This is the first study to demonstrate a possible association between TMPRSS2 p.Val160Met polymorphism and the degree of SARS-CoV-2 viral load as indicated by the Ct value of NAAT in patients with COVID-19. Patients with a CC genotype, which corresponds to the presence of valine amino acid, tend to display a lower Ct value (high viral load). We also found a trend of association between a CC genotype and mortality in a group of patients with severe COVID-19.
It is widely known that the SARS-CoV-2 virus enters the host cells via binding with ACE2, which acts as the main receptor for the viral particles [6, 9, 12]. The spike (S) protein of the SARS-CoV-2 virus consists of two sub-units: the S1 sub-unit, which is important for virus attachment and the S2 sub-unit, which is essential for membrane fusion. ACE2 molecule can bind to the S1 protein to promote virus invasion into the host cells [6, 13]. In human, ACE2 is expressed in many organs, such as the upper respiratory tract, alveolar epithelial cells, vascular endothelial cells and macrophages .
In addition to ACE2 several other molecules are involved in SARS-CoV-2 virus binding and cell penetration. The S protein needs to be cleaved to activate the endocytic route of virus entry and to enable membrane fusion. It has been reported that several host proteases are involved in the process of S protein breakdown. These include TMPRSS2, cathepsin L, furin  and NRP1 .
TMPRSS2 is a serine protease that can prime the S protein of SARS-CoV-2 to enable cell penetration [9, 14]. The expression of TMPRSS2 in VeroE6 cells facilitates SARS-CoV-2 virus entry and promotes virus invasion . Notably, treatment with the TMPRSS2 inhibitor (camostat mesylate) significantly reduced SARS-CoV-2 virus infection . Moreover, TMPRSS2 is also involved in SARS-CoV-1 virus infection , supporting the idea of the critical role of this molecule in mediating virus entry.
The human TMPRSS2 gene is located in chromosome 21.q22.3. It encodes protein that contains a transmembrane domain, low-density lipoprotein receptor class A (LDLRA) domain, scavenger receptor cysteine-rich (SRCR) domain and serine protease catalytic domain . At least six nucleotide variants within the human TMPRSS2 coding region that cause amino acid substitutions have been identified. These include p.Val160Met, p.Gly181Arg, p.Arg240Cys, p.Gly259Ser, p.Pro335Leu and p.Gly432Ala . Of these variants, the p.Val160Met variant is often associated with diseases, notably prostate cancer. A study conducted on a Japanese population indicated that the TMPRSS2 p.Val160Met variant (also known as Met160Val polymorphism) was associated with the risk of sporadic prostate cancer . Also, a study conducted on 214 patients with prostate cancer demonstrated that the T allele of this variant, which is associated with the presence of Met amino acid, was associated with TMPRSS2-ERG fusion and, thus, might be important in prostate cancer pathogenesis .
Interestingly, recent bioinformatic analysis studying the functional effects of nucleotide variants within the human TMPRSS2 gene revealed that the p.Val160Met variant was the most likely variant that might affect TMPRSS2 protein function and stability . Furthermore, a computational analysis to predict the effects of polymorphism on protein structure suggested that the Val160Met substitution might create a pocket protein by influencing several amino acid residues, which might affect TMPRSS2 structure and its role in SARS-CoV2 cell entry . Possible changes in TMPRSS2 function and/or structure due to the Val160Met substitution might explain our findings on the association of this SNP with the viral load in COVID-19 patients. Alteration of TMPRSS2 function/structure will likely affect binding of the S protein to ACE2 or the membrane fusion process. Reduction in TMPRSS2 enzymatic activity may decrease the furin cleavage of the S1 protein, which may subsequently decrease S2 fusion to the host’s cell membrane. However, further studies at the molecular level are required to prove this hypothesis, for example, by generating recombinant TMPRSS2 proteins bearing the variants and testing them in an in vitro model of SARS-CoV-2 cell infection.
Despite the association with the Ct value, we did not find any correlation between the pVal160Met variant and COVID-19 severity. This might be due to other confounding factors that strongly contribute to the severity of COVID-19. It is believed that factors, such as age , gender  and pre-existing diseases (hypertension, diabetes, CVD and lung disease) , strongly correlate with the risk of severe COVID-19. Further analysis with a larger study population is required to control these confounding variables. Interestingly, in patients with severe COVID-19, we observed a trend of association between this polymorphism and the mortality of COVID-19 patients. However, this requires further confirmation in studies with a larger sample size.
Several studies have found associations between genetic variations in the patient’s genome and COVID-19 severity. Many of the reported polymorphisms were related to genes involved in the development of inflammatory response, for example, polymorphisms in genes related to type 1 interferon immunity , polymorphisms in X-chromosomal TLR7  and polymorphisms within genes involved in the interleukin 1 signalling pathway . Our finding indicates a correlation between polymorphism in the gene encoding the virus receptor complex, i.e. TMPRSS2, and COVID-19 severity. This will contribute to the growing body of evidence on the crucial involvement of the host’s genetic factor in determining susceptibility to and/or severity of COVID-19. Furthermore, our data are consistent with those of a previous report that observed a decreasing allele frequency of the TMPRSS2 rs12329760 variant among patients with severe COVID-19 compared with patients with mild COVID-19 , indicating the importance of TMPRSS2 in COVID-19.