Characteristics and Prognostic Value of the RS2070744 Polymorphism in the NOS3 Gene in Patients with Heart Failure with Reduced Ejection Fraction

2.1. Study Design and Population

This study was a cross-sectional descriptive analysis, utilizing convenience sampling to include all patients with heart failure with reduced ejection fraction (HFrEF) who were hospitalized in the Cardiology Department of Bac Lieu General Hospital between April 2023 and June 2024. Inclusion and exclusion criteria were established to select appropriate participants for the study. Patients were diagnosed with HFrEF according to the 2021 ESC guidelines, meeting the following criteria: (1) clinical manifestations of heart failure, (2) elevated NT-proBNP levels ≥125 pg/mL, and (3) LVEF less than 40% on echocardiography [7]. Exclusion criteria included: (1) chronic kidney disease with an estimated glomerular filtration rate (eGFR) <30 mL/min/1.73m², (2) hyperkalemia >5 mmol/L, (3) active cancer, (4) psychiatric disorders or dementia impairing the ability to respond to questions, and (5) loss to follow-up. After the follow-up period, a total of 90 HFrEF patients meeting the criteria were included in the study for analysis.

2.2. Data Collection

All patients were assessed for demographic characteristics, including age, gender, and body mass index (BMI), with overweight and obesity defined as BMI ≥23 kg/m² for Asian populations [22]. Waist circumference was measured using a measuring tape at the level of the anterior superior iliac spine, with abdominal obesity defined as >90 cm in men and >80 cm in women for Asian populations [23]. A clinical examination was conducted by a specialist physician immediately thereafter to document clinical symptoms of heart failure, blood pressure levels, and the NYHA classification at the time of admission [24] to assess the severity of heart failure. Comorbid conditions, such as hypertension, type 2 diabetes, dyslipidemia, and coronary artery disease, were also documented, and smoking status was defined according to the criteria set in the COMMIT (Community Intervention Trial) study [25].

2.3. Sample Sizes

We calculated the sample size using the proportion estimation formula, with α = 5%, d = 0.07, and p = 11.0%, which represents the prevalence of the CC genotype in the study by Terzi S et al. on the impact of rs2070744 polymorphism in the NOS3 gene on long-term mortality in patients with chronic heart failure [26]. The minimum required sample size was determined to be 77 patients; however, our study included a total of 90 patients. The sample size was calculated using the standard proportion formula (Eq. 1):

(1)

2.4. Genotyping

Previous studies have reported a thymine-to-cytosine point mutation at position –786T/C (rs2070744) in the NOS3 gene, which may alter its expression levels and enzymatic activity [19-21]. Based on this, to characterize this polymorphism in patients with heart failure, we conducted the following procedure: Two milliliters of blood were drawn from each patient into an EDTA anticoagulant tube, transported to the laboratory within 24 hours, and stored at -20°C until use. The NOS3 gene sequencing to detect rs2070744 polymorphism was performed using the RFLP-PCR technique at the Molecular Biology Laboratory, Can Tho University of Medicine and Pharmacy. The DNA extraction procedure involved using 200 µl of whole blood from each sample, which was processed with the QIAamp DNA Blood Mini Kit, including three main steps: (1) cell and nuclear membrane lysis to release DNA; (2) washing away proteins and impurities; and (3) DNA collection. To ensure the quality of the extracted DNA, the concentration was measured, and its purity was assessed using a spectrophotometer, with the following parameters: concentration >10 ng/µl; purity OD260/280 = 1.8 – 2.0.

The RFLP-PCR technique, with the PCR amplification process, was performed using the following primers:

Forward primer: 5'- TGG AGA GTG CTG GTG TAC CCC A -3'

Reverse primer: 5'- GCC TCC ACC CCC ACC CTG TC -3'

Each PCR reaction was performed with a final volume of 25 µl, consisting of 3 µl of DNA template, 4 µl of primers (2 µl for each forward and reverse primer), 0.5 µl of Taq DNA polymerase (Gen Fanavaran, Iran), 12.5 µl of Taq DNA Polymerase Master Mix Red (Ampliqon, Denmark), and 5 µl of double-distilled water (nuclease-free). The thermal cycling process consisted of an initial denaturation at 94°C for 2 minutes, followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 62°C for 1 minute, and extension at 72°C for 1 minute. The amplification process concluded with a final extension at 72°C for 7 minutes. After the thermal cycling process, the PCR products were digested with restriction enzymes according to the manufacturer's instructions. The restriction enzyme-digested products were then analyzed by electrophoresis on a 2% agarose gel, and the results were recorded and analyzed.

2.5. Follow-up Outcomes

All patients with HFrEF were treated according to the ESC guidelines, including medications that improve heart failure symptoms and prognosis, such as agents targeting the renin-angiotensin-aldosterone system (ARNI/ACEi/ARB), beta-blockers, mineralocorticoid receptor antagonists, and sodium-glucose cotransporter-2 inhibitors (SGLT2i) [7]. After discharge, patients were followed up for 3 months and 6 months. Survival status and clinical events were assessed by contacting patients and their family members via telephone, with verification through the review of hospital medical records. The clinical events monitored included readmission due to heart failure and mortality from all causes. According to the 2021 ESC diagnostic criteria, heart failure rehospitalization is defined as a hospital readmission following discharge due to acute clinical manifestations of heart failure, including acute dyspnea and fluid overload, requiring intravenous therapeutic intervention [7]. Mortality included both cardiovascular and all-cause death, recorded based on medical records. At the end of the follow-up period, data were compiled and statistically analyzed.

2.6. Statistical Analysis

Data was analyzed using SPSS 20.0 software. Qualitative variables were presented as frequencies (percentages), while mean (± standard deviation, SD) was used for normally distributed quantitative variables, and median (interquartile range, IQR) for non-normally distributed quantitative variables. The normality of the distribution was assessed using the Kolmogorov-Smirnov test, and it was considered normal if the significance level (Sig.) was greater than 0.05. The differences between qualitative variables were described using the Chi-square test. The prognostic ability of the genotype characteristic, rs2070744 polymorphism allele, was assessed based on Kaplan-Meier survival curves. Finally, univariate and multivariate Cox regression analyses were conducted to identify independent prognostic factors for patients with reduced ejection fraction failure.

2.7. Ethical Approval

All patients were informed about the study objectives and methods, and provided written consent before participation. They were also allowed to withdraw from the study at any stage. All information collected was used solely for research purposes and will not be published without the patient's consent. The costs for genetic sequencing and any other expenses arising during the study were fully covered by the research team. The study was approved by the Biomedical Research Ethics Committee of the Can Tho University of Medicine and Pharmacy and adhered to the ethical principles outlined in the Helsinki Declaration.

4.1. Principal’s Findings

Our study observed that 22.8% of patients with reduced ejection fraction heart failure carried the C allele. The CC genotype was associated with a more severe prognosis, with a higher probability of heart failure-related readmission and lower cumulative survival at 3 months post-discharge compared to the TT and CT genotypes. Regarding mortality events, after adjusting for risk factors, the CC/CT genotype and dyslipidemia were independent predictors of mortality at 3 months. However, at 6 months, only hypertension as a comorbid condition remained an independent prognostic factor.

4.2. Strengths and Weaknesses of the Study

Our study involved a clearly defined sample collection process with specific inclusion and exclusion criteria. All participants were enrolled voluntarily, and the gene sequencing was conducted in a modern molecular biology laboratory, ensuring reliable results with well-characterized genotypes and alleles of the rs2070744 NOS3 gene, which can be reproduced. The event follow-up period was relatively long, at 3 and 6 months, providing valuable information regarding long-term prognosis. Notably, the C allele was identified as a risk allele, with patients carrying the CC genotype having a more severe prognosis. In contrast, the CC/CT genotype was an independent predictor of mortality.

However, our study was conducted in a single hospital or region with a limited sample size, which may lead to differences in certain demographic and clinical characteristics. Additionally, we focused on only two outcomes: readmission due to heart failure and mortality, thus not covering all aspects of prognosis. Therefore, further research with a larger sample size, conducted across multiple centers, considering treatment aspects and evaluating a broader range of outcomes, is necessary to fully describe the characteristics of rs2070744 and capture the full prognostic potential of this polymorphism.

4.3. Possible Explanations and Comparison with other Studies

The study recorded a mean age of 64.5 ± 13.3 years, with most patients being male, accounting for 53.3% (Table 1). Regarding the rs2070744 polymorphism, in 2016, Agata Bielecka-Dabrowa reported the distribution in heart failure with preserved ejection fraction (HFpEF) patients as 49.0% for the TT genotype, 51.0% for CT, and no cases with the CC genotype. In contrast, patients with heart failure with reduced ejection fraction due to dilated cardiomyopathy had a genotype distribution of 18.0% TT, 78.0% CT, and 4.0% CC, while HFrEF patients with ischemic heart disease had TT and CT genotypes at 53.0% and 47.0%, respectively [27]. These results differ significantly from our study, where the TT genotype was the most common at 61.1%, followed by CT and CC at 32.2% and 6.7%, respectively (Table 2). In 2017, this author observed in patients with HFpEF and HFmrEF that the genotypic distribution was 43.0% TT, 56.0% CT, and 1.0% CC, with allele frequencies of 43.0% for T and 57.0% for C. Clinically, patients carrying the risk allele C exhibited lower EF, eGFR (MDRD), and both systolic and diastolic blood pressure, while having a higher NYHA class [28]. Similarly, Abdullayeva et al. assessed the rs2070744 polymorphism in Uzbekistani patients with chronic heart failure aged 41 to 70 years, finding a significant deviation from Hardy-Weinberg equilibrium, with an overrepresentation of heterozygotes. The genotypic distribution was 55.6% TT, 43.2% CT, and 1.2% CC [18]. Additionally, in a study of type 2 diabetes patients, M. Boronat found genotype frequencies of 28.5% TT, 41.1% CT, and 30.5% CC, with the risk allele C occurring in 51.0% of patients, compared to 49.0% for allele T [29]. Similarly, Andrzej Pawlik analyzed 246 patients with a history of coronary artery disease, comparing them with a control group. The genotype frequencies of CC and CT in the control group were 17.55% and 44.68%, respectively, while in the disease group, the frequencies were 13.93% and 43.03%. Regarding the disease-causing allele, the author found that the C allele appeared in the control group at a rate of 39.89%, compared to 35.45% in the disease group [19]. Sahar Gamil studied Sudanese individuals with hypertension, finding genotype frequencies of CC, CT, and TT at 6.6%, 46.1%, and 47.4%, respectively, compared to 6.1%, 28%, and 65.9% in the control group [30]. Although several preliminary studies have evaluated the rs2070744 polymorphism in heart failure patients (Table 7), there is still limited evidence regarding heart failure with reduced ejection fraction. Differences in race and geographic region have influenced genetic stability, leading to varying genotype and allele frequency distributions among different authors [18].

In terms of prognosis, we found that at the 3-month time point, patients with the CC genotype had a higher probability of heart failure-related readmission and a lower cumulative survival rate compared to those with the CT and TT genotypes (p<0.05) (Figs. 1 and 2). Furthermore, the CC/CT genotypes, along with dyslipidemia, were identified as independent predictors of mortality (Table 5). Currently, there is limited research on the prognostic value of the rs2070744 polymorphism in heart failure patients. Sait Terzi's preliminary study on the effect of this polymorphism on long-term mortality in patients with chronic heart failure found a significant increase in mortality among patients with the CC genotype compared to those with the TT genotype (HR=2.4; 95% CI=1.01-5.60; p=0.04). In particular, the Kaplan-Meier analysis showed that the CC genotype was associated with a worse long-term prognosis, with a lower survival rate compared to the TT genotype (10% vs. 39%) [26]. Previous studies have suggested that the rs2070744 polymorphism is likely to impact NOS3 gene expression and serum nitric oxide levels. The –786T/C substitution in the promoter region reduces its activity by approximately 50%, which may lead to decreased NOS3 mRNA expression and a reduction in serum nitric oxide levels. Evidence indicates that this effect is closely associated with the development of coronary artery disease, vasospastic angina, and myocardial infarction, which are among the leading causes of heart failure or exacerbate existing heart failure conditions [19-21]. In addition to its role in regulating vascular tone, this polymorphism is also associated with endothelial dysfunction, promoting the development of dilated cardiomyopathy [27, 31]. Furthermore, the thymidine-to-cytosine substitution at rs2070744 in the nitric oxide synthase gene leads to an imbalance in the autonomic nervous system within the heart, which manifests as heart rate variability. Philip F. Binkley suggests that this factor may allow the rs2070744 polymorphism to serve as a prognostic marker for patients at risk of sudden cardiac death and rapid progression of heart failure [29]. Regarding dyslipidemia, no studies have yet provided evidence on the impact of dyslipidemia on prognosis in patients with HFrEF that specifically involves the rs2070744 polymorphism of the NOS3 gene. The prognostic value of dyslipidemia in HFrEF patients has been reported in several clinical studies; however, these studies did not examine genetic polymorphisms. Solmaz Norouzi evaluated factors influencing mortality in HFrEF patients using a prognostic model based on the Bayesian method, identifying hyperlipidemia as a significant variable associated with HFrEF-related mortality, with a hazard ratio (HR) of 0.34 (95% CI: 0.13–0.64), and with non-HFrEF-related mortality, with an HR of 0.60 (95% CI: 0.37–0.90). This suggests that hyperlipidemia may be closely associated with reduced survival time in heart failure patients [32]. Additionally, statin therapy has been reported to be associated with a reduction in both short-term and long-term all-cause mortality in patients with severe heart failure [33]. Regarding the interaction between NOS3 and dyslipidemia, Andrii Sydorchuk et al. investigated the rs5443 polymorphism of the GNB3 gene and the rs2070744 polymorphism of the NOS3 gene in patients with hypertension. Their findings indicated that rs5443, rather than rs2070744, was the polymorphism associated with hyperlipidemia in hypertensive patients [34]. These findings highlight the existing knowledge gap regarding the interaction between the rs2070744 polymorphism of NOS3 and dyslipidemia in predicting cardiovascular events in HFrEF patients. Further studies are needed to clarify this relationship in future research.