key: cord-0894353-sg4i99ar authors: Torre‐Fuentes, Laura; Matías‐Guiu, Jorge; Hernández‐Lorenzo, Laura; Montero‐Escribano, Paloma; Pytel, Vanesa; Porta‐Etessam, Jesús; Gómez‐Pinedo, Ulises; Matías‐Guiu, Jordi A. title: ACE2, TMPRSS2, and Furin variants and SARS‐CoV‐2 infection in Madrid, Spain date: 2020-07-28 journal: J Med Virol DOI: 10.1002/jmv.26319 sha: e1dbd5f0470448f1f6ac393e5c8f4fb3adf60334 doc_id: 894353 cord_uid: sg4i99ar It has been suggested that some individuals may present genetic susceptibility to SARS‐CoV‐2 infection, with particular research interest in variants of the ACE2 and TMPRSS2 genes, involved in viral penetration into cells, in different populations and geographic regions, although insufficient information is currently available. This study addresses the apparently reasonable hypothesis that variants of these genes may modulate viral infectivity, making some individuals more vulnerable than others. Through whole‐exome sequencing, the frequency of exonic variants of the ACE2, TMPRSS2, and Furin genes was analyzed in relation to presence or absence of SARS‐CoV‐2 infection in a familial multiple sclerosis cohort including 120 individuals from Madrid. The ACE2 gene showed a low level of polymorphism, and none variant was significantly associated with SARS‐CoV‐2 infection. These variants have previously been detected in Italy. While TMPRSS2 is highly polymorphic, the variants found do not coincide with those described in other studies, with the exception of rs75603675, which may be associated with SARS‐CoV‐2 infection. The synonymous variants rs61735792 and rs61735794 showed a significant association with infection. Despite the limited number of patients with SARS‐CoV‐2 infection, some variants, especially in TMPRSS2, may be associated with COVID‐19. Canada 12 ; and rs776995986, rs769062069, rs765152220, and rs750145841 in the Middle East 13 ; and the combination of ACE I/D and ACE2 polymorphisms in Brazil. 14 The data reported by Benetti et al 15 in Italy are particularly relevant to the Spanish setting. These findings appear to demonstrate the influence of geographic or ethnic differences in the presence of these variants. [16] [17] [18] Studies into genetic susceptibility to SARS-CoV-2 infection have focused on genetic variants of ACE2 in different populations; currently, evidence on the subject is scarce and contradictory. 19 The gene encoding transmembrane protease serine 2 (TMPRSS2) is another candidate gene studied in relation to COVID-19. TMPRSS2 expression increases ACE2-mediated invasion of cells by SARS-CoV-2. [20] [21] [22] [23] Therefore, the hypothesis that ACE2 and TMPRSS2 variants may modulate viral infectivity in humans, 24 making some individuals more vulnerable than others, seems reasonable. Recently, other host factors including Furin, TMPRSS4, and lyosomal cathepsins have been shown to be relevant for SARS-CoV-2 entry into host cells. [25] [26] [27] In this study, One relevant consideration was how infection should be defined, given that, in accordance with the instructions of the Spanish healthcare authorities, biological studies were not routinely used to confirm the diagnosis. Both in patients with MS and in the remaining participants, diagnosis of SARS-CoV-2 infection was established based on the criteria described below, obtained using the questionnaire. An individual was considered to be infected if they met any of the following criteria: (a) compatible symptoms, with positive PCR results obtained during the episode or positive serology results obtained afterwards; (b) episode of at least 7 days' duration of fever associated with at least 2 of the following symptoms: dry cough, diarrhea, pneumonia, and chest pain; (c) episode of at least 7 days' duration of fever and olfactory alterations 29 associated with at least one of the following symptoms: dry cough, diarrhea, pneumonia, and chest pain; (d) episode similar to those described in (b) and (c), of less than 7 days' duration, in an individual living with somebody with PCR confirmation of infection; and (e) episode of symptoms related to the infection, with or without fever, motivating a physician to order selfisolation. The WES methodology followed is published elsewhere, 30 but is also included in the Supplemental material. Sequencing information is included in the European Genome-Phenome Archive. Variants were described using the dbSNP database, which includes data on nucleotide and amino acid sequence changes. Data are also provided on minor allele frequency (MAF) and combined annotation-dependent depletion (CADD) score. with the infection presented TMPRSS2 variants. No difference was observed in distribution between men and women. Eleven variants were detected: three missense (rs75603675, rs12329760, and rs200291871) and eight synonymous variants (rs17854725, rs61735789, rs2298659, rs3787950, rs61735794, rs61735792, rs142750000, and rs141788162) ( Table 1) been functionally linked to rs8134378. None of these variants were detected in this cohort. The second haplotype contains rs2070788, rs9974589, and rs7364083; none of these variants were detected in the present study. However, the missense variants rs12329760 and rs200291871 did appear in the cohort, but showed no association with infection. The first variant is potentially pathogenic. The synonymous variants rs61735794 and rs61735792 were also detected at significantly different frequencies in the groups of individuals with and without SARS-CoV-2 infection. Synonymous variants are not usually pathogenic, although we may consider that the risk may be not related to the production of a pathological protein, as with missense mutations, but to smaller modifications. SARS-CoV-2 -(n = 113) SARS-CoV- 2+ (n = 7) ACE2 rs35803318 c.2247G>A 2 (4.8%) 0 (0.0%) 6 (8.5%) 0 (0.0%) 8 (7.1%) 0 (0.0%) NS ACE2 rs41303171 c.2158A>G 1 (2.4%) 0 (0.0%) 1 (1.4%) 1 (25.0%) 2 (1.8%) 1 (14.3%) NS TMPRSS2 Furin has been implicated in the SARS-CoV-2 infectivity. S protein is cleaved by TMPSS2 with the collaboration of furin, which has been linked to the entry of the virus in the respiratory tract and also with an increased risk of contagion. 43 To our knowledge, genetic variants of furin and its association with COVID-19 have not been examined. In our study, we did not found association between Furin variants and COVID-19. MS has previously been linked to coronavirus infections 44, 45 ; therefore, it would be beneficial to establish whether the pandemic has a specific impact on these patients. Furthermore, polymorphisms of genes related to the renin-angiotensin system, although not ACE2 specifically, have been studied in these patients. 46 There is also debate around the effect of MS treatment on COVID-19 severity, 47, 48 and it has been suggested that some drugs acting on the immune system may protect against the infection. [49] [50] [51] [52] While this issue lies beyond the scope of the current study, comparison of variants between patients with MS and unaffected individuals seems not to show that these genes play a pathogenic role. In this study, WES findings were used to analyze the frequency of Finally, WES only analyses exonic variants; therefore, the study may have failed to detect potentially related intronic variants. Some strengths of the study are that it identified the frequency of variants of these genes in a geographic region where this had not previously been analyzed, and that human subjects were tested, rather than samples from a gene bank, enabling more precise analysis of the results. The results show that ACE2 presents a low level of polymorphism, with only two variants (rs41303171 and rs35803318) being identi- The authors wish to thank the participating patients and their families for the information and the DNA samples provided, the Spanish Society of Neurology's Research Operations Office for assisting with the English-language version of the manuscript, and Sara Álvarez and Pablo Maietta for their assistance with sequencing and the advice given. The authors declare that there are no conflict of interests. Lead researchers: JAMG, JMG; study design: VP, JAMG, and JMG. The data that support the findings of this study are available from the corresponding author upon reasonable request. Ulises Gómez-Pinedo http://orcid.org/0000-0003-3097-5557 Jordi A. 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