Angiotensin-converting enzyme 2 SNPs as the common genetic loci and optimal early identication genetic markers for COVID-19

signicantly related to COVID-19. For rs4646142, there were also signicant differences between GC and GG genotypes (OR = 0.58, 95% CI; 0.34-0.96, P = 0.035). These results suggested that ACE2 SNPs rs4646142, rs6632677 and rs2074192 were different between normal subjects and COVID-19 patients in our population.


Background
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes Coronavirus disease 2019 (COVID-19) via respiratory infection, was rst discovered in Wuhan, China, in December 2019. Since then, the virus has become deeply entrenched in many countries and territories, and is an enormous threat to global public health and the economy. As of June 02, 2021, over 170 million cases of COVID-19 have been con rmed worldwide and 3.56 million deaths have resulted from SARS-CoV-2 infection (1). The most common clinical symptoms of COVID-19 are fever (2), dry cough, headache (3), sore throat, nausea, breathlessness, loss of taste or smell (4) while some remain asymptomatic (5, 6). Morbidity and mortality caused by COVID-19 are also closely associated with past co-existing diseases, especially cardiovascular diseases (7). Meanwhile, human genetic factors may lead to the increased transmissibility of SARS-CoV-2 and contribute to persistent progressive disease observed in a part of infected people, but these genetic factors are largely unknown (8). Firstly, four mathematical models were constructed to predict SARS-CoV-2 transmission and the effectiveness of eradication strategies (9). The effective detection rate and detection time of SARS-CoV-2 real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) analysis with the sensitivity and speci city of various antibody detection methods were reviewed (10). We have further investigated that the determinants of disease severity seem to be mainly derived from host factors, such as age and lymphopenia, while genetic variation of the virus has no signi cant effect on the outcomes of COVID-19 (11). Furthermore, abundant studies have indicated the possible associations of ACE2 single-nucleotide polymorphisms (SNPs) and cardiovascular risks in patients with cardiovascular disease (12)(13)(14). These observations suggest how much of the variation in COVID-19 disease severity may be explained by genetic types. Hence, early warning of in ammatory infections and cardiovascular disease risks by identifying host genetic DNA polymorphisms (variants), including excessive immune responses to viruses, will greatly promote the development of new prevention and/or treatment strategies for COVID-19.
At present, the cognition of the physicochemical property of SARS-CoV-2 mostly derived from research on SARS-CoV and MERS-CoV. As is well-known, angiotensin-converting enzyme 2 (ACE2) was highly expressed in human pneumocytes, intestinal epithelial cells and endothelial cells (15,16). Given the importance of 3D structure of proteins in protein-protein interaction (PPI) it's not surprising that any process resulting in ACE2s' 3D structure change could in uence COVID-19s' cell entry. Accumulating evidence has shown that 3D structure of ACE2 might be in uenced at both transcriptional and posttranscriptional levels. SNPs are able to impact on protein function, structure, stability, and abundance (17). Therefore, the fundamental role of ACE2 in virus infection created this hypothesis that different complications to SARS-CoV-2 might be due to different ACE2 SNPs (18). Since the outbreak of the COVID-19 pandemic several authors have speculated about the role of the ACE and ACE2 gene polymorphisms in disease susceptibility and severity. However, as to what extent the variable response to complications (in ammatory infections and cardiovascular disease risks) with COVID-19 is in uenced by the variability of the hosts' genetic background was almost unclear.
In this study, we speculated that genetic factors in the ACE2 gene are likely to impact the susceptibility to COVID-19 with previous coexisting disease. By establishing the SNP genotyping method and collecting literature to ACE2 polymorphisms, we chose rs2074192, rs6632677, rs4646142, rs2048683, rs4240157, ve intronic SNP in ACE2, based on different genotyping, the analysis of clinical indicators is performed in COVID-19 patients. We veri ed the pathological degree of the COVID-19 was associated with hypertension and diabetes. Furtherly, our observations illustrated that ACE2 SNPs rs4646142 and rs6632677 may be an optimal genetic susceptibility marker for COVID-19 related cardiovascular complications. In summary, this study aims to present possible variants in the regulatory regions of ACE2, which may lead to marked in ammatory infections and cardiovascular disease clinical indicators variations in the prevalence and mortality of COVID-19. These may be further used in the genetic association study of patients with SARS-CoV-2 infection.

Statistical analysis
Allele frequencies were calculated following the genotypes. categorical variables were presented as number (%) and continuous variables were presented as mean ± standard deviation (SD). Categorical variables were compared using Chi-square-test and continuous variables were compared using one-way analysis of variance. The Odds ratio (OR) between control genotype and SARS-CoV-2 risk genotype for each ACE2 SNP among categorical variables was evaluated using binary logistic regression. a 95% con dence interval. A P value less than 0.05 was considered statistically signi cant. All probabilities are two-tailed.

Characteristics of the study participants
Among 196 patients who had a laboratory-rmed SARS-CoV-2 infection, there were two cases of mild type, 184 cases of common, three cases of critical disease, and seven cases of severe COVID-19 (Table  1), which were classi ed according to the latest WHO regulations (23). The correlation between the COVID-19 severity degree and baseline clinical characteristics were showed in Table 1. We found that the proportion of older patients (≥ 60 years old) with critical and severe COVID-19 were higher than common (P = 0.029). The prevalence of major coexisting illnesses such as heart disease (P = 0.021) and Pulmonary disease (P < 0.001) were also more likely to exist differently in these patients with COVID-19. Interestingly, lower percentage of patients with drug allergy were discovered in the patients with wild, critical disease and severe COVID-19. From the disease origin perspective, there was signi cantly different disease types of COVID-19 in the inland and oversea patient (P < 0.001). The most-reported symptoms of COVID-19 were fever (84.2%), dry cough (48.9%) and are shown in Table 2. Furthermore, the most common initial therapies were oxygen and arbidol antiviral treatment (Table 2).
Sore/Itchy/dry throat 0 (0) 12 (6.5%) 0 (0) 0 (0) 12 (6.1%) Others symptoms 0 (0) 23 (12.5%) 0 (0) To nd out further relationships between the 5 ACE2 polymorphisms and COVID-19, we used LightCycler®480 II system for TaqMan ®SNP Genotyping Assays and LightCycler®480SW1.5.1 software for data analysis (Figure 1). Among the results of rs2048683 (T > G) genotyping (Fig. 1A, F), FAM uorescent labeled probe targeted at base T and VIC uorescent labeled probe targeted at base G. The prediction analysis of rs2048683 locus of 210 normal subjects (Fig. 1A) in peripheral blood was as follows: TT genotype 0 cases, TG genotype 0 cases, GG genotype 192 cases (red dots), and 18 cases were not classi ed into different genotypes (blue dots).The predicted analysis of the locus of 196 patients with COVID-19 ( Fig. 1F)were as follows: TT genotype 1 case (blue dots), TG genotype 22 cases (green dots), GG genotype 168 cases (red dots), and 18 cases were not classi ed into different genotypes (purple dots). Among the results of rs4240157 (C > T) genotyping (Fig. 1B, G), FAM uorescent labeled probe targeted at base T and VIC uorescent labeled probe targeted at base C. The prediction analysis of rs4240157 locus of 210 normal subjects (Fig. 1B) in peripheral blood was as follows: CC genotype 7 cases (red dots), CT genotype 11 cases (green dots), TT genotype 181 cases (blue dots), and 11 cases were not classi ed into different genotypes (purple dots). The predicted analysis of the locus of 196 patients with COVID-19 (Fig. 1G) were as follows: CC genotype 7 case (red dots), CT genotype 4 cases (green dots), TT genotype 183 cases (blue dots), and 2 cases were not classi ed into different genotypes (purple dots). Among the results of rs4646142 (G > C) genotyping (Fig. 1C, H), FAM uorescent labeled probe targeted at base G and VIC uorescent labeled probe targeted at base C. The prediction analysis of rs4646142 locus of 210 normal subjects (Fig. 1C) in peripheral blood was as follows: CC genotype 81 cases (red dots), GC genotype 73 cases (green dots), GG genotype 53 cases (blue dots), and 3 cases were not classi ed into different genotypes (purple dots).The predicted analysis of the locus of 196 patients with COVID-19 (Fig. 1H) were as follows: CC genotype 76 case (red dots), GC genotype 51 cases (green dots), GG genotype 64 cases (blue dots), and 5 cases were not classi ed into different genotypes (purple dots). Among the results of rs6632677 (G > C) genotyping (Fig. 1D, I), FAM uorescent labeled probe targeted at base G and VIC uorescent labeled probe targeted at base C. The prediction analysis of rs6632677 locus of 210 normal subjects (Fig. 1D) in peripheral blood was as follows: CC genotype 37 cases (red dots), GC genotype 43 cases (green dots), GG genotype 122 cases (blue dots), and 3 cases were not classi ed into different genotypes (purple dots).The predicted analysis of the locus of 196 patients with COVID-19 (Fig. 1I) were as follows: CC genotype 12 case (red dots), GC genotype 15 cases (green dots), GG genotype 169 cases (blue dots), and 5 cases were not classi ed into different genotypes (purple dots). Among the results of rs2074192 (C > T) genotyping (Fig. 1E, J), FAM uorescent labeled probe targeted at base T and VIC uorescent labeled probe targeted at base C. The prediction analysis of rs2074192 locus of 210 normal subjects (Fig. 1E) in peripheral blood was as follows: CC genotype 84 cases (red dots), CT genotype 69 cases (green dots), TT genotype 53 cases (blue dots), and 19 cases were not classi ed into different genotypes (purple dots).The predicted analysis of the locus of 196 patients with COVID-19 (Fig. 1J) were as follows: CC genotype 84 case (red dots), CT genotype 48 cases (green dots), TT genotype 59 cases (blue dots), and 5 cases were not classi ed into different genotypes (purple dots).

Association of ACE2 SNPs and clinical indicators
C-reactive protein (CRP) is the main acute phase protein with a rapid and sharp increase in plasma concentration during infection and tissue damage, which can activate complement system and strengthen the phagocytosis of phagocytes. Acid glycoprotein (AGP) is the main acute response protein, which is increased during acute in ammation. Obviously, CRP and AGP play a vital protective role in the body's innate immune process. As shown in Fig. 2A.F, COVID-19 related ACE2 SNP rs4646142 was associated with increased CRP (P < 0.001) and AGP (P < 0.001), and rs6632677 may also be associated with increased CRP (P = 0.052). High-density lipoprotein (HDL) is a plasma lipoprotein that resists atherosclerosis and is a protective factor for coronary heart disease. Apolipoprotein A (ApoA) is also an important component of plasma HDL, which can effectively remove lipids from tissues and is considered ananti-atherosclerosis factor. As shown in Fig. 2C.D. ACE2 SNP rs4646142 were correlated with the abnormality of ApoA (P = 0.003) and HDL (P = 0.009). For Fig. 2B. ACE2 SNP rs4646142 was signi cantly associated with prealbumin (PAB) (P = 0.044), and PAB is used as a sensitive nutritional protein indicator. For Fig. 2E. ACE2 SNP rs6632677 was further associated with HPT (P = 0.027), and rs4646142 may also be associated with HPT although there is no signi cant difference (P = 0.083). Dramatically, ACE2 SNP rs2074192 was not correlated with any clinical indicators.

Discussion
Notably different responses to SARS-CoV-2 infection among different populations raise the possibility that different SNPs pro le might be responsible for the risk of acute in ammation, cardiovascular, hypertension, diabetes, and stroke in COVID-19patients. In the present study, we identi ed in our population ACE2 SNPs rs4646142, rs6632677 and rs2074192, are associated with COVID-19 following SARS-CoV-2 infection. As far as we know, this is the rst genetic study that has shown rs4646142, rs6632677 and rs2074192 may increase the odds of COVID-19 infection with previous coexisting disease in Chinese. Recent reports in Caucasian individuals determined the ACE2 rs2285666 variant was related to hypertension in elderly population (24). Taken together, the ndings indicate a prominent role of ACE2 polymorphisms in the pathogenesis of COVID-19. Previous studies have reported that among patients with COVID-19 and coexisting hypertension, the mortality was lower through taking ACE inhibitors or angiotensin II receptor blockers (ARBs), when compared with COVID-19 patients with hypertension not taking such drugs (27). One study put forward a hypothesis on the role of Renin-Angiotensin-System (RAS) pathway genes including ACE2 (rs2285666, rs1978124, rs714205) in COVID-19 prognosis: inherited genetic predispositions can forecast the degree of severity of COVID-19 (28). Besides, the rs2285666 allele (T or A) was signi cantly positively correlated with lower infection and mortality among Indian population (29).
As can be seen from the baseline clinical characteristics (  (Table S1). Next, through the sequence of UCSC hg19 (exome) and the reference gene Homo sapiens angiotensin I converting enzyme 2 (ACE2, NG_012575.1:6110-46037) on chromosome X, 5 ACE2 SNPs (rs4646142 G > A or C, rs2048683 T > A or G, rs4240157 C > G or T, rs6632677 G > C, rs2074192 C > T) known to be associated with human cardiovascular diseases and diabetes were checked. Notably, research showed that SARS-CoV-2 is most closely associated with bat coronaviruses, 100% amino acid resemblance to bat SL-CoVZC45 in the E proteins and nsp7. That is, bats are a possible host of SARS-CoV-2 virus (30). Interestingly, rs4646142, rs2048683, rs4240157, rs6632677 and rs2074192 were completely consistent with bats ACE2 SNPs, while the SNPs in snakes were signi cantly different (Fig. S1), suggesting that these SNPs may be associated with the infection of SARS-CoV-2. We hypothesized that ACE2 variants rs2074192 and rs6632677 could modi ed the disease outcome.
In this study, we investigated possible associations between ACE2 polymorphisms and COVID-19 in our collected population. we found that ACE2 SNPs rs4646142, rs6632677, rs2074192 might correlated with the susceptibility of COVID-19 related cardiovascular risk and acute in ammatory infection. So why exactly do these SNPs impact susceptibility to COVID-19? We found that rs4646142, rs6632677 and rs2074192 were located in the intron region and could not encode amino acids. This may be related to the adjustment mode of the intron sequence. For instance, existing research shows that NS1 protein-RNA interactome, and NS1 primarily binds intronic sequences as a multifunctional virulence factor of the in uenza a virus, thereby inhibiting cellular processes to accelerate viral gene expression (31). Human transformer 2 alpha homolog (huTRA2A) can inhibit mRNA splicing by binding to the intron silencer motif in the NS mRNA of the PR8/H1N1 virus (32). Some SNPs in tonicity-responsive enhancer binding protein (TonEBP) can impact transcription process as cis-expressed quantitative trait loci. Meanwhile, these SNPs are associated with increased risk of T2D, in ammation, hypertension, indicating that changes in TONEBP expression may be related to these phenotypes (33). In other words, these SNPs may be able to impact SARS-Cov2 infection process by binding to regulatory proteins or RNAs.

Conclusions
In conclusion, our study showed that the pathological degree of the COVID-19 may be associated with hypertension and diabetes. And, ACE2 gene variants were associated with the degree of severity in COVID-19 patients. Furthermore, ACE2 SNPs rs4646142 and rs6632677 may be an optimal genetic susceptibility marker for COVID- 19