Sex-Specific HLA Alleles Contribute to the Modulation of COVID-19 Severity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript “Sex specific HLA alleles contribute to the modulation of Covid- 2 19 severity” investigates the association between Covid-19 severity (severe vs. asympto- 30 matic/oligosymptomatic healed individuals) and HLA gene variants, analyzed by next-generation 31 sequencing.The results have reference value, but there are many problems in the writing and the presentation of the results.

1. The information in the abstract is not specific enough, and detail information about important results should be listed.

2. Some directly related articles were not referenced in the article, such as PMID38251811.

3. Males in cases group is 67.55%, high than 42.47% in control.

4. In line 97-97, the content in this section does not match the sub-title. The results did not mention the impact of gender differences.

5. In line 101-102, there is no result of HLA-F, HLA-DQA 1, HLA-DRB5 in table 2. In “Whole population” column, why marker A and DPA1 lines? In “Females” column, why marker DPB1 line?

6. In line 120-121, why there are two different titles for table 3? What does bold font in Table 3 represent?

7. Did the author analyze the impact of gender on patients with same age?

8. In table 5, “n.210” change to “n=210”.

9. In line 120-121, inappropriate expression for “our data provide a first evi- 254 dence of biological sex specific differences in disease susceptibility related to HLA genes 255 in humans. ”. There have been multiple articles discussing and analyzing this point.

Comments on the Quality of English Language

Language is acceptable. But the writing of the results section needs to be improved.

Author Response

”Sex specific HLA alleles contribute to the modulation of COVID-19 severity”.

 

 

Response to Reviewer 1 Comments
1. Summary
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.
2. Questions for General Evaluation	Reviewer’s Evaluation	Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	Can be improved	Introduction has been extended and some references added
Are all the cited references relevant to the research?	Yes
Is the research design appropriate?	Yes
Are the methods adequately described?	Can be improved	Methods have been extended and further detailed
Are the results clearly presented?	Must be improved	Results have been extended, pointing on comments on tables
Are the conclusions supported by the results?	Can be improved	The discussion has been deeply revised and rephrased
3. Point-by-point response to Comments and Suggestions for Authors
Comments 1: The information in the abstract is not specific enough, and detail information about important results should be listed.
Response 1: following the suggestions of the reviewer, the abstract has been rephrased
Comments 2: Some directly related articles were not referenced in the article, such as PMID38251811.
Response 2: Thank you very much for the suggestion. We missed this reference that has been now included in the manuscript.   Comments 3: Males in cases group is 67.55%, high than 42.47% in control. Response 3: Thank you very much for the comment. We are well aware that this is one of the limitations of our study, as pointed in the Discussion section. Unfortunately, due to the progression of the pandemics (as stated in the manuscript) we could not balance the number of individuals in the two cohorts. For this reason, biological sex was included as one of the co-variates in the comparison of patients and controls.   Comments 4: In line 97-97, the content in this section does not match the sub-title. The results did not mention the impact of gender differences Response 4: We thank very much the Reviewer for the comment. The title of the paragraph has been simplified by mentioning only the name of the HLA genes associated with the phenotype. Part of the text, reporting the sex differences, was deleted by mistake and now restored. The last sentence of the paragraph was a repetition of what indicated in the title and has been deleted; additionally, some genes were named inappropriately, due to a typo. The correct data were those indicated in Table 2. The HLA-C*15 allele is protective in males: the corresponding table cells were shadowed in gray as significant. As reported in the methods, we considered as significant associations with a p-value<0.05.      Comments 5: In line 101-102, there is no result of HLA-F, HLA-DQA 1, HLA-DRB5 in table 2. In “Whole population” column, why marker A and DPA1 lines? In “Females” column, why marker DPB1 line? Response 5: We apologize for the several mistakes of the paragraph that have been edited. See Response to Comment 4.   Comments 6: In line 120-121, why there are two different titles for table 3? What does bold font in Table 3 represent? Response 6: We apologize for the mistake. The title of Table 3 was slightly modified and the table legend extended and detailed to make the interpretation more clear to the readers.    Comments 7: Did the author analyze the impact of gender on patients with same age? Response 7: Due to the sample size, it was not possible to perform a group analysis, stratifying the cohorts by age or age-range. The subgroups would have been too small to get significant results. For this reason, we performed a multinomial logistic regression analysis, including age and sex as co-variates. In table 3, we indicated in bold the HLA-SNPs that, following the multinomial approach remained significant. These aspects were detailed also in the text.     Comments 8: In table 5, “n.210” change to “n=210”. Response 8: Thank you, we have modified the text accordingly.   Comments 9: In line 120-121, inappropriate expression for “our data provide a first evidence of biological sex specific differences in disease susceptibility related to HLA genes in humans”. There have been multiple articles discussing and analyzing this point. Response 9: In spite of an intensive research in Pubmed and publicly available databases, we could not find studies specifically analysing the frequency of risk HLA alleles in males and females, beside the very well-known different prevalence of many diseases in the two biological sexes. We would be very grateful to the reviewer if they could provide us with such references that we would be very happy to include in our manuscript. However, also based on the suggestion of Reviewer 3, we have rephrased the sentence as follows:” From the immunology point of view, we provide here evidence of biological sex specific differences in disease susceptibility related to HLA genes in Humans. It is well known that the immune response is different in males and females, and that there are clear differences in the epidemiology of some infectious and autoimmune diseases.[18] Some of the differences in immune response have been very recently attributed to the effect of sex-hormones, namely androgens.[19] However, we could not find in the Literature studies including the differential analysis of HLA genotype according to biological sex. Similarly, we could not find raw data of published association studies on autoimmune or other HLA-related diseases, including sex-specific allelic frequencies, to perform meta-analyses. It could be of extreme interest to assess whether our findings are replicated in other disease models, beyond COVID-19, since this could shed some additional hints on the functional diversity in the immune system function between males and females”.
4. Response to Comments on the Quality of English Language
Point 1: Language is acceptable. But the writing of the results section needs to be improved.
Response 1: Thank you, we have modified the text accordingly.

 

 

 

 

 

 

 

 

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks for the opportunity to review the manuscript by Serena Spartano and Cols. 

 

The authors aimed to investigate the association between COVID-19 severity and HLA alleles analyzed by NGS. They identified alleles, SNPs, and haplotypes associated with COVID-19 severity related to biological sexes. Also, they identified specific haplotypes ("super-haplotypes") shared by different HLA alleles that, they argue, have a sex-specific impact on COVID-19 risk.

The research was impressive: methodological, hands-on, and bioinformatics analyses are well-designed and described adequately.

I have some comments that I hope the authors find helpful.

Throughout the entire manuscript, please correct Covid-19 by COVID-19.

Supplemental Figure 1 is really depicting, I recommend include in the main manuscript file.

In line 112, you stated: We prioritized coding and intronic variants that could affect exon splicing: 28 variants. Was this independent of the allele frequency of the selected variants? Please comment on the text and its meaning and importance, if any.

In Table 4, better than gDNA ID, include the "rs" identifier in Table 4.

In different sections, you propose that your study has been conceived as retrospective with prospective recruitment. So, could it be appropriate to mention it as prolective? Please consider it.

In the discussion section, move the limitations paragraph to the end of the section.

A couple of lines explaining the meaning of cis— / trans— variants would be helpful for non-expert readers. Please add it.

In line 303, include the rs ID for the SNP in TBC1D4.

The conclusion paragraph, "In conclusion, our study provides some hints that will likely open new scenarios in understanding HLA-mediated immune modulation in response to environmental triggers, beyond the SARS-CoV2 pandemics." should be:

a) Separated from the rest of the remaining paragraph, and

b) Reformulated, these lines can be part of the conclusion as perspectives but are not your conclusions. This should include the theoretical (biological, clinical, evolutionary, etc.) interpretation of your main results, and maybe later "...provides some hints that will likely open new scenarios in understanding HLA-mediated immune modulation in response to environmental".

 

Author Response

”Sex specific HLA alleles contribute to the modulation of COVID-19 severity”.

 

 

Response to Reviewer 2 Comments
1. Summary
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.
2. Questions for General Evaluation	Reviewer’s Evaluation	Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	Yes
Are all the cited references relevant to the research?	Yes
Is the research design appropriate?	Yes
Are the methods adequately described?	Yes	Methods have been extended and further detailed
Are the results clearly presented?	Can be improved	Results have been extended, pointing on comments on tables
Are the conclusions supported by the results?	Can be improved	The discussion has been deeply revised and rephrased
3. Point-by-point response to Comments and Suggestions for Authors
Comments 1: Throughout the entire manuscript, please correct COVID-19 by COVID-19.
Response 1: Thank you, we have modified the text accordingly.
Comments 2: Supplemental Figure 1 is really depicting, I recommend include in the main manuscript file.
Response 2: We appreciate the comment of the reviewer. The figure has been moved to the main text. Comments 3: In line 112, you stated: We prioritized coding and intronic variants that could affect exon splicing: 28 variants. Was this independent of the allele frequency of the selected variants? Please comment on the text and its meaning and importance, if any. Response 3: we agree with the reviewer that selecting variants based on the allelic frequency could have introduced a bias for the identification of relevant SNPs. We modified the text as follows: “Among the significant variants, we prioritized only those in the coding region and the intronic that could affect exon splicing, irrespective of the allelic frequency, thus we did not focus the analysis on rare variants only”.   Comments 4: In Table 4, better than gDNA ID, include the "rs" identifier in Table 4. Response 4: Table 4 was modified according to the suggestion. Since for not all variants rsID was available, we kept both, indicating the rsID in parentheses     Comments 5: In different sections, you propose that your study has been conceived as retrospective with prospective recruitment. So, could it be appropriate to mention it as prolective? Please consider it. Response 5: We are very greatful to the reviewer for the suggestion. Indeed we were not even aware of the existence of the term “prolective”. We evaluated the opportunity to replace “prospective” with “prolective” but, as far as we could understand from the literature, “prolective” is not a commonly used definition and, apparently, refers more to studies with a prospective follow-up, rather than to the design of our study. We thus felt more appropriate and less confounding for the general readership to keep the original definition.   Comments 6: In the discussion section, move the limitations paragraph to the end of the section. Response 6: The text was modified according to the reviewer’s suggestion.     Comments 7: A couple of lines explaining the meaning of cis— / trans— variants would be helpful for non-expert readers. Please add it. Response 7: The text was modified according to the reviewer’s suggestion.   Comments 8: In line 303, include the rs ID for the SNP in TBC1D4.   Response 8: The text was modified according to the reviewer’s suggestion Comments 9: The conclusion paragraph, "In conclusion, our study provides some hints that will likely open new scenarios in understanding HLA-mediated immune modulation in response to environmental triggers, beyond the SARS-CoV2 pandemics." should be:   a) Separated from the rest of the remaining paragraph, and   b) Reformulated, these lines can be part of the conclusion as perspectives but are not your conclusions. This should include the theoretical (biological, clinical, evolutionary, etc.) interpretation of your main results, and maybe later "...provides some hints that will likely open new scenarios in understanding HLA-mediated immune modulation in response to environmental". Response 9:  thank you very much for suggestion. The Discussion paragraph has been re-organised according to the suggestions.

 

 

 

 

 

 

 

 

 

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

The work presented by Spartano et al is very well structured and interesting. However, I have a few minor comments for the authors.

Major:

1)      Line 139: Table3D is missing. Is this a typo and do you mean table 3?

2)      I would prefer the whole ‘materials and methods’ section to be written in more detail.

Minor:

1)      The text must be organised according to the journal guidelines.

2)      Covid-19 should be standardised throughout the text. I would ask the authors to write it in all caps (COVID-19).

3)      Line 38: write ‘immuno-responses’, the hyphen improves reading.

4)      Lines 49-50: write SARS-CoV-2, not write SARS-CoV2.

5)      All tables appear with two descriptions. Please standardise according to the style of the paper.

6)      Line 117: OR and IC acronyms appear without first describing what they are. They also appear in the tables. These acronyms, however well known, should also be explained in the notes of the tables.

7)      Line 145: the word ‘haplotypes’ already appears previously. Fix the position of the acronym.

8)      Lines 165-166 and 181: extra spaces.

9)      Lines 254-256: writing in an article that it is the first evidence is not correct even though it actually is. Please reword the sentence.

10)   Line 344: a space is missing between ‘>2.The’.

11)   Line 388: the acronym OR has already been used (see comment 6 minor).

Extra:

1)      Lines 187-197: The topic is very interesting. I look forward to future insights from you.

Author Response

 ”Sex specific HLA alleles contribute to the modulation of COVID-19 severity”.

 

 

Response to Reviewer 3 Comments
1. Summary
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.
2. Questions for General Evaluation	Reviewer’s Evaluation	Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	Yes
Are all the cited references relevant to the research?	Yes
Is the research design appropriate?	Yes
Are the methods adequately described?	Must be improved	Methods have been extended and further detailed
Are the results clearly presented?	Yes	Results have been extended, pointing on comments on tables
Are the conclusions supported by the results?	Yes	The discussion has been deeply revised and rephrased
3. Point-by-point response to Comments and Suggestions for Authors MAJOR:
Comments 1: Line 139: Table3D is missing. Is this a typo and do you mean table 3?
Response 1: We apologize for the typo. Table 2 was inappropriately indicated as Table 3D
Comments 2: I would prefer the whole ‘materials and methods’ section to be written in more detail.
The text was extended and further detailed as requested.   MINOR:   Comments 1: The text must be organised according to the journal guidelines. Response 1: we apologize for misunderstanding. The manuscript should now fit the Journal’s guidelines, especially for table formatting and legends   Comments 2: COVID-19 should be standardised throughout the text. I would ask the authors to write it in all caps (COVID-19). Response 2: Thank you, we have modified the text accordingly.   Comments 3: Line 38: write ‘immuno-responses’, the hyphen improves reading. Response 3: Thank you, we have modified the text accordingly.   Comments 4: Lines 49-50: write SARS-CoV-2, not write SARS-CoV2. Response 4: Thank you, we have modified the text accordingly.     Comments 5: All tables appear with two descriptions. Please standardise according to the style of the paper. Response 5: The mistake has been edited, we apologize.   Comments 6:  Line 117: OR and IC acronyms appear without first describing what they are. They also appear in the tables. These acronyms, however well known, should also be explained in the notes of the tables. Response 6: Thank you, we have modified the text accordingly.   Comments 7: Line 145: the word ‘haplotypes’ already appears previously. Fix the position of the acronym. Response 7: we have removed the acronym “hap”, since finally non-functional to the reading of the text. We thank the reviewer for the comment   Comments 8: Lines 165-166 and 181: extra spaces. Response 8: Thank you, we have modified the text accordingly.   Comments 9: Lines 254-256: writing in an article that it is the first evidence is not correct even though it actually is. Please reword the sentence. Response 9: the sentence has been rephrased as follows: “From the immunology point of view, we provide here evidence of biological sex specific differences in disease susceptibility related to HLA genes in Humans. It is well known that the immune response is different in males and females, and that there are clear differences in the epidemiology of some infectious and autoimmune diseases.[18] Some of the differences in immune response have been very recently attributed to the effect of sex-hormones, namely androgens.[19] However, we could not find in the Literature studies including the differential analysis of HLA genotype according to biological sex. Similarly, we could not find raw data of published association studies on autoimmune or other HLA-related diseases, including sex-specific allelic frequencies, to perform meta-analyses. It could be of extreme interest to assess whether our findings are replicated in other disease models, beyond COVID-19, since this could shed some additional hints on the functional diversity in the immune system function between males and females”.       Comment 10: Line 344: a space is missing between ‘>2.The’. Response 10: Thank you, we have modified the text accordingly. Comment 11: Line 388: the acronym OR has already been used (see comment 6 minor). Response 11: Thank you, we have modified the text accordingly. Extra: 1)      Lines 187-197: The topic is very interesting. I look forward to future insights from you. Response: We really appreciate the Reviewer’s comment. We are seeking for the collaboration of our colleagues from the immunology department of our Institution to conceive a collaborative project on this matter and to get the adequate fundings. In this view, in our opinion, it will be crucial 1) to identify the more appropriate disease model (studying homogenous cohorts will be of key relevance) and 2) to obtain preliminary information on the physiologic variability of the alternative splicing of HLA genes. We can postulate that different variables may impact on the dynamic regulation of HLA expression, including the genotype but also the possible (likely?) variability among the different leukocyte sub-populations, and/or the immune-response activation status. HLA-focused transcriptomic analyses by NGS in different white cell sub-populations would provide important preliminary datasets that, to our knolwedge, are not available.

 

 

 

 

 

 

 

 

 

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks for attending to my previous concerns.

Reviewer 3 Report

Comments and Suggestions for Authors

The article is greatly improved and can be accepted.