Abstract

We aimed to analyze the expression profile of ACE2 and similar genes with ACE2, predict the number of variations in ACE2, detect the suspected SNPs on ACE2 gene, and perform the pathway analysis of renin-angiotensin system (RAS) and protein absorption-digestion. Moreover, we have predicted the gene-related diseases with ACE2. STRING was used to analyze functionally similar genes with ACE2. Exome Variant Server, SIFT, Polyphen2 were used to predict the number of variations in ACE2 and detect the suspected SNPs on ACE2. KEGG database and STRING were used to draw pathway of ACE2. Then, DISEASES resource, FitSNPs, UniProt, BioXpress, IGV Browser, Ensembl Genome Browser, and UCSC Genome Browser were used to predict the ACE2 gene-related diseases and expression profile in human normal and cancer tissues. We have shown that expression of ACE2 was correlated with AGT, REN, AGTR1, AGRT2, MME2, DPP4, PRCP, MEP1A, XPNPEP2, MEP1BandACE2 is expressed in testis, kidney, heart, thyroid, colon, esophagus, breast, minor salivary gland, pancreas, lung, liver, bladder, cervix, and muscle tissues. We found 99 variations in ACE2 gene, in which no previous study has been performed. In the future, this in silico analysis should be combined with other pieces of evidence including experimental data to assign function.

Keywords:
COVID-19; ACE2; Sars-CoV-2; small intestine; kidney failure; testis; heart; renin angiotensin system

INTRODUCTION

According to the World Health Organization Coronavirus disease 2019 (COVID-19) Situation Report - 116, the total number of confirmed cases hit almost 4.3 million in the world by May 15^th, 2020 [¹1 WHO, Coronavirus disease 2019 (COVID-19) Situation Report - 116 [cited: May-15-2020]. https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200515-covid-19-sitrep-116.pdf?sfvrsn=8dd60956_2
https://www.who.int/docs/default-source/... ]. The severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) is a positive-strand RNA virus that caused 297,119 deaths over the world which makes this disease quite serious at the global level [²2 WHO, Coronavirus disease 2019 (COVID-19) Situation Report - 75 [cited: Apr-09-2020]. https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200404-sitrep-75-covid-19.pdf?sfvrsn=99251b2b_4
https://www.who.int/docs/default-source/... ], [³3 Yan R, Zhang Y, Li Y, Xia L, Guo Y, Zhou Q. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science. 2020;367:6485-1444-5]. SARS-CoV-2 infection is a pandemic that has continued to cause serious death in the world but still, no drug or vaccine was found to completely eliminate SARS-CoV-2 [⁴4 Jin Z, Du X, Xu Y, Deng Y, Liu M, Zhao Y, et al. Structure of Mpro from COVID-19 virus and discovery of its inhibitors. Nature. 2020. doi: 10.1038/s41586-020-2223-y.
https://doi.org/10.1038/s41586-020-2223-...

5 On being human in the face of a pandemic. Nat Cancer (2020). Editorial doi: 10.1038/s43018-020-0062-2.
https://doi.org/10.1038/s43018-020-0062-... -⁶6 Xia S, Liu M, Wang C, Xu W, Lan Q, Feng S, et al. Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion. Cell Res. 2020;30:343-55. Doi: 10.1038/s41422-020-0305-x.
https://doi.org/10.1038/s41422-020-0305-... ].

Angiotensin-converting enzyme II (ACE2) is well-known as a host receptor for SARS-COV-2 in human so studies in recent days have shown that ACE2 has been the center of interest by researchers for SARS-CoV-2 [³3 Yan R, Zhang Y, Li Y, Xia L, Guo Y, Zhou Q. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science. 2020;367:6485-1444-5], [⁷7 Zhao Y, Zhao Z, Wang Y, Zhou Y, Ma Y, Zuo W. Single-cell RNA expression profiling of ACE2, the putative receptor of Wuhan 2019-nCov. Bio Rxiv. 2020. doi:10.1101/2020.01.26.919985
https://doi.org/10.1101/2020.01.26.91998... ], [⁸8 Lan J, Ge J, Shan S, Zhou H, Fan S, Zhang O et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature. 2020. https://doi.org/10.1038/s41586-020-2180-5
https://doi.org/10.1038/s41586-020-2180-... ]. ACE2 is composed of three parts which are distinct signal peptide, a single metalloprotease active site, and a transmembrane domain. [⁹9 Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N et al. A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circ Res. 2000;1;87:1-9. Doi: 10.1161/01.res.87.5.e1
https://doi.org/10.1161/01.res.87.5.e1... ] ACE is one of the significant parts of the Renin-Angiotensin System (RAS) that is a monomeric, membrane-bound, zinc- and chloride-dependent peptidyl dipeptidase. [¹⁰10 Riordan JF, Angiotensin-I-converting enzyme and its relatives. Genome Biol. 2003; 4:8:225. Doi: 10.1186/gb-2003-4-8-225
https://doi.org/10.1186/gb-2003-4-8-225... ] Moreover, it catalyzes the translation of the decapeptide angiotensin I to the octapeptide angiotensin II, by eliminating a carboxy-terminal dipeptide. RAS plays a role as a homeostatic regulator of vascular function. [¹¹11 Tikellis C and Thomas MC. Angiotensin-Converting Enzyme 2 (ACE2) Is a Key Modulator of the Renin Angiotensin System in Health and Disease. Int J Pept. 2012;2012:256294. Doi: 10.1155/2012/256294
https://doi.org/10.1155/2012/256294... ] The expression and distribution of the ACE2 can have an important point at the probable infection mechanism of SARS-CoV-2. Recent studies have shown that the expression of ACE2 is fundamentally in lung [¹²12 Hamming I, Timens W, Bulthuis ML, Lely AT, Navis G, van Goor H. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol.2004;203:2:631-7. Doi: 10.1002/path.1570
https://doi.org/10.1002/path.1570... ], intestine [¹³13 Vuille-dit-Bille RN, Camargo SM, Emmenegger L, Sasse T, Kummer E, Jando J et al. Human intestine luminal ACE2 and amino acid transporter expression increased by ACE-inhibitors. Amino Acids.2015;47:693-705. Doi: 10.1007/s00726-014-1889-6
https://doi.org/10.1007/s00726-014-1889-... ], kidney [¹⁴14 Mizuiri S, Hemmi H, Arita M, Ohashi Y, Tanaka Y, Miyagi M, et al. Expression of ACE and ACE2 in individuals with diabetic kidney disease and healthy controls. Am J Kidney Dis. 2008;51(4):613-23. Doi: 10.1053/j.ajkd.2007.11.022.
https://doi.org/10.1053/j.ajkd.2007.11.0... ], and heart [¹⁵15 Goulter AB, Goddard MJ, Allen JC, Clark KL. ACE2 gene expression is up-regulated in the human failing heart. BMC Med. 2004 May 19;2:19. Doi:10.1186/1741-7015-2-19.
https://doi.org/10.1186/1741-7015-2-19... ]. A group of researchers investigated RNA expression profile of ACE2 at single-cell [⁷7 Zhao Y, Zhao Z, Wang Y, Zhou Y, Ma Y, Zuo W. Single-cell RNA expression profiling of ACE2, the putative receptor of Wuhan 2019-nCov. Bio Rxiv. 2020. doi:10.1101/2020.01.26.919985
https://doi.org/10.1101/2020.01.26.91998... ] and they have reported that high-level expression of ACE2 gene has been found on type II alveolar cells (AT2) [⁷7 Zhao Y, Zhao Z, Wang Y, Zhou Y, Ma Y, Zuo W. Single-cell RNA expression profiling of ACE2, the putative receptor of Wuhan 2019-nCov. Bio Rxiv. 2020. doi:10.1101/2020.01.26.919985
https://doi.org/10.1101/2020.01.26.91998... ,¹⁶16 Zhang Z, Kang Z, Gong H, Xu D, Wang J, Li Z et al. The digestive system is a potential route of 2019-nCov infection: a bioinformatics analysis based on single-cell transcriptomes. 2020. Doi: https://doi.org/10.1101/2020.01.30.927806.
https://doi.org/10.1101/2020.01.30.92780... ,¹⁷17 Zou X, Chen K, Zou J, Han P, Hao J, Han Z. Single-cell RNA-seq data analysis on the receptor ACE2 expression reveals the potential risk of different human organs vulnerable to 2019-nCoV infection. Front. Med. Doi: 10.1007/s11684-020-0754-0.
https://doi.org/10.1007/s11684-020-0754-... ], together with several genes promoting reproduction and transmission of the virus. Esophagus upper and stratified epithelial cells [¹⁸18 Chai X, Hu L, Zhang Y, Han W, Lu Z, Ke A et al. Specific ACE2 Expression in Cholangiocytes May Cause Liver Damage After 2019-nCoV Infection. Doi: https://doi.org/10.1101/2020.02.03.931766
https://doi.org/10.1101/2020.02.03.93176... ], absorptive enterocytes from ileum and colon [⁷7 Zhao Y, Zhao Z, Wang Y, Zhou Y, Ma Y, Zuo W. Single-cell RNA expression profiling of ACE2, the putative receptor of Wuhan 2019-nCov. Bio Rxiv. 2020. doi:10.1101/2020.01.26.919985
https://doi.org/10.1101/2020.01.26.91998... ], cholangiocytes [¹⁸18 Chai X, Hu L, Zhang Y, Han W, Lu Z, Ke A et al. Specific ACE2 Expression in Cholangiocytes May Cause Liver Damage After 2019-nCoV Infection. Doi: https://doi.org/10.1101/2020.02.03.931766
https://doi.org/10.1101/2020.02.03.93176... ], myocardial cells, kidney proximal tubule cells, and bladder urothelial cells [¹⁷17 Zou X, Chen K, Zou J, Han P, Hao J, Han Z. Single-cell RNA-seq data analysis on the receptor ACE2 expression reveals the potential risk of different human organs vulnerable to 2019-nCoV infection. Front. Med. Doi: 10.1007/s11684-020-0754-0.
https://doi.org/10.1007/s11684-020-0754-... ] are other types of cells which have found to be positive for the high level of ACE2 expression.

Another study has also shown that attenuated expression of ACE2 is correlated with cardiovascular diseases [¹⁹19 Junyi Guo, Huang Z, Lin L, Jiagao LV. Coronavirus Disease 2019 (COVID-19) and Cardiovascular Disease: A Viewpoint on the Potential Influence of Angiotensin-Converting Enzyme Inhibitors/Angiotensin Receptor Blockers on Onset and Severity of Severe Acute Respiratory Syndrome Coronavirus 2 Infection. Journal of the American Heart Association.2020;9:7. Doi: 10.1161/JAHA.120.016219
https://doi.org/10.1161/JAHA.120.016219... ]. In the light of these findings, we thought that some organs with the high number of ACE2-expressing cells are directly linked to the probable high risk for SARS-CoV-2 infection. Discoveries about ACE2 have the greatest value for therapeutic approaches for the current infection because of acting ACE2 as a host receptor for SARS-CoV-2. Prediction of the expression profile of ACE2 on normal human tissues, similar genes to ACE2, ACE2 related pathways and physiological features of ACE2 is significant for developing anti-ACE2 antibodies or blocking SARS-CoV-2 to prevent binding the virus to the receptor [²⁰20 Xuo H, Zhong L, Deng J, Peng J, Dan H, Zeng X et al. High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa. Int. J. Oral Sci. 2020; 12:1:1-5. Doi: 10.1038/s41368-020-0074-x
https://doi.org/10.1038/s41368-020-0074-... ]. In this study, we aimed to analyze the expression profile of ACE2 and similar genes to ACE2, predict the number of variations in ACE2 gene and detect the suspected SNPs on ACE2 gene, perform pathway analysis of the renin-angiotensin system, protein absorption and digestion. Also, the purpose is to predict the connection between ACE2 and gene-related diseases.

MATERIAL AND METHODS

Collection of the ACE2 gene dataset

Information on ACE2 was collected by Online Mendelian Inheritance in Man (OMIM) [²¹21 OMIM - Online Mendelian Inheritance in Man [accessed Apr. 09, 2020].https://omim.org/
https://omim.org... ] and Entrez Gene on National Center for Biological Information (NCBI) web site [²²22 National Center for Biotechnology Information. https://www.ncbi.nlm.nih.gov/ (accessed Apr. 09, 2020).
https://www.ncbi.nlm.nih.gov... ]. The Single Nucleotide Polymorphism (SNPs) information (Protein accession number and SNP ID) of the ACE2 gene was retrieved from the NCBI dbSNP [²³23 Home - SNP - NCBI. https://www.ncbi.nlm.nih.gov/snp/ (accessed Apr. 09, 2020).
https://www.ncbi.nlm.nih.gov/snp... ], and SWISS Prot databases [²⁴24 ExPASy: SIB Bioinformatics Resource Portal -Home [accessed Apr. 09, 2020].https://www.expasy.org/
https://www.expasy.org... ].

Analyzing of Functionally Similar Genes with ACE2

STRING is a functional protein associations network database that shows the relationship between the input gene and other genes. Protein-protein interactions, co-expression, co-localization and genetic interactions of ACE2 gene with other genes were shown by STRING [⁹9 Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N et al. A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circ Res. 2000;1;87:1-9. Doi: 10.1161/01.res.87.5.e1
https://doi.org/10.1161/01.res.87.5.e1...

10 Riordan JF, Angiotensin-I-converting enzyme and its relatives. Genome Biol. 2003; 4:8:225. Doi: 10.1186/gb-2003-4-8-225
https://doi.org/10.1186/gb-2003-4-8-225...

11 Tikellis C and Thomas MC. Angiotensin-Converting Enzyme 2 (ACE2) Is a Key Modulator of the Renin Angiotensin System in Health and Disease. Int J Pept. 2012;2012:256294. Doi: 10.1155/2012/256294
https://doi.org/10.1155/2012/256294... -¹²12 Hamming I, Timens W, Bulthuis ML, Lely AT, Navis G, van Goor H. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol.2004;203:2:631-7. Doi: 10.1002/path.1570
https://doi.org/10.1002/path.1570... ,²⁵25 STRING: functional protein association networks [accessed Apr. 09, 2020].https://string-db.org/.
https://string-db.org... ].

Prediction of Number of Variation in ACE2 and Detection of Suspected SNPs on ACE2

National Heart, Lung, and Blood Institute (NHLBI) has supported the Exome Variant Server. Exome Variant Server was used to determine the number of variations according to the European African population (EA) and American African Population (AA) alleles [¹³13 Vuille-dit-Bille RN, Camargo SM, Emmenegger L, Sasse T, Kummer E, Jando J et al. Human intestine luminal ACE2 and amino acid transporter expression increased by ACE-inhibitors. Amino Acids.2015;47:693-705. Doi: 10.1007/s00726-014-1889-6
https://doi.org/10.1007/s00726-014-1889-... ,¹⁴14 Mizuiri S, Hemmi H, Arita M, Ohashi Y, Tanaka Y, Miyagi M, et al. Expression of ACE and ACE2 in individuals with diabetic kidney disease and healthy controls. Am J Kidney Dis. 2008;51(4):613-23. Doi: 10.1053/j.ajkd.2007.11.022.
https://doi.org/10.1053/j.ajkd.2007.11.0... ]. In our study, suspected SNPs were identified for ACE2 gene and allele frequency were evaluated with this database [²⁶26 Exome Variant Server [accessed Apr. 09, 2020]. https://evs.gs.washington.edu/EVS/
https://evs.gs.washington.edu/EVS... ]. SIFT algorithm uses to find a nonsynonymous variant [⁷7 Zhao Y, Zhao Z, Wang Y, Zhou Y, Ma Y, Zuo W. Single-cell RNA expression profiling of ACE2, the putative receptor of Wuhan 2019-nCov. Bio Rxiv. 2020. doi:10.1101/2020.01.26.919985
https://doi.org/10.1101/2020.01.26.91998... ,¹⁵15 Goulter AB, Goddard MJ, Allen JC, Clark KL. ACE2 gene expression is up-regulated in the human failing heart. BMC Med. 2004 May 19;2:19. Doi:10.1186/1741-7015-2-19.
https://doi.org/10.1186/1741-7015-2-19... ,²⁷27 PROVEAN Home [accessed Apr. 09, 2020]. http://provean.jcvi.org/index.php.
http://provean.jcvi.org/index.php... ] and Polyphen-2 algorithm uses to predict damaging effects of nonsynonymous variant and it predicts benign or possibly damaging of variants of genes [¹⁶16 Zhang Z, Kang Z, Gong H, Xu D, Wang J, Li Z et al. The digestive system is a potential route of 2019-nCov infection: a bioinformatics analysis based on single-cell transcriptomes. 2020. Doi: https://doi.org/10.1101/2020.01.30.927806.
https://doi.org/10.1101/2020.01.30.92780... ,¹⁷17 Zou X, Chen K, Zou J, Han P, Hao J, Han Z. Single-cell RNA-seq data analysis on the receptor ACE2 expression reveals the potential risk of different human organs vulnerable to 2019-nCoV infection. Front. Med. Doi: 10.1007/s11684-020-0754-0.
https://doi.org/10.1007/s11684-020-0754-... ,²⁸28 PolyPhen-2: prediction of functional effects of human nsSNPs. http://genetics.bwh.harvard.edu/pph2/
http://genetics.bwh.harvard.edu/pph2... ]. Consequently, our findings were confirmed with each other.

Pathway Analysis of ACE2

KEGG is a database that is used to determine the methodical searching of gene functions. It is related to genomic information with other advanced classify functional parts [¹⁸18 Chai X, Hu L, Zhang Y, Han W, Lu Z, Ke A et al. Specific ACE2 Expression in Cholangiocytes May Cause Liver Damage After 2019-nCoV Infection. Doi: https://doi.org/10.1101/2020.02.03.931766
https://doi.org/10.1101/2020.02.03.93176... ,²⁹29 Guo J, Huang Z, Lin L, Lv J, Cardiovascular Disease: A Viewpoint on the Potential Influence of Angiotensin-Converting Enzyme Inhibitors/Angiotensin Receptor Blockers on Onset and Severity of Severe Acute Respiratory Syndrome Coronavirus 2 Infection. 2020. doi: 10.1161/JAHA.120.016219.,³⁰30 KEGG PATHWAY Database [accessed Apr. 09, 2020]. https://www.kegg.jp/kegg/pathway.html
https://www.kegg.jp/kegg/pathway.html... ]. We used the KEGG database for mapping of Renin-Angiotensin System. The other database was employed for mapping system and pathway analysis is the Reactome Pathway Database. The results were compared to each other and validation was done by STRING.

Prediction of ACE2 Gene-Related Diseases and Expression Profile in Human Normal and Cancer Tissues

DISEASES resource was used to predict gene-related diseases [³¹31 DISEASES - Search [accessed Apr. 09, 2020]. https://diseases.jensenlab.org/Search
https://diseases.jensenlab.org/Search... ]. Then our results were compared with FitSNPs, UniProt, BioXpress [³²32 fitSNPs Server - Functionally interpolating Single Nucleotide Polymorphism [accessed Apr. 09, 2020]. http://fitsnps.ucsf.edu/
http://fitsnps.ucsf.edu...

33 UniProt [accessed Apr. 09, 2020]. https://www.uniprot.org/
https://www.uniprot.org... -³⁴34 Wan Q, Dingerdissen H, Fan Y, Gulzar N, Pan Y, Wu TJ, et al.BioXpress: an integrated RNA-seq-derived gene expression database for pan-cancer analysis. Database (Oxford). 2015:28;2015:bav019, doi: 10.1093/database/bav019.
https://doi.org/10.1093/database/bav019... ]. FitSNPs depends on human microarray data that also shows gene-related diseases. Gene/miRNA expression relationship with cancer was investigated by BioXpress. SwissProt and The Hive Lab also was used to analyze for expression profile in human normal tissues and human cancer tissues. IGV Browser, Ensembl Genome Browser, and UCSC Genome Browser were used to find gene expression, exon expression, and junction expression [³⁵35 Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nat Biotechnol. 2011;29(1):24-6. doi: 10.1038/nbt.1754.
https://doi.org/10.1038/nbt.1754...

36 Ensembl genome browser 99 [accessed Apr. 09, 2020]. https://www.ensembl.org/index.html
https://www.ensembl.org/index.html... -³⁷37 UCSC Genome Browser Home [accessed Apr. 09, 2020]. https://genome.ucsc.edu/
https://genome.ucsc.edu... ]. Thus, all results confirmed with each other and the GTEx portal [³⁸38 GTEx Portal [accessed Apr. 09, 2020]. https://gtexportal.org/home/
https://gtexportal.org/home... ].

RESULTS

Analyzing of Functionally Similar Genes with ACE2

We analyzed that ACE2 relationship with ten different genes (Figure 1).

Predicting the Number of Variations in ACE2 and Detection of Suspected SNPs on ACE2

Some of these genes play key rol in RAS. Then we showed that suspected variations in ACE2 gene with three different programme (in Table 1, Table 2).

Pathway Analysis of ACE2

Then the importance of ACE2 gene (Figure 2) and protein secretion and absorption pathways throughout the digestion system (Figure 3) were described by KEGG pathway.

Prediction of ACE2 Gene-Related Diseases and Expression Profile in Human Normal and Cancer Tissues

BioXpress has shown that ACE2 gene had over-expression in kidney cancer (P-Value: 0.014, log2FC: 0.71) and esophageal cancer (P-Value: 0.012, log2FC: 1.61) and it had under expression in prostate cancer (P-Value 0.001, log2FC: -0.77) and head and neck cancer (P-Value 0.02, log2FC: -0.56).

When we predicted of ACE2 Gene-Related Diseases and Expression Profile in Human Normal and Cancer Tissues, we have shown that ACE2 gene had over-expression in kidney cancer, esophageal cancer and it had under expression in prostate cancer and head and neck cancer. And we had seen that ACE2 gene were mostly expressed in testis, small intestine, kidney, heart, and adipose tissues (Figure 4, 5 and 6).

Finally we analyzed ACE2 with gene-related diseases and these diseases showed in Table 3.

DISCUSSION

One of the type I integral glycoprotein is ACE2 that has a role in RAS. RAS is responsible for organizing physiological processes in the cell which are cycle progression, survival, and apoptosis [³⁹39 Gurung AB and Bhattacharjee A. Significance of Ras Signaling in Cancer and Strategies for its Control. Oncology & Hematology Review, 2015;11(2):147-52. Doi:10.17925/OHR.2015.11.02.147
https://doi.org/10.17925/OHR.2015.11.02.... ] and with RAS activation, it can initiate various pathological processes such as hypertension [⁴⁰40 Navar LG. Counterpoint: Activation of the intrarenal renin-angiotensin system is the dominant contributor to systemic hypertension. J Appl Physiol (1985). 2010;109(6):1998-2000; discussion 2015. Doi: 10.1152/japplphysiol.00182.2010a.
https://doi.org/10.1152/japplphysiol.001... ], heart failure [⁴¹41 Dostal DE and Baker KM. The Cardiac Renin-Angiotensin System Conceptual, or a Regulator of Cardiac Function? Which RAS Components Have Been Identified in Cardiac Tissue?. Circulation Research.1999;85:643-650 doi:10.1161/01.RES.85.7.643
https://doi.org/10.1161/01.RES.85.7.643... ] and kidney disease [⁴²42 Hall JE, Mizelle HL, Woods LL. The renin-angiotensin system and long-term regulation of arterial pressure. J Hypertens. 1986;4(4):387-97. Doi:10.1097/00004872-198608000-00001
https://doi.org/10.1097/00004872-1986080... ]. Thus; we analyzed functionally similar genes with ACE2. We found that ACE2 was correlated with Angiotensinogen (AGT), Renin (REN), Angiotensin II Receptor Type 1 (AGTR1), Angiotensin II Receptor Type 1 (AGRT2), NADP Malic Enzyme (MME2), Dipeptidyl Peptidase 4 (DPP4), Prolylcarboxypeptidase (PRCP), Meprin A Subunit Alpha (MEP1A), X-prolyl Aminopeptidase 2 (XPNPEP2), Meprin A Subunit Beta (MEP1B) (Figure1). Especially AGT, AGTR1, AGTR2, and REN have a key role in the RAS such as sodium homeostasis, fluid balance and blood pressure [⁴³43 Laragh JH. Renin-angiotensin-aldosterone system for blood pressure and electrolyte homeostasis and its involvement in hypertension, in congestive heart failure and in associated cardiovascular damage (myocardial infarction and stroke). J Hum Hypertens. 1995;9(6):385-90.], [⁴⁴44 Urushihara M, Kobori H. Intrarenal renin-angiotensin system activation in end-stage renal disease. Hypertens Res. 2017 Apr;40(4):351-2. Doi: 10.1038/hr.2017.7.
https://doi.org/10.1038/hr.2017.7... ] although, all constituents of the RAS have expressed in a lot of tumor tissues [⁴⁵45 Campbell CY, Fang BF, Guo X, Peralta CA, Psaty BM, Rich SS, et al. Associations between genetic variants in the ACE, AGT, AGTR1 and AGTR2 genes and renal function in the Multi-ethnic Study of Atherosclerosis. Am J Nephrol. 2010;32(2):156-62. Doi: 10.1159/000315866.
https://doi.org/10.1159/000315866... ]. Thus; Cambell and coauthors have reported that ACE, AGT, AGTR1, and AGTR2 genes can promote renal function variation [⁴⁵45 Campbell CY, Fang BF, Guo X, Peralta CA, Psaty BM, Rich SS, et al. Associations between genetic variants in the ACE, AGT, AGTR1 and AGTR2 genes and renal function in the Multi-ethnic Study of Atherosclerosis. Am J Nephrol. 2010;32(2):156-62. Doi: 10.1159/000315866.
https://doi.org/10.1159/000315866... ]. MME encodes neutral endopeptidase and PRCP encodes prolyl carboxypeptidase, and they are also under-expressed in tumor tissue, although they are highly expressed in normal lung tissues [⁴⁶46 Goldstein B, Trivedi M, Speth RC. Alterations in Gene Expression of Components of the Renin-Angiotensin System and Its Related Enzymes in Lung Cancer. Lung Cancer Int. 2017;2017:6914976. Doi: 10.1155/2017/6914976.
https://doi.org/10.1155/2017/6914976... ]. DPP4 is also known as CD26, was associated as a cellular [⁴⁷47 Lu G, Hu Y, Wang Q, Qi J, Gao F, Li Y, et al. Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26. Nature. 2013:8;500(7461):227-31. Doi: 10.1038/nature12328.
https://doi.org/10.1038/nature12328... ] and functional receptor [⁴⁸48 Raj VS, Mou H, Smits SL, Dekkers DH, Müller MA, Dijkman R, et al. Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature. 2013 Mar 14;495(7440):251-4. Doi: 10.1038/nature12005.
https://doi.org/10.1038/nature12005... ] for MERS-CoV. Moreover, we thought that the important point in the function of these macromolecules were mostly peptidase activities. According to our results; we also think that DPP2 and ACE2 work together in the pathogenesis of 2019-nCoV infection as it is a direct functional partner. Last studies have shown that ACE2 variations can be a probable candidate in many diseases such as cardiovascular diseases [⁴⁹49 Burrell LM, Harrap SB, Velkoska E, Patel SK. The ACE2 gene: its potential as a functional candidate for cardiovascular disease. Clin Sci (Lond). 2013;124(2):65-76. Doi: 10.1042/CS20120269.
https://doi.org/10.1042/CS20120269... ], 2019-nCoV infection [⁵⁰50 Asselta R, Paraboschi EM, Mantovani A, and Duga S. ACE2 and TMPRSS2 variants and expression as candidates to sex and country differences in COVID-19 severity in Italy. medRxiv.2020. Doi: 10.1101/2020.03.30.20047878.
https://doi.org/10.1101/2020.03.30.20047... ], essential hypertension [⁵¹51 Pan Y, Wang T, Li Y, Guan T, Lai Y, Shen Y, et al. Association of ACE2 polymorphisms with susceptibility to essential hypertension and dyslipidemia in Xinjiang, China. Lipids Health Dis. 2018;20;17(1):241. Doi: 10.1186/s12944-018-0890-6.
https://doi.org/10.1186/s12944-018-0890-... ] in different populations. We found that there were a total number of 99 variants in ACE2 gene that 53 of them belong to European American populations and 46 of them belong to African American populations. There were 17 suspected variants, 53 unknown variants, 29 benign variants in ACE2 gene and exome variant server has shown that these mutations were possibly damaging (Table.1).

We analyzed the pathway of RAS and the protein digestion and absorption by KEGG analysis (Figure 2 and Figure 3). Blood pressure regulation is implemented by RAS which also employes in the regulation of electrolyte and fluid balance in the body. The enzyme-substrate mechanism starts with the renin enzyme by cleaving Agt into decapeptide angiotensin I. ACE takes place to form Angiotensin II (AngII) from decapeptide angiotensin I. AngII further employes on the activation of the main receptor responsible for the regulation of AngII function in kidney, AT1 (AT1R). Therefore, RAS signaling pathways including (ACE)/Ang II/AT1R, and AT2R pathway, and ACE2/angiotensin-(1-7)/Mas and Ang IV/IRAP pathway have been shown in Figure 2.

Protein digestion route through the digestive system with digestive enzymes results in the generation of some amino acids and relatively small peptides. Amino acids transportation to intestinal epithelial cells by amino acids transporters is performed according to the pH level of amino acids. Moreover, PEP1 is a transporter that employes in the transportation of small peptides into the enterocytes where peptides hydrolyzation takes place. Amino acids released after hydrolyzation are transported to the bloodstream by amino acid transporters. (Figure 3).

We investigated the ACE2 gene-related diseases and expression profiles in human normal and cancer tissues. BioXpress has shown that ACE2 gene have over-expression in kidney cancer (P-Value: 0.014, log2FC: 0.71) and esophageal cancer (P-Value: 0.012, log2FC: 1.61) and it have under expression in prostate cancer (P-Value 0.001, log2FC: -0.77) head and neck cancer (P-Value 0.02, log2FC: -0.56). After COVID-19, maybe over-expression of ACE2 can trigger some serious diseases and these can cause damage to kidney or esophageal tissues and promote cancer. In addition to this, underexpression of ACE2 can cause prostate cancer and head and neck cancer. One of the most common symptoms is a severe sore throat, headache, nasal congestion, runny nose and maybe in the future recovered cancer patient may get serious illnesses again, after COVID-19. When we analyzed ACE2 in human normal tissues, we saw that ACE2 has mostly expressed in the small intestine. ACE2 is over-expressed in the small intestine, suggesting that it may cause symptoms of diarrhea and abdominal pain in COVID-19. We found that ACE2 is also expressed in testis, kidney, heart, thyroid, colon, esophagus, breast, minor salivary gland, pancreas, lung, liver, bladder, cervix and muscle (Figure4). On the other hand, the exon expression of ACE2 analysis was described and it emphasizes far genomic variations related to splicing regulation. Respectively testis, small intestine, kidney, heart, and adipose tissues correlate with exon expression of ACE2. Another interesting point is the expression of ACE2 in testis. This may be due to the aldosterone synthesis and secretion property of RAS. According to KEGG analysis; RAS has a role in vasodilation, anti-fibrosis, apoptosis, natriuresis, anti-inflammation, aldosterone synthesis, and secretion. We confirmed that our results were consistent with each other.

CONCLUSION

The role of ACE2 is important in preventing, treating or developing drugs for COVID19. Defining why certain diseases are a high-risk group will help to clarify the pathogenesis of the disease. In the future, this in silico analysis should be combined with other pieces of evidence including experimental data in order to assign function.

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