PONE-D-22-21304Population-specific positive selection on low CR1 expression in malaria-endemic regionsPLOS ONE

Dear Dr. Gusareva,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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ACADEMIC EDITOR:The reviewers have raised some concerns about the selection signals identified against malaria, and authors are suggested to clarify these concerns in detail.

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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: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

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.

Reviewer #1: Yes

Reviewer #2: Yes

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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: Yes

Reviewer #2: Yes

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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: The novelty of this manuscript is two fold as mentioned below

One is finding of NEW SNPs related to protection of malaria and the other, more important than the first one, is finding a population-specific manner of positive selection.

Similar result for the latter case is reported by an example of LCT persistence, which shows different SNPs are target of this selection between African and European. However, such a case of population specific selection is not commonly observed.

However, in this manuscript, there are several unclear points commented as follows.

I will recommend revision of this manuscript for its publication.

Major comments:

1) This manuscript described four SNPS, rs2274567, rs3811381, rs12034598, and rs3886100. But they did not mention the latter two SNPs are novel ones to protect people from malaria, when they appeared at the first time. Later, on line 221 they mentioned “two novel variants” are rs12034598 and rs3886100. If the authors wanted to emphasize this result, description of these two SNPs would be separated the previously known ones and the authors are recommended to make a new paragraph to describe this. The current description of theses alleles may lead confusion to readers.

2) TMRCA estimation (Adaptive haplotypes and their age): RELATE identified 17 haplotype blocks in the region. On line 180, “the haplotype trees show similar pattern of phylogeny, especially for the trees of the haplotype blocks containing the regions from exon2 to exon 33”. When I looked at the S7 Fig., based on the legend the trees are from intron 27 to exon 35. There is no clear correspondence of each tree with exons/introns. On line 196, “suggest LD throughout the region between the two exons” is not appropriately expressed. This should be “suggest strong LD (r2=0.89) throughout the region between the two SNPs (rs2274567 in exon 22, rs3811381 in exon 33)”.

Minor comments:

1) On page6 line 109-110: top-ranked values were selected for iHS: Significant signals for iHS appears either positive and negative. In this case the test should be both sided. If so, the top-ranked values should be 2.5% for each side. But in the present description, we can read 5% for each side because of the sentence of line 107-108. You should distinguish “one-side test” or “ both-side test”.

2) On page 7 line 124: to avoid inaccurate inference. The word of “inaccurate” is vague. Please rewrite this phrase by clear expression.

3) On page 7 line 128-129: “for 100 different individual sets of 120 randomly sampled genomes”. This sentence may be confusing for readers. What do the authors mean “100 different individual sets”? I guess that “100 (different) sets of 120 randomly sampled genomes” is clearer.

4) On page 8 line 155: “have the top 1.12% and 1.28% of the percentile rank in the whole genome”. I am not sure about “percentile rank of what” and “whose whole genome”.

5) On page 9 line 182-183: “defined by six SNPs that include rs2274567. Thus, we designated the two haplogroups as low(L) and high(H) expression level haplogroups”. If the authors use “Thus”, the authors should mention that rs2274567 is involved in the expression level of CR1.

6) On page 10 line 186: "the most frequent subnode". For me this expression reads somewhat strange. This phrase may be “the most frequent subclade”.

7) On page 10 line 192-193: "based on 100 repeated estimates of random sampling". First, Di this mean that this estimation based on 100 bootstrap resampling? Second, resampling is from the total samples or from only L or LS haplogroups.

8) On page 10 line 204: Do the authors mean “minimum selection constraints” as “relaxation of functional constraints” or “minimum selection coefficients”?

9) On page 12 line 238,239,241: The frequency of LS 75% to 100% does not seem to agree to 9.6 per 100,000 people (0.0096%). What is the base of this LS frequency? The same argument is for line 239 and 241.

Reviewer #2: Despite CR1 gene has been extensively studied to be in association with rosseting of red blood cells that subsequently cause microvascular obstruction and eventual severe/cerebral malaria, the complete spectrum of variations across the whole gene and its flanking regulatory regions are remained unknown. This work harnesses the power of next generation sequencing and has sequenced the complete CR1 gene and identify all variations from individuals residing in malaria-endemic and non-endemic regions. Further analyses of positive selection identified SNPs that could act as protective biomarkers against malaria infection / development toward severe/cerebral malaria.

A number of comments on this manuscript are as follow:

1. Previous studies always select a few SNPs of CR1 gene for testing its association to malaria infection. You pointed out that the association could be contrasting. Thus, this should be overcome. In addition, as a gene could have a few hundred variations across human populations, the power of sequencing and subsequent analyses should be harnessed to select candidate SNPs with strong protective effects. Please have an in-depth literature review over this and articulate the specific objective you wish to work out.

2. Do you specify that the force of selection is the lethal cerebral malaria or severe types of malaria (in exception to cerebral one). Some species might be more pathogenic and lethal e.g. P. falciparum infection commonly lead to cerebral malaria, attributed to different pathogenesis pathways. And the distribution of the different species could be different, e.g. P. knowlesi in ISEA. In addition, using malaria case numbers as a measure of endemicity and force of selection is an appropriate proxy, but unfortunately these case numbers do not specify which species of Plasmodium that caused which severe types of malaria. Judging on that, these could serve to drive the selection force differently. Looking at the high number of cases in the current days, I wonder has this force of selection yet been fixed, and thus the method of analysis should be revised? Please clarify these inter-related problems in detail.

3. Since you inferred that the positive selection already occurred before human migrating out of Africa, I suppose that majority of the extant human racial groups should carry the same polymorphic SNPs which are almost fix, in both endemic and non-endemic extant populations. But your data did not find so. What could be the reason?

4. Some samples are carrying more variations in the gene. How do you test and remove the mentioned ‘less admixed’ individuals? How do you affirm that the positively selected variants are not due to ancestry / anthropology, instead of natural selection?

5. Based on the GTEX paper published in 2020, a great majority of 85% of the dataset are of European American. Since European countries are not in the Malaria endemic region, and you also found that there is no signal of selection on CR1 gene among the European samples, you thereby used this public gene expression data (low CR1 expression) as your strong support that infers that this gene is also expressed low among the other multi-racial / ancestries individuals in Asia & Africa. Unless it is tested empirically by any measure of gene expression using RNA from your studies samples, this inference is invalid.

6. The outcome of this work is derived from bioinformatic analyses of individuals with unknown history of malaria infection, and thus the obtained genotypic and allelic frequencies should represent the general polymorphisms in each regional population. However, the conclusion can only be made after a well-designed case-control association test is conducted. As such, your previous work (Gusareva et al., 2021) should have already identified similar results as this current manuscript. I wonder why CR1 analysis was split out from the previous work?

7. The conclusion states that the current findings could be helpful in precision medicine of malaria medical management. Unfortunately, this bioinformatic findings have yet to be extensively tested but the authors already gave strong conclusive statements. I find this misleading and should be removed.

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Reviewer #1: No

Reviewer #2: No

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Population-specific positive selection on low CR1 expression in malaria-endemic regions

PONE-D-22-21304R1

Dear Dr. Gusareva,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Hoh Boon-Peng, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. 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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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4. 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: Yes

Reviewer #2: Yes

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5. 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: Yes

Reviewer #2: Yes

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6. 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: (No Response)

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

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