Expression profiles of the SARS-CoV-2 host invasion genes in nasopharyngeal and oropharyngeal swabs of COVID-19 patients

Abstract

We collect the nasopharyngeal and oropharyngeal swabs of 63 subjects with severe symptoms or contacts with COVID-19 confirmed cases to perform a pilot-study aimed to verify the "in situ" expression of SARS-CoV-2 host invasion genes (ACE2, TMPRSS2, PCSK3, EMILIN1, EMILIN2, MMRN1, MMRN2, DPP4). ACE2 (FC = +1.88, p ≤ 0.05) and DPP4 (FC = +3, p < 0.01) genes showed a significant overexpression in COVID-19 patients. ACE2 and DPP4 expression levels had a good performance (AUC = 0.75; p < 0.001) in distinguishing COVID-19 patients from negative subjects. Interestingly, we found a significant positive association of ACE2 mRNA and PCSK3, EMILIN1, MMRN1 and MMRN2 expression and of DPP4 mRNA and EMILIN2 expression only in COVID-19 patients. Noteworthy, a subgroup of severe COVID-19 (n = 7) patients, showed significant high level of ACE2 mRNA and another subgroup of less severe COVID-19 patients (n = 6) significant raised DPP4 levels. These results indicate that a group of SARS-CoV-2 host invasion genes are functionally related in COVID-19 patients and suggests that ACE2 and DPP4 expression level could act as genomic biomarkers. Moreover, at the best of our knowledge, this is the first study that shows an elevated DPP4 expression in naso- and oropharyngeal swabs of COVID-19 patient thus suggesting a functional role of DPP4 in SARS-CoV-2 infections.

Keywords: ACE2; Biochemistry; Clinical genetics; Covid-19; DPP4; Genetics; Genomic biomarkers; Host invasion genes; Molecular biology; SARS-CoV-2; Virology.

Figure 1

Expression level of the eight genes involved in the SARS-CoV-2 entry into the host cell and infection. Kolmogorov-Smirnov test was used to analyze the distribution of expression data. Mann-Whitney test was used for data analysis. Expression data (2^−ΔCt) are represented as mean and range. A) Expression level of ACE2 gene in nasopharyngeal and oropharyngeal swabs of COVID-19 vs negative patients (FC = +1.88, p ≤ 0.05); H) Expression level of DPP4 gene in nasopharyngeal and oropharyngeal swabs of COVID-19 vs negative patients (FC = +3.0; p < 0.01); B-G) Expression level of TMPRSS2, PCSK3, EMILIN1, EMILIN2, MMRN1, MMRN2 genes in nasopharyngeal and oropharyngeal swabs of COVID-19 vs negative patients.

Figure 2

Expression level of ACE2 and DPP4 mRNA in COVID-19 patients, negative subjects, ACE2h-COVID-19 and DPP4h-COVID-19 patients. Panel A (left) ACE2 expression level in negative subjects, COVID-19 patients and ACE2h-COVID-19 patients; Panel A (right) DPP4 expression level in negative subjects, COVID-19 patients and DPP4h-COVID-19 patients. Panel B (left) ACE2 expression level in negative subjects, COVID-19 patients and DPP4h-COVID-19 patients; Panel B (right) DPP4 expression level in negative subjects, COVID-19 patients and ACE2h-COVID-19 patients. Kolmogorov-Smirnov test was used. Kruskal-Wallis test was used for data analysis. Expression data (2^−ΔCt) are represented as mean and range. Negative subjects n = 28; COVID-19 patients n = 28–29; ACE2h-COVID-19 patients n = 7; DPP4h-COVID-19 patients n = 6.

Figure 3

Receiver operator characteristic (ROC) curves. Receiver operator characteristic (ROC) curves between COVID-19 patients and negative subjects (set as control group). Area under the ROC curve (AUC) 0.75, 95% confidence interval 0.63 to 0.87, p-value < 0.001.

Figure 4

Correlation analysis of ACE2 and DPP4 expression in nasopharyngeal and oropharyngeal swabs. Scatter plots depicting the relationship between ACE2 and (A) PCSK3 mRNA expression (R² = 0.139, p < 0.05, Pearson r = 0.372); (B) EMILIN1 mRNA expression (R² = 0.147, p < 0.05, Pearson r = 0.383); (C) MMRN1 mRNA expression (R² = 0.331, p < 0.0005, Pearson r = 0.575); (D) MMRN2 mRNA expression (R² = 0.339, p < 0.0005, Pearson r = 0.582). Scatter plot depicting the relationship between DPP4 and (E) EMILIN2 mRNA expression (R² = 0.114, p < 0.05, Pearson r = 0.338).

Figure 5

Correlation analysis of ACE2 and DPP4 m-RNA levels with clinical data in nasopharyngeal and oropharyngeal swabs of COVID-19 patients. Scatter plots depicting the relationship between (A) ACE2 mRNA and number of lymphocytes (Lym) (R² = 0.190, p < 0.005, Pearson r = -0.436; (B) DPP4 mRNA and CRP levels (R² = 0.118, p < 0.05, Pearson r = -0.344).

Figure 6

Correlation analysis of ACE2 m-RNA expressions with clinical data in nasopharyngeal and oropharyngeal swabs of ACE2h-COVID-19 patients. Scatter plots depicting the relationship between (A) ACE2 and number of neutrophils (neu) (R² = 0.645, p < 0.05, Pearson r = -0.803); (B) ACE2 and LDH levels (R² = 0.578, p < 0.05, Pearson r = -0.760).