key: cord-0793887-5zershyl
authors: Gómez, Juan; Albaiceta, Guillermo M; García-Clemente, Marta; López-Larrea, Carlos; Amado-Rodríguez, Laura; Lopez-Alonso, Inés; Hermida, Tamara; Enriquez, Ana I.; Herrero, Pablo; Melón, Santiago; Alvarez-Argüelles, Marta E.; Boga, José A.; Rojo-Alba, Susana; Cuesta-Llavona, Elías; Alvarez, Victoria; Lorca, Rebeca; Coto, Eliecer
title: Angiotensin-converting enzymes (ACE, ACE2) gene variants and COVID-19 outcome
date: 2020-08-31
journal: Gene
DOI: 10.1016/j.gene.2020.145102
sha: a9538d1fc0ae4ac3da5e39c77535020bf144e352
doc_id: 793887
cord_uid: 5zershyl

The Angiotensin system is implicated in the pathogenesis of COVID-19. First, ACE2 is the cellular receptor for SARS-CoV-2, and expression of the ACE2 gene could regulate the individuaĺs susceptibility to infection. In addition, the balance between ACE1 and ACE activity has been implicated in the pathogenesis of respiratory diseases and could play a role in the severity of COVID-19. Functional ACE1/ACE2 gene polymorphisms have been associated with the risk of cardiovascular and pulmonary diseases, and could thus also contribute to the outcome of COVID-19. We studied 204 COVID-19 patients (137 non-severe and 67 severe-ICU cases) and 536 age-matched controls. The ACE1 insertion/deletion and ACE2 rs2285666 polymorphism were determined. Variables frequencies were compared between the groups by logistic regression. We also sequenced the ACE2 coding nucleotides in a group of patients. Severe COVID-19 was associated with hypertension male gender (p<0.001), hypertension (p=0.006), hypercholesterolaemia (p=0.046), and the ACE1-DD genotype (p=0.049). In the multiple logistic regression hypertension (p=0.02, OR=2.26, 95%CI=1.12-4.63) and male gender (p=0.002; OR=3.15, 95%CI=1.56-6.66) remained as independent significant predictors of severity. The ACE2 polymorphism was not associated with the disease outcome. The ACE2 sequencing showed no coding sequence variants that could explain an increased risk of developing COVID-19. In conclusion, an adverse outcome of COVID-19 was associated with male gender, hypertension, hypercholesterolemia and the ACE1 genotype. Our work suggested that the ACE1-I/D might influence COVID-19 severity, but the effect was dependent on the hypertensive status. This result requires further validation in other large cohorts.

The Angiotensin system is implicated in the pathogenesis of COVID-19. First, ACE2 is the cellular receptor for SARS-CoV-2, and expression of the ACE2 gene could regulate the individual´s susceptibility to infection. In addition, the balance between ACE1 and ACE activity has been implicated in the pathogenesis of respiratory diseases and could play a role in the severity of COVID-19. Functional ACE1/ACE2 gene polymorphisms have been associated with the risk of cardiovascular and pulmonary diseases, and could thus also contribute to the outcome of COVID-19.

We studied 204 COVID-19 patients (137 non-severe and 67 severe-ICU cases) and 536 age-matched controls. The ACE1 insertion/deletion and ACE2 rs2285666 polymorphism were determined. Variables frequencies were compared between the groups by logistic regression. We also sequenced the ACE2 coding nucleotides in a group of patients.

Severe COVID-19 was associated with hypertension male gender (p<0.001), hypertension (p=0.006), hypercholesterolaemia (p=0.046), and the ACE1-DD genotype (p=0.049). In the multiple logistic regression hypertension (p=0.02, OR=2.26, 95%CI=1.12-4.63) and male gender (p=0.002; OR=3.15, 95%CI=1.56-6.66) remained as independent significant predictors of severity. The ACE2 polymorphism was not associated with the disease outcome. The ACE2 sequencing showed no coding sequence variants that could explain an increased risk of developing COVID-19.

In conclusion, an adverse outcome of COVID-19 was associated with male gender, hypertension, hypercholesterolemia and the ACE1 genotype. Our work suggested that the ACE1-I/D might influence COVID-19 severity, but the effect was dependent on the hypertensive status. This result requires further validation in other large cohorts.

Keywords: COVID-19; angiotensin converting enzyme; gene polymorphism; genetic association.

The SARS-CoV-2 responsible for the COVID-19 pandemic is a new coronavirus of the subgenus Sarbecovirus (Gorbalenya et al., 2020) . Like SARS-CoV, the SARS-CoV-2 is an Angiotensin I converting enzyme 2 (ACE2)-tropic virus, and the "spike" (S) protein of the viral envelope would thus bind to the nasopharyngeal mucosa and alveolar pneumocytes that express ACE2 at their surface (Yan et al., 2020; Shang et al., 2020; Hoffman et al., 2020) . The clinical spectrum of this disease, termed COVID-19, ranges from mild to very severe cases (Fu et al., 2020; Rivieccio et al., 2020) . It has been hypothesized that viral infection drives an exacerbated inflammatory response, leading to severe lung injury that may require ICU admission, mechanical ventilation and increases the risk of multi-organ failure and death (Jose et al., 2020) .

The Renin-Angiotensin-Aldosterone system (RAAS) seems to play an important role in the pathogenesis of COVID-19 (Ingraham et al., 2020) . The angiotensin-converting enzyme (ACE or ACE1) catalyzes the synthesis of Angiotensin-II (Ang-II) from Ang-I, and ACE2 hydrolyzes Ang-II into Ang-1-7. Ang-II binds to the AT1-receptor driving vasoconstriction, fibrosis, inflammation, thrombosis, among other responses; while Ang-1-7 binds to the AT2-receptor with increased vasodilation and reduced fibrosis, inflammation, and thrombosis. The ACE and ACE2 are thus seen as opposite players in the balance that determines the risk of developing hypertension and cardiovascular disease. In the lung, ACE2 drives a protective response by reducing oedema, permeability Both, acquired and inherited factors associated with differences in the expression and function of the RAAS components could explain the risk of developing COVID-19 and its adverse events. For instance, ACE2 expression in the lungs markedly decreases with age and is greater in men than in women (Xie et al., 2006) . This could explain the higher risk for adverse outcomes in elderly and male. In general, conditions related with a reduced ACE2 expression would increase the risk for hypertension, cardiac hypertrophy, and heart failure (Patel et al., 2012; Luo et al., 2019) . In opposition, a high activity of ACE would increase the risk of lung and cardiovascular disease by increased activity of the Ang-II/AT1R axis. Common variants in the two ACE genes have been associated with the risk of hypertension, heart disease, renal failure, and pulmonary disease. In fact, the ACE insertion/deletion (I/D) is one of the best characterised human polymorphisms.

Individuals with a D/D genotype showed the highest blood ACE levels, and this increased expression would explain the higher risk for cardiovascular and respiratory disease among individuals who are deletion-homozygous. This polymorphism has been related with the outcome in acute respiratory distress syndrome (ARDS) by some authors, and also with the progression of pneumonia in SARS (Marshall et Our current knowledge supports a role for the ACE/ACE2 imbalance in the pathogenesis of COVID-19. In this context, variants at these genes associated with differences in gene expression and protein function might explain the individual´s predisposition to manifest the disease symptoms and the risk for hospitalization and adverse events. Moreover, some authors have hypothesised that regional differences in allele frequencies could explain the different rate of the incidence and mortality (Yamamoto et al., 2020; Devaux et al., 2020; Delanghe et al., 2020; Cao et al., 2020; Hatami et al., 2020) .

Our purpose was to determine whether two common functional ACE and ACE2 variants were associated with susceptibility and outcome in COVID-19.

Study cohorts.We collected the anthropometric and clinical data of 204 patients who required hospitalization due to COVID-19 (mean age 64.77 years, range 24-95). All the study participants were Caucasian from the region of Asturias (Northern Spain, total population 1 million), and positive for SARS-Cov-2 (PCR test from nasal swabs or tracheobronchial aspirates). Severe cases (n=53) were defined as those in need of critical care support, including high-flow oxygen, positive-pressure ventilation (either invasive or non-invasive) or vasoactive drugs. We also studied 536 healthy population controls matched with the patients for age (n=536; mean age 70.01 years, range 50-81). The presence of comorbidities (hypertension, diabetes, hypercholesterolaemia) was obtained from the participants medical records. The study was approved by the Ethics Committee . table   1 ). These fragments were sequenced with Sanger BigDye chemistry in a capillary ABI3130xl equipment, and the sequences for each patient compared with the ACE2 reference sequence (www.ensembl.org).

All the patients and controls data were collected in an excel file and following the requirements of the Ethical Committee. The statistical analysis was performed with the R-project free software (www.r-project.org). The logistic regression (linear generalized model, LGM) was used to compare mean values and frequencies between the groups.

Compared to age-matched controls, patients with COVID-19 did not differ in the frequency of diabetes, hypertension, and the ACE-DD genotype (table 1). We found a non-significantly lower frequency of hypercholesterolemia in the patients. We compared these variables in severe COVID-19 (patients who required mechanical ventilation and/or ICU supportive care) and mild-disease patients. Male sex, hypertension, hypercholesterolemia, and the ACE-DD genotype frequencies were significantly higher in the severe group (table 1). The multiple logistic regression-LGM showed that hypertension (p=0.02; OR=2.26, 95%CI=1.12-4.63) and male gender (p=0.002;

OR=3.15, 95%CI=1.56-6.66) remained as independent significant predictors of severity.

We then compared the values in men and women. In men, hypertension, hypercholesterolemia and the ACE-DD were significantly increased in severe patients (table 2). The ACE2 rs2285666 alleles did not differ between the two patients groups and were non-significantly higher to the control frequencies. The same analysis performed among female patients revealed no differences between the severe and mild cases, although we observed a trend toward a higher risk for severity among hypertension and hypercholesterolaemia, and a non-significantly higher frequency of the A-allele in the two patients groups compared to controls (suppl. table 2). The results in this female cohort would be limited by the reduced size of the severe cohort (n=14).

To better understand the relationship between the two ACE polymorphisms and hypertension, we compared the genotype frequencies between hypertensives and normotensives in male and female patients and controls (suppl. table 3). In males, the ACE-DD genotype had a higher frequency among hypertensives in the three groups, without statistically significant differences (figure 1). The ACE2-A allele was significantly increased in the hypertensive controls (21% vs. 10%; p=0.02), with no significant differences between severe and non-severe COVID-19 cases. In the female cohort, the DD frequencies did not differ between hypertensive and normotensive in the three groups. The rs2285666 A allele was significantly more frequent among the hypertensive controls (p=0.04). These results suggested that the ACE-DD genotype and ACE2-A carriers could be at a higher risk for hypertension in our population, and genotype and allele differences between the severe and non-severe COVID-19 cases could be attribute to their association with hypertension.

We sequenced the ACE2 coding and immediate intronic flanking regions in a group of patients, and the only variant identified was rs2285666. This suggested a very low genetic variability in the SARS-CoV-2 receptor, and a significant absence of common variants that could increase the risk for infection in our population.

Since the outbreak of the COVID-19 pandemic several authors have speculated about the role of the ACE and ACE2 gene polymorphisms in disease susceptibility and severity.

Because ACE2 is the SARS-CoV-2 receptor, a functional variant that increased gene expression could be associated with a higher number of membrane-bound viral binding sites, increasing the vulnerability of carriers to infection. These risk variants might be particularly adverse in males, who carry only one copy of X-linked ACE2 gene. The ACE2 rs2285666 SNP was in the intronic-consensus splicing nucleotides and could thus affect the processing of ACE2 total RNA to mRNA and, eventually, the amount of the protein. At least one study has reported higher levels of circulating ACE2 levels in men than in women (Sama et al., 2020) . One study has investigated the effect of rs2285666 on serum ACE2 levels, and found significantly higher levels in A-carriers compared to G-homozygotes (Wu et al., 2017) . A different intronic SNP in strong linkage disequilibrium with rs2285666 (rs879922) has been associated with ACE2 gene expression (Zhang et al., 2018) . We thus hypothesised that these functional ACE2 variants could modify the disease outcome. In our study, the A-allele frequency was nonsignificantly higher inpatients vs. controls but was associated with hypertension in both, male and female controls. Lower ACE2 levels should be harmful for patients with lung disease, and the frequency observed in COVID-19 patients could thus be the balance between the negative association with viral infection (lower expression in the airway epithelia) and a positive association with respiratory and cardiovascular disease (lower expression in lung and other organs).

The ACE2 A allele has frequencies of 0.15 and 0.19 in our elderly controls. According to the human gene variation databases, this frequency is lower that the reported among unselected Caucasians (0.20-0.25) including the Spanish population (0.24). This lower frequency could be characteristic of our population, but might also reflect a reduction of the lifespan for rs2285666 A carriers.

Functional variants in receptors for other viruses confer resistance to infection. One the best characterised is CCR5, the cellular receptor for HIV. A common variant that determines the absence of the receptor (CCR5-Δ32) confers a complete resistance to HIVinfection among homozygotes, while reduces the disease progression in heterozygotes.

Approximately 1% of the Caucasians are CCR5-Δ32 homozygotes and they have a nonsignificant reduction in lifespan (Alvarez et al., 1998) . On the contrary, ACE2 pathogenic variants are very rare at a population scale, and the complete absence of the receptor would be incompatible with life in humans. Moreover, according to the human genome variation databases, there are no common missense changes in the coding ACE2 sequence. At a minor allele frequency >0.01% only four missense changes have been reported (all with global frequencies <1%), and rs2285666 was the only variant that could affect splicing (suppl. table 4) . We sequenced the exon and intron-flanking sequence in 60 patients and rs2285666 was the only identified variant. Therefore, it is unlikely that the ACE2 coding variants have a significant effect on susceptibility to SARS-CoV-2 infection. Of course, this does not exclude that variants in other gene regions are related with gene expression and the amount of protein. SARS cases and 103 healthy controls who had been exposed to SARS-CoV and 50 controls without contact with SARS-patients (non-exposed) (Itoyama et al., 2004) . There were no significant differences for DD-frequency among the groups, suggesting that this polymorphism has no effect on the risk for SARS-CoV infection. However, the frequency of the D allele was significantly higher in hypoxemic than in the non-hypoxemic patients, and could thus contribute to the progression of pneumonia in SARS. In our study we did not find differences for DD-frequencies between COVID-19 and controls, thus confirming the lack of association with the risk of developing COVID-19 symptoms.

However, we did not study individuals exposed who remained asymptomatic and we could thus not exclude an effect in the resistance to viral infection. We found a significant higher risk for a severe form of COVID-19 in males. This association was not found among females, although the number of severe-disease women was very low (n=14). We confirmed that this genotype was associated with the risk for hypertension in our male controls, with a trend for association with hypertension in the patients. Thus, we concluded that the deleterious effect of the ACE polymorphism on COVID-19 outcome was likely due to its association with hypertension.

Our study has several limitations, mainly the reduced sample size of the patients and of female severe cases in particular. This limits the statistical interpretation of the significant and non-significant associations. Also, we did not study subjects exposed to the virus who did not show disease symptoms. These individuals would be resistant to SARS-CoV-2 infection and are crucial for the identification of gene variants associated with disease susceptibility.

In conclusion, our study suggested that the ACE-ID polymorphism was associated with the risk of developing severe COVID-19 depending on the hypertension status. The ACE2 rs2285666 variant was associated with hypertension in our elderly population, without significant difference between mild and severe COVID-19 patients.

Contributorship. All the authors contributed to this work by recruiting the patients and performing the genetic and statistical analysis. JG, GMA and EC wrote the ms. All the authors approved the submission of this ms.

Competing interests. None of the authors have competing interests related to this work. 

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All the authors contributed to this work by designing the study, recruiting the study cohorts including the anthropometric, clinical and analytical data, isolating the DNA, performing the genetic studies, and/or performing the statistical analysis. EC and JG wrote the ms, and this was revised and approved by all the authors

mgclemen@gmail.com Carlos López-Larrea, inmuno@hca.es Laura Amado, lar@crit-lab.org Inés López-Alonso

es Marta Elena Alvarez-Arguelles, martaealvarez@gmail.com Susana Rojo-Alba, ssnrj4@gmail.com Pablo Herrero, pabloherrero71@gmail.com Victoria Alvarez, victoria.alvarez@sespa.es Rebeca Lorca, lorcarebeca@gmail.com Elías-Cuesta-LLavona

Data curation; Formal analysis; Funding acquisition; Investigation

Authorship statements should be formatted with the names of authors first and CRediT role(s) following. Covid19: coronavirus disease 19 ACE: angiotensina converting enzyme RAAS: renin-angiotensin aldosterone system Ang: angiotensin RFLP: restriction fragment length polymorphism Highlights -The Angiotensin system has been implicated in the pathogenesis of COVID-19. -Functional ACE/ACE2 polymorphisms might contribute to the outcome of COVID-19. -Severe COVID-19 was associated with hypertension, male gender, and ACE-DD genotype

-ACE2 showed no coding variants that could explain an increased risk of COVID-19

Acknowledgements. This work was supported by a grant from the Spanish Plan Nacional de I+D+I Ministerio de Economía y Competitividad and the European FEDER, grant ISCIII-Red de Investigación Renal-REDINREN RD16/9/5 (EC).