Polymorphisms of FDPS, LRP5, SOST and VKORC1 genes and their relation with osteoporosis in postmenopausal Romanian women

Alina Deniza Ciubean; Rodica Ana Ungur; Laszlo Irsay; Viorela Mihaela Ciortea; Ileana Monica Borda; Gabriela Bombonica Dogaru; Adrian Pavel Trifa; Stefan Cristian Vesa; Anca Dana Buzoianu

doi:10.1371/journal.pone.0225776

Peer Review History

Original SubmissionAugust 22, 2019
21 Oct 2019 Decision Letter - Sakamuri V. Reddy, Editor PONE-D-19-23734 Polymorphisms of FDPS, LRP5, SOST and VKORC1 genes and their relation with osteoporosis in postmenopausal Romanian women PLOS ONE Dear Dr. Ciubean, 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. ACADEMIC EDITOR: Though the reviewers finds the study is interesting, the authors should address the editorial comments as noted below to further improve the manuscript. Particularly, they should provide specifics/details and clarify rationale appropriately to study the gene polymorphisms associated with bone anabolic and antiresorptive agents used to treat postmenopausal osteoporosis. We would appreciate receiving your revised manuscript by Dec 05 2019 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable 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. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Dr. Sakamuri V. Reddy Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional 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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please provide a sample size and power calculation in the Methods, or discuss the reasons for not performing one before study initiation. Additional Editor Comments (if provided): The authors have examined the occurrence of polymorphisms in FDPS, LRP5, SOST and VKORC1 genes with respect to bone mineral density (BMD) in postmenopausal Romanian women. The results showed a strong correlation of BMD and polymorphisms in FDPS (farnesyl diphosphate synthase) gene, but a modest association with LRP5 and SOST genes in postmenopausal Romanian women with osteoporosis and no association with VKORC1. Specific comments to further improve the manuscript are: Introduction-noted very briefly. Please clarify the source and function of SOST and LRP5 genes in bone remodeling with citations appropriately. For ex., a rationale for genes examined ie anti-resorptive and anti-anabolic. Also, clarify SOST gene product Sclerostin protein is primarily produced by the osteocyte has anti-anabolic effect on bone formation. Methods-SNP genotyping is described very briefly, and no details of PCR conditions given. Results-no rationale given for experiments conducted and this section is noted briefly with no details. References-correct ref#29, follow the journal format ex., remove the month, number in refs etc.. [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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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: No ****** 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 ****** 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: This is an interesting report on distribution of 4 osteoporosis genes and their association with osteoporosis in a Romanian women cohort. Overall results are interesting and confirmatory. The paper is well written and contributes to the field of genetics of osteoporosis ****** 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 [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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0225776.r001
Revision 1
11 Nov 2019 Author Response Dear Reviewers, Below you have the responses to the points raised during the review process. 1. Introduction-noted very briefly. Please clarify the source and function of SOST and LRP5 genes in bone remodeling with citations appropriately. For ex., a rationale for genes examined ie anti-resorptive and anti-anabolic. Also, clarify SOST gene product Sclerostin protein is primarily produced by the osteocyte has anti-anabolic effect on bone formation. Author response: The introduction has been updated accordingly. “ One of the most important signaling pathways in bone in Wnt, which is crucial for bone development during embryogenesis and has a dual role in bone mass regulation, influencing both bone formation and resorption. The components of the Wnt pathway are proteins involved in cell proliferation, differentiation, and apoptosis of bone cells.[5] When cells are stimulated through the membrane receptors low-density lipoprotein receptor-related protein 5/6 (LRP5/6), the architecture of the multiprotein complex is modified, which inhibits b-catenin, leading to its translocation to the nucleus, where it initiates the transcription of target genes.[6] LRP5 is the most important membrane receptor of the Wnt signaling pathway and it was previously tagged in a genome-wide association study to be associated with OP.[7] LRP5 inactivation caused by mutation is responsible for osteoporosis-pseudoglioma syndrome, in which low bone mass and fractures occur.[8] Also, there are several naturally occurring inhibitors of Wnt signaling, such as Dickkopf (DKK) and sclerostin (SOST) proteins that inactivate signaling from LRP5/6 receptors. Sclerostin, encoded by the SOST gene, antagonizes Wnt signaling in both osteocytes and osteoblasts by binding to the LRP5/6 coreceptor and preventing bone formation.[9.10] High-bone-mass syndromes are thought to be caused by inactivating mutations of SOST (sclerosteosis and van Buchem’s disease).[11] Animal studies have indicated that sclerostin inhibition increases bone mass by stimulating bone formation and inhibiting bone resorption.[12]” 2. Methods-SNP genotyping is described very briefly, and no details of PCR conditions given. Author response: The PCR conditions has been added to the Material and Methods section “Genomic DNA was obtained from peripheral blood withdrawn on EDTA, using commercially available kits (Quick gDNA MiniPrep kit, Zymo Research, USA; PureLink Genomic DNA Mini Kit, Invitrogen, Thermo Fisher, USA). We genotyped four SNPs (FDPS rs2297480, LRP5 rs3736228, SOST rs1234612, VKORC1 rs9934438) in all patients and controls using the real-time PCR technique. Standard, predesigned TaqMan SNP genotyping assays, containing all the primers and probes needed for genotyping, were purchased from Thermo Fisher (codes C___2737970_10, C__25752205_10, C___7566033_10, C__30204875_10). All the genotyping were performed according to manufacturer’s instructions. The reaction mix contained 10 μl of 2xTaqMan Genotyping Master Mix (Applied Biosystems, Thermo Fisher, USA), 0.5 μl of the corresponding 40xTaqMan SNP genotyping assay, approximately 25 ng of genomic DNA and free-nucleases water to the final volume of 20 μl. The same amplification program was used for all the genotyping, consisting in a pre-read stage of 30 seconds (s) at 60°C, hold stage of 10 minutes (min) at 95°C, followed by the PCR stage, consisting of 40 cycles, each comprising 15 s at 95°C and 1 min at 60°C, and a post-read stage of 30 s at 60°C. All the experiments were run on a QuantStudio 3 real-time PCR machine (Applied Biosystems, Thermo Fisher, USA).” 3. Results-no rationale given for experiments conducted and this section is noted briefly with no details. Author response: The rationale behind the experiments have been added ” After dividing the patients into groups, analysis of the codominant model was performed. Codominant models hypothesize that the major allele homozygotes, the heterozygotes or the minor allele homozygotes are associated with the lowest, the intermediate, and the highest risk, respectively, or they are associated with the highest, the intermediate, and the lowest risk, respectively.” 4. References-correct ref#29, follow the journal format ex., remove the month, number in refs etc.. Author response: all references have been modified accordingly. 5. Have the authors made all data underlying the findings in their manuscript fully available? Reviewer response: No Author response: An Excel file with the raw data has been uploaded with the manuscript files In the name of all the authors, we thank you for the peer-review process, and we hope the modifications are sufficient and the article is ready to be published in its currents form. If any further clarifications are required, we are happy to give them. Sincerely, Dr. Alina Deniza Ciubean Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0225776.r002
13 Nov 2019 Decision Letter - Sakamuri V. Reddy, Editor Polymorphisms of FDPS, LRP5, SOST and VKORC1 genes and their relation with osteoporosis in postmenopausal Romanian women PONE-D-19-23734R1 Dear Dr. Ciubean, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Dr. Sakamuri V. Reddy Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0225776.r003
Formally Accepted
19 Nov 2019 Acceptance Letter - Sakamuri V. Reddy, Editor PONE-D-19-23734R1 Polymorphisms of FDPS, LRP5, SOST and VKORC1 genes and their relation with osteoporosis in postmenopausal Romanian women Dear Dr. Ciubean: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Sakamuri V. Reddy Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0225776.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .