Influence of Neuropeptide Y and Neuropeptide Y 2 Receptor Variants in Acute Coronary Syndrome

Background: The neuropeptide Y (NPY) is one of the most abundant neurotransmitters in the nervous system. NPY acts as a potent stimulator of angiogenesis, inflammation, and adipogenesis, through the NPY 2 receptor (NPY2R). Changes in the NPY signaling pathway have been linked to Acute Coronary Syndrome (ACS). Objectives: The purpose of this study is to determine the association between variants in the NPY and NPY2R genes, as well as the severity of acute coronary syndrome (ACS). Methods: Approximately 221 ACS patients and 278 healthy controls were selected for this study. Four variants in NPY and two variants in NPY2R genes were genotyped using Taqman allelic discrimination and sequencing. The Chi-square and Fisher's exact tests were used to verify the genotype frequencies. The logistic regression analyses were used for the evaluation of the studied variables. Haplotype analysis was used to evaluate the linkage disequilibrium (LD) between the variants (p<0.05). Results: An association of NPY c.20T>C variant was found with the ACS group when compared to the healthy group. In the analysis between variants and risk factors in the ACS group, NPY c.84G>A was associated with hypertension. The analysis between TIMI risk showed a significance for NPY c.20T>C between the low and intermediate/high TIMI risk groups. In the haplotype analysis, strong linkage disequilibrium (LD) was found between the variants NPY c.150G>A and NPY c.-485T>C. Conclusion: The NPY c.20T>C variant appears to contribute to the development of ACS. The NPY2R c.-1116A>G variant may contribute to the early development of ACS and the NPY c.84G>A variant appears to contribute to the development of hypertension. In addition, the NPY c.20T>C is associated with a protective effect in ACS severity.

Adult ACS patients (n = 221), over 18 years of age, of both sexes, underwent an electrocardiogram, and if necessary, cineangiocardiography was also performed. The myocardial injury markers, creatine kinase-MB (CK-MB) and troponin, were measured. Non-ACS patients admitted to the RHH (n = 95) with atrial fibrillation were also included in this study. The sample size was defined by convenience. A total of 278 healthy blood donors (healthy group), over 18 years of age, of both sexes, were recruited from the Hematology and Hemotherapy Foundation of Pernambuco (Hemope) and participated in interviews on the presence of acute or chronic diseases or significant comorbidities and laboratory tests (HIV, hepatitis C, syphilis, Human T-lymphotropic virus type 1 and 2, and Chagas' disease) to identify infectious parasitic diseases.

Inclusion criteria
This study enrolled patients who were diagnosed with ACS, after having undergone a physical examination, an electrocardiogram, and a measurement of myocardial injury markers. The non-ACS patients were those hospitalized without a diagnosis of coronary disease and typically who presented atrial fibrillation.

Exclusion Criteria
Individuals taking anti-inflammatory drugs, those who had suffered a recent trauma, those with a history of active infectious diseases or neoplasms, and those who refused to participate in the study were excluded from the ACS and non-ACS groups.

Ethical considerations
The Real Heart Hospital/Realcor Research Ethics Committee of the Royal Portuguese Charity Hospital in Pernambuco approved this study (CAAE: 03187512.2.0000.5202). Before sample collection, each subject was informed about the research and signed an informed consent form.

Level of ACS severity in patients
In all patients, the severity level was determined by the left ventricular ejection fraction, which was determined by the echocardiogram. Additionally, the risk score for Thrombolysis in Myocardial Infarction (TIMI) was used. The TIMI Risk Score is used to categorize patients as having an intermediate and high risk of suffering acts through its receptors NPYR1, NPYR2, NPY4R, NPY5R, and NPY6R. The NPY1R, NPY2R, NPY4R, and NPY5R receptors are functional in all mammals, but Y6 is a pseudo-gene in humans and other primates, and is also absent in the mouse genome. 4 The NPY c.20T>C mutation, which results in the substitution of Leucine for Proline (Leu7Pro) in the pre-pro-NPY signal peptide, is associated with an increase in serum lipid levels. NPY c.20T>C increased the risk of accelerated and early progression of atherosclerosis, 5-7 acute myocardial infarction and stroke, 8 coronary artery disease, 9 hypertension, 10,11 and obesity in children. 12 The NPY c.84G>A and c.150G>A variants were investigated for their association with obesity and hypertension, 10-11 while c.150G>A was studied for its association with atherosclerosis. 7 By contrast, the NPY c.-485T>C (rs16147) variant, which is located in the promoter region, 13 has proven to alter NPY expression in vitro, and is likely to affect mRNA expression levels in vivo. [14][15] Numerous studies have established a link between the c.-485T>C variant and the development of early atherosclerosis 7 and stroke. [16][17] Other studies have established a link between this variant and a reduction of insulin resistance and type 2 diabetes. [18][19] Through the NPY 2 receptor (NPY2R), NPY may also act as a stimulator of angiogenesis, inflammation, and adipogenesis in the abdominal region. 20 NPY2R variants c.-1088C>T and c.-1116A>G are associated with obesity, [21][22][23][24] as well as with low HDL-C serum levels. 25 Since the earliest experimental studies with NPY, its association with cardiovascular diseases has been established, with the evidence that cardiac NPY is released from sympathetic nerves during acute myocardial infarction. 26 Plasma levels of NPY were found to be high in human suffering from Acute Coronary Syndromes (ACS), such as acute myocardial infarction and during left ventricular failure. [27][28][29] NPY and the NPY2R may serve as biomarkers for ACS prognosis, risk stratification for death or cardiovascular events, or even a potential therapeutic target in other types of treatments. This purpose of this study was to evaluate the influence of NPY and NPY2R variants on the severity of ACS.

Population
The study enrolled patients who were admitted to the Real Heart Hospital (RHH), which is affiliated with the Royal Portuguese Charity Hospital in Pernambuco (RPHCP), located in Recife, Pernambuco, Brazil. a cardiovascular event. For the patients without the ST-elevation myocardial infarction, seven risk factors were considered. 30 For the patients with an ST-elevation myocardial infarction (STEMI), eight factors are considered, in accordance with Morrow et al. (2000). 31

Blood samples and DNA extraction
During the routine of cardiologic exams performed on RPHCP, peripheral blood samples were collected in a 5ml EDTA tube to perform molecular analysis at the Oswaldo Cruz Foundation (Fiocruz-PE). Purification of the DNA was carried out according to manufacturer's instructions, using the Illustra blood genomicPrep Mini Spin Kit (GE Healthcare, Buckinghamshire, UK).

Identification and genotyping of variants
DNA sequencing was used to screen for the NPY gene variants 20T>C (rs16139), c.84G>A (rs5572), and c.150G>A (rs5573). Initial denaturation at 95°C for 5 minutes was followed by 35 cycles of denaturation (95°C for 1 minute), annealing (60°C for 30 seconds), and extension (72°C for 45 seconds), followed by a final extension at 72°C for 7 minutes32. According to manufacturer's recommendations, Platinum® Taq DNA polymerase (Invitrogen Life Technologies) was used. The reagents were used without adding DNA as a negative control. The amplified fragments were viewed on a 1.5% agarose gel. The PCR products were sequenced and analyzed using the Chromas Lite 2.01 program at the Aggeu Magalhes Institute's Technological Platform Centers (NPT/IAM). The NPY variants c.-485T>C (rs16147) (Assay ID C 2267279 10), NPY2R c.-1088C>T (rs6857715) (Assay ID C 29013142 10), and NPY2R c.-1116A>G (rs6857530) (Assay ID C 44837 30) were identified by applying realtime PCR with Genotyping, which was carried out using the 7500 RealTime PCR System (Applied Biosystems, Foster City, CA, USA) under the following conditions: initial denaturation at 95°C for 10 minutes, followed by 40 cycles of denaturation (92°C for 15 seconds), annealing (60°C for 60 seconds), extension (60°C for 60 seconds), and final extension at 60°C for 60 seconds.

Statistical analysis
To determine whether the genotypic frequencies agree with the Hardy-Weinberg proportions, the Chi-square and Fisher's exact tests were used. The clinical variables, inflammatory markers, ischemia, and variants were all evaluated using logistic regression analyses. The data were analyzed using The R Project for Statistical Computing (R Development Core Team), version 2.10. The haplotype analysis and evaluation of linkage disequilibrium (LD) between variants were performed using the HaploView 4.2 software. When the p-value was less than 0.05, the data were considered statistically significant.

Characterization of the study population
A total of 221 patients with ACS were selected, the majority of whom (76.02%) was male. This distribution was also observed for non-ACS patient and health patient groups, comprised of 52.69% (49/93) female individuals and 84.17% (234/278) male individuals, respectively. The median age ACS, non-ACS, and healthy patients was respectively 60, 6,3 and 45 years. No significant differences in age were found between the ACS and non-ACS groups (p = 0.7775), but significant differences were observed between the ACS and the healthy groups (p < 0.001). According to the TIMI risk, patients with ACS were classified as having a low risk (34.84%), an intermediate risk (46.61%), or a high risk (18.55%). Multiple logistic regression analysis revealed that gender, smoking, diabetes, and dyslipidemia are contributing factors to the increased risk of ACS (Supplementary Table S1).

Analysis of the variants
The groups of patients with ACS and healthy patients showed Hardy-Weinberg equilibrium (HWE) (p > 0.05) for all variants in NPY and NPY2R. In the group of non-ACS patients, NPY c.150G>A, NPY2R c.-1088C>T, and c.-1116A>G showed HWE, while NPY c.20T>C and NPY c.84G>A were not in HWE. Therefore, no statistical analyses involved these two populations.
The NPY c.20T>C showed a majority of ancestral homozygous genotype TT (94.57%) in the ACS group, while 5.43% of the patients were heterozygous. In the healthy group, 96.40% of the samples showed the TT genotype, while only 3.60% presented the TC or CC genotypes. When the analysis after adjusting for age and gender was performed, there was an association between this variant and ACS. Previously, it presented p = 0.3251 for the TC/CC and p = 0.3307 for the C allele, later showing a p = 0.0256 for the TC/CC and p = 0.0892 for the C allele, with an odds ratio (OR) of approximately three-fold that of the individual having the TC/CC genotype or the C allele to develop ACS, as can be seen in Table 1.  Table S2). In the healthy group, 21.50% showed the AA genotype, while 78.50% presented the AG/GG genotypes. When the logistic regression analysis was performed, an association between c.-1116A>G and ACS (p = 0.0054 for the AG/GG genotypes and p = 0.0109 for the G allele) was observed between the healthy and the ACS groups. After adjustment for age and gender, this association continued (p = 0.4619 for the AG/GG genotypes and p = 0.2982 for the G allele), as can be observed in Table 1.

Relationship between variants and clinical and biological characteristics and habit
The association of the variants with the following variables of ACS was also analyzed: gender/time, smoking, diabetes, hypertension, dyslipidemia, levels of C-reactive protein (CRP), number of arterial lesions, troponin levels, levels of CK-MB mass, and ejection fraction.
The results showed that in the ACS group, the NPY c.84G>A variant was associated with hypertension, indicating a 3.57-fold chance of developing this disease (p = 0.0223) in individuals who do not have GA/AA genotypes ( Table 3).
The NPY2R c.-1116A>G variant showed an association with the onset time of ACS (Table 4), indicating that the individual who has this variant has almost twice the chance to developing the early syndrome (p = 0.0253; OR = 1.91). However, no association was found for other variants (Supplementary Tables S3, S4, S5, and S6).   Figure S7).

Discussion
Genotyping of NPY and NPY2R polymorphisms in 221 ACS patients and 278 healthy controls indicated that the NPY c.20T>C polymorphism significantly raised the risk of ACS. The NPY2R c.-1116A>G contributes to the development of the early stages of the syndrome. In addition to this, the NPY c.20T>C is associated with a protective effect in ACS severity.
NPY has been linked to hypertension, congestive heart failure, and other cardiovascular diseases due to its high sympathetic nervous system activity. 33 The NPY c.20T>C variant has been linked to cardiovascular pathologies, such as accelerated and early progression of atherosclerosis, 5,6 acute myocardial infarction, and cerebrovascular disease in hypertensive patients. 8 The results of the present study were similar to those reported by Masoudi-Kazemabad et al., 9 which showed an association between NPY c.20T>C and coronary artery disease in an Iranian population. 9 Wallerstedt et al., 8 demonstrated the association of this variant with acute myocardial infarction in a Swedish hypertensive population. 8 The NPY c.20T>C variant was initially associated with increased cholesterol and lowdensity lipoprotein (LDL) levels, 5 in obese and healthy individuals. 6 In a subsequent study in Finnish women with coronary heart disease, the NPY c.20T>C is associated with total cholesterol, but not with LDL. 34 Further, the  NPY c.20T>C is associated with altered lipid metabolism 18 and increased the susceptibility to type 2 diabetes mellitus (T2DM) and diabetic retinopathy in T2DM. 35 Bhaskar et al., 11 evaluated the NPY c.20T>C variant with hypertension and found an association. 11 The variations in the associations are due to variations in allele frequencies across populations. The carrier frequency of the C allele of the NPY c.20T>C ranges from 6-15% in Caucasian populations, but it is very low or absent in Eastern populations. 36,3 The highest allele frequencies were found in the Nordic countries. Moreover, the NPY c.20T>C could originate in northern Europe and then spread to neighboring regions. 36  In the present study, pairwise LD values between NPY c.20T>C and c.150G>A variants indicated that there is no strong LD between these markers. These findings were corroborated by a few other studies that did not report a significant LD between these markers. 10,11 Further, a weak LD between the NPY c.20T>C and c.-485T>C variants was demonstrated in this study, corroborating with Patel et al., 18 The possible explanation for the weak LD between these markers is due to the lack of recombination between adjacent markers or low frequency of the alleles. 3 The study's primary limitation is that the evaluated population was from Brazil, a country well-known for its high genetic heterogeneity. Genetic diversity studies in five Brazilian geopolitical regions revealed that European ancestry contributed the most (77.1%), followed by African (14.3%) and Amerindian (8.5%) contributions. 38 Pena et al. ,39 also showed that the European ancestry was predominant in all regions studied in Brazil, with proportions ranging from 60.6% in the Northeast to 77.7% in the South. Further, Ferreira et al., 40 demonstrated that 79% of contributions to a population in the state of São Paulo came from Europeans, 14% from Africans, and 7% from indigenous Brazilian Amerindians. Furthermore, the Rio de Janeiro population had a predominantly European genetic influence (from 55.2 to 58.6%), followed by African (from 31.1 to 30.3%) and Amerindian (from 13.7 to 11.0%) contributions. 41 Further, the number of individuals studied may limit the conclusions of our results.

Conclusions
In summary, the NPY c.20T>C variant appears to contribute to the development of ACS. The NPY2R c.-1116A>G variant may contribute to the early development of ACS, and the NPY c.84G>A variant appears to contribute to the development of hypertension. In addition, the NPY c.20T>C is associated with a protective effect in ACS severity. This information contributes to a better understanding of the effect of NPY and NPY2R variants in the population under study. Further research with a larger sample size is necessary to confirm our results.

Potential Conflict of Interest
No potential conflict of interest relevant to this article was reported.

Sources of Funding
This study was funded by FIOCRUZ CNPq Proep APQ 1620 4.01/15.

Study Association
This article is part of the thesis of master submitted by Fábia Carla Silva Soares, from Aggeu Magalhães Institute (FIOCRUZ/PE).