1. Genetics and Genomics
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Association of Toll-like receptor 7 variants with life-threatening COVID-19 disease in males: findings from a nested case-control study

  1. Chiara Fallerini
  2. Sergio Daga
  3. Stefania Mantovani
  4. Elisa Benetti
  5. Nicola Picchiotti
  6. Daniela Francisci
  7. Francesco Paciosi
  8. Elisabetta Schiaroli
  9. Margherita Baldassarri
  10. Francesca Fava
  11. Maria Palmieri
  12. Serena Ludovisi
  13. Francesco Castelli
  14. Eugenia Quiros-Roldan
  15. Massimo Vaghi
  16. Stefano Rusconi
  17. Matteo Siano
  18. Maria Bandini
  19. Ottavia Spiga
  20. Katia Capitani
  21. Simone Furini
  22. Francesca Mari
  23. GEN-COVID Multicenter Study
  24. Alessandra Renieri  Is a corresponding author
  25. Mario U Mondelli
  26. Elisa Frullanti
  1. Medical Genetics, University of Siena, Italy
  2. Med Biotech Hub and Competence Center, Department of Medical Biotechnologies, University of Siena, Italy
  3. Division of Infectious Diseases and Immunology, Department of Medical Sciences and Infectious Diseases, Fondazione IRCCS Policlinico San Matteo, Italy
  4. Department of Mathematics, University of Pavia, Italy
  5. University of Siena, DIISM-SAILAB, Italy
  6. Infectious Diseases Clinic, Department of Medicine 2, Azienda Ospedaliera di Perugia and University of Perugia, Santa Maria Hospital, Italy
  7. Infectious Diseases Clinic, "Santa Maria" Hospital, University of Perugia, Italy
  8. Genetica Medica, Azienda Ospedaliero-Universitaria Senese, Italy
  9. Department of Internal Medicine and Therapeutics, University of Pavia, Italy
  10. Department of Infectious and Tropical Diseases, University of Brescia and ASST Spedali Civili Hospital, Italy
  11. Chirurgia Vascolare, Ospedale Maggiore di Crema, Italy
  12. Department of Biomedical and Clinical Sciences Luigi Sacco, University of Milan, Italy
  13. III Infectious Diseases Unit, ASST-FBF-Sacco, Italy
  14. Department of Preventive Medicine, Azienda USL Toscana Sud Est, Italy
  15. Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Italy
  16. Molecular Mechanisms of Oncogenesis, ISPRO Core Research Laboratory (CRL), Italy
Short Report
Cite this article as: eLife 2021;10:e67569 doi: 10.7554/eLife.67569
3 figures, 2 tables and 2 additional files

Figures

Rare TLR7 variants and association with COVID-19.

LASSO logistic regression on boolean representation of rare variants of all genes of the X chromosome is presented. TLR7 is picked up by LASSO logistic regression as one of the most important genes on the X chr (Panel A). The LASSO logistic regression model provides an embedded feature selection method within the binary classification tasks (male patients with life-threatening COVID-19 vs infected asymptomatic male participants). The upward histograms (positive weights) reflect a susceptible behavior of the features to the target COVID-19, whereas the downward histograms (negative weights) a protective action. Panel B represents the cross-validation accuracy score for the grid of LASSO regularization parameters; the error bar is given by the standard deviation of the score within the 10 folds; the red circle (1.26) corresponds to the parameter chosen for the fitting procedure. Performances are evaluated through the confusion matrix of the aggregated predictions in the 10 folds of the cross-validation (Panel C) and with the boxplot (Panel D) of accuracy (60% average value), precision (59%), sensitivity (75%), specificity (43%), and ROC-AUC score (68%). The box extends from the Q1 to Q3 quartile, with a line at the median (Q2) and a triangle for the average.

Gene expression profile analysis in peripheral blood mononuclear cells (PBMCs) and in HEK293 cells transfected with the functional variants after stimulation with a TLR7 agonist for 4 hr.

(A) 5 × 105 PBMCs from COVID-19 patients and six unaffected male and female controls were stimulated for 4 hr with the TLR7 agonist imiquimod at 5 μg/mL or cell culture medium. Quantitative PCR assay was performed and the 2-ΔΔCt calculated using HPRT1 as housekeeping gene. Fold change in mRNA expression of TLR7 and type 1 IFN-related genes ISG15, IRF7, IFN-ɑ and IFN-γ induced by TLR7 agonist imiquimod was compared with cell culture medium. Ctl indicates healthy controls (white bar); C1, the asymptomatic mutated control (diagonal lines bar); P2, P5, cases with neutral variants (vertical lines bar); P1, P3, P8, P7 cases with functional variants (gray bar) (as in Table 2). (B) Histograms of intracellularly expressed TLR7 protein in HEK293 cells transfected with the different TLR7 plasmids. (C) Gene expression profile analysis of IFN-ɑ in transfected cells after stimulation with the TLR7 agonist imiquimod. WT indicates cells transfected with WT TLR7 plasmid. Quantitative PCR assay was performed and the 2-ΔΔCt calculated using HPRT1 as housekeeping gene. Fold change in mRNA expression induced by imiquimod was compared with cell culture medium. Error bars show standard deviation. p values were calculated for the reduction using an unpaired t test: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Segregation analysis.

Fold change in mRNA expression following Imiquimod stimulation of TLR7 itself and its main effectors, IRF7, ISG15, IFN-alpha, and IFN-gamma is shown in Panel A. Gray columns represent individuals harboring the TLR7 variant and black columns are severely affected SARS-CoV-2 cases. Pedigree (Panel B) and respective segregation of TLR7 variant and COVID-19 status (Panel C) are also shown. Squares represent male family members; circles, females. Individuals infected by SARS-CoV-2 are indicated by a virus cartoon close to the individual symbol ().

Tables

Table 1
Fisher exact test of the overall combined cohorts in young males (<60 years).
Clinical categoryN. wild-type variants (97.84%)N. pathological variants (2.15%)Total
Severely affected males1296135
Asymptomatic males1040104
Total2336239 (Grand Total)
  1. p-value=0.0037.

Table 2
TLR7 variants in severely affected Italian males -all ages- (cases).
Nucleotide changeAmino acid changedbSNPCADDExAC_
NFE
Function*N. of patientsClinical category†AgeCohortPatient ID
 c.901T>CSer301Pro-26.4N/ALOF1346ItalianP3
 c.2759G>AArg920Lysrs18968181116.520.0002LOF‡1449ItalianP6
 c.3094G>AAla1032Thrrs14724466222.30.0006LOF2365/66ItalianP7/P8
 c.655G>AVal219Ilers14931402312.280.0003HYPO1432ItalianP1
 c.863C>TAla288Valrs20014665815.370.000012Neutral1357ItalianP2
 c.1343C>TAla448Valrs574378113.080.00465Neutral2353/58ItalianP4/P5
  1. CADD, Combined Annotation Dependent Depletion; ExAC, Exome Aggregation Consortium; NFE, Non-Finnish European;

    *Function: HYPO, hypomorphic; LOF, loss-of-function;

  2. †Clinical category: 4, Hospitalized and intubated; 3, Hospitalized and CPAP-BiPAP and high-flows oxygen treated; 2, Hospitalized and treated with conventional oxygen support only; 1, Hospitalized without respiratory support; 0, Not hospitalized oligo/asymptomatic individuals.

    ‡based on in silico prediction.

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