key: cord-1010883-vee44eei authors: Mahmood, Zainab S.; Fadhil, Hula Y.; Abdul Hussein, Thaer A.; Ad'hiah, Ali H. title: Severity of coronavirus disease 19: Profile of inflammatory markers and ACE (rs4646994) and ACE2 (rs2285666) gene polymorphisms in Iraqi patients date: 2022-01-10 journal: Meta Gene DOI: 10.1016/j.mgene.2022.101014 sha: 17a720abf100e94dd58856f832356931c4910e3f doc_id: 1010883 cord_uid: vee44eei Susceptibility to coronavirus disease 2019 (COVID-19) and disease severity has recently been associated with inflammatory markers and genetic polymorphisms of ACE (angiotensin-converting enzyme) and ACE2 genes, but the evidence has been inconclusive. This case-control study (99 COVID-19 patients and 96 controls) sought to assess the significance of age, C-reactive protein (CRP), neutrophil-to-lymphocyte ratio (NLR) and SARS-CoV-2 RT-PCR cycle threshold (Ct) in severity of COVID-19. Besides, two variants of ACE and ACE2 genes (rs4646994 and rs2285666, respectively) were analyzed to determine their role in COVID-19 susceptibility and/or disease severity. Results revealed that age, CRP and NLR were significantly elevated in severe cases compared to moderate cases, while RT-PCR Ct value was significantly decreased. Allele and genotypes of both variants were not associated with COVID-19 risk, with the exception of rs2285666 A allele. It showed a significantly higher frequency in female patients than in female controls (probability = 0.041). In conclusion, the study indicated the role of age, CRP, NLR and SARS-CoV-2 RT-PCR Ct in susceptibility to COVID-19 severity. However, analysis of the ACE and ACE2 gene variants (rs4646994 and rs2285666, respectively) showed that the two variants were not associated with the risk of developing COVID-19. ACE2 gene expression and age and was independent of gender. It has been suggested that lower ACE2 expression in children compared to adults may explain the lower prevalence of COVID-19 in children (Bunyavanich et al., 2020) . Further, it has been proposed that RAS, and through the opposite functions of ACE and ACE2, plays a significant role in the development of acute lung diseases, particularly acute respiratory distress syndrome (Gemmati and Tisato, 2020) . Common variants (single nucleotide polymorphisms; SNPs) in the ACE and ACE2 genes have been described, and their association with the risk of various diseases has been indicated. The ACE gene (Gene ID: 1636) is located in the long arm of human chromosome 17 (17q23.3) and consists of 26 exons (https://www.ncbi.nlm.nih.gov/gene/1636). Insertion/deletion (I/D: rs4646994) is among the distinct genetic polymorphisms of this gene, and besides being associated with the risk of hypertension, heart disease, kidney failure, acute respiratory distress syndrome, SARS and COVID-19, it has been shown that DD genotype may influence ACE serum levels (Gómez et al., 2020) . The ACE2 gene (Gene ID: Gene ID: 59272) is located in the short arm of human X chromosome (Xp22.2) and consists of 22 exons (https://www.ncbi.nlm.nih.gov/gene/59272). Several SNPs of the gene, for instance rs4240157, rs4646155, and rs4830542, have been associated with the risk of hypertension, but recent studies in COVID-19 have focused on SNP rs2285666 (Cafiero et al., 2021; Devaux et al., 2020; Gómez et al., 2020; Karakaş Çelik et al., 2021; Pouladi and Abdolahi, 2021; Singh et al., 2021) . Although these studies proposed a role for SNPs rs4646994 and rs2285666 in COVID-19 susceptibility and/or disease severity, the evidence has not been conclusive as some findings have not been replicated. This study sought to assess the significance of age, C-reactive protein (CRP), neutrophil-to-lymphocyte ratio (NLR) and SARS-CoV-2 RT-PCR cycle threshold (Ct) in severity of COVID-19. Besides, two variants of ACE and ACE2 genes (rs4646994 and rs2285666, respectively) were analyzed to determine their role in COVID-19 susceptibility and/or disease severity. To the best knowledge of investigators, the second part of study has not been conducted on Iraqi patients with COVID-19. severe cases (68 and 31, respectively). Criteria established by the WHO Interim Guidance was followed to define the case as moderate (patient with symptoms of pneumonia and no signs of severe pneumonia) or severe (severe respiratory distress, respiratory rate ≥ 30 breaths/minute or pulse oxygen saturation (SpO 2 ) ≤ 93% on resting state) (World Health Organization, 2020). According to the patient's clinical history, some had diabetes and some had hypertension (51.5 and 53.5%, respectively). A control group of 96 individuals was also included (mean age ± SD: 43.1 ± 10.3 years; range: 24 -70 years; 50.0% males). They were healthy and had no respiratory infection in the past 12 months or chronic diseases (cardiovascular and diabetes). Besides, their serum tested negative for COVID-19 IgG and IgM antibodies (FREND COVID-19 IgG/IgM Duo kit, NanoEntek, South Korea). Nasopharyngeal swabs were obtained from patients 4 -5 days after their hospitalization. The QIAamp Viral RNA Mini kit was used to isolate viral RNA (Qiagen, Germany). Real-time polymerase chain reaction (RT-PCR) analysis was performed to diagnose SARS-CoV-2 using commercial kit (DIAGNOVITAL SARS-COV-2 real time PCR kit, RTA laboratories Biological products, Turkey), and manufacturer's instructions were followed. Based on this analysis, the SARS-CoV-2 RT-PCR Ct value was recorded for each patient. In combination with molecular testing, the diagnosis was confirmed by chest computerized tomography (CT). The only patients included were those who showed a positive molecular test and had a CT scan indicating COVID-19. Five milliliters of venous blood were collected and distributed into plain and ethylene-diamine-tetra-acetic-acid (EDTA) tubes (3 and 2 mL, respectively). Plain tube was left to clot and then centrifuged (15 minutes at 4 °C for 15 minutes) to collect serum. Serum was tested for CRP using electro-chemiluminescence immunoassay system (Roche Cobas Integra 400 plus, Switzerland). EDTA blood was used to count white blood cells (WBC: total, neutrophils and lymphocytes) using automated hematology analyzer (ABX Micros ES 60, Horiba, USA). The NLR was obtained by dividing the absolute neutrophil count by the absolute lymphocyte count. 2014). Briefly, the PCR was performed in a total volume of 25 µL, with 5 µL AccuPower PCR PreMix (Bioneer, Korea), 1µl forward primer (5'-CTGGAGACCACTCCCATCCTTTCT-3'), 1µL reverse primer (5'-GATGTGGCCATCACATTCGTCAGAT-3'), 3 µL DNA and 15 µL deionized distilled water. The tube was transferred to thermal cycler (Eppendorf, Germany) that was programmed for the following optimized conditions: an initial denaturation cycle (94 °C for 5 minutes), followed by 35 cycles of denaturation (94 °C for 30 seconds), annealing (58 °C for 30 seconds) and extension (72 °C for 45 seconds), and a final extension cycle (72 °C for 5 minutes). The PCR products were electrophoresed in agarose gel (1.5%; 5 V/cm 2 for 55 minutes), and migrating bands were visualized using gel documentation system. Two bands of different molecular sizes were encountered; 490bp (I allele) and 190bp (D allele) (Supplementary Figure) . For SNP rs2285666, also a previous method was adopted (Wu et al., 2017) . Briefly, the PCR was performed in a total volume of 25 µL, with 5 µL AccuPower PCR PreMix (Bioneer, Korea), 1µl forward primer (5'-CATGTGGTCAAAAGGATATCT -3'), 1µL reverse primer (5'-AAAGTA AGGTTGGCAGACAT -3'), 3 µL DNA and 15 µL deionized distilled water. The tube was transferred to thermal cycler (Eppendorf, Germany) that was programmed for the following optimized conditions: an initial denaturation cycle (95 °C for 1 minute), followed by 35 cycles of denaturation (94 °C for 30 seconds), annealing (50.6 °C for 30 seconds) and extension (72 °C for 45 seconds), and a final extension cycle (72 °C for 7 minutes). The PCR products were digested with the restriction enzyme Alu I for 4 hours at 37 °C. Then, the digested PCR products were electrophoresed in agarose gel (1.5%; 5 V/cm 2 for 55 minutes), and migrating bands were visualized using gel documentation system. Allele G was presented with a single band with a molecular size of 466bp, while allele A had two bands with molecular sizes of 281bp and 185bp (Supplementary Figure) . Number and percentage were used to describe categorical variables and significant differences were assessed by two-tailed Fisher exact test. Continuous variables were tested for normality (Kolmogorov-Smirnov and Shapiro-Wilk test). Normally distributed variables were given as mean and standard deviation (SD), and significant differences were evaluated with These analyses were performed using IBM SPSS Statistics 25.0 (Armonk, NY: IBM Corp.). G*Power software (version 3.1.9.7) was used to determine power of sample size. The power of sample size was estimated for COVID-19 patients (N = 99) and controls (N = 96) at an α error probability of 0.05 and effect size of 0.3. The actual power (1-β error probability) was 0.66, which is less than 0.80; statistically acceptable power. 6.5 ± 1.4 *10 9 /L; p < 0.001), neutrophil (2.4 ± 0.7 vs. 3.7 ± 0.8*10 9 /L; p < 0.001) and lymphocyte (1.0 ± 0.3 vs. 2.6 ± 0.7*10 9 /L; p < 0.001) counts decreased significantly in severe cases compared to moderate cases, while the NLR increased Genotype frequencies of rs4646994 and rs2285666 SNPs were in good agreement with HWE in COVID-19 patients and controls, as there were no significant differences between observed and expected frequencies. When comparing patients to also correlated with oxygen saturation, and patients with SpO 2 ≤ 90% were presented with markedly elevated CRP levels compared to patients with SpO 2 > 90% (76.51 vs. 12.70 mg/L) (Xie et al., 2020) . Thus, CRP has been linked to more severe disease, lung injury and worse prognosis in COVID-19 patients. NLR is an additional marker of inflammation, and it has been indicated that NLR can be used as an early warning marker for exacerbation of severe COVID-19 and can provide an objective basis for the identification and management of severe pneumonia in COVID-19 patients (Imran et al., 2021) . Furthermore, the NLR has been considered as an independent predictor of poor clinical outcome and mortality in COVID-19 patients (Ciccullo et al., 2020) . In addition to the immune system, comorbidities associated with aging such as diabetes and cardiovascular disease have been shown to be associated with the risk of contracting COVID-19 and disease severity. In the current study, most severe cases already had diabetes and hypertension and their prevalence was significantly higher in patients with severe disease than in moderately ill patients. Previous studies have also shown that diabetes and hypertension are the most prevalent comorbidities among COVID-19 patients, and have been observed to increase the risk of morbidity and mortality (Barrera et al., 2020; De Almeida-Pititto et al., 2020) . However, it is not clear whether the two comorbidities were independent predictors of COVID-19 severity or might have synergistic effects (Tadic and Cuspidi, 2021) . To shed light on this issue, a study was conducted to evaluate the contributions of diabetes alone, hypertension alone, or their combination to the risk of severe COVID-19 infection and other clinical complications. The data obtained revealed that hypertension was not independent risk factors for respiratory failure but slightly increased the risk of severe COVID-19, and the risk associated with hypertension may be due to the confounding effects of diabetes (Sun et al., 2021) . However, further recent study showed that diabetes and hypertension were the most common co-morbid conditions that occurred in COVID-19 patients with an approximate prevalence (45.1 and 48.1%, respectively) and based on the Framingham risk score, most of them were classified within the high-risk group (Alshaikh et al., 2021) . This study also pointed out for the significance of SARS-CoV-2 RT-PCR Ct value in determining severity of COVID-19. A Ct value represents the number of the cycle at which the signal crosses the positivity threshold, and thus a lower Ct value White blood cells; *10 9 /L 6.5 ± 1.4 3.5 ± 0.8 < 0.001 Neutrophils; *10 9 /L 3.7 ± 0.8 2.4 ± 0.7 < 0.001 Lymphocytes; *10 9 /L 2.6 ± 0.7 1.0 ± 0.3 < 0.001 Neutrophil-to-lymphocyte ratio 1. 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