key: cord-0716441-dwn856hr authors: Khayat, A. S.; Assumpcao, P. P. d.; Khayat, B. C. M.; Araujo, T. M. T.; Batista-Gomes, J. A.; Imbiriba, L. C.; Ishak, G.; Assumpcao, P. B. d.; Moreira, F. C.; Burbano, R. R.; Ribeiro-dos-Santos, A. M.; Ribeiro-dos-Santos, A. K.; Santos, N. P. C. d.; Santos, S. E. B. d. title: ACE2 polymorphisms as potential players in COVID-19 outcome date: 2020-05-29 journal: nan DOI: 10.1101/2020.05.27.20114843 sha: a80790da163620612ef0154b9bed21d686151627 doc_id: 716441 cord_uid: dwn856hr The clinical condition COVID-19, caused by SARS-CoV-2, was declared a pandemic by the WHO in March 2020. Currently, there are more than 5 million cases worldwide, and the pandemic has increased exponentially in many countries, with different incidences and death rates among regions/ethnicities and, intriguingly, between sexes. In addition to the many factors that can influence these discrepancies, we suggest a biological aspect, the genetic variation at the viral S protein receptor in human cells, ACE2 (angiotensin I-converting enzyme 2), which may contribute to the worse clinical outcome in males and in some regions worldwide. We performed exomics analysis in native and admixed South American populations, and we also conducted in silico genomics databank investigations in populations from other continents. Interestingly, at least ten polymorphisms in coding, noncoding and regulatory sites were found that can shed light on this issue and offer a plausible biological explanation for these epidemiological differences. In conclusion, ACE2 polymorphisms should influence epidemiological discrepancies observed among ancestry and, moreover, between sexes. At the end of 2019, a new outbreak caused by SARS-CoV-2 (a coronavirus) started in 28 Hubei Province, China. The clinical condition, COVID-19, probably arose from natural selection 29 in bat reservoirs [1] . There are currently more than five million cases worldwide, and the 30 pandemic has been increasing exponentially in many countries since the disease was deemed a 31 pandemic by WHO in March 2020 [2] . 32 The lethality rate is influenced by the speed of contagion, idiosyncrasies of the affected 33 populations according to the containment policies adopted, socioeconomic conditions and the 34 absorption limit of the health system [3] . Due to the high rate of transmission of the virus by air 35 and the novelty of the infection to humans, the disease has become a global emergency problem, 36 forcing periods of social confinement to contain the pandemic, in addition to hygiene habits [4] . 37 Epidemiological data show a tendency towards slightly greater contamination in men; 38 however, male mortality is significantly higher [5] , representing an increase from 20% to 70% in 39 European countries, approximately 65% in some Asian countries, and, even more peculiarly, 40 Dominican Republic citizens showed three times more deaths in men than women [6,2,7], even 41 considering factors related to behavioral issues. Certainly, there are biological aspects that 42 contribute to this more adverse clinical condition. 43 Studies indicate that cell-virus interaction is mediated by the connection of the 44 transmembrane glycoprotein spike (S), present in the form of homotrimers on the viral surface, to 45 angiotensin I-converting enzyme 2 (ACE2, also called hACE2), which is responsible primarily 46 for inducing vasodilation [8] . . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 29, 2020. It is noteworthy that the ACE2 is located on the X chromosome, causing the impossibility of 60 heterozygosity in men. Therefore, polymorphisms in their single copy could be related to the 61 worst outcomes observed in males [16] . Considering the above, we sought explanations for an intrinsic factor that differed 63 between sexes and populations that may justify the differences observed in the incidence and 64 lethality of SARS-CoV-2 infection among the different regions of the world, as well as between 65 sexes. We analyzed global data in the 1000 Genomes Database and, in addition, we conducted 66 studies of exomes in two population groups in the Brazilian Amazon (Indians and miscegenated), 67 without description in public genomic banks, and we compared this information with a public 68 databank from a population in southeastern Brazil. These comparisons are important because 69 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 29, 2020. Brazil has a continental size and an admixed population in the North (more Amerindians among 70 all regions), Northeast (more Africans), and South and Southeast (more Europeans) [17] . Analyses in the 1000 Genomes Project 73 The analysis was performed on data from the 1000 Genomes Phase 3 database (1000G), Analyzing the polymorphisms contained in the ACE2 locus, in addition to ten thousand 128 base pairs upstream, we found 2266 polymorphisms, of which 199 were contained in the region 129 5´ upstream of the gene, 85 were located in exonic regions, and the others were located in the 130 introns. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 29, 2020. . https://doi.org/10.1101/2020.05.27.20114843 doi: medRxiv preprint 132 In the exonic region, 15 SNPs of the 85 polymorphisms found in 1000G present 133 differences greater than 1% between some of the populations (all between exons 17 to 21). Another three polymorphisms (rs889263894, rs1027571965, rs147464721) appear mainly in the 135 Brazilian population. Nine of these exonic polymorphisms are present in the most common 136 isomorphs (v1 and v2) and show important differences in populational frequency (Fig 1, 137 additional data on S1 Table) . Polymorphisms in upstream regions that may influence disease outcome 158 Differences greater than 1% of MAF in the region 10,000 base pairs 5´ upstream to the 159 gene were observed in 57 polymorphisms (Fig 2) . . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 29, 2020. It is also worth mentioning that the rs2097723 SNP presents a very heterogeneous 168 distribution, oscillating between 7% in Africans, 32% in Americans, 42% in East Asians, 28% in 169 Europeans and 22% in South Asians. Africans through EUR, AMR, BAP, EAS, ABM and SAS (Fig 3) . Furthermore, considering the 179 possibility of influence in determining isoforms v2, it is worth noting rs190614788 on intron 1 180 (with a difference of more than 0.11 between EUR and EAS). The main findings of our study are concatenated in Table 1 (additional information in S3 182 Table) , and they are discussed below. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 29, 2020. The ACE2 gene is mainly composed of two isoforms with 18 or 19 exons (v1 and v2) that 208 encode the same protein (805 amino acids) and three other smaller variants: x1, x2 and x3, which 209 have rarely been studied [19, 23] . Thus, we searched for genotypic information in these exons that 210 could allow us to infer a disruption with the potential to culminate in impacts on the disease 211 process. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 29, 2020. In conclusion, considering this genetic aspect involving ACE2 in the complex relationship 290 between SARS-CoV-2 and humans, we emphasize the following: There are genetic markers that could influence the unequal rates of aggravation and death 292 observed between men and women. Considering the polymorphisms as harmful when in 293 homozygosity (genotypic frequency equal to allelic frequency squared) or hemizygosity 294 (genotypic frequency equal to allelic frequency), men would have this conditions from 1·4 to 77 295 times more (uniallelic) than women (Table 1) . Thus, a relevant contribution to the understanding 296 of higher mortality in males is presented, as reported in the various populations affected by 297 COVID-19. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 29, 2020. . https://doi.org/10.1101/2020.05.27.20114843 doi: medRxiv preprint The rates of contagion and death fluctuate greatly; in this sense, ACE2 polymorphisms 299 could contribute to these differences. The rs182366225 and rs2097723 polymorphisms are more 300 frequent in the East Asian population and are potentially unfavorable to the individual, as they 301 increase the expression of the enzyme. These allele frequencies are even higher in Chinese and 302 Vietnamese populations. Such markers are on the order of 30% to 180% more frequently in East 303 Asians than in other populations. Indigenous populations from Amazon have exclusive genetic polymorphisms 305 (rs1027571965 and rs889263894) or with higher frequencies (rs2285666 and rs35803318) than 306 other populations. These polymorphisms are related to increased expression of the ACE2 gene in 307 brain tissues, among others. This is an extremely relevant finding because they may influence the 308 outcomes in these populations, whose involvement by COVID-19 was recently reported. This 309 population group, due to its genetic peculiarity and less previous exposure to viral infections, 310 represents a major challenge in understanding and handling this pandemic. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 29, 2020. . https://doi.org/10.1101/2020.05.27.20114843 doi: medRxiv preprint CK2 phosphorylates the 350 angiotensin-converting enzyme and regulates its retention in the endothelial cell plasma 351 membrane Comparative genetic analysis of the novel coronavirus nCoV/SARS-CoV-2) receptor ACE2 in different populations A pneumonia outbreak associated with a new coronavirus of 355 probable bat origin The ACE2 expression in human heart indicates 357 new potential mechanism of heart injury among patients infected with SARS-CoV-2 Physiological and pathological regulation of ACE2, the 360 SARS-CoV-2 receptor Interaction of the spike protein RBD from 362 hot-spot analysis and effect of the 363 receptor polymorphism Role of changes in SARS-coV-2 spike protein in the 367 interaction with the human ACE2 receptor: an in silico analysis Structural basis for the recognition of SARS-370 CoV-2 by full-length human ACE2 Structure, Function, and 372 Antigenicity of the SARS-CoV-2 Spike Glycoprotein Assessing individual interethnic 376 admixture and population substructure using a 48-insertion-deletion (INSEL) ancestry-377 informative marker (AIM) painel The International Genome Sample Resource. IGRS ABraOM Online Archive of Brazilian Mutations Molecular cloning: a laboratory manual Predicting functional effect of human missense mutations 387 using PolyPhen-2 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted CoV2 Receptor ACE2 Gene Expression and Regulation The Genotype-Tissue Expression (GTEx) Predicting effective microRNA target sites in 394 mammalian mRNAs Angiotensin II-regulated 398 microRNA 483-3p directly targets multiple components of the renin-angiotensin system African gene pool in the Americas Association of Genetic Variants in the Apelin-APJ System and 403 ACE2 With Blood Pressure Responses to Potassium Supplementation: The GenSalt Study Association of ACE2 genetic polymorphisms with hypertension-406 related target organ damages in south Xinjiang The minor allele frequency (MAF) of SNPs in exonic regions of ACE2 that 409 showed differences between the investigated populations AFR: African; NAM: Native American; BAP: Brazilian Admixed Population Archive of Brazilian Mutations; GLO: Global (1000 Genomes) S2 Table. Chromosome position, reference (RS) and minor allele of SNPs (MAF>0.01) in 415 ten thousand base pairs 5´ upstream of ACE2. *Insertion of an Alu mobile element relative to the 416 reference 417 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 29, 2020. . https://doi.org/10.1101/2020.05.27.20114843 doi: medRxiv preprint