key: cord-330384-yujbcwg5
authors: Al-Mulla, Fahd; Mohammad, Anwar; Al Madhoun, Ashraf; Haddad, Dania; Ali, Hamad; Eaaswarkhanth, Muthukrishnan; John, Sumi Elsa; Nizam, Rasheeba; Channanath, Arshad; Abu-Farha, Mohamed; Ahmad, Rasheed; Abubaker, Jehad; Thanaraj, Thangavel Alphonse
title: A comprehensive germline variant and expression analyses of ACE2, TMPRSS2 and SARS-CoV-2 activator FURIN genes from the Middle East: Combating SARS-CoV-2 with precision medicine
date: 2020-05-16
journal: bioRxiv
DOI: 10.1101/2020.05.16.099176
sha: 
doc_id: 330384
cord_uid: yujbcwg5

The severity of the new COVID-19 pandemic caused by the SARS-CoV-2 virus is strikingly variable in different global populations. SARS-CoV-2 uses ACE2 as a cell receptor, TMPRSS2 protease, and FURIN peptidase to invade human cells. Here, we investigated 1,378 whole-exome sequences of individuals from the Middle Eastern populations (Kuwait, Qatar, and Iran) to explore natural variations in the ACE2, TMPRSS2, and FURIN genes. We identified two activating variants (K26R and N720D) in the ACE2 gene that are more common in Europeans than in the Middle Eastern, East Asian, and African populations. We postulate that K26R can activate ACE2 and facilitate binding to S-protein RBD while N720D enhances TMPRSS2 cutting and, ultimately, viral entry. We also detected deleterious variants in FURIN that are frequent in the Middle Eastern but not in the European populations. This study highlights specific genetic variations in the ACE2 and FURIN genes that may explain SARS-CoV-2 clinical disparity. We showed structural evidence of the functionality of these activating variants that increase the SARS-CoV-2 aggressiveness. Finally, our data illustrate a significant correlation between ACE2 variants identified in people from Middle Eastern origins that can be further explored to explain the variation in COVID-19 infection and mortality rates globally.

The global pandemic COVID-19 caused by SARS-CoV-2 virus is life-threatening and has become a significant concern to humanity. Notably, the severity of this disease is highly variable in different populations across the world 1 . Since the outbreak, several studies have reported specific factors including age, gender, and pre-existing health conditions that could have contributed to the increased severity of the disease [2] [3] [4] . The genetic susceptibility to has also been explored by scrutinizing Angiotensin converting enzyme 2 (ACE2) genetic variations in different populations. ACE2 is the functional receptor mediating entry of SARS-CoV-2 into the host cells 5 , which is facilitated by FURIN cleavage [6] [7] [8] . Transmembrane serine protease 2 (TMPRSS2) is another candidate gene that has been linked to COVID-19 disease [8] [9] [10] . TMPRSS2 expression enhances ACE2-mediated SARS-CoV-2 cell invasion by operating as a co-receptor 10 .

The increased cleavage activity of this protease was suggested to diminish viral recognition by neutralizing antibodies and by activating SARS spike (S) protein for virus-cell fusion 11 and facilitates the active binding of SARS-CoV-2 through ACE2 receptor, which is a risk factor for a more serious COVID-19 presentation [8] [9] [10] .

The hallmark of the novel SARS-CoV-2, as compared to other SARS viruses, is the presence of a polybasic FURIN cleavage site. FURIN has been reported to facilitate the transport of SARS-CoV-2 into or from the host cell [6] [7] [8] . Notably, a recent study has highlighted the presence of a unique functional polybasic FURIN cleavage consensus site between the two spike subunits S1 and S2 by the insertion of 12 nucleotides encoding PRRA in the S protein of SARS-CoV-2 virus 12 . The FURIN-like cleavage-site is cleaved during virus egress, which primes the S-protein providing a gain-of-function for the efficient spreading of the SARS-CoV-2 among humans 13, 14 .

It is, therefore, likely that the presence of a deleterious ACE2, TMPRSS2 and FURIN gene variants may modulate viral infectivity among humans, making some people less vulnerable than others.

Recent studies, assessed the genetic variations and eQTL (expression quantitative trait locus) expression profiles in the candidate genes ACE2, TMPRSS2, and FURIN to demonstrate the sex and population-wise differences that may influence the pathogenicity of SARS-CoV-2 9, [15] [16] [17] [18] [19] . It is to be noted that these studies focused only on the European and East Asian populations. Given the extremely high prevalence of obesity (80%), hypertension (28%) and diabetes (20%) of the population in the Gulf states [20] [21] [22] which are considered as risk factors for mortality from COVID- 19 4,23 , the witnessed low infectivity and mortality rates registered in this area of the world are intriguing. Even though this could be due to various factors that are not well accounted for yet such as testing, hot weather or extreme measures taken early on by some countries, it could also be due to ethnic genetic variations in the ACE2-TMPRSS2-FURIN genes that are key regulators for orchestrating SARS-CoV-2 cellular access.

As a result, it is crucial to study the variation of these candidate genes ACE2, TMPRSS2

and FURIN in Middle Eastern populations to better understand possible natural genetic components that can be responsible for these differences. Here, we present a comprehensive comparative assessment of deleterious or gain of function mutations of ACE2, TMPRSS2, and 

We first examined ACE2 gene variation frequencies in Kuwait, Qatar, East Asia, and Africa where the impact of SARS-CoV-2 has been modest and compared it to Iran, which is moderately affected and then to Europe, the continent with the most deaths per population. Overall, we found human ACE2 gene variations and the probability of loss of function mutations (pLOF=0.1, C.I. 0.04-0.25) to be low in comparison to ACE (pLOF=0.87, C.I. 0.71-1.08), which is a gene of similar size, indicating that the ACE2 gene is highly intolerant to loss of function mutations. Additionally, we identified 19 missense variants in the ACE2 gene from Kuwait, Qatar and Iran (Table 1 ). All were rare variants defined by minor allele frequency (MAF) of less than 1%. The genetic variants included four novel variants from Kuwait, and six from Iran.

We identified four deleterious variants (rs776995986, rs769062069, rs765152220, and rs750145841), causing R708W, R708Q, D494V, Y199C missense amino acid substitutions in the ACE2 gene using risk prediction tools as described in the methods section (Table 1) . All the ACE2 gene deleterious variants were absent from the African, East Asian and Qatari data and were very rare in Europeans but were present at MAF of 0.063-0.5% in the Iranian population (Table 1) . This suggests a more protective effect and a significant decrease in the disease burden in Iran compared to Europe (p<0.05; Table 2 ). The positions of the ACE2 receptor polymorphisms on the linearized ACE2 protein model are shown in Supplementary Fig. 1 , and 3D-models for the same are shown in Supplementary Fig. 2 and Supplementary Fig. 3 . It is noteworthy that none of the ACE2 polymorphisms identified in this study involved the three ACE2 regions known to directly bind the SARS-CoV-2 S-Protein Receptor Binding Domain (RBD), namely amino acids 30-41, 82-84, and 353-357 ( Supplementary Fig. 1 ).

Next, we examined whether natural ACE2 gene variations that increase the affinity of ACE2 to the S-protein or facilitate viral entry/viral load exist more frequently in high-burden compared to low-burden populations. Two such genetic variants existed in our data ( Table 1 ). The first, rs4646116, is a missense variant that changes a lysine amino acid at position 26 to arginine (K26R) ( Table 1 ). The K26, which is just proximal to the first region of the ACE2 receptor involved in S-protein binding, has been shown previously to bind the sterically hindering first mannose in the glycan that is linked to N90 and thus stabilizes the glycan moiety hindering the binding of S-protein RBD to ACE2 24 (Fig. 1a) . The missense variant R26 creates a new hydrogen bond with D30, which is then poised to build a salt-bridge with the S-protein RBD K417 that increases the affinity of SARS-CoV-2 to the ACE2 receptor 18 (Fig. 1b) . Indeed, the ACE2 K26R activating variant was extremely rare in East Asian (MAF=0.007%), Africans (MAF=0.095%), but the second most common variant in Europeans with MAF of 0.587% (shown in green fonts in Table 1 ). The MAF of this variant in the Kuwaiti population was nearly half that of Europeans (MAF=0.29%), and it was absent from the Qatari and Iranian exome data (Table 1 ). Our structural modeling supports the notion that K26R is an ACE2 receptor activating variant (Fig. 1a, b) .

Consistent with these findings, using a synthetic human ACE2 mutant library, a recent study reported that the R26 variant increased S-protein binding and susceptibility to the virus significantly 25 .

We also subjected novel and known ACE2 variants to structural predictions that may impact the binding of SARS-CoV-2 to the host cells. These changes (A372G, Y199C, D225G, F452V and V485L) were located proximal to the protein residues that mediate its activity. A previous study indicated that the three amino acid (aa) regions 30-41, especially the residue near lysine 31 and tyrosine 41, 82-84 and 353-357 in ACE2 were essential for the binding of S-protein in coronavirus 26 Fig. 1 ).

The second activating, and by far the most common ACE2 gene variant in Europeans (MAF=2.52%) and Italians (MAF=1.6%; detected in 105 of 6984 exomes) 17 was rs41303171, which replaces the amino acid asparagine at position 720 to aspartic acid (N720D) (Green font in Table 1 ). This ACE2 variant was absent from the East Asian population (13,784 exomes) and was significantly rarer in the Middle East and Africa (Table 1 ). This particular variant has been reported before, but its clinical relevance was persistently dismissed because the codon 720, being far from ACE2-Spike protein interface, does not appear to be an obvious candidate for ACE2 receptor binding to the S-protein of SARS-CoV-2 18, 29 . However, we noted that N720D is located 4 amino acids proximal to the TMPRSS2 cleavage site (aa 697-716) as shown in Supplementary Fig. 1 .

Recent studies demonstrated that TMPRSS2 cleavage of the ACE2 receptor increases SARS-CoV-2 cellular entry 30, 31 . It is not unreasonable to suggest that this activating variant may play a similar role in SARS-CoV-2, rendering people who harbor it more prone to severe infection and higher viral load.

ACE2 cleavage by TMPRSS2 enhances the S-protein viral entry 5 . TMPRSS2 cleaves ACE2 between residues 697 and 716, which is the third and fourth helices in the C-terminal collectrin neck domain of the dimer interface of ACE2 30 . To further dissect the mechanisms underlying the enhanced ACE2-TMPRSS2 accessibility, we performed structural analysis of the recently published ACE2 domain bound to B0AT1 (PDB ID:6M18) 5 and showed that N720 is located on the same interface of the loop region in close vicinity to the TMPRSS2 cleavage site (Fig. 2a) .

Since loop region of protein are unordered and display conformational dynamics, a mutation close to the cleavage site can affect the binding affinity of TMPRSS2. Therefore, we used DynaMut web server to predict the effect of mutation N720D on the stability and flexibility of ACE2 32, 33 .

Whereby, the predicted stability change was (ΔΔG): -0.470 Kcal/mol, which indicated the destabilization of ACE2 receptor after the introduction of D720 (Fig. 2b) . The N720D mutation has resulted in an increase in entropy in the loop region (ΔΔSVib: 0.070 kcal.mol -1 .K -1 ) 33 (Fig.   2b ), depicting a more unstable state, which makes ACE2 more readily cleavable by TMPRSS2.

In addition, we modelled in the various non-covalent interactions for both N720 and mutant D720 and other amino acids in the loop region ( Fig. 2c, d) . In Fig. 2c , N720 formed a backbone hydrogen-bond with N718, this conformation also resulted in an NH-NH between E723 and S721.

Whereas, with the activating variant D720 (Fig. 2d ) the back-bone hydrogen bond with N718 is still established, however, the conformational change has resulted in a polar interaction between the backbone D720 COO-group and backbone NH of L722, forming a weak polar interaction (through water-mediated hydrogen bond). As such, the D720 variant altered the conformation of the loop, and the NH-NH bond between E723 and S721 cannot be formed. Whereby, such intermolecular amide interactions are significant for protein stability 34 . Such a change in interatomic interactions between amino acids near the cleavage region can decrease the stability and increase the flexibility of the loop 35, which makes it easier for TMPRSS2 to cleave.

To gain insights that are pathologically and clinically relevant, we then asked whether a correlation exists between the N720D MAF data and mortality rates reported in corresponding regions. We identified a significant correlation between N720D MAF and deaths per 1 million Table 1 ). There were few variants in the Kuwaiti and Iranian populations that influenced ACE2 expression. In the Qatari population the variants modifying ACE2 gene expression were similar in frequencies to Europeans (Supplementary Table 1 ) with most upregulating ACE2 expression in the brain and tibial nerve tissues (https://gtexportal.org/home/).

Notably, the tibial nerves were affected in diabetic conditions 36 , which inflicts a high number of patients in the Middle East. The most downregulating eQTL variant, rs112171234, was present in 20% and 6% of the African and Qatari populations respectively. Overall, the eQTL data pertaining to ACE2 expression were not significantly predictive nor informative.

Five rare and one common deleterious variants were identified in the TMPRSS2 gene in the Middle Eastern population ( Supplementary Fig. 4 , Supplementary Table 2, Supplementary   Table 3 ). Overall, there was no significant conclusion that may be withdrawn from the TMPRSS2 genetic variation data. On the expression level, we discerned four TMPRSS2 eQTL variants, which were detected only in Qataris among the Middle Eastern populations (Supplementary Table 4 ).

One of these variants, rs6517673 is intronic and downregulates the expression of TMPRSS2 in the prostate. Two of the eQTL variants, rs79391937 and rs79566442, decrease the expression of the TMPRSS2 gene in the thyroid and ovary tissues, respectively. While the variant rs11701542 upregulates the TMPRSS2 gene expression in the testis (https://gtexportal.org/home/). It is worth noting that two-third of the mortality due to COVID-19 disease affects males 15, 37 .

Genetic variation analysis of the FURIN gene resulted in the identification of 13 known missense variants (Table 3 , Supplementary Fig. 5 ). Like ACE2, all the identified variants were rare in the Middle Eastern populations. However, unlike the ACE2 gene, no novel variants were observed in FURIN in the Middle Eastern population (Table 3) . Among the 13, we detected seven deleterious variants suggesting a possible decrease in FURIN protease function, which can potentially reduce the risk of SARS-CoV-2 in the studied populations. In this context, deleterious FURIN gene variations were observed least in East Asians, Africans, followed by Europeans then Iranians (Table 4 ). Interestingly, both in Qatar and Kuwait the deleterious FURIN genetic variations were more common, suggesting a possible protective effect against the SARS-CoV-2 (p<0.05; Table 4 ). For example, the MAF of R37C, and R81C in Kuwait and Qatar were 0.4%/0.7% and 2.4%/3.4% respectively compared to significantly lower MAF in corresponding variants in Europeans (p<0.05; Table 4 ). Together, these data support the premise that FURIN gene variants may play important roles in protection against SARS-CoV-2 in the Middle East. It is worth noting, however, that in Africa FURIN gene variants may not be a contributing factor in viral protection (Table 4) . We next sought to determine the extent of FURIN expression in the Middle East. We detected 16 FURIN eQTL variants in the Middle Eastern populations, most of which were reported in the Qatar population (Supplementary Table 5 ). We observed a high frequency of the FURIN upregulating variants, rs6226 (93%) and rs8039305 (81%) in the African populations compared to the Middle Eastern, European and East Asian populations (p<0.05) (Supplementary Table 5 ). In the GeneATLAS PheWAS database 38 40 circulating between Qatar, Kuwait and Europe, which was attributed to traveling and repatriation between these countries (Manuscript in preparation; GISAID 40 ). For these reasons, we argued that the most likely explanation for the differences observed in mortality rate among countries may be attributed to genetic variation in human genes involved in SARS-CoV-2 processing and cellular entry or exit. Therefore, we screened the genetic variations and eQTL expression of the SARS-CoV-2 candidate genes, ACE2, TMPRSS2 and FURIN in three Middle Eastern populations: Kuwaiti, Iranian, and Qatari and compared them to available MAF data in the gnomAD database 41 .

In this study we showed that amongst the nine known ACE2 missense variants, N720D (rs41303171) and K26R (rs4646116) were the most frequent in the global datasets 18 . In agreement with our analysis, structural predictions by Stawiski and colleagues revealed that the K26R missense variant enhanced the affinity of ACE2 for SARS-CoV-2 whereas N720D had little involvement in the SARS-CoV-2 S-protein interaction 18 . Our data suggest that the ACE2 receptor Notably, the ACE2 novel variant Q661P is close to the aa region 652-659, which is important for cleavage by the metalloprotease ADAM17. R716H and the two deleterious variants R708W and R708Q are located within residues 697-716 essential for cleavage by TMPRSS11D and TMPRSS2. In fact, the mutation of arginine -such as R708-and lysine residues within aa residues 697-716 markedly reduced ACE2 cleavage by TMPRSS2 43 . However, it should be mentioned that sequentially distant aa residues in the ACE2 receptor can be seen brought structurally proximal to each other to create active sites for catalysis 28 . We illustrated this in Supplementary Fig. 6 to show that the novel aa changes (colored green) are proximal to the protein residues that mediate its activity (colored blue and red). Further studies are needed to directly assess the functional aspects of the reported missense variants. We urge the international community to assess ACE2 variation differences among people with mild/asymptomatic disease versus patients presenting with severe respiratory distress syndrome.

Interestingly, there was not a single individual in the Middle East with ACE2 receptor variations in amino acids known to be crucial for SARS-CoV-2 S-protein binding (K31, E35, D38, M82, K353) 44 . This may indicate that natural immunity conferred by the ACE2 receptor variations is lacking or extremely rare. The rareness is evident from a recent comprehensive study that scrutinized ACE2 gene variations in more than 290,000 individuals from 400 different worldwide population groups and in which only eight rare ACE2 gene missense variants (K31R, E35K, E37K, D38V, N33I, H34R, Y83H, and Q388L) with reduced binding to the S-protein of SARS-CoV-2 were reported 18 . Notably, this study has disproved the claim of Cao and colleagues on the absence of such protective ACE2 variants in human populations 15 .

Finally, according to the Human Protein Atlas portal, the mRNA and protein expression of the ACE2 gene is predominant in the human testis, cardiovascular and type II pneumocytes (https://www.proteinatlas.org/ENSG00000130234-ACE2/tissue). Further, a recent study that profiled scRNA-seq from the human testicles revealed the presence of this receptor in Spermatogonia, Leydig and Sertoli Cells 45 . These findings may indicate that human testis is a potential target for coronavirus conforming to male prevalence in infected cases. Notably, in our data, Kuwaiti individuals carrying ACE2 missense variants were all males. Also, a Chinese population study observed a higher hemizygous mutation rate in males than females 15 . It is also possible that the high hemizygosity seen in males can be responsible for male prevalence in infected cases. This hypothesis can be further supported by the recent detection of SARS-CoV-2 virus in the semen of infected people 46, 47 .

In summary, we report the differential occurrence of gene variants in the Middle Eastern 

The whole-exome sequences data of 473 Kuwaitis 48 , 800 Iranians 49 and 100 Qataris 42 published previously were used in this analysis. The genetic data of non-Finnish European, East Asian and African American populations were obtained from the gnomAD repository 41 , which contain data on a total of 125,748 exomes and 71,702 genomes (https://gnomad.broadinstitute.org/).

The expression data for ACE2, TMPRSS2, and FURIN were obtained from the genotype-tissue expression (GTEx) database (https://gtexportal.org/home/). The same database portal was used to extract quantitative trait loci (eQTLs) for the three genes.

The missense variants were defined as deleterious when predicted to be damaging, probably damaging, disease causing and deleterious by the five algorithms applied, SIFT 50 , PolyPhen-2 HumVar, PolyPhen-2 HumDiv 51 , MutationTaster 52 and LRT score 53 and/or CADD (Combined Annotation-Dependent Depletion) score of more than 20 54 . We considered only deleterious variants with minor allele frequency (MAF) less than 1% in the burden analysis. The significance of the differences in MAFs between different populations was calculated using Chi-Square test, using the R software (https://www.r-project.org/). All the p-values presented in the tables are not corrected for multiple testing. P values ≤0.05 were considered significant.

All the identified ACE2 missense exon variants were mapped, modeled, and analyzed using Pymol modeling software (https://pymol.org/2/). DynaMut web server was used to predict the effect of genetic variants on the stability and flexibility of ACE2 receptor 32,33 . The missense variants were defined as deleterious when predicted to be damaging, probably damaging, disease causing and deleterious by the five algorithms applied (SIFT, PolyPhen-2 HumVar, PolyPhen-2 HumDiv, MutationTaster and LRT score) and/or CADD score of more than 20. We considered only deleterious variants with minor allele frequency less than 1% in the burden analysis. The missense variants were defined as deleterious when predicted to be damaging, probably damaging, disease causing and deleterious by the five algorithms applied (SIFT, PolyPhen-2 HumVar, PolyPhen-2 HumDiv, MutationTaster and LRT score) and/or CADD score of more than 20. We considered only deleterious variants with minor allele frequency less than 1% in the burden analysis. P values are calculated using Chi-square test. KWT-Kuwaitis; IRN-Iranians; QAR-Qataris; EUR-Europeans (non-Finnish); EAS-East Asians; AFR-Africans. The missense variants were defined as deleterious when predicted to be damaging, probably damaging, disease causing and deleterious by the five algorithms applied (SIFT, PolyPhen-2 HumVar, PolyPhen-2 HumDiv, MutationTaster and LRT score) and/or CADD score of more than 20. We considered only deleterious variants with minor allele frequency less than 1% in the burden analysis. The missense variants were defined as deleterious when predicted to be damaging, probably damaging, disease causing and deleterious by the five algorithms applied (SIFT, PolyPhen-2 HumVar, PolyPhen-2 HumDiv, MutationTaster and LRT score) and/or CADD score of more than 20. We considered only deleterious variants with minor allele frequency less than 1% in the burden analysis. P values are calculated using Chi-square test. KWT-Kuwaitis; IRN-Iranians; QAR-Qataris; EUR-Europeans (non-Finnish); EAS-East Asians; AFR-Africans. The mutant R26, magenta, forms one H-bond with mannose. A process that could provoke the moiety-ACE2 stability and increases the affinity of the ACE2 a-helix to S protein RBD binding, where, R26 functions as a backbone and interacts with D30 which in turn dignified to build a saltbridge with the S protein RBD K417, yellow stick. 

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DUET: a server for predicting effects of mutations on protein stability using an integrated computational approach

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scRNA-seq Profiling of Human Testes Reveals the Presence of the ACE2 Receptor, A Target for SARS-CoV-2 Infection in Spermatogonia

SARS-CoV-2 and the testis: similarity with other viruses and routes of infection

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Assessment of coding region variants in Kuwaiti population: implications for medical genetics and population genomics

Iranome: A catalog of genomic variations in the Iranian population

Predicting amino acid changes that affect protein function

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Identification of deleterious mutations within three human genomes

CADD: predicting the deleteriousness of variants throughout the human genome

This work was supported by Coronavirus emergency resilience grant from Kuwait Foundation for the Advancement of Sciences. 

All the authors declare no competing interests.

Correspondence and requests for materials should be addressed to F.A-M. Figure 6 . ACE2 protein structure (open form, yellow; and closed form with substrate, gray). The active site residues are coded in red color. The zinc binding residues are coded in blue color. The identified novel amino acid changes in the Middle Eastern populations are coded in green color. The novel changes are proximal to the protein residues that mediate its activity.