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Introduction

Coronary artery disease (CAD) is established as a major cause of mortality worldwide (1). Pathogenesis of CAD involves the formation of atherosclerotic plaques, which consist of endothelial cells, leukocytes, inflamed smooth muscle cells, necrotic cores, accumulated modified lipids and calcified regions, all of which indicate that CAD is an inflammatory disease in which immune mechanisms interact with metabolic risk factors (2). Polymorphonuclear neutrophils may modulate and signal in inflammatory pathways through the secretion of enzymes including myeloperoxidase (MPO) (3). To date, findings have implicated MPO as a prominent participant in the link between inflammation and cardiovascular diseases such as CAD (4).

MPO is an enzyme expressed by leukocytes, particularly neutrophils and monocytes, that catalyzes the formation of numerous reactive oxidant species at sites of inflammation (5). The human MPO gene is located on chromosome 17q23.1 and consists of 11 introns and 12 exons (5). A common single nucleotide polymorphism (SNP) of MPO is MPO-463G/A, which consists of a substitution from G to A at position 463 bp. Meanwhile, MPO-129G/A, as another SNP, is located in the MPO gene promoter. Both of these SNPs have been reported to affect the binding of the transcription factor specificity protein 1, and thus, the protein expression of MPO (6,7).

Under physiological conditions, MPO-derived oxidation products serve an important role in host defense, though continuous activation of MPO results in increased levels of reactive chlorine species, and MPO-derived oxidants have been linked with atherosclerosis (8,9). However, whether MPO levels may serve as a marker of plaque vulnerability in the assessment of cardiovascular risk remains uncertain. Therefore, the present study aimed to investigate the association between the MPO SNPs -463G/A and -129G/A and CAD risk in a Turkish population. Additionally, the effect of these SNPs on patient survival rate was examined, and an evaluation of the possible relationships between the SNPs and demographic parameters including sex, age and rates of hypertension, diabetes and hypercholesterolemia, was a further study aim.

Patients and methods

Study population

The study group consisted of 201 patients with CAD (case group; age, 61.06±6.81; 115 male, 86 female) and 201 healthy individuals (control group; age, 59.87±7.14; 98 male, 103 female) enrolled from Cumhuriyet University Hospital (Sivas, Turkey) between June 2011 and December 2011. In all patients with CAD, coronary angiography identified >50% stenosis in at least one major coronary vessel as a result of atherosclerosis. The control group consisted of healthy individuals with a negative family history of CAD. To select the control population, findings of routine clinical tests, physical and laboratory examinations and electrocardiography and echocardiography were evaluated; individuals with negative test results indicative of the absence of pathology were chosen. Participants in the case and control groups were born in Turkey. Information concerning sex, age, the presence of hypertension (>130-140/80-90 mmHg) (10), diabetes and/or hypercholesterolemia (≥240 mg/dl) (11) and smoking habits was collected using a standardized questionnaire. Informed consent was obtained from the patients prior to the study, and the study was approved by the Ethics Committee of the Medical School of Cumhuriyet University (approval no. 2011-02/04).

Genotyping

Genomic DNA of the case and control groups was extracted from blood leukocytes (in 1 ml blood samples) in collection tubes with EDTA using a standard phenol-chloroform method (12). MPO genotypes were determined using a polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) assay. The PCR-RFLP was performed according to the method reported by Arslan et al (13). The primers for MPO-463G/A were forward, 5′-CGGTATAGGCACACAATGGTGAG-3′ and reverse, 5′-CAATGGTTCAAGCGATTCTTC-3′; and for MPO-129G/A were forward, 5′-CCTCCACAGCTCACCTGATAT-3′ and reverse, 5′-CGCTTGAACCATTGCACATCA-3′. The MPO-463 and -129G/A SNP amplicon sizes were 350 and 278 bp, respectively. The PCR products were digested with SsiI (for MPO-463G/A) and ApaI (for MPO-129G/A), according to the manufacturer's instructions (Fermentas; Thermo Fisher Scientific, Inc., Waltham, CA, USA), and fragment sizes were determined by 3% agarose gel electrophoresis and ethidium bromide staining, using a UV transilluminator for visualization. The -463G/A genotypes following SsiI digestion were GG (169, 120 and 61 bp), GA (289, 169, 120 and 61 bp) and AA (289 and 61 bp). The -129G/A genotypes following ApaI digestion were GG (278 bp), GA (278, 154 and 124 bp) and AA (154 and 124 bp). To confirm the MPO genotypes, 15 samples of each genotype (homozygous wild-type, heterozygous and homozygous mutation) were selected for detection using an ABI 310 DNA sequencing system (Applied Biosystems; Thermo Fisher Scientific, Inc.), which was performed externally by Life Technologies, Ltd. (Thermo Fisher Scientific, Inc.).

Statistical analysis

Data are representative of three independent repeat experiments. All statistical analyses were performed using SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA). The statistical significance of differences in the MPO genotypes between the case and control groups were evaluated using χ2 tests. Multivariate logistic regression analyses were performed for comparisons regarding demographic and clinical parameters (sex, age, presence of hypertension, diabetes and/or hypercholesterolemia and smoking habits), for which odds ratios (ORs) and 95% confidence intervals were calculated. Kaplan-Meier analysis was used to compare the survival curves of the subjects determined from a 20-week follow-up. In all cases, P<0.05 was considered to indicate statistical significance.

Results

Demographic and clinical parameters

The present study involved 201 patients with CAD (115 male, 86 female) and 201 healthy controls (98 male, 103 female). The demographic and clinical parameters of the CAD and control populations are summarized in Table I. There was a significantly higher frequency of hypertension in the CAD population compared with the control group (OR=5.15, P<0.001). However, no significant differences were observed between the patients and controls regarding sex distribution, smoking status and the presence of diabetes or hypercholesterolemia (P>0.05).

Table I. Demographic and clinical parameters of CAD patients and healthy controls.

Table I.

Demographic and clinical parameters of CAD patients and healthy controls.

Variable	CAD cases, n (%)	Controls, n (%)	OR (95% CI)	P-value
Total	201 (100)	201 (100)
Age, mean ± SD	61.06±6.81	59.87±7.14		0.088
Sex
Female	86 (42.79)	103 (51.24)
Male	115 (57.21)	98 (48.76)	1.40 (0.94–2.04)	0.089
Smoking status
Non smoker	92 (45.77)	110 (54.73)
Smoker	109 (52.23)	91 (45.27)	1.43 (0.96–2.12)	0.073
Hypertension
>130-140/80-90 mmHg
Absent	73 (36.32)	150 (74.63)
Present	128 (63.68)	51 (25.37)	5.15 (3.36–7.91)	<0.001
Diabetes
Absent	124 (61.69)	130 (64.68)
Present	77 (38.31)	71 (35.32)	1.13 (0.75–1.70)	0.535
Hypercholesterolemia
≥240 mg/dl
Absent	129 (64.18)	146 (72.64)
Present	72 (35.82)	55 (27.36)	1.48 (0.97–2.26)	0.068

[i] CAD, coronary artery disease; SD, standard deviation; OR, odds ratio; CI, confidence interval.

Allele and genotype frequencies

The allele and genotype distributions of MPO-463G/A and -129G/A in the CAD cases and controls are presented in Table II. The MPO-463GG, GA and AA genotype frequencies were 48.25, 40.79 and 10.94%, respectively, in the patients with CAD; and 57.71, 31.84 and 10.44%, respectively, in the healthy controls. The MPO-463A allele was identified in 31.34% of CAD patients and 26.36% of controls. Comparing the genotype frequency distributions of MPO-463GG and MPO-463GA revealed that patients with MPO-463GA had a significantly higher risk of developing CAD (OR=1.53, P=0.048). Meanwhile, no significant differences were observed regarding disease risk of the AA genotype and A allele frequencies compared with the GG genotype (OR=1.25, P=0.500) and G allele (OR=1.27, P=0.120) frequencies, respectively.

Table II. Risk estimates and frequency distributions of alleles and genotypes of MPO polymorphisms (-463G/A and -129G/A) in association with demographic and clinical parameters.

Table II.

Risk estimates and frequency distributions of alleles and genotypes of MPO polymorphisms (-463G/A and -129G/A) in association with demographic and clinical parameters.

	MPO-463			MPO-129
Variable	Cases/controls, n (%)	OR (95% CI)	P-value	Cases/controls, n (%)	OR (95% CI)	P-value
Total
G	276 (68.65)/296 (73.63)	Ref.		292 (72.63)/325 (80.84)	Ref.
A	126 (31.34)/106 (26.36)	1.27 (0.93–1.73)	0.120	110 (27.36)/77 (19.15)	1.59 (1.14–2.21)	0.006
GG	97 (48.25)/116 (57.71)	Ref.	–	91 (45.27)/124 (61.69)	Ref.
GA	82 (40.79)/64 (31.84)	1.53 (1.00–2.34)	0.048	110 (54.72)/77 (38.30)	1.94 (1.30–2.89)	0.001
AA	22 (10.94)/21 (10.44)	1.25 (0.65–2.41)	0.500	Undetected	–	–
Female
GG	48 (55.81)/53 (51.45)	Ref.		44 (51.16)/70 (67.96)	Ref.
GA	30 (34.88)/36 (34.95)	0.92 (0.49–1.71)	0.793	42 (48.83)/33 (32.03)	2.02 (1.12–3.65)	0.019
AA	8 (9.3)/14 (13.59)	0.63 (0.24–1.63)	0.341	–	–	–
Male
GG	52 (44.44)/63 (64.28)	Ref.		48 (41.73)/55 (56.12)	Ref.
GA	50 (42.73)/29 (29.59)	2.08 (1.16–3.75)	0.013	67 (58.26)/43 (43.87)	1.78 (1.03–3.07)	0.036
AA	15 (12.82)/6 (6.12)	3.02 (1.09–8.36)	0.027	–	–	–
Smoking
GG	53 (48.18)/51 (56.04)	Ref.		43 (39.44)/58 (63.73)	Ref.
GA	42 (38.18)/30 (32.96)	1.34 (0.73–2.47)	0.335	66 (60.55)/33 (36.26)	2.69 (1.51–4.79)	0.001
AA	14 (12.72)/10 (10.98)	1.34 (0.54–3.30)	0.515	–	–	–
Hypertension
GG	74 (57.81)/24 (47.05)	Ref.		58 (45.31)/29 (56.86)	Ref.
GA	43 (33.59)/21 (41.17)	0.66 (1.33–1.33)	0.248	70 (54.68)/22 (43.13)	1.59 (0.82–3.06)	0.163
AA	11 (8.59)/6 (11.76)	0.59 (0.19–1.77)	0.349	–	–	–
Diabetes
GG	45 (58.44)/45 (63.38)	Ref.		37 (48.05)/40 (56.33)	Ref.
GA	25 (32.46)/21 (29.57)	1.19 (0.58–2.42)	0.631	40 (51.94)/31 (43.66)	1.39 (0.73–2.66)	0.313
AA	7 (9.09)/5 (7.04)	1.40 (0.41–4.74)	0.588	–	–	–
Hypercholesterolemia
GG	32 (44.44)/29 (52.72)	Ref.		29 (40.27)/33 (51.56)	Ref.
GA	30 (41.66)/20 (36.36)	1.35 (0.63–2.89)	0.426	43 (59.72)/31 (48.43)	2.22 (1.08–4.55)	0.028
AA	10 (13.88)/6 (10.90)	1.51 (0.48–4.67)	0.473	–	–	–
Haplotype
MPO
-463    -129
G    G	114 (56.71)/132 (65.67)	Ref.
G    A	30 (14.92)/21 (10.44)	1.65 (0.89–3.04)	0.105
A    G	32 (15.92)/30 (14.92)	1.23 (0.70–2.15)	0.458
A    A	25 (12.43)/18 (8.95)	1.60 (0.83–3.09)	0.153

[i] MPO, myeloperoxidase; OR, odds ratio; CI, confidence interval.

The MPO-129GG, GA, AA genotype frequencies were 45.27, 54.72 and 0.00%, respectively, in the patients with CAD; and 61.69, 38.30 and 0.00%, respectively, in the healthy controls. The MPO-129A allele was identified in 27.36% of patients and 19.15% of controls (Table II), and comparing the A and G allele distributions indicated that -129A conferred a significantly greater risk of developing CAD (OR=1.59, P=0.006). Additionally, comparing the GG and GA genotype distributions revealed that the GA genotype was a significant risk factor of CAD (OR=1.94, P=0.001).

On analysis of genotype distribution regarding patient characteristics, MPO-463AA was identified to be a significant risk factor in males compared with male GG carriers (OR=3.02, P=0.027; Table II). It was also indicated that male patients with the MPO-463GA genotype had a significantly higher risk of CAD compared with male GG carriers (OR=2.08, P=0.013). With regard to hypertension, MPO-463G/A was not a significant risk factor as GA (OR=0.66, P=0.248) or AA (OR=0.59, P=0.349). For MPO-129G/A, the GA genotype was a significant risk factor regarding sex (female: OR=2.02, P=0.019; male: OR=1.78, P=0.036), smoking status (OR=2.69, P=0.001) and the presence of hypercholesterolemia (OR=2.22, P=0.028), but not regarding the presence of hypertension (OR=1.59, P=0.163) or diabetes (OR=1.39, P=0.313; Table II).

Haplotype and survival analysis

The haplotypes for all probable haplotypes were examined. All of the four possible haplotypes determined for the two SNPs were observed in the study samples. As presented in Table II, the differences between the frequency distributions of all possible haplotypes did not differ significantly (P>0.05). Furthermore, on Kaplan-Meier analysis to compare the survival curves of subjects with regard to MPO-463G/A and MPO-129G/A, it was determined that the SNPs had no significant effect on the survival of patients with CAD (for MPO-463G/A, P=0.516 and for MPO-129G/A, P=0.220; data not shown).

Additionally, the risk estimates and frequency distributions of the MPO polymorphisms were adjusted for age, sex, smoking status and the presence of hypertension, diabetes and hypercholesterolemia (Table III). There was a marked association between MPO-463G/A and CAD risk on adjustment for hypertension (adjusted OR=8.38), which was deemed to be significant to P=0.001. Furthermore, patients with hypertension exhibited a marked association between MPO-129G/A and disease risk (adjusted OR=9.78) significant to P=0.001.

Table III. Risk estimates and frequency distributions of MPO polymorphisms adjusted for age, sex, smoking habit, hypertension, diabetes and hypercholesterolemia.

Table III.

Risk estimates and frequency distributions of MPO polymorphisms adjusted for age, sex, smoking habit, hypertension, diabetes and hypercholesterolemia.

	MPO-463G/A		MPO-129G/A
Variable	Adjusted OR (95% CI)	P-value	Adjusted OR (95% CI)	P-value
Genotype
GA	1.13 (0.59–2.18)	0.695	1.23 (0.69–2.18)	0.478
AA	1.03 (0.40–2.67)	0.937
Age	1.07 (1.05–1.10)	<0.001	1.07 (1.05–1.09)	<0.001
Sex	3.69 (1.83–7.44)	<0.001	3.43 (1.75–6.70)	0.001
Smoking	0.50 (0.25–1.00)	0.051	0.48 (0.25–0.92)	0.028
Hypertension	8.38 (4.38–16.05)	0.001	9.78 (5.26–18.21)	0.001
Diabetes	0.47 (0.24–0.90)	0.024	0.58 (0.31–1.07)	0.084
Hypercholesterolemia	0.85 (0.43–1.67)	0.645	0.90 (0.47–1.72)	0.767

[i] Adjusted ORs are relative to the GG genotype (reference). The mean ORs of the GA and AA genotypes are presented for age, sex, smoking, hypertension, diabetes and hypercholesterolemia. MPO, myeloperoxidase; OR, odds ratio; CI, confidence interval.

Discussion

CAD is a leading cause of mortality worldwide (14), and results from interactions between numerous genes and environmental factors (15). It is considered that oxidative stress serves a key role in the initiation and progression of atherosclerosis (16). In particular, MPO has been identified to influence the incidence of CAD, and polymorphisms in genes such as MPO are potential modifiers of individual predisposition to CAD (17).

The present study aimed to research the association between CAD and two MPO SNPs, -463G/A and -129G/A, in a Turkish population. For this, the allele and genotype distribution frequencies for MPO-463G/A and -129G/A were determined in the study population. These data were also investigated in association with the demographic and clinical parameters of the subjects. Previous studies have indicated that MPO-463A allele frequency in cases and controls varied from 8 to 47% and 16 to 56%, respectively, in different ethnic populations (18–21). However, it has been reported that MPO-463A frequency in control subjects was 26.7% in French-Canadian (15), 22.4% in Swedish (19) and 43.5% in Turkish (20) populations. In the present study, it was identified in 31.34% of CAD patients and 26.36% of controls. According to a previous meta-analysis (21), the MPO-463AA and GA genotypes were associated with a 63 and 27% decreased risk of CAD, respectively, relative to the GG genotype. However, in the current study, the distributions of the MPO-463G/A allele and genotypes did not differ significantly between the CAD and control populations except for GA.

MPO is an important factor of the innate immune response, and forms diffusible oxidative substances with antimicrobial activity, though also promotes oxidative damage of host tissues at sites of inflammation (22). This may lead to endothelial dysfunction and unstable plaque formation, which potentially impacts on atherosclerosis formation (23). Previous studies have indicated that MPO may be used as a marker of inflammation in the coronary artery and after myocardial infarction (24,25). Additionally, high plasma levels of MPO have been correlated with cardiovascular events (26). Notably, MPO levels were significantly increased in CAD, though there was no effect of MPO-463G/A polymorphism on MPO levels (20). Conversely, according to a another clinical study (4), MPO-463G/A lead to a decrease in MPO expression in the genotype AA, intermediate levels in GA, and higher levels of intracellular MPO in the genotype GG. Furthermore, serum cholesterol levels and smoking have been reported as contributing factors in the upregulation of MPO enzymes (20). However, for MPO-463G/A in the present study, the majority of demographic and clinical parameters did not differ significantly between the case and control populations; only male individuals with the GA and AA genotypes were implicated to have a higher risk of CAD. Accordingly, previous study indicated that males developed CAD more frequently than females (27).

CAD susceptibility may be modulated by polymorphisms in oxidative enzymes such as MPO (28). For MPO-129G/A in the present study, regarding A allele and GA genotype frequency distributions, individuals with the GA genotype had ~2-fold higher risk of developing CAD than those carrying GG (OR=1.94). In a study in 2016 (28), no significant association was observed between -129G/A and CAD in an Indian population. Additionally, in the same study, haplotype analysis revealed that -463G/A of the AA genotype and -129G/A of the GG genotype significant improved the clinical condition of disease; however, this relationship was not supported by haplotype analysis in the present study. In a Swedish population, the A allele of the MPO-129G/A promoter polymorphism may serve a protective role against myocardial infarction in women (19). In the current study, both females and males with MPO-129GA were implicated to be at significantly higher risk of CAD. Therefore, the MPO-129G/A genotype may be associated with upregulation of MPO expression.

Associations of -463G/A and -129G/A polymorphisms with CAD have previously been reported (28); however, results vary depending on the ethnicity of the study population. Therefore, the current study evaluated the association between these polymorphisms of the MPO gene with CAD risk in a Turkish population. Additionally, to the best of our knowledge, survival curves of the patients were analyzed for the first time; however, the survival rates of subjects with or without the SNPs did not differ significantly. As sex, smoking status, and the presence of hypertension, diabetes and/or hypercholesterolemia influence CAD and MPO levels (29), these factors were adjusted for each SNP.

In conclusion, the MPO-129A allele was implicated as a risk factor for CAD in the present study. Additionally, significant associations were identified in smoking individuals and in subjects with hypertension between MPO-129G/A and CAD risk. Indeed, hypertension and smoking have previously been implicated as independent predictors for CAD (28). Meanwhile, in the total cohort, MPO-463G/A was not significantly associated with CAD; however, individuals with hypertension carrying MPO-463G/A had a markedly greater risk of developing CAD. Further comprehensive studies are now required to determine whether MPO-129A is a marker of CAD.

Acknowledgements

The present study was supported by the Research Council of Cumhuriyet University, Sivas, Turkey (grant no. F-340).

References