key: cord-0729196-nj115xpw
authors: Papadopoulou, Anna; Fragkou, Paraskevi C.; Maratou, Eirini; Dimopoulou, Dimitra; Kominakis, Antonis; Kokkinopoulou, Ioanna; Kroupis, Christos; Nikolaidou, Athina; Antonakos, Georgios; Papaevangelou, Vasiliki; Armaganidis, Apostolos; Tsantes, Argirios; Polyzogopoulou, Eftychia; Tsiodras, Sotirios; Antoniadou, Anastasia; Moutsatsou, Paraskevi
title: Angiotensin‐converting‐enzyme insertion/deletion polymorphism, ACE activity, and COVID‐19: A rather controversial hypothesis. A case‐control study
date: 2021-11-05
journal: J Med Virol
DOI: 10.1002/jmv.27417
sha: 70ea0d48bdf04f383c6e09b1cf307cbf49e53a6b
doc_id: 729196
cord_uid: nj115xpw

Accumulating data has shown a contribution of the renin‐angiotensin system in COVID‐19 pathogenesis. The role of angiotensin‐converting enzyme (ACE) insertion (I)/deletion (D) polymorphism as a risk factor in developing COVID‐19 disease comes from epidemiological data and is controversially discussed. We conducted a retrospective case‐control study and assessed the impact of ACE I/D genotype in COVID‐19 disease prevalence and severity. In 81 COVID‐19 patients explicitly characterized and 316 controls, recruited during the first wave of COVID‐19 pandemic, ACE I/D genotype, and ACE activity were determined. A generalized linear model was used and Poisson regression analysis estimated the risk ratios (RRs) of alleles and genotypes for disease severity. DD patients had almost 2.0‐fold increased risk (RR: 1.886, confidence limit [CL] 95%: 1.266–2.810, p = 0.0018) of developing a more severe disease when contrasted to ID and II individuals, as did D allele carriers compared to I carriers (RR: 1.372; CL 95%: 1.051–1.791; p = 0.0201). ACE activity (expressed as arbitrary units, AU/L) was lower in patients (3.62 ± 0.26) than in controls (4.65 ± 0.13) (p < 0.0001), and this reduction was observed mainly among DD patients compared to DD controls (3.97 ± 0.29 vs. 5.38 ± 0.21; p = 0.0014). Our results demonstrate that ACE DD genotype may predispose to COVID‐19 increased disease severity via a mechanism associated, at least in part, with the significant fall in their ACE activity. Our findings suggest a more complex pattern of synergy between this polymorphism and ACE activity in COVID‐19 patients compared to healthy individuals and set the grounds for large‐scale studies assessing ACE genotype‐based optimized therapies with ACE inhibitors and angiotensin receptor blockers.

The renin-angiotensin system (RAS) has been at the forefront in the quest of genetic factors involved in the pathogenesis of COVID-19. The "proposed" imbalance between the angiotensin-converting enzyme 2 (ACE2) which serves as an anchor for the pathogenic corona virus to the target cells, 1 and the ACE in COVID-19 patients, attempted to explain, at least in part, the progression of the disease. 2, 3 To this direction, several studies supported a potential benefit of the use of ACE inhibitors (ACEi) and angiotensin II receptor blockers (ARBs) in COVID-19 outcome, however, their efficacy as a treatment for COVID-19 remains to be seen. 4, 5 It has been hypothesized that ACE gene polymorphisms that affect ACE activity may be used as a host genetic factor for COVID-19 patients triage regarding the severity of the disease and their response to ACEi and ARBs treatment. 2 The ACE insertion (I)/deletion (D) polymorphism (rs1799752) is a 287-bp Alu sequence in intron 16 of the ACE gene which accounts for the majority of interindividual variation in ACE activity in circulation and shows an important geographic variation. 6 ACE activity in ACE II healthy individuals is half of that in ACE DD individuals. 6 In the early pandemic, an extensive debate has been launched regarding the effect of the ACE I/D alleles on the prevalence and the outcome of the COVID-19 infection. 7, 8 First, Delanghe et al. 8 In COVID-19 patients of variable disease severity, we performed a case-control study and determined the ACE I/D genotypes and ACE activity in samples collected during the first wave of the pandemic in Greece. A cohort of blood-donors and health workers non-COVID volunteers of Greek origin were recruited at the same period and served as controls. We correlated ACE activity and ACE I/D genotypes and allelic frequencies with clinical features, disease laboratory markers, and serum anti-SARS-CoV-2S.

We conducted a single-centre retrospective case-control study. The samples had been collected in a tertiary referral hospital for COVID-19, "Attikon" University General Hospital, Athens, Greece, between 15th March 2020 and 30th June 2020 during the early pandemic in Greece. Eligible COVID-19 cases were Caucasian adult patients of

Greek ancestry. No exclusion criteria regarding hospitalization requirement, the magnitude of symptoms, disease severity, and outcomes were applied. Samples from 316 sex-matched adult blood/ blood product donors and volunteer healthcare workers of Greek ancestry, with no history of COVID-19-related symptoms or relevant epidemiological history within the past month, were collected and served as controls.

This study was approved by the institutional Research Bioethics Committee and was conducted according to the STrengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelines. 9 Patients' data were collected and analyzed under strict anonymity in agreement with the Helsinki Declaration. Written informed consent had been obtained by all participants.

Clinical data of patients were collected by reviewing the medical files.

Routine blood tests including disease laboratory markers, namely, neutrophil to lymphocyte ratio (NLR), C-reactive protein (CRP), ferritin, d-dimers and interleukin-6 (IL-6) were retrieved by the institutional electronic system. 10 Patients were stratified as mild, moderate, severe, and critical COVID-19 cases according to the World Health Organization (WHO) definitions for COVID-19 severity classification (Table S1 ) upon completion of their hospitalization (until discharge or death) or recovery from COVID-19 (for patients with mild symptoms and outpatients). The maximum Sequential Organ Failure Assessment (SOFA) Score was calculated in hospitalized patients. 11 Comorbid conditions were summarized based on the Charlson Comorbidity Index (CCI). 12 In patients, ACE I/D genotype, serum ACE activity, and serum anti-SARS-CoV-2S were determined in leftover patient serial serum samples.

Investigation of the ACE I/D polymorphism in intron 16 of the ACE gene was performed by PCR in genomic DNA isolated from whole blood. A second internal PCR of the DD genotypes was performed to avoid mistyping of ID as DD genotype (details in Supporting Information Appendix).

Serum ACE activity was determined in a subgroup of patients (n = 52)

according to the manufacturer's instructions (Sentinel Diagnostics) and analyzed on ROCHE Cobas 801 analyzer (Roche Diagnostics).

The inter-and intra-assay coefficient variations were <2% and <2.6%, respectively. In a subgroup of patients (n = 10) ACE activity was measured in sequential samples (3-6 for each patient) collected at different time points of the disease course (details in Supporting Information Appendix).

Serum samples from patients taken on or after the 10th day of the disease course were analyzed for anti-SARS-CoV-2S antibody titer using Elecsys Anti-SARS-CoV-2S assay in Cobas 801 analyzer (Roche Diagnostics). The assay sensitivity and specificity were 98.8% and 100%, respectively. Values <0.8 U/ml are considered negative for anti-SARS-CoV-2S (details in Supporting Information Appendix).

We estimated overall (pooled data) and per group (controls and patients) allelic and genotypic frequencies and tested for significance for differences of allelic and genotypic frequencies between groups using exact G-tests. A generalized linear model 3 | RESULTS Since laboratory data were available for only two mild cases, mild patients were not included in our analysis.

Results of Poisson regression analysis are shown in Table 3 and in As shown in Table 4 , the frequency of the D allele was higher (Table S2 ).

Serum ACE activity (expressed as arbitrary units, AU/L) was lower in patients than in controls (3.62 ± 0.26 vs. 4.65 ± 0.13, p < 0.001) (Figure 2 top, and Table S3 ). ACE activity in the pooled population was highest in 

Regarding other disease laboratory parameters, DD patients displayed the highest levels of inflammatory markers (NLR (7.97 ± 8.08), ferritin (907 ± 1027.0), and IL-6 (55.2 ± 77.1) ( The initial hypothesis, based on epidemiological data, was that ACE DD genotype may be associated with a better COVID-19

prognosis. 8 However, the accumulation of data from additional epidemiological studies, showed that the ACE D allele was involved in the susceptibility to SARS-CoV-2 infection and the exacerbation of symptoms such as pneumonia. 7 Gomez et al., 13 working on casecontrol samples, showed that ACE DD genotype was more frequent in severely affected COVID-19 patients compared to mild patients, Furthermore, no differences in ACE activity were seen between the different disease severity cases, while sequential measurements of ACE activity (within each patient), showed no intra-individual variability and remained consistently low throughout the disease course.

In line with our findings, Zhu et al. 19 found lower ACE activity in COVID-19 patients which, however, increased after recovery. Low ACE activity has also been detected in other lung disorders such as ARDS and has been correlated with the severity of lung injury and hypoxemia. 20, 21 We hypothesize that the decreased ACE activity in our COVID-19 patients could be due to their hypoxemic respiratory failure or to disease induced-molecules (proteolytic enzymes and natural ACE inhibitors). 19 It should be noted that the reduced ACE activity in our patient group was observed mainly among DD subjects. In an attempt to explain this finding, we refer to the modulation of ACE amino (N-) and carboxy (C-) terminal domain activities by the ACE I/D polymorphism.

At the protein level, the ACE enzyme presents two functional active sites that belong to two independent catalytic domains (N-and C-) which share 60% amino acid homology and differ in substrate and inhibitor specificities and activity. 22 It has been reported that in DD individuals only the C-domain converts Ang I to AngII 22 while the N-activity of DD subjects is higher than that of ID (133%) and even higher (228%) than that of II. 23 

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Angiotensin-converting-enzyme insertion/ deletion polymorphism, ACE activity, and COVID-19: A rather controversial hypothesis. A case-control study

The authors would like to thank Roche Diagnostics for kindly donating laboratory testing kits and consumables and for secretarial support.

The authors declare that there are no conflict of interests. 

The data that support the findings of this study are available on request from the corresponding author.

http://orcid.org/0000-0002-0463-4321