Abstract
Background/Aim: Renin-angiotensin system (RAS) is present in a diverse type of cells and plays an important role in lung physiology and pathophysiology. Angiotensin converting enzymes (ACE) are part of the RAS system. There are still controversies about the association of I/D polymorphisms of ACE1 with COVID-19 severity. The goal of the study was to determine whether there is an association of the I/D polymorphism with severity of COVID-19 in Mexican patients. Patients and Methods: The study included voluntary participants: 53 healthy individuals negative to RT-PCR COVID-19 (control), and 165 patients positive to COVID-19. Severity was defined by the need of hospitalization, invasive ventilation, shock, or multiple organ failure. The patient group consisted of 28 asymptomatic, 82 with mild, and 55 with severe COVID-19. I/D polymorphism was determined by PCR. Rutinary laboratory tests were performed in all the participants. Results: DD polymorphism was significantly associated with severe COVID-19, independently of comorbidities, or any other variable. Receiver operator characteristic curves demonstrated association of low total cholesterol, low high-density lipoproteins, and high c-reactive protein with severity of COVID-19. Conclusion: The DD polymorphism was associated with the course of the infection and severity of COVID-19 in a sample of Mexican patients.
The renin-angiotensin system (RAS) is present regulates diverse cellular processes. Angiotensin converting enzymes (ACE), are part of RAS. ACE1 has two main functions: production of angiotensin II and degradation of bradykinin, whereas the main functions of ACE2 are degradation of angiotensin II and production of ang1-7. ACE1 and ACE2 have opposite functions (1). The ACE1 gene maps on chromosome 17, locus 17q23 (1). The main polymorphisms of ACE1 are insertion (I) or deletion (D) of a 287-base pair segment, which produces three polymorphisms, DD, ID, and II (2). ACE activity and ACE levels in plasma depend on the D polymorphism (1). DD is related to higher concentrations of angiotensin II in plasma and has been associated with hypertension, cerebrovascular disease, myocardial infarction, and increased mortality in people younger than 65 years (1, 3). The D allele is associated with diabetic nephropathy and left ventricular hypertension, whereas the I allele is associated with high performance exercise (1). The ID polymorphism has been associated with lung acute and chronic diseases such as chronic obstructive pulmonary disease (COPD), pulmonary hypertension, asthma, lung cancer, and pulmonary sarcoidosis (3).
It is known that ACE2 is the receptor of SARS-COVID-19 (4). Therefore, regulation of ACE1 and ACE2 has been extensively explored in COVID-19. The association of DD polymorphism with severity of COVID-19 seems to depend on ethnicity, sampling bias, biological factors, study design, data analysis, and time of sampling (wave of COVID-19) (2).
Prevalence of the D allele has been reported to be 39.1% in Asians and 56.2% in Caucasians, whereas distribution of the DD polymorphism is 17.9% and 32.5%, respectively (5). In another study, the D allele was reported to be 89% in African Americans, 69% in American Indian/Caucasian, and 87% in European Caucasian populations (6). The prevalence of DD polymorphism in the Mexican population is 40% (7).
There are epidemiological studies providing indirect evidence of the relationship between I/D polymorphisms and severity of COVID-19. In these studies, the authors report prevalence of I/D polymorphisms before the pandemic and associate such prevalence with severity of COVID-19. In one of these studies, an analysis of 25 countries representing different parts or the world was performed and showed association of the prevalence of II polymorphism with decreased COVID-19 mortality, with no association of the DD polymorphism with mortality (8). Another study showed that the prevalence of the D allele was associated with COVID-19 infection and mortality in the Asian population (9). A review based on the DNA bank of a Czech population concluded that the II polymorphism could be associated with symptomatic COVID-19 (10).
The prevalence of I/D polymorphism and the severity of COVID-19 in patients is controversial. A study performed in the Spanish population showed that there was no difference in the prevalence of the DD polymorphism between controls and COVID-19 patients (5). The authors concluded that DD was not associated with the risk of developing COVID-19 symptoms. However, the study did not include asymptomatic patients (5). Another study performed in the Iranian population showed association of the DD polymorphism with COVID-19 severity in patients without comorbidities (11). In a Saudi Arabian cohort, the prevalence of DD was higher in patients with COVID-19 compared with controls and associated with severity and mortality (12). Two studies performed in the Turkish population showed controversial results. One of them associated DD polymorphism with higher levels of c-reactive protein (CRP), fibrinogen and ferritin, and D allele with mortality of COVID-19 patients (2). The other study showed that the DD polymorphism was more frequent in asymptomatic compared with patients with severe COVID-19 (13). A review including 786 patients concluded that DD polymorphism was a risk factor for the development of severe COVID-19 in Caucasian hospitalized patients (14).
A recent review (15) highlights the importance of the genetic factor, including ACE1 polymorphisms, on clinical evolution, transmission, virus mutation and responses to vaccination of COVID-19.
COVID-19 severity has been determined in different ways, one of them includes the need of assisted ventilation, a pO2 <90 mm-hg, presence of shock or organ failure (13). The aim of the present study was to examine whether there is an association between ACE1 polymorphisms and the severity of COVID-19 in Mexican patients.
Patients and Methods
Study population. The study was approved by the ethical committee of the Central Military Hospital (CMH) and performed according to the Helsinki declaration and Title 45, US Code of Federal Regulations, part 46, Protection of human rights. All the volunteers or their legal representatives voluntarily signed the informed consent. Demographic characteristics, comorbidities, and clinical laboratory results were obtained from all the volunteers.
Patients and controls (age ≥18 years) were recruited at the CMH and the Institute of Health of the State of Mexico -Tlalnepantla (HIS-T) from August 2020 to February 2021. Severe COVID-19 was evidenced by the need of invasive mechanical ventilation, pO2<90 mmHg, shock or multiorgan failure (n=55). COVID-19 was classified as mild when patients were symptomatic and had oxygen saturation ≥94% and there was no need for hospitalization (n=82). Asymptomatic patients had no symptoms, and their oxygen saturation was ≥94% (n=28). All COVID-19 patients had a positive RT-PCR test. Controls had negative test and did not have any symptom or morbidity (n=53). Body mass index (BMI), leukocytes, lymphocytes, c-reactive protein (CRP), eosinophils, neutrophils, platelets, glucose, urea, creatinine, total cholesterol (TC), high density lipoproteins (HDL), triglycerides (TGD), and the ratio TGD/HDL were obtained from all the participants.
For COVID-19 detection, nasopharyngeal swab samples were kept in viral transport medium according to the technique reported by Juarez-Castelan et al. (16). RNA extraction was performed in a Magna Pure 96 instrument (Roche Diagnostic Corporation, Indianapolis, IN, USA) employing the RNA pure LC lysis kit/RNA isolation kit-high performance (Roche Diagnostic Corporation). COVID-19 RNA was detected through RT-PCR using the one-Step Real time RT-PCR kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturers’ instructions.
Samples, DNA extraction and ACE genotyping. Venous blood samples were obtained from the antecubital vein in EDTA tubes. Total genomic DNA was isolated using the commercial kit DNeasy®Blood (Qiagen, Redwood City, CA, USA). The quantity and quality of the purified DNA were evaluated using the spectrophotometer NanoDrop One® (Thermo Fisher Scientific). The genomic DNA samples were stored at −20°C until genotyping. PCR was used to investigate the insertion/deletion (I/D) rs4646994 polymorphism of ACE. The forward primer was 5′-CTG GAG ACC ACT CCC ATC CTT TCT-3, and the reverse primer was 5′-GAT GTG GCC ATC ACA TTC GTC AGAT-3′. A Veriti 96-well thermal cycler (Applied Biosystems LLC Company, Bedford, MA, USA) was used for amplification. The amplification included incubation at 95°C for 1 min, 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 40 s, first extension at 72°C for 90 s, and final extension at 72°C for 50 s. PCR products were separated on 2.5% agarose gel stained with Red Gel (Biotium Inc. Freemont, CA, USA) for 50 min and visualized in a ChemiDoc MP imaging system (Bio-Rad Laboratories, Hercules CA, USA).
Statistical analysis. Prism 9 software (GraphPad Software, San Diego, CA, USA) was used for analysis. Normality of data was analyzed with the Kolmogorov-Smirnov test. Data are presented as mean standard error of the mean (SEM). Categorical variables are presented as numbers or percentages. Demographic and laboratory test results were compared using one way ANOVA or Kruskal-Wallis tests (K-W) with post hoc Tukey (T) or Dunnett’s multiple comparison (DMC), respectively) depending on the distribution. Categorical variables were analyzed with the chi-square test (χ2).
Receiver operator characteristic (ROC) curves were built to associate laboratory variables with severity. Spearman r was used to evaluate correlation of ACE1 polymorphisms with all the other variables. Differences were considered significant at p<0.05.
Results
The control group (n=53) consisted of military personnel who agreed to participate in the study. They were healthy and negative to RT-PCR COVID-19 test.
Patients with COVID-19 were consecutively recruited at the HIS-T: 28 asymptomatic, 82 with mild and 55 with severe COVID-19. Demographic and laboratory results are shown in Table I. Comorbidities and polymorphism distribution are shown in Table II. An example of the results of the genotypic analysis of the polymorphisms is shown in Figure 1. Sex distribution was not significantly different between groups (χ2). Patients with mild and severe COVID-19 were older than control and asymptomatic groups (p<0.0001, One-way ANOVA, T post hoc). Even though there were no differences in BMI between asymptomatic and control groups; obesity, hypertension, and diabetes were equally distributed in patients with COVID-19 (χ2).
Demographic characteristics and laboratory results.
Comorbidities and distribution of ACE polymorphisms.
Genotypes of ACE1 I/D (II: 481 bp, ID: 481,194 bp, DD: 194 bp). M: DNA ladder (100 bp). Electrophoretic conditions: agarose gel 2.5%, TAE 1X, 70 V, 50 min. Data were obtained at Institute of Health of the State of Mexico-Tlalnepantla.
Differences in hemoglobin (p<0.001 compared with all the other groups, KW-DMC), erythrocytes (p<0.001 compared with control, KW-DMC), and eosinophils compared with all the other groups (p<0.0001, KW-DMC) were seen in the asymptomatic group, who had a higher level. Glycemia (One Way ANOVA-T), leukocytes (KW-DMC), and HDL (KW-DMC) differences were seen in patients with mild and severe COVID-19, who had higher concentrations of glycemia and leukocytes, and lower levels of HDL compared with control and asymptomatic patients (p<0.0001). There was no difference in leukocytes and HDL between mild and severe COVID-19 patients. Urea and CRP were higher in patients with COVID-19 compared with the control (p<0.01, KW-DMC). Even though urea in asymptomatic patients was higher than that in control patients, the concentration was within normal standards. There was no difference in glucose and urea between mild and severe groups. CRP was lower in asymptomatic compared with mild and severe COVID-19 patients, but all COVID-19 patients had higher levels compared with control group. Lymphocytes were higher in COVID-19 patients compared with controls (KW-DMC, p<0.000). Neutrophils were higher in mild and severe COVID-19 patients compared with control and asymptomatic groups (p<0.0001, KW-DMC). Asymptomatic patients had lower counts of neutrophils compared to the other three groups (p<0.001). HDL differences were observed in mild and severe groups who had lower levels than asymptomatic patients and controls (p<0.0001). Differences in the TGD/HDL ratio were observed in both the mild and severe groups who had the higher ratios (p<0.0001, KW-DMC). ROC curves (Figure 2) significantly associated severity with HDL, total cholesterol, and CRP.
The figure shows receiver operator characteristic (ROC) curves of the associations with severity. ROC curve for high density lipoproteins (A): area=0.66, sensitivity=59.4%, specificity=60%, for a value of <31.1 mg/dl (p=0.01). ROC curve for total cholesterol (B): area 0.66, sensitivity=59.39%, specificity of 60.0% for a value of <145 mg/dl (p=0.002). ROC curve for c-reactive protein (C): area 0.62, sensitivity 63.5%, specificity of 56.0%, for a value >40.8 mg/l (p=0.01).
There was no difference (χ2) in the distribution of DD, ID, and II polymorphisms between control (26, 36 and 38%, respectively), asymptomatic (14, 39, and 46%, respectively) and mild COVID-19 patients (37, 41, and 44%, respectively). Whereas the percentage of DD was significantly higher in the severe COVID-19 group (59%), which had the same percentage of ID and II polymorphisms (20%) (Table II).
No correlation was found between ACE1 polymorphisms and any of the other variables.
Discussion
The main finding of the present study was that Mexican patients with severe COVID-19 had a higher proportion of DD polymorphism compared with control, asymptomatic, and mild COVID-19 groups. Our results are in accordance with previous studies (2, 11, 12). The limitation in these studies and our study is the small number of patients. However, the significant higher percentage of DD polymorphism in the sample of Mexican patients with severe COVID-19 evidences the association. Our study is also in accordance with reports that highlight the role of the genetic factor with the evolution of COVID-19 (14, 15). To the best of our knowledge, this is the first report regarding the I/D polymorphism in Mexican patients with COVID-19 severity. Martínez Gomez et al. (17) found that the ACE2 polymorphism rs2285666 was a risk factor in men regardless of morbidity. In the present study, comorbidities and sex were similar in COVID-19 patients, independently of their clinical evolution.
Further follow up studies should be performed to analyze whether the DD polymorphism is related to long-term lung complications of COVID-19.
Another limitation of our study is that it was transversal. There were no medical records of the patients prior to acquisition of COVID-19, thus it is difficult to interpret differences in laboratory results. Even though the main goal of the study was to analyze ACE1 polymorphisms, the study showed that mild and severe COVID-19 patients had the lowest concentrations of TC and HDL. Low HDL concentrations in COVID-19 have been reported in patients from different countries including Mexico (18, 19). It is postulated that the fall in HDL is part of the inflammation and cytokine storm experienced in severe COVID-19 (20). Moreover, it has been proposed that low HDL is a risk and prognostic factor in COVID-19 (18). Hu et al. (21) found a significant association (ROC curve) between HDL and severity in patients with COVID-19. In the present study, ROC curves showed significant association of HDL, TC, and CRP with COVID-19 severity. Alcantara Alonso et al. (22) found association of the ratio TGD/HDL with severity in Mexican patients with COVID-19. The present study did not show such association. Changes in lipid profiles and their association with severity have been postulated to be a good way for evaluating COVID-19 patients in middle-low-income countries, since quantification of lipids is affordable (23). In the present study, the DD polymorphism was not associated with any of the other variables.
In conclusion, the DD polymorphism was associated with severity in the sample of Mexican patients with COVID-19 with no correlation in respect to sex, age, morbidity, or clinical laboratory results. Low HDL, high TC, and high CRP were associated with severity of COVID-19, independently of the I/D polymorphism.
Acknowledgements
The study was financed by Grants CONACYT-611866, ERM received a scholarship from CONACYT 311866 to do her Ph.D., and the study was part of her thesis.
Footnotes
Authors’ Contributions
PBZ, FBG, CV: Design, analysis of results, discussion, conclusion. ERM: Introduction, polymorphism detection, analysis of results, discussion, conclusion. MJPM, EDG: Polymorphism detection, analysis of results.
AGG, RSS, CMJJ, SCH, LMPF: Treatment and selection of patients, discussion, conclusion. All the Authors contributed to the writing and reviewed the manuscript.
Conflicts of Interest
The Authors declare that there are no conflicts of interest in relation to this study.
- Received November 10, 2022.
- Revision received November 26, 2022.
- Accepted November 28, 2022.
- Copyright © 2023 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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