ORIGINAL RESEARCH published: 23 July 2021 doi: 10.3389/fimmu.2021.719115 Genetic Screening for TLR7 Variants Edited by: in Young and Previously Healthy Men Antonio Condino-Neto, University of São Paulo, Brazil With Severe COVID-19 Reviewed by: Alessandro Aiuti, Xavier Solanich 1*†, Gardenia Vargas-Parra 2,3†, Caspar I. van der Made 4,5,6†, Annet Simons 4, Vita-Salute San Raffaele University, Janneke Schuurs-Hoeijmakers 4, Arnau Antolı́ 1, Jesús del Valle 2,3, Italy Gemma Rocamora-Blanch 1, Fernando Setién 7, Manel Esteller 3,7,8,9, Alexandra Freeman, National Institutes of Health (NIH), Simon V. van Reijmersdal 4, Antoni Riera-Mestre 1,10, Joan Sabater-Riera 11, United States Gabriel Capellá 2,3, Frank L. van de Veerdonk 5,6, Ben van der Hoven 12, Xavier Corbella 1,13, Giuseppe Novelli, Alexander Hoischen 4,5,6* and Conxi Lázaro 2,3* University of Rome Tor Vergata, Italy 1 Department of Internal Medicine, Hospital Universitari de Bellvitge, Bellvitge Biomedical Research Institute (IDIBELL), *Correspondence: L’Hospitalet de Llobregat, Barcelona, Spain, 2 Hereditary Cancer Program, Catalan Institute of Oncology, Program in Xavier Solanich Molecular Mechanisms and Experimental Therapy in Oncology (Oncobell), Bellvitge Biomedical Research Institute (IDIBELL), xsolanich@bellvitgehospital.cat L’Hospitalet de Llobregat, Barcelona, Spain, 3 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Alexander Hoischen Spain, 4 Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands, 5 Department of Alexander.Hoischen@radboudumc.nl Internal Medicine and Radboud Center for Infectious Diseases (RCI), Radboud Institute for Molecular Life Sciences (RIMLS), Conxi Lázaro Radboud University Medical Center, Nijmegen, Netherlands, 6 Radboud Expertise Center for Immunodeficiency and clazaro@iconcologia.net Autoinflammation and Radboud Center for Infectious Disease (RCI), Radboud University Medical Center, Nijmegen, † Netherlands, 7 Josep Carreras Leukaemia Research Institute (IJC), Badalona, Spain, 8 Institucio Catalana de Recerca i These authors have contributed equally to this work and share Estudis Avançats (ICREA), Barcelona, Spain, 9 Physiological Sciences Department, School of Medicine and Health Sciences, first authorship University of Barcelona (UB), Barcelona, Spain, 10 Faculty of Medicine and Health Sciences, Universitat de Barcelona, Barcelona, Spain, 11 Department of Intensive Care, Hospital Universitari de Bellvitge, Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, Barcelona, Spain, 12 Department of Intensive Care, Erasmus MC, Rotterdam, Specialty section: Netherlands, 13 School of Medicine, Universitat Internacional de Catalunya, Barcelona, Spain This article was submitted to Primary Immunodeficiencies, a section of the journal Introduction: Loss-of-function TLR7 variants have been recently reported in a small Frontiers in Immunology number of males to underlie strong predisposition to severe COVID-19. We aimed to Received: 01 June 2021 Accepted: 08 July 2021 determine the presence of these rare variants in young men with severe COVID-19. Published: 23 July 2021 Methods: We prospectively studied males between 18 and 50 years-old without Citation: Solanich X, Vargas-Parra G, predisposing comorbidities that required at least high-flow nasal oxygen to treat van der Made CI, Simons A, COVID-19. The coding region of TLR7 was sequenced to assess the presence of Schuurs-Hoeijmakers J, Antolı´ A, potentially deleterious variants. del Valle J, Rocamora-Blanch G, Setién F, Esteller M, Results: TLR7 missense variants were identified in two out of 14 patients (14.3%). van Reijmersdal SV, Riera-Mestre A, Sabater-Riera J, Overall, the median age was 38 (IQR 30-45) years. Both variants were not previously Capellá G, van de Veerdonk FL, reported in population control databases and were predicted to be damaging by in silico van der Hoven B, Corbella X, predictors. In a 30-year-old patient a maternally inherited variant [c.644A>G; Hoischen A and Lázaro C (2021) Genetic Screening for TLR7 Variants p.(Asn215Ser)] was identified, co-segregating in his 27-year-old brother who also in Young and Previously Healthy contracted severe COVID-19. A second variant [c.2797T>C; p.(Trp933Arg)] was found Men With Severe COVID-19. Front. Immunol. 12:719115. in a 28-year-old patient, co-segregating in his 24-year-old brother who developed mild doi: 10.3389/fimmu.2021.719115 COVID-19. Functional testing of this variant revealed decreased type I and II interferon Frontiers in Immunology | www.frontiersin.org 1 July 2021 | Volume 12 | Article 719115 Solanich et al. Rare TLR7 Variants in COVID-19 responses in peripheral mononuclear blood cells upon stimulation with the TLR7 agonist imiquimod, confirming a loss-of-function effect. Conclusions: This study supports a rationale for the genetic screening for TLR7 variants in young men with severe COVID-19 in the absence of other relevant risk factors. A diagnosis of TLR7 deficiency could not only inform on treatment options for the patient, but also enables pre-symptomatic testing of at-risk male relatives with the possibility of instituting early preventive and therapeutic interventions. Keywords: COVID-19, SARS-CoV-2, host genetics, TLR7, immunodeficiency, genetic screening INTRODUCTION METHODS A proportion of patients with COVID-19 develop fatal lung This is a joint study performed at the Hospital Universitari de injury and multi-organ failure due to systemic host-immune Bellvitge – IDIBELL, L’Hospitalet de Llobregat, Barcelona, Spain; inflammatory processes triggered by the viral infection (1). and the Radboud University Medical Center, Nijmegen, The Advanced age, male sex and chronic disease such as diabetes Netherlands and the Erasmus Medical Center, Rotterdam, and obesity are common in patients with more severe forms of The Netherlands. COVID-19 (2–4). However, these risk factors cannot explain why critical disease also occurs in young (below 50 years of age) The Barcelona Cases and apparently healthy individuals. From March to July 2020, researchers from Hospital Universitari In the past months, several publications have identified loci de Bellvitge - IDIBELL prospectively collected biological samples and genes associated with COVID-19 susceptibility for severe from young patients without comorbidities related to severe COVID-19 by using comprehensive GWAS studies or genome, COVID-19. Selection criteria were: 1) patients aged between 18 exome or candidate gene analyses (5). However, most of these and 50 years old; 2) absence of known comorbidities associated susceptibility alleles showed risk values too low (odds ratio <2) to with most severe forms of COVID-19 (BMI ≥ 30kg/m2, diabetes be regarded as predictive genomic markers. Some of the loci mellitus, hypertension, chronic heart, pulmonary or kidney reported include ABO blood group (6, 7), ACE2 (8), TMPRSS2 disease, or an immunocompromised state); and 3) SARS-CoV- (8), several HLA alleles (5, 9), APOE (10), and IFITM3 (11). On 2 related lung injury requiring high flow oxygen devices or the other hand, rare variants in genes encoding for members of mechanical ventilation. Ten male patients fulfilled these the type I/III interferon (IFN) pathway showed a higher selection criteria (Table 1). Eight patients (patients 1-8) were estimated risk to severe COVID-19 (12, 13). also evaluated by the COVID Human Genetic Effort, although no In July 2020 rare, deleterious germline variants in the X- pathogenic variants were identified in any of the 13 type I IFN chromosomal Toll-like receptor 7 (TLR7) gene were reported in pathway genes studied (12). young and, otherwise, healthy males with severe COVID-19. In Informed consent was obtained from all patients and relatives, these two brother pairs, rapid whole-exome sequencing and the IDIBELL Research Ethics Committee approved this study identified both a maternally inherited 4-nucleotide deletion (PR152/20). Demographic, epidemiological, laboratory and [c.2129_2132del; p.(Gln710Argfs*18)] and a missense variant clinical data were collected. Treatments specifically used to treat [c.2383G>T; p.(Val795Phe)]. Both variants were associated with COVID-19 at any time during admission were also documented. impaired type I and II IFN responses upon stimulation with the DNA was isolated from total blood either using a Maxwell TLR7 agonist imiquimod, supporting the importance of intact instrument RSC (Promega, Madison, WI, USA) or QIAGEN TLR7 signaling in COVID-19 pathogenesis (13). Most recently Flexigene DNA kit (Qiagen, Germany). Nine PCR primer pairs these findings were replicated in an independent Italian cohort (Sigma-Aldrich, MO, USA) were designed to cover the whole study of males <60 years of age with severe COVID-19 (79 severe coding region of TLR7 (HGNC ID:15631). PCR was performed cases versus 77 control cases), showing that 2.1% of severely using DreamTaq MasterMix (ThermoFisher Scientific, affected males harbored deleterious TLR7 variants compared to Waltham, MA, USA), products were purified using EXO-SAP none of the asymptomatic participants. These variants were (New England Biolabs) and sequenced using the BigDye demonstrated to decrease transcription of type I and II IFN- Terminator v.3.1 Sequencing Kit (Applied Biosystems, CA, related genes in patient peripheral blood mononuclear cells USA) in an ABI Prism 3730 XL Genetic Analyzer (Applied (PBMC) treated with imiquimod, supporting a loss of function Biosystems CA, USA). Primers and PCR conditions are available effect (14). upon request. Mutation Surveyor software was used to detect In follow-up of the recent discovery of TLR7 deficiency in variants and nomenclature was given according to HGVS patients with severe COVID-19, we aimed to prospectively guidelines. All variants identified were submitted to Alamut determine the presence of TLR7 variants in young and Software Suite v2.15.0 (Interactive Biosoftware) to retrieve previously healthy men with severe COVID-19. population frequency and in silico prediction data. Frontiers in Immunology | www.frontiersin.org 2 July 2021 | Volume 12 | Article 719115 Solanich et al. Rare TLR7 Variants in COVID-19 TABLE 1 | Demographic and clinical findings of investigated patients. Patient Sequencing Gender Age (y) Ethnicity Comorbidities ARDSa ICU ECMO 1 Sanger M 31 Caucasian no yes no no 2 Sanger M 44 Caucasian no yes yes no 3 Sanger M 41 Latin (Venezuela) no yes yes yes 4 Sanger M 40 Caucasian no yes yes no 5 Sanger M 50 Latin (Peru) no yes yes no 6 Sanger M 48 Caucasian no yes yes no 7 Sanger M 45 Latin (Peru) no yes yes no 8 Sanger M 31 Latin (Honduras) no yes yes no 9 Sanger M 47 Latin (Peru) no yes no no 10b Sanger M 30 Latin (Dominican Republic) no yes yes no 11 Sanger M 21 Caucasian no yes yes no 12 Sanger M 36 Caucasian no yes yes no 13b WES M 28 Caucasian no yes yes no 14 WES M 31 Caucasian no yes no no ARDS, acute respiratory distress syndrome; ECMO, extracorporeal membrane oxygenation; ICU, intensive care unit; F, female; M, male; WES, whole-exome sequencing; Y, years. a ARDS Definition Task Force. Acute Respiratory Distress Syndrome - The Berlin Definition. JAMA. 2012;307(23):2526-2533. doi:10.1001/jama.2012.5669. b Patients 10 and 13 resulted carriers of TLR7 variants and belong to family 1 and 2, respectively. The Dutch Cases experiments. Apart from a negative medium (RPMI) control, At the Radboud University Medical Center in Nijmegen and the the TLR7 agonist imiquimod (imidazoquinoline compound, Erasmus Medical Center in Rotterdam, the Netherlands, patients Invivogen) was used at a concentration of 5 mg/mL. After the 4 were screened prospectively in a clinical setting from December hour incubation period, the supernatants were discarded and 2020 to February 2021 with the following criteria: 1) males the remaining cell pellets were resuspended in RLT buffer between 18 and 40 years of age; 2) absence of comorbidities (Qiagen) and snap frozen to be stored at −80°C until known to be associated with severe COVID-19 (BMI ≥ 30kg/m2, processing for RNA isolation. In addition, after the 7 day diabetes mellitus, hypertension, chronic heart, pulmonary or incubation and a centrifugation step, supernatants were kidney disease, or an immunocompromised state); and 3) PCR- collected and stored at −20°C until measured using enzyme- confirmed SARS-CoV-2 infection requiring high-flow oxygen linked immunosorbent assay in case of the 7 day timepoint. therapy or ICU admission. A total of 4 patients (patients 11-14) fulfilled these inclusion criteria and underwent clinical Sanger sequencing (patients 11 and 12) or rapid whole-exome sequencing (patient 13 and 14) to specifically assess genetic RESULTS variants in TLR7 (Table 1). Written informed consent was obtained from patient 13 whose clinical data has been included in TLR7 Sequencing Results this study. Rapid whole-exome sequencing was performed similar A total of 14 patients were included, with a median age of 38 to previous reports (13). Sanger sequencing was done according to (IQR 30-45) years. Putative deleterious TLR7 missense variants standard diagnostic procedures at the Department of Human were identified in two patients (patient 10 and 13). Both variants, Genetics, Radboud University Medical Center, protocols and c.644A>G; p.(Asn215Ser) in patient 10 and c.2797T>C; primers sequences are available upon request. p.(Trp933Arg) in patient 13, were not previously reported in For patient 13, in whom a rare TLR7 missense variant was our in-house databases nor in the population database gnomAD identified, functional testing was performed to evaluate impaired (15). The p.(Asn215Ser) variant affects a highly conserved type I and II IFN responses due to TLR7 loss-of-function, as nucleotide and amino acid in the TLR7 leucine-rich region described previously (13). In brief, venous blood was drawn and domain and is predicted damaging or possibly damaging by in collected in EDTA tubes (Monject). Subsequently, peripheral silico software (Table S1). The p.(Trp933Arg) variant is also mononuclear blood cells (PBMC) were isolated by density located at an evolutionarily highly conserved position within the centrifugation of blood, diluted 1:1 in pyrogen-free saline over TIR domain, important for downstream signaling via adapter Ficoll-Paque (Pharmacia Biotech). Cells were washed twice in proteins, and is considered deleterious by in silico effect saline and resuspended in cell culture medium (Roswell Park predictors (Table S1). These TLR7 variants and other Memorial Institute [RPMI] 1640, Gibco) supplemented with previously reported variants in COVID-19 patients are shown gentamicin, 10 mg/mL; L-glutamine, 10 mM; and pyruvate, 10 schematically in Figure 1. mM. PBMC were then stimulated at 5 × 105 cells/well in round- bottom 96-wells plates (Greiner) for either 4 hours (for Patients’ Characteristics transcription of type I IFN genes) and 7 days [for production Patient 10 (family 1, proband) was a 30-year-old man from Latin of the type II IFN, (IFNg)] in the presence of 10% human pool origin (Dominican Republic) with no general risk factors serum at 37°C and 5% carbon dioxide, in two separate predisposing to severe COVID-19. He developed pneumonia Frontiers in Immunology | www.frontiersin.org 3 July 2021 | Volume 12 | Article 719115 Solanich et al. Rare TLR7 Variants in COVID-19 A B FIGURE 1 | (A) displays pedigrees of families 1 (patient 10) and 2 (patient 13) with segregation analysis. (B) shows schematic representation of TLR7 (HGNC ID:15631) variants reported to date in severely affected COVID-19 cases. Variants in cDNA (top) and protein (bottom). Color code: orange, variants found in the present series; blue, previously reported in van der Made (13); black, previously reported in Fallerini (14). Shape code: circle, missense variants; square, frameshift variants. Line code: single, reported in one case; double, reported in 2 cases. with bilateral consolidations on a computed tomography (CT) radiological findings of the three relatives are summarized scan and fulfilled the criteria of acute respiratory distress in Table 2. syndrome (ARDS) secondary to PCR-proven COVID-19 Patient 13 (family 2, proband) was a 28-year-old male from (Table 2). The patient received antiviral treatment with Caucasian origin (The Netherlands) without previous medical remdesivir and immunosuppressive therapy with dexamethasone. history or comorbidities. The patient complained of progressive Due to respiratory insufficiency, the patient was intubated and dyspnea and shortness of breath. Following rapid clinical admitted in ICU. The patient was successfully extubated after 4 days deterioration and respiratory insufficiency, the patient was of mechanical ventilation and was discharged from ICU after 6 days. intubated and hospitalized in ICU at a peripheral hospital. A After the identification of the TLR7:p.(Asn215Ser) variant in the CT-scan showed multiple diffuse ground-glass opacities and patient, segregation analysis confirmed co-segregation in both his consolidations in all lung segments, meeting the criteria for brother and mother, in hemi- and heterozygous state, respectively ARDS. Treatment consisted of mechanical ventilation with (Figure 1). The eldest brother lives in the Dominican Republic and prone positioning, intravenous dexamethasone, and the was therefore not available for testing. According to relatives, there antibiotics ceftriaxone and ciprofloxacin. Despite this is no evidence that he has been infected with SARS-CoV-2. The therapeutic regiment, the patient’s condition further youngest brother had no previous medical history but also deteriorated and he was referred to the Erasmus Medical contracted severe COVID-19, requiring mechanical ventilation Center for possible ECMO treatment. However, with and ICU admission at another hospital (Table 2). These brothers continuing prone positioning he gradually improved before therefore represent the third brother pair with severe COVID-19, ECMO was required. A repeated CT scan also showed following the initial report (13). The 65 year-old mother suffered subsegmental pulmonary embolisms for which intravenous from obesity, dyslipidemia, type 2 diabetes and hypertension, and heparin was started. In the following weeks, the patient was also admitted in ICU due to critical respiratory failure caused by gradually recovered and was successfully extubated and COVID-19. She was discharged from the ICU 16 days after eventually discharged from the hospital. The patients’ first- admission. Main demographic, clinical, laboratory, and degree family contracted COVID-19 at the time the patient Frontiers in Immunology | www.frontiersin.org 4 July 2021 | Volume 12 | Article 719115 TABLE 2 | Demographic, clinical, laboratory, and radiological findings of investigated patients. Frontiers in Immunology | www.frontiersin.org Solanich et al. Family 1 TLR7:c.644A>G Family 2 TLR7:c.2797T>C Reference ranges Proband Brother Mother Proband Demographic characteristics Date of hospitalization July/2020 March/2020 August/2020 January/2021 Age, y 30 27 65 28 Sex Male Male Female Male Medical history None None Obesity, dyslipidemia, Vasovagal syncope hypertension, type 2 diabetes, Clinical characteristics at presentation Time from symptom onset to hospitalization, d 7 6 12 2 Symptoms at disease onset Dyspnea, cough, fever, myalgia Dyspnea, cough, fever, headache Dyspnea, cough, fever, myalgia Dyspnea, cough, fever, respiratory arrest Imaging features (CT scan) Bilateral pulmonary consolidations Bilateral pulmonary consolidations Bilateral pulmonary consolidations Multiple ground glass opacities and consolidations in all lung segments ICU admission Time from symptom onset to ICU admission, d 8 7 12 7 Medical reason for ICU admission Respiratory insufficiency Respiratory insufficiency Respiratory insufficiency Respiratory failure, respiratory arrest, resuscitation at home. Disease severity status on admission, SOFA score* 3 3 4 6 Laboratory findings at ICU admission Chemistry 5 Alanine aminotransferase, U/L 135 14 23.5 41 <40 Albumin, g/L 37 31 28.1 23 35 - 52 Alkaline phosphatase, U/L 131 66 84.0 222 ≤ 129 Aspartate aminotransferase, U/L 92 22 73.8 37 ≤ 39 Cardiac troponin, high sensitivity, ng/L NA 7 NA NA ≤ 13 Creatine kinase, U/L 51 35 180 NA ≤ 189 Creatinine, mmol/L 60 57 57.1 84 44-97 eGFR, mL/min/1.73 m2 >90 >90 >90 >90 >90 g-Glutamyltransferase, U/L 243 27 34.8 263 ≤ 70 Lactate dehydrogenase, U/L 381 432 1088.4 201 <250 Blood count Hemoglobin, g/L 112 114 110 121 130 - 165 Lymphocyte count, ×109/L 1.8 1.47 2.19 1.64 1.3-3.4 White blood cell count, ×109/L 18 10.7 14.74 8.4 3.9-9.5 July 2021 | Volume 12 | Article 719115 Platelet count, ×109/L 385 408 416 369 149 - 303 Coagulation Activated partial thromboplastin time ratio 0.95 0.98 1.00 37 0.8-1.2 Rare TLR7 Variants in COVID-19 D-dimer, ng/mL <250 463 2400 3660 <250 Fibrinogen, g/L >7 >7 5.5 4,2 2.76-4.71 Prothrombin time ratio 1.38 1.32 1.10 NA 0.8-1.2 Inflammatory markers C-reactive protein, mg/L 346.6 267 203.98 196 <3 Ferritin, mg/L 1957.6 920 384.9 845 30 - 400 Procalcitonin, mg/L 0.28 NA 0.1 4.31 <0.5 IL-6, ng/L 48.4 1.5 24 NA ≤ 6.9 (Continued) Solanich et al. Rare TLR7 Variants in COVID-19 Reference COVID-19, coronavirus disease 2019; CT, computed tomography; ICU, intensive care unit; eGFR, estimated glomerular filtration rate; NA, not assessed; PCR, polymerase chain reaction; SARS-CoV-2; severe acute respiratory syndrome intravenously; L-R, Lopinavir 400mg-Ritonavir 100mg orally twice daily for three days; H, Hydroxychloroquine orally 400mg twice daily the first day and 200mg twice daily the next 10 days; I, Interferon b1b 0.25mg every other day *The SOFA score is calculated using 6 systems: respiratory, coagulation, hepatic, cardiovascular, central nervous, and kidney. Scores range from 0 for normal function to 4 for most abnormal and are summed for a final range of 0 to 24. An The treatments were administered to the patients as follows: D, Dexamethasone 8 mg daily for 10 consecutive days; R, Remdesivir 200mg intravenously the first day and 100mg daily the next four days; T, Tocilizumab 600 mg single dose developed symptoms, including his 24-year-old brother, who ranges had only minor symptoms but was shown to be a carrier of the TLR7 variant (Figure 2B). Information on the exact SARS-CoV- 2 viral titers during infections were unavailable. In addition, two Positive before admission, PCR admission after resuscitation at male cousins, sons of a maternal aunt, were proven to be carriers of Small ventral pneumothorax at home. Bilateral subsegmental Family 2 TLR7:c.2797T>C this variant. These individuals had not contracted (symptomatic) pulmonary embolisms. negative at day 29 24 days (ongoing) 24 days (ongoing) COVID-19. Based on the TLR7 variant carriership, these individuals Proband were fast-tracked for early vaccination. NA NA D Functional Testing in Primary Immune Cells In PBMC isolated from patient 13, the type I and II IFN responses were evaluated upon stimulation with the TLR7 agonist imiquimod, in order to assess the functional impact Positive at admission, no follow- of the p.(Trp933Arg) variant. It was shown that TLR7 stimulation in the patient resulted in a defective upregulation up measurement None reported None reported of TLR7 mRNA expression, as well as that of the type I IFN- Mother R, D, T 16 related genes involved in the TLR7 pathway IRF7, IFNB1 and 7 5 ISG15, as compared to a healthy male control (Figures 2A, B). Expression of IRF3, a transcription factor that is induced by TLR3 and not TLR7, showed a modest increase of almost 1.7 fold in the patient, but was not induced in the healthy control. Moreover, the production of IFNg upon exposure with Positive at admission, no follow- catheter-related bloodstream imiquimod was deficient in the patient, compared to two Family 1 TLR7:c.644A>G healthy male controls (Figure 2C). up measurement H, L-R, MP, I, T None reported Brother infection 10 12 11 DISCUSSION initial score of 2 to 3 is associated with 6% mortality; an initial score of 4 to 5 is associated with 20% mortality. In this cohort of young males affected with severe COVID-19 rare TLR7 variants were prospectively identified in 2 out of 14 cases (14.3%). In 10 cases from Spain, one patient was shown to Positive at admission, no follow- carry a new missense variant [c.644A>G; p.(Asn215Ser)]. The variant was predicted as damaging by in silico programs and up measurement None reported None reported segregated in a brother who also contracted severe COVID-19. In Proband R, D addition, four Dutch cases were studied, leading to the 4 6 3 identification of another patient with a novel, unique missense variant [c.2797T>C; p.(Trp933Arg)] located in the highly conserved TIR domain. Functional analysis of the latter variant demonstrated defective type I and II IFN responses, similar to those documented in the previous reports (13, 14). coronavirus 2; SOFA, Sequential Organ Failure Assessment. A role for the TLR7 receptor has been described in the host Time from ICU discharge to hospital discharge, d defense against single-stranded RNA viruses as well as in the Duration of viral shedding after COVID-19 onset pathogenesis of autoimmune disease such as SLE (16). However, no human mutations in TLR7 had been reported before the Complications during follow-up period SARS-CoV-2 pandemic. It should be noted that complete TLR7 deficiency is estimated to be extremely rare, because endosomal Duration of ventilatory support, d (positive SARS-CoV-2 PCR), d TLRs (TLR3, TLR7, TLR8, and TLR9) play an essential, non- redundant biological role in host survival (17, 18). Therefore, Secondary complications subcutaneously for 3 days. Duration of ICU stay, d TABLE 2 | Continued these rare TLR7 variants are unlikely to be an explanation for severe COVID-19 in the general population. Distinct from the previously published studies that have identified (enrichment of) Treatments Follow-up rare TLR7 variants in severe COVID-19 who were 1) males <35 years of age without comorbidities (13), 2) males <60 years of age with or without comorbidities (14) or 3) a group of unselected Frontiers in Immunology | www.frontiersin.org 6 July 2021 | Volume 12 | Article 719115 Solanich et al. Rare TLR7 Variants in COVID-19 A B C FIGURE 2 | Assessment of ex vivo/in vitro type I and II interferon responses in peripheral blood mononuclear cells. In all panels, patient 13 is indicated in red and the healthy controls in black. (A) shows the fold change in TLR7 mRNA expression in patient 13 and a healthy male control after stimulation for 4 hours with the TLR7 agonist imiquimod, compared to the negative medium control RPMI. (B) displays the fold change in mRNA expression of type I IFN–related genes IRF3, IRF7, IFNB1 and ISG15 induced by imiquimod as compared to RPMI. (C) shows the production of IFN-g production after imiquimod stimulation for 7 days as compared with unstimulated control cells (RPMI) from patient 13 and two healthy male controls. The dotted line indicates the assay’s detection limit. Abbreviations: IRF3, interferon regulatory factor 3; IRF7, interferon regulatory factor 7; ISG15, interferon-stimulated gene 15; and IFNB1, interferon beta 1. patients (19), in this study we have selected males <50 years of healthy. Affected young brother pairs, as well as pedigrees age without comorbidities predisposing to severe COVID-19. suggestive for X-linked segregation, should be further prioritized. Despite the small cohort, the yield of TLR7 variants was We furthermore hypothesize that some rare variants unexpectedly high (14.3%) encouraging that screening for compromise essential functional domains in TLR7 and lead to TLR7 rare variants in severely affected men may be fruitful. full TLR7 deficiency (20), while other less rare variants might Elderly individuals also carry rare, loss of function TLR7 variants lead only to a partial TLR7 deficiency, and consequently impact a (14), but are more difficult to identify due to other predisposing larger group of individuals, but exert a lower relative risk to general risk factors for severe disease in people of advanced age. develop severe COVID-19. Accordingly, these low effect size Therefore, we suggest the following screening criteria: young genetic variants in TLR7 have been proposed as a possible men (<50 year of age) suffering from severe COVID-19 requiring explanation of the male sex bias in COVID-19 severity because at least high-flow oxygen therapy, and who were previously of its localization on the X chromosome and well-established Frontiers in Immunology | www.frontiersin.org 7 July 2021 | Volume 12 | Article 719115 Solanich et al. Rare TLR7 Variants in COVID-19 function in innate immunity (21). Furthermore, it is possible, but which is further corroborated by impaired type I and II IFN only speculative, that the addition of factors that deteriorate responses in the patient with the p.(Trp933Arg) variant. These TLR7 function [e.g. circulating 25-hydroxy vitamin D [25OHD] findings reinforce the notion that TLR7 plays a critical role in the levels decline with age (22) and may be accompanied by a lower recognition of SARS-CoV-2 and the subsequent initiation of an TLR7 expression and defective function (23)] in patients with early antiviral immune response that could prevent the these low effect variants may be a common cause of progression development of severe COVID-19. More importantly, a better to the most severe stages of COVID-19 in male. The same understanding of strong genetic factors predisposing to severe speculative hypothesis could be applicable to other genes COVID-19 may help physicians to identify patients at risk. We involved in immune response regulation after SARS-CoV-2 therefore propose clinical screening criteria for the identification infection (4, 24). of TLR7 variants in male patients with severe COVID-19. This Strong host genetic factors that confer an increased risk to could not only enable targeted therapeutic management of the develop severe COVID-19 might serve as genomic biomarkers, patient, but could also offer relatives the option of pre- along with other factors, that could be used for early diagnosis symptomatic testing and by extension preventative measures and preventative measures, and could allow the identification of such as early vaccination. possible molecular targets for treatment. In this respect, there would be a strong argument to offer hemizygous TLR7 deficient males that have not had COVID-19 direct access to early DATA AVAILABILITY STATEMENT vaccination as an effective preventative measure, similar to other patients with primary immunodeficiencies. This option The raw data supporting the conclusions of this article will be has indeed been offered to the hemizygous carriers in the family made available by the authors, without undue reservation. of patient 13. Although no data is yet available to support specific management of TLR7 deficiency or the at-risk hemizygous carriers, early hospitalization and IFN-based therapies in ETHICS STATEMENT inborn errors of IFN signaling form rational treatment options (18, 25). The studies involving human participants were reviewed and This study has several limitations. First, no biological samples approved by IDIBELL Research Ethics Committee (approval were available to functionally validate the pathogenicity of the number PR152/20). The patients/participants provided their p.(Asn215Ser) variant and therefore the functional impact of this written informed consent to participate in this study. Written variant remains unknown. Nevertheless, the absence of this informed consent was obtained from the individual(s) for the variant in population databases, the high prediction scores for publication of any potentially identifiable images or data pathogenicity and the general scarcity of rare variants in TLR7 included in this article. (13) suggest this finding is unlikely due to chance. Second, the TLR7 variant identified in patient 13 segregates in a brother who only experienced mild COVID-19. There are some explanations AUTHOR CONTRIBUTIONS that may explain why a pathogenic variant in TLR7 does not become fully penetrant, since protection against SARS-CoV-2 XS, GV-P, and CM contributed equally to this work. XS, AH, and depends on other genetic and environmental factors (e.g. CL devised the study. GC, CM, AR-M, FS, ME, and XC provided exposure to a lower initial viral load, previous exposure to input on the study design. AA, BH, JS-H, FV, and G-RB assisted common cold coronaviruses) (26). Moreover, TLR7 function in patient management. GV-P, AS, JV, and SR designed and might be specifically influenced by epidemiological factors and performed the sequence and functional analysis. XS, GV-P, CM, certain comorbidities (e.g. 25OHD). Thirdly, the size of this AH, and CL had full access to all data and take responsibility for study does not permit to draw any firm conclusions on the the integrity and the accuracy of the data. XS, GV-P, CM, AH, prevalence of loss of function TLR7 variants in males with severe and CL drafted the manuscript. All authors contributed to the COVID-19. Hence, larger cohort studies are required. Most article and approved the submitted version. recently, in pan-ancestry whole-exome sequencing data of 586,157 individuals, encompassing 20.592 patients who contracted COVID-19 and 1.266 of those who had severe FUNDING disease, TLR7 was the only gene in which the burden of rare variants was significantly increased among patients with severe Contract grant sponsor: Supported by La Marató de TV3 COVID-19, using a less conservative significance threshold [OR foundation (202115-30-31), the Carlos III National Health 4.53 (2.64, 7.77)] (19). This is one of the few occasions in which a Institute funded by FEDER funds – a way to build Europe – Mendelian disease gene is replicated through the use of an [PI19/00553 and CIBERONC]; the Government of Catalonia association study, even without stratifying for male gender or [2017SGR1282 and 2017SGR496]. AH is supported by the Solve- younger age. RD project. The Solve-RD project has received funding from the In summary, we have identified two novel germline variants European Union’s Horizon 2020 research and innovation in the X chromosomal TLR7 that likely lead to TLR7 deficiency, programme under grant agreement No. 779257. FV was Frontiers in Immunology | www.frontiersin.org 8 July 2021 | Volume 12 | Article 719115 Solanich et al. Rare TLR7 Variants in COVID-19 supported by a ZonMW (The Netherlands Organization for thank CERCA Programme/Generalitat de Catalunya for Health Research and Development) Vidi grant (No. 91718351). institutional support, and the unstoppable campaign of the This research was part of the Netherlands X-omics Initiative and Josep Carreras Leukaemia Foundation and the Cellex partially funded by NWO (The Netherlands Organization for Foundation. We also thank the Radboud Genomics Technology Scientific Research; project 184.034.019). Center for their technical support. ACKNOWLEDGMENTS SUPPLEMENTARY MATERIAL We sincerely thank the patients and their families for their The Supplementary Material for this article can be found online participation. With the support of COVID-19 funding from at: https://www.frontiersin.org/articles/10.3389/fimmu.2021. the Departament de Salut de la Generalitat de Catalunya. We 719115/full#supplementary-material 19 Disease in Males: Findings From a Nested Case-Control Study. Elife (2021) REFERENCES 10:e67569. doi: 10.7554/eLife.67569 1. Siddiqi HK, Mehra MR. COVID-19 Illness in Native and Immunosuppressed 15. Karczewski KJ, Francioli LC, Tiao G, Cummings BB, Alföldi J, Wang Q, et al. States: A Clinical-Therapeutic Staging Proposal. J Heart Lung Transplant The Mutational Constraint Spectrum Quantified From Variation in 141,456 (2020) 39(5):405–7. doi: 10.1016/j.healun.2020.03.012 Humans. Nature (2020) 581(7809):434–43. doi: 10.1038/s41586-020-2308-7 2. 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