Most frequent South Asian haplotypes of ACE2 share identity by descent with East Eurasian populations.

It was shown that the human Angiotensin-converting enzyme 2 (ACE2) is the receptor of recent coronavirus SARS-CoV-2, and variation in this gene may affect the susceptibility of a population. Therefore, we have analysed the sequence data of ACE2 among 393 samples worldwide, focusing on South Asia. Genetically, South Asians are more related to West Eurasian populations rather than to East Eurasians. In the present analyses of ACE2, we observed that the majority of South Asian haplotypes are closer to East Eurasians rather than to West Eurasians. The phylogenetic analysis suggested that the South Asian haplotypes shared with East Eurasians involved two unique event polymorphisms (rs4646120 and rs2285666). In contrast with the European/American populations, both of the SNPs have largely similar frequencies for East Eurasians and South Asians, Therefore, it is likely that among the South Asians, host susceptibility to the novel coronavirus SARS-CoV-2 will be more similar to that of East Eurasians rather than to that of Europeans.

Introduction

The novel coronavirus SARS-CoV-2, the causative agent of the ongoing pandemic of COVID-19, today presents one of the major challenges to humanity [1]. Recent studies have effectively demonstrated that the Angiotensin-converting enzyme 2 (ACE2) encoded by a gene located on the X-chromosome is the host receptor for the virus [1, 2]. A decreased level of ACE2 expression mitigates the severity of the disease. The over-expression or a unique genetic polymorphism of the receptor among Asians have been ruled out in a recent study [3, 4]. ACE2 also maintains cardiovascular homeostasis and electrolyte balance and protects against lung injury by acid aspiration [5]. A comprehensive understanding of ACE2 variations among various ethnic groups has hitherto been largely unknown.

The South Asia subcontinent harbours diverse and endogamous ethnic groups [6]. Most of the genomes of South Asia are autochthonous but show a considerable amount of sharing with East and West Eurasia [7]. However, when we compare overall genome sharing with East vs. West Eurasia, South Asians show greater genetic affinity with West Eurasia [8-10]. The only exception is Tibeto-Burman speaking populations, who share a large amount of ancestry with East Eurasia [11]. The genetic structure of ACE2 haplotypes among South Asian populations is not known. Therefore, we have analysed the whole genome data of South Asians with respect to various world populations for ACE2 published elsewhere [12, 13] (S1 Table).

Materials and methods

The research has been approved by the Institutional Ethical Committee of Banaras Hindu University, Varanasi, India. To analyse the ACE2 among various populations, we have extracted the sequences from the published datasets [12, 13], by using PLINK 1.9 [14]. It has been shown that the 1000 genome dataset for South Asia does not capture the complete South Asian variation, mainly due to unsampled Austroasiatic populations [15]. Hence, we analysed Pagani et al. [12] by way of primary data and further confirmed the results with the 1000 genome data [13]. We extracted 447 samples designated as a diversity set panel in the Pagani et al. data [12]. After excluding samples from Africa, Sahul and relatives up to the second degree, we used 393 samples in all our analyses (S1 Table). A total of 248 polymorphisms were observed in the Pagani et al. data [12] (S2 Table). LD maps for each of the groups were analysed from Haploview [16] (S1 Fig). For both of the datasets, we converted plink file to fasta file (ped to IUPAC) from customised script. Phasing of the data, the calculation of population-wise genetic distances, and Arlequin and Network input files were generated by DnaSP v 6 [17]. The neighbour joining (NJ) tree was constructed by MEGA-X [18] (Fig 1A). Nei’s genetic distances and pairwise differences were calculated from Arlequin 3.5 [19] and plotted by R v 3.1 [20] (Fig 1B and S2 Fig). Network v5 [21] and Network publisher were used to construct the median joining (MJ) networks (Fig 2 and S3 Fig). The spatial map of rs4646120 and rs2285666 were drawn from PGG toolkit (S4 Fig) [22].

Fig 1

a) The Neighbour-Joining (NJ) tree showing the genetic relationship of the studied populations. The figure was drawn from the Fst distances obtained from the haplotype analysis of Eurasian populations. b) Heat map showing the intra- and inter-population variation measured by average pairwise sequence differences of the ACE2 gene. The average pairwise differences between populations are shown in the upper triangle of the matrix (green). The average number of pairwise differences within each population group are shown along the diagonal (orange). The differences between populations based on Nei's genetic distances are depicted in lower triangle of the matrix (blue). The obtained values of various parameters have been shown at the color scales.

Fig 2

The median joining (MJ) network of 142 haplotypes belonging to gene ACE2.

Circle sizes are proportional to the number of samples with that haplotype. The three most common haplotypes are marked. The three important SNPs studied in details have been marked in the figure. We used median joining method implemented in the NETWORK programme ver. 5.

Result and discussion

Our pooled data have yielded 248 high quality polymorphisms (S2 Table). In the LD (linkage disequilibrium) plot analysis, significant LD blocks of different sizes were present among Caucasus, Central Asians, South Asians, mainland Southeast Asians, insular Southeast Asians and Siberians (S1 Fig). Europeans showed the lowest level of LD. We have used a haplotype based approach for the comparison. In contrast with the genome-wide analysis [8-10], the NJ (Neighbour Joining) tree based on Fst distances clustered South Asians together with insular and mainland Southeast Asian populations (Fig 1A). This unexpected result suggested closer a genetic affinity of South Asians with East Eurasians for ACE2. The pairwise difference analysis suggested lower diversity for South Asian, Southeast Asian and Siberian populations (Fig 1B). Similarly, the 1000 genome populations showed the lowest diversity for East Asian populations (S2 Fig).

The phylogenetic analysis of various haplotypes among studied populations helped to identify the SNPs responsible for the affinity of South Asians with East Eurasians (Fig 2 and S3 Fig). Three major distinct haplotypes were observed. Haplotype 1 (ht1) was more common in West Eurasians, including Central Asian populations, whereas haplotype 2 (ht2) was frequent among East Eurasians, South Asians and Americans (Fig 2 and S3 Fig). Haplotype 3 (ht3) was harboured mainly by East Eurasians and South Asians. The haplotype 2 (ht2) originated from SNP rs4646120, whereas ht3 was derived from SNP rs2285666. Phylogenetically both of these SNPs play a key role in the distinction between East and West Eurasian populations (Fig 2 and S3 and S4 Figs). Interestingly, the most frequent haplotypes of South Asia involve these SNPs. A recent study has also highlighted the highest frequency of this SNP (rs 2285666) among Chinese populations (0.5) as well as significant frequency differences among 1000 genome populations (S4 Fig) [4]. In our study, we also found high frequency (0.6) of this SNP among South Asians (S2 Table and S4 Fig). Moreover, we also found that a synonymous coding region variant rs35803318 was most frequent among Americans (0.15), followed by Europeans (0.055), Caucasians (0.051) and Central Asians (0.021), whilst this site was not polymorphic for West Asians, South Asians, Southeast Asians and Siberians (S2 Table).

Phylogenetic analysis has suggested that the majority of South Asian samples share with East Eurasians the monophyletic haplotypes 2 and 3 by the unique polymorphism events (rs4646120) and (rs2285666). Recent studies have suggested that the reference allele has a reduced ACE2 expression of up to 50%, resulting in greater severity of a SARS-CoV-2 infection [23-25]. Additionally, a synonymous coding region variant rs35803318 was also significantly more polymorphic among Americans and Europeans than among South Asians. Hence, it is likely that among South Asians, the host susceptibility to the novel coronavirus SARS-CoV-2 more closely resembles that of East/Southeast Asians rather than that of Europeans or Americans.

The LD (linkage disequilibrium) plots of ACE2 gene of various studied populations.

Shading from white to red indicates the intensity of r from 0 to 1. Strong LD is represented by a high percentage (>80) and a darker red square.

(TIF)

Click here for additional data file.

Heat map showing the intra- and inter-population variation measured by average pairwise sequence differences of the ACE2 gene among 1000 genome populations.

The average pairwise differences between populations are shown in the upper triangle of the matrix (green). The average number of pairwise differences within each population group are shown along the diagonal (orange). The differences between populations based on Nei's genetic distances are depicted in lower triangle of the matrix (blue). The populations are grouped in to superpopulations e.g. European, South Asian, East Asian and American.

(TIF)

Click here for additional data file.

The median joining (MJ) network of 491 haplotypes belonging to gene ACE2 among 1000 genome populations.

Circle sizes are proportional to the number of samples with that haplotype. The three most common haplotypes are marked. All three SNPs studied in detail have been marked by arrow. We used median joining method implemented in the NETWORK programme ver. 5.

(TIF)

Click here for additional data file.

The spatial distribution of alleles of rs4646120 and rs2285666 among 1000 genome populations.

The map was obtained from the PGG toolkit implemented in the https://www.pggsnv.org/.

(TIF)

Click here for additional data file.

The number of samples from each of the region used in the analysis.

The number of South Asian groups shown with their linguistic affiliations.

(PDF)

Click here for additional data file.

The details of 248 polymorphic loci extracted from analysed data.

The frequencies of alternate alleles for each loci and group have been mentioned in the table. The three important SNPs studied in details have been highlighted.

(XLSX)

Click here for additional data file.

Transfer Alert

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2 Jun 2020

PONE-D-20-12219

Most frequent South Asian haplotypes of ACE2 share identity by descent with East Eurasian populations

PLOS ONE

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Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

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Reviewer #2: Yes

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Reviewer #1: Srivastava et al present “Most frequent South Asian haplotypes of ACE2 share identity by descent with East Eurasian populations”. While the basic concept is interesting and timely given the current SARS-CoV-2 pandemic, the manuscript unfortunately can not be published in its current form:

- Unfortunately important information is missing, reproducibility is not entirely given

- the manuscript was written quickly and includes several errors, which make it hard to read but also trust the results, e.g. wrong SNP name.

- Unfortunately the images in Figure1 where hardly readable, with the figure legends not very informative and helpful

Major Issues:

The main issue I see with this manuscript is that it was written in a hustle, which a reader can easily tell, as there is information missing, one would typically expect, besides several errors (typos but also sentences, issues with references and figures). Comparison to 1000G data is very limited; as the main focus is on South Asia, with only 25 samples included, whereas 1000G includes 662 samples. Therefore one of my main concerns is, how representative are the 25 (mostly males, as ACE2 on X?) samples for South Asia. How do the results differ to the one reported by Cao et al 2020, what is the frequency of the two main SNPs in South-Asians for 1000G data?

The file in the provided data includes 402 samples, Pagani et al describe 483 samples. This work according the haplotypes in Suppl.Materials a total of 393 samples. Can you describe the discordance?

Unfortunately (technical issue here) the figures can not be read well – can you please elaborate and include a larger font? Also the figure legends are not very informative.

Please check the manuscript with help of a native english speaker

Minor Issues:

Abstract:

- include sample size of your data set, including for super populations once mentioned (EAS, SAS, EUR).

- the human Angiotensin-converting enzyme 2 (ACE2)

- more related TO West Eurasians

-focusing ON South Asia

-The second SNP – without talking about the first SNP? Why only the second one, what is with the first one?

Introduction:

- Please recheck sentence: ...has been one of the serious threat to humanity now

- encoded by A gene located ON

- expression of ACE2 receptor? relate to the severity OF THE disease

- Confusing sentence: Growing evidence are accumulating, however, still the expression of ACE2 variations affecting host susceptibility among various world population is not known.

- South Asia is a country ...really? It is a region consisting of countries Afghanistan, Bangladesh, Bhutan, Maldives, Nepal, India, Pakistan and Sri Lanka

- Please recheck, very confusing: For the ACE2, it is not known that how the polymorphisms present in South Asia is shared with the East and West Eurasian populations?

-Reference 10 and 11 are identical, Reference 3 missing journal,

Material and Methods:

- Reference 10 vs 11. a [11] (Supplementary Table 1) here I was searching the data in Paganis Supplementary table – please make clearer

- Supplementary Figure 3: one can clearly see that the frequency of unique polymorphisms is strongly correletad to the sample size with one exception (SEA_M, this gets obvious when you order by sample size) – what is the purpose of the barplot? Same with Figure 1, how useful is this plot, can the sample size difference also be biasing the main finding?

- What is the purpose of supplemental table 1 and figure 3 – as figure 3 reports the same data?

- Please check sentence: … were constructed from the Haploview.

ResultS and discussion

- Please check: Europeans and Siberians had THE highest number of private polymorphisms which ARE likely due to their large number of samples from these groups (Supplementary Figure 1, Figure 3, Table 1)

- what are the snps defining ht1?

- LD patters → LD patterns

- this unexpected result – wasn’t it reported by Cao et al 2020 yet?

- LD Plots – why are different amount of SNPs used for the subfigures on population level? However no chance to read. Can you please provide a better description of how the LD

- consistent naming of haplotypes ht1 … either with space or none

- rs446120 is on chr8 – should be rs4646120 according figure 1c. 2X wrong in text.

- was more common in West Eurasians

- What leads to the conclusion that rs2285666 has lower severity for SARS-CoV-2?

Reviewer #2: Comments on the manuscript entitled “Most frequent South Asian haplotypes of ACE2 share identity by descent with East Eurasian populations”, by Srivastava et al.

Srivastava et al. have analysed whole genome data of the populations around the world, focusing South Asia, and extracted 248 ACE2 variations. Phylogenetic analysis of the above data suggested that the majority of South Asian ACE2 haplotypes are closer to the East Eurasians in the background of two unique event polymorphisms. Hence, the authors have suggested that it is highly likely that among the South Asians, the host susceptibility to the novel coronavirus SARS-CoV-2 will be more similar to East/Southeast Asians rather than the Europeans.

Considering the ongoing pandemic, this manuscript is appropriate now. However, I suggest the authors to take care of the following points.

1. No author has affiliation No. 5!

2. No mention about the total number of samples, analysed; although supplementary Table 1 has the information. I suggest the authors to mention the numbers in the methods.

3. It may be useful to discuss in the manuscript about the linguistic background of South Asian populations that were included in the study.

**********

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30 Jun 2020

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

________________________________________

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Srivastava et al present “Most frequent South Asian haplotypes of ACE2 share identity by descent with East Eurasian populations”. While the basic concept is interesting and timely given the current SARS-CoV-2 pandemic, the manuscript unfortunately can not be published in its current form:

We have substantially revised the manuscript by addressing all the concerns of the reviewers.

- Unfortunately important information is missing, reproducibility is not entirely given

- the manuscript was written quickly and includes several errors, which make it hard to read but also trust the results, e.g. wrong SNP name.

- Unfortunately the images in Figure1 where hardly readable, with the figure legends not very informative and helpful

We have separated the figures now in the revised version.

Major Issues:

The main issue I see with this manuscript is that it was written in a hustle, which a reader can easily tell, as there is information missing, one would typically expect, besides several errors (typos but also sentences, issues with references and figures). Comparison to 1000G data is very limited; as the main focus is on South Asia, with only 25 samples included, whereas 1000G includes 662 samples. Therefore one of my main concerns is, how representative are the 25 (mostly males, as ACE2 on X?) samples for South Asia. How do the results differ to the one reported by Cao et al 2020, what is the frequency of the two main SNPs in South-Asians for 1000G data?

We have replicated our analysis with the 1000G data phase 3 data now in the revised version (Supplementary Figs 2-4). Results from both of the data has yielded similar results and conclusions drawn by us is supported by the analysis of independent datasets (Pagani et al. 2016 and 1000G).

We differ from Cao et al 2020 paper substantially. Cao et al have just looked the frequency of coding region substitutions among 1000G as well as Chinese dataset. They also didn’t add any analysis by the ancestry sharing point of view. Whereas, we used more robust haplotype based approach and moved a step ahead than Cao et al.

To have a better understanding of these SNPs worldwide, in the revised version, we have added the spatial distribution of two main SNPs in 1000G data (Supplementary Fig 4).

The file in the provided data includes 402 samples, Pagani et al describe 483 samples. This work according the haplotypes in Suppl.Materials a total of 393 samples. Can you describe the discordance?

According to the Supplementary Table 1. Pagani used 447 samples for their diversity panel. We have used the same data. After excluding regions e.g. Africa and Sahul as well as close relatives, we landed to 393 samples. We have explicitly mentioned this in the material and method section.

Unfortunately (technical issue here) the figures can not be read well – can you please elaborate and include a larger font? Also the figure legends are not very informative.

We have made separate figures now and also improved the legends.

Please check the manuscript with help of a native english speaker

We have included a co-author who has improved the quality of writing as well as edited the manuscript for the language.

Minor Issues:

Abstract:

- include sample size of your data set, including for super populations once mentioned (EAS, SAS, EUR).

- the human Angiotensin-converting enzyme 2 (ACE2)

- more related TO West Eurasians

-focusing ON South Asia

We have revised the manuscript as per the suggestions of the reviewer.

-The second SNP – without talking about the first SNP? Why only the second one, what is with the first one?

We have expanded the discussion about both of the SNPs in the main text.

Introduction:

- Please recheck sentence: ...has been one of the serious threat to humanity now

- encoded by A gene located ON

- expression of ACE2 receptor? relate to the severity OF THE disease

We have revised the sentences accordingly.

- Confusing sentence: Growing evidence are accumulating, however, still the expression of ACE2 variations affecting host susceptibility among various world population is not known.

We have rephrased the sentence.

- South Asia is a country ...really? It is a region consisting of countries Afghanistan, Bangladesh, Bhutan, Maldives, Nepal, India, Pakistan and Sri Lanka

We are sorry for this error! We have corrected it in the revised text.

- Please recheck, very confusing: For the ACE2, it is not known that how the polymorphisms present in South Asia is shared with the East and West Eurasian populations?

The sentence is revised now.

-Reference 10 and 11 are identical, Reference 3 missing journal,

We have rechecked all the references for the consistency.

Material and Methods:

- Reference 10 vs 11. a [11] (Supplementary Table 1) here I was searching the data in Paganis Supplementary table – please make clearer

These errors have been corrected.

- Supplementary Figure 3: one can clearly see that the frequency of unique polymorphisms is strongly correletad to the sample size with one exception (SEA_M, this gets obvious when you order by sample size) – what is the purpose of the barplot? Same with Figure 1, how useful is this plot, can the sample size difference also be biasing the main finding?

We agree with the reviewer and excluded these figures. To verify the main finding we used 1000G data as well as 26 random chromosomes for each of the groups. We didn’t get any deviation from the main finding in these analyses.

- What is the purpose of supplemental table 1 and figure 3 – as figure 3 reports the same data?

We agree with the reviewer. Since in the revised version we excluded Supp Fig. 3, we retained the Supplementary Table 1.

- Please check sentence: … were constructed from the Haploview.

It has been corrected now.

ResultS and discussion

- Please check: Europeans and Siberians had THE highest number of private polymorphisms which ARE likely due to their large number of samples from these groups (Supplementary Figure 1, Figure 3, Table 1)

We have excluded the figures as well as the sentence.

- what are the snps defining ht1?

We have updated Supp Table 1 SNPs with their location in different haplotypes.

- LD patters → LD patterns

It has been removed.

- this unexpected result – wasn’t it reported by Cao et al 2020 yet?

Cao et al. have looked the coding SNPs and didn’t analyse ancestry sharing, whereas we looked haplotypes and their sharing.

- LD Plots – why are different amount of SNPs used for the subfigures on population level? However no chance to read. Can you please provide a better description of how the LD

The program Haploview only consider the polymorphic SNPs for LD reconstruction, the polymorphic SNPs vary in different regions, therefore, the different amount of SNPs.

- consistent naming of haplotypes ht1 … either with space or none

It has been consistent now.

- rs446120 is on chr8 – should be rs4646120 according figure 1c. 2X wrong in text.

We are sorry for this typo, it is corrected in the manuscript.

- was more common in West Eurasians

Revised accordingly.

- What leads to the conclusion that rs2285666 has lower severity for SARS-CoV-2?

Recent studies have suggested that the Reference allele has reduced the ACE2 expression upto 50%, resulting the higher severity for SARS-CoV-2 (Singh et al. 2020; Wu et al., 2017; Asselta et al., 2020)

Reviewer #2: Comments on the manuscript entitled “Most frequent South Asian haplotypes of ACE2 share identity by descent with East Eurasian populations”, by Srivastava et al.

Srivastava et al. have analysed whole genome data of the populations around the world, focusing South Asia, and extracted 248 ACE2 variations. Phylogenetic analysis of the above data suggested that the majority of South Asian ACE2 haplotypes are closer to the East Eurasians in the background of two unique event polymorphisms. Hence, the authors have suggested that it is highly likely that among the South Asians, the host susceptibility to the novel coronavirus SARS-CoV-2 will be more similar to East/Southeast Asians rather than the Europeans.

Considering the ongoing pandemic, this manuscript is appropriate now. However, I suggest the authors to take care of the following points.

Thanks for the good words.

1. No author has affiliation No. 5!

We are sorry for this error. We have corrected it in the revised manuscript.

2. No mention about the total number of samples, analysed; although supplementary Table 1 has the information. I suggest the authors to mention the numbers in the methods.

We have revised the manuscript to reflect these informations.

3. It may be useful to discuss in the manuscript about the linguistic background of South Asian populations that were included in the study.

Thanks. We have added a supplementary table (table S1) with the linguistic information of South Asian samples.

________________________________________

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Reviewer #1: No

Reviewer #2: No

We hope that in the revised version you will find everything in order.

Sincerely,

G Chaubey (on behalf of coauthors)

Submitted filename: Reply to Reviewers.docx

Click here for additional data file.

4 Aug 2020

PONE-D-20-12219R1

Most frequent South Asian haplotypes of ACE2 share identity by descent with East Eurasian populations

PLOS ONE

Dear Dr. Chaubey,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: I thank the authors for addressing my concerns and for the additional work carried out. The manuscript is now almost ready for publication - only few minor issue:

In the abstract there are 383 samples while you mentioned 393 in material and methods (which I can replicate now)

S1 Table - as we are on Chromosome X - Chromosomes (2n) describes the data incorrectly, as there are 258 males and only 135 females - therefore suggest to write number of individuals

S2 Table - highlight the three SNPs described in the main text - would prefer an Excel file over the PDF file here.

Figure S3: can you please indicate the SNP rs3503318 in Figure 2 and S3?

Figure 1b and S2: can you please specify the axis values on the legends for diversity and describe in the figure legend respectively?

Reviewer #2: I have gone through the revised manuscript and the pint-by-point reply to my comments. I am satisfied with all the reply to my previous questions. I have no more question.

**********

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Reviewer #1: No

Reviewer #2: No

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5 Aug 2020

To,

Dr. Alessandro Achilli,

Academic Editor

PLOS ONE

Date: 05/08/2020

Dear Dr Achilli,

We have revised our manuscript considering the comments from reviewer1. Our pointwise answers are given below (in bold letters).

///////////////////////////////////////////////////////////////////////////////////////////////////////////////

Reviewer #1: I thank the authors for addressing my concerns and for the additional work carried out.

We are grateful to reviewers for their constructive suggestions which has helped us to improve our manuscript substantially.

The manuscript is now almost ready for publication - only few minor issue:

In the abstract there are 383 samples while you mentioned 393 in material and methods (which I can replicate now)

We have updated the number accordingly.

S1 Table - as we are on Chromosome X - Chromosomes (2n) describes the data incorrectly, as there are 258 males and only 135 females - therefore suggest to write number of individuals

Thanks for the great suggestion. We have corrected the table accordingly.

S2 Table - highlight the three SNPs described in the main text - would prefer an Excel file over the PDF file here.

We have followed the suggestions and updated the manuscript accordingly.

Figure S3: can you please indicate the SNP rs3503318 in Figure 2 and S3?

We have indicated the SNP in both of the figures.

Figure 1b and S2: can you please specify the axis values on the legends for diversity and describe in the figure legend respectively?

The values have been already mentioned in the each of the color scale.

We hope that you will find everything in order.

Sincerely,

G. Chaubey (on behalf of co-authors)

13 Aug 2020

Most frequent South Asian haplotypes of ACE2 share identity by descent with East Eurasian populations

PONE-D-20-12219R2

Dear Dr. Chaubey,

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Kind regards,

Alessandro Achilli, Ph.D.

Academic Editor

PLOS ONE

19 Aug 2020

PONE-D-20-12219R2

Most frequent South Asian haplotypes of ACE2 share identity by descent with East Eurasian populations

Dear Dr. Chaubey:

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on behalf of

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Academic Editor

PLOS ONE