key: cord-0852950-mc97ln8f
authors: Coto, Eliecer; Albaiceta, Guillermo M.; Amado‐Rodríguez, Laura; García‐Clemente, Marta; Cuesta‐Llavona, Elías; Vázquez‐Coto, Daniel; Alonso, Belén; Iglesias, Sara; Melón, Santiago; Alvarez‐Argüelles, Marta E.; Boga, José A.; Rojo‐Alba, Susana; Pérez‐Oliveira, Sergio; Alvarez, Victoria; Gómez, Juan
title: FURIN gene variants (rs6224/rs4702) as potential markers of death and cardiovascular traits in severe COVID‐19
date: 2022-04-12
journal: J Med Virol
DOI: 10.1002/jmv.27748
sha: dd544585d38f55e75af6e9c9e2f46499e2039f7a
doc_id: 852950
cord_uid: mc97ln8f

Furin is a protease that plays a key role in the infection cycle of SARS‐CoV‐2 by cleaving the viral proteins during the virus particle assembly. In addition, Furin regulates several physiological processes related to cardio‐metabolic traits. DNA variants in the FURIN gene are candidates to regulate the risk of developing these traits as well as the susceptibility to severe COVID‐19. We genotyped two functional FURIN variants (rs6224/rs4702) in 428 COVID‐19 patients in the intensive care unit. The association with death (N = 106) and hypertension, diabetes, and hyperlipidaemia was statistically evaluated. The risk of death was associated with age, hypertension, and hypercholesterolemia. The two FURIN alleles linked to higher expression (rs6224 T and rs4702 A) were significantly increased in the death cases (odds ratio= 1.40 and 1.43). Homozygosis for the two high expression genotypes (rs6224 TT and rs4702 AA) and for the T‐A haplotype was associated with an increased risk of hypercholesterolemia. In the multiple logistic regression both, hypercholesterolemia and the TT + AA genotype were significantly associated with death. In conclusion, besides its association with hypercholesterolemia, FURIN variants might be independent risk factors for the risk of death among COVID‐19 patients.

cut preactivates the viral particles and might explain the higher infective capacity of SARS-CoV-2 compared to other viruses that require two protease-cuts. Interestingly, some of the SARS-CoV-2 variants of concern could enhance the cleavage by furin, likely increasing the infection capacity. [7] [8] [9] Furin is a membrane-bound protease of the subtilisin-like proprotein convertase family whose natural cellular function is to process precursor proteins but is also utilized by a number of pathogens to process their envelope proteins. [10] [11] [12] [13] [14] The furin polybasic insertion endows SARS-CoV-2 with a selective advantage. 15 In ferrets the lack of the S1/S2 furin site shed to lower titres among infected animals, that were unable to transmit the virus to cohoused animals. 16 COVID-19 symptoms range from none or mild symptoms to severe pneumonia with admission to the intensive care unit (ICU), and this heterogeneity might be partly explained by host genetic variants that influence vulnerability to infection or modulate the immunological responses. The FURIN gene is a candidate to regulate the risk of COVID-19 severity. A single-nucleotide polymorphism (SNP, rs4702 A/G) located in the 3′-untranslated region of the gene influences alveolar and neuron infection by SARS-CoV-2 in vitro. 17 FURIN expression would be down-regulated by the rs4702 G allele, which is located in the binding site of a micro RNA (miR-338-3p). 17, 18 FURIN variants associated with differences in gene expression could modify the individual´s susceptibility to developing severe COVID-19 pneumonia with an increased risk of death. Our aim was to determine the association between two common FURIN variants and adverse outcomes in COVID-19 patients.

We obtained the demographic and clinical data of 428 patients who required admission in the ICU due to COVID-19 (mean age: 64, range: 24-95). All the participants were of European ancestry and from the region of Asturias (Northern Spain, total population of 1 million). The study was approved by the Ethics Committee of Principado de Asturias (Oviedo, Spain). All the patients or their next of kin gave their consent to participate in the study.

These patients were hospitalized between March (2020) and February (2021), a period in which three pandemic waves took place in our community. They were followed till disease remission with hospital discharge or death.

The hypercholesterolemia, diabetes, and hypertension status were obtained from the medical records. We measured the D-Dimer and IL6 blood levels at ICU admission. Based on previous reports values <2000 ng/ml (D-Dimer) or <15 pg/ml (IL6) were considered the predictors of severity. 19, 20 The FURIN rs6224 T/G and rs4702 A/G were determined by real-time PCR with Taqman assays (Thermo Fisher; Figure S ). The rs6224 SNP was genotyped instead of rs17514846 A/C because the two were in almost complete linkage disequilibrium (D´= 0.98; r 2 = 0.94) and there was no custom Taqman assay for rs17514846.

The rs17514846 A/C (intron 1 of FURIN gene) was associated with differential expression, with cultured vascular endothelial cells with AA genotype showing higher FURIN expression than CC cells. 21 

We determined the association between the study variables and death in the COVID-19 patients ( 

The two FURIN SNPs were in linkage disequilibrium with two major haplotypes, T-A (higher expression) and G-A ( Figure S As protease furin plays a role in the activation of proteins involved in multiple cellular pathways, including some that contribute to cardiometabolic traits. A cross-sectional analysis of 2312 participants (mean age: 53 years) found that individuals with the lowest serum furin had average systolic, diastolic, and mean arterial blood pressures higher than the corresponding pressures in individuals with the highest serum quartile. 26 Furin cleaves the pro-renin receptor PRR to generate soluble sPRR. The PRR is a regulator of the renin-angiotensin system (RAS), and the sPRR is a candidate biomarker that reflects the status of the tissue RAS and the risk for cardiovascular traits such as hypertension. 27 Therefore, the role of furin in the regulation of RAS function might explain the association between FURIN variants and hypertension, a major determinant of COVID-19 severity. In this regard, the rs4702 GG genotype (linked to lower FURIN expression) has been associated with an increased risk of hypertension. [26] [27] [28] [29] [30] Furin is a key regulator of the PCSK9 expression, a protein that regulates the number of LDL receptors and the level of blood lipids. 31, 32 Higher circulating furin has been associated with increased LDL-cholesterol in plasma. 33 Furin might be also associated with COVID-19 severity through platelet activation and the risk of thrombotic events. Patients with coronary artery disease and SARS-CoV-2 positive displayed a significantly enhanced platelet activation. 34 Moreover, furin plasma levels were significantly increased in patients with a poor clinical prognosis.

The association between FURIN variants and the risk for viral infections has been reported. [35] [36] [37] In the SARS-CoV-2 infection these variants could explain the heterogeneous response, with individuals remaining asymptomatic while others develop severe pneumonia with an increased risk of death. Variants that increase the furin expression could be associated with worse outcomes ( Figure 2 ). Our data were in agreement with this hypothesis because the rs6224 T and rs4702 A (high expression alleles) were significantly increased in the deceased patients. Furin has been associated with the risk of developing cardiovascular traits, such as hypertension, hypercholesterolemia, and diabetes, which are risk factors for COVID-19 severity and increased mortality. In our study, the two FURIN high-expression genotypes were associated with a significantly higher risk of hypercholesterolemia. Blood furin concentration has been associated with LDL cholesterol. [31] [32] [33] The association between circulating furin and lipid values could be mediated by its capacity to regulate the expression and activity of pcsk9, a protein that competes with the LDL-cholesterol particles by binding to the LDL-receptor. 38, 39 Also, furin converts the propeptide form of Bmp1 into an active form that cleaves the LDL-R making it unable to bind the LDL particles. 40 Thus, the effect of FURIN variants on the risk of death might be secondary to hypercholesterolemia, which was significantly associated with death. However, the fact that the risk genotypes were associated with death among the COVID-19 patients independently of hypercholesterolemia suggested that these FURIN gene variants might contribute to disease severity by themselves.

Our study has several limitations, mainly the reduced sample size.

Additional studies with larger cohorts from different populations are necessary to confirm the association with COVID-19 severity. Also, we studied patients with the most severe disease manifestation represented by the ICU patients that are at increased risk of death.

Patients who did not require ICU admission, or who showed symptoms that did not require hospitalization were not studied and no conclusion about the effect of the FURIN variants on these manifestations can be concluded. The studied variants might regulate the risk of hypercholesterolemia and COVID-19 severity through a F I G U R E 1 Haplotype frequencies in the different groups. rs6224 T-rs4702 A was significantly increased in the deceased ICU patients. ICU, intensive care unit differential expression of the protein, but the level of blood circulating furin was not measured in our patients.

The FURIN high expression alleles rs6224 T and rs4702 A were associated with an increased risk of death in COVID-19. These variants were also associated with hyperlipaemia, a risk factor for disease severity and mortality. However, both FURIN variants were independently associated with death suggesting that furin is a risk factor for COVID-19 severity beyond its role in cholesterol hemostasis.

Our work was based on a limited number of patients and requires validation in larger cohorts from different regions. 

The authors declare no conflicts of interest.

The data that support the findings of this study are available from the corresponding author upon reasonable request. An excel file with the raw data would be available for meta-analysis research.

This study was approved by the clinical research ethics committee of Hospital Universitario Central Asturias (HUCA). All the participants or they next of keen gave written or verbal consent. Data were handled in observance of Spanish legislation on data protection. The study complies with the principles of the Declaration of Helsinki ("Recommendations guiding doctors in biomedical research involving human subjects").

Eliecer Coto http://orcid.org/0000-0002-8649-9150

Marta E. Alvarez-Argüelles http://orcid.org/0000-0002-

F I G U R E 2 Proposed mechanism for the association between FURIN gene variants and increased risk of COVID-19 severity. Variants linked to increased furin expression might enhance SARS-CoV-2 infectivity by facilitating the viral particles' assembly in the host cell. In addition, these FURIN variants would increase the risk of developing cardiovascular traits, such as hypercholesterolemia. These variants have been also associated with increased vascular inflammation, a hallmark of atherosclerosis. The FURIN gene maps to chromosome 15, and the position of the SNPs relative to the coordinate sequence (Ensembl reference gene) is indicated. The two genotyped variants were located in intron 13 (rs6224) and the 3´-UTR (rs4702). SNP, single-nucleotide polymorphism; UTR, untranslated region

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