Gene Expression and Methylation Levels of PCSK9 Gene in Iraqi Patients with Coronary Artery Disease

: The expression of the Proprotein Convertase Subtilisin/Kexin Type 9 gene ( PCSK9 ) is inextricably related to lipid levels and a risk of atherosclerotic coronary artery disease (ASCAD). The present study aims to measure the quantity of PCSK9 gene expression and the effect of methylation on its expression level taking part in the pathogenesis of acute coronary artery disorder. A current study included 150 subjects from the Iraqi population, 100 ASCAD patients and 50 healthy controls. The concentration of PCSK9 in each serum sample was determined by the ELISA technique, the expression levels of the PCSK9 gene in whole blood were estimated by RT-qPCR – Quantitative Reverse Transcription PCR method, and DNA methylation level detection in PCSK9 gene by using High Resolution Melting Analysis (HRM) technique. The expression level of the PCSK9 gene was 6.06 ± 1.84 in ASCAD patients group compared with a control group 1.00 ±0.19. That indicates the up-regulation of the PCSK9 gene in patient with atherosclerosis. In addition, the PCSK9 concentration was higher in patient group in comparison to the healthy controls group P= < 0.01. However, PCSK9 methylation levels, a highly significant distinction between the two study groups p=0.002. In conclusion, serum PCSK9 levels are associated with coronary artery lesions as shown in receiver operating characteristic (ROC) analysis. Suggests that, serum PCSK9 might be a good indicator of coronary artery disease development.


Introduction:
Atherosclerosis (AS) coronary artery disease is a lipid-derived inflammatory disease that includes multicellular alteration it is characterized by plaque formation and it reasons millions of death globally 1 .These plaques are primarily composed of lipids, which cause an inflammatory response, resulting in disorderly flow and ASCAD 2 .Many risk factors for ASCAD, some of which can be controlled, while others cannot.Risk factors that can be controlled (modifiable) are: High blood pressure, high levels of cholesterol, smoking, diabetes, being obese, insufficient physical activity, unhealthy eating habits, and stress are all risk factors.Those who are uncontrollable (conventional) are: Gender (men are generally at higher risk of CAD); age (simply getting older raises the risk); as well as family history 3 .The initial soft lesion is composed of extracellular fat deposits, foam cells, and a small number of platelets 4 .In Iraq, as stated by the world health organization (WHO), CVD is the major cause of mortality in Iraq exceeding 18.50% of total mortality in 2017 and Iraq ranked the 19th internationally 5 .According to a previous study documented by 6 , who reported that an unexpectedly high prevalence of coronary atherosclerosis in Iraqi men is higher than in women, which is the highest in Asia when compared to results of other similar studies conducted in other countries.The PCSK9 is a serine protease that has recently received a lot of attention due to its important role in the regulation of plasma low density lipoprotein-cholesterol (LDL-C) levels and in determining (ASCAD) risk 7,8 .PCSK9 promotes low density lipoprotein-receptor (LDL-R) degradation in the liver via an unknown posttranscriptional mechanism.Some sequence variations of the PCSK9 gene were found in the large long-term atherosclerosis risk in communities study, this is related to low LDL-C levels and a lower risk of coronary events.Other sequence variants, on the other hand, have been linked to the development of premature atherosclerosis, since mutations in PCSK9 can cause severe hypercholesterolemia.
In addition, the measurement of PCSK9 serum levels is a further important biomarker for atherosclerosis to detect the disease course and respond to therapy 9 .DNA methylation is an epigenetic alteration in human DNA that involves a chemical modification (substitution mutation) by the additional methyl group CH3 to 5′ carbon of the pyrimidine ring (5′position of cytosine residues) and because of the changes caused by this process, the necessary proteins are unable to bind to the DNA molecule for initiate transcription and translation 10 .Compatible with a recent study, we discovered that methylated PCSK9 gene at promoter region in CAD patients has 3.72-fold lower expression and 85 percent lower serum protein levels of PCSK9 than CAD patients with unmethylated DNA 11 .This study aimed to evaluate PCSK9 gene methylation, gene expression and quantification of serum levels of PCSK9 regulatory protein (affect lipid levels) in patients with ASCAD.

Materials and Procedures:
Participant in the study: This study included one hundred fifty participants, ages between 30 to 65 years both male and female.Hundreds of patients had the clinical appearance of ASCAD, as well as undergoing a medical and physical examination and documented through coronary angiography, and fifty healthy controls were chosen at random, clinical examination revealed no signs of cardiovascular disease.Subjects were enlisted to help with diagnosis and treatment from the Iraqi Center of Heart Disease in Baghdad, Iraq.The study was ethically approved by the Ministry of Health in Iraq (decree order 3310 on 08/21/2012), through the Scientific Committee of the College of Biotechnology, Al-Nahrain University, carried out between December 2021 and May 2022.Patients diagnosed with ASCAD with TC usually greater than 200mg/dl and LDL-C greater than 160mg/dl (hypercholesterolemia), triglycerides (TG) less than 200 mg/dl whose other lipid levels were normal according to the American heart association (AHA) guidelines, coronary angiography technique should be included with criteria equal to or more than 50 percent stenosis of at least one major coronary vessel due to atherosclerosis and confirmed by two experienced cardiologists ASCAD, and normolipidemic subjects with LDL-C less than 130 mg/dl and TG less than 150 mg/dl 4 .Exclusion criteria were: subjects with the presence of hypertension were previously diagnosed to have hypertension by a clinician.Diabetes mellitus was defined as the use of diabetes medications or a fasting blood glucose level equal to or more than 126 mg/dl.To avoid inaccuracy in the results, hypertriglyceridemia, renal, liver, or thyroid disease, autoimmune disease, malignant tumor, congenital heart disease (CHD), and Patients who lacked basic information were barred from participation.

Collection of blood samples:
The blood samples were separated into two groups; First group put into the serum separating tube was used to measure the concentration of PCSK9 protein by ELISA test.

Biochemical analysis:
Enzyme Linked Immunosorbent Assay (ELISA) test is a method of quantitative sandwich enzyme immunoassay, the microplate has been pre-coated with a monoclonal antibody (mAb) specific for human PCSK9 11 .The PCSK9 concentration in each serum sample was represented as ng/ml for the comparison with the results with those of controls concentration and the detection ranges: 177ng/ml-460ng/ml, ELISA kits used in this study produced by (R&D/ USA).

Quantitative Reverse Transcriptase.PCR.(RT-qPCR) for. Relative PCSK9 Gene Expression:
Quantitative reverse transcription PCR (RT-qPCR) involves two steps, the first convert in total RNA to cDNA was performed by using EasyScript®.One-Step.gDNA.Removal and cDNA Synthesis SuperMix Kit through utilizing PCR (thermocycler) machine.The PCR Condition for cDNA synthesis is programmed as, 25 ºC for 10 min for random primer binding, 42°C for 15 min for reverse transcriptase enzyme activation and Oligo(dT) primer binding and 85 ºC for 5 sec for enzyme inactivation.The second step RT-qPCR analysis was used to evaluate the gene expression levels of target gene by utilizing Qiagen Rotor gene Q Real-time PCR System.The components for RT-qPCR analysis were the.cDNA.wasmixed with.the SYBER Green master mix provided by TransStart® Top.Green.qPCRSuperMix Kit, in addition specific forward and reverse primers (primers and housekeeping genes utilized in present study with their sequences are summarized in Table 1) and complete the volume of reaction to 20 μl by using nuclease free water and the RT-qPCR program of PCSK9, and GAPDH genes as the following condition: the Enzyme activation step 94 ºC for 90 sec for 1cycle in hold 1, followed denaturation step 94 ºC for 5 sec, then annealing 58 ºC for PCSK9 and 64 ºC for GAPDH genes for 15 sec and extension 72 ºC for 20 sec for 40 cycles in hold 2, followed dissociation 55-95 ºC for 1 min for 1 cycle.Each reaction was performed in duplicate and included a non-template control (NTC), nonamplification control(NAC) and non-primer control (NPC) as negative controls were done in separate run to ensure efficacy of (primers , template and master mix).The relative cycle threshold (2 -∆∆Ct ) method was used to calculate fold changes in the quantified expression of mature RNAs 13 .The target gene expressions were normalized by using housekeeping gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a reference gene to obtain accurate Ct values from the Quantitative real time PCR (qPCR) instrument 14 .In addition, the Kolmogorov-Smirnov and Shapiro-Wilk tests were used to determine normality.Frequencies and proportions were used to express categorical variables.The Chi-square test 2 was utilized to compare proportions.A mean and standard deviation of continuous variables were calculated (SD).The student's t-test was applied to compare group differences.Variables with nonnormal data distributions were log-transformed for analysis before being converted back to standard units.Mann-Whitney.The U nonparametric test was exercised to compare the two groups.Moreover, the Pearson Correlation produces a correlation coefficient (r) which measures the relationships between the studied parameters.The area under the curve (AUC) of some studied parameters is calculated using receiver operator characteristics (ROC).In addition, the best cut-off value (CV),.sensitivity,and specificity were studied.A p-value <0.05 was considered statistically significant 15,16,17 .

Results and Discussion:
The study population's anthropometric distribution: The result of anthropometric factors used in this study as shown in Table 3, was assessed according to a question addressed to the participants in this study.According to the age mean±SD of subjects, of a study group was 51.86 ± 9.76 for the patient group and 48.62 ± 10.09 for the control group, the result found that there was no significant association between both groups studied p= 0.06.The results displayed there was a statistically significant association between the two groups of study based on height 170.37 ± 8.38 vs. 174.80± 9.29, systolic pressure 13.52 ± 2.14 vs. 11.94 ± 1.03, diastolic pressure 8.27 ± 1.37 vs. 7.40 ± 1.04, ejection fraction 55.72 ± 10.29 vs. 63.28 ± 4.04 respectively P< 0.05, while no significant association P= 0.7 between two study groups based on weight 81.41 ± 12.47 vs. 80.76 ± 13.70.In addition, upon comparing the results of patient and control groups according to gender found the control group was made up of 74% men and 26% women, while the patient group was made up of 79% men and 21% women, there was no significant association between case and control groups P= 0.4.Furthermore, the smoking percentage in the patient group was 39% compared with the control group 16% and the non-smoker percentage in the case group was 61% compared with the control group 84%, there was a significant association between two groups p= 0.004, all males and females included either smokers or not smokers depending on patient data form.The percentage of the patient group depending on alcohol-consuming was 4% compared with the control group that was 0%, while the patient group non-consumed 96 % more than the control group 100 %, but there were no significant differences between the two groups P=0.15.
The previous study displayed there was a significant association between smoking and the development of atherosclerosis, that risk was proportional to the number of cigarettes smoked per day 18 .Smoking contributed to the deaths of 2 million adults in Asia their age over 45 in 2004, which corresponded to a 1.4-fold increase in mortality 19 .Furthermore, 31% of smoking-related deaths were attributable to CVDs, the smokingrelated CVD through many pathological processes including endothelial dysfunction, inflammation, altered lipid metabolism, and hypoxia because the chemicals in cigarette smoke cause the blood to thicken and form clots inside veins and arteries, blockage from a clot can lead to a heart attack and sudden death 20 .Also, alcohol consumption (light to moderate) in the patient with CVD is associated with a lower incidence of cardiovascular and allcause mortality 21 .Furthermore, study in 2009 found that 86.3% of the lower risk of CVD observed in moderate drinkers was explained by alcohol's effects on lipids, glucose metabolism, inflammatory/hemostatic factors, and blood pressure.Nearly, 20% of the reduced risk of either total or CVD death among moderate drinkers was accounted for by these intermediate factors 22 The relationship between the mean of ASCAD patients and controls according to age there was no significant association, however, there was a significant relationship between the two study groups based on BMI means 23 .In this study, the highest percentage of gender distribution was found in males than females, this is the same result of study prepared in 2015 that showed in their study the prevalence of ASCAD in Iraqi patients is more in men than women 6 .Men are more likely than women to develop atherosclerosis at a younger age because of female sex hormones' protective function, but this protection is lost after menopause such as progesterone, but other factors such as the way men deal with stress might also be of significance.However, as women get older their risk of developing atherosclerosis soon catches up with that of their male counterparts, especially after menopause 24 .On the other hand, higher systolic (the pressure in the arteries when a heart beats) and diastolic blood pressure (the pressure in the arteries when a heart rests between beats) were associated with a higher risk of CVD, but higher systolic blood pressure was associated with a higher risk of cardiovascular disease and little association of diastolic blood pressure with ASCAD, but not with CVD 25 .According to ejection fraction (EF) Patients with EF ≤30% but no other risk factor have low predicted mortality risk.Patients with EF >30% and other risk factors may have higher mortality and a higher risk of sudden death than some patients with EF ≤ 30%.Thus, risk of sudden death in patients with coronary disease depends on multiple variables in addition to EF 26 .Ejection fraction is a measurement of the percentage of blood leaving a heart each time it contracts, it is usually measured only in the left ventricle.The left ventricle is the heart's main pumping chamber.It pumps oxygenrich blood up into the body's main artery (aorta) to the rest of the body 27 .

Biochemical analysis for estimation of Serum PCSK9 Level:
Evaluation of serum PCSK9 level between two study groups as shown in Fig. 1.The results showed significant association between patient and healthy controls group according to PCSK9 serum levels P= < 0.01, the mean for the case group 303.82 ± 49.94 and the controls group 181.15 ± 31.85.The increase in PCSK9 levels belongs to transcription factors SREBP-2 is a protein that binds the SRE in a PCSK9 proximal promoter, which then causes upregulation of the PCSK9 gene and leads to increased circulating PCSK9 levels 28 .In another similar study, 29,30 showed that statins medication inhibiting a rate-limiting enzyme in the synthesis of cholesterol, hydroxyl methylglutarylcoenzyme A (HMG-CoA) reductase, reduces ER cholesterol, increases SREBP-2 processing, stimulates hepatic LDLR expression, and promotes plasma LDL-C clearance.However, under these conditions, SREBP-2-induced PCSK9 expression likely reduces statin efficacy by stimulating PCSK9-mediated LDLR degradation.

Expression Level of PCSK9 Gene:
As shown in Fig. 2 amplification curves for PCSK9 and GAPDH genes by Quantitative real time PCR (qPCR) included all samples of study groups that were important for determining the Ct value of each sample, the image was taken directly from Rotor-Gene qPCR machine.The gene expression for the PCSK9 gene was analyzed by qPCR and used reference gene (GAPDH) for quantification and compare Ct-value between two study groups in different stages of gene expression levels (Ct, ∆Ct, ∆∆Ct, and Fold Change) as shown in Table 4.The findings of present study showed the Ct value of the PCSK9 gene were 33.19 for patient group and 35.18 for control group and the Ct-value for GAPDH was 20.45 for patient group and 19.96 for control group.The ΔCt-value was calculated as result of the difference in Ct-value of PCSK9 gene for patient and control and the Ct of GAPDH gene, these results found the expression level of PCSK9 gene that was 6.06 ± 1.84 in the ASCAD patients' group depending on 2 -∆∆Ct and in the control group 1.00 ±0.19, that indicates the up-regulation of PCSK9 gene in patients with atherosclerosis.The concentration of intracellular cholesterol is the major regulator of the transcription of PCSK9 via SREBP2 but insulin has also been shown to regulate the expression of PCSK9 via SREBP-1c 7,31 .

Methylation of PCSK9 Using HighiResolutioniMeltingiAnalysis:
The results of the HRM-PCR analysis for PCSK9 gene methylation levels for patients and healthy control groups presented in percentages graduating from 100% methylation to un-methylation that was used for normalization, as shown in Fig. 3A.While, Fig. 3B   The results of DNA methylation patterns are displayed according to the diagnosis of a methylated pattern as shown in Fig. 4 For PCSK9 methylation levels mean ± SD, a highly significant relationship between two study groups p=0.002, the mean for patients group 51.28±21.32 that leading to indicate that hypomethylation has happened, while in the controls group hypermethylation was happened 62.72±20.67.DNA methylation regulates gene transcription where DNA hypermethylation usually leads to a long-term and stable gene suppression and DNA hypomethylation of the gene leads to gene expression 11,32 .The results of recent study found that were patients with methylated PCSK9 promoter, the PCSK9 gene expression and serum circulating levels are lower in those with unmethylated DNA, indicating a lower risk of disease 11 .

Receiver Operating Characteristic (ROC) Analysis of study parameter:
A Receiver Operator Curve (ROC) was used to discover if the study parameter could be predictive of ASCAD 4 .As displayed in Table 5, the serum PCSK9 level in a patient with atherosclerotic coronary artery disease has a high predictor of ASCAD with AUC=0.98, with the best Cutioffivalue = 226.29ng/ml,sensitivity 93.20%, and specificity 100%, and displayed a significant difference P= 0.00.While the PCSK9 methylation level in a patient with ASCAD has a good predictor of ASCAD with AUC=0.70,Cut off value = 55.95,sensitivity 64.20% and specificity 64.60%, with a significant difference P= 0.002.In addition, the PCSK9 gene expression fold in a patient with ASCAD has a good predictor of ASCAD with AUC=0.79,Cut off value = 2.00, sensitivity 75.5% and specificity 76%, A result showed a significant difference P= 0.00.

Conclusion:
In summary, the findings revealed that serum PCSK9 levels are associated with the severity of coronary artery lesions, with an increase in ASCAD patients compared to control.The parameters employed in this study served as a sensitive marker for precise diagnosis of ASCAD and as a guide for future PCSK9 inhibitor strategies.

Figure 1 .
Figure 1.Serum PCSK9 level of the two study groups.

Figure 2 .
Figure 2. Amplification plots by qPCR included all study samples, A: for PCSK9; B: for GAPDH, that important for normalization to calculate ∆Ct.
displayed the PCSK9 gene comparative as a result of the change in fluorescence observed when double-stranded DNA with incorporated dye molecules dissociates into single-stranded DNA as the temperature of the reaction is raised.In contrast, Fig.3C, show amplification plots of PCSK9 gene is useful for identifying value of methylation levels in the samples of ASCAD patient and control groups.

Figure 3A .Figure 3B .Figure 3C .
Figure 3A.Normalization graph for identification of a methylated and unmethylated pattern of PCSK9 gene was done by HMR-PCR technique.

Figure 4 .
Figure 4. DNA methylation level of PCSK9 gene of the studied groups.

Table 1 . Primers of PCSK9 gene expression used in the study.
11A Methylation of PCSK9 Gene:The extracted genomic DNA was utilized for DNA Methylation of PCSK9 by using the High-Resolution Melting (HRM) technique.The HRM-PCR template was prepared by Bisulfite conversion assay, utilizing the EpiTect-Fast DNA Bisulfite Kit.Sodium bisulfite was used to modify genomic DNA, converting all unmethylated cytosine to uracil, while methylated cytosine remain unchanged.After completion of the bisulfite conversion, the converted DNA is ready to be used in the HRM-PCR technique for determining the percentage of methylated and un-methylated cytosine in DNA11.A primer sequence utilized in the present study as shown in Table2, was designed for current study by using bioinformatics tools (Meth Primer and Beacon Designer software 8.21) (http://epidesigner.Com) for methylation of the human PCSK9 promoter region and a region was selected from 2768-2997 bp provided by Alpha-DNA Company (Canada).The choice of this region in the promoter of PCSK9 is because this region within the transcription start site (TSS) is rich in CpG island=15.