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.

Keywords: ACE-2; B vitamins; COVID-19; expression regulation; hypomethylation.

FIGURE 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).

FIGURE 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).

FIGURE 3

(A) miR‐200c expression rate in the studied samples, as seen miR‐200c significantly downregulated in ADRS patients (p < 0.0001). (B) Let‐7b showed no significant difference among the different studied samples (p > 0.05). (C) Also there was no a significant difference in the miR‐1246 expression ratio between patients and controls (p > 0.05).

FIGURE 4

(A) A comparison of ACE‐2 expression rate between severe and nonsevere ARDS patients, as seen ACE‐2 significantly upregulated in severe ARDS patients (p < 0.001). (B) Significant downregulation of miR‐200c in severe ARDS patients (p < 0.0001). (C) Significant hypomethylation of ACE‐2 gene among the severe ARDS patients (p < 0.001).

FIGURE 5

The frequency of different mechanisms involved in ACE‐2 expression. As seen in the figure, the highest percent of ARDS patients showed hypomethylation of the ACE‐2 gene.

FIGURE 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, S‐adenosyl methionine; THF, tetra hydro folate.