Dear Dr Millischer,

Thank you very much for submitting your Research Article entitled 'Proteome-wide Mendelian randomization identifies causal links between blood proteins and severe COVID-19' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.

Should you decide to revise the manuscript for further consideration here, your revisions should address the specific points made by each reviewer. We will also require a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

If you decide to revise the manuscript for further consideration at PLOS Genetics, please aim to resubmit within the next 60 days, unless it will take extra time to address the concerns of the reviewers, in which case we would appreciate an expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments are included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool.  PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, use the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.

Yours sincerely,

Chris Cotsapas, PhD

Associate Editor

PLOS Genetics

Scott Williams

Section Editor: Natural Variation

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: In this manuscript, Palmos, Millischer et al. describe a mendelian randomization study between ~4000 blood protein levels and a sever COVID-19 (incidence vs. population controls). Obviously this is a pressing are of research, although insights from host genetic analyses have arguably been limited to date.

Overall, the manuscript follows standard practices in the MR literature, the presentation was clear, and the results might be important. However, the mendelian randomization approach has serious limitations, and my comments are focused on how the authors might engage with those limitations. I don’t think it is necessary for every one of the suggested analyses to unambiguously support a causal relationship.

The most important limitation of MR is that pleiotropy, and in particular genetic correlations, lead to false positive “causal” relationships. This is extremely common, even with commonly-used sensitivity analyses (e.g. MR-Egger) (O’Connor and Price 2018, Verbanck et al 2018). Two recent methods are more robust (LCV and CAUSE, Morrison et al 2020), but I expect that both will be underpowered for these phenotypes. It is encouraging that the authors tested for a reverse causal effect and did not find one, but this is not unexpected given the phenotypes involved (and limited power in the COVID study). I have three suggestions to determine the level of confidence in the putative causal relationships:

1. Please show scatter plots for the effect size estimates of your instrumental variables on the protein level and on COVID risk, respectively. These are helpful to evaluate whether the correlation is uniform, with all variants affecting protein level also affecting risk, or whether it is driven by just one or two shared variants.

2. Please report the effect, if any, of variants that are in the cis region of the protein that they encode. Such variants make relatively strong instrumental variables, because their effects are unlikely to be mediate by a confounder in trans.

3. Please check if any of your instrumental variables are pleiotropically associated with BMI, which would be an obvious confounder. (It may affect both protein levels and severe COVID risk). Also, please check which of your results change if the HLA region (which has very long-range LD and unusually strong associations with numerous phenotypes) is excluded from the analysis.

I don’t think it is necessary that these pieces of evidence provide unambiguous evidence of a causal relationship for all, or even any, of the inferred relationships; it is appropriate to publish equivocal evidence when it is the best that is available, if it is communicated accurately.

I’ll just highlight one particular result as needing more attention and discussion: the ABO finding seems concordant with early reports of a blood-type association, but my impression was that recent evidence has shown this to be a false positive, possibly due to stratification. Is the ABO association supported by any variants outside of the disputed locus?

Reviewer #2: The authors seek to understand if protein levels are causally associated to severe COVID19 infection. They use the Mendelian Randomisation framework in which SNPs associated with protein levels are instrument variable used to test for the causal relationship between proteins as exposure traits and COVID-19 hospitalization as outcome. This is an interesting and potentially important question as such proteins could be targeted by drugs. The MR framework is a very cost-effective approach to address this question.

The analyses use published methods and results from published studies. The analyses are likely to be repeated over time as GWAS sample sizes become larger, but it makes sense to conduct the analyses now if suitable SNP instruments exist.

To be useful to the research community the study needs to clearly build on other studies and be reproducible.

Major comments

1. Poor choice of COVID GWAS

a) The primary phenotype is taken from the host genetic initiative release 5, which makes sense as these were presented in the Nature paper (reference 7) published earlier this year. From this paper, the authors have selected the GWAS results: “A2_ALL_leave_UKBB: Very severe respiratory confirmed covid (5,870) vs. population (1,155,203)” incorrectly calling it the European only sample. I agree it makes sense to use the European sample, they should have selected: “A2_ALL_eur: Very severe respiratory confirmed covid (5,101) vs. population (1,383,241)”

b) The authors have chosen to label this phenotype “hospitalized-COVID-19 GWAS” even though Ganna et al called this phenotype “critically ill COVID-19+” ; Ganna et al used the name “Hospitalised COVID” for a less severe phenotype 13K case 2M controls (or just Europeans B2_ALL_eur: (9,986) vs. population (1,877,672)). I don’t think it is helpful to mix up names in this way. No doubt the reason for this was because the authors tried to select a what they thought was a more phenotype selecting from release 4, “Very severe respiratory confirmed covid vs. not hospitalized covid” 269 cases vs 688 controls (A1_ALL). It is not clear why this sample was chosen it was very small, has no GWS SNPs, and while I don’t think this analysis is needed (the very large population control makes more sense) release 5 has much bigger samples for this phenotype 4,829 and 11,816 (B1_ALL_eur).

Summary: Use only A2_ALL_eur release 5 (or 6 if now available).

2. Non-reproducibility of protein GWAS.

From what is written or available in the supplement I am not sure if others could repeat the analysis.

a) Supplementary Table 1 lists 10 studies but five of these use the OLINK panel – were these meta-analysed?

b) “We tested 4,366 associations with hospitalized-COVID-19 as the exposure and the blood proteins as outcome”. So is 4,366 the number of proteins or the number of SNPs? Suggest you provide a supplementary data file listing the SNPs used as instruments for each protein.

Minor comments:

1) Referring to the differences in GWAS results severe vs hospitalised a statement is made “The findings suggest that one set of individual genetic variants may be responsible for hospitalization, and once hospitalized, another set of genetic variants may be responsible for respiratory failure and death.” This statement and elsewhere provides a feeling that the researchers have not fully engaged with the severe-COVID19 phenotype. This statement makes these findings sound novel, but this difference is well known clinically. For example, the GENOMICC GWAS (Pairo-Castineira et al) said “In the UK, the group of patients admitted to critical care is relatively homogeneous, with profound hypoxaemic respiratory failure being the archetypal presentation “ and “Patients admitted to intensive care units in the UK during the first wave of COVID-19 were, on average, younger and less burdened by comorbid illnesses than the hospitalized population” – citing Docherty et al.

2) What are the units of the Beta and odds ratios. Are the units the same for all analyses?

3) gwasrapidd not gwasrapid

4) Statements such as “the genetic liability for exposure X is causal for outcome Y” are made on several occasions. Tthese statements are commonly found in MR publications but in my opinion miss the point about MR. The point is that using genetic instruments and under the assumptions of MR results are consistent with exposure X being causal for outcome Y. There is a subtle difference in meaning here

5) In this study, the 1,000 Genomes dataset was the reference dataset (18). Do you mean the European ancestry samples to match the EUR ancestry GWAS?

6) To get sufficient SNP instruments to conduct MR, the significance threshold was set at p < 5 x 10-6. This decision is justified through reference to 5 papers Ref 26-30, which includes one paper by the authors, and three rather old papers. Just because other papers have passed through peer review it does not justify the approach, this must be justified by a methodological paper. Generally, I believe use of weak instruments is not supported.

7) Given that the incorrect GWAS was selected I haven’t focussed on the results.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: No: See major point 2. Suggest that a supplementary data file listing the SNPs used as instruments for each protein is provided.

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Luke O'Connor

Reviewer #2: No

Dear Dr Millischer,

Thank you very much for submitting your Research Article entitled 'Proteome-wide Mendelian randomization identifies causal links between blood proteins and severe COVID-19' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated your responses to their previous comments, but identified some minor remaining concerns that we ask you address in a revised manuscript. In particular, reviewer 1 had a handful of remaining comments. We also want to draw your attention to reviewer 2's initial minor comment 4, about the language of causality. There are some remaining references to causality of genetic liability of an exposure, rather than the exposure itself, in the abstract, and on pp 25, 31 etc. We ask that you correct these in your revisions.

We therefore ask you to modify the manuscript according to the review recommendations. Your revisions should address the specific points made by each reviewer.

In addition we ask that you:

1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

2) Upload a Striking Image with a corresponding caption to accompany your manuscript if one is available (either a new image or an existing one from within your manuscript). If this image is judged to be suitable, it may be featured on our website. Images should ideally be high resolution, eye-catching, single panel square images. For examples, please browse our archive. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License. Note: we cannot publish copyrighted images.

We hope to receive your revised manuscript within the next 30 days. If you anticipate any delay in its return, we would ask you to let us know the expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments should be included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

Please let us know if you have any questions while making these revisions.

Yours sincerely,

Chris Cotsapas, PhD

Associate Editor

PLOS Genetics

Scott Williams

Section Editor: Natural Variation

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors have performed analyses to address my comments, and I have only minor follow up comments remaining:

1. In Supplementary Figures 1-4, the authors show scatterplots for their significant associations. Most of these look quite good, better than a lot of MR analyses, indicating that most protein-associated SNPs have proportional effects on hospitalization risk. For example slCAM1, which also passed the cis-variant sensitivity test, looks like it is supported by a large number of instruments with consistent effects. In contrast the ABO plot looks fairly poor, with many ABO-associated SNPs having discordant effects on the outcome. I think these results merit some discussion in the main text. Also, please fix the figure labels in Supp Fig 1, some of which are cut off.

2. The BMI analyses do seem to indicate that confounding due to BMI is potentially quite important. Please add some discussion about whether the direction of effect that you observed was consistent with potential confounding – i.e., when BMI-associated variants are associated with increased protein levels, are those the same proteins for which increased protein-increasing variants are associated with increased COVID risk?

3. Regarding the cis analysis, you might report that the total number of proteins that had testable cis variants (which was smaller than I expected, if I understand correctly); initially I got the impression that most proteins failed this sensitivity analyses, when it was actually not applicable. Please add a caption to Supplementary Table 6, and cite it in the text as a Table not as a Note.

Reviewer #2: The authors have addressed my comments, except Point 4, I leave in the hands of the editor.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Luke J O'Connor

Reviewer #2: No

Dear Dr Millischer,

We are pleased to inform you that your manuscript entitled "Proteome-wide Mendelian randomization identifies causal links between blood proteins and severe COVID-19" has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional acceptance, but your manuscript will not be scheduled for publication until the required changes have been made.

Once your paper is formally accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you’ve already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosgenetics@plos.org.

In the meantime, please log into Editorial Manager at https://www.editorialmanager.com/pgenetics/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production and billing process. Note that PLOS requires an ORCID iD for all corresponding authors. Therefore, please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field.  This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

If you have a press-related query, or would like to know about making your underlying data available (as you will be aware, this is required for publication), please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Chris Cotsapas, PhD

Associate Editor

PLOS Genetics

Scott Williams

Section Editor: Natural Variation

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

----------------------------------------------------

Comments from the reviewers (if applicable):

----------------------------------------------------

Data Deposition

If you have submitted a Research Article or Front Matter that has associated data that are not suitable for deposition in a subject-specific public repository (such as GenBank or ArrayExpress), one way to make that data available is to deposit it in the Dryad Digital Repository. As you may recall, we ask all authors to agree to make data available; this is one way to achieve that. A full list of recommended repositories can be found on our website.

The following link will take you to the Dryad record for your article, so you won't have to re‐enter its bibliographic information, and can upload your files directly: 

http://datadryad.org/submit?journalID=pgenetics&manu=PGENETICS-D-21-01126R2

More information about depositing data in Dryad is available at http://www.datadryad.org/depositing. If you experience any difficulties in submitting your data, please contact help@datadryad.org for support.

Additionally, please be aware that our data availability policy requires that all numerical data underlying display items are included with the submission, and you will need to provide this before we can formally accept your manuscript, if not already present.

----------------------------------------------------

Press Queries

If you or your institution will be preparing press materials for this manuscript, or if you need to know your paper's publication date for media purposes, please inform the journal staff as soon as possible so that your submission can be scheduled accordingly. Your manuscript will remain under a strict press embargo until the publication date and time. This means an early version of your manuscript will not be published ahead of your final version. PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via plosgenetics@plos.org.