The coronavirus disease-2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first detected in Wuhan, China, in 2019, followed by a widespread trajectory throughout the world (1), making COVID-19 one of the 21st-century pandemics owing to its high morbidity and mortality rates worldwide (2). Historically, several viruses from the same coronavirus family have emerged, including the famous SARS-CoV and MERS-CoV. However, the characteristics of the strain that caused the COVID-19 pandemic made the resulting infection more severe and the spread of the disease faster (1). SARS-CoV-2 has affected over 759 million individuals worldwide and caused over 6.8 million mortalities.

The severity and clinical outcomes of COVID-19 vary markedly among individuals, with some remaining asymptomatic, whereas others develop severe respiratory illnesses, organ damage, or even death. Identifying the factors that predispose individuals to COVID-19 susceptibility and poor outcomes has been the subject of intense research. One such area of investigation is the role of genetics in COVID-19 susceptibility and its exacerbation. Genetic factors have been proposed to contribute to the susceptibility to and severity of COVID-19.

Genome-wide association studies (GWAS) have been conducted to investigate the genetic factors that may contribute to the susceptibility and exacerbation of COVID-19 (3,4). These studies identified genetic variations associated with an increased risk of developing COVID-19 and severe clinical outcomes. These genetic variations affect various aspects of the immune response, including cytokine regulation, immune cell responses, and susceptibility to viral infections (4). One GWAS investigated different genetic mechanisms associated with COVID-19 in a large cohort of individuals. The study highlighted several single-nucleotide polymorphisms (SNPs) in several genes, such as interleukin-6 (IL6), tumor necrosis factor (TNF) and genes involved in angiotensin-converting enzyme 2 (ACE2) regulation, which have been proven to be involved in the immune response. Other significant SNPs were identified in the NOTCH4 (rs3131294) and TYK2 (rs74956615) genes, which have been proven to be associated with critical illness in COVID-19 patients and an exacerbating effect in these patients (3).

TYK2 encodes a tyrosine kinase involved in cytokine signaling pathways, including those of type I and III interferons (IFN) (5). TYK2 is also associated with the IFNAR1 subunit of type 1 IFN receptors (6). Interferons play a critical role in the antiviral immune response by inducing the expression of genes that inhibit viral replication and promote the destruction of infected cells. Several studies have identified TYK2 variants that are associated with an increased risk of severe COVID-19 symptoms. For example, Pairo-Castineira et al (3,4) reported that a missense variant in TYK2 (p.Arg203His) is associated with a higher risk of respiratory failure in COVID-19 patients (3,4). Another study reported that TYK2 haploinsufficiency is associated with an increased risk of severe COVID-19 in a cohort of patients with innate immunity defects. A GWAS conducted by the COVID-19 Host Genetics Initiative found that genetic variations in TYK2 were associated with an increased risk of severe COVID-19. Specifically, a study found that a rare variant of TYK2 is associated with a 3.5-fold increased risk of severe COVID-19(7). Another study identified that the rs2304257C>T polymorphism in TYK2 is associated with increased susceptibility to severe COVID-19 in the Chinese population (8). These findings highlight the importance of TYK2 in the immune response to SARS-CoV-2 infection, and suggest that TYK2 variants may serve as biomarkers for predicting COVID-19 outcomes.

Another genetic factor implicated in COVID-19 susceptibility and exacerbation is NOTCH4, which encodes a transmembrane receptor involved in immune system regulation. NOTCH signaling plays a critical role in the differentiation and function of immune cells, including T, B, and myeloid cells. NOTCH4 gene expression is increased in patients with COVID-19(9). This increased expression results in the disruption of the repair mechanism in Regulatory T cells, leading to the exacerbation of respiratory infections in COVID-19 patients (10). This also indicates that NOTCH4 signaling is an obstacle to tissue repair and a promoter of more severe respiratory infections (10,11). Several studies have reported an association between NOTCH4 variants and the risk of developing severe COVID-19 (3,10,11). These findings suggest that NOTCH4 plays a critical role in the immune response to SARS-CoV-2 infection and may serve as a potential therapeutic target for COVID-19.

The present study aimed to investigate the association between genetic variants of TYK2 (rs74956615) and NOTCH4 (rs3131294) and the severity and mortality of COVID-19 in a Jordanian cohort.

A total of 362 individuals diagnosed with COVID-19, comprising diverse populations from different geographic regions, were included in the analysis. Participants were categorized into three groups based on disease severity: Non-severe, severe and critical, and into two groups based on clinical outcomes: non-survivors and survivors. The clinical characteristics of the study participants were presented in Table I.

Genotypic and allelic distributions of TYK2 (rs74956615) and NOTCH4 (rs3131294), along with Hardy-Weinberg equilibrium testing and association measures, were summarized in Table II. The distribution of genotypes showed a predominance of the wild-type alleles for both SNPs, with relatively low frequencies of homozygous variant genotypes. HWE analysis based on complete-case data indicated no major deviation for TYK2 (P>0.05), whereas a slight deviation was observed for NOTCH4, which may reflect small genotype counts or missing data rather than genotyping error.

The present study addressed the association between genetic variants of TYK2 and NOTCH4 and the severity and clinical outcomes of COVID-19. The analysis encompassed a diverse population of 362 individuals from different geographic regions, who were categorized based on disease severity and clinical outcomes. The findings revealed significant associations between specific gene variants and the severity and mortality of COVID-19. The present study found that variant alleles of TYK2 and NOTCH4 were increased in individuals with COVID-19 as a function of disease severity and were associated with heightened mortality, demonstrating that this mechanism is effective in SARS-CoV-2 infection and may be critically involved in the pathogenesis of acute respiratory failure.

TYK2 produces tyrosine kinase, which is essential for cytokine signaling, particularly in the type I and III interferon pathways (5). TYK2 plays an essential role in the stable expression of IFNAR1 on the cell surface (17,18). Thus, appropriate TYK2 activity is a key step in the initiation of the type I IFN response (17,18). IFNs are important antiviral cytokines that induce the expression of genes that inhibit viral replication during the early stages of viral infection (6,17,18). Studies have shown that COVID-19 infection does not elicit a competent IFN response, leading to decreased disease severity (19,20). The analysis of TYK2 variants revealed a strong association between them and COVID-19 severity. This association is congruent with that reported by Pairo-Castineira et al (3), who reported that rs74956615 increases the severity of COVID-19 by 1.6 and has a very strong P-value (OR=1.6, discovery P=2.3x10^-8). Located within the chromosome 19 genomic sequence (NCBI Accession: NC_000019.10), this regulatory eQTL locus (at position 10,317,045) is highly critical because its risk allele correlates with upregulated TYK2 expression, functioning as an inflammatory driver that promotes the hyper-activation of downstream JAK-STAT cytokine signaling during severe infection (3). The genotype distribution varied markedly among the non-severe, severe and critical groups, with the critical group exhibiting a higher proportion of heterozygous genotypes than those in the other groups. This finding suggests a potential role for TYK2 variants in influencing the progression of critical illness (7). The observed association aligns with the existing literature implicating TYK2 in immune response regulation, supporting the idea that genetic variations in this gene may contribute to diverse clinical outcomes in COVID-19 patients (7). These findings may contribute to an improved understanding of the mechanisms underlying the disease progression.

Several studies have identified TYK2 variants that are associated with an increased risk of severe COVID-19 symptoms. For example, Pairo-Castineira et al (3) reported that a missense variant in TYK2 (p.Arg203His) is associated with a higher risk of respiratory failure in COVID-19 patients (4,17). Another study reported that TYK2 haploinsufficiency is associated with an increased risk of severe COVID-19 in patients with inborn errors of immunity (5). Notably, a study found that a rare variant of TYK2 is associated with a 3.5-fold increased risk of severe COVID-19 disease (7). Another study identified that the rs2304257C>T polymorphism in TYK2 is associated with increased susceptibility to severe COVID-19 in the Chinese population (8). These findings highlight the importance of TYK2 in the immune response to SARS-CoV-2 infection, and suggest that TYK2 variants may serve as biomarkers for predicting COVID-19 outcomes. As aforementioned, TYK2 prevents the accumulation of IFNAR1 in the intracellular compartment and increases its stabilization at the cell surface (17,18). However, another route of IFNAR1 stabilization at the cell surface is TYK2 independent. For example, the RNA-binding protein RBM47 stabilizes IFNAR1 transcripts (21).

NOTCH4 is a critical regulator of disease severity in various respiratory viral infections. NOTCH4 encodes a transmembrane receptor crucial for immune system regulation. NOTCH signaling is vital for the differentiation and function of immune cells, such as T, B and myeloid cells. Patients with COVID-19 exhibit elevated NOTCH4 gene expression levels, indicating its potential involvement in the disease (9).

The findings of the current study revealed that NOTCH4 variants were substantially associated with the severity of COVID-19. The critical group displayed a higher prevalence of heterozygous genotypes, indicating a potential correlation between NOTCH4 variants and critical illness development. This finding was consistent with the role of NOTCH4 in immune regulation and suggested that genetic variations in this gene may contribute to the variability in COVID-19 severity. Studies have reported that NOTCH4 expression increases in tissue Treg cells early in lung inflammation and promotes the innate immune response when adaptive immunity has not yet been effectively mobilized (10,11). In the context of severe virus-mediated damage, NOTCH4 promotes the development of an intrinsic protective response driven by lung epithelium-derived danger signals in the host. This physiological function of NOTCH4 is lost in severe respiratory viral diseases, leading to uncontrolled and excessive activation of innate immunity, which harms lung tissue (10). Moreover, several studies have reported an association between NOTCH4 variants and the risk of severe COVID-19 (3,22,23). These findings suggest that NOTCH4 plays a critical role in the immune response to SARS-CoV-2 infection and may serve as a potential therapeutic target for COVID-19. These findings could aid in the development of targeted therapeutic approaches for managing COVID-19 based on genetic profiles.

Examining the association between gene variants and clinical outcomes, the present study found that specific TYK2 variants were markedly associated with COVID-19 mortality. TA and AA variants were more prevalent in the non-survivor group, whereas the TT variant was predominant in the survivor group. This emphasized the potential prognostic value of TYK2 variants in predicting mortality outcomes in COVID-19 patients. Similarly, specific NOTCH4 variants were found to be markedly associated with COVID-19 mortality. The non-survivor group exhibited a higher prevalence of TA and AA variants, indicating the potential role of NOTCH4 gene variants as predictors of adverse clinical outcomes in COVID-19 patients.

GWAS examining genetic factors in COVID-19 susceptibility and severity have identified variations associated with an increased risk of contracting the virus and experiencing severe clinical outcomes (3,4,8). These genetic variations affect diverse aspects of the immune response, including cytokine regulation, immune cell responses, and susceptibility to viral infections (4). Notably, specific SNPs in genes such as NOTCH4 (rs3131294) and TYK2 (rs74956615) have been linked to critical illnesses in COVID-19 patients, underscoring the genetic basis of disease exacerbation (3,7).

The methodology for accurately stratifying the exacerbation level of COVID-19 patients can be based on a combination of clinical prediction scores, radiographic imaging and biomarkers. Studies have shown that combining these methodologies can lead to a more accurate and holistic assessment of a patient's condition (24-26). Binomial logistic regression analysis confirmed the significant effects of TYK2 and NOTCH4 variants on COVID-19 severity and mortality. The model explained a considerable proportion of the variance in both the severity and mortality, highlighting the potential predictive power of these genetic variants. Notably, individuals with heterozygous or variant alleles of NOTCH4 have markedly higher odds of exhibiting severe or critical disease, emphasizing the clinical relevance of this gene in determining disease severity. Additionally, heterozygous or variant alleles of both NOTCH4 and TYK2 were associated with increased odds of mortality, highlighting the potential utility of genetic information in identifying patients at a higher risk of adverse outcomes.

It is important to note that the methods mentioned in the present study have been utilized in several studies; however, it is recommended that further validation studies be conducted on diverse populations worldwide, including the population in which the present study was conducted. Additionally, these methods should not be used in isolation, but as part of an integrated approach that considers other risk factors, such as environmental and lifestyle factors.

Collapsing the heterozygous and mutant genotypes into a single ‘variant-carrier’ category is justified and consistent with the biological rationale and statistical characteristics of the present study dataset. First, the homozygous mutant genotype was extremely rare in the cohort, accounting for <1% of patients. Separate estimation of the effects of the mutant genotype would yield unstable odds ratios with wide confidence intervals and limited interpretability. Combining heterozygous and mutant individuals into a single ‘variant-present’ category is an accepted approach for rare alleles in genetic epidemiology and preserves the statistical power without introducing artificial comparisons. Second, previous GWAS and functional studies have demonstrated that both heterozygous and homozygous non-reference forms of TYK2 and NOTCH4 produce a similar directional biological effect on interferon signaling and immune dysregulation. Therefore, expanding the analysis to include additive, recessive and codominant models is unlikely to provide additional meaningful insights and may instead introduce unstable estimates due to sparse cell counts. Thus, the current modelling strategy remains justified.

Furthermore, the multiple-testing strategy employed in the present study must be understood within the framework of a hypothesis-driven design. The analysis concentrated on two predetermined SNPs (TYK2 rs74956615 and NOTCH4 rs3131294) derived from previous GWAS findings, rather than employing an exploratory genome-wide or multi-locus screening methodology. The Bonferroni correction for the small number of planned comparisons (eight tests) is therefore considered appropriate and sufficiently conservative. Extending the correction to encompass all potential genetic inheritance models (additive, recessive, and codominant) or implementing false discovery rate (FDR) adjustments would not conform to the established analytical framework and could introduce superfluous statistical penalties, especially in the context of sparse genotype frequencies.

The present study had some limitations. The sample size of the present study was relatively small. Future studies with larger sample sizes could provide more definitive insights into the roles of TYK2 and NOTCH4 in COVID-19 susceptibility and exacerbation. Additionally, the genetic factors examined accounted for only a small proportion of the COVID-19 susceptibility and severity. Other genes, environmental and clinical factors (such as treatment strategies) may also serve a role in COVID-19 severity and mortality; however, these factors were not analyzed in the present study, which is a limitation. Finally, data were collected during early 2020-2021; therefore, the dominant variant circulating in Jordan was the ancestral strain. Another limitation of the present study is the use of a dominant genetic model, whereby heterozygous and homozygous variant genotypes were combined due to the very low frequency of the mutant genotype; although this approach preserved statistical power, it precluded evaluation of additive or recessive effects, which could be explored in larger cohorts with sufficient genotype representation.

Another limitation pertains to the multiple-testing correction strategy. The present study applied the Bonferroni correction to the predefined comparisons, but did not apply it to all possible genetic models (additive, recessive and codominant) or adjust for FDR. This approach aligns with the study's hypothesis-driven design; however, it may increase the risk of residual type I error when interpreting the reported associations.

A further constraint is the absence of functional validation for the identified genetic associations within the study cohort. While rs74956615 has established eQTL regulatory roles (3), no independent mechanistic analyses (such as eQTL or sQTL evaluations) were conducted here to ascertain their specific biological impacts in the sample population. As a result, while these associations align with known expression pathways, direct causality within the cohort has yet to be determined.

In conclusion, the present study provided valuable insights into the roles of TYK2 and NOTCH4 variants in the severity and clinical outcomes of COVID-19. Understanding the genetic determinants of disease progression may contribute to the development of personalized and effective management strategies for COVID-19 patients. Further research and validation studies are warranted to confirm these associations and to explore the mechanisms underlying the interplay between host genetics and COVID-19 outcomes. Accurately stratifying the exacerbation level of COVID-19 patients is crucial for identifying high-risk patients who require intensive care and for guiding treatment and management strategies. A scientific methodology based on clinical data, including clinical prediction scores, radiographic imaging and biomarkers, combined with a multidisciplinary approach, can be used to accurately stratify COVID-19 patients based on the severity of their illness. Further research is needed to validate these methods and determine the most effective approach for stratifying COVID-19 patients based on disease severity. The present study provided evidence that genetic factors, specifically TYK2 and NOTCH4 variants, may contribute to COVID-19 susceptibility and severity. These findings have important implications for understanding the underlying mechanisms of COVID-19 and developing more targeted prevention and treatment strategies.