COVID-19 susceptibility, severity, clinical outcome and Toll-like receptor (7) mRNA expression driven by TLR7 gene polymorphism (rs3853839) in middle-aged individuals without previous comorbidities

Abstract

Background: Toll-like receptors are implicated in the pathophysiology of the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory disease (MERS), according to several studies. The whole-genome sequencing of SARS-CoV-2 revealed that the TLR7 gene could be implicated in the virus's pathogenesis since the virus includes ssRNA patterns that could bind to TLR7.

Aim: The purpose of this study was to look into the function of the TLR7 (rs3853839) C/G polymorphism and the expression of TLR7 mRNA transcript in the development, severity and progression of COVID-19.

Subjects and methods: A case-control study included 285 participants who were divided into two groups: 150 middle-aged people with COVID 19 who had no previous co-morbidities and 135 healthy volunteers who served as controls. TaqMan test was used to genotype the TLR7 (rs3853839) C/G polymorphism, and real-time PCR was used to determine the relative expression of its mRNA transcript. The level of IL-6 in serum was determined using the ELISA method as an indicator of cytokine storm and COVID-19 severity.

Results: The GG genotype was shown to be much more common in COVID-19 patients (38.7%) than controls (4.4%), with an OR of 19.86 (95% CI: 7.85; 50.22) and was linked to disease severity and poor clinical outcomes (hospitalization, respiratory failure, cardiac complications, ICU admission and mechanical ventilation).As a result, the G allele was considerably higher in cases (57.0%), while the C allele was significantly higher in controls (p = 0.001). The GG genotype was found to be substantially more common in patients who were severely/critically unwell. TLR7 mRNA expression levels were significantly higher in COVID-19 patients (2.44 ± 0.89) than in controls (1.06 ± 0.46) (p = 0.001). TLR7 mRNA levels were highest in COVID 19 patients with the GG genotype (rs3853839). Patients with the GG genotype had considerably lower WBC counts, but significantly higher serum ferritin, CRP, IL-6 and D dimer levels (P = 0.045, 0.001, 0.023, 0.033, 0.001, respectively).

Conclusion: The GG form of the TLR7 SNP (rs3853839) could be a genetic risk factor for COVID-19 infection, severe illness and poor clinical outcome. TLR7 mRNA expression was also elevated in COVID-19 patients who were severely/critically unwell and had a bad outcome, suggesting that they could be used as COVID-19 prognostic biomarkers.

Keywords: ARDS, Acute respiratory distress syndrome; CBC, complete blood count; COVID-19; CRP, C-reactive protein; IL-6; MERS, Middle East respiratory disease, according to several studies; PCR; RT-PCR, reverse transcriptase polymerase chain reaction; SARS-CoV, Severe acute respiratory syndrome coronavirus; SNP; TLR7; TLR7, Toll Like Receptor 7; WHO, World Health Organization; mRNA; ssRNA, single-strand RNA.

Graphical abstract

Fig. 1

A - Allelic discrimination plot showing different genotypes of TLR 7 Gene SNP (rs3853839). An allelic discrimination plot or “cluster plot” shows three clusters. The points in each cluster are grouped closely together and express one genotype. At the lower right corner of the plot the cluster represents allele C (homozygote), labelled with VIC® dye. At the Upper left corner of the plot the cluster represents allele G (homozygote), labelled with FAM™ dye. Approximately midway between the allele C and allele G clusters represent both (allele C and allele G—heterozygote). B - Amplification plot of TLR7 gene expression displaying fluorescence versus cycle number. Amplification plots are created when the fluorescent signal from each sample is plotted against cycle number; therefore, amplification plots represent the accumulation of product over the duration of the real-time PCR experiment.

Fig. 2

A - Melting curve of TLR7 gene showing the specificity of the chosen primers. Melting curve can detect the presence of nonspecific products, such as primer-dimer, if they present, will appear as additional peaks to the left and right of the main peak for the amplified product in the melt curve. B - Association between TLR7 mRNA expression and SNP rs3853839 genotyping among cases.