K121Q Polymorphism of the ENPP1 Gene is Related to Acute Coronary Syndrome in Ukrainian Patients with Normal but not Enhanced Body Mass Index

Department of Physiology, Pathophysiology and Medical Biology, Sumy State University, Sumy, Ukraine Scientific Laboratory of Molecular Genetic Research, Sumy State University, Sumy, Ukraine Department of Family Medicine with Propaedeutic of Internal Diseases and Endocrinology, Sumy State University, Sumy, Ukraine Department of Biology, Medical Genetics and Ecology, Kursk State Medical University, Kursk, Russian Federation


Introduction
Ectonucleotide Pyrophosphatase Phosphodiesterase 1 (ENPP1), also known as plasma cell membrane glycoprotein 1 (PC-1), belongs to a class II membrane glycoprotein which is widely expressed in many organs and tissues of the human and animal organisms (liver, skeletal muscle, heart, brain, kidney, lung, adipose tissue, etc.). Nowadays, the biological role of ENPP1 is not fully understood, but there are two groups of evidences concerning the ENNP1 significance in pathogenesis of some pathological processes and diseases.
The first group is about the ability of ENPP1 to influence insulin sensitivity by downregulating insulin receptor signaling (Dong et al., 2005). It was shown that over-expression of ENPP1 inhibits tyrosine kinase activity with subsequent diminishing insulin receptor autophosphorylation in various cells (Maddux and Goldfine, 2000). This property of ENPP1 is considered to be in association with insulin resistance and type 2 diabetes (Goldfine et al., 2007).
The second group of data is related to the processes of ectopic calcification. It is known that inorganic Pyrophosphate (PPi) is one of the most important inhibitor of soft tissues mineralization (Abedin et al., 2004;Shao et al., 2006). The main way of PP i generating is hydrolysis of extracellular nucleoside triphosphates, particular ATP, due to ENPP1 specific enzymatic activity (Johnson et al., 2005;Towler, 2005). When the activity of ENNP1 falls off the formation of PP i is disturbed and calcification of arteries may develop in many cases. The massive arterial calcification is the most impressive feature in genetically knocked out mice (ENPP1 -/-) and in humans who have defective ENNP1 gene (Johnson et al., 2005).
As a general rule, Acute Coronary Syndrome (ACS) is the consequence of atherosclerotic lesions appearing in arterial walls. It is well known that one of the main risk factors of atherosclerosis is type 2 diabetes which is frequently and tightly associated with insulin resistance. On the other hand, the coronary arteries calcification, as shown in many studies, is an adverse prognostic feature with regard to myocardial infarction in patients with atheromatous plaques (Lehto et al., 1996).
Taking into consideration the stated above, a relation of various Single-Nucleotide Polymorphisms (SNPs) of ENPP1 gene to ACS is of great interest. The most widely investigated ENPP1 SNP in genotype-phenotype association studies is the polymorphism K121Q. In this SNP located in exon 4, a lysine (K) is substituted by a glutamine (Q) at codon 121 (Pizzuti et al., 1999).
There are some studies in which association of the ENPP1 K121Q polymorphism with early onset of coronary artery disease in Caucasians has been investigated (Endler et al., 2002;Bacci et al. 2005), but the data obtained in various ethnic groups remain controversial (Chen et al., 2006).
The aim of the present study was to perform a casecontrol study on representatives of the Ukrainian population in order to assess the possible association of the ENPP1 K121Q polymorphism with ACS in subjects of both genders who had normal and overweight levels of Body Mass Index (BMI).
Diagnosis of ACS was established on the basis of clinical, electrocardiography and biochemical examinations according to the recommendations of WHO experts and also according to recommendations of European and American cardiologic societies (Antman et al., 2004;Thygesen et al., 2007). Patients with hereditary and congenital diseases, severe metabolic pathologies including a severe form of diabetes mellitus, marked renal and liver failures, deficiencies of the haemostatic system, oncology and systemic pathologies, chronic heart failure of IIB-III stage, true cardiogenic shock were excluded from the study group.
The control group consisted of 110 clinically healthy individuals with the absence of cardio-and cerebrovascular pathologies, as confirmed by medical history, ECG and measurement of arterial pressure and biochemical data. The study had been previously approved by the Ethic Committee of the Medical Institute of Sumy State University. Appropriate informed consent was obtained from all patients and control subjects. The participants were unrelated Ukrainian people from the northeastern region of Ukraine.
The subjects of both groups were divided into subgroups by gender and by BMI (BMI<25 kg/m 2 and ≥25 kg/m 2 ).
Blood sampling for genotyping was performed under sterile conditions into 2.7 ml tubes (SMonovette [Sarstedt, Germany]) containing EDTA potassium salt as an anticoagulant, samples were frozen and stored at -20°C.

Genotyping of SNP
DNA for genotyping was extracted from the venous blood using commercially available kits (Isogene Lab Ltd, Russia) according to the manufacturer's protocol. To identify ENPPI K121Q polymorphism (rs1044498) the Polymerase Chain Reaction (PCR) with subsequent Restriction Fragment Length Polymorphism (RFLP) analysis was performed. Specific region of the ENPPI gene was amplified using a pair of specific primers: Upstream (sense)-5' CTGTGTTCACTTTGGACATGTTG 3' and downstream (antisense)-5' GACGCTGGAAGATACCAGGCTG 3'. Primers were provided by Metabion (Germany). PCR was performed for 33cycles in a 25 µL volume containing 50-100 ng of DNA, 5 µL 5X PCR-buffer, 1.5 mM magnesium sulfate, 200 µM of each dNTP, 15pM of each primer and 0.75U of Taq DNA polymerase (Thermo Scientific, USA). PCR was carried out in a termocycler GeneAmp PCR System 2700 (Applied Biosystems, USA). Six microlitres (6 µL) of the PCR products (238bp) were subjected to digestion with 5U Eco47I (AvaII) (Thermo Scientific, USA) and incubated at 37°С for 18 h. In case of the presence of cytosine at the position 48213 of the exon 4, Eco47I restriction enzyme produces two fragments of 148 and 90bp in length. Substitution of cytosine for adenine prevents restriction and the amplified fragment of the exon 4 (238bp) can not be cleaved. The restriction fragments were separated by electrophoresis and analysed on an ethidium bromide-stained 2.5% agarose gel visualized using ultraviolet transillumination.

Statistical Analysis
The normal distribution and homogeneity of variances were tested before further statistical analyses. The comparison of variables between the groups of genotypes was performed using two-tailed Student's ttest. The χ2-test was used for comparison of the allele and genotype frequencies between different studied groups and subgroups. Odds ratio was evaluated by using the logistic regression method. The differences were considered statistically significant with a P-value <0.05. All statistical analyses were performed using the Statistical Package for Social Science program (SPSS for Windows, version 17.0, SPSS Inc., Chicago, IL).

Results
The clinical characteristics of 118 patients with ACS and 140 healthy controls are summarized in Table 1. No differences between the groups were noted with respect to sex, age or body mass index. Atherogenic risk factors (including cigarette smoking, hypertension, total cholesterol and glucose concentration) were significantly more prevalent in the ACS patient group.
In Figure 1 the results of RFLP analysis of ENPP1 K121Q polymorphism are demonstrated. In both groups we studied, the genotype distributions of the ENPP1 K121Q polymorphism were in Hardy-Weinberg equilibrium. The minor allele frequencies of SNP were not significantly different between the ACS (0.18) and control (0.12) groups. These frequencies were comparable with other studies using the same SNP in populations of European descent (Bottcher et al., 2006;Morandi et al., 2009).
For genotype case-control analysis we took into consideration two subgroups (KK and KQ+QQ) since there were very few minor allele homozygotes QQ both in ACS (3 individuals) and in control (0 subjects) groups. As shown in Table 2, major allele homozygotes KK and carriers of minor allele KQ+QQ were detected in 66.9 and 33.1% of the ACS group, respectively (control group: 75.5 and 24.5%). The differences in the distribution of allelic variants between the ACS and control groups were not statisticaly significant. Such conclusion was true when subjects of both groups were divided into subgroups by gender but not by BMI.

Discussion
In the present study, we explored associations between genetic variation in the ENPP1 gene and ACS risk. Analyzing the SNP of this gene we found that K121Q polymorphism in exon 4 was associated with ACS in the representatives of Ukrainian population only with normal (BMI<25 kg/m 2 ), but not enhanced weight. The risk of ACS in not-overweight persons who were carriers of minor Q-allele was 3.9 fold greater as compared with main K-allele homozygotes.
A functional missense DNA polymorphism in exon 4 causes an amino acid change from lysine to glutamine at codon 121 (K121Q) (Pizzuti et al., 1999). Studies in vitro have shown that the Q variant of ENPP1 binds insulin receptors more strongly than the K variant and reduces insulin receptor autophosphorylation (Costanzo et al., 2001). It is therefore a stronger inhibitor of insulin signaling.
In this connection wide range of case-control studies were conducted to asses the possible association between the ENPP1 gene K121Q polymorphism and (1) insulin resistance, (2) Type 2 Diabetes Mellitus (T2DM) and (3) overweight/obesity in many Caucasian and not-Caucasian populations (Goldfine et al., 2007). Although the published results are rather contradictory, it can be concluded that in many cases there are positive associations between Q variant of ENPP1 gene and aforementioned pathological conditions.
All of these conditions are considered to be tightly pathogenetic connected with atherosclerotic process which is the main cause of ACS. This should be taken into account when studying relationship of the ENPP1 gene SNPs to cardiovascular diseases.
There is evidence suggesting that the Q allele is associated with an increased risk of earlier onset of myocardial infarction (Endler et al., 2002;Bacci et al., 2005). This association may be secondary to the effect of the Q allele on insulin resistance, T2DM and overweight/obesity which all predispose to atherosclerosis (Fig. 2).
In our study, we showed that ENPP1 gene K121Q polymorphism is related to ACS only in nondiabetic patients with normal weight and fasting glucose levels. It suggested the possible impact of this SNP on ACS not only by overweight/obesity and T2DM, but also by processes witch cause coronary insufficiency, i.e., atherosclerosis and arterial calcification.
The influence of ENPP1 overexpression on arterial wall may be in two different ways (Fig. 3). On the one hand, downregulating insulin receptor signaling by ENPP1 leads to insulin resistance and contribute to atherosclerotic lesions development. On the other hand, enhanced enzymatic activity of ENPP1 should increase the formation of PP i which is one of the most potent anticalcinogenic factor that can prevent arterial wall calcification.
It is yet not known how K121Q polymorphism impacts on enzymatic feature of ENPP1. This is a question that needs to be under further investigation.

Conclusion
In the present study, genotypes with minor allele (KQ+QQ) for ENPP1 K121Q polymorphism were observed as a possible genetic risk factor for ACS only in persons with BMI <25 kg/m 2 . It is more likely that these genotypes affect ACS not through the traditional risk factors (overweight/obesity, insulin resistance and type 2 diabetes) but by direct or undirect influence on pathologic processes in the wall of the coronary vessels (atherosclerosis and arterial calcification).

Acknowledgement
We thank all the consultants for their invaluable help during sample collection. We appreciate the participation of all the subjects who volunteered for this study. The authors declare that they have no conflict of interest.

Funding Information
The study was a part of scientific project "Association of ectopic calcification genes polymorphisms with widespread cardiovascular diseases and their complications" supported by the Ministry of Education and Science of Ukraine, 2013-2014 (No 0113U000132).

Ethics
The study has been approved by the Ethic Committee of the Medical Institute of Sumy State University.