Causal effect of porphyria biomarkers on alcohol-related hepatocellular carcinoma through Mendelian Randomization

Xiaoyu Yang; Shuomin Wang; Chen Sun; Yunhong Xia

doi:10.1371/journal.pone.0299536

Peer Review History

Original SubmissionNovember 10, 2023
15 Jan 2024 Decision Letter - Ashwani Singal, Editor PONE-D-23-37115Casual effect of porphyria biomarkers on alcohol-related hepatocellular carcinoma through Mendelian RandomizationPLOS ONE Dear Dr. Xia, 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. Please submit your revised manuscript by Feb 29 2024 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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Thank you for stating the following financial disclosure: "This study was funded by the National Natural Science Foundation of China (No. 81472331), Natural Science Foundation of Anhui Province (No. 2108085MH289), and the Project of Scientific Research Foundation of Anhui Medical University (No. 2019xkj146)." Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 4. We note that your Data Availability Statement is currently as follows: [All relevant data are within the manuscript and its Supporting Information files.] Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition). For example, authors should submit the following data: - The values behind the means, standard deviations and other measures reported; - The values used to build graphs; - The points extracted from images for analysis. Authors do not need to submit their entire data set if only a portion of the data was used in the reported study. If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access. [Note: HTML markup is below. Please do not edit.] 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: I Don't Know 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: Yes 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: Yes 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: 1. Novelty: this study fulfils criteria for novelty. 2. Biological plausibility: The acute hepatic porphyrias are associated with a risk of primary liver cancer especially among older patients of 50 years with active porphyria. Previous studies (ref 14 & 15) have shown that risk of primary liver cancer is increased in symptomatic patients with Acute Intermittent Porphyria (AIP) due to accumulation of porphyrin precursors. 3. Methodology: The authors have used a novel technique called Mendelian Randomization-a research method that provides evidence about putative causal relations between modifiable risk factors and disease, using genetic variants as natural experiment. This methodology is less likely to be affected by biases such as confounding or reverse causation. 4. Conclusion: Conclusion is misleading as nowhere in the manuscript there is description of cases with alcohol-related hepatocellular carcinoma (AR-HCC). comments: 5. Spelling correction for manuscript title on line 4. It should be “Causal” instead of Casual. 6. Spelling correction on line 26 “porphyria” 7. The authors should remove statement in lines 83-85 of the introduction section, which implies that they have already concluded their hypothesis even before presenting the data. 8. Increased risk of primary liver cancer in AIP and other hepatic porphyrias is well-documented, however it is unclear if congenital erythropoietic porphyria (CEP), an even rare form of cutaneous porphyria, is associated with primary liver cancer. What prompted the authors to look for UROS SNPs in CEP cases? 9. Mechanism of liver injury and progression to AR-HCC in porphyrias was briefly mentioned, it may be worthwhile to expand on it. 10. The authors write that “the causal effect of PBGD and UROS on AR-HCC were confirmed using MR”, as the title also suggests. However, nowhere the causes of HCC are described. If anything, this study establishes a causal associated with HCC but not specifically AR-HCC. 11. Previous studies reported increased urinary PBD in symptomatic patients with AIP and incident cases of AR-HCC. It is unclear from this study whether GWAS cases were symptomatic or had AR-HCC. If that is case, it needs to be stated clearly. 12. Also unclear is why there is increased risk of AR-HCC as opposed to other causes of HCC. This needs to be explained in introduction. Reviewer #2: In this study, Dr. Xia et al. investigated causal relationships between single-nucleotide polymorphisms (SNPs) associated with porphobilinogen deaminase (PBGD) and uroporphyrinogen-III synthase (UROS), and alcohol related HCC (ARHCC) using the public genome-wide association studies (GWAS). The results found that both PBGD (effect estimate = 1.51; 95% CI, from 1.08 to 2.11, p = 0.016) and UROS (effect estimate = 1.53; 95% CI, from 1.08 to 2.18, p = 0.018) have a significant causal effect on AR-HCC. The authors concluded that both AIP and CEP have a causal association with AR-HCC1. The study investigates a fairly novel concept and technqiue, but there are several limitations which need to be addressed. These are: Abstract should clarify how many HCC and non-HCC cases extracted the data on SNP for the two genes of heme metabolism. The authors should clarify what formed their basis for the porphyria genes f0r their association with AR-HCC. This should be clarified in the introduction of the manuscript and purpose of the abstract section. It should be clarified whether any of these cases also had porphyria (biochemical and/or symptomatic). Further, the manuscript should be read by an expert in English language for syntax and grammatical errors. ****** 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: Yes: Ashwani K. Singal ******** [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. Attachments Attachment Submitted filename: Reviewer comments.docx https://doi.org/10.1371/journal.pone.0299536.r001
Revision 1
9 Feb 2024 Author Response Dear Dr Singal, Plos One Thank you very much for giving us an opportunity to resubmit the enclosed manuscript entitled “Casual effect of porphyria biomarkers on alcohol-related hepatocellular carcinoma through Mendelian Randomization” (PONE-D-23-37115) for possible publication in Plos One. On behalf of my coauthors, I would like to thank you and the reviewers for your valuable suggestions and comments. I have carefully incorporated all comments of reviewers into the newly revised manuscript. All major changes are in red in this revised version. Below are my responses to the reviewers’ comments. I believe all concerns have been addressed, and the quality of the revised manuscript has been improved significantly. I sincerely hope the revised manuscript will be acceptable to you and the reviewers. If you have any further questions about the manuscript, please feel free to contact me. Your consideration for this manuscript to be published in Plos One is much appreciated. Best regards, Yunhong Xia Department of Oncology, the First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China. Email: yhxia21@sina.com Journal Requirements 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Response: Thank you for your attention to detail. We have carefully reviewed the requirement and made the necessary changes to the names of supporting information figures and tables in accordance with the MANUSCRIPT BODY FORMATTING GUIDELINES. Additionally, we have addressed several other minor errors that were present in the manuscript. 2. Note from Emily Chenette, Editor in Chief of PLOS ONE, and Iain Hrynaszkiewicz, Director of Open Research Solutions at PLOS: Did you know that depositing data in a repository is associated with up to a 25% citation advantage (https://doi.org/10.1371/journal.pone.0230416)? If you’ve not already done so, consider depositing your raw data in a repository to ensure your work is read, appreciated and cited by the largest possible audience. You’ll also earn an Accessible Data icon on your published paper if you deposit your data in any participating repository (https://plos.org/open-science/open-data/#accessible-data). Response: Thank you for your valuable information. We believe that all the data relevant to our study are already available and relatively accessible. Nevertheless, we will certainly consider the idea of using a repository for our future research. Again, we appreciate your valuable suggestion. 3. Thank you for stating the following financial disclosure: "This study was funded by the National Natural Science Foundation of China (No. 81472331), Natural Science Foundation of Anhui Province (No. 2108085MH289), and the Project of Scientific Research Foundation of Anhui Medical University (No. 2019xkj146)." Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. Response: Your suggestion is well received. Now we would like to state that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 4. We note that your Data Availability Statement is currently as follows: [All relevant data are within the manuscript and its Supporting Information files.] Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition). For example, authors should submit the following data: - The values behind the means, standard deviations and other measures reported; - The values used to build graphs; - The points extracted from images for analysis. Authors do not need to submit their entire data set if only a portion of the data was used in the reported study. If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access. Response: Your suggestion is well received. Now we would like to confirm that our submission contains all raw data required to replicate the results of your study. Review Comments to the Author Reviewer #1: comments: Spelling correction for manuscript title on line 4. It should be “Causal” instead of Casual. Response: Thank you very much. We are very sorry for our negligence of this typo. The mistake has been corrected. Please see line 4. Spelling correction on line 26 “porphyria” Response: Thank you very much for your thorough review. We apologize for the incorrect writing. The error has been corrected. Please see line 27. The authors should remove statement in lines 83-85 of the introduction section, which implies that they have already concluded their hypothesis even before presenting the data. Response: Thank you very much for your helpful suggestion. We have removed this statement in the original line 83-85. Increased risk of primary liver cancer in AIP and other hepatic porphyrias is well-documented, however it is unclear if congenital erythropoietic porphyria (CEP), an even rare form of cutaneous porphyria, is associated with primary liver cancer. What prompted the authors to look for UROS SNPs in CEP cases? Response: Thank you for your question. CEP is a result of an error in heme synthesis caused by UROS. Currently, there is limited research on the relationship between CEP and AR-HCC. Therefore, we hypothesized that CEP biomarkers SNPs could serve as a potential entry point for further investigation. To conduct our study, we utilized preexisting GWAS dataset that provided data on UROS SNPs. Mechanism of liver injury and progression to AR-HCC in porphyrias was briefly mentioned, it may be worthwhile to expand on it. Response: Thank you very much for your constructive and helpful suggestion. The mechanism of liver injury and progression to AR-HCC in porphyrias remains poorly understood due to limited studies. However, this also indicates that our research is somewhat innovative. We hope that our findings will help overcome barriers on this topic. Despite the limited current research, we have added some contents regarding AR-HCC and porphyrias. Please see line 59-61 and line 77-78. The authors write that “the causal effect of PBGD and UROS on AR-HCC were confirmed using MR”, as the title also suggests. However, nowhere the causes of HCC are described. If anything, this study establishes a causal associated with HCC but not specifically AR-HCC. Response: Thank you very much. The trait of the outcome GWAS dataset (ebi-a-GCST90092003) in our study is documented as AR-HCC (PMID: 34902334). The methodological basis of our conclusion was provided. Additionally, MR research is often restricted to the population region of the exposure and outcome data. Currently, GWAS research on porphyria biomarkers primarily focuses on the European population, while research on HCC still requires further expansion in the future. Therefore, we are also eagerly anticipating the availability of more high quality GWAS data concerning HCC, which would greatly benefit our future study. Please see line 109-111. Previous studies reported increased urinary PBD in symptomatic patients with AIP and incident cases of AR-HCC. It is unclear from this study whether GWAS cases were symptomatic or had AR-HCC. If that is case, it needs to be stated clearly. Response: Thank you for your valuable suggestion. The PBGD and UROS datasets related to our study were extracted from a genomic atlas of the human plasma proteome study, without records of AR-HCC cases numbers (PMID: 29875488). We have mentioned it in the revised manuscript. Please see line 229-233. Although the absence of this aspect does not impact the process of reaching the conclusions of this study, it is our hope that there will be an increase in the number of high-quality studies in the future to enhance research in this particular field. Also unclear is why there is increased risk of AR-HCC as opposed to other causes of HCC. This needs to be explained in introduction. Response: Thank you very much for your helpful suggestion. Up to today, the GWAS dataset of HCC in European population is focused on AR-HCC only. Due to limited research, future research on other causes of HCC is needed to expand on it. Please see line 79-83. Special thanks to you for your good comments. Reviewer #2: The study investigates a fairly novel concept and technique, but there are several limitations which need to be addressed. These are: Abstract should clarify how many HCC and non-HCC cases extracted the data on SNP for the two genes of heme metabolism. Response: Thank you very much for your helpful suggestion. The PBGD and UROS datasets in our study were extracted from a genomic atlas of the human plasma proteome study, without records of AR-HCC cases numbers (PMID: 29875488). We have mentioned it in the revised manuscript. Please see line 239-243. Although this lack does not affect the process of reaching the conclusions of this study, we hope that further high-quality research can be conducted in the future to strengthen research in this specific area. The authors should clarify what formed their basis for the porphyria genes for their association with AR-HCC. This should be clarified in the introduction of the manuscript and purpose of the abstract section. Response: Thank you very much for your helpful suggestion. Former studies showed a possible association between AIP and liver cancer was identified through investigation on porphyria biomarkers. PBGD and UROS are important biomarkers of AIP and CEP, respectively. GWAS studies on porphyria biomarkers are still in the emerging stage, however, high-quality data of PBGD and UROS is already available. We have rewritten these sections following your valuable suggestion. Please see line 28-29 and line 73-83. It should be clarified whether any of these cases also had porphyria (biochemical and/or symptomatic). Response: Thank you very much for your constructive suggestion. The original study did not record whether these cases also had porphyria (PMID: 34902334). We have mentioned it in the revised manuscript. Please see line 239-243. Again, we hope that high-quality research can be conducted in the future to improve research in this specific area. Further, the manuscript should be read by an expert in English language for syntax and grammatical errors. Response: Thank you very much for your helpful suggestion. We invited native English speakers to polish the language of this paper. The wrong words have been removed in revised version. Special thanks to you for your kind comments. Attachments Attachment Submitted filename: Response_to_Reviewers.docx https://doi.org/10.1371/journal.pone.0299536.r002
13 Feb 2024 Decision Letter - Ashwani Singal, Editor Casual effect of porphyria biomarkers on alcohol-related hepatocellular carcinoma through Mendelian Randomization PONE-D-23-37115R1 Dear Dr. Xia, 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, Ashwani Singal Academic Editor PLOS ONE Additional Editor Comments (optional): Authors have appropriately revised the paper with point by point responses to the reviewers comments. No further comments from this reviewer. Reviewers' comments: https://doi.org/10.1371/journal.pone.0299536.r003
Formally Accepted
11 Mar 2024 Acceptance Letter - Ashwani Singal, Editor PONE-D-23-37115R1 PLOS ONE Dear Dr. Xia, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Ashwani Singal Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0299536.r004

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