Functional variation (Q63R) in the cannabinoid CB2 receptor may affect the severity of COVID-19: a human study and molecular docking

Archives of Virology https://doi.org/10.1007/s00705-021-05223-7 ORIGINAL ARTICLE Functional variation (Q63R) in the cannabinoid CB2 receptor may affect the severity of COVID‑19: a human study and molecular docking Mostafa Rastegar1 · Saeed Samadizadeh1 · Mohammad Yasaghi1 · Abdolvahab Moradi1 · Alijan Tabarraei1 · Vahid Salimi2 · Alireza Tahamtan1,3 Received: 8 May 2021 / Accepted: 16 July 2021 © The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature 2021 Abstract Evidence supports a role of host genetic diversity in the clinical course of coronavirus disease 2019 (COVID-19). Variation in the cannabinoid CB2 receptor gene (CNR2) could affect the regulatory action of endocannabinoids on the immune system, resulting in an increased risk of various inflammatory diseases. The present study investigated the relationship between the CNR2-Q63R variant and COVID-19 severity. A total of 200 Iranian COVID-19 patients were enrolled in the study and genotyped using a TaqMan assay. The co-dominant, dominant, recessive, over-dominant, and additive inheritance models were analyzed using SNPStats software. In silico molecular docking was also performed to simulate the effects of the Q63R variation on CB2 binding with a ligand and with the G-protein. A significant difference in the Q63R allele and genotype distribution was found between expired and discharged COVID-19 patients in co-dominant, recessive, and additive inheritance models. The molecular docking results showed that the predicted structure of mutant CB2 (63R type) could not bind to the G-protein in the correct position. The data indicated that the Q63R variation in the CNR2 gene may affect the severity of COVID-19. Identification of genes related to susceptibility and severity of COVID-19 may lead to specific targets for drug repurposing or development. Abbreviations SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 COVID-19 Coronavirus disease 2019 EC Endocannabinoid CB2 Cannabinoid receptor 2 CNR2 Cannabinoid CB2 receptor gene RT-PCR Reverse transcription polymerase chain reaction HWE Hardy-Weinberg equilibrium 3D 3-Dimensional PDB Protein Data Bank OR Odds ratios Handling Editor: Tim Skern. * Alireza Tahamtan ; 1 Department of Microbiology, Faculty of Medicine, Golestan University of Medical Sciences, Gorgan, Iran 2 Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 3 Infectious Diseases Research Center, Golestan University of Medical Sciences, Gorgan, Iran CI Confidence interval AIC Akaike information criterion RMSD Root-mean-square deviation ACE2 Angiotensin-converting enzyme 2 RSV Respiratory syncytial virus GPCRs G-protein-coupled receptors Introduction Severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) is a newly emerging virus that causes mild-to-severe respiratory disease, which has been named "coronavirus disease 2019" (COVID-19) [1, 2]. All people are susceptible to the virus infection, but there is considerable variation in the course of disease and outcome among infected individuals [3]. While many infected individuals do not experience any symptoms, others proceed to develop COVID-19; however, severe illness and death occur only in a small minority of patients [4]. Although our understanding of SARS-CoV-2 and COVID-19 is still in its infancy, there is now strong evidence supporting a role of host genetic diversity alongside with other host, viral, and environmental factors in the clinical course of disease [5–13]. Host genetic diversity could 13 Vol.:(0123456789) M. Rastegar et al. dictate the clinical response to respiratory viruses through susceptibility to viral infection and the propensity for developing harmful pulmonary inflammation [14]. Finding a relationship between host genetics and the clinical outcome of SARS-CoV-2 infection may be necessary for identifying high-risk individuals. The endocannabinoid (EC) system is a biological system composed of endogenous cannabinoids and their respective receptors, CB1 and CB2 [15]. The system has been identified as a critical endogenous regulator of immune system homeostasis due to its effects on immune cell development, migration, proliferation, and effector functions [16]. Cannabinoids have been proposed for use as immunomodulators to reduce the inflammatory effects of SARS-CoV-2 [17, 18]. Variations in the cannabinoid CB2 receptor gene (CNR2) could affect intracellular signaling and reduce the effects of ECs, which has been associated with an unbalanced immune response and an increased risk of various inflammatory diseases [19–25]. The mammalian CB2 gene is highly conserved in most of the regions, but not at amino acid position 63 [26]. The CB2-Q63R polymorphism is a missense mutation of the second and third bases at codon 63 of the CNR2 gene, which leads to a Q/R substitution, causing a different polarization state of the protein [27]. This variation has been shown to affect the response of CB2 to cannabinoids and differently modulate the EC-induced inhibition of lymphocyte proliferation [28]. While evidence has indicated that the mutation does not affect receptor-ligand binding [27], the exact mechanism behind this action is still unknown. Focusing on the immunopathogenesis of SARS-CoV-2 and the effects of ECs on the immune system, we describe here how variability in the CNR2 gene can conceivably explain variability in COVID-19 clinical phenotype. In addition, in silico molecular docking was performed to simulate the effects of the CB2-Q63R variation on receptor-ligand and receptor-G-protein interactions. The data imply the involvement of the CNR2 gene in the severity of COVID-19 in Iranian patients. from all patients, including gender, age, clinical symptoms, and comorbidities at admission. Nasopharyngeal samples were collected from all patients and divided into two groups according to their disease outcome. The samples were collected until the number of subjects reached 100 (50 women and 50 men) for each group. All of the subjects in this study were from Golestan Province and had the same geographical origin, and none were related. Genomic DNA was extracted from the collected nasopharyngeal samples using a DNA extraction kit following the manufacturers' instructions (GeneAll, South Korea). Extracted samples were genotyped for the CNR2 rs35761398 (Q63R) variant using a TaqMan assay with commercial primers and probes (Thermo Fisher, USA): 5’CTGTGA AGGTCATAGTCACGCT3’ (F primer), 5’CTCTTCTGG GCCTGCTAAGTG3’ (R primer), and CAGGTATGAGGG CTTCCGGCGGAG [CC/TT] GGTGGGAGGACAGGA TCAGATAGA[VIC/FAM] (Probes). The reaction conditions were as follows: 95 °C for 4 min, followed by 50 cycles of 95 °C for 15 s and 60 °C for 90 s. Both PCR and post-PCR allelic discrimination was performed on an ABI PRISM 7300 system (Applied Biosystems, USA). Genotypes of ten percent random samples per group were confirmed by direct PCR sequencing as (...truncated)