Several studies have expressed that the hyper-inflammatory response induced by SARS-CoV-2 is a major cause of disease severity and death [6, 12, 13]. Levels of interleukin TNF-α were reported in patients with severe COVID-19, although other reports [14, 15] suggest that other cytokines are involved in the pathogenesis of the illness. In order to explore the factors in the host, with the strongest effect on the clinical progression of COVID-19, we studied the − 308A > G polymorphism of TNFα gene located at the promoter region, which has been ascribed to the polymorphisms within the regulatory regions [16]. It results in two allelic forms, one in which guanine defines the common allele (TNFα.G) and the other in which guanine is replaced by adenosine, which behaves as the rarer allele (TNFα.A) at position − 308. The presence of the rarer allele was found to correlate with enhanced spontaneous or stimulated TNF-α production [17].
When comparing genotype and allele frequencies of TNFα− 308A > G polymorphism in individuals having SARS-CoV-2 infection, with those of the reference population, no relevant differences were found. This led us to infer that persons carrying the A allele are not more susceptible to SARS-CoV-2 infection. This contrasts with the report by Ahmed Saleh et al., who found that the A allele is expressed more frequently in patients than in the controls, and that individuals with AA and AG genotypes are more susceptible to COVID-19 [18]. We acknowledge that a limitation in our study is the fact that we did not control the allele and genotype frequencies according to ethnic background.
However, when the reference frequencies in Holguín province were compared to the frequencies of our study in that province, the distribution of genotypes differed, mainly due to an increased frequency of TNFα.G allele homozygotes. The low frequency of the TNFα.AA genotype in this population may be due to natural selection favoring individuals with a low production potential instead of those with a high secretion of TNFα. An important implication of the Hardy–Weinberg Law is that, for a rare allele, the frequency of heterozygotes is much higher than the unusual homozygote. This argument proves that it is difficult to eliminate recessive deleterious alleles from the population, since most of them are in a heterozygous state.
To better understand the differences in the clinical forms of SARS-CoV-2 infection, we compared the TNFα − 308A > G polymorphism genotypes according to disease severity. Our results suggest that the TNFα.AG genotype seems to be related to symptomatic forms of the disease, which could be explained considering that TNFα mediates in many symptoms such as fever [19], dry cough [20], muscle weakness [21, 22] and multiple organ disfunction [23], all of which are present in COVID-19 [24]. However, we found that the TNF.AA genotype may not be a relevant factor in more severe forms, although the number of homozygotes for the A allele was very low in individuals with severe forms of the disease. In addition, we found a higher tendency of being asymptomatic in the G allele homozygous individuals infected with SARS-CoV-2. When the genotype frequencies of the TNFα − 308A > G polymorphism in asymptomatic, mild symptomatic and severe symptomatic groups were compared to those of the reference population, random distribution was not found in the asymptomatic cluster, where the frequency of the TNFα − 308.AG genotype was lower and there was an increase of the TNFα − 308.GG genotype. This led us to infer that individuals with the GG genotype could be predisposed to develop asymptomatic forms of SARS-CoV-2 infection.
Reports show that patients with SARS-CoV-2 infection can develop a symptomatic or asymptomatic form of the disease, but most COVID-19 cases are symptomatic with a moderate fatality rate [25]. A large proportion of COVID-19 patients present common symptoms including fever, cough, sore throat, nasal congestion, weakness, fatigue or myalgia, dizziness, shortness of breath, muscle pain, arthralgia, chest tightness, overproduction of mucus with expectoration, hemoptysis and dyspnea [24] Our study agrees with others concluding that fever and cough are the most prevalent symptoms of the disease, in that order, although we did not find similar results regarding fatigue as the third most frequent symptom [26, 27]. Smell and taste loss were also frequent symptoms in our population, which agrees with others reporting these as the fourth most common symptom of SARS-Cov-2 infection [28]. The approximate proportion of severe versus mild symptomatic patients in our study was estimated as 1:8, it differs from that reported by other researchers with estimates of 1:4 [29].
COVID-19 has an average duration of approximately 6 weeks [24]. However, we observed a high variability in our COVID-19 patients. Recovery time was highly variable, with an average duration of 17.1 ± 6.8 days, which differs from other studies reporting a duration of more than 20 days [30, 31]. This finding may be related to the early start of therapeutic protocols in Cuba, as a result of an efficient active tracing of cases in the communities for the epidemiological control of active cases and their contacts. However, it is noteworthy that in the eastern region of the country, the median of the number of days from diagnosis to hospital discharge (after a negative test by RT-PCR) was higher than in the other regions. The presence of the A allele increases the number of days hospitalized in this region, but because of sample size, new studies with a larger number of individuals are recommended. This result is similar to that reported by Angioni et al. [14] who relates the high levels of TNFa with hospitalization time.
The analysis of the risk of a severe COVID-19 clinical form, according to region, with the presence of the A allele, showed a relationship between the polymorphism and disease severity only for the eastern region of Cuba, which is similar to what was reported in Mansoura (Egypt) where it was associated with a more severe disease [18]. Our results suggest that the presence of the A allele in the eastern Cuban population could be associated to a more symptomatic form of the disease and this may be related to variations in TNF-ɑ levels in the serum as a key mediator of the inflammatory response. However, the risk of developing COVID-19 symptoms had a tendency to increase in all regions, although not dramatically, in individuals carrying the − 308.A allele, even when analyzing this polymorphism accompanied by comorbidities [32] associated to the severity of the disease. Although plasma levels of TNF-α may be submitted to a multifactorial regulatory process, the local TNF-α concentration involved in the pathogenesis of COVID-19 [15] may be under a major control by the A allele of the TNFα.− 308 polymorphism. The use of anti-TNF therapy in individuals carrying the A allele may contribute to the reduction of disease symptoms. We consider that further studies are necessary to recommend this treatment in patients with specific symptoms of COVID-19, although this therapy has already been recommended in patients with acute respiratory distress syndrome (ARDS) within 2 days following hospital admission [33]. The use of this therapy has also been suggested for outpatients, as a treatment in high-risk individuals with COVID-19, such as elderly patients with comorbidities that can be appropriately monitored [34].