Effect of B12 and folate deficiency in hypomethylation of Angiotensin I converting enzyme 2 gene and severity of disease among the acute respiratory distress syndrome patients

Abstract Background Angiotensin I converting enzyme 2 (ACE‐2) is the most important receptor and has important role in the entry of corona virus to the host cells. The present study aimed to investigate the different mechanisms involved in the expression regulation of this gene among the COVID‐19 patients. Methods A total of 140 patients with COVID‐19 (n = 70 mild COVID‐19, n = 70 ARDS) and 120 controls were recruited. The expression of ACE‐2 and miRNAs was evaluated by quantitative real‐time PCR (QRT‐PCR), and methylation of CpG dinucleotides in the ACE2 promoter was quantified using bisulfite pyro‐sequencing. Finally, different polymorphisms of the ACE‐2 gene were studied by Sanger sequencing. Results Our results showed a significant high expression of the ACE‐2 gene in the blood samples of acute respiratory distress syndrome (ARDS) patients (3.8 ± 0.77) in comparison with controls (0.88 ± 0.12; p < 0.03). The methylation rate of the ACE‐2 gene in ARDS patients was 14.07 ± 6.1 compared with controls (72.3 ± 5.1; p < 0.0001). Among the four studied miRNAs, only miR200c‐3p showed significant downregulation in ARDS patients (0.14 ± 0.1) in comparison with controls (0.32 ± 0.17; p < 0.001). We did not see a substantial difference in the frequency of rs182366225 C>T and rs2097723 T>C polymorphisms between patients and controls (p > 0.05). There was a significant correlation between B12 (R = 0.32, p < 0.001), folate (R = 0.37, p < 0.001) deficiency, and hypo‐methylation of the ACE‐2 gene. Conclusion These results for the first time indicated that among the different mechanisms of ACE‐2 expression regulation, its promoter methylation is very crucial and can be affected by factors involved in one‐carbon metabolisms such as B9 and B12 vitamins deficiency.


| INTRODUC TI ON
Coronavirus disease  has become a pandemic today. All researchers from all over the world are conducting numerous studies to treat this disease, while the definitive cure for this disease is not yet known. The disease is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 1 Patients with severe symptoms usually show an immune system storm, and one of the organs involved is the lung, which eventually leads to respiratory distress. 2 Studies have shown that different molecular mechanisms are involved in the hosts' response to the virus. The virus generally enters the host cell in five ways: adhesion, penetration, biosynthesis, maturation, and release.
SARS-CoV-2 enters the host cells through the angiotensin-converting enzyme 2 (ACE-2) receptor. 3 In this regard, after binding of virus Spike protein to the receptor, the virus enters the host cells. ACE-2 is involved in SARS-CoV-2 by entering the cells and finally cytokine storms. 3 In general, few studies have been performed on the expression of ACE-2 in patients with COVID-19, and the results are slightly contradictory. 4,5 There are also very few studies on the mechanisms of regulation of ACE-2 expression in these patients. Some polymorphisms, methylation, and microRNAs are the three mechanisms in the expression regulation of the different genes; therefore, in this study, we decided to evaluate the role of the mentioned mechanisms in regulating the ACE-2 gene expression among the COVID-19 patients.
Based on our results, hypo-methylation was the vital mechanism in the regulation of expression of this gene. Studies on various organisms, including humans, have suggested roles for nutrient metabolism in regulating the epigenetic state in normal and disease states. 6,7 Accordingly, in this study, the effect of the amount of B9 and B12 vitamins, which are present in natural foods, are involved in the one-carbon metabolism cycle, and play an essential role in the methylation of various genes, was investigated in the ACE-2 gene methylation pattern.

| Sample collection
Whole blood samples were obtained from COVID-19 patients at Velayat Hospital and Khatam Pathobiology and Genetics lab, Qazvin, Iran. These patients were divided into two groups: The first group were people with mild symptoms who did not need to be hospitalized, and only their PCR test was positive (n = 70). The second group had acute respiratory distress syndrome (ARDS) and were hospitalized in the intensive care unit (ICU; n = 70). n = 120 samples were taken as controls whose PCR test was negative and had no clinical signs. The patients were informed about the sample collection process and had signed informed consent forms. The recruited patients were men between 50 and 55 years old. Patients with obesity, diabetes, hypertension, heart, kidney, and lung diseases were not included in the study due to many factors involved in the expression and methylation of ACE-2 gene. The study was approved by the Ethics Committee of Qazvin University of Medical Sciences (Qazvin, Iran).

| RNA extraction and QRT-PCR for ACE-2 expression detection
RNAs were extracted using RNeasy Mini Kit (Qiagen) and were frozen at −80°C. We used Nano-Drop 2000c (Thermo) to evaluate the quality and quantity of isolated total RNAs. In this regard, RNA samples with A260/A280 ratios of >1. 8

| ACE-2 gene polymorphisms evaluation
To analyze the polymorphisms of the ACE-2 gene, we designed the following primers: rs18236622 5′-CAT CCC TAT TGG CAG GTT AC -3′ as forward primer and 5′-GAC GGT GCG GTG AGA GTG-3′ as reverse primer. For rs2097723 polymorphism, these primers were 5′-CTTTGG GGAGC TGA AGG ACT ACTA C-3′ and 5′-CAC TTT GTG ACC ATT CCG GTT TG-3′ as forward and reverse primers, respectively. For doing PCR, first, we isolated genomic DNA from the blood cells by using Dyna Bio™ Blood/Tissue DNA Extraction Mini Kit (Takapouzist). Our PCR condition was primary denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 2 min, annealing at 60°C for 1 min, extension at 70°C for 1 min, and a final extension at 70°C for 5 min. After the PCR procedure, we sequenced PCR products based on the Sanger method using ABI 3730XL Capillary Sequencer. The ACE-2 normal sequences were obtained from NCBI website: http://www.ncbi.nlm.nih.gov, then were assembled by using Chromas software (version 2.4).

| DNA extraction and bisulfite modification
The standard phenol-chloroform method was used to extract DNA from the blood samples. 8 Then, for the methylation study, 10 ng of genomic DNA was bisulfited using the EpiJET™ Bisulfite Conversion kit (Thermo Fisher Scientific, Inc) according to the kit instructions. Bisulfite treatment converted nonmethylated cytosine 'C' bases to thymine 'T' bases; however, the methylated cytosine 'C' was left unchanged. The methylation levels were determined by using the Pyrosequencing method. In this regard, first, CpG islands of the ACE-2 gene were identified using MethPrimer

| QRT-PCR for microRNA expression evaluation
We used TRIZOL reagent to isolate the total blood RNAs based on kit protocols (Invitrogen Life Technology Co). In order to separate the proteins, three steps of phenol/chloroform purification were performed. To evaluate the concentration and purity of RNA, we used Nano Drop w ND-1000. miRCURY LNA Universal RT microRNA kit was used to reverse the transcription of RNA (Exiqon, Denmark). The final volume was 10 μL containing 1 ng/μL of purified total RNA, 5× reaction buffer, Enzyme mix, and nuclease-free water. The mix was incubated at 42°C for 60 min, then at 95°C for 5 min (for enzyme inactivation). It was then quickly cooled to 4°C. The three miRNAs examined in our present study were hsa-miR-1246 (MIMAT 0005898), hsa-miR-200c (MIMAT0000617), and hsa-miR-let7-b (MIMAT 0000063). Also, we used hsa-miR30a-5p (MIMAT0000087) and hsa-miR100-5p (MIMAT 0000102) as internal controls.
Subsequently, real-time quantification was performed using Rotor gene-Q real-time PCR system (Qiagen). Each real-time PCR (10 μL) included 1 μL of reverse and forward primers (Exiqon), 5 μL of Ampliqon real Q plus 2× master mix green (Ampliqon), and 4 μL of diluted cDNA. The reactions were incubated in a 72-well optical strip at 95°C for 15 min (enzyme activation), followed by 95°C for 20 s and 60°C for 60 s (40 cycles). All reactions were run in triplicate. After the reactions, the mean Ct was determined from the triplicate PCRs. We used Ct values to evaluate the expression levels of the three miRNAs. Note that hsa-miR30a-5p and hsa-miR100-5p are the endogenous control genes to normalize RNA contents among different samples. The expression value of miR-NAs relative to internal controls was determined using the 2 −ΔCt method.

| Determination of vitamin B12 and folate in serum
Blood samples were collected from fasting ARDS individuals (since recent food intake may increase the folic acid level appreciably) in 5-or 10-mL evacuated glass tubes. We allowed the blood to clot at room temperature, and the serum was collected by centrifugation. Vitamin B12 and folate were measured in serum samples of patients simultaneously using a vitamin B12/folate RIA kit (IBL). In this kit, B12 vitamin <120 pg/mL is low, 120-160 pg/mL is intermediate, 160-970 pg/mL is normal, and >970 pg/mL is high. Regarding the folate, the normal range is >1.5 ng/mL.

| Statically analysis
The results of this research were analyzed by Graph Pad software (GraphPad PRISM V 5.04 analytical software). The difference in the expression and methylation levels of ACE-2 among the studied samples was calculated with ANOVA one-way test. The association between different genotypes of the mentioned polymorphisms and disease was assessed by computing the odds ratio and 95% confidence intervals (95% CI) from logistic regression analyses. We also used the Hardy-Weinberg equilibrium test for polymorphism frequency evaluation. For further investigation, the correlation between the prevalence of ACE-2 expression and methylation was analyzed by Spearman's rank correlation. All p-values were two-tailed, with p < 0.05 considered statistically significant. 3.1.1 | ACE-2 expression and methylation level in patients and controls ACE-2 expression level in mild COVID-19 patients was 1.32 ± 0.88 as compared to controls (0.88 ± 0.12), and there was no significant difference (p = 0. 12; Figure 1); although the expression had been increased, this increase was not significant. In ARDS patients, the ACE-2 levels were significantly increased (3.8 ± 0.77) compared with controls (0.88 ± 0.12; p < 0.00001; Figure 1). Also, there was a significant difference in ACE-2 expression ratio between mild and ARDS patients (p < 0.001). We ex-  Table 1).

| Expression of miRNAs
The expression of the three studied miRNAs was estimated by qRT-PCR. The expression of hsa-miR-200c-3p in the mild COVID-19 group

F I G U R E 1
The ACE-2 expression ratio comparison among the three studied groups, as shown there was a significant over-expression in ARDS patients in comparison with controls (p < 0.00001) and mild COVID-19 patients (p < 0.001).

F I G U R E 2
Methylation rate of the ACE-2 gene between the controls and patients. As it can be seen in the graph, there was a significant hypomethylation rate in ARDS patients (p < 0.0001).

TA B L E 1
Genotypes frequency of the ACE-2 gene polymorphisms among the studied groups.

| Evaluation of polymorphisms frequency, miRNA expression, and ACE-2 hypo-methylation rate between two ARDS groups
In another section of the study, we divided ARDS patients into two groups: patients who used only oxygen masks (n = 45) and patients who had more severe symptoms and were connected to ventilators and eventually died (n = 25). The results of ARDS patients' ACE-2 expression showed that in severe samples, expression ratio was 4.93 ± 2 ± 0.2 and in nonsevere samples was 2.13 ± 0.98; this difference was significant (p < 0.001; Figure 4A).
Then, the three factors that effected ACE-2 gene expression (polymorphism, miRNA expression, and methylation) in these two ARDS groups were evaluated. The frequency of different genotypes of rs182366225 and rs182366225 polymorphisms between two ARDS patient groups showed no significant difference (p > 0.05).

| B12 and folate concentration
The interesting point about this research was that a high vitamin concentration between the two ARDS groups (p < 0.001).
Folate concentration in severe ARDS patients was 2 ± 0.99 and in nonsevere was 8.8 ± 0.99, and this difference was significant (p < 0.01; Table 2). There was a significant positive correlation between hypo methylation rate and B12, folate deficiency (R = 0.32, p < 0.001 and R = 0.37, p < 0.000, respectively; Table 3).  with comorbidities. 13 Since studies have shown the role of increased expression of the ACE-2 gene in disease severity, it is essential to investigate the mechanism of increased expression for treatment.

| DISCUSS ION
In this research, frequency of some ACE-2 gene polymorphisms, its

F I G U R E 6
The folate and methionine cycles are interconnected and are required for many cellular processes, and the methionine from this cycle provides the methyl group needed for the methylation of various genes. As seen in this diagram, B9 and B12 are two important components of these cycles. A simplified schematic drawing of the folate and methionine cycles. Metabolites and enzymes: MAT, methionine adenosyl transferees; MT, methyl tansferase; MTHF, Methylene tetra hydro folate; SAH, S-adenosylhomocysteine; SAM, Sadenosyl methionine; THF, tetra hydro folate.
first time we showed, deficiency of B 9 and B 12 vitamins has an important role in hypo-methylation and over-expression of the ACE-2 gene and severity of symptoms of the COVID-19 disease. There has been no report regarding this finding, and it seems that taking B vitamins, which are involved in monocarbon metabolism, can play a vital role in reducing the symptoms of COVID-19. A few reports have shown that taking B vitamins can reduce the symptoms of COVID-19, 28 but the exact mechanism has not been discussed. In conclusion, the results of this study showed that among the various mechanisms involved in regulating the ACE-2 expression (such as polymorphism, miRNAs, and methylation), the reduction of methylation plays a critical role in the overexpression of this gene and the severity of symptoms; moreover, there was a significant and direct correlation between B 9 and B 12 deficiency with hypomethylation and overexpression ( Figure 6).

ACK N OWLED G M ENTS
We appreciate all of the participants in the current study. The funding source has no involvement in the study.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare no conflicts of interest for this work.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author [SM]. The data are not publicly available because they contain information that could compromise the privacy of research participants.