Role of the alpha-1 antitrypsin towards progression and severity of COVID- 19 infection among Saudi patients

Role of the alpha-1 antitrypsin towards progression and severity of COVID- 19 infection among Saudi patients | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Role of the alpha-1 antitrypsin towards progression and severity of COVID- 19 infection among Saudi patients Rabab A. Ali, Walaa A. Mohammedsaeed, Hesham A. Fakher, Hala K. Noor, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4725061/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Oct, 2025 Read the published version in BMC Infectious Diseases → Version 1 posted 4 You are reading this latest preprint version Abstract Background Alpha-1 antitrypsin (A1AT) is involved in pathophysiology of severe COVID-19, including thrombosis expansion. A1AT has anti-inflammatory, tissue-protective, and anticoagulant capabilities. We aimed to screen frequencies of A1AT gene polymorphism among COVID-19 Saudi patients and its relation to severity. Methods Through cross-sectional study, we examined 100 COVID-19 Saudi patients to explore possible correlation between A1AT/interleukin 6 (IL-6) ratio and COVID-19 severity. The COVID-19 patients grouped as severe (31 patients) and non-severe (69 patients) cases. A1AT gene polymorphism was conducted using the PCR technique (ARMS) and ELISA. Results A1AT, IL-6, and vitamin D (VIT-D) showed extreme statistical significance among COVID-19 patients (severe, mild, and asymptomatic). The prevalence of A1AT gene mutation was higher among COVID-19 cases compared with non-mutated patients (56% vs. 44%). Moreover, serum A1AT levels were lower while serum IL-6 levels were higher than reference range and highly significant among mutated cases compared with non-mutated cases. Also, IL-6/A1AT ratio in severe COVID-19 patients (mean 1.4) was significantly higher compared with asymptomatic or moderate patients (0.16, 0.21; respectively). Strictly, all COVID-19 patients have severed deficiency of VIT-D level significant among mutated and non-mutated cases ( p <0.04 and p <0.03; respectively). The frequency of MM (wild type) was substantially high among asymptomatic cases compared with severe cases (67.2% vs. 16.1%). Heterozygous MS+MZ genotypes showed lower frequency among asymptomatic cases compared with severe and mild cases (27.6% vs. 48.4% and 72.7%; respectively). On the other hand, the more severe forms of SS+ZZ+SZ genotypes were all relatively rare with lower frequency among asymptomatic compared with mild and severe COVID-19 cases (5.2%, 27.3% and 35.5%; respectively). Interestingly, homozygous SS genotype elicited higher frequency among severe cases compared with mild or asymptomatic cases (22.6% vs. 0% and 5.2%). The more severe forms homozygous ZZ genotype vanished among asymptomatic and mild cases. This extensively illuminated that, severe COVID-19 patients have diminished A1AT response towards inflammation. Conclusion Two haplotypes (S) and (Z) alleles of A1AT have higher frequency and were clearly recognized among severe COVID-19 cases suggesting that SS and ZZ genotypes may be associated with an increased risk, while MM genotype may be protective against severe COVID-19 infection. α1-Antitrypsin (A1AT) Polymorphism COVID-19 Interleukin 6 Vitamin D (VIT-D) Figures Figure 1 Figure 2 Background Severe COVID-19 exerts excessive inflammatory response, which results in extensive alveolar and endothelial injuries and eventually leads to hypoxemia and respiratory failure [ 1 ]. Alpha-1 antitrypsin (A1AT), is a serpin protease inhibitor. It is most widely distributed serpin and third most abundant protein in plasma [ 2 ]. A1AT is an acute phase protein, and systemic infection or inflammation causes its levels to rise 3–5 times [ 2 ] and can be generated by macrophages, hepatocytes, intestinal epithelial cells, and bronchial epithelium. A1AT has a knack to scavenge free radicals and govern development and release of cytokines and chemokines [ 3 ]. The majority of A1AT haplotype distribution in human population is composed of genotype combinations consisting of M, S, and Z. Additionally, PI*MM genotype may yield 100% normal A1AT and is present in 85–95% globally. The PI*MS, PI*MZ, PI*SS, PI*SZ, and PI*ZZ constitute 5–15% [ 4 ]. Most A1AT gene mutations lead to production of mutant proteins, which is dysfunctional and causes cell injuries [ 5 ]. The PI*ZZ genotype is linked to increased risk of illnesses concurrent with A1AT unworthy, whereas PI*SZ, PI*SS, and PI*MZ genotypes are only correlated with a possible increased risk [ 5 ]. The reason for A1AT's inability to prevent SARS-CoV-2 entry could be unprocessed ACE2-mediated cell entry in lack of TMPRSS2 or presence of other proteases that could cleave S protein. It has been demonstrated that A1AT inhibits SARS-CoV-2 viral replication in cell lines, including human airway epithelial preparations [ 6 ]. The alpha-1 antitrypsin deficiency (A1ATD) has been identified as one of the biochemical and clinical predictors of COVID-19 in Italy [ 7 ]. The goal of this research was to ascertain whether Saudi Arabia's gene frequency of A1AT alleles and the severity of the COVID-19 pandemic are correlated. Furthermore, we aimed to ascertain if the ratio of A1AT to IL-6 levels is related to COVID-19 severity. Methods and materials Study subjects The study recruited 100 COVID-19 Saudi individuals (32–87 years-old) chosen randomly from King Fahd hospital in Madinah, Saudi Arabia (69 males and 31 females), divided into 3 categories (asymptomatic, mild, and severe) according to their clinical characteristics, symptoms, and chest radiography [ 10 ]. A signed informed consent was obtained from participants. Sampling Peripheral blood samples were obtained from each participant and divided into 2 tubes: one for EDTA tubes used for DNA extraction using Gentra® kits, and the other for plain tube used for enzyme linked immunosorbent assay (ELISA). Enzyme linked immunosorbent assay (ELISA) The A1AT and IL-6 concentrations were determined using ELISA development kits (CUSABIO®, USA). The microplate reader with wavelength 450nm was used to validate optical density. The experiment was repeated twice, each time with identical materials contained within the individual experiments themselves. Genotyping of A1AT polymorphism The amplification refractory mutation system (ARMS) technique was used to analyze A1AT subsequent mutation. The PI gene locus that encodes A1AT (14q32.1). PCR was done for either S or Z allele in reaction volume 50µl, including 15µl (2X) PCR master mix (Promega®, Germany), 26µl distilled water, 2µl of each primer (Metabion®, Germany) ( Table 1A) , mixed with 3µl DNA. Each DNA was separately amplified for S allele using primers 1, 2 and 3 (wild tube) and primers 1, 2 and 3a (mutant tube). Similarly for Z allele using primer 5,6 and 7 (wild tube) and primers 5, 6 and 7a (mutant tube). PCR conditions are shown in Table 1B . Using 1.5% agarose gel, PCR products were electrophoresed and visualized under a UV illuminator. Data analysis The statistical package for social science (SPSS) version 25 was used to analyze data obtained. To determine statistical significance of non-parametric variable's difference between more than 2 study groups, the Kruskal-Wallis test was employed. The receiver operating characteristic (ROC) curve is used for assessing specificity and sensitivity of quantitative diagnostic measures. Significant P values are defined as < 0.05 or 0.001 at 95% confidence Interval. Results ARMS interpretation of the A1AT S locus The 220 bp as internal control (primer 1 & 2) was generated; and the 152 bp product was observed in both tube or only one tube according to homozygous or heterozygous (S) DNA ( Fig. 1). ARMS interpretation of the A1AT Z locus The 360 bp as internal control (primers 5 & 6) was generated; and the 150 bp product was observed either in both tube or only one tube according to homozygous or heterozygous Z DNA ( Fig. 2) . Biochemical data of the COVID-19 patients Table 2A shows the biochemical data of participants. A total of 100 COVID-19 patients. Out of them (2%) with asthma; 15% have Diabetes mellitus; 4% with cardiovascular disease; 3% with chronic kidney disease and no cases with chronic obstructive pulmonary disease (COPD) or liver diseases. The neutrophil-to-lymphocyte ratio (NLR) is determined. In individuals with severe COVID-19, NLR was found to be higher compared to those with asymptomatic or mild cases, with mean NLRs of (10, 2.5 and 7.11; respectively). Table 2B shows association between A1AT, IL-6, and VIT-D and different status (asymptomatic, mild, and severe) COVID-19 patients. The three-biomarkers showed extreme statistical significance through comparing the 3 groups of patients. All cases have deficient VIT-D, which was statistically significant through comparing asymptomatic with severe patients. A1AT showed low levels below reference range and statistically significant through comparing each pair of groups. This elucidate that deficiency of both A1AT, and VIT-D are associated with inflammation and autoimmunity. As a result, severe patients exhibited elevation in IL-6 levels than asymptomatic or moderate patients, despite the latter group experiencing reduction in A1AT levels. Potential risk factors and frequency analysis of the A1AT genotypes Table 2C shows that prevalence of A1AT gene mutation was higher among COVID-19 cases compared with non-mutated patients (56% vs. 44%). Serum A1AT levels were lower than reference range and highly significant among mutated cases ( p =0.01) compared with non-mutated augmenting the severity of illness among 3 groups of COVID-19 patients. In addition, serum IL-6 levels were significantly higher than reference range among third COVID-19 group but extreme higher among mutated compared with non-mutated boosting role of A1AT. Also, VIT-D levels were more interesting all COVID-19 cases were significantly lower than reference range among mutated and non-mutated cases ( p =0.04 and p =0.03; respectively). Analysis of the binary logistic regression analysis of A1AT genotypes Table 3A shows the logistic regression analysis, with the status of various cases serving as dependent variable and presence or absence of S/Z allele as primary independent variable. It reveals higher frequency with extremely significant difference of MM genotype among asymptomatic patients compared with severe cases (67.2% vs. 16.1%), p =0.001, [OR=0.09, 95% CI (0.03-0.28)], while the severe form MZ genotype appeared only among mild and severe states and have higher frequency of mild compared with severe (72.7% vs. 9.7%), p =0.001, [OR= 0.04, 95% CI (0.01 to 0.24)]. The disappear of Z allele from asymptomatic cases and appearance of high frequency of Z allele among mild cases compared with severe cases (50% vs. 12.9%) p =0.001, [OR= 0.15, 95% CI (0.05 - 0.45)] may be attributed to lack of proper follow up for the cases due to their hospital discharge without precise follow up after they exhibited severe complications later. In addition, patients own the more severe forms SS+ ZZ+SZ genotypes have higher frequency statistically significant among sever compared with asymptomatic patients (35.5% vs . 5.2%), p =0.001, [OR=10.08, 95%CI (2.55-39.9)]. The S allele was associated with greater incidence about 3 times among severe compared with asymptomatic patients (46.8% vs . 19%) p =0.001, [OR= 3.75, 95% CI (1.9-7.42)]. This means that people with either Z or S allele boost severity of COVID-19 infection than those with the other alleles and increased risk many times. Analysis of the association between A1AT genotypes and A1AT, IL-6 and VIT-D Table 3B shows that the A1AT, IL-6 levels showed statistically significant with A1AT genotypes, where patients carrying MM genotype had highest level of A1AT and lowest IL-6 level. On the other hand, low A1AT and high IL-6 levels noticed among the heterozygous group carrying MS, MZ, SS, ZZ, or SZ genotypes. Interestingly, only asymptomatic COVID-19 patients showed statistically significant association between VIT-D levels and A1AT genotypes this may be attributed to large number of cases while other groups have small number and nearly equal in levels. Extrapolated increased probability for infection with COVID-19 is associated primary with VIT-D deficiency. Table 4A shows that 15 individuals have diabetes, exhibited varying degrees of severity in their COVID-19 infection. 6 out of 31 patients were severe COVID-19 and 9 out of 58 were asymptomatic. Despite 6 out of 15 (46.6%) carrying MM genotype of A1AT, but they were positively associated with low levels of circulated A1AT. In addition, severe group have significantly low A1AT level ( P =0.02) compared with asymptomatic. Furthermore, the frequency of mutant A1AT genotypes (PI*MZ and PI*SS) was high in severe cases (n=3 and n=1; respectively) and (n=1 and n=2; respectively) among asymptomatic individuals with diabetes. Table 4B revealed linear regression analysis for A1AT, IL-6, and VIT-D to predict potential risk factors for COVID-19 patients having different A1AT genotypes. In terms of A1AT and IL-6 levels, MM genotype was associated with high A1AT levels and low IL-6, while homozygous or hetero genotype was associated with low A1AT levels. On the other hand, all forms of genotypes weren’t statistically significant predictor of VIT-D levels. Table 4C reveals that receiver operating characteristic (ROC) curve of A1AT, IL-6, and VIT-D data was executed for discrimination between healthy subjects and COVID-19 patients. A1AT and VIT-D showed highest accuracy AUC (1.0) as a diagnostic ability for COVID-19. We could explain that IL-6 has variable levels influenced by the severity of case and treatment. Table 4D showed the ROC curve of A1AT, IL-6, and VIT-D data was conducted for discrimination between severe and non-severe COVID-19 patients. A1AT and IL-6 showed high accuracy AUC 0.955 and 0.953; respectively. The VIT-D showed low accuracy AUC (0.665) as diagnostic ability for severe COVID-19. Discussion The COVID-19 is a multifactorial illness as genetic and environmental factors may contribute to incidence rates observed in various nations and ethnic groups. Age and comorbid disorders like diabetes, hypertension, and liver and kidney ailments are examples of non-genetic variables [8].Acute respiratory distress syndrome (ARDS) is the most severe COVID-19 symptoms [9].The global percentage of the population infected with COVID-19 grew because of combined impact of these variables. One of strength of this study is that it is considered the first publication included A1AT gene polymorphism among infected Saudi COVID-19 patients. This cross-sectional study showed that 24% of cases were associated with other diseases. About half of them were classified severe cases. A1AT gene polymorphism was examined in this study. This is only one example of a hereditary disorder that could make individuals more prone to COVID-19. A hereditary illness referred to as A1ATD causes liver and/or lung damage. The symptoms of A1ATD are often misinterpreted due to their overlapping with pulmonary and hepatic disorders. This has led to a significant underdiagnosis of A1ATD globally. The symptoms of A1ATD-related lung ailments are like those of other obstructive lung conditions, and patients are often misdiagnosed for years [10]. Comorbid conditions linked to A1ATD, such as diabetes, hypertension, COPD, and chronic renal disease, are also independently linked to a higher incidence of COVID-19 [8]. Several studies hypothesized that severe impact of COVID-19 is influenced by areas of high prevalence of A1ATD [3] . A1AT has been revealed to block this protease, which might hinder SARS-CoV-2 cell entry and thus minimize viral replication[6].Additionally, the study demonstrated that sera from participants with an A1AT-deficient genotype have a decreased capacity to prevent the entry of Wuhan-Hu-1 (WT) and B.1.617.2 (Delta), but not B.1.1.529 (Omicron). The results illustrated potential involvement of these serum parameters in controlling viral entry in vivo [11]. Table 2C suggested that SS and ZZ genotypes may be tied with an increased risk of severe COVID-19 infection, while the MM genotype may be protective against severe COVID-19 infection. Furthermore, number of A1ATD individuals among Saudi population is probably not clear or not accurate because some patients with A1ATD -such as children- have not yet been diagnosed with COPD or liver illness linked to A1ATD. These preliminary findings inspired us to move forward with additional plans to ascertain the frequency of A1AT in sizable portion of Saudi population and additionally seek out other genotypes, including VIT-D. Another study that investigated healthy Saudi population, registered frequency of mutant S and Z A1AT alleles (9.49% and 3.19%; respectively) [12]. The PI*ZZ A1AT genotype's global prevalence in patients with COPD was evaluated in a recent study that comprised 48 nations. According to the study, which covered 11 Asian nations, rate of COPD in persons over 40 years is 9%. The greatest PI*ZZ weighted average prevalence among COPD individuals. Saudi Arabia was found among high values [13]. Similarly, another study revealed high prevalence of Z allele has been observed among Saudi Arabia (15:1000) [10]. Further confirmatory investigations conducted among Italians from different locations revealed that the region most impacted by COVID-19, northern Italy, had higher prevalence of the deficient linked phenotypes SZ, MZ, and ZZ than Southern region, with 47% of all cases only documented [14]. It was also reported that, a robust correlation was noticed between the PI*Z variant and the number of COVID-19 cases (r = 0.8584) and deaths (r = 0.8713) in 68 countries [15]. The PI*ZZ, PI*SZ, and PI*MZ rates are projected to be approximately 12% higher for Europeans and Latinos, who also make up the majority ethnic group in the nations with the largest number of COVID-19 cases and deaths [16]. On the other hand, a recent retrospective analysis bizarre in small cohort of hospitalized subjects revealed that only 2 were found to be PI*MZ and none for PI*MS, PI*SZ, PI*SS, or PI*ZZ individuals who are heterozygotes for A1AT alleles are not at high risk of acquiring severe COVID-19 [11]. Furthermore, it can be challenging to distinguish out baffling effect of patients' awareness of their A1ATD status. According to a survey performed in Germany, individuals suffering from A1ATD were 65% less likely to have been infected than the general population, and they were more likely to restrict their social groupings as a result to their greater concern about infection [12]. It was revealed that, there was no increment in either SARS-CoV-2 infection or death rates in presence of S- or Z-A1AT alleles among British patients [16]. Patients with PI*MM and PI*SS alleles, and non-smoking patients with the PI*MZ allele, do not have an abundant risk of lung ailments [10]. People with PI*MM genotype, concomitant with defective S and Z alleles have serum A1AT levels that are reduced by around 40% and 85%; respectively [17]. Because COVID-19 is linked to increased oxidative stress status [18], higher levels of A1AT may become useless. This fact was emphasized in (Table 4 ) that revealed 6 out of 15 (46.6%) diabetics were carrying MM A1ATgenotype, but they were positively associated with low levels of circulated A1AT, and this can be due to uncontrolled glucose levels. VIT-D deficiency level may link with type 2 diabetics to A1ATD which may cause a higher frequency COVID-19. We could explain the non-significance of certain genotypes due to small numbers and nearly similar levels of the parameters. Interestingly that all our COVID-19 patients have VIT-D deficiency. In addition, A1AT homeostasis in diabetic nephropathy leads to reduced levels of A1AT [19]. The non-enzymatic glycosylation of A1AT or oxidation of methionine in A1AT active site, may be the trigger for the decline in serum trypsin inhibitory capacity [20]. It was revealed that, VIT-D active form drives CD4 + T cells to emit A1AT, via direct contact with complement C3a, strengthens IL-10 secretion; implying that A1AT is crucial for active VIT-D to induce IL-10. linked with inflammation status and autoimmunity [21]. It was evident that there is a probable link between VIT-D levels and COVID-19 severity and lethality [22]. Our study revealed strong association with VIT-D, as there was high significant between asymptomatic and severe patients (Tables 2B,2C,3B). VIT-D has robust safeguard against acute lung injury and ARDS via regulating RAS and limiting bradykinin buildup. On other hand, COVID-19 patients utilize cytokine storm and bradykinin storm to counterfeit the virus. In agreement with our results, is well-known that COVID-19 patients have substantial declines in VIT-D levels [23]. In our study, severe patients exhibited a greater elevation in circulating IL-6 levels than asymptomatic or moderate patients, despite the latter group experiencing a reduction in A1AT levels. Hyper-inflammation and coagulopathy are hallmarks of COVID-19, and in more severe instances, an intensifying "cytokine storm" may trigger respiratory failure, sepsis, and even death [23]. Extrapolated the severity of COVID-19 patients is linked to a rise in the IL-6/A1AT ratio, which suggests an inadequate response of A1AT to IL-6 production. The IL-6/A1AT ratio in severe COVID-19 patients (mean 1.4) was significantly higher compared to asymptomatic or moderate patients (0.16, 0.21, respectively, P=0.001). This extensively elucidated that severe patients have a decreased A1AT response to inflammation (Table 2B,2C). It was postulated that, innate immune defense relies heavily on A1AT, whose plasma concentration may spike 2-4 times during the acute phase protein response. Protease-antiprotease disparities may emerge during the COVID-19 clinical course of the virus [24]. It was clearly reported that, patients in the intensive care unit (ICU) demonstrated a higher IL-6/A1AT ratio. Furthermore, clinical improvement was observed in ICU patients whose IL-6/A1AT ratio declined during treatment; no improvement was noted in patients whose ratio remained larger [25]. Another study emphasized that primary cytokine storm tracked in COVID-19 individuals exhibiting mild to severe symptoms is serum IL-6 [26]. A1AT can alter actions resulting in descending IL-6 inhibition, which has a crucial role in COVID-19 pathogenicity [27]. Within a multi-center investigation involving more than two thousand COVID-19 patients, the Pi*Z allele and/or a plasma A1AT level less than 116 mg/dl were significantly linked to severe COVID-19 as opposed to non-severe cases [26]. We could have concluded from table (8 A, B) that IL-6 and ATT can be used as predictor for severity of COVID-19; While VIT-D was a fuzzy (ambiguous) risk factor for COVID-19 especially overall patients have VIT-D deficiency. Results from another study showed that, in comparison to Italian population, patients with A1ATD had a considerably increased relative risk (8.8; P=0.0001) of symptomatic severe SARS-COV-2. 209 persons with significantly lower serum A1AT levels and A1AT-deficient genotypes participated in the survey [28]. Furthermore, in our severe COVID-19 patients, the NLR was found to be higher compared to those with asymptomatic or mild cases, with mean NLRs of 10, 2.5, and 7.11; respectively (Table 2 and 3). Severe COVID-19 progression is predicted by incremented absolute neutrophil count, elevated neutrophil percentage, and high blood neutrophil: lymphocyte ratio [29]. One of the postulates of the mechanisms by which A1AT can alleviate COVID-19 is the creation of neutrophil extracellular traps (NETs), a complex extracellular structure made of neutrophil-derived DNA, histones, and proteases that is linked to the immune-thrombosis of COVID-19, can be resisted by A1AT suppression of elastase [8]. Notably, dysregulated neutrophil elastase activity has been linked to diminished plasma concentrations or A1AT function, which might occur in pulmonary emphysema [30]. It was declared that significant higher severity and mortality were linked to NLR ratios of ≥4.5 and ≥6.5; respectively [2]. In addition, under hypoxic conditions, ZZ-A1ATD neutrophils produce more ROS than MM control subjects [30]. Conclusion A1AT may be viewed as a protective host factor against COVID-19. A1AT deficient allele was shown to be highly prevalent in Saudi Arabian population. It is likely justified to implement a screening program for those who are at risk, enabling them to take appropriate preventive action. Abbreviations A1AT Alpha-1 Antitrypsin A1ATD Alpha-1 Antitrypsin Deficiency ARDS Acute Respiratory Distress Syndrome ARMS Amplification-Refractory Mutation System COPD Chronic Obstructive Pulmonary Disease COVID-19 Coronavirus Disease 19 ELISA Enzyme Linked Immunosorbent Assay ICU Intensive Care Unit IL-6 Interleukin 6 PCR Polymerase Chain Reaction Saudi Arabia SA VIT-D Vitamin D Declarations Ethics approval and consent to participate The Saudi Arabia Ministry of Health's (MOH) General Administration for Research & Studies (IRB) and faculty of Applied Medical Sciences at Taibah University provided ethical approval (approved IRB-452) for the study to be conducted. Consent for publication Not applicable. Availability of data and material Data related to this manuscript is available on the hand of corresponding author and will be obtained under a reasonable request. Declaration of competing interests The authors affirm that no conflict of interest exists that might be seen as compromising objectivity of the study they have published. Funding This work was fully financially supported by the Deanship of Scientific Research (DSR)- Taibah University- Saudi Arabia under the project number RC-442/11. Author contributions RA: lead, study design, data analysis, writing introduction, methodology, results, and discussion parts. WM: data analysis and manuscript revision. HF: samples gathering. YA: data analysis, interpretations, and help in writing discussion part. HN: help in the practical work. All authors read and authorized the final manuscript. Acknowledgments The authors extend their appreciations to the Deanship of Scientific Research (DSR)-Taibah University, Saudi Arabia for their sincere continuous enormous help. Also, we thank the staff of the hospital for their help and support throughout the study. References Smadja DM, Mentzer SJ, Fontenay M, Laffan MA, Ackermann M, Helms J, et al. COVID-19 is a systemic vascular hemopathy: insight for mechanistic and clinical aspects. Angiogenesis 2021; 24(4): 755-88. Bai X, Schountz T, Buckle AM, Talbert JL, Sandhaus RA, Chan ED, et al. Alpha-1-antitrypsin antagonizes COVID-19: a review of the epidemiology, molecular mechanisms, and clinical evidence. Biochem Soc Trans 2023; 51(3):1361-75. Teckman JH, Lindblad D. Alpha-1-antitrypsin deficiency: diagnosis, pathophysiology, and management. Curr Gastroenterol Rep 2006; 8(1):14-20. Blanco I, Bueno P, Diego I, Pérez-Holanda S, Casas-Maldonado F, Esquinas C, et al. Alpha-1 antitrypsin Pi*Z gene frequency and Pi*ZZ genotype numbers worldwide: an update. Int J Chron Obstruct Pulmon Dis 2017; 12:561-9. de Serres FJ, Blanco I. Prevalence of α1-antitrypsin deficiency alleles PI*S and PI*Z worldwide and effective screening for each of the five phenotypic classes PI*MS, PI*MZ, PI*SS, PI*SZ, and PI*ZZ: a comprehensive review. Ther Adv Respir Dis 2012; 6(5):277-95. Azouz NP, Klingler AM, Callahan V, Akhrymuk IV, Elez K, Raich L, et al. Alpha 1 antitrypsin is an inhibitor of the SARS-CoV-2-priming protease TMPRSS2. Pathog Immun 2021; 6 (1):55-74. Yang C, Keshavjee S, Liu M. Alpha-1 antitrypsin for COVID-19 treatment: Dual role in antiviral infection and anti-Inflammation. Front Pharmacol 2020; 11: 615398. Jordan RE, Adab P, Cheng KK. Covid-19: risk factors for severe disease and death. BMJ 2020; 368: m1198. Zhou F, Yu T, Du R, Fan G, Liu Y, Liu Z, et al. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet 2020; 395(10229):1054-62. Craig TJ, Corbett ML, Meadows JA. Improving detection of alpha-1 antitrypsin deficiency: Role of the allergist. J Allergy Clin Immunol Pract 2023; 11(8): 2348-54. Nygren D, Mölstad U, Thulesius H, Hillman M, Broman LM, Tanash H, et al. Low prevalence of mild alpha-1-antitrypsin deficiency in hospitalized COVID-19-patients. Int J Gen Med 2022; 15: 5843-8. Shapira G, Shomron N, Gurwitz D. Ethnic differences in alpha-1 antitrypsin deficiency allele frequencies may partially explain national differences in COVID-19 fatality rates. FASEB J 2020; 34(11):14160-5. Ferrarotti I, Ottaviani S, Balderacchi AM, Barzon V, De Silvestri A, Piloni D, et al. COVID-19 infection in severe Alpha 1-antitrypsin deficiency: Looking for a rationale. Respir Med 2021;183:106440. Blanco I, de Serres FJ, Fernandez-Bustillo E, Lara B, Miravitlles M. Estimated numbers and prevalence of PI*S and PI*Z alleles of α1-antitrypsin deficiency in European countries. Eur Respir J 2006; 27(1): 77-84. Yoshikura H. Epidemiological correlation between COVID-19 epidemic and prevalence of α-1 antitrypsin deficiency in the world. Glob Health Med 2021; 3(2): 73-81. Schneider CV, Strnad P. SARS-CoV-2 infection in alpha1-antitrypsin deficiency. Respir Med 2021;184: 106466. Blanco I, Bueno P, Diego I, Pérez-Holanda S, Lara B, Casas-Maldonado F, et al. Alpha-1 antitrypsin Pi*SZ genotype: estimated prevalence and number of SZ subjects worldwide. Int J Chron Obstruct Pulmon Dis 2017; 12: 1683-94. Wang JZ, Zhang RY, Bai J. An anti-oxidative therapy for ameliorating cardiac injuries of critically ill COVID-19-infected patients. Int J Cardiol 2020; 312:137-8. Piwowar A, Knapik-Kordecka M, Warwas M. Concentration of leukocyte elastase in plasma and polymorphonuclear neutrophil extracts in type 2 diabetes. Clin Chem Lab Med 2000; 38(12):1257-61. Yaghmaei M, Hashemi, Shikhzadeh MA, Mokhtari M, Niazi A, Ghavami S. Serum trypsin inhibitory capacity in normal pregnancy and gestational diabetes mellitus. Diabetes Res Clin Pract 2009; 84(3): 201-4. Dimeloe S, Rice LV, Chen H, Cheadle C, Raynes J, Pfeffer P, et al. Vitamin D (1,25(OH)2D3) induces α-1-antitrypsin synthesis by CD4+ T cells, which is required for 1,25(OH)2D3-driven IL-10. J Steroid Biochem Mol Biol 2019; 189: 1-9. Kazemi A, Mohammadi V, Aghababaee SK, Golzarand M, Clark CT, Babajafari S. Association of vitamin D status with SARS-CoV-2 infection or COVID-19 severity: A systematic review and meta-analysis. Adv Nutr 2021; 12(5):1636-58. Jain A, Chaurasia R, Sengar NS, Singh M, Mahor S, Narain S. Analysis of vitamin D level among asymptomatic and critically ill COVID-19 patients and its correlation with inflammatory markers. Scientific Reports 2020; 10(1): 20191. O'Brien ME, Murray G, Gogoi D, Yusuf A, McCarthy C, Wormald MR, et al. A review of alpha-1 antitrypsin binding partners for immune regulation and potential therapeutic application. Int J Mol Sci 2022; 23(5):2441. Farcas M, Csernik F, Popp R-A, Trifa A, Crisan T, Petrisor F, et al. The interleukin 4 VNTR polymorphism frequency in a romanian population group. Annals of the Romanian Society for Cell Biology 2009,14(1):97-101. McElvaney OJ, McEvoy NL, McElvaney OF, Carroll TP, Murphy MP, Dunlea DM, et al. Characterization of the inflammatory response to severe COVID-19 illness. Am J Respir Crit Care Med 2020; 202(6): 812-21. Pedersen SF, Ho YC. SARS-CoV-2: a storm is raging. J Clin Invest 2020; 130(5): 2202-05. Rodríguez Hermosa JL, Vargas Centanaro G, González Castro ME, Miravitlle M, Lázaro-Asegurado, Jiménez-Rodríguez BM, et al. Severe COVID-19 illness and α1-antitrypsin deficiency: COVID-AATD study. Biomedicines 2023; 11(2):516. Philippe A, Puel M, Narjoz C, Gendron N, Durey-Dragon MA, Vedie B, et al. Imbalance between alpha-1-antitrypsin and interleukin 6 is associated with in-hospital mortality and thrombosis during COVID-19. Biochimie 2022; 202:206-11. Magallón M, Castillo-Corullón S, Bañuls L, Pellicer D, Romero T, Martínez-Ferraro C, et al. Hypoxia enhances oxidative stress in neutrophils from ZZ alpha-1 antitrypsin deficiency patients. Antioxidants 2023;12(4):872. Tables Tables 1 to 8 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files TablesBMCID.docx figuresuppl.docx Cite Share Download PDF Status: Published Journal Publication published 28 Oct, 2025 Read the published version in BMC Infectious Diseases → Version 1 posted Editorial decision: Revision requested 17 Jul, 2024 Editor assigned by journal 17 Jul, 2024 Submission checks completed at journal 15 Jul, 2024 First submitted to journal 11 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4725061","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":328160515,"identity":"98fa5f24-973d-47c5-80bb-57d7b7f76b4a","order_by":0,"name":"Rabab A. Ali","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArElEQVRIiWNgGAWjYDCCA2DShoFBgkQtaaRrOUyCFr7bB9g+/Kg5n9g/u/ngA4Yam2iCWiTPJTDP7Dl2O3HGnWPJBgzH0nIbCGkxOMPAzMDDdjux4UaOmQRjw2HitDD++XcucT5JWph52w4kbiBaiyRIi2xfsvHGG2nJBgnE+IUP5LA33+xk591IPvjgQ40NYS0MDPwfQKQjWGUCYeUIYE+K4lEwCkbBKBhhAACoqD/d5KyxtgAAAABJRU5ErkJggg==","orcid":"","institution":"Taibah University","correspondingAuthor":true,"prefix":"","firstName":"Rabab","middleName":"A.","lastName":"Ali","suffix":""},{"id":328160516,"identity":"a3115b47-f76a-40e1-8ac4-92f0f912ecad","order_by":1,"name":"Walaa A. Mohammedsaeed","email":"","orcid":"","institution":"Taibah University","correspondingAuthor":false,"prefix":"","firstName":"Walaa","middleName":"A.","lastName":"Mohammedsaeed","suffix":""},{"id":328160517,"identity":"4fd44e04-f9c1-4d7b-b6ca-efd6e1591ea5","order_by":2,"name":"Hesham A. Fakher","email":"","orcid":"","institution":"Ministry of Health","correspondingAuthor":false,"prefix":"","firstName":"Hesham","middleName":"A.","lastName":"Fakher","suffix":""},{"id":328160518,"identity":"b7645b63-2113-449c-bbe0-b05a4cab8bee","order_by":3,"name":"Hala K. Noor","email":"","orcid":"","institution":"Fakeeh College for Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hala","middleName":"K.","lastName":"Noor","suffix":""},{"id":328160519,"identity":"6b6af4bc-43ae-4434-9617-f523a5c0b8cb","order_by":4,"name":"Yasir M. Al Qurashi","email":"","orcid":"","institution":"Taibah University","correspondingAuthor":false,"prefix":"","firstName":"Yasir","middleName":"M. Al","lastName":"Qurashi","suffix":""}],"badges":[],"createdAt":"2024-07-11 15:12:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4725061/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4725061/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12879-025-11699-4","type":"published","date":"2025-10-28T15:58:44+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62222337,"identity":"57798526-5214-4890-99db-550653debd04","added_by":"auto","created_at":"2024-08-11 12:36:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":54776,"visible":true,"origin":"","legend":"\u003cp\u003ePCR-ARMS product on agarose gel for A1AT gene polymorphism in COVID-19 patients. Lane (M): Shows DNA ladder (100 -1200 bp). Lanes (1 \u0026amp; 2): Patient 1 M/M homozygote polymorphism of A1AT where Lane (1): shows internal control at 220 bp and M at 152 bp while Lane (2): shows only internal control at 220 bp. Lanes (3, 4): Patient 2 S/S homozygote polymorphism of A1AT where Lane (3): shows only internal control at 220 bp; while Lane (4) shows internal control at 220 bp and S at 152 bp. Lanes (5 \u0026amp; 6): Patient 3 M/S heterozygote polymorphism of A1AT where Lane (5): shows internal control at 220 bp and M at 152 bp while Lane (6): shows internal control at 220 bp and S at 152 bp.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4725061/v1/2dda3ab1b5c5c078515b68f9.png"},{"id":62222340,"identity":"c8bff54b-d436-49f1-b979-cf649cbf2610","added_by":"auto","created_at":"2024-08-11 12:36:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":54508,"visible":true,"origin":"","legend":"\u003cp\u003ePCR-ARMS product on agarose gel for A1AT gene polymorphism in COVID-19 patients. Lane (M): Shows DNA ladder (100-1200 bp). Lanes (1,2): Patient 4 M/M homozygote polymorphism of A1AT where Lane (1): shows internal control at 360 bp and M at 150 bp while Lane (2): shows only internal control at 360 bp. Lanes (3, 4): Patient 5 Z/Z homozygote polymorphism of A1AT where Lane (3): shows only internal control at 360 bp, while Lane (4) shows internal control at 360 bp and Z at 150 bp. Lanes (5 \u0026amp; 6): Patient 6 M/Z heterozygote polymorphism of A1AT where Lane (5): shows internal control at 360 bp and M at 150 bp while Lane (6): shows internal control at 360 bp and Z at 150 bp. Lanes (7, 8,9,10): Patient 7 S/Z heterozygote polymorphism of A1AT where Lane (7): shows internal control at 220 bp and M at 152 bp while Lane (8): shows internal control at 220 bp and S at 152 bp. Lane(9): shows internal control at 360 bp and M at 150 bp while Lane (6): shows internal control at 360 bp and Z at 150 bp.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4725061/v1/66005a8feac26f6ef314979e.png"},{"id":95040057,"identity":"2bf99ba0-b057-411e-a98c-1bef120f0121","added_by":"auto","created_at":"2025-11-03 16:08:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":836501,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4725061/v1/8e0c7926-6c47-4f06-b1a4-75e6a6f87d53.pdf"},{"id":62222341,"identity":"35c3db58-049b-45b3-8140-cc29a1aaadb1","added_by":"auto","created_at":"2024-08-11 12:36:22","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":63612,"visible":true,"origin":"","legend":"","description":"","filename":"TablesBMCID.docx","url":"https://assets-eu.researchsquare.com/files/rs-4725061/v1/3912de8b7e9cfb4a745db67e.docx"},{"id":62222339,"identity":"28971f86-0514-4ef0-87f6-3738dd209a7b","added_by":"auto","created_at":"2024-08-11 12:36:22","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":694630,"visible":true,"origin":"","legend":"","description":"","filename":"figuresuppl.docx","url":"https://assets-eu.researchsquare.com/files/rs-4725061/v1/a59cf40bff9d1aa825175bff.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Role of the alpha-1 antitrypsin towards progression and severity of COVID- 19 infection among Saudi patients","fulltext":[{"header":"Background","content":"\u003cp\u003eSevere COVID-19 exerts excessive inflammatory response, which results in extensive alveolar and endothelial injuries and eventually leads to hypoxemia and respiratory failure [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Alpha-1 antitrypsin (A1AT), is a serpin protease inhibitor. It is most widely distributed serpin and third most abundant protein in plasma [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. A1AT is an acute phase protein, and systemic infection or inflammation causes its levels to rise 3\u0026ndash;5 times [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and can be generated by macrophages, hepatocytes, intestinal epithelial cells, and bronchial epithelium. A1AT has a knack to scavenge free radicals and govern development and release of cytokines and chemokines [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe majority of A1AT haplotype distribution in human population is composed of genotype combinations consisting of M, S, and Z. Additionally, PI*MM genotype may yield 100% normal A1AT and is present in 85\u0026ndash;95% globally. The PI*MS, PI*MZ, PI*SS, PI*SZ, and PI*ZZ constitute 5\u0026ndash;15% [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Most A1AT gene mutations lead to production of mutant proteins, which is dysfunctional and causes cell injuries [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The PI*ZZ genotype is linked to increased risk of illnesses concurrent with A1AT unworthy, whereas PI*SZ, PI*SS, and PI*MZ genotypes are only correlated with a possible increased risk [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The reason for A1AT's inability to prevent SARS-CoV-2 entry could be unprocessed ACE2-mediated cell entry in lack of TMPRSS2 or presence of other proteases that could cleave S protein. It has been demonstrated that A1AT inhibits SARS-CoV-2 viral replication in cell lines, including human airway epithelial preparations [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The alpha-1 antitrypsin deficiency (A1ATD) has been identified as one of the biochemical and clinical predictors of COVID-19 in Italy [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe goal of this research was to ascertain whether Saudi Arabia's gene frequency of A1AT alleles and the severity of the COVID-19 pandemic are correlated. Furthermore, we aimed to ascertain if the ratio of A1AT to IL-6 levels is related to COVID-19 severity.\u003c/p\u003e"},{"header":"Methods and materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy subjects\u003c/h2\u003e \u003cp\u003eThe study recruited 100 COVID-19 Saudi individuals (32\u0026ndash;87 years-old) chosen randomly from King Fahd hospital in Madinah, Saudi Arabia (69 males and 31 females), divided into 3 categories (asymptomatic, mild, and severe) according to their clinical characteristics, symptoms, and chest radiography [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. A signed informed consent was obtained from participants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSampling\u003c/h2\u003e \u003cp\u003ePeripheral blood samples were obtained from each participant and divided into 2 tubes: one for EDTA tubes used for DNA extraction using Gentra\u0026reg; kits, and the other for plain tube used for enzyme linked immunosorbent assay (ELISA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eThe A1AT and IL-6 concentrations were determined using ELISA development kits (CUSABIO\u0026reg;, USA). The microplate reader with wavelength 450nm was used to validate optical density. The experiment was repeated twice, each time with identical materials contained within the individual experiments themselves.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eGenotyping of A1AT polymorphism\u003c/h2\u003e \u003cp\u003eThe amplification refractory mutation system (ARMS) technique was used to analyze A1AT subsequent mutation. The PI gene locus that encodes A1AT (14q32.1). PCR was done for either S or Z allele in reaction volume 50\u0026micro;l, including 15\u0026micro;l (2X) PCR master mix (Promega\u0026reg;, Germany), 26\u0026micro;l distilled water, 2\u0026micro;l of each primer (Metabion\u0026reg;, Germany) (\u003cb\u003eTable\u0026nbsp;1A)\u003c/b\u003e, mixed with 3\u0026micro;l DNA. Each DNA was separately amplified for S allele using primers 1, 2 and 3 (wild tube) and primers 1, 2 and 3a (mutant tube). Similarly for Z allele using primer 5,6 and 7 (wild tube) and primers 5, 6 and 7a (mutant tube). PCR conditions are shown in \u003cb\u003eTable\u0026nbsp;1B\u003c/b\u003e. Using 1.5% agarose gel, PCR products were electrophoresed and visualized under a UV illuminator.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003e The statistical package for social science (SPSS) version 25 was used to analyze data obtained. To determine statistical significance of non-parametric variable's difference between more than 2 study groups, the Kruskal-Wallis test was employed. The receiver operating characteristic (ROC) curve is used for assessing specificity and sensitivity of quantitative diagnostic measures. Significant \u003cem\u003eP\u003c/em\u003e values are defined as \u0026lt;\u0026thinsp;0.05 or 0.001 at 95% confidence Interval.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eARMS interpretation of the A1AT S locus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eThe 220 bp as internal control (primer 1 \u0026amp; 2) was generated; and the 152 bp product was observed in both tube or only one tube according to homozygous or heterozygous (S) DNA (\u003cstrong\u003eFig. 1).\u003c/strong\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eARMS interpretation of the A1AT Z locus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 360 bp as internal control (primers 5 \u0026amp; 6) was generated; and the 150 bp product was observed either in both tube or only one tube according to homozygous or heterozygous Z DNA (\u003cstrong\u003eFig. 2)\u003c/strong\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical data of the COVID-19 patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2A\u003c/strong\u003e shows the\u0026nbsp;biochemical data of participants. A total of 100 COVID-19 patients. Out of them (2%) with asthma; 15% have Diabetes mellitus; 4% with cardiovascular disease; 3% with chronic kidney disease and no cases with chronic obstructive pulmonary disease (COPD) or liver diseases. The neutrophil-to-lymphocyte ratio (NLR) is determined. In individuals with severe COVID-19, NLR was found to be higher compared to those with asymptomatic or mild cases, with mean NLRs of (10, 2.5 and 7.11; respectively).\u0026nbsp;\u003cstrong\u003eTable 2B\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eshows association between A1AT, IL-6, and VIT-D and different status (asymptomatic, mild, and severe) COVID-19 patients. The three-biomarkers showed extreme statistical significance through comparing the 3 groups of patients. All cases have deficient VIT-D, which was statistically significant through comparing asymptomatic with severe patients. A1AT showed low levels below reference range and statistically significant through comparing each pair of groups. This elucidate that deficiency of both A1AT, and VIT-D are associated with inflammation and autoimmunity. As a result, severe patients exhibited elevation in IL-6 levels than asymptomatic or moderate patients, despite the latter group experiencing reduction in A1AT levels.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePotential risk factors and frequency analysis of the A1AT genotypes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2C\u003c/strong\u003e shows that\u0026nbsp;prevalence of A1AT gene mutation was higher among COVID-19 cases compared with non-mutated patients (56% \u003cem\u003evs.\u003c/em\u003e 44%). Serum A1AT levels were lower than reference range and highly significant among mutated cases (\u003cem\u003ep\u003c/em\u003e=0.01) compared with non-mutated augmenting the severity of illness among 3 groups of COVID-19 patients. In addition, serum IL-6 levels were significantly higher than reference range among third COVID-19 group but extreme higher among mutated compared with non-mutated boosting role of A1AT. Also, VIT-D levels were more interesting all COVID-19 cases were significantly lower than reference range among mutated and non-mutated cases (\u003cem\u003ep\u003c/em\u003e=0.04 and \u003cem\u003ep\u003c/em\u003e=0.03; respectively).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of the binary logistic regression analysis of A1AT genotypes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3A\u003c/strong\u003e shows the logistic regression analysis, with the status of various cases serving as dependent variable and presence or absence of S/Z allele as primary independent variable.\u0026nbsp;It reveals higher frequency with extremely significant difference of MM genotype among asymptomatic patients compared with severe cases (67.2% \u003cem\u003evs.\u003c/em\u003e 16.1%), \u003cem\u003ep\u003c/em\u003e=0.001, [OR=0.09, 95% CI (0.03-0.28)], while the severe form MZ genotype appeared only among mild and severe states and have higher frequency of mild compared with severe (72.7% vs. 9.7%), \u003cem\u003ep\u003c/em\u003e=0.001, [OR= 0.04, 95% CI (0.01 to 0.24)]. The disappear of Z allele from asymptomatic cases and appearance of high frequency of Z allele among mild cases compared with severe cases (50% vs. 12.9%) \u003cem\u003ep\u003c/em\u003e =0.001, [OR= 0.15, 95% CI (0.05 - 0.45)] may be attributed to lack of proper follow up for the cases due to their hospital discharge without precise follow up after they exhibited severe complications later. In addition, patients own the more severe forms SS+ ZZ+SZ genotypes have higher frequency statistically significant among sever compared with asymptomatic patients (35.5% \u003cem\u003evs\u003c/em\u003e. 5.2%),\u0026nbsp;\u003cem\u003ep\u003c/em\u003e=0.001, [OR=10.08, 95%CI (2.55-39.9)]. The S allele was associated with greater incidence about 3 times among severe compared with asymptomatic patients (46.8% \u003cem\u003evs\u003c/em\u003e. 19%)\u0026nbsp;\u003cem\u003ep\u003c/em\u003e =0.001, [OR= 3.75, 95% CI (1.9-7.42)]. This means that people with either Z or S allele boost severity of\u0026nbsp;COVID-19 infection than those with the other alleles and increased risk many times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of the association between A1AT genotypes and A1AT, IL-6 and VIT-D\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3B\u003c/strong\u003e shows that the A1AT, IL-6 levels showed statistically significant with A1AT genotypes, where patients carrying MM genotype had highest level of A1AT and lowest IL-6 level. On the other hand, low A1AT and high IL-6 levels noticed among the heterozygous group carrying MS, MZ, SS, ZZ, or SZ genotypes. Interestingly, only asymptomatic COVID-19 patients showed statistically significant association between VIT-D levels and A1AT genotypes this may be attributed to large number of cases while other groups have small number and nearly equal in levels.\u0026nbsp;Extrapolated increased probability for infection with COVID-19 is associated primary with\u0026nbsp;VIT-D deficiency.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4A\u003c/strong\u003e shows that 15 individuals have diabetes, exhibited varying degrees of severity in their COVID-19 infection. 6 out of 31 patients were severe COVID-19 and 9 out of 58 were asymptomatic. Despite 6 out of 15 (46.6%) carrying MM genotype of A1AT, but they were positively associated with low levels of circulated A1AT.\u0026nbsp;In addition, severe group have significantly low A1AT level (\u003cem\u003eP\u003c/em\u003e=0.02) compared with asymptomatic. Furthermore, the frequency of mutant A1AT genotypes (PI*MZ and PI*SS) was high in severe cases (n=3 and n=1; respectively) and (n=1 and n=2; respectively) among asymptomatic individuals with diabetes.\u0026nbsp;\u003cstrong\u003eTable 4B\u0026nbsp;\u003c/strong\u003erevealed linear regression analysis for A1AT, IL-6, and VIT-D to predict potential risk factors for COVID-19 patients having different A1AT genotypes. In terms of A1AT and IL-6 levels, MM genotype was associated with high A1AT levels and low IL-6, while homozygous or hetero genotype was associated with low A1AT levels. On the other hand,\u0026nbsp;all forms of genotypes weren\u0026rsquo;t statistically significant predictor of VIT-D levels.\u0026nbsp;\u003cstrong\u003eTable 4C\u003c/strong\u003e reveals that receiver operating characteristic (ROC) curve of A1AT, IL-6, and VIT-D data was executed for discrimination between healthy subjects and COVID-19 patients. A1AT and VIT-D showed highest accuracy AUC (1.0) as a diagnostic ability for COVID-19. We could explain that IL-6 has variable levels influenced by the severity of case and treatment.\u003cstrong\u003e\u0026nbsp;Table 4D\u003c/strong\u003e showed the ROC curve of A1AT, IL-6, and VIT-D data was conducted for discrimination between severe and non-severe COVID-19 patients. A1AT and IL-6 showed high accuracy AUC 0.955 and 0.953; respectively. The VIT-D showed low accuracy AUC (0.665) as diagnostic ability for severe COVID-19.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe COVID-19 is a multifactorial illness as genetic and environmental factors may contribute to incidence rates observed in various nations and ethnic groups. Age and comorbid disorders like diabetes, hypertension, and liver and kidney ailments are examples of non-genetic variables [8].Acute respiratory distress syndrome (ARDS) is the most severe COVID-19 symptoms [9].The global percentage of the population infected with COVID-19 grew because of combined impact of these variables. One of strength of this study is that it is considered the first publication included A1AT gene polymorphism among infected Saudi COVID-19 patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis cross-sectional study showed that 24% of cases were associated with other diseases. About half of them were classified severe cases. A1AT gene polymorphism was examined in this study. This is only one example of a hereditary disorder that could make individuals more prone to COVID-19. A hereditary illness referred to as A1ATD causes liver and/or lung damage. The symptoms of A1ATD are often misinterpreted due to their overlapping with pulmonary and hepatic disorders. This has led to a significant underdiagnosis of A1ATD globally. The symptoms of A1ATD-related lung ailments are like those of other obstructive lung conditions, and patients are often misdiagnosed for years [10]. \u0026nbsp;Comorbid conditions linked to A1ATD, such as diabetes, hypertension, COPD, and chronic renal disease, are also independently linked to a higher incidence of COVID-19 [8].\u003c/p\u003e\n\u003cp\u003eSeveral studies hypothesized that severe impact of COVID-19 is influenced by areas of high prevalence of A1ATD [3]\u003cstrong\u003e.\u003c/strong\u003eA1AT has been revealed to block this protease, which might hinder SARS-CoV-2 cell entry and thus minimize viral replication[6].Additionally, the study demonstrated that sera from participants with an A1AT-deficient genotype have a decreased capacity to prevent the entry of Wuhan-Hu-1 (WT) and B.1.617.2 (Delta), but not B.1.1.529 (Omicron). The results illustrated potential involvement of these serum parameters in controlling viral entry \u003cem\u003ein vivo\u003c/em\u003e[11].\u003c/p\u003e\n\u003cp\u003eTable 2C suggested that SS and ZZ genotypes may be tied with an increased risk of severe COVID-19 infection, while the MM genotype may be protective against severe COVID-19 infection. Furthermore, number of A1ATD individuals among Saudi population is probably not clear or not accurate because some patients with A1ATD -such as children- have not yet been diagnosed with COPD or liver illness linked to A1ATD. These preliminary findings inspired us to move forward with additional plans to ascertain the frequency of A1AT in sizable portion of Saudi population and additionally seek out other genotypes, including VIT-D. Another study that investigated healthy Saudi population, registered frequency of mutant S and Z A1AT alleles (9.49% and 3.19%; respectively) [12].\u003c/p\u003e\n\u003cp\u003eThe PI*ZZ A1AT genotype\u0026apos;s global prevalence in patients with COPD was evaluated in a recent study that comprised 48 nations. According to the study, which covered 11 Asian nations, rate of COPD in persons over 40 years is 9%. The greatest PI*ZZ weighted average prevalence among COPD individuals. Saudi Arabia was found among high values [13]. Similarly, another study revealed high prevalence of Z allele has been observed among Saudi Arabia (15:1000) [10]. Further confirmatory investigations conducted among Italians from different locations revealed that the region most impacted by COVID-19, northern Italy, had higher prevalence of the deficient linked phenotypes SZ, MZ, and ZZ than Southern region, with 47% of all cases only documented [14]. It was also reported that, a robust correlation was noticed between the PI*Z variant and the number of COVID-19 cases (r = 0.8584) and deaths (r = 0.8713) in 68 countries [15]. The PI*ZZ, PI*SZ, and PI*MZ rates are projected to be approximately 12% higher for Europeans and Latinos, who also make up the majority ethnic group in the nations with the largest number of COVID-19 cases and deaths [16].\u003c/p\u003e\n\u003cp\u003eOn the other hand, a recent retrospective analysis bizarre in small cohort of hospitalized subjects revealed that only 2 were found to be PI*MZ and none for PI*MS, PI*SZ, PI*SS, or PI*ZZ individuals who are heterozygotes for A1AT alleles are not at high risk of acquiring severe COVID-19 [11]. Furthermore, it can be challenging to distinguish out baffling effect of patients\u0026apos; awareness of their A1ATD status. According to a survey performed in Germany, individuals suffering from A1ATD were 65% less likely to have been infected than the general population, and they were more likely to restrict their social groupings as a result to their greater concern about infection [12]. It was revealed that, there was no increment in either SARS-CoV-2 infection or death rates in presence of S- or Z-A1AT alleles among British patients [16]. Patients with PI*MM and PI*SS alleles, and non-smoking patients with the PI*MZ allele, do not have an abundant risk of lung ailments [10].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePeople with PI*MM genotype, concomitant with defective S and Z alleles have serum A1AT levels that are reduced by around 40% and 85%; respectively [17]. Because COVID-19 is linked to increased oxidative stress status [18], higher levels of A1AT may become useless. This fact was emphasized in (Table 4\u003cstrong\u003e)\u0026nbsp;\u003c/strong\u003ethat revealed 6 out of 15 (46.6%) diabetics were carrying MM A1ATgenotype, but they were positively associated with low levels of circulated A1AT, and this can be due to uncontrolled glucose levels. VIT-D deficiency level may link with type 2 diabetics to A1ATD which may cause a higher frequency COVID-19. We could explain the non-significance of certain genotypes due to small numbers and nearly similar levels of the parameters. Interestingly that all our COVID-19 patients have VIT-D deficiency. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition, A1AT homeostasis in diabetic nephropathy leads to reduced levels of A1AT [19]. The non-enzymatic glycosylation of A1AT or oxidation of methionine in A1AT active site, may be the trigger for the decline in serum trypsin inhibitory capacity [20]. It was revealed that, VIT-D active form drives CD4\u003csup\u003e+\u003c/sup\u003e T cells to emit A1AT, \u003cem\u003evia\u003c/em\u003e direct contact with complement C3a, strengthens IL-10 secretion; implying that A1AT is crucial for active VIT-D to induce IL-10. linked with inflammation status and autoimmunity [21]. It was evident that there is a probable link between VIT-D levels and COVID-19 severity and lethality [22]. Our study revealed strong association with VIT-D, as there was high significant between asymptomatic and severe patients (Tables 2B,2C,3B). VIT-D has robust safeguard against acute lung injury and ARDS \u003cem\u003evia\u003c/em\u003e regulating RAS and limiting bradykinin buildup. On other hand, COVID-19 patients utilize cytokine storm and bradykinin storm to counterfeit the virus. In agreement with our results, is well-known that COVID-19 patients have substantial declines in VIT-D levels [23].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In our study, severe patients exhibited a greater elevation in circulating IL-6 levels than asymptomatic or moderate patients, despite the latter group experiencing a reduction in A1AT levels. Hyper-inflammation and coagulopathy are hallmarks of COVID-19, and in more severe instances, an intensifying \u0026quot;cytokine storm\u0026quot; may trigger respiratory failure, sepsis, and even death [23]. Extrapolated the severity of COVID-19 patients is linked to a rise in the IL-6/A1AT ratio, which suggests an inadequate response of A1AT to IL-6 production. The IL-6/A1AT ratio in severe COVID-19 patients (mean 1.4) was significantly higher compared to asymptomatic or moderate patients (0.16, 0.21, respectively, P=0.001). This extensively elucidated that severe patients have a decreased A1AT response to inflammation (Table 2B,2C). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt was postulated that, innate immune defense relies heavily on A1AT, whose plasma concentration may spike 2-4 times during the acute phase protein response. Protease-antiprotease disparities may emerge during the COVID-19 clinical course of the virus [24]. It was clearly reported that, patients in the intensive care unit (ICU) demonstrated a higher IL-6/A1AT ratio. Furthermore, clinical improvement was observed in ICU patients whose IL-6/A1AT ratio declined during treatment; no improvement was noted in patients whose ratio remained larger [25]. Another study emphasized that primary cytokine storm tracked in COVID-19 individuals exhibiting mild to severe symptoms is serum IL-6 [26]. A1AT can alter actions resulting in descending IL-6 inhibition, which has a crucial role in COVID-19 pathogenicity [27]. Within a multi-center investigation involving more than two thousand COVID-19 patients, the Pi*Z allele and/or a plasma A1AT level less than 116 mg/dl were significantly linked to severe COVID-19 as opposed to non-severe cases [26]. We could have concluded from table (8 A, B) that IL-6 and ATT can be used as predictor for severity of COVID-19; While VIT-D was a fuzzy (ambiguous) risk factor for COVID-19 especially overall patients have VIT-D deficiency.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResults from another study showed that, in comparison to Italian population, patients with A1ATD had a considerably increased relative risk (8.8; P=0.0001) of symptomatic severe SARS-COV-2. 209 persons with significantly lower serum A1AT levels and A1AT-deficient genotypes participated in the survey [28]. Furthermore, in our severe COVID-19 patients, the NLR was found to be higher compared to those with asymptomatic or mild cases, with mean NLRs of 10, 2.5, and 7.11; respectively (Table 2 and 3). Severe COVID-19 progression is predicted by incremented absolute neutrophil count, elevated neutrophil percentage, and high blood neutrophil: lymphocyte ratio [29]. One of the postulates of the mechanisms by which A1AT can alleviate COVID-19 is the creation of neutrophil extracellular traps (NETs), a complex extracellular structure made of neutrophil-derived DNA, histones, and proteases that is linked to the immune-thrombosis of COVID-19, can be resisted by A1AT suppression of elastase [8]. Notably, dysregulated neutrophil elastase activity has been linked to diminished plasma concentrations or A1AT function, which might occur in pulmonary emphysema [30]. It was declared that significant higher severity and mortality were linked to NLR ratios of \u0026ge;4.5 and \u0026ge;6.5; respectively [2]. In addition, under hypoxic conditions, ZZ-A1ATD neutrophils produce more ROS than MM control subjects [30]. \u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eA1AT may be viewed as a protective host factor against COVID-19. A1AT deficient allele was shown to be highly prevalent in Saudi Arabian population. It is likely justified to implement a screening program for those who are at risk, enabling them to take appropriate preventive action.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.43399638336347%\" valign=\"top\"\u003e\n \u003cp\u003eA1AT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.56600361663652%\" valign=\"top\"\u003e\n \u003cp\u003eAlpha-1 Antitrypsin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.43399638336347%\" valign=\"top\"\u003e\n \u003cp\u003eA1ATD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.56600361663652%\" valign=\"top\"\u003e\n \u003cp\u003eAlpha-1 Antitrypsin Deficiency\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.43399638336347%\" valign=\"top\"\u003e\n \u003cp\u003eARDS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.56600361663652%\" valign=\"top\"\u003e\n \u003cp\u003eAcute Respiratory Distress Syndrome\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.43399638336347%\" valign=\"top\"\u003e\n \u003cp\u003eARMS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.56600361663652%\" valign=\"top\"\u003e\n \u003cp\u003eAmplification-Refractory Mutation System\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.43399638336347%\" valign=\"top\"\u003e\n \u003cp\u003eCOPD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.56600361663652%\" valign=\"top\"\u003e\n \u003cp\u003eChronic Obstructive Pulmonary Disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.43399638336347%\" valign=\"top\"\u003e\n \u003cp\u003eCOVID-19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.56600361663652%\" valign=\"top\"\u003e\n \u003cp\u003eCoronavirus Disease 19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.43399638336347%\" valign=\"top\"\u003e\n \u003cp\u003eELISA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.56600361663652%\" valign=\"top\"\u003e\n \u003cp\u003eEnzyme Linked Immunosorbent Assay\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.43399638336347%\" valign=\"top\"\u003e\n \u003cp\u003eICU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.56600361663652%\" valign=\"top\"\u003e\n \u003cp\u003eIntensive Care Unit\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.43399638336347%\" valign=\"top\"\u003e\n \u003cp\u003eIL-6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.56600361663652%\" valign=\"top\"\u003e\n \u003cp\u003eInterleukin 6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.43399638336347%\" valign=\"top\"\u003e\n \u003cp\u003ePCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.56600361663652%\" valign=\"top\"\u003e\n \u003cp\u003ePolymerase Chain Reaction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.43399638336347%\" valign=\"top\"\u003e\n \u003cp\u003eSaudi Arabia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.56600361663652%\" valign=\"top\"\u003e\n \u003cp\u003eSA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.43399638336347%\" valign=\"top\"\u003e\n \u003cp\u003eVIT-D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.56600361663652%\" valign=\"top\"\u003e\n \u003cp\u003eVitamin D\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Saudi Arabia Ministry of Health\u0026apos;s (MOH) General Administration for Research \u0026amp; Studies (IRB) and faculty of Applied Medical Sciences at Taibah University provided ethical approval (approved IRB-452) for the study to be conducted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData related to this manuscript is available on the hand of corresponding author and will be obtained under a reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors affirm that no conflict of interest exists that might be seen as compromising objectivity of the study they have published.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was fully financially supported by the Deanship of Scientific Research (DSR)- Taibah University- Saudi Arabia under the project number RC-442/11.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRA: lead, study design, data analysis, writing introduction, methodology, results, and discussion parts. WM: data analysis and manuscript revision. HF: samples gathering. YA: data analysis, interpretations, and help in writing discussion part. HN: help in the practical work. All authors read and authorized the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors extend their appreciations to the Deanship of Scientific Research (DSR)-Taibah University, Saudi Arabia for their sincere continuous enormous help. Also, we thank the staff of the hospital for their help and support throughout the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSmadja DM, Mentzer SJ, Fontenay M, Laffan MA, Ackermann M, Helms J, et al. COVID-19 is a systemic vascular hemopathy: insight for mechanistic and clinical aspects. \u003cem\u003eAngiogenesis\u003c/em\u003e 2021; 24(4): 755-88.\u003c/li\u003e\n\u003cli\u003eBai\u003cspan dir=\"RTL\"\u003e \u003c/span\u003eX, Schountz T, Buckle AM, Talbert JL, Sandhaus RA, Chan ED, et al. Alpha-1-antitrypsin antagonizes COVID-19: a review of the epidemiology, molecular mechanisms, and clinical evidence. \u003cem\u003eBiochem Soc Trans\u003c/em\u003e 2023; 51(3):1361-75.\u003c/li\u003e\n\u003cli\u003eTeckman JH, Lindblad D. Alpha-1-antitrypsin deficiency: diagnosis, pathophysiology, and management. Curr Gastroenterol Rep 2006; 8(1):14-20.\u003c/li\u003e\n\u003cli\u003eBlanco I, Bueno P, Diego I, P\u0026eacute;rez-Holanda S, Casas-Maldonado F, Esquinas C, et al. Alpha-1 antitrypsin Pi*Z gene frequency and Pi*ZZ genotype numbers worldwide: an update. \u003cem\u003eInt J Chron Obstruct Pulmon Dis\u003c/em\u003e 2017; 12:561-9.\u003c/li\u003e\n\u003cli\u003ede Serres FJ, Blanco I. Prevalence of \u0026alpha;1-antitrypsin deficiency alleles PI*S and PI*Z worldwide and effective screening for each of the five phenotypic classes PI*MS, PI*MZ, PI*SS, PI*SZ, and PI*ZZ: a comprehensive review. \u003cem\u003eTher Adv Respir Dis \u003c/em\u003e2012; 6(5):277-95.\u003c/li\u003e\n\u003cli\u003eAzouz NP, Klingler AM, Callahan V, Akhrymuk IV, Elez K, Raich L, et al. Alpha 1 antitrypsin is an inhibitor of the SARS-CoV-2-priming protease TMPRSS2. \u003cem\u003ePathog Immun\u003c/em\u003e 2021; 6 (1):55-74.\u003c/li\u003e\n\u003cli\u003eYang C, Keshavjee S, Liu M. Alpha-1 antitrypsin for COVID-19 treatment: Dual role in antiviral infection and anti-Inflammation. \u003cem\u003eFront Pharmacol\u003c/em\u003e 2020; 11: 615398.\u003c/li\u003e\n\u003cli\u003eJordan RE, Adab P, Cheng KK. Covid-19: risk factors for severe disease and death. \u003cem\u003eBMJ\u003c/em\u003e 2020; 368: m1198.\u003c/li\u003e\n\u003cli\u003eZhou F, Yu T, Du R, Fan G, Liu Y, Liu Z, et al. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. \u003cem\u003eLancet\u003c/em\u003e 2020; 395(10229):1054-62.\u003c/li\u003e\n\u003cli\u003eCraig TJ, Corbett ML, Meadows JA. Improving detection of alpha-1 antitrypsin deficiency: Role of the allergist. \u003cem\u003eJ Allergy Clin Immunol Pract\u003c/em\u003e 2023; 11(8): 2348-54.\u003c/li\u003e\n\u003cli\u003eNygren D, M\u0026ouml;lstad U, Thulesius H, Hillman M, Broman LM, Tanash H, et al. Low prevalence of mild alpha-1-antitrypsin deficiency in hospitalized COVID-19-patients. \u003cem\u003eInt J Gen Med\u003c/em\u003e 2022; 15: 5843-8.\u003c/li\u003e\n\u003cli\u003eShapira G, Shomron N, Gurwitz D. Ethnic differences in alpha-1 antitrypsin deficiency allele frequencies may partially explain national differences in COVID-19 fatality rates. \u003cem\u003eFASEB J\u003c/em\u003e 2020; 34(11):14160-5.\u003c/li\u003e\n\u003cli\u003eFerrarotti I, Ottaviani S, Balderacchi AM, Barzon V, De Silvestri A, Piloni D, et al. COVID-19 infection in severe Alpha 1-antitrypsin deficiency: Looking for a rationale. \u003cem\u003eRespir Med\u003c/em\u003e 2021;183:106440.\u003c/li\u003e\n\u003cli\u003eBlanco I, de Serres FJ, Fernandez-Bustillo E, Lara B, Miravitlles M. Estimated numbers and prevalence of PI*S and PI*Z alleles of \u0026alpha;\u0026lt;sub\u0026gt;1\u0026lt;/sub\u0026gt;-antitrypsin deficiency in European countries. \u003cem\u003eEur Respir J\u003c/em\u003e 2006; 27(1): 77-84.\u003c/li\u003e\n\u003cli\u003eYoshikura H. Epidemiological correlation between COVID-19 epidemic and prevalence of \u0026alpha;-1 antitrypsin deficiency in the world. \u003cem\u003eGlob Health Med\u003c/em\u003e 2021; 3(2): 73-81.\u003c/li\u003e\n\u003cli\u003eSchneider CV, Strnad P. SARS-CoV-2 infection in alpha1-antitrypsin deficiency. Respir Med 2021;184: 106466. \u003c/li\u003e\n\u003cli\u003eBlanco I, Bueno P, Diego I, P\u0026eacute;rez-Holanda S, Lara B, Casas-Maldonado F, et al. Alpha-1 antitrypsin Pi*SZ genotype: estimated prevalence and number of SZ subjects worldwide. \u003cem\u003eInt J Chron Obstruct Pulmon Dis\u003c/em\u003e 2017; 12: 1683-94.\u003c/li\u003e\n\u003cli\u003eWang JZ, Zhang RY, Bai J. An anti-oxidative therapy for ameliorating cardiac injuries of critically ill COVID-19-infected patients. \u003cem\u003eInt J Cardiol\u003c/em\u003e 2020; 312:137-8.\u003c/li\u003e\n\u003cli\u003ePiwowar A, Knapik-Kordecka M, Warwas M. Concentration of leukocyte elastase in plasma and polymorphonuclear neutrophil extracts in type 2 diabetes. \u003cem\u003eClin Chem Lab Med\u003c/em\u003e 2000; 38(12):1257-61.\u003c/li\u003e\n\u003cli\u003eYaghmaei M, Hashemi, Shikhzadeh MA, Mokhtari M, Niazi A, Ghavami S. Serum trypsin inhibitory capacity in normal pregnancy and gestational diabetes mellitus. \u003cem\u003eDiabetes Res Clin Pract\u003c/em\u003e 2009; 84(3): 201-4.\u003c/li\u003e\n\u003cli\u003eDimeloe S, Rice LV, Chen H, Cheadle C, Raynes J, Pfeffer P, et al. Vitamin D (1,25(OH)2D3) induces \u0026alpha;-1-antitrypsin synthesis by CD4+ T cells, which is required for 1,25(OH)2D3-driven IL-10. \u003cem\u003eJ Steroid Biochem Mol Biol\u003c/em\u003e 2019; 189: 1-9.\u003c/li\u003e\n\u003cli\u003eKazemi A, Mohammadi V, Aghababaee SK, Golzarand M, Clark CT, Babajafari S. Association of vitamin D status with SARS-CoV-2 infection or COVID-19 severity: A systematic review and meta-analysis. \u003cem\u003eAdv Nutr\u003c/em\u003e 2021; 12(5):1636-58.\u003c/li\u003e\n\u003cli\u003eJain A, Chaurasia R, Sengar NS, Singh M, Mahor S, Narain S. Analysis of vitamin D level among asymptomatic and critically ill COVID-19 patients and its correlation with inflammatory markers. \u003cem\u003eScientific Reports\u003c/em\u003e 2020; 10(1): 20191.\u003c/li\u003e\n\u003cli\u003eO\u0026apos;Brien ME, Murray G, Gogoi D, Yusuf A, McCarthy C, Wormald MR, et al. A review of alpha-1 antitrypsin binding partners for immune regulation and potential therapeutic application. \u003cem\u003eInt J Mol Sci\u003c/em\u003e 2022; 23(5):2441.\u003c/li\u003e\n\u003cli\u003eFarcas M, Csernik F, Popp R-A, Trifa A, Crisan T, Petrisor F, et al. The interleukin 4 VNTR polymorphism frequency in a romanian population group. \u003cem\u003eAnnals of the Romanian Society for Cell Biology\u003c/em\u003e 2009,14(1):97-101.\u003c/li\u003e\n\u003cli\u003eMcElvaney OJ, McEvoy NL, McElvaney OF, Carroll TP, Murphy MP, Dunlea DM, et al. Characterization of the inflammatory response to severe COVID-19 illness. \u003cem\u003eAm J Respir Crit Care Med\u003c/em\u003e 2020; 202(6): 812-21.\u003c/li\u003e\n\u003cli\u003ePedersen SF, Ho YC. SARS-CoV-2: a storm is raging. \u003cem\u003eJ Clin Invest\u003c/em\u003e 2020; 130(5): 2202-05.\u003c/li\u003e\n\u003cli\u003eRodr\u0026iacute;guez Hermosa JL, Vargas Centanaro G, Gonz\u0026aacute;lez Castro ME, Miravitlle M, L\u0026aacute;zaro-Asegurado, Jim\u0026eacute;nez-Rodr\u0026iacute;guez BM, et al. Severe COVID-19 illness and \u0026alpha;1-antitrypsin deficiency: COVID-AATD study. \u003cem\u003eBiomedicines\u003c/em\u003e 2023; 11(2):516.\u003c/li\u003e\n\u003cli\u003ePhilippe A, Puel M, Narjoz C, Gendron N, Durey-Dragon MA, Vedie B, et al. Imbalance between alpha-1-antitrypsin and interleukin 6 is associated with in-hospital mortality and thrombosis during COVID-19. \u003cem\u003eBiochimie\u003c/em\u003e 2022; 202:206-11.\u003c/li\u003e\n\u003cli\u003eMagall\u0026oacute;n M, Castillo-Corull\u0026oacute;n S, Ba\u0026ntilde;uls L, Pellicer D, Romero T, Mart\u0026iacute;nez-Ferraro C, et al. Hypoxia enhances oxidative stress in neutrophils from ZZ alpha-1 antitrypsin deficiency patients. \u003cem\u003eAntioxidants \u003c/em\u003e2023;12(4):872.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 8 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"α1-Antitrypsin (A1AT), Polymorphism, COVID-19, Interleukin 6, Vitamin D (VIT-D)","lastPublishedDoi":"10.21203/rs.3.rs-4725061/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4725061/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eAlpha-1 antitrypsin (A1AT) is involved in pathophysiology of severe COVID-19, including thrombosis expansion. A1AT has anti-inflammatory, tissue-protective, and anticoagulant capabilities. We aimed to screen frequencies of A1AT gene polymorphism among COVID-19 Saudi patients and its relation to severity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e Through cross-sectional study, we examined 100 COVID-19 Saudi patients to explore possible correlation between A1AT/interleukin 6 (IL-6) ratio and COVID-19 severity. The COVID-19 patients grouped as severe (31 patients) and non-severe (69 patients) cases. A1AT gene polymorphism was conducted using the PCR technique (ARMS) and ELISA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eA1AT, IL-6, and vitamin D (VIT-D) showed extreme statistical significance among COVID-19 patients (severe, mild, and asymptomatic). The prevalence of A1AT gene mutation was higher among COVID-19 cases compared with non-mutated patients (56% \u003cem\u003evs.\u003c/em\u003e 44%). Moreover, serum A1AT levels were lower while serum IL-6 levels were higher than reference range and highly significant among mutated cases compared with non-mutated cases. Also, IL-6/A1AT ratio in severe COVID-19 patients (mean 1.4) was significantly higher compared with asymptomatic or moderate patients (0.16, 0.21; respectively). Strictly, all COVID-19 patients have severed deficiency of VIT-D level significant among mutated and non-mutated cases (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.04 and \u003cem\u003ep\u003c/em\u003e\u0026lt;0.03; respectively). \u0026nbsp;The frequency of MM (wild type) was substantially high among asymptomatic cases compared with severe cases (67.2% \u003cem\u003evs.\u003c/em\u003e16.1%). Heterozygous MS+MZ genotypes showed lower frequency among asymptomatic cases compared with severe and mild cases (27.6% \u003cem\u003evs.\u003c/em\u003e48.4% and 72.7%; respectively). On the other hand, the more severe forms\u003cstrong\u003e \u003c/strong\u003eof SS+ZZ+SZ genotypes were all relatively rare with lower frequency among asymptomatic compared with mild and severe COVID-19 cases (5.2%, 27.3% and 35.5%; respectively). Interestingly, homozygous SS genotype elicited higher frequency among severe cases compared with mild or asymptomatic cases (22.6% \u003cem\u003evs.\u003c/em\u003e0% and 5.2%). The more severe forms homozygous ZZ genotype vanished among asymptomatic and mild cases. This extensively illuminated that, severe COVID-19 patients have diminished A1AT response towards inflammation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion \u003c/strong\u003eTwo haplotypes (S) and (Z) alleles of A1AT have higher frequency and were clearly recognized among severe COVID-19 cases suggesting that SS and ZZ genotypes may be associated with an increased risk, while MM genotype may be protective against severe COVID-19 infection.\u003c/p\u003e","manuscriptTitle":"Role of the alpha-1 antitrypsin towards progression and severity of COVID- 19 infection among Saudi patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-11 12:36:12","doi":"10.21203/rs.3.rs-4725061/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-17T10:46:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-17T10:38:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-15T06:30:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Infectious Diseases","date":"2024-07-11T15:11:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d7136baa-611b-4411-ba2b-829a50fc26b7","owner":[],"postedDate":"August 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-03T16:03:36+00:00","versionOfRecord":{"articleIdentity":"rs-4725061","link":"https://doi.org/10.1186/s12879-025-11699-4","journal":{"identity":"bmc-infectious-diseases","isVorOnly":false,"title":"BMC Infectious Diseases"},"publishedOn":"2025-10-28 15:58:44","publishedOnDateReadable":"October 28th, 2025"},"versionCreatedAt":"2024-08-11 12:36:12","video":"","vorDoi":"10.1186/s12879-025-11699-4","vorDoiUrl":"https://doi.org/10.1186/s12879-025-11699-4","workflowStages":[]},"version":"v1","identity":"rs-4725061","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4725061","identity":"rs-4725061","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}