1 Introduction

Corona-virus disease19 (COVID-19), a global pandemic initiated in Wuhan city of China in December 2019 responsible for 578,142,444 human infections with 6,405,080 deaths worldwide [1]. The etiological agent SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) belongs to the family beta-Coronaviridae [2]. Symptoms associated with COVID-19 include pneumonia, bronchitis, cough, sore throat, stuffy nose, body weakness and high fever as like a cold. However, in some cases, it might be asymptomatic [3]. Various characteristics like geographical region, hypertension, obesity, race, male gender and older age are vital risk factors for COVID-19 infection [4]. Furthermore, several genetic factors also contribute to COVID-19 infection, where variations among individuals are inherited. The genetic polymorphism (variants) in those genes that code for the entry receptors of SARS-CoV-2 change the pattern of susceptibility to COVID-19 infection [5]. Polymorphism in DNA can be transferred from one generation to another one and is only identified in 1% of the population; however, involved in the variation of susceptibility to different complex and multigenic infections like COVID-19 among other individuals [6].

SARS-CoV-2 contains four vital proteins, including nucleocapsid (N), envelope (E), membrane (M), and spike (S) proteins in their envelope where transmembrane serine protease 2 (TMPRSS2) splits up the S proteins into two parts S1 and S2 upon successful attachment with host receptor ACE2 [7]. One of the host essential proteins named interferon-induced transmembrane (IFITM) proteins play a vital role in the antiviral defense mechanism during both innate and adaptive arm of immunity [8]. Population genetics has a significant role in the variation of susceptibility to COVID-19 infection where genetic polymorphism [9] in various key genes involved in infection pathways is of great interest [10]. There are two ways to assess the association of genetic polymorphism and risk of infection development; the first is to study the genetic linkage while the second is to evaluate their association with diseases that is candidate gene and genome-wide association studies [11].

Angiotensin-converting enzyme 2 (ACE2) receptors in humans are generally used by RBD (receptor binding domain) of the S1 subunit of SARS-Cov-2, which activates the S2 subunits, which express fusion peptides responsible for the entry of the virus into the infected cell membrane [12]. SARS-CoV-2 fuses with exfoliated ACE2, degrading it, which leads to a decline of free ACE2 and, thus, the homeostasis of ACE/ACE2 disturbs responsible for the development of aggressive inflammatory reactions which promotes endothelial and interstitial fibrosis as well as myocardial hypertrophy [13]. Polymorphism in ACE2 is frequently observed [14], where approximately 1700 polymorphism has been reported to date, which mostly correlated with high protein expression and are most popularly found in the East-Asian populations [15]. Previous studies have reported association of ACE2 polymorphisms with increased neurological complications among the infected population of COVID-19 [16]. Transmembrane protease, serine 2 (TMPRSS2) is a vital host protease involved in the entry of SARS-CoV-2 into the cell, where polymorphism is strongly correlated with frequent COVID-19 infection [17]. Polymorphism in TMPRSS2 and ACE2 genes ultimately leads to the disturbance in the structure and functions of proteins which might be a risk factor for severity of various viral infections of respiratory system like COVID-19 and influenza [18]. In the current study previously reported polymorphisms of ACE2 rs2285666 and TMPRSS rs2070788 were screened in Pakistani COVID-19 patients to determine their role in COVID-19 susceptibility in Pakistan.

2 Material and methods

2.1 Criteria for patients collection

In the preset work, protocol was approved by Ethical Review Board of Isra University Islamabad, Pakistan, COMSATS University Islamabad under approval Ethical approval number F-2/IUIC-ANMC/EC-245/2022 in accordance with the Declaration of Helsinki for medical research involving human subjects.

A total of 300 (normal control n = 150; COVID-19 positive n = 150) individuals visited the Isolation Hospital and Infectious Treatment Center (IHITC), Islamabad, Pakistan, that visited the hospital between 2020–2021 were selected/included for the study. Patients with COPD, Pneumonia, Asthma and Chronic Medical illnesses e.g. liver disease, lung Malignancy, & heart diseases like congestive Cardiac Failure, Chronic Renal disease and those attending hospitals other than IHITC were not included in this study.

2.2 Data and sample collection of COVID-19 patients

The demographics including age, gender, and physiological factors like cough, fever, and shortness of breath were recorded through a comprehensive questionnaire for physical and physiological assessment of the patients (Table S1). In addition, a total of 10 cc of arterial blood was collected through a sterilized syringe, stored in EDTA vacutainer tubes and stored at 4 °C temperature till further processing.

2.3 DNA extraction and genotype determination

Leukocytes in the whole blood were used for DNA extraction through standardized phenol–chloroform extraction method, as previously described by Sambrook & Maniatis, [25]. The quantity of DNA was evaluated through NanoDrop® and samples were stored till amplification of targeted DNA sequence of ACE II (rs2285666) and TMPRSS2 (rs12329760) using primers [19] and amplification condition listed in along with annealing temperature and amplicon size listed in Table 1. The Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR) was carried out in a total of 25 µl of PCR mixture containing each primer of 1 pmol, 50 ng genomic, and 12.5 µl Master Mix (1X) (Ampliqon, Denmark) was prepared. A 35-cycle PCR was performed where each cycle comprised of denaturation for 30 s at 95 °C, annealing for 30 s (for respective primers in Table 1), extension for 30 s, and final extension for 10 min at 72 °C. UV trans-illuminator was used for the visualization of bands after successful running at 100 V for 30 min at 3% agarose gel.

Table 1 The amplification refractory mutation system polymerase chain reaction (ARMS-PCR) primers along with annealing temperature and amplicon size
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2.4 Statistical analysis

The entire categorical data was analyzed using SPSS® 23.0 (SPSS Inc., Chicago, Illinois) software. The categorical parameters were compared between groups, and genotypic distribution was done by performing Chi-Square test (χ2). Odds Ratio (OR) was estimated by keeping Confidence Interval (CI) at 95% for evaluation of the association of allele frequencies and genotypes, as well as other parameters with the possibility and severity of COVID-19 disease, through multiple logistic regression analyses. The test was considered statistically significant, where p values were less than 0.05.

3 Results

3.1 Demographics of the studied cohort

The Age Median & IQR of the non-COVID-19 to COVID-19 group was 50 and 52 respectively. The samples that were collected during the study period showed a higher percentage of male being affected with COVID-19 than females (Table 2). In addition, among the infected patients, a significantly higher (p = 0.03) number belonged to asymptomatic individuals 58% as compared to symptomatic 42% in the COVID-19 infected patients (Table 2).

Table 2 Descriptive statistics including gender, age and symptomatology in COVID-19 patients and non-COVID-19 group
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3.2 Association analysis of ACE2 rs2285666

In Table 3 genotype and allele frequency distribution analysis for ACE2 rs2285666, in COVID-19 patients and healthy control is represented. Out of 150 COVID-19cases 6.6% are C/C homozygous,34.6% are C/T are heterozygous and 58% are T/T homozygous. Out of 150 controls 33.3% are C/C homozygous, 23.3% are C/T heterozygous and 43.3% are T/T homozygous. The Allelic frequencies of both alleles C and T in the COVID-19patients were 24% and 76% respectively, whereas in healthy controls the frequencies were 45% for C and 55% for T allele, respectively. A significant association of the genotypes under both dominant and recessive models [DM: OR ratio = 7.000, 95% CI (3.387–14.465), p = 0.000]; [RM: OR = 1.856, 95% CI (1.174–2.935), p = 0.011] were observed with the COVID-19 infection. In addition, the risk allele T association was also found with COVID-19 infection [OR = 2.591, 95% CI (1.827–3.674), p = 0.000]. A similar trend of association was observed with male and female genders in gender-based analysis (Table 3). Our data therefore indicated that there is a significant association of the risk allele T with the COVID-19 patients’ infection in Pakistani population.

Table 3 Statistical analysis of overall cohort and gender-based analysis of ACE2 rs2285666
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3.3 Association analysis of TMPRSS2 rs2070788

In Table 4, genotype, and allele frequency distribution analysis for TMPRSS2 rs2070788, in COVID-19patient and healthy control is represented. Out of 150 COVID-19cases 88.6% were A/A homozygous,11.33% were A/G heterozygous and 0% were homozygous. Out of 150 Controls 80.6% were A/A homozygous, 18% were A/G heterozygous and 1.33% were G/G homozygous. The Allelic frequencies of both alleles A and G in the COVID-19 patients were 94% and 6%, respectively, whereas in healthy control the frequencies were 90% for A and 10% for G allele respectively. Though no genotype association was observed of the SNP under dominant and recessive logistic analysis [DM: OR = 0.533, 95%CI = 0.279–1.019 and p = 0.077; RM = OR = 0.493 95% CI = 0.044–5.499 and p = 0.498]. However, a marginal G allele protective association was observed [OR = 0.521, 95% CI = 0.282–0.964 and p = 0.049]. In the gender-based analysis the polymorphism did not show association with any of the gender (Table 4). The data of the current study revealed no association of TMPRSS2 rs2070788 with COVID-19 infection in Pakistani population.

Table 4 Statistical analysis of overall cohort and gender-based analysis of TMPRSS2 rs2070788 (N = 300)
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4 Discussion

COVID-19 is an infectious viral disease of SARS-CoV-2 etiology responsible for cytokine storm because of the elevated level of anti-inflammatory and pro-inflammatory cytokines [20]. COVID-19 pandemic has also hit Pakistan, increasing with the emerging number of COVID-19 cases, 20th May 2022; COVID-19 has caused 1,529,541 confirmed cases with 30,379 deaths in Pakistan[21].

In the current study male population was highly infected with SARS-CoV-2 as compared to female population. In agreement with our findings two previous studies also reported similar trend of male being infected more than females [22]. This is presumably because men are more vulnerable to COVID-19-1919 due to several reasons. Females have unique features such as their genome showing lower ACE 2 receptor expression and differences in sex hormones, making them more resistant to this viral infection [23]. Other possible reasons may include that males have high morbidity rates due to diabetes and heart problems contributing to their sex-based severity to COVID-19 [24]. Age wise distribution shows that older individuals are more prone to SARS-CoV-2 infection. This is probably due to a weak immune system, a higher prevalence of comorbidities increasing with age [25]. Another reason might be extended family type, mainly due to crowded living conditions that favors viral disease spread very quickly. The same is being concluded in various relevant studies [26].

In the current study, most of the COVID-19 individuals were with moderate symptoms. Similar results were reported by previous study [27]. The possible reason might be that there is a need for comprehensive screening to determine asymptomatic carrier prevalence. Comprehensive analysis of asymptomatic carriers would provide circulating hidden viral strain important information in the community. The number of asymptomatic carriers in high high-density urban areas is highly varied [28]. Han et al., (2020) [29] find that during hospitalization, milder clinical manifestation is observed in asymptomatic patients, though symptoms severity in asymptomatic carriers among all confirmed cases highly varies. Current study finds cough (90%) followed by fever (80%) and shortness of breath (75%) clinical features more common in severely infected SARS-CoV-2 hospitalized patients as this disease affects the respiratory system directly. Petersen et al., (2020) [30] show a similar finding in their scientific report. The similarities in signs and symptoms include abnormal chest x-rays, pneumonia and shortening of breath among severe COVID-19 patients due to a similar study design. A common type of symptoms like difficulty in breathing, fatigue, cough, and fever has been reported also previously [31]. Various studies suggested that lower respiratory system infection is strongly associated with the severity of COVID-19 [32].

The significantly elevated level of ACE2, which works as a SARS-CoV-2 entry receptor, is observed considerably in endothelial cells, alveolar epithelial type II cells, and airway cells of the cardiovascular and respiratory system [33]. This study also observed that COVID-19 patients containing GG genotype in ACE2 receptors were more prone to COVID-19 infection. This study also observed another genotype (AA) (rs2285666). This SNP is found in the intronic region of the ACE2 where it can change mRNA splicing and affect the expression of the gene on the protein level [34]. Similar to our results of wild type genotype [35], the GG genotype is strongly linked with risk and hyper prevalence of COVID-19 infection likewise found in the Indian and Caucasian populations [36]. In contrast to our study no association of GG genotype and mortality and severity rate of SARS-CoV-2 infection [37]. Similar to the current study results, a study found a strong association and -expression of AA genotype in ACE2 in diabetes mellitis Mellitus type II patients compared to other genotypes [34]. The strong binding between ACE2 and SARS-CoV-2, followed by increased virus replication in host cells, is responsible for imbalance in the renin-angiotensin system (RAS) [38]. The imbalance in RAS in such a way that local Ang-II upregulation while downregulation of Ang-(1–7) starts inflammation and coagulation inside lung tissue [39]. Besides the increased expression of Ang-II, which causes inflammation during COVID-19 [40], the Ang- (1–7) is responsible for the regulations of various intracellular signaling pathways which facilitates anti-fibrotic, anti-inflammatory, anti-proliferative and vasodilation effects upon binding to the Mas receptor [41]. Ang-II also activates the AT receptors which is responsible for vasoconstriction, endovascular thrombosis, and endothelial damage by starting coagulation pathway, observed during COVID-19 infection [42]. As per publications, ACE receptors play multiple physiological roles, and that’s why the cross-model of ACE/ACE2 were causing an imbalance in RAS, which is involved in the pathogenesis of respiratory, diabetes, and cardiovascular diseases may exacerbate COVID-19 infection [43].

The current study observed various genotype in TMPRSS2 involved in various physiological and pathological conditions like pain, digestion, tissue remodeling, cell death, cell invasion, and inflammation [44]. TMPRSS2 is an androgen-regulated cell surface serine protease responsible for activating the S-protein of several highly pathogenic coronaviruses, including SARS-CoV-2 [45]. The report observed that engineered VeroE6/TMPRSS2 cells were ten times more susceptible to SARS-coV-2 attack as compared to VeroE6 cells [46]. A decreased rate of SARS-CoV-2 replication in TMPRSS2 deficit mice has been observed elsewhere [47]. The genotype TT allelic forms where dominant model (TT + CT vs. CC), codominant model (TT vs. CC), and recessive model (TT vs. CT + CC) significantly leads to hyper-infection by COVID-19. Other studies also suggest the human genetic initiative after observing the association of risk and genetic susceptibility for SARS-CoV-2 associated with polymorphism of TMPRSS2, including p.Val160Met (rs12329760) in order to fight this pandemic [48]. As a key host protease for the replication of influenza, MERS, HCoV-229E, SARS-CoV-1, and SARS-CoV-2, TMPRSS2 is expressed in respiratory tracts [46]. Prior research revealed a correlation between intronic SNPs in the TMRPSS2 gene [49] and heightened severity of SARS-CoV-2 and influenza A virus infection [49]. Other studies also observed a link between COVID-19 susceptibility and severity with overexpression and variant of TMPRSS2 and ACE2 [8], although there is a need for further research to evaluate the association of TMPRSS2 polymorphisms in the severity and infectivity of SARS-CoV-2.

It has long been established that Mendelian and complex diseases are largely influenced by genetic variations and mutations in the coding regions of human DNA [50]. Accordingly, the genome-wide microarrays [51] and genome technologies of WES [52] have been widely used for the molecular diagnosis and gene discovery of harmful CNVs and SNVs for human diseases, particularly congenital abnormalities. However, recent advancement in genomic and genetic investigations has found functional genetic variants in non-coding areas of the human genome. Whole genome sequencing (WGS) may therefore be instructive and promising for clinical practice in the future [53] not only for its capacity to identify SV, including copy number neutral SV and CNV like translocations and inversions, is noteworthy but also makes robust identification of SV possible. The need for technological and methodological advancements to support data mining and functional interpretation from the millions of non-coding variants present in a single human genome is another requirement of this research trend [54].

5 Conclusion

The current study concluded that the male population of old age is more prone to infection where cough, fever, and shortness in breath are commonly observed symptoms among COVID-19 patients. The single nucleotide polymorphism (SNPs) in various vital genes like ACE1/ACE2 and TMPRSS2 can significantly change the outcome of the disease. One of limitations of the study was small sample size which might have resulted in not finding significant association of TMPRSS2 in the current cohort. As previous GWAS studies on larger cohort have reported strong association of TMPRSS2 SNP with COVID-19 infection. Further research is needed to evaluate the association of genetic polymorphisms in various COVID-19 19-associated genes and the severity and infectivity of SARS-CoV-2.