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PONE-D-21-26920

Effect of PCSK9 inhibition on lipoprotein subfractions in familial dysbetalipoproteinemia (type III hyperlipidemia)

PLOS ONE

Dear Dr. Parhofer,

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

Please submit your revised manuscript by Jan 02 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Laura Calabresi

Academic Editor

PLOS ONE

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“I have read the journal's policy and the authors of this manuscript have the following competing interests: KGP has received research funding and/or honoraria for consultancy and/or speaker’s bureau and/or DMC activity from:

Akcea, Amarin, Amgen, Berlin-Chemie, Biomarin, Boehringer-Ingelheim, Dr. Schär, Daiichi-Sankyo, MSD, Novartis, Regeneron, Sanofi, and Silence Therapeutics”

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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

Reviewer #3: Partly

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

Reviewer #1: I Don't Know

Reviewer #2: N/A

Reviewer #3: No

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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

Reviewer #3: No

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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

Reviewer #3: 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: Although I believe the study is interesting, it contains some issues that need to be sorted out.

The introduction misses the part relative to PCSK9, e.g., the role of PCSK9 on the expression of LDL-R, and the general impact of PCSK9 inhibition in terms of LDL-C lowering and atherosclerotic cardiovascualr disease (ASCVD) risk reduction. Concerning the biology of PCSK9, the Authors can refer to a recent review (PMID: 34019847). A similar comment is valid in the case of lipoprotein(a). Relative to this, the discussion lacks to mention the impact of PCSK9 inhibition on lipoprotein(a). Moreover, lipoprotein(a) levels seem to be very high and in the hypercholesterolemic group, they overtake the ASCVD risk threshold of 50 mg/dL. The Authors should evaluate the contribution of cholesterol carried by lipoprotein(a), and calculate, in addition, the corrected LDL-C (PMID: 34450317). Considering that, for lipoprotein(a), conversion between mg/dL and mmol/L is not advisable, cholesterol should be expressed both in mmol/L and mg/dL. Please report the assay that has been used to measure lipoprotein(a).

Relative to ultracentrifugation, is it correct “48 hours at 158°C?”

Which statistical test has been used to evaluate differneces? My apology if I missed it.

The impact of PCSK9 on triglycerides should be discussed in more details. Please refer to the review by Dijk W et al (PMID: 29665987) or Baragetti et al (PMID: 29428206).

Which is the percentage of recurrence of FDBL? This could be reported both in the introduction and in the limitation section. Please report that it is a single centre study. The title should report that data have been obtained with evolocumab.

Reviewer #2: In this study, the Authors investigated the impact of PCSK9 inhibition on lipoproteins in patients with FDBL. A comparison with patients with LDL-hypercholesterolemia and mixed hyperlipidemia has been made. PCSK9 inhibitors reduced cholesterol and apoB in all patients. In FDBL they specifically reduced VLDL-cholesterol. The Authors concluded that PCSK9-inhibition reduces the concentration of a wide variety of apoB-containing lipoproteins with the dominant particle size being reduced the most.

The study is original and interesting, however the paper is only descriptive, since sample size is too small and statistical analysis could not be performed.

This reviewer is aware of the rareness of familial dysbetalipoproteinemia, however three patients are not so much to draw robust conclusions, mostly because of the huge variability in lipid and lipoprotein profile.

Thus, more efforts should be dedicated to enroll additional subjects and increase sample size.

Other comments:

Tables: how are data reported? Please provide whether the number are mean ± SD or median ± SEM.

Table 1: which is the prevalence of metabolic syndrome in these patients.

Figure 2 is confusing. I would suggest using 3 different panels.

Non si reduce solo il picco più alto. Si riduce tutto in proporzione

State which inhibitor has been used also in method section, since it is reported only in the abstract.

Reviewer #3: The study is interesting but some consideration has to be done.

Please consider revising the description of the disease.

Aim. From your introduction, I do not understand what you want to test by comparing these population. Which differences do you expect? Why this study is relevant?

LDL-hypercholesterolemia, I imagine that these patients are heterozygous FH. Please use the correct terminology.

I do not understand the population selection:

1) Please do not give in methods any information on the demographic or clinical characteristics of these patients. These are reported in Table 1.

2)Did you genotype your patients? Both ApoE2/E2 patients and HeFH have already received a molecular confirmation? If yes, was it done for scientific purpose for this study or for clinical reasons?

3)Nevertheless, how did you choose patients with mixed dyslipidemia and HeFH to be compared with FDBL. Did you take all your patients taking PCKS9i?

“All had the indication for treatment with PCSK9-inhibitors according to German

regulatory standards (not reaching treatment goals recommended by the European guidelines despite

maximally tolerated oral lipid-lowering therapy)” please give a reference otherwise it is difficult to understand patient’s characteristics.

Where PCKS9i given in a study protocol? I do not understand the study design. I guess if it is an observational real-world study or an interventional trial.

As you assess only lipoprotein fraction at baseline and 3 months a suggest you consider in your discussion that your results might be in “acute” phase of treatment.

Add significance to Table 1 (BMI is significantly different between groups)

Please define CVD, CAD and PVD. Did you consider only acute events or all the events?

Please also give statistics and the methodology used to analyze the data (reporting it in a separate chapter.

Inferential statistics is completely lacking. You should add it throughout the manuscript (including Table 2 and figures)

Moreover, there was any changes in the background lipid lowering therapies in the 3 months of treatment? Please add lipid lowering therapies to table 2.

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6. 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: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-21-26920R1Effect of PCSK9 inhibition with evolocumab on lipoprotein subfractions in familial dysbetalipoproteinemia (type III hyperlipidemia)PLOS ONE

Dear Dr. Parhofer,

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.

Authors must solve issues raised by Reviewer #3, who I agree with.

Please submit your revised manuscript by Mar 04 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Laura Calabresi

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

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: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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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: (No Response)

Reviewer #2: Partly

Reviewer #3: Partly

**********

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

Reviewer #1: (No Response)

Reviewer #2: N/A

Reviewer #3: N/A

**********

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: (No Response)

Reviewer #2: (No Response)

Reviewer #3: 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: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

**********

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: Almost all comments have been addressed. Sample size remains a major issue that cannot be resolved.

Minor comments:

Please revise abbreviations and typos (e.g. Celcius, PCSK-9 or PCSK9)

Reviewer #3: Dear author my major concearn is the lack of an accurate definition of the patients included in the study.

In fact, I am awared that familial dysbetalipoproteinemia is rare but for this reason, patients should be very well characterized. Similarly, the "control" population lack of definition as for example, you have included patients with FH with those suffering with other form of hypercholesterolemia in a unique group. I believe that these could be potentially confounders. I suggest publishing this data as a short communication rather than as an original article.

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7. 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: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Effect of PCSK9 inhibition with evolocumab on lipoprotein subfractions in familial dysbetalipoproteinemia (type III hyperlipidemia)

PONE-D-21-26920R2

Dear Dr. Parhofer,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Laura Calabresi

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 #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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 #2: Partly

Reviewer #3: Yes

**********

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

Reviewer #2: N/A

Reviewer #3: Yes

**********

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 #2: Yes

Reviewer #3: Yes

**********

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 #2: Yes

Reviewer #3: Yes

**********

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 #2: (No Response)

Reviewer #3: Thank you for your replay and having added further information on patients. I still believe that molecular diagnosis could have added some further interesting to this observation.

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7. 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 #2: No

Reviewer #3: No