ACE2 and TMPRSS2 variants and expression as candidates to sex and country differences in COVID-19 severity in Italy

As the outbreak of coronavirus disease 2019 (COVID-19) progresses, prognostic markers for early identification of high-risk individuals are an urgent medical need. Italy has one of the highest numbers of SARS-CoV-2-related deaths and one of the highest mortality rates. Worldwide, a more severe course of COVID-19 is associated with older age, comorbidities, and male sex. Hence, we searched for possible genetic components of COVID-19 severity among Italians by looking at expression levels and variants in ACE2 and TMPRSS2 genes, crucial for viral infection. Exome and SNP-array data from a large Italian cohort were used to compare the rare-variants burden and polymorphisms frequency with Europeans and East Asians. Moreover, we looked into gene expression databases to check for sex-unbalanced expression. While we found no significant evidence that ACE2 is associated with disease severity/sex bias, TMPRSS2 levels and genetic variants proved to be possible candidate disease modulators, prompting for rapid experimental validations on large patient cohorts.


INTRODUCTION
As we write, Italy, Europe, and the entire world are facing one of the worst medical emergencies spanning centuries, the coronavirus disease 2019 (COVID- 19) pandemia due to infection by SARS-CoV-2 virus. The early identification of risk factors for COVID-19 is an urgent medical need to provide the appropriate support to patients, including access to intensive care units.
Presently, Italy has one of the highest rate of SARS-CoV-2 infection in the world among large countries, with 371 cases per 100,000 people, one of the highest number of deaths and apparently also one of the highest mortality rates, 14.1% vs. an average value of 6.6% (as of May 16th, 2020, data from https://coronavirus.jhu.edu/ map.html). These data may have different explanations, including: 1) the number of tests performed, 2) the structure of the population (Italy has the oldest population in Europe) [https://ec.europa.eu/eurostat/data/ database], 3) the percentage of smokers, even though no significant association was found between smoking and severity of COVID-19 in a very recent study on the Chinese population [1], 4) the possible existence of a different virus strain [2], 5) a high population density in some hot spot areas of the infection, 6) the concentration of severe cases in a limited region of the country, potentially overwhelming the available intensive care units, 7) differences in environmental factors (e.g. air pollution), as well as 8) social factors, such as trust in the institutions and tendency to socialize [3]. However, there could also be some peculiar genetic characteristics of the Italian population that may have an impact on the susceptibility to viral infection, the AGING disease severity, and the number of patients shedding huge amounts of virus.
What is unquestionable is a more severe course of the disease associated with older age and high number of comorbidities and with the male sex (male:female ratio in case fatality rate among Italians 1.75, data from the Italian National Institute of Health: https://www.epicentro.iss.it/coronavirus/), a feature shared with the 2003 SARS epidemic and MERS [4][5][6]. Indeed, while males and females have similar susceptibility to both SARS-CoV-2 and SARS-CoV, males are more prone to have higher severity and mortality, independently of age [4]. Among the many possible factors impacting on sex-related differences in disease manifestations, including the fact that females are known to mount a stronger immune response to viral infections compared to males due to more robust humoral and cellular immune responses [7], we decided to center our attention on possible genetic components, with a particular focus on the Italian population.
It was recently demonstrated that both angiotensin I converting enzyme 2 (ACE2) and the transmembrane protease, serine 2 (TMPRSS2) are crucial for SARS-CoV-2 entry into host cells [8,9]. As previously described for SARS-CoV, ACE2 is the main receptor also for the spike (S) protein of SARS-CoV-2, mediating viral attachment to target cells. Moreover, both coronaviruses use TMPRSS2 for protein S priming, i.e. the cleavage of protein S at the S1/S2 and the S2' sites, allowing fusion of viral and cellular membranes [9]. Both genes have been proposed to modulate susceptibility to SARS-CoV [10,11], and are good candidates to mediate sex-related effects: ACE2 is located on the X chromosome, while TMPRSS2 expression is responsive to androgen/estrogen stimulation [12]. Controversial data have been reported on the level of expression of ACE2 in the lung of males and females [13][14][15], however, it must also be taken into account the effect of estrogen drop in postmenopausal life and the possible compensating effect of hormone replacement therapy in some females.
With this background, we searched for possible genetic components of COVID-19 severity among Italians by looking at expression levels and genetic variants in ACE2 and TMPRSS2, two crucial genes for viral infection.

ACE2
For most X-chromosome genes, the double allelic dosage in females is balanced by the epigenetic silencing of one of the X chromosomes in early development [16].
However, the X-chromosome inactivation (XCI) is incomplete in humans and up to one third of genes are expressed from both alleles, with the degree of XCI escape varying between genes and individuals [17]. ACE2 is one of the genes escaping X inactivation, but it belongs to a subgroup of X-chromosome genes escaping XCI showing an uncharacteristically heterogeneous pattern of male-female expression, with higher expression in males in several tissues [13]. Specifically concerning the lung, a recent analysis on published expression data, reported a substantial similar level of ACE2 transcript in males and females [14], however, another study, using single-cell sequencing, found a higher expression of ACE2 in Asian males [15]. Figure 1 reports data on ACE2 mRNA expression levels in the lung as retrieved from the largest datasets available in the literature; no substantial differences were found between males and females, nor between younger and older females, thus confirming what already observed by Cai and colleagues [14].
Another possible sex-related effect might be due to the fact that males are hemizygous for the gene, therefore, in the presence of an ACE2 allelic variant increasing disease susceptibility or severity, males will have all cells expressing the risk variant. Based on this hypothesis, we looked into the genetic variation in ACE2. A recent manuscript explored this same topic in different populations using data from public databases [18]. However, a specific analysis of the Italian population is lacking.
We have therefore exploited the available data on 3,984 exomes obtained from an Italian cohort representative of the whole country [19,20] to extract the variants in exons and splice junctions of ACE2. Variants were filtered for quality and classified according to their predicted effect at protein level and on splicing. Concerning rare variants (i.e. those with a minor allele frequency, MAF, <1%; to be used in burden tests), we considered only null variants, abolishing or significantly impairing protein production (nonsense, out-of-frame ins/dels, and splicing variants), and missense variants predicted to be deleterious or possibly deleterious by all the 5 prediction algorithms used (see Supplementary Methods, paragraph "Definition of disrupting variants and statistical analysis"). Concerning common variants (i.e., MAF>5%), all were retained for comparing their frequency with those of the European (non Finnish) and East Asian populations, retrieved from the GnomAD repository.
No significant differences in the burden of rare deleterious variants were observed comparing the Italian population with Europeans and East Asians (Table 1A). Concerning common exonic variants, the AGING only striking difference, as also noticed by Cao and colleagues [18], was observed for the single nucleotide polymorphism (SNP) rs2285666 (also called G8790A), with the frequency of the rare A allele being 0.2 in Italians and Europeans, and 0.55 in East Asians (uncorrected P=2.2*10-16 for difference in Italians vs East Asians; corrected P=7.9*10-15; Table 1B). This variant was extensively studied as a potential risk factor Figure 1. ACE2 expression levels. All panels show ACE2 mRNA expression levels in human normal lung samples stratified according to sex (or on sex and age). On left panels, data were retrieved for a total of 578 RNAseq experiments from the GTex repository. Expression levels are reported as transcripts per kilobase million (TPM). On the right, data were collected from two different datasets (GSE66499 and GSE19804) from the GEO database. Expression levels are reported as normalized signal intensities. P values were calculated by using either the Kruskal-Wallis or the student t test, using the R software (https://www.r-project.org/).  for hypertension, type 2 diabetes, and coronary artery disease [21,22], hence possibly constituting a predisposing factor also for the comorbidities observed in COVID-19 patients. A single paper reports the association of the three rs2285666 genotypes with ACE2 protein level measured in serum by ELISA, with the A/A genotype having an expression level almost 50% higher than the G/G genotype, while heterozygous G/A individuals had intermediate levels [23]. Given the position of the variant, at nucleotide +4 in the donor splice site of intron 3 (c.439+4G>A), we calculated the predicted effect on splicing and indeed the substitution of G with an A is predicted to increase the strength of the splice site of about 9.2% (calculation made through the Human Splicing Finder v.3.1 webtool, http://www.umd.be/HSF/), consistently with the higher level of ACE2 protein in serum. It would be crucial to compare the frequency of this variant with ACE2 expression in the lung and with susceptibility to viral infection and severity of COVID-19 manifestations. Of note, no eQTL for ACE2 in the lung has been described so far in the GTEx database, and investigations on this topic are recommended.

TMPRSS2
TMPRSS2 is a gene well known to oncologists as genetic rearrangements producing a fusion between TMPRSS2 and ERG (or, more rarely, other members of the ETS family) are the most frequent genetic lesions in prostate cancer patients [24]. As TMPRSS2 is an androgen responsive gene, the fusion results in androgen dependent transcription of ERG in prostate tumor cells. Therefore, we can hypothesize that males might have higher TMPRSS2 expression also in the lung, which might improve the ability of SARS-CoV-2 to enter cells by promoting membrane fusion. Looking into GTEx and GEO data, the overall expression of TMPRSS2 in the lung is only slightly increased in males (P=0.029; Figure 2A). However, TMPRSS2 expression is also promoted by estrogens [12], and therefore the situation might be different when considering individuals above 60 years, who are at higher risk of fatal events due to COVID-19, as in this group females will all be postmenopausal. According to this hypothesis, we checked the expression of the gene in lungs of males and females at different ages, but no AGING AGING substantial differences emerged between males and females (neither below, nor above 60 years of age; data not shown).
Finally, we explored genetic variation in TMPRSS2 in search of variants, possibly already annotated as eQTL in the lung, which might have an impact on the serine protease expression as well as on its catalytic activity. Again, we used the available Italian exome data, as well as data deposited in GnomAD [25].
Firstly, we looked at the overall burden of deleterious rare variants, using the variant classification described above. Italians had a nominally significant decrease in the burden of deleterious variants compared to Europeans (uncorrected P=0.039, not significant after correction for multiple testing; Table 2A). This decrease was even more evident for the East Asian population (corrected P=8.6*10-4); however, in this case, we must consider that the number of individuals over 65 years of age in Italy is more than double the one in the Hubei province (22.7 vs. 10%, respectively) and this is a major determinant of disease lethality.
Focusing specifically on common exonic variants, 4 SNPs showed significantly (P<2.2*10-16) different frequencies when comparing the Italian population with East Asians (and with Europeans) ( Table 2B); 3 of them are synonymous variants, whereas one is the missense substitution p.Val160Met, which impacts on a residue far from the serine protease catalytic triad. This variant was previously found significantly associated with genomic rearrangements involving TMPRSS2, with the risk of prostate cancer [26] and with shorter time to prostate cancer diagnosis for high-risk patients [27].
Concerning eQTLs, a number of variants significantly impacting on TMPRSS2 expression in the lung (GTex data) are reported in the 3' region of the gene ( Figure  2B). In Table 2C, a list of the most significant (P<1*10-8), together with their GnomAD frequencies in the East Asian and European populations, are reported. As for the Italian frequencies, we took advantage of the genome-wide association study (GWAS) performed on the above-described cohort (for a total of 3,284 individuals) [28]; in this case, we had to infer genotype frequencies by an imputation approach (for details, see Supplementary Methods). Interestingly, all these eQTLs appear to have extremely different frequencies among populations. In particular, 2 different haplotypes can be inferred from frequency data: 1) A frequent "European" haplotype (composed at least of SNPs rs463727, rs34624090, rs55964536, rs734056, rs4290734, rs34783969, rs11702475, rs35899679, and rs35041537), which is totally absent in the Asian population. Interestingly, this haplotype has been functionally linked to another eQTL (rs8134378), located at a known androgen-responsive enhancer for TMPRSS2, 13 kb upstream of the TMPRSS2 transcription start site [29] ( Figure 2B). Hence, this haplotype is expected to up regulate TMPRSS2 gene expression in an androgen-specific way.
2) A second haplotype, predicted to be associated with higher TMPRSS2 expression, is characterized by 3 SNPs (rs2070788, rs9974589, rs7364083), whose MAF is significantly increased in Europeans (9% increase in Italians respect to East Asians, corrected P<6.8*10 -9 ). Importantly, a small-scale GWAS, comparing the distribution of genetic variants in severe and mild cases of patients with A(H1N1)pdm09 influenza, identified rs2070788 as being associated with increased risk to both human A(H7N9) and severe A(H1N1)pdm09 influenza [11]. Of note, also in A(H7N9) influenza, the proportion of male patients was more than double that of female patients [30].

LIMITATIONS AND CONCLUSIONS
We are aware of the limitations of our study: first of all we focused our attention only to two candidate genes identified on the basis of their crucial role in viral infection and on the a priori probability that they might mediate sex-specific effects. A number of other Xlinked genes (such as IL13, IL4, IL10, XIST, TLR7, FOXP3) and Y-linked genes (SRY, SOX9) may underlie sexually dimorphic immune responses [31]. Moreover, the number of non-genetic determinants of sex-biased severity and case fatality rates is huge and probably has to do not only with sex differences in both innate and adaptive immune responses [7], but also with gender and cultural habits in different countries. In particular, important gender-related factors might concern the social role of women (job, maternal and childcare role), the propensity to smoke, the hand hygiene compliance, as well as differences in the impact of the social role of women in the different countries.
In conclusion, we have explored possible genetic components impacting on COVID-19 severity, focusing on effects mediated by ACE2 and TMPRSS2 genes in the Italian population. From available data, it seems unlikely that sex-differences in ACE2 levels can explain sex differences in disease severity. However, it remains to be evaluated if changes in ACE2 levels in the lung correlate with susceptibility and severity of SARS-CoV-2 infection. Experimental data from patients with different disease manifestations are urgently needed. Among the analyzed hypotheses, the most interesting signals refer to sex-related differences in TMPRSS2 expression and in genetic variation in TMPRSS2. In particular, we AGING   AGING identified an exonic variant (p.Val160Met) and 2 distinct haplotypes showing profound frequency differences between East Asians and Italians. The rare alleles of these haplotypes, all predicted to induce higher levels of TMPRSS2, are more frequent in the Italian than in the East Asian population; in one case, the haplotype could be regulated through androgens, thus possibly explaining the sex bias in COVID-19 severity, in the other case, a SNP belonging to the haplotype has been associated with increased susceptibility to influenza, possibly related to a higher susceptibility in Italians and Europeans.
Our data, beside suggesting possible explanations for the unusually high, relative to known data, lethality rates among Italians, provide reference frequencies in the general Italian population for candidate variants that can be compared to genetic data from patients infected by SARS-CoV-2 with different disease manifestations, as soon as they will be available on large numbers of patients. These studies will hopefully be of help in predicting the individual risk of infection and susceptibility to CoV-2 and in recognizing in advance infected individuals being at higher risk of poor prognosis.

Gene expression data
Expression data for ACE and TMPRSS2 genes were obtained through the: 1) genotype-tissue expression (GTEx) database (https://gtexportal.org/home/), which was also used to extract quantitative trait loci (eQTLs) for the two genes (all data based on RNAseq experiments); and 2) Gene Expression Omnibus (GEO) repository (https://www.ncbi.nlm.nih.gov/geo/). In particular, two GEO datasets were extracted and analyzed: 1) GSE66499, reporting microarray data on 152 normal lung samples from Caucasian individuals; 2) GSE19804, reporting microarray data on 60 normal lung samples from Taiwanese females (see also Supplementary Methods, paragraph "Datasets and statistical power estimations").

Genetic data
Genetic data for general European and East Asian populations were retrieved through the GnomAD repository, which contains data on a total of 125,748 exomes and 71,702 genomes (https://gnomad.broad institute.org/).
As for Italians, details on whole-exome sequencing (on 3,984 individuals) and genome-wide microarray genotyping (on 3,284 individuals) of the analyzed cohort are specified elsewhere [19,20,28], as well as in Supplementary Methods (paragraphs "Sequencing" and "Datasets and statistical power estimations"). Imputation procedures are detailed in Supplementary materials (paragraph "Dataset imputation").

Statistical analysis
Expression levels were compared by using either the Kruskal-Wallis test (RNAseq data) or the student t test (microarray data). Allele frequencies were compared using the chi square test. All calculations were performed using the R software (https://www.r-project.org/). P values are presented as non-corrected for multiple testing, but the Bonferroni-corrected threshold of significance is indicated below each set of comparisons presented in Tables. Power calculations have been described in Supplementary Methods (paragraph "Datasets and statistical power estimations").

Sequencing
Whole-exome sequencing (WES) was performed at the Broad Institute (Boston, MA). Demographic characteristics, as well as exome capture methods, sequencing, variant annotation, and data processing of the samples were described previously [1].

Definition of disrupting variants and statistical analysis
Using WES data, we searched the ACE2 and TMPRSS2 genes for loss-of-function variants (nonsense, frameshift, splicing, or disrupting missense mutations).
The positions of mutations were based on the cDNA reference sequence for ACE2 and TMPRSS2 (NM_021804 and NM_005656) with the ATG initiation codon numbered as residue 1 (p.Met1).
Burden test analyses were performed considering only those variants having a minor allele frequency (MAF) <1%. Significance in the differences of MAFs between different populations were calculated using chi-square tests, with the R software (https://www.r-project.org/). A P<0.05 was considered to indicate statistical significance.

Dataset imputation
When missing from exome data, intronic variant frequencies in TMPRSS2 were retrieved from SNParray data obtained from the same Italian cohort. Genome-wide genotyping was performed at the Broad Institute. Genotyping details and data processing of the samples have been already described [6].

Datasets and statistical power estimations
For expression data analyses, we took advantage of microarray data reported in the GEO repository (https://www.ncbi.nlm.nih.gov/geo/). We specifically searched for the wider datasets reporting expression data on normal lung tissues derived from individuals whose sex and geographical origin were specified (search done by keywords, filters based on the number of available samples in the dataset, and by a final manual inspection of the retrieved data). This search allowed the identification of two datasets: GSE66499 and GSE19804, for a total of 115 samples from male individuals, and 135 samples from female subjects. Indeed, it is difficult to provide an accurate power estimate for a microarray study. Among others, [9] suggested that a sample size of 20 is necessary, at a P value of 0.01 and 90% power, to detect a two-fold change in the 75% least variable genes in a microarray study. Based on this observation, the data available through the GSE66499 and GSE19804 datasets were considered reasonably powered to identify possible altered levels in the ACE2 and TMPRSS2 genes.
As for genotype data, from one side we took advantage of exome and SNP-array in-house data on ~3,500 individuals; [1,6], from the other of exome and genome data on the largest dataset freely accessible online, i.e. the GnomAD repository (https://gnomad.broadinstitute.org/). For GnomAD data, we extracted allele/genotype frequencies available for East Asian and European individuals, for a total of at least 9,967 and 64,302 subjects, respectively. The use of such large cohorts ensured us to be sufficiently powered to detect significant differences in allele frequencies between the analyzed populations. As an example, a sample size of 2,000 pairs has an approximately 80% power of detecting a significant allele difference at P<0.05 if the frequency of the rare allele is 2%. For higher frequencies of 10% or more, the power of detection increases to more than 90%.