Pain perception genes, asthma, and oral health: A reverse genetics study

Rosany O. Lisboa; Raymond F. Sekula; Mariana Bezamat; Kathleen Deeley; Luiz Carlos Santana-da-Silva; Alexandre R. Vieira

doi:10.1371/journal.pone.0277036

Peer Review History

Original SubmissionApril 26, 2022
4 Jul 2022 Decision Letter - JJ Cray Jr., Editor PONE-D-22-12279Pain Perception Genes, Asthma, and Oral Health: A Reverse Genetics StudyPLOS ONE Dear Dr. Vieira, 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. There are significant request to elaborate on experimental design, subject and target selection, and the novel approach used by the grup. Please submit your revised manuscript by Aug 18 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. 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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 2. Thank you for stating the following financial disclosure: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” At this time, please address the following queries: a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution. b) State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” c) If any authors received a salary from any of your funders, please state which authors and which funders. d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: "We would like to acknowledge the Dental Registry and DNA Repository project, which is supported by the University of Pittsburgh School of Dental Medicine. Also, we thank the Graduate Program in Oncology and Medical Sciences at the Federal University of Pará, Brazil, and the National Center for Advancing Translational Sciences of the National Institutes of Health, for the support to Lisboa, RO, and Bezamat, M, respectively.” We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. 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We will update your Data Availability statement to reflect the information you provide in your cover letter. 5. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. [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. 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And in similar clinical situations? Example: in third molar extractions or class I restorations or endodontic treatment, etc. It is necessary to describe what types of dental treatment were used as a parameter in anesthesia, and what anesthetics were used. 2. In table 01, include what the numbers inside the parentheses mean (maximum and minimum?). If so, it is important to distinguish adults from children, since the parameter used to evaluate the groups was the amount of anesthetic during the dental procedure; in children, fear and anxiety greatly influence pain, especially during procedures involving needles, as is the case with anesthesia. 3. Was it the same person who anesthetized all participants? Has any calibration been done between dentists? What kind of treatment was done on everyone to compare anesthesia? 4. The presence of some inflamed tissue can influence the amount of anesthetic tubes to be used in each procedure, was this taken into account? 5. How were these phenotypes evaluated? More detail is needed. 6. It is necessary to mention which studies were used as a reference in the choice of SNPs. 7. It is necessary to include the background of the selected SNPs in the introduction. 8. Add in table 02 information such as “increase or decrease pain sensitivity” other than just “Intensity or pain sensitivity”. So we would know what exactly the polymorphism effect on pain perception. 9. Was an epistasis analysis performed? Some studies demonstrate that the GCH1 gene can modulate the effect of COMT on the pain response. Patients who have SNP in both genes usually have a protective effect (analgesia) by GCH1, even with the COMT SNP still present. 10. I suggest increasing the quality of the images, it is difficult to understand what is written. 11. Results need to be organized more clearly, it's confusing. 12. I suggest further specifying the purpose of the work. Through the methodology of the work, the objective was to investigate these clinical phenotypes and some genetic markers in the response/efficacy of anesthetic or analgesia and not necessarily pain. It is necessary to clarify the justification and background of these objectives during the introduction, as it is not clear exactly what this work is about, only during the reading of the methodology. There are numerous ways to work with or assess pain: acute, spontaneous, chronic, stimulated/induced. It is necessary to specify and better describe the type of pain being investigated in this study, since one of the objectives is precisely to make this correlation between pain-related SNPs and other clinical phenotypes. 13. Importantly, the SNPs investigated here are not exclusively related to nociceptive pathways, but also to other non-painful conditions. It is necessary to describe each one of them better in this sense, depending on the amount of clinical phenotypes that were included in the analysis and are not directly related to nociceptive processes. Furthermore, it is necessary to include this observation in the discussion of the work. 14. The title is a few confusing: “Pain Perception Genes, Asthma, and Oral Health: A Reverse Genetics Study”. What does it mean “reverse genetics study” ? 15. The only relationship with pain in this work was the way the sample was divided into two groups – and this division, and the genes that are really related to painful processes, still need to be more detailed. However, the immense amount of clinical phenotypes evaluated are not directly related to pain, it is necessary to establish a link between these phenomena and describe them very well in the study. 16. It should also be discussed that there was not found any difference between the both groups after Bonferroni correction. Perhaps the method of using the amount of anesthetic as a parameter for dividing the groups was not satisfactory for evaluating these clinical phenotypes (pain-related? What exactly?) with genetic markers of pain. There are many factors that influence the amount of anesthetic used. This was a decisive criterion in the selection of the two groups, however, the way in which it was made was neither mentioned nor described. If there was calibration between the dentists who performed the procedure (both anesthetic and dental – surgical/restorative/endodontic/periodontal – which should also be described), nor if there was any nervous branch of predilection and region that was anesthetized – maxilla or mandible, if there was previous pain or local inflammation/infection. These are all factors that influence the anesthesia procedure, and that influenced the choice/division of the study sample. 17. The final considerations state that “Nevertheless, our study provides a pioneering approach to the study of pain perception”. What tool was used to evaluate pain perception? Visual analogue scale (VAS)? Sound, eye, and motor (SEM) indexes? Quantitative Sensory Testing? 18. Which previous study was used to determine the cut-off point for the amount of anesthetic? So it could be used as parameter to dived the sample into two groups. Reviewer #2: The authors performed PheWAS-like analysis to examine the associations of 58 phenotypes with 7 pain related SNPs. A total of 1289 subjects were selected and grouped based on the dose of anesthesia received during the dental treatment procedure. They found an association between the rs1799971 in OPRM1 and asthma, which was in agreement with a recent report. In addition, the study identified three associations of oral phenotypes with variants of pain-related genes. Overall, the study is carefully designed and performed. Major critiques: 1. The authors applied novel criteria to recruit subjects - individuals who feel more pain (received more anesthesia) or less pain (who received less anesthesia). However, using “anesthesia utilization” as a surrogate indicator of pain severity raised concerns. It is unknown if other factors were taken into consideration, e.g. medication history, especially opioids and other analgesic drugs, dental procedure types (location, duration), as well as pain assessment (score, type). Whether these variants were adjusted in the statistical analysis was not mentioned in the manuscript. 2. The sample size of "black – less anesthesia" group is small. The authors should address how they controlled the power of analysis to ensure the association detected between rs4818 of COMT and diseases of the tongue was not false-positive. 3. The authors should address the rationale of selecting these 7 SNPs, in terms of their relationships with orofacial pain perception, specifically. Minor issues: Line 233, Appendix Table 1 is not included in the manuscript. Line 143, reference is missing. ****** 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: Yes: Flávia Fonseca Carvalho Soares Reviewer #2: 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. Attachments Attachment Submitted filename: PLOS ONE - Comments to the author.docx https://doi.org/10.1371/journal.pone.0277036.r001
Revision 1
11 Aug 2022 Author Response 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 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 All information has been revised as requested. 2. Thank you for stating the following financial disclosure: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” At this time, please address the following queries: a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution. b) State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” c) If any authors received a salary from any of your funders, please state which authors and which funders. d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. The additional information in the acknowledgment section was excluded. It is marked in yellow and crossed out with a line. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: "We would like to acknowledge the Dental Registry and DNA Repository project, which is supported by the University of Pittsburgh School of Dental Medicine. Also, we thank the Graduate Program in Oncology and Medical Sciences at the Federal University of Pará, Brazil, and the National Center for Advancing Translational Sciences of the National Institutes of Health, for the support to Lisboa, RO, and Bezamat, M, respectively.” We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. The additional information in the acknowledgment section was excluded. It is marked in yellow and crossed out with a line. We added it in the cover letter as asked. 4. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. We added a supporting information file with our raw data. 5. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. We changed the cover letter to reflect that we are uploading all raw data as an appendix. Reviewers' comments: Reviewer #1: 1. Was only one type of anesthetic compared? Example: lidocaine with vasoconstrictor. And in similar clinical situations? Example: in third molar extractions or class I restorations or endodontic treatment, etc. It is necessary to describe what types of dental treatment were used as a parameter in anesthesia, and what anesthetics were used. The information of the local anesthetics used were added to the manuscript in the methods section (line 134) and also the duration of anesthesia according to the article reference. 2. In table 01, include what the numbers inside the parentheses mean (maximum and minimum?). If so, it is important to distinguish adults from children, since the parameter used to evaluate the groups was the amount of anesthetic during the dental procedure; in children, fear and anxiety greatly influence pain, especially during procedures involving needles, as is the case with anesthesia. The number inside the parentheses mean the range of age in each group. As you can see in the table 1 the groups were matched for age, so there are no significant variations between groups. Both children and adults may display some fear and anxiety. 90% of pediatric dentistry is done under local anesthesia. Comparison groups were originally matched by age to mitigate the influence of age. 3. Was it the same person who anesthetized all participants? Has any calibration been done between dentists? What kind of treatment was done on everyone to compare anesthesia? Data for the study comes from our registry, that currently spans over 17 years. It was not the same operator that anesthetized all participants. All dentists in training perform anesthesia under the same protocol. Treatments done were typical operative dentistry procedures. We added these clarifications in the Methods section (line 137) to address this concern. 4. The presence of some inflamed tissue can influence the amount of anesthetic tubes to be used in each procedure, was this taken into account? To mitigate this aspect, comparison groups were matched by dental procedure. But beyond that, it was not possible to fully account for levels of inflammation at the day treatments were performed. We added a clarification in the method section (line 139) to acknowledge this detail. 5. How were these phenotypes evaluated? More detail is needed. Phenotypes were defined based on coding used in the registry, which reflects descriptions at the dental records. We added this information in the methods section (line 159) to address this concern. 6. It is necessary to mention which studies were used as a reference in the choice of SNPs. The studies used as references for each SNP were originally mentioned in table 2. 7. It is necessary to include the background of the selected SNPs in the introduction. The background of the SNPs was inserted in the introduction. 8. Add in table 02 information such as “increase or decrease pain sensitivity” other than just “Intensity or pain sensitivity”. So we would know what exactly the polymorphism effect on pain perception. The SNPs in the chosen genes act mainly in the general context of the pain perception process and unfortunately in the literature there are no specific concrete associations of these SNPs regarding their participation in the increase or decrease of pain, many of them seem to act in both situations depending on some factors, therefore, to avoid future mistakes when conditioning them with a specific association, it is preferable to use the term “Intensity or pain sensitivity” as used in the reference articles. Among the suggested explanations for the discrepancy found in the literature is that this may indicate that these polymorphisms affect pain sensations of some, but not all, stimuli. This information will be added to the manuscript to explain why we used the term. 9. Was an epistasis analysis performed? Some studies demonstrate that the GCH1 gene can modulate the effect of COMT on the pain response. Patients who have SNP in both genes usually have a protective effect (analgesia) by GCH1, even with the COMT SNP still present. No. For this specific paper epistasis analysis was not performed because the study's main objective was to search associations using the PheWAS methodology and not the influence that could exist between the studied markers. 10. I suggest increasing the quality of the images, it is difficult to understand what is written. All images were submitted to the PACE tool and the quality of the images was enhanced. 11. Results need to be organized more clearly, it's confusing. The organization was done according to the specific results of each PheWAS analysis performed and following the presentation standard for this methodology. 12. I suggest further specifying the purpose of the work. Through the methodology of the work, the objective was to investigate these clinical phenotypes and some genetic markers in the response/efficacy of anesthetic or analgesia and not necessarily pain. It is necessary to clarify the justification and background of these objectives during the introduction, as it is not clear exactly what this work is about, only during the reading of the methodology. There are numerous ways to work with or assess pain: acute, spontaneous, chronic, stimulated/induced. It is necessary to specify and better describe the type of pain being investigated in this study, since one of the objectives is precisely to make this correlation between pain-related SNPs and other clinical phenotypes. The information in the main sections of the manuscript has been improved for a better understanding of the purpose of the study and how it was done. 13. Importantly, the SNPs investigated here are not exclusively related to nociceptive pathways, but also to other non-painful conditions. It is necessary to describe each one of them better in this sense, depending on the amount of clinical phenotypes that were included in the analysis and are not directly related to nociceptive processes. Furthermore, it is necessary to include this observation in the discussion of the work. Most of phenotypes included in the analysis have a pain sensitivity component. SNPs were chosen because they have some association with the pain perception processor regardless of whether it is nociceptive pain or not. And since the perception of pain is something extremely complex with the involvement of several genes, it is expected that these SNPs participate in other processes and are also associated with other non-painful conditions. 14. The title is a few confusing: “Pain Perception Genes, Asthma, and Oral Health: A Reverse Genetics Study”. What does it mean “reverse genetics study” ? In reverse genetics, genetic marker data are used to drive, or form the basis of, new phenotype definitions, that is, the analysis starts from genotype to phenotype. We addeda clarification in the Introduction section (line 83)/ 15. The only relationship with pain in this work was the way the sample was divided into two groups – and this division, and the genes that are really related to painful processes, still need to be more detailed. However, the immense amount of clinical phenotypes evaluated are not directly related to pain, it is necessary to establish a link between these phenomena and describe them very well in the study. Most phenotypes included actually have potentially a pain sensitivity component associated with them. We added this clarification in the methods section (line 162). 16. It should also be discussed that there was not found any difference between the both groups after Bonferroni correction. Perhaps the method of using the amount of anesthetic as a parameter for dividing the groups was not satisfactory for evaluating these clinical phenotypes (pain-related? What exactly?) with genetic markers of pain. There are many factors that influence the amount of anesthetic used. This was a decisive criterion in the selection of the two groups, however, the way in which it was made was neither mentioned nor described. If there was calibration between the dentists who performed the procedure (both anesthetic and dental – surgical/restorative/endodontic/periodontal – which should also be described), nor if there was any nervous branch of predilection and region that was anesthetized – maxilla or mandible, if there was previous pain or local inflammation/infection. These are all factors that influence the anesthesia procedure, and that influenced the choice/division of the study sample. Bonferroni is quite a strict method of multiple comparion correction. One result survived this correction. Results that do not survive a Bonferroni correction were also presented and have been added to the Discussion section, since they may represent true biological relationships and deserve further study. The reviewer's other considerations were supplied in the previous comments. 17. The final considerations state that “Nevertheless, our study provides a pioneering approach to the study of pain perception”. What tool was used to evaluate pain perception? Visual analogue scale (VAS)? Sound, eye, and motor (SEM) indexes? Quantitative Sensory Testing? The final consideration was re-elaborated to suit the proposed objectives and results of the study. 18. Which previous study was used to determine the cut-off point for the amount of anesthetic? So it could be used as parameter to dived the sample into two groups. It is common clinical practice to give up to one tubete of anesthetic for dental treatments. Hence, the cutoff used. Reviewer #2: The authors performed PheWAS-like analysis to examine the associations of 58 phenotypes with 7 pain related SNPs. A total of 1289 subjects were selected and grouped based on the dose of anesthesia received during the dental treatment procedure. They found an association between the rs1799971 in OPRM1 and asthma, which was in agreement with a recent report. In addition, the study identified three associations of oral phenotypes with variants of pain-related genes. Overall, the study is carefully designed and performed. Major critiques: 1. The authors applied novel criteria to recruit subjects - individuals who feel more pain (received more anesthesia) or less pain (who received less anesthesia). However, using “anesthesia utilization” as a surrogate indicator of pain severity raised concerns. It is unknown if other factors were taken into consideration, e.g. medication history, especially opioids and other analgesic drugs, dental procedure types (location, duration), as well as pain assessment (score, type). Whether these variants were adjusted in the statistical analysis was not mentioned in the manuscript. Groups were matched by dental procedures, but chronic use of opioids or other analgesic oral drugs, was not considered. No pain assessments were performed. 2. The sample size of "black – less anesthesia" group is small. The authors should address how they controlled the power of analysis to ensure the association detected between rs4818 of COMT and diseases of the tongue was not false-positive. Test power was controlled using the simulation study by Verma et al. (2018) and according to this study for the group that required less anesthetic, there was sufficient power if the allele frequency was 20% and the penetrance was 20% or higher. 3. The authors should address the rationale of selecting these 7 SNPs, in terms of their relationships with orofacial pain perception, specifically. SNPs were selected for their involvement with the perception of the general participation of genes in fundamental processes. Those SNPs that are related to orofacial pain were described in the introduction for better understanding. Per reviewer 1, rationale was added to the introduction section. Minor issues: Line 233, Appendix Table 1 is not included in the manuscript. The appendix table1 was inserted in the main text as table 5. Line 143, reference is missing. The missing reference was added. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0277036.r002
9 Sep 2022 Decision Letter - JJ Cray Jr., Editor PONE-D-22-12279R1Pain Perception Genes, Asthma, and Oral Health: A Reverse Genetics StudyPLOS ONE Dear Dr. Vieira, 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. There are some outstanding issues mostly with interpretation that should be addressed in a revision. Please submit your revised manuscript by Oct 24 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, JJ Cray Jr., Ph.D. Academic Editor PLOS ONE Journal Requirements: 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. [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: All comments have been addressed Reviewer #2: 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 #1: Partly Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 #1: Yes Reviewer #2: 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 #1: Yes Reviewer #2: 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: The authors answered most of the questions raised and significantly improved the quality of the manuscript. However, there are still major concerns that need to be addressed. 1. Lidocaine and articaine have very different particularities. For instance, The duration of effect in a maxillary infiltration of 2% lidocaine with 1:100,000 epinephrine ranges from 60 to 90 minutes in pulp tissue and 170 to 300 minutes in soft tissue, while the duration of 4% articaine with 1:100,000 epinephrine encompasses 60 to 75 minutes in the pulp and 170 to 360 minutes in the soft tissues. Palate anesthesia is unpleasant for many patients because it is a very sensitive site, so when the anesthetic used is articaine, it is not necessary to perform infiltration in the palatal location for extraction procedures, as it is very effective only with injection in the region maxillary vestibular. Articaine presents tissue diffusion when compared to the lidocaine in areas other than the place where the anesthetic was deposited, avoiding excessive punctures and patient discomfort. Therefore, depending on the dental procedure, the type of anesthetic could influence the number of tubetes to be used. This should have been considered in the methodological strategy of the study. 2. Regarding including children in the sample, I still have concerns about the parameter of “quantity of anesthetic used”. Since we use weight calculate the maximum amount of anesthetic. In this way, children necessarily must use less tubetes then adults. Since the parameter of “pain perception” is the amount of anesthetic used, these variables involved in metabolization, duration effect, toxicity and others, should be addressed accordingly. 3. The final consideration state that “Through this powerful strategy, it was possible to identify new associations between SNPs in genes related to pain and oral phenotypes in the groups of individuals who feel more pain (those who received more anesthesia) or less pain (who received less anesthesia)”. Actually, it is not possible to establish this type of equivalence. Since the amount of anesthetic used can be influenced by several factors, such as anatomical variations, metabolism rate, type of anesthetic used, local tissue pH (which can be modified by the presence of inflammation), technique used, among others, and not just pain. Reviewer #2: The authors have carefully addressed the questions from the reviewers. The revised manuscript is in better shape. However, there are couple places need additional inputs. First of all, based on the analysis scale (58 phenotypes, 7 SNPs, 1289 subjects), it can not be called as a real PheWAS study. The authors should be careful when using the term in the manuscript. Second, the authors should revise the Introduction to be more concise and focused. This is not a review paper. ****** 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: Yes: Flávia Fonseca Carvalho Soares Reviewer #2: 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. https://doi.org/10.1371/journal.pone.0277036.r003
Revision 2
19 Sep 2022 Author Response Response to Reviews Reviewer #1: The authors answered most of the questions raised and significantly improved the quality of the manuscript. However, there are still major concerns that need to be addressed. 1. Lidocaine and articaine have very different particularities. For instance, The duration of effect in a maxillary infiltration of 2% lidocaine with 1:100,000 epinephrine ranges from 60 to 90 minutes in pulp tissue and 170 to 300 minutes in soft tissue, while the duration of 4% articaine with 1:100,000 epinephrine encompasses 60 to 75 minutes in the pulp and 170 to 360 minutes in the soft tissues. Palate anesthesia is unpleasant for many patients because it is a very sensitive site, so when the anesthetic used is articaine, it is not necessary to perform infiltration in the palatal location for extraction procedures, as it is very effective only with injection in the region maxillary vestibular. Articaine presents tissue diffusion when compared to the lidocaine in areas other than the place where the anesthetic was deposited, avoiding excessive punctures and patient discomfort. Therefore, depending on the dental procedure, the type of anesthetic could influence the number of tubetes to be used. This should have been considered in the methodological strategy of the study. RESPONSE: We added discussion to acknowledge this limitation. 2. Regarding including children in the sample, I still have concerns about the parameter of “quantity of anesthetic used”. Since we use weight calculate the maximum amount of anesthetic. In this way, children necessarily must use less tubetes then adults. Since the parameter of “pain perception” is the amount of anesthetic used, these variables involved in metabolization, duration effect, toxicity and others, should be addressed accordingly. RESPONSE: We added discussion and acknowledged this limitation. 3. The final consideration state that “Through this powerful strategy, it was possible to identify new associations between SNPs in genes related to pain and oral phenotypes in the groups of individuals who feel more pain (those who received more anesthesia) or less pain (who received less anesthesia)”. Actually, it is not possible to establish this type of equivalence. Since the amount of anesthetic used can be influenced by several factors, such as anatomical variations, metabolism rate, type of anesthetic used, local tissue pH (which can be modified by the presence of inflammation), technique used, among others, and not just pain. RESPONSE: We rewrote the statement to address this concern. Reviewer #2: The authors have carefully addressed the questions from the reviewers. The revised manuscript is in better shape. However, there are couple places need additional inputs. First of all, based on the analysis scale (58 phenotypes, 7 SNPs, 1289 subjects), it can not be called as a real PheWAS study. The authors should be careful when using the term in the manuscript. RESPONSE: The definition of PheWAS is a study design in which the association between single nucleotide polymorphisms or other types of DNA variants is tested across a large number of different phenotypes. Based on the definition, our study is a PheWAS study. We added this definition in the discussion section to address this concern. Second, the authors should revise the Introduction to be more concise and focused. This is not a review paper. RESPONSE: We shortened the introduction as suggested. Attachments Attachment Submitted filename: Response to Reviews.docx https://doi.org/10.1371/journal.pone.0277036.r004
11 Oct 2022 Decision Letter - JJ Cray Jr., Editor PONE-D-22-12279R2Pain Perception Genes, Asthma, and Oral Health: A Reverse Genetics StudyPLOS ONE Dear Dr. Vieira, 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. There is one outstanding concern that potentially some citations have been ommitted. Please submit your revised manuscript by Nov 25 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, JJ Cray Jr., Ph.D. Academic Editor PLOS ONE Journal Requirements: 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. [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: All comments have been addressed Reviewer #2: 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 #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 #1: Yes Reviewer #2: 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 #1: Yes Reviewer #2: 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: The authors have carefully addressed the questions from the reviewers, and significantly improved the quality of the manuscript. However, there are still minor issues that need to be addressed. All the new data included are lacking references. It is necessary to cite the sources from which the information was obtained. Reviewer #2: (No Response) ****** 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: Yes: Flávia Fonseca Carvalho Soares Reviewer #2: 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. https://doi.org/10.1371/journal.pone.0277036.r005
Revision 3
17 Oct 2022 Author Response Reviewer #1: The authors have carefully addressed the questions from the reviewers, and significantly improved the quality of the manuscript. However, there are still minor issues that need to be addressed. All the new data included are lacking references. It is necessary to cite the sources from which the information was obtained. RESPONSE: We added the references as requested. Attachments Attachment Submitted filename: Response to Reviews2.docx https://doi.org/10.1371/journal.pone.0277036.r006
19 Oct 2022 Decision Letter - JJ Cray Jr., Editor Pain Perception Genes, Asthma, and Oral Health: A Reverse Genetics Study PONE-D-22-12279R3 Dear Dr. Vieira, 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, JJ Cray Jr., Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0277036.r007
Formally Accepted
9 Nov 2022 Acceptance Letter - JJ Cray Jr., Editor PONE-D-22-12279R3 Pain Perception Genes, Asthma, and Oral Health: A Reverse Genetics Study Dear Dr. Vieira: I'm 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 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 plosone@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. JJ Cray Jr. Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0277036.r008

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