Association of allelic polymorphisms of the Matrix Gla-protein system genes with acute coronary syndrome in the Ukrainian population

Calcifi cation of the vascular wall is a prognostic factor for the outcome of acute coronary syndrome (ACS). Matrix Gla-protein system, which includes MGP, VDR, VKOR, GGCX, BMP-2 is an important factor in vessels protection of ectopic calcifi cation. Polymorphisms of genes, which encode the structure of these proteins, determine their activity and may affect the intensity of calcifi cation and the consequences of ACS. Aim. The association between ACS and polymorphic variants of Matrix Gla-protein system genes: MGP (rs1800802, rs1800801, rs4236), VDR (rs2228570, rs1544410, rs7975232, rs731236), GGCX (rs699664), VKORC1 (rs2359612), BMP-2 (rs2273073), was analyzed. Methods. Venous blood of 118 patients with ACS and 234 healthy individuals (control group) was used for genotyping. Polymorphisms of Matrix Glaprotein system genes were examined by PCR-RFLP methodology. Results. The risk of ACS in carriers of minor allele A/A (rs1800801) is 2.8 times higher; G/G (rs1544410) 2.1 times higher; A (rs699664) and C (rs2359612) 2 times higher than in carriers of the major allele. The best classifi cation model is a two-component model that includes polymorphisms rs1800801 and rs4236 of the MGP gene (predictive ability is 63 % for MDR and 68 % for the Random forest method). The coincidence of similar orientation genotype variants for chosen polymorphism[s] was associated with a high risk of developing ACS: in the heterozygote genotype it increased by 2.1 times, and in the homozygote for the minor allele genotype, by 6.3 times. Conclusion. There is an association between ACS and some polymorphic variants of Matrix Gla-protein system genes: MGP (rs1800801), VDR (rs1544410), GGCX (rs699664), VKORC1 (rs2359612). This indicates a higher risk of complications in the ACS patients with the following genotypes: A/A (rs1800801), G/G (rs1544410), A/A (rs699664) and C/C (rs2359612).


Introduction
Vascular calcifi cation has a pivotal role among the factors which relate to the complications of atherosclerosis, including acute coronary syndrome, that can occur either in the intima (mineralization of atherosclerotic plaques) or in the middle layer of the vascular wall (Mönckeberg's sclerosis) [1][2][3][4].The deposition of calcium phosphate crystals in arteries structure, according to many authors, is a poor prognostic factor that indicates a high probability of fatal complications [5][6][7].The matrix Gla-protein plays an important role in the vessel protection against ectopic calcifi cation; its presence in tissues prevents initiation and spread of a pathological calcifi cation [8][9][10][11].A large number of studies managed to fi nd the factors involved in the regulation of the MGP gene expression, and identifi ed possible mechanisms, through which the corresponding protein realizes its anti-calcifi cation properties.It gave a reason to talk of the Matrix Gla-protein functional system that yields, except for the matrix Gla-protein, ISSN 0233-7657 Biopolymers and Cell. 2015. Vol. 31. N 1. P. 46-56 doi: http://dx.doi.org/10.7124/bc.0008CCsuch factors as vitamin D receptor (VDR), the enzymes involved in biochemical transformations of the matrix Gla-protein, vitamin K epoxide reductase (VKOR) and vitamin K-dependent gamma-glutamyl carboxylase (GGCX), as well as potential targets for the matrix Gla-protein, including bone morphogenetic protein-2 (BMP-2) (Fig. 1).The effective operation of the system may depend on many factors, including gene polymorphisms that encode corresponding protein structures.
Today the connection between the different MGP gene allelic variants and the cardiovascular diseases (atherosclerosis, myocardial infarction, heart stroke) [12][13][14][15][16][17][18], osteoporosis [19][20], urolithiasis [21], tooth loss [22], plumbum intoxication [23][24] is studied.The published data on this subject are ambiguous and contradictory.The com prehensive studies on the role of genetic polymorphism of MGP and related genes in the development of cardiovascular diseases have not been carried out yet.Therefore, the purpose of this study was to explore the allele's frequency of polymorphisms of Matrix Gla-protein system genes (MGP, VDR, VKORC1, GGCX, BMP-2) in patients with acute coronary syndrome in the Ukrainian population and to analyze the complex infl uence of studied polymorphisms on the disease development.The main components of the process that ensures the functioning of the matrix Gla-protein in tissues ommendations of WHO experts and the recommendations of European and American cardiologic societies [25][26].

Study subjects
The control group consisted of 234 clinically healthy individuals.The absence of cardio-vascular pathologies was confi rmed by anamnesis, ECG examination, measurement of arterial pressure and biochemical data.The group of healthy individuals and patients with ACS differed in the ratio of men and women (P = 0.034), the average age in the fi rst group (66.0 ± 0.95 years) was signifi cantly higher than in the second group (P < 0.001) (Table 1).The significantly higher average age in the control group contributes to the reliability of selection procedure, ex-cluding the infl uence of age as a risk factor for acute coronary syndrome development.

Amplifi cation and genotyping
Blood sampling was performed under sterile conditions in 2.7 ml monovettes containing EDTA potassium salt (11.7 mM) as an anticoagulant (Sar stedt, Germany), sample were frozen and stored at -20 C.DNA for genotyping was extracted from the samples using Isogene kits (Russian Federation) according to manufacturer's protocol.10po lymorphisms were studied (Table 2).
In the present study, a polymerase chain reaction with subsequent analysis of restriction fragment length (PCR-RFLP) was used for genotyping a single nucleotide polymorphism (Table 3).Primers were synthesized by «Metabion» (Germany), and enzymes (Taq-polymerase, and restriction enzyme) from «Thermo Scientifi c» (USA).PCR was performed in a thermocycler Gene Amp PCR System 2700 («Applied Biosystems», USA).Restriction products were separated in 2.5% agarose gel containing etidium bromide, which formed a stable compound with DNA fragments, visualized as stripes in the UV-illuminated gel with the transilluminator UVT-a Biocom (Russian Federation).

Statistical analysis
The normal distribution and homogeneity of variances were tested before further statistical analyses.The distribution of genotypes of studied SNPs was analyzed using the Pearson`s chi-square test.Statistically signifi cant results were presented by the corresponding exact P-values.Statistical signifi cance of differ-

Results and Discussion
Genotyping patients with ACS for ten sites of the Matrix Gla-protein system genes and comparison of the data with the results of the restriction analysis in the control group allowed determining the frequency of some variants of these genes (Table 4).
There was a statistically signifi cant difference in distribution of alleles in patients with ACS and healthy individuals only for rs1800801 of MGP gene (P = = 0.040) and for rs2359612 of VKORC1 gene (P = = 0.038).By this means, there was a link between these polymorphisms and the development of ACS.Using a logistic regression confi rmed this conclusion: females of A/A genotype (rs1800801) had 5.6 times higher risk to suffer from ACS than carriers of the major allele (G/A + G/G) (Table 5).
To identify a possible association of genetic markers of the disease risk there were used conventional statistical models (codominant, dominant, overdominant, recessive, additive).The best model for each polymorphism was characterized by the lowest Akaike information criterion.The association of SNPs (rs1800801 of MGP gene, rs 1544410 of VDR gene, rs2355612 of VKORC1 gene, rs 699664 of GGCX gene) with ACS was proven (Table 6).
Similar studies in this area are limited and contradictory.For example, the connection between six variants of the MGP gene and the development of myocardial infarction (MI) was analyzed in ECTIM Study (ECTIM Study, Northern Ireland, and France) [16].It was shown that allele and genotype frequen-cies were the same for all SNP types in MI patients and in the control group.Only in one subgroup, where patients and control subjects were divided into groups of high and low risk of coronary heart disease, it was found that the frequency of minor alleles (-7A and 83G) in MI patients with low-risk factors was higher than in the corresponding control subgroup.Ortlepp et al. determined an association between the G/G genotype of VDR gene BsmI polymorphism and the risk of myocardial infarction in patients under the age of 65 [31].The contradictory data were obtained by Shanker et al. [32] and Pan et al. [33] -they detected no association between polymorphic variants and haplotypes of the VDR gene and the development of coronary heart disease.fi cation.The authors found that a polymorphism of the fi rst intron of the C1173T VKORC1 gene was associated with calcifi cation of the blood vessels and was an important genetic factor in the development of atherosclerosis [34].Wang et al., studying the distribution of the VKORC1 T2255C polymorphism, found that the presence of the C allele more than twice increased the risk of coronary heart disease and stroke, and more than three times -the risk of aortic dissection [35].Nevertheless, Hindorff et al., who studied the association of VKORC1 gene polymorphisms, including T2255C, with the development of myocardial infarction and other cardiovascular diseases, showed that none of the studied SNPs were associated with the development of diseases of heart and blood vessels [36].

Porojan et al. examined the relation of VKORC1 allelic polymorphisms with atherosclerosis and calci-
The next step was to study the combined effects of polymorphisms of the Matrix Gla-protein system genes and to clarify their common contribution to the development of ACS.A similar analysis made it possible to identify the most informative combinations of polymorphic loci; in case of its combined effect they had a pivotal role in development of ACS.Random forest was the method of fi rst choice in the algorithm of data analysis.It allowed ranking all the studied polymor-phisms (predictors) according to their importance level (degree of contribution to the development of ACS and the ability to predict the risk of its occurrence).According to this method, rs1800801 of the MGP gene was the most important among all predictors (Fig. 2).
The method of selection of the most signifi cant predictors, proposed by C. Strobl, was used to improve the classifi cation model [29].This method is based on the principle that randomization importance of the risk factors which have a minor impact on the risk of disease, hovers around zero.According to this method, the absolute value is taken as the factor with the lowest value of randomization and this value becomes a threshold and reference for the vertical line, as all signifi cant predictors have to cross this limit (Fig. 3).Polymorphism rs1800801 of the MGP gene was confi rmed to be the most signifi cant predictor of ACS like in the previous analysis.Only the polymorphic variants located to the right of the dotted line were selected for further study.The classifi cation model with 75 % predictive ability in training sample and 68 %in testing sample was designed on this basis.
Simulation of intergenic interaction of studied polymorphisms implemented by Multifactorial Dimensionality Reduction (MDR) allowed selection of the most pathogenic combinations of polymorphic loci signifi cant for ACS.
Two-loci pattern of rs1800801 and rs4236 was statistically signifi cant.It had 63 % classifi cation abi lity with 8/10 cross-validation consistency.MDR method eliminated some of those predictors that were identifi ed by the «noise fi ltering», whereas the predictive ability of the model decreased by only 4 %.The combination of polymorphisms rs1800801 and rs4236 of the MGP gene is refl ected in Fig. 4. It was found that the coincidence of both SNP genotypes is associated with the increased risk of ACS.Attention is drawn to the fact that even the coincidence of two homozygotes for the major allele leads to a significant increase of the risk of ACS.It was found by means of MDR that the largest proportion of entropy (the biggest independent effect) with connection to the status of «case-control» was associated with rs1 800801 and rs2359612 loci and was 1.26 %, 1.33 %, respectively (Fig. 5).At the same time an analysis of Fig. 4. Display the combination of rs1800801 and rs4236 genotypes associated with high-and low-risk ACS.Left column within each cell represents the number of cases, right columnthe number of controls.Dark gray cells correspond to high risk, and light-gray -low risk of ACS Association of allelic polymorphisms of the Matrix Gla-protein system genes intergenic interactions revealed that the greatest phenotypic entropy was accounted for the interaction of rs1800801 & rs4236 loci and it amounted to 8 %.It showed a pronounced synergistic effect for both SNPs and once again proved that rs1800801 was the most important predictor of ACS.The synergy was also observed between polymorphisms rs731236 and rs1544410 of VDR gene (5.66 %), although they Fig. 5. Diagram of cluster analysis of intergenic interaction results by MDR simulation in ACS group.Synergistic interaction is marked by a bold line, and lack of interaction is marked by a thin line were not included in the composition of the most informative classifi cation model.All other polymorphisms showed mainly independent effects.Finally, we applied permutation (randomized) tests, which showed that the two-component model was statistically signifi cant at p < 0.05.
In order to confi rm our MDR results, the interaction between polymorphisms rs1800801 and rs4236 of the MGP gene was modeled, using the binary logistic regression (Table 7).Analysis of the results showed that two heterozygotes and two homozygotes for the minor allele and homozygous for the major allele genotypes were associated with the risk of disease.Heterozygote genotype was associated with the increased risk of disease by 2.1 times and homozygote for the minor allele genotype -by 6.3 times.
Analyzing the ROC-curve that describes the link between the sensitivity and specifi city of the model, we found out that the area under the curve (AUC) is 63 %, which proves satisfactory prognostic significance of the created model (Fig. 6).
To answer the question why MDR method chose two-component model as the best one, though Random Forest showed a greater number of predictors, the model was constructed once again using the Random Forest method that contained only polymorphisms rs1800801 and rs4236 of the MGP gene.The predictive ability of the model did not change.We had the same 68 % predictive ability while analyzing all ten loci.It was the evidence that the rs1800801 and rs4236 loci were indeed statistically signifi cant.All other predictors to the right of the dotted line -were important, but did not reach the level of statistical signifi cance to affect considerably the ability of classifi cation models.

Conclusions
There is a connection between ACS and polymorphic variants of the genes: MGP (rs1800801), VDR (rs1544410), GGCX (rs699664) and VKORC1 (rs23 59612).The risk of ACS in carriers of the minor allele A/A (rs1800801) is 2.8 times higher; G/G (rs1544410) 2.1 times higher; A (rs699664) and C (rs2359612) twice higher than in the carriers of the major allele.The best classifi cation model is a two-component model that includes polymorphisms rs1800801 and rs4236 of the MGP gene (predictive ability is 63 % for MDR and 68 % for the Random forest method).The coincidence of similar orientation genotypes variants for chosen polymorphism was associated with the high risk of ACS: in he terozygotes it increased by 2.1 times, and homozygotes for the minor allele -by 6.3 times.

Table 4 . The distribution of genotypes by the polymorphisms of Matrix Gla-protein system genes in patients with ACS and control group
BMP-2, rs2273073 50.0 : 32.2 : 17.8 45.3 : 40.2 : 14.5 0.327 N o t e: 1/1 1/2 2/2 -ratio for major allele homozygotes to heterozygotes and to homozygotes for the minor allele, P -an indicator of the signifi cance of differences in distribution of genotypes between the ACS group and control groups.

Table 5 . Analysis of ACS risk that depends on G-7A polymorphism genotype of MGP gene in females and males
N o t e: homozygotes for the minor allele (A/A) are compared with carriers of the principal allele (G/A + G/G); CR -regression coeffi cient; SE -standard error; WS -Wald statistics; P -statistical signifi cance; OR -odds ratio ; CI -confi dence interval

Table 6 . Analysis of ACS risk associated with variation in genes of Matrix Gla-protein system
in probability estimates was checked by Student's t-test.In order to predict the risk of acute coronary syndrome the logistic regression was used.
N o t e: O -the best inheritance model is overdominant, R -the best inheritance model is recessive, A -the best inheritance model is additive.*-the best model has been chosen with the help of AIC (Akaike Information Criteria).ence

Table 7 . Analysis of ACS risk, that depends on the polymorphisms rs4236 and rs1800801of MGP gene
N o t e: The comparison was carried out among homozygotes for the major allele; CR -regression coeffi cient; SE -standard error; WS -Wald statistics; P -statistical signifi cance; OR -odds ratio; CI -confi dence interval.