
Total cholesterol (mmol/l) HDL cholesterol (mmol/l
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Triglycerides (mmol/l)
a
61 ± 15.6
< 0.05
12.3 ± 7.6
< 0.05
104 (27)
0.043
108 (28)
0.007
72 (19)
0.074
0 247 (64)
0.144
7.9 ± 2.4
7.7 ± 2.7
0.345
4.9 ± 1.2
4.7 ± 1.1
< 0.001
1.2 ± 0.7
1.6 ± 0.8
< 0.001
2.2 ± 1.2
1.9 ± 0.7
0.01
28.8 ± 5.9
27.6 ± 4.9
0.01
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BMI (kg/m2)
0.325
379 (71)
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HbA1c (%)
170 / 216
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Male / female
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Variable
Pearson’s χ2 test for categorical variables, Mann-Whitney test for continuous data.
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T2DM, type 2 diabetes mellitus. CVD, cardiovascular disease. Values are presented as mean ± SD or numbers (%).
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ACCEPTED MANUSCRIPT Table 2 Genotype and allele distribution of miRNA-196a2 SNP in type 2 diabetes patients and controls p Subjects Genotype Allele OR (95 % CC
CT
TT
C
CI)
T
396
110
= 920)
(45)
(43)
(12)
Controls
292
417
125
(n = 834)
(35)
(50)
(15)
0.67
0.33
0.66 (0.54-
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414
< 0.0001
0.79)
0.60
0.40
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T2 DM (n
T
for T allele
1.0
(reference)
T2DM, type 2 diabetes mellitus. Genotype distributions are shown as numbers (%).
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HWE test for Controls Х2 = 1.46, p = 0.227; for T2DM patients Х2 = 1.04, p = 0.308. For T allele frequency p = 0.153. The nominal p value was adjusted according to
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Bonferroni correction.
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ACCEPTED MANUSCRIPT Table 3 Genotype and allele distribution of miRNA-196a2 SNP in type 2 diabetes patients with and without CVD p Subjects Genotype Allele OR (95 % CC
CT
TT
C
CI)a for T
T
209
240
CVD+ (n
(39)
(45)
T2DM
205
156
CVD- (n
(54)
(40)
85 (16)
0.62
0.38
1.76 (1.35-
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T2 DM
T
allele
< 0.0001
2.30)
25 (6)
0.27
1.0
(reference)
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= 386)
0.73
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= 534)
CVD+ versus CVD-
OR (95 % CI) for dominant model (CC vs. CT+TT) = 0.56 (0.43-074), p < 0.0001 OR (95 % CI) for recessive model (TT vs. CT+CC) = 2.73 (1.71-4.36), p < 0.0001 OR (95 % CI) for additive model (CT vs. CC) = 1.509 (1.14-1.99), p = 0.0038 a
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(TT vs. CC) = 3.33 (2.05-5.42), p < 0.0001 Adjusted for age, sex and hypertension. T2DM, type 2 diabetes mellitus; CVD,
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cardiovascular disease. Genotype distributions are shown as numbers (%). For T allele frequency CVD+ vs. CVD- p = 0.013. At α = 0.05 statistical power for T2DM
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CVD+ vs. T2DM CVD- = 97 %. The nominal p values were adjusted according to
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Bonferroni correction.
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S1056-8727(14)00134-2 doi: 10.1016/j.jdiacomp.2014.05.006 JDC 6270
To appear in:
Journal of Diabetes and Its Complications
Received date: Revised date: Accepted date:
18 January 2014 3 May 2014 17 May 2014
Please cite this article as: Buraczynska, M., Zukowski, P., Wacinski, P., Ksiazek, K. & Zaluska, W., Polymorphism in microRNA-196a2 contributes to the risk of cardiovascular disease in type 2 diabetes patients, Journal of Diabetes and Its Complications (2014), doi: 10.1016/j.jdiacomp.2014.05.006
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ACCEPTED MANUSCRIPT
Polymorphism in microRNA-196a2 contributes to the risk of
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cardiovascular disease in type 2 diabetes patients
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Monika Buraczynska a*, Pawel Zukowski a, Piotr Wacinski b, Katarzyna Ksiazek a,
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Wojciech Zaluska a
Department of Nephrology, Medical University of Lublin, Lublin, Poland
b
Department of Cardiology, Medical University of Lublin, Lublin, Poland
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a
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Running title :
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miRNA-196a2 gene polymorphism in type 2 diabetes patients
*Corresponding author : Monika Buraczynska, Department of Nephrology, Medical University of Lublin, Dr K. Jaczewskiego 8, 20-954 Lublin, Poland, tel: +48 (81) 7244 716, fax: +48 (81) 7244 357, e-mail: [email protected]
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ACCEPTED MANUSCRIPT ABSTRACT
Aims: To investigate the effect of the microRNA-196a2 gene polymorphism
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(rs11614913) on risk of cardiovascular disease in type 2 diabetes patients.
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Methods: We examined 920 patients with diabetes and 834 healthy controls. All subjects were genotyped for the miRNA-196a2 SNP by polymerase chain reaction (PCR) and restriction analysis.
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Results: The genotype distribution among controls and patients was in HardyWeinberg equilibrium (p = 0.227 and 0.308, respectively). The frequency of the T
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allele was lower in patients than in controls (p = 0.044). The odds ratio 0.66 (95 % CI 0.54-0.79) suggests an association of the T allele with decreased risk of T2DM. For the main purpose of the study, T2DM patients were stratified into patients with CVD and those without it. The T allele and TT genotype were significantly more frequent in patients with CVD compared to those without CVD
(p = 0.013, p < 0.001,
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respectively). The odds ratio for the T allele in the CVD+ subgroup vs. CVD- was 1.76 (1.35-2.30), p < 0.0001, mostly due to the overrepresentation of TT
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homozygotes. The highest risk of development of CVD was observed in the additive model for TT homozygotes (OR 3.33, 95 % CI 2.05-5.42, p < 0.0001).
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Conclusion: Our findings suggest that miRNA-196a2 T/C polymorphism (rs11614913) is associated with an increased risk of CVD
in type 2 diabetes patients. This
provides further insights on pathogenesis of cardiovascular disease in type 2
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diabetes patients.
Keywords Type 2 diabetes, miRNAs, Single nucleotide polymorphism, Genetics, Cardiovascular disease
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ACCEPTED MANUSCRIPT
1. Introduction
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Diabetes mellitus (DM), a complex multifactorial disease, increases the risk for
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many serious vascular complications, such as retinopathy, nephropathy and neuropathy. The macrovascular complications are associated with cardiovascular diseases, mainly coronary artery disease, atherosclerosis, hypertension and stroke
SC
(Beckman, Creager & Libby, 2002; He & King, 2004; Villeneuve, Reddy & Natarajan, 2011) . The increased evidence of these complications in diabetic population has attributed
to
several
pathological
factors
such
as
hyperglycemia,
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been
hyperlipidemia, growth factors, inflammatory cytokines and chemokines, and advanced glycation end products (Winer & Sowers, 2004; Brownlee, 2005). Recently, advances in genomics and proteomics have generated many new candidate gene markers of cardiovascular disease (May & Wang, 2008; Gianfagna,
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Cugino, Santimone & Jacoviello, 2012). Recent studies have unveiled a novel class of gene regulators called microRNAs (miRNAs) that play important roles in diverse
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aspects of cardiac development and functions (Bartel, 2004; Barringhaus & Zamore, 2009; Small & Olson). MicroRNAs are small non-protein-coding RNAs, 18-25
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nucleotides in length. They have been shown to play an important role in regulating mammalian gene expression and therefore involved in several key cellular functions (Bartel, 2004; Ambros, 2004; He & Hannon, 2004; Bentwich et al., 2005). MicroRNAs
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have made an enormous impact on our understanding of gene regulation at the posttranscriptional level, and numerous studies have documented their role in cell differentiation, growth, proliferation and apoptosis (Filipowicz, Bhattaharya & Sonenberg, 2008). Dysregulation of miRNAs can lead to a variety of human diseases. MicroRNAs are expressed in the cardiovascular system and could have a crucial role in normal development and physiology, as well as in disease development. They have been shown
to
participate
in
cardiovascular
disease
pathogenesis
including
atherosclerosis, coronary artery disease, myocardial infarction, heart failure and cardiac arrhythmias (Kartha & Subramanian, 2010; Papageorgiou et al., 2012; Quiat & Olson, 2013). Single nucleotide polymorphisms (SNPs) or mutations may alter miRNA expression and/or maturation and make a significant contribution to
3
ACCEPTED MANUSCRIPT phenotypic variation, including the susceptibility to cardiovascular diseases (Georges, Coppieters & Charlier, 2007). According to recent data, miR-196a2 T/C polymorphism (rs11614913) contributes
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to pathogenesis of variety of diseases and is possible genetic predisposing factor. It
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is closely associated with regulation of annexin A1 (ANXA1) that is connected to decreased TNF-α levels. This miRNA target is related to thrombosis and inflammation pathway in the circulation system (Luthra et al., 2008). Recently, a pre-
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miRNA SNP in his-mir196a2 was found to be associated with occurrence and prognosis in coronary artery disease (Zhi et al., 2012), and with increased risk of
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cardiovascular disease, contributing to susceptibility and/or unfavorable prognosis (Xu et al., 2009; Zhou et al., 2010).
Since cardiovascular complications are very common in diabetes, the aim of our study was to investigate the potential effect of the rs11614913 SNP in miRNA-196a2 on the risk of cardiovascular disease in type 2 diabetes patients. We have performed
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a case-control association study in a cohort of 920 T2DM patients and 834 healthy
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controls.
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2. Materials and methods
2.1 Study subjects
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The study was designed to analyze the miRNA-196a2 gene polymorphism in type 2 diabetes (T2DM) patients . To compare the prevalence of polymorphism in patients with a healthy population, we included a control group of healthy individuals. The study population consisted of 920 unrelated T2DM patients, consecutively enrolled between 2007 and 2011 from the Departments of Nephrology and Cardiology of Medical University of Lublin and outpatient diabetes clinic.
All subjects were
Caucasians of Polish origin. Diabetes was diagnosed according to American Diabetes Association criteria. One or more of the following conditions were met for diagnosis : the presence of classic symptoms of hyperglycemia (polyuria, polydipsia, weight loss), fasting plasma glucose ≥ 7 mmol/L or random plasma glucose ≥ 11 mmol/L, the use of insulin or oral hypoglycemic agents. Age at diagnosis of type 2 diabetes was > 35 years in all subjects. The mean duration of diabetes, estimated from time of the first symptoms attributable to the disease or from time of first 4
ACCEPTED MANUSCRIPT detection of glycosuria, was 13.2 years (range 7-31). Glycemic control was evaluated by measuring glycated HbA1c levels by turbidimetric inhibition immunoassay (TINIA) using Tina-quant hemoglobin A1cII (Roche-Hitachi 747). consecutive determinations in the
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albuminuria ≥ 300 mg/24 hrs in at least two
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Diabetic nephropathy was diagnosed clinically in the presence of persistent
absence of hematuria or infection. Diabetic retinopathy was diagnosed according to the Early Treatment Diabetic Retinopathy Study (ETDRS) criteria : the presence of
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microaneurysms, hemorrhages, cotton wool spots, intraretinal microvascular abnormalities, hard exudates, venous beading and new vessels.
as one or the combination of several
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disease was diagnosed and documented
Cardiovascular
pathological states: congestive heart failure (with left ventricular rejection fraction < 45 %) (mainly from atherosclerotic heart disease or dilated cardiomyopathy), left ventricular hypertrophy, angina pectoris, ischemic heart disease, myocardial infarction, ischemic cerebral stroke, vascular calcifications or atheromatous lesions. of CVD were confirmed
by appropriate biochemical,
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Clinical manifestations
radiographic, echocardiographic and vascular diagnostic criteria. There was a
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substantial overlap between categories. All cases of congenital heart disease were excluded from the study.
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Among diabetic patients 635 individuals (69 %) were hypertensive, as defined according to World Health Organization criteria. Hypertensive patients had persistent systolic blood pressure > 140 mm Hg and diastolic blood pressure > 90 mm Hg
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and/or were currently receiving anti-hypertensive treatment. Healthy control subjects of Polish origin (n = 834) were unrelated volunteers (mostly blood donors and hospital staff members) with no history of diabetes or cardiovascular events and no signs of disease or dysfunction of cardiovascular system upon examination.
Subjects with a positive family history of diabetes or
cardiovascular disease in first degree relatives were excluded from the study. A written informed consent was obtained from all subjects in accordance with principles of the Declaration of Helsinki. The protocol of the study was approved by the institutional ethics committee.
2.2 Genotype determination
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ACCEPTED MANUSCRIPT Genomic DNA was extracted from peripheral blood leukocytes (obtained from EDTA anticoagulated blood) using standard procedures. All subjects were genotyped by PCR-RFLP method for the miRNA-196a2 T/C polymorphism (rs11614913). The
-
3’
and
antisense
primer
5’
-
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CCCCTTCCCTTCTCCTCCAGATA
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following primers were used for amplification reaction : sense primer 5’ CGAAAACCGACTGATGTAACTCCG - 3’. Genomic DNA (300 ng) was amplified in a final volume of 30 μl , using the following conditions : initial denaturation at 96 oC for
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6 min, followed by 35 cycles of 94oC for 30 sec, annealing at 58oC for 30 sec and extension at 72oC for 45 sec. A final extension step was at 72oC for 7 min. Ten μl of
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the PCR product were digested with Msp I restriction endonuclease (Fermentas, Vilnius, Lithuania) and digestion products were separated on 3 % agarose gel. The T allele was detected as a 149 bp fragment and the C allele as 125 bp and 24 bp fragments. The quality of genotyping was controlled by using blind DNA duplicates for some samples). In addition, 20
samples were randomly selected for each
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genotype of studied SNP and the PCR products were sequenced in CEQ 8000 Genetic Analysis System (Beckman Coulter, High Wycombe, England). Observed
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concordance between genotyping assays was 100 %.
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2.3 Statistical analysis
Statistical calculations were performed using SPSS version 11.0 for Windows
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(SPSS, Inc., Chicago, IL, USA). For baseline characteristics the normally distributed continuous variables are presented as means ± SD. The Hardy-Weinberg equilibrium was evaluated with the Χ2 test. Genotype distribution and allele frequencies were compared between groups using a Pearson Χ2 test of independence with 2x2 contingency and z statistics. ANOVA was used to compare average values of biochemical parameters. Student’s t-test and Mann-Whitney test were used for comparing discrete and continuous variables. For significant allelic and genotyping associations the adjusted odds ratios (OR) with corresponding 95 % confidence intervals (CI) were calculated. Power calculations were performed with the program of
Purcell
et
al.
(Purcell,
Cherny
&
Sham,
2003)
(available
at
http://pngu.mgh.harvard.edu/~purcell/gpc/ ). The prevalence of CVD was 58 % and the frequency of the miRNA-196a2 T allele was 0.33. The study had 97 % power (α = 0.05) to detect an association (OR vs CVD- patients 1.76 , 95 % CI 1.35-2.30). An 6
ACCEPTED MANUSCRIPT interaction of the polymorphisms with diabetic complications and various risk factors was examined with multiple logistic regression analysis. The Bonferroni correction was applied for multiple comparisons to control type 1 error. Statistical significance
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was set at P < 0.05.
3. Results
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The genotypes of the miRNA-196a2 T/C polymorphism were determined in 920 type 2 diabetes patients and 834 healthy individuals. The baseline clinical and laboratory
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parameters of studied T2DM patients are summarized in Table 1. The CVD+ patients presented an average age 4 years older than patients without CVD and longer diabetes duration. Both diabetic retinopathy and nephropathy were more frequent in CVD+ subgroup (p = 0.043 and 0.007, respectively). Patients with CVD had also higher levels of total cholesterol and triglycerides and a lower level of HDL
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cholesterol. BMI in CVD+ subgroup was higher than in CVD- (p = 0.01). The frequencies of the genotypes and alleles in the control group were similar to
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those reported in other studies of European populations (Catucci et al., 2010; Hezova et al., 2012). The genotype distribution among the controls and T2DM patients was in
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Hardy-Weinberg equilibrium (p = 0.227 and p = 0.308, respectively). Table 2 presents the genotypes of the miRNA-196a2 polymorphism in patients and controls.
The
frequency of the T allele was lower in T2DM patients than in controls (for T allele
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carriers p = 0.044). The odds ratio 0.66 (95 % CI 0.54-0.79) might suggest an association of the T allele with decreased risk of type 2 diabetes. For the main purpose of the study T2DM patients were divided into subgroups with and without CVD (n = 534 and n = 386, respectively). The comparison between these subgroups showed that theT allele and TT genotype were significantly more frequent in patients with CVD compared to those without CVD (p = 0.013, p < 0.001, respectively). A multiple logistic regression analysis was performed to evaluate the association of the miRNA-196a2 polymorphism adjusted for variables such as age, sex and hypertension. The odds ratio for the T allele in the CVD+ subgroup of patients was 1.76 (1.35-2.30), p < 0.0001 vs. CVD- subgroup. The difference in allele distribution was mostly due to the overrepresentation of TT homozygotes. The CT heterozygote was present at the similar levels (p = 0.307) in CVD+ and CVD-
7
ACCEPTED MANUSCRIPT subgroup. The highest risk of development of CVD was observed in the additive
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model for TT homozygotes (OR 3.33, 95 % CI 2.05-5.42, p < 0.0001).
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4. Discussion
MicroRNAs are involved in a wide range of biological processes such as
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development, differentiation, cell growth and proliferation, metabolism, immunity and diseases such as cancer, diabetes and cardiovascular diseases (Catalucci, Gallo &
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Condorelli, 2009; Roberts & Steer, 2010). In the cardiovascular field numerous studies have documented the implication of miRNAs in nearly every pathological process
of
cardiovascular
system,
including
ischemia,
heart
failure
and
atherosclerosis (Kartha & Subramanian, 2010; Catalucci, Gallo & Condorelli, 2009). Genetic polymorphisms of miRNAs (SNPs) can potentially influence the processing
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or selection of miRNA targets.
To our knowledge, this is the first study to provide evidence that common SNP in
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miRNA might play a role in the risk of cardiovascular disease in type 2 diabetes patients.
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The T allele and TT genotype of the miRNA-196a2 T/C polymorphism (rs11614913) were significantly more frequent in type 2 diabetes patients with CVD compared to those without CVD (p = 0.013, p < 0.001, respectively). The T allele
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was associated with a 1.76-fold increased risk of CVD in type 2 diabetes patients. At α = 0.05 the statistical power for T2DM CVD+ vs. T2DM CVD- was 97 %. There are few reports available that demonstrated the effect of the miRNA-196a2 gene polymorphism on cardiovascular disease. Xu et al. (Xu et al., 2009) found that miR-196a2 polymorphism plays a role in sporadic congenital heart disease in Chinese population. It was associated with significantly increased expression of mature mir-196a in cardiac tissue specimens from congenital heart disease. In another Chinese study, the T allele of miRNA-196a2 polymorphism was associated with significantly increased risk of dilated cardiomyopathy (Zhou et al., 2010). In the study of Zhi et al. (Zhi et al., 2012) the authors did not find any main effect of the hsa-mir-196a2 polymorphism on the risk of coronary artery disease. They demonstrated, however, that CC and CC/CT genotypes of this polymorphism were significantly associated with the unfavorable prognostic factors compared with TT 8
ACCEPTED MANUSCRIPT genotype. The discrepancies between obtained results might be explained by the population-specific factors such as different genetic background or other causative variants in linkage. There are also some recent studies evaluating a role of the
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miRNA-196a2 gene polymorphism in cerebrovascular events. Liu at al. (Liu et al., on risk of
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2013) investigated the effect of three miRNA gene polymorphisms
ischemic stroke in a Chinese population. The hsa-mir-196a2 polymorphism was not associated with increased risk of stroke in studied subjects. In contrast, in a Korean
196a2
polymorphism
among
them)
associated
with
ischemic
stroke
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pathogenesis (Jeon et al., 2013).
was
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study, the combination of alleles of four miRNA gene polymorphisms (with miRNA-
The mechanism of effect of miRNA gene polymorphism on cardiovascular disease is unknown. The miR-196a2 is operative in several biological pathways. It was found to be associated with regulation of annexin A1 (ANXA1) that in turn is connected to decreased TNF-α levels. This mRNA target is involved in thrombosis and
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inflammation pathway in the circulation system (Luthra et al., 2008). Yekta et al. (Yekta et al., 2004) reported that miR-196a is expressed from HOX gene clusters loci
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in mammals and that HOX genes in turn are targets of miR-196a. Xu and colleagues (Xu et al., 2009) showed that
a SNP (rs11614913) in miR-196a2 affects its
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expression and consequently, expression of the downstream gene HOXB8. They hypothesized that rs11614913 might influence miR-196a-HOXB8-Shh signaling and thus be associated with congenital heart disease susceptibility. The rs11614913
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variant of miRNA-196a2 has been shown to alter the expression of mature miRNA196a2 and binding activity of target mRNA (Hoffman et al., 2009). It might modulate the expression level of miRNA-196a2 that in turn might affect the risk and/or progression of the disease (Saunders, Liang & Li, 2007). Cardiovascular diseases are conditions that often develop over many years of life and even small effects on gene expression patterns can play a role in the regulation processes occurring during CVD development. As in most of the association studies, one has to consider potential limitations. Although our study has a considerable statistical power, some differences might be due to a chance. Also some weak associations may have been missed. A survival and selection bias cannot be ruled out in this retrospective case-control study. We included consecutive patients and tried to adjust for known confounding risk factors. The comorbidities, however, might still represent a confounding factor. The strengths 9
ACCEPTED MANUSCRIPT of the study are the relatively large size of studied groups and the same ethnic origin of all patients and controls. Furthermore, all subjects were examined in a standardized manner with well defined diagnostic criteria. The genotyping was
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performed blind with respect to case-control status. The occurrence of diabetes and
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comorbidities such as CVD depends on interaction between different risk alleles, environmental factors and the lifestyle. Since the influence of single polymorphisms is rather small, an interactive effect of several factors may lead to an underestimation or
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overestimation of a role of polymorphism in determining the phenotype. These results might not therefore apply to populations with different genetic or environmental
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background. Additional studies in other populations should be performed, since the potential contribution of the polymorphism to cardiovascular disease might differ between ethnic populations.
In conclusion, the results of this study suggest that miRNA-196a2 T/C polymorphism (rs11614913) is associated with an increased risk of CVD in type 2
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diabetes patients. Our findings provide further insights on pathogenesis of
Acknowledgments
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cardiovascular disease in type 2 diabetes patients.
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This study was supported by grants DS 379/11 (MB) and DS 379/13 (MB) from Medical University of Lublin. The authors thank Ms. Bozenna Jarzabkowska for
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skillful technical help. Conflict of interest
The authors declare no conflict of interest.
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Table 1 Demographic and clinical profile of T2DM subjects with and without cardiovascular disease
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T2DM CVD+
T2DM CVD-
(534)
(386)
P valuea
262 / 272 65 ± 16
Diabetes duration (years)
14.6 ± 7.5
Diabetic retinopathy (%)
191 (36)
Diabetic nephropathy (%)
214 (40)
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Age at study (years)
Diabetic neuropathy (%)
139 (26)
Cardiovascular disease (%)
534 (100)
Hypertension (%)
Total cholesterol (mmol/l) HDL cholesterol (mmol/l
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Triglycerides (mmol/l)
a
61 ± 15.6
< 0.05
12.3 ± 7.6
< 0.05
104 (27)
0.043
108 (28)
0.007
72 (19)
0.074
0 247 (64)
0.144
7.9 ± 2.4
7.7 ± 2.7
0.345
4.9 ± 1.2
4.7 ± 1.1
< 0.001
1.2 ± 0.7
1.6 ± 0.8
< 0.001
2.2 ± 1.2
1.9 ± 0.7
0.01
28.8 ± 5.9
27.6 ± 4.9
0.01
CE
BMI (kg/m2)
0.325
379 (71)
ED
HbA1c (%)
170 / 216
SC
Male / female
RI P
T
Variable
Pearson’s χ2 test for categorical variables, Mann-Whitney test for continuous data.
AC
T2DM, type 2 diabetes mellitus. CVD, cardiovascular disease. Values are presented as mean ± SD or numbers (%).
15
ACCEPTED MANUSCRIPT Table 2 Genotype and allele distribution of miRNA-196a2 SNP in type 2 diabetes patients and controls p Subjects Genotype Allele OR (95 % CC
CT
TT
C
CI)
T
396
110
= 920)
(45)
(43)
(12)
Controls
292
417
125
(n = 834)
(35)
(50)
(15)
0.67
0.33
0.66 (0.54-
RI P
414
< 0.0001
0.79)
0.60
0.40
SC
T2 DM (n
T
for T allele
1.0
(reference)
T2DM, type 2 diabetes mellitus. Genotype distributions are shown as numbers (%).
MA NU
HWE test for Controls Х2 = 1.46, p = 0.227; for T2DM patients Х2 = 1.04, p = 0.308. For T allele frequency p = 0.153. The nominal p value was adjusted according to
AC
CE
PT
ED
Bonferroni correction.
16
ACCEPTED MANUSCRIPT Table 3 Genotype and allele distribution of miRNA-196a2 SNP in type 2 diabetes patients with and without CVD p Subjects Genotype Allele OR (95 % CC
CT
TT
C
CI)a for T
T
209
240
CVD+ (n
(39)
(45)
T2DM
205
156
CVD- (n
(54)
(40)
85 (16)
0.62
0.38
1.76 (1.35-
RI P
T2 DM
T
allele
< 0.0001
2.30)
25 (6)
0.27
1.0
(reference)
MA NU
= 386)
0.73
SC
= 534)
CVD+ versus CVD-
OR (95 % CI) for dominant model (CC vs. CT+TT) = 0.56 (0.43-074), p < 0.0001 OR (95 % CI) for recessive model (TT vs. CT+CC) = 2.73 (1.71-4.36), p < 0.0001 OR (95 % CI) for additive model (CT vs. CC) = 1.509 (1.14-1.99), p = 0.0038 a
ED
(TT vs. CC) = 3.33 (2.05-5.42), p < 0.0001 Adjusted for age, sex and hypertension. T2DM, type 2 diabetes mellitus; CVD,
PT
cardiovascular disease. Genotype distributions are shown as numbers (%). For T allele frequency CVD+ vs. CVD- p = 0.013. At α = 0.05 statistical power for T2DM
CE
CVD+ vs. T2DM CVD- = 97 %. The nominal p values were adjusted according to
AC
Bonferroni correction.
17
