384 J Contemp Med Sci | Vol. 7, No. 6, November-December 2021: 384–391 Original Angiotensin-Converting Enzyme Receptor Genotype and its Activity Level as Potential Predictors of the Severity COVID-19 among Iraqi Patients Suzan Haleem Kamel1 , Fadhil Jawad Al-Tu’ma1*, Riyadh Mohi Al-Saegh2 1Department of Chemistry and Biochemistry, College of Medicine, University of Kerbala, Kerbala, Iraq. 2Section of Nephrology, Department of Internal Medicine, College of Medicine, University of Kerbala, Kerbala, Iraq. *Correspondence to: Fadhil Jawad Al-Tu’ma (E-mail: fadhil.jawad@uokerbala.edu.iq) Introduction More than 140 million cases of infection with Severe acute res- piratory syndrome coronavirus 2 (SARS-CoV-2) have been reported worldwide by April 19, 2021, with over 3 million deaths due to the virus1 coronavirus disease situation dashboard. Coronavirus disease 2019 (Covid-19) which emerged in Wuhan, China, has spread to almost all countries and regions of the world, becoming one of the most lethal pandemic after the Spanish flu in 1918–1920. It is caused by an RNA virus (2019 novel coronavirus or 2019-nCoV or SARS-CoV-2). As of July 13, 2020, a total of 12,768,307 confirmed Covid-19 cases and 566,654 related deaths have been reported. Beyond its impor- tant morbidity and mortality and the huge burden of health care systems, Covid-19 has a massive societal and economic impact globally.1,2 This pathogen was later renamed as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the coronavirus study group (Gorbalenya, 2020),3 and the dis- ease was named coronavirus disease 2019 (Covid-19) by the WHO. Covid-19 can be either silent (asymptomatic) or associ- ated with many symptoms, such as familiar cold symptoms (fever, stuffy nose, cough, weakness) bronchitis and pneu- monia.4 Covid-19 is moderately infectious with a relatively high mortality rate, but the information available in public reports and published literature is rapidly increasing. Many risk factors have been described for this corona- virus such as elderly age, male gender, race, obesity, hyperten- sion, diabetes and geographic region.5,6 Clinically, most of Covid-19 cases (80%) are either asymp- tomatic or have mild forms.7 However, about 13.8 and 6.1% have severe and critical life-threatening disease that requires admission to hospital and sometimes in the intensive care unit. In the context of outstretched heath care systems and limited resources, risk stratification is pivotal to identify patients who the most need in-hospital and intensive management. Bio- markers along with some clinical factors might help to predict adverse outcomes among Covid-19 patients. Hence, we con- ducted this systematic review and meta-analysis to summarize available data on the association between some common hematological, inflammatory, biochemical parameters and the severity of Covid-19.7,8 To date, there is no established curative treatment for Covid-19. Although some drugs such as hydrox- ychloroquine are integrated in treatment guidelines or under investigation in interventional studies, the management of Covid-19 is mostly supportive.7,9 Identifying biological abnor- malities induced by Covid-19 may contribute to a better under- standing of the pathophysiology of the disease and ultimately guide the development of targeted adjuvant therapies besides antivirals drugs. Furthermore, such information on the biolog- ical profile of Covid-19 can guide clinicians in the assessment and treatment of these patients.10 Genetic factors also play a major role in Covid19 infection. Inter-individual inherited dif- ferences in susceptibility to SARS-CoV-2 infection is linked to the presence of genetic polymorphisms (variants) in many Abstract Objectives: The purpose of this study is to determine whether the angiotensin-converting enzyme activity and various biomarkers are used to investigate the severity of Covid-19 and to study the genetic variation occurs in angiotensin-converting enzyme-2 (ACE-2) receptor in severe Covid-19-related genes in the Iraqi population of Kerbala Province. Methods: This case-control study was conducted on 176 subjects who survived hospitalization and diagnosed by physician. Various biomarkers including ferritin, C-reactive protein, lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, angiotensin- converting enzyme-2 activity levels were determined. Accordingly, they were divided into three groups: 59 of them were infected with severe Covid-19, 54 of them were infected with moderate Covid-19 and 63 of them were checked and obtained as apparently healthy control. Severe and moderate patients were collected from Al-Hayat tertiary center at Al-Hussein Medical City, Kerbala Health Directorates, Kerbala – Iraq during Oct., 2020-July, 2021 with matched age ranged between (23–88) years. Blood samples of apparently healthy and Covid-19 samples were subjected to genomic DNA extraction within 24–48 hours of aspiration. The genomic DNA extracted was subjected to electrophoresis through 1.5% of agarose gels which was detected by staining with the fluorescent dye ethidium bromide and then visualized by illumination with UV light to confirm the presence and integrity of the extracted DNA. Results: Genotyping of ACE-2 (I/D) polymorphism (rs4646994), which has a high prevalence, was performed by polymerase chain reaction assay. The amplification of an Alu repetitive element in an intron of the ACE-2 has shown three potential genotypes of I/I and D/D as homozygous, and I/D as heterozygous. Individuals with normal homozygous (DD) revealed band of (190 bp), while individuals with normal (II) revealed band of (490 bp), and the individuals with heterozygous (ID) revealed two bands (190, 490 bp) respectively. Every severe Covid-19 group carried (DD) allele genotype, moderate group carried (ID and II) alleles and finally the control group carried (DD, ID, II) alleles genotype. Conclusion: In the ACE-2 polymorphism, the D/D genotype allele is implicated as a risk factor for severe Covid-19 patients, in Iraqi population. Keywords: COVID-19, homozygote, ferritins, genotype ISSN 2413-0516 (Submitted: 09 September 2021 – Revised version received: 16 September 2021 – Accepted: 12 October 2021 – Published online: 26 December 2021) 385J Contemp Med Sci | Vol. 7, No. 6, November-December 2021: 384–391 S.H. Kamel et al. Original ACE Receptor Genotype and its Activity Level as Potential Predictors of the Severity COVID-19 among Iraqi Patients genes especially in those that code for the host receptors involved in viral entry process. These DNA changes are trans- missible from one generation to another, detectable in at least 1% of individuals in a population and could explain the differ- ences between individuals in the susceptibility to some multi- genic, complex diseases like Covid19.11,12 Two main approaches can be used in genetic epidemiology to establish a link between genetic variations and the risk of developing a disease: Genetic linkage analysis and association studies (candidate gene and genome-wide association studies).13 Angiotensin-converting enzyme (chromosome Xp22.2) is the enzyme responsible for converting angiotensin-2 to angiotensin (1–7) form,14 it is expressed in most organs such as, thyroid and lungs, heart, eso- phagus, kidney, adipose tissue, liver, retina, the vascular system, the small intestine, nasal and bronchial tissue, and alveolar type II epithelial cells.15,16 ACE-2 is also known as a host cell receptor that contributes to the viral infection by corona viruses. The COVID-19 virus binds to the target cells through ACE-2 receptor which makes the COVID-19 attachment, invasion and penetration processes easier.17 There are other receptors that can be used but, the virus has greater affinity for ACE-2 and weaker affinities for two other receptors, CD147 and Grp78 (Glucose-Regulate Protein 78).16 The ACE-2 expression level has been reported to be significantly increased among men than women, which could explain the male predominance of COVID-19.18,19 However, in another study ACE-2 expression was not significantly associated with gender/disease severity bias among Covid-19 Italian patients.20 Similar results were obtained by another author, who didn’t observe a disparity between age groups and gender groups (male vs female) in ACE-2 gene expression.21 ACE-2 is very polymorphic gene with about 1700 polymorphisms and their frequencies vary between populations, some of these polymorphisms were cor- related with increased expression of ACE-2 protein and were more frequent among the East-Asian populations.17,22,23 Not only that, ACE deletion allele that is linked to alterations of ACE expression, also influences the spread of the virus and outcomes of infection with COVID-19 especially in the Asian populations.24,25 Angiotensin converting enzyme-2 receptor (ACE-2) is a protein on the surface of many cell types. It is an enzyme that generates small proteins – by cutting up the larger protein angiotensinogen – that then go on to regulate functions in the cell. Using the spike-like protein on its surface, the SARS-CoV-2 virus binds to ACE-2 – like a key being inserted into a lock – prior to entry and infection of cells. Hence, ACE-2 acts as a cellular doorway – a receptor – for the virus that causes Covid-19. ACE-2 acts as the receptor for the SARS-CoV-2 virus and allows it to infect the cell. ACE-2 receptor is present in many cell types and tissues including the lungs, heart, blood vessels, kidneys, liver and gastro-intestinal tract. It is present in epithelial cells, which line certain tissues and create protective barriers. The purpose of the presented study is to investigate the activity levels of angiotensin-converting enzyme and var- ious biomarker levels in adults infected with Covid-19 and study the molecular basis of angiotensin-converting enzyme-2 (ACE-2) receptor in Covid-19-related gene polymorphism in Iraqi populations of Kerbala Province. Materials and Methods The current case-control study was conducted on 176 subjects who survived hospitalization and diagnosed by physician. Various biomarkers including ferritin, C-reactive protein, lac- tate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), angiotensin-converting enzyme-2 (ACE-2) activity were determined. Accordingly, they were divided into three groups: 59 of them were infected with severe Covid-19, 54 of them were infected with moderate Covid-19 and 63 of them were checked and obtained as appar- ently healthy control. Severe and moderate patients were col- lected from Al-Hayat tertiary center at Al-Hussein Medical City, Kerbala Health Directorates, Kerbala–Iraq during Oct., 2020-July, 2021 with matched age ranged between (23–88) years. Blood samples of apparently healthy and Covid-19 sam- ples were obtained from each case and used for some bio- markers determination (ferritin, ALT, AST and ALP activity levels) and molecular studies. One ml of whole blood was col- lected in EDTA containing tube and subjected for genomic DNA extraction within 24–48 hours of aspiration. Then DNA concentration and purity were measured by UV absorption at 260 and 280 nm (Bio Drop, U.K.). The genomic DNA extracted was subjected to electropho- resis through 1.5% of agarose gels electrophoresis in 1X (TBE) buffer at 100 volts for 75 minutes or until dye markers have migrated an appropriate distance, depending on the size of the DNA to be visualized. The percentage of agarose used depends on the size of fragments to be resolved, where an agarose gels percentage are normally in the range of 0.5% to 2%; the ethidum bromide staining was done according to Robinson and Lafleche method.26 DNA ladder 100 bp bands were used as standard for comparison with bands that resulted for allelic gene migration through gel electrophoresis and then detected at staining with the fluorescent dye ethidium bromide and then visualized by illumination with UV light to confirm the presence and integrity of the extracted DNA. Genotyping for ACE-2 receptor gene polymorphism was performed by the polymerase chain reaction- Amplification Refractory Mutation System (PCR-ARMS) method using thermocycler (Biometra, Germany). The primers were taken in a lyophilized state; their units are known as a mass in pico- moles then mixed by suitable vortex and their sequence of ACE-2 receptor gene was listed in Table 1. The subsequent steps were done for the reconstitution and dilution of the primers: The tube was centrifuged at 10,000 rpm for 5–10 min before de-capping. The chosen volumes from nuclease free water were added according to the manu- facturer to obtain a 100 P-moles/μl (master stock). Ten micro- liters of the master stock were transported to a 0.5 ml eppendorf tube that contained 90 μl of nuclease free water to obtain a 10 pmoles/μl a working primer stock solution. The master stock and working stock were kept at –20°C. The working stock was warmed up and kept on ice for use in PCR and then stored at –20°C after each use. Table 1. Primers used in molecular study of angiotensin- converting enzyme receptor-2 gene polymorphism Primer name Sequence Annealing temp. (°C) Product size (bp) ACE-rs4646994- F 5`-CTGGAGACCA CTCCCATCCTTTCT-3` 58 190 ACE-rs4646994-R 5`-GATGTGGCCATCA CATTCGTCAGAT-3` 58 490 386 J Contemp Med Sci | Vol. 7, No. 6, November-December 2021: 384–391 ACE Receptor Genotype and its Activity Level as Potential Predictors of the Severity COVID-19 among Iraqi Patients Original S.H. Kamel et al. The reaction setup and thermal cycling protocol of the PCR components and program for ACE-2 receptor gene show in Table 2. Electrophoresis involves running a current through a gel loaded with the molecules of interest. The movement of the samples is directed based on the charge that the molecule car- ries. Based on the size and charge, the molecules will travel through the gel at different speeds, allowing them to be sepa- rated from one another as shown in Figure 1. Since, all the DNA molecules possess same amount of charge per mass, the gel electrophoresis separates them on the basis of size only. Body mass index (BMI) was used to define obesity. The range of BMI (18.5–24.99 kg/m2) that set it WHO but it does Table 2. Program of polymerase chain reaction of ACE-2 receptor gene in Covid-19 and healthy control Steps Temp., °C Time Cycle Initial denaturation 95 5 min. 1 Denaturation 95 30 sec. 30 Annealing 58 30 sec. Extension 72 30 sec. Final extension 72 5 min 1 Table 3. Comparison of age and body mass index and angiotensin-converting enzyme between moderate and severe Covid-19 groups as compared with control group Parameters Moderate Covid-19 N = 54 Mean ± SD Control group N = 63 Mean ± SD P-value Control group N = 63 Mean ± SD Severe Covid-19 N = 59 Mean ± SD P-value Age, year 52.06 ± 14.35 40.03 ± 11.86 <0.001 40.03 ± 11.86 59.1 ± 12.64 <0.001 BMI, kg/m2 32.59 ± 5.52 33.31 ± 5.83 0.495 33.31 ± 5.83 33.07 ± 5.72 0.817 ACE-2 activity, ng/ml 3.83 ± 0.82 3.03 ± 0.82 0.721 3.03 ± 0.82 4.74 ± 0.85 0.001 BMI, Body mass index; N, Number; Significant P < 0.05; SD, Standard Deviation. not accurately indicate the degree of fatness. The body mass index was measure by dividing weight in kilograms by length of individual in square meter. Results Main features of 176 individuals included in this study were divided into 63 cases as apparently health control and the other 113 cases of them were infected with Covid-19 as sum- marized in Table 3. The Covid-19 patients were sub-divided into two groups depending on the severity of the disease and some biomarker levels changes. Mild Covid-19 includes 54 patients and the remaining 59 patients were infected with severe Covid-19 and its complications. The mean ± SD of age and BMI of each groups were indicated in Table 3. In this table a significant differences between each of severe and moderate Covid-19 group as compared with con- trol group was observed with respect to the age (P < 0.001), while BMI data show a non-significant results (P = 0.817 and P = 0.495) respectively. The mean ± SD values of angiotensin- converting enzyme-2 (ACE-2) activity levels was higher in the severe and moderate Covid-19 as compared with that found in healthy control (4.74 ± 0.85, 3.83 ± 0.82, and 3.03 ± 0.82 ng/ml) respectively. A non-significant differences between moderate and control groups (P = 0.721) was obtained and Fig. 1 The amplification of rs4646994 region of human samples were fractionated on 1.5% agarose gel electrophoresis stained with ethidium bromoide. M: DNA Marker (Ladder 100 bp). Lanes 1-19 resemble 190, 490 bp of PCR products. 387J Contemp Med Sci | Vol. 7, No. 6, November-December 2021: 384–391 S.H. Kamel et al. Original ACE Receptor Genotype and its Activity Level as Potential Predictors of the Severity COVID-19 among Iraqi Patients Table 4. Comparison of age and body mass index in severe Covid-19 cases according to ACE-2 gene polymorphism allele, ID and II obtained by polymerase chain reaction amplification as compared with control DD allele Parameters Moderate Covid-19, N = 54 P-valueID allele N = 46 Mean ± SD II allele N = 8 Mean ± SD Age, Year 52.91 ± 14.62 47.13 ± 12.32 0.297 BMI, (kg/m2) 33.07 ± 5.6 29.83 ± 4.38 0.126 N, Number; BMI, Body Mass Index; SD, Standard Deviation; P-value, Prober value; ID, Insertion/Deletion; II, Insertion/Insertion. significant differences between severe Covid-19 and appar- ently health control group (P < 0.001). The angiotensin-converting enzyme gene (ACE-2) was located on chromosome 17q23.3, spansv21 kb, and comprises 26 exons and 25 introns. Exon 26 encodes for the functionally important membrane-anchoring domain of the ACE-2 protein. Insertion (I allele) polymorphism had band in (490 bp) of an Alu repetitive element in an intron of the ACE-2 receptor gene that called homozygotes. Deletion genotype (D allele) had band in (190 bp) that lack the repetitive element also called homozygotes, while (ID) genotype had two band I and D in the same gene that mean in the same gene had insertion and deletion of an Alu repetitive element in intron of the ACE-2 receptor gene and it is called heterozygotes, so the two band in the same location differ in speed of migration as shown in Figure 1. The (DD) genotype showed band at 190 bp, (II) genotype showed band at 490 bp while (ID) genotype showed both bands at 190 and 490 bp. The size of each of (DD, II and ID) genotypes was determined as indicated in Table 4. Therefore, individuals with normal homozygous deletion/ deletion (DD) revealed band size of (190 bp), while individ- uals with normal insertion/insertion (II) revealed band size of (490 bp), and the individuals with heterozygous insertion/ deletion (ID) revealed two bands size at each of (190, 490 bp) respectively, Figure 1. Table 4 shows the comparison of age and body mass index in moderate Covid-19 group with the ACE-2 receptor gene allele types by unpaired t test. In this table show age and BMI are non-significant correlated with moderate Covid-19 group according to the allele (ID) and (II). Table 5 shows a significant result for mean ± SD of age between severe Covid-19 as compared with apparently control groups according to the ACE-2 receptor gene polymorphism allele deletion/deletion (DD), but the relation with BMI was non-significant. Table 6 show the results obtained for the relation between hypertension and T2DM associated with Covid-19 are signifi- cant as compared with control groups according to the ACE-2 receptor gene polymorphism allele (DD) but for moderate Covid-19 group the relation was non-significant with ACE-2 receptor gene polymorphism alleles (ID) and (II). As shown in Table 6 the effect of smoking nicotine on the renin-angiotensin system was observed which indicated that 8.7% of angiotensin-converting enzyme receptor gene poly- morphism allele (ID) in moderate Covid-19 and 8.5% of (DD) allele in severe Covid-19 infection were smokers respectively Table 5. Shows a comparison between severe Covid-19 and control groups according to the type of ACE-2 receptor gene allele deletion/deletion for age and BMI Parameters Severe Covid-19 DD allele N = 59 Mean ± SD Control DD allele N = 27 Mean ± SD P-value Age, Year 59.1 ± 12.64 41.96 ± 12.62 <0.001 BMI, (kg/m2) 33.07 ± 5.72 33.93 ± 6.41 0.537 N, Number; BMI, Body Mass Index; SD, Standard Deviation; P-value, Prober value; DD, Deletion/Deletion allele found in sever Covid-19 and control group. Table 6. Comparison of gene polymorphism of ACE-2 alleles (ID and II) with hypertension, type 2 diabetes mellitus and smoking in moderate Covid-19 cases and compared with severe and control alleles (DD) Parameters Moderate Covid-19 P-value Severe Covid-19 Control P-valueID allele N = 46 (%) II allele N = 8 (%) DD allele N = 59 (%) DD allele N = 27 (%) Hypertension with 25 (54.3%) 5 (62.5%) 0.720 26 (44.1%) 0 (0.0) <0.001 without 21 (45.7%) 3 (37.5%) 33 (55.9%) 27 (100%) T2DM with 16 (34.8%) 4 (50.0%) 0.450 28 (47.5%) 0 (0.0) <0.001 without 30 (65.2%) 4 (50.0%) 31 (52.5%) 27 (100%) Smoking Yes 4 (8.7%) 0 (0.0) 1.000 5 (8.5%) 0 (0.0) 0.320 No 42 (91.3%) 8 (100%) 54 (91.5%) 27 (100%) N, Number; T2DM, Type 2 Diabetes Mellitus; ID, Insertion/Deletion; II, Insertion/Insertion; P value, Prober value. as compared with control group. Nicotine can impact the angiotensin-converting enzyme (ACE-2), which is relevant because coronaviruses bind to ACE-2. Current and past tobacco smoking is associated with changes in ACE-2 receptor expression. Table 6 indicated that most of patients whether it is severe or moderate infection with Covid-19 nonsmokers, the reason is due to nicotine may bind with the ACE-2 receptor and decrease levels of ACE-2 in multiple organs.27 Smoking is associated with increased susceptibility and mortality in MERS‐CoV infection, potentially due to up-regulation of dipeptidyl peptidase‐IV, the host receptor for MERS‐CoV, in smokers. In the context of respiratory viruses, smoking has been reported to cause increased hospital and ICU admissions with influenza infection, greater severity with respiratory syn- cytial virus bronchiolitis and increased mortality with viral pneumonia.28 In Table 7 all parameters (ferritin, LDH, CRP, ACE2, ALT and AST) are significantly elevated in moderate group according to the allele ID compared with control group expect ALP, while they are significantly elevated in severe Covid-19 group as compared with control according to the DD allele expect ACE-2 which was non-significantly changes. 388 J Contemp Med Sci | Vol. 7, No. 6, November-December 2021: 384–391 ACE Receptor Genotype and its Activity Level as Potential Predictors of the Severity COVID-19 among Iraqi Patients Original S.H. Kamel et al. Table 7. Comparison of biochemical in ACE-2 Alleles, ID allele in moderate Covid-19 group as compared to ID allele in control and also the DD allele in severe Covid-19 as compared with control DD allele Parameters ID allele in moderate Covid-19 group as compared with control DD allele in severe Covid-19 group as compared with control Group Mean ± SD P-value Group Mean ± SD P-value Ferritin, (ng/ml) Moderate 602.24 ± 509.41 <0.001 Severe 765.04 ± 359.37 <0.001 Control 81.36 ± 47.31 Control 70.08 ± 26.96 LDH activity, (U/l) Moderate 395.78 ± 240.34 <0.001 Severe 432.9 ± 211.86 <0.001 Control 132.93 ± 37.95 Control 140.98 ± 39.41 CRP, (mg/dl) Moderate 9.56 ± 3.26 <0.001 Severe 15.02 ± 23.53 <0.001 Control 0.34 ± 0.15 Control 0.36 ± 0.13 ACE-2 activity, (ng/ml) Moderate 3.14 ± 0.83 0.001 Severe 3.74 ± 0.85 0.345 Control 2.55 ± 0.62 Control 3.66 ± 0.64 ALT activity, (U/l) Moderate 60.81 ± 19.25 <0.001 Severe 101.11 ± 266.69 <0.001 Control 29.56 ± 11.71 Control 26.54 ± 9.7 AST activity, (U/l) Moderate 41.28 ± 12.32 <0.001 Severe 51.6 ± 53.43 <0.001 Control 22.77 ± 8.33 Control 23.54 ± 8.78 ALP activity, (U/l) Moderate 89.87 ± 30.73 0.063 Severe 109.48 ± 39.34 <0.001 Control 77.12 ± 23.52 Control 73.66 ± 24.41 LDH: Lactate dehydrogenase; CRP: C- Reactive protein; ACE-2: Angiotensin Converting Enzyme; ALT: Alanine Aminotransferase; ALP: Alkaline phosphatase; AST: Aspartate Aminotransferase; S.D: Standard Deviation; DD: Deletion/Deletion; ID: Insertion/Deletion. Table 8 show the comparison of biochemical in control group according to allele. In this table all parameters in control group are non-significant according to the allele DD and ID expect ACE-2 is significant. Discussion As indicated previously, age and BMI are non-significantly in moderate Covid-19 group according to the (ID) and (II) allele as shown in Table 2, whereas age is significant between severe and control groups according to the deletion/deletion allele (DD), as shown in Table 3 but BMI was non-significantly. The DD allele found in severe and control group but was absent in moderate group. The ACE-2 receptor gene polymorphism allele (DD) in severe Covid-19 was significantly associated with hyperten- sion and T2DM as compared with control groups P < 0.001, but the (ID) and (II) alleles in moderate Covid-19 group was association non-significantly, P = 0.720 and P = 0.450 respec- tively as shown in Table 4. The total 113 cases of Covid-19 patients were selected with demographic and clinical charac- teristics. We observed that high frequency of hypertensive patients was (54.3%) as compared to diabetic (34.8%) as shown in Table 4. In contrast, de Abajo et al., found higher prevalence of diabetes in 1339 Covid-19 cases as compared with 13,390 matched controls (27.2% vs 20.3%; crude odds ratio, OR, 1.50).29 Similarity, Singh et al., pooled different studies (N = 2209) and found higher percentage of hypertension (21%) as compared to diabetes (11%) and CVD (7%).30 In con- trast, a meta-analysis of Covid-19 patients (N = 1576 patients) reported percentage of different comorbid conditions such as hypertension (17%) > diabetes (8%).31 To date, few studies have been published that investigate the relationship between ACE-2 gene polymorphism and Covid-19 severity, but we are Table 8. Comparison of biochemical in control group according to allele Parameters Group Mean ± SD P-value Ferritin, (ng/ml) DD allele 70.08 ± 26.96 0.581 ID allele 81.36 ± 47.31 LDH activity, (U/l) DD allele 140.98 ± 39.41 0.441 ID allele 132.93 ± 37.95 CRP, (mg/dl) DD allele 0.36 ± 0.13 0.278 ID allele 0.34 ± 0.15 ACE-2 activity, (ng/ml) DD allele 3.66 ± 0.64 <0.001 ID allele 2.55 ± 0.62 ALT activity, (U/l) DD allele 26.54 ± 9.7 0.305 ID allele 29.56 ± 11.71 AST activity, (U/l) DD allele 23.54 ± 8.78 0.994 ID allele 22.77 ± 8.33 ALP activity, (U/l) DD allele 73.66 ± 24.41 0.420 ID allele 77.12 ± 23.52 LDH, Lactate dehydrogenase; CRP, C- Reactive protein; ACE-2, Angiotensin Converting Enzyme-2; ALT, Alanine aminotransferase; ALP, Alkaline phosphatase; AST, Aspartate aminotransferase; SD, Standard Deviation; DD, Deletion/Deletion; ID, Insertion/Deletion. still lacking definite results.17,32–34 In present study, we observed that individual with ‘DD’ genotype showed significantly 3.69- fold higher risk of Covid-19 severity. Similarly, other studies found that ACE-1 D/D-genotype showed association with Covid-19 related mortality.35,36 Gomez et al. found D allele was significantly associated with hypoxemic as compared to non-hypoxemic patients; however, ‘DD’ genotype individuals did not show any association with Covid-19 infection.37 389J Contemp Med Sci | Vol. 7, No. 6, November-December 2021: 384–391 S.H. Kamel et al. Original ACE Receptor Genotype and its Activity Level as Potential Predictors of the Severity COVID-19 among Iraqi Patients Some studies have reported an association of the DD gen- otype of ACE-2 polymorphism and higher circulating levels and activity of ACE-2,38 which could explain the higher sus- ceptibility to develop severe forms of the disease in patients with the DD genotype, in addition to hypertension and T2DM. SARS-CoV-2 sequesters ACE-2 to invade cells, decreasing the bioavailability of ACE-2 which entails a reduction in the deg- radation of ANG II and an exacerbation of the damaging effects of ANG II.39 Thus, the lung injury and inflammation caused by the reduced ACE-2 levels due to the viral infection, and also by the hypertension and diabetes may be worsened by ACE-2 genotypes that further increase ACE-2 levels, and hence ANG II levels, such as the DD genotype of ACE-2 ana- lyzed. In our study we confirmed that ACE-2 levels are associ- ated with the DD genotype of both polymorphisms, which in turn is associated with greater severity of the disease in hyper- tensive and T2DM. Although according to our data we could not affirm a direct association with Covid-19 severity. Covid-19 is a highly contagious disease characterized by high mortality, especially for patients with severe comorbidi- ties such as diabetes, hypertension, CVD and CKD.9 The doc- umented history of diabetes has been stated to be an independent indicator of morbidity and death in SARS patients.40,41 Diabetes hyperglycemia is suspected to cause immune response dysfunction, which fails to regulate the spread of invasive pathogens. Therefore, diabetic individuals are considered to be more prone to infections and incidence of infectious diseases and it will increase associated comorbidi- ties.42 A study was released by the Chinese Centre for Disease Control and Prevention, which showed elevated mortality rate in people with diabetes (2.3%, total and 7.3%, diabetes patients) study was performed in 72,314 cases of Covid-19.43 Blood pressure homeostasis maintained by renin–angio- tensin system (RAS).44,45 RAS system modulated by ACE-1 and ACE-2, angiotensin I is converted into angiotensin II (ATII) by ACE1 and degraded bioactive bradykinin.46 Insertion/dele- tion (I/D) polymorphism has been correlated with levels of circulating and tissue ACE-1 and influences almost half of the variability of serum ACE levels in the general population. The ‘D’ allele of ACEI/D is associated with higher ACE activity.47 This means individuals with DD genotype showed approxi- mately twice ACE activity levels as compared to II genotype individuals.48 The ‘D’ allele of ACE-1 gene is significantly asso- ciated with hypoxemic group as compared to non-hypoxemic group.49 Recently, Delanghe et al. found that the prevalence of Covid-19 in 33 countries has been substantially associated with ACE1 I/D polymorphism.50 However, our results clearly revealed that polymorphisms of the ACE-2 gene are related to the risk of developing severe Covid-19 (ICU admission) in hypertension and diabetic patients. Our study confirms with previously reported find- ings of another ACE polymorphism on Covid-19 patients with hypertension37 however; it did not corroborate the association with Covid-19 severity found by Gomez et al. and other authors for the ACE I/D polymorphism, regardless of comor- bidities. Unlike these studies, we also found an additional association of D Allele with enhanced severity in hypertension and diabetic patients, and interestingly, we further found a higher prevalence of the DD, ID genotypes (all containing the D allele) among deceased ICU-patients, not described so far, confirming the deleterious effect of the D allele in Covid-19 outcomes. Precisely most of these patients were hypertensive. In Table 7 all parameters (Ferritin, LDH, CRP, ACE-2, ALT and AST) are significant in moderate group according to the allele ID compared with control group expect ALP. All these parameters are significantly elevated in severe group according to the allele DD as compared with control group expect ACE-2, because D allele responsible for activity of ACE-2 and this allele found homogenous at form DD in severe and control group that carry DD genotype, so that non-significant between severe and control according to the allele DD. Genetic polymorphisms in ACE-2 indicate that the ACE I/D polymorphism, have been shown to affect ACE-2 activity levels and confer susceptibility to hyperten- sion type 2 diabetes,38 overweight nephropathy and certain cardiovascular and autoimmune diseases. More specifically, the DD genotype of the ACE I/D polymorphism has been associated to higher levels of serum ACE and higher levels of circulating IL-6 in patients with myocardial infarction. In contrast, the II genotype has been associated to lower circu- lating ACE levels. Since some of these processes have been reported to be involved in the pathogenesis of Covid-19, the DD genotype could predispose to complications of Covid-19 due to higher baseline ACE levels and its consequences.37 Indeed, an association of the DD genotype of the ACE I/D polymorphism with severe Covid-19 has been reported in hypertensive males.37 However, analyzing the I/D polymor- phism is laborious and time-consuming and some authors have described a preferential amplification of the D allele. The ACE-2 gene polymorphisms are in complete linkage dis- equilibrium with the ACE I/D polymorphism, therefore they could be better prognostic markers. Table 8 show all parameter are non-significant correlated because there were healthy peoples. ACE-2 was significantly to DD and ID alleles because the D allele is associated with higher ACE-2 activity. Mean ACE activity levels in DD car- riers were approximately twice that in II genotype individuals. Therefore, it may be propose a hypothesis that ACE-2 gene polymorphism may play an important role in patients with Covid-19 who are susceptible to develop severe lung injury or ARDS.48 Conclusions The severity of Covid-19 patients may depend on age, dia- betes, hypertension and ACE-2 gene polymorphism. The observed results suggest that a prospective paradigm of DD genotype that has the potential help to explain the suscepti- bility of the host response to SARS-Cov-2 infection and involve in numerous pathological. Thus, ACE-2 I/D polymor- phism may be act as a useful tool to predict the development of disease and may have an influence on the treatment out- comes against the Covid -19 to establish a population-based therapeutic development. Acknowledgments All authors would like to thank the participated patients and the team of Covid-19 Centers for their support during this study. 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