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

Abstract

Background: Recently, loss-of-function variants in TLR7 were identified in two families in which COVID-19 segregates like an X-linked recessive disorder environmentally conditioned by SARS-CoV-2. We investigated whether the two families represent the tip of the iceberg of a subset of COVID-19 male patients.

Methods: This is a nested case-control study in which we compared male participants with extreme phenotype selected from the Italian GEN-COVID cohort of SARS-CoV-2-infected participants (<60 y, 79 severe cases versus 77 control cases). We applied the LASSO Logistic Regression analysis, considering only rare variants on young male subsets with extreme phenotype, picking up TLR7 as the most important susceptibility gene.

Results: Overall, we found TLR7 deleterious variants in 2.1% of severely affected males and in none of the asymptomatic participants. The functional gene expression profile analysis demonstrated a reduction in TLR7-related gene expression in patients compared with controls demonstrating an impairment in type I and II IFN responses.

Conclusions: Young males with TLR7 loss-of-function variants and severe COVID-19 represent a subset of male patients contributing to disease susceptibility in up to 2% of severe COVID-19.

Funding: Funded by private donors for the Host Genetics Research Project, the Intesa San Paolo for 2020 charity fund, and the Host Genetics Initiative.

Clinical trial number: NCT04549831.

Keywords: COVID-19; LASSO Logistic Regression Analysis; TLR7; genetics; genomics; human; medicine.

Conflict of interest statement

CF, SD, SM, EB, NP, DF, FP, ES, MB, FF, MP, SL, FC, EQ, MV, SR, MS, MB, OS, KC, SF, FM, AR, MM, EF No competing interests declared

© 2021, Fallerini et al.

Figures

Figure 1.. 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.

Figure 2.. 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.

Figure 3.. 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 ().