The oxytocin receptor gene polymorphism rs2268491 and serum oxytocin alterations are indicative of autism spectrum disorder: A case-control paediatric study in Iraq with personalized medicine implications

Zainab Al-Ali; Akeel Abed Yasseen; Arafat Al-Dujailli; Ahmed Jafar Al-Karaqully; Katherine Ann McAllister; Alaa Salah Jumaah

doi:10.1371/journal.pone.0265217

Peer Review History

Original SubmissionSeptember 27, 2021
29 Nov 2021 Decision Letter - Elsayed Abdelkreem, Editor PONE-D-21-31116The oxytocin receptor gene polymorphism rs2268491 and serum oxytocin alterations are indicative of autism spectrum disorder: A case-control paediatric study in Iraq with personalized medicine implicationsPLOS ONE Dear Dr. McAllister, 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. Specifically, the authors should address the following points: Criterion 3. “Experiments, statistics, and other analyses are performed to a high technical standard and are described in sufficient detail”. Experiments must have been conducted rigorously, with appropriate controls and replication. Sample sizes must be large enough to produce robust results, where applicable. Methods and reagents must be described in sufficient detail for another researcher to reproduce the experiments described. (1) Authors should clarify sampling method and justify sample size. (2) Define severity of ASD. (3) Provide required details for all used kits and reagents, such as manufacturer, city, and country. (4) Experimental details on Isolation of genomic DNA and Agarose gel electrophoresis are commonly used techniques so that they should be summarized in the main text and the technical details can be better provided as supplementary. (5) Did the authors test data for normality? (6) Did the author consider confounders “e.g., the Median age of control is lower than that of ASD, which may affect serum OXT level”. (7) In table 2, authors should describe which statistical tests were used; moreover, data are presented as mean (SD) but some appear to be not normally distributed. (8) In addition “The ANOVA test found statistically significant difference (p=0.032) in OXT levels according to severity of ASD, as shown in Table 2”. Please, specify the difference? (9) In table 2, it seems that Oxytocin levels are lower in severe ASD (higher in mild ASD), which may be associated with older age. (10) It is interesting to see that healthy controls had highest OXT levels in the CC genotype, while ASD subjects had the highest OXT in the TT genotype; it would be meaningful to explain/discuss such findings. Criterion 4. “Conclusions are presented in an appropriate fashion and are supported by the data.” Authors have to revise the conclusion of their study to be based on study findings (e.g., “The measurement of peripheral OXT levels and OXTR genetic alterations provide a simple dual-biomarker test to gauge social functioning in the ASD patient for diagnostic, monitoring and support purposes”; however, this study didn’t investigate monitoring and support aspects). Other suggested implications/recommendations based on study findings/conclusion should be separated from the conclusion itself. Criterion 5. “The article is presented in an intelligible fashion and is written in standard English.” (1) Table 1: better to show ASD and controls in columns and variables as rows. Please, provide other important data, such as gender and comorbid conditions. (2) The article contain several errors related to English language, spelling, and grammar. PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. We may reject papers that do not meet these standards. If the language of a paper is difficult to understand or includes many errors, we may recommend that authors seek independent editorial help before submitting a revision. Criterion 6. “The research meets all applicable standards for the ethics of experimentation and research integrity.”. The authors repeat the ethics statement twice, once under study populations “Formal parental consent was obtained from families who expressed interest in the study, who were provided with consent sheets and a questionnaire to complete” and under Ethical approval “All patients were informed about the purpose of the work, and a written consent obtained from each patient.”. Authors should provide this information once. Moreover, did they obtain consent from the patients “children?”, parents, or both? Criterion 7. “The article adheres to appropriate reporting guidelines and community standards for data availability.” (1) In the abstract and discussion, authors seem to selectively report some findings in an inappropriate manner, such as “study characteristics in the ASD population revealed a high level of consanguinity (36.66%)” although the percentage of consanguinity was higher among control group (40%). (2) The authors stated that “all data are fully available without restriction” but they did not provide unidentified patients’ data. Authors are required to make all data underlying the findings described fully available, without restriction, and from the time of publication. PLOS allows rare exceptions to address legal and ethical concerns, but this has to be completely explained. Please, review PLOS Data Policy. Please submit your revised manuscript by Jan 13 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). 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Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. [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: I Don't Know ****** 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 ****** 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 important study from clinical point of view. The whole article seemed to be written in readers friendly language, well articulated and intelligently structured. It is publishable, though I would like to suggest for further evaluation of statistical issues by an expert in this field. ****** 6. 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Revision 1
19 Jan 2022 Author Response 1. Criterion 3: a. (1) Authors should clarify sampling method and justify sample size. Answer: (lines 126 paragraph manuscript) literature searches were performed to assess the prevalence of autism spectrum disorder. An estimated 1 % of the world’s population has autism spectrum disorder, with recent figures of 1.5%. Since the prevalence of the disease is a relatively uncommon event at 1 out of 100 cases, a case control study was selected as the most efficient sampling method to investigate autism among the Iraqi Arab population. A study sample size of N=120 (60 ASD and 60 controls) was feasible to manage in the face of opposition to the study from local tribal and religious restrictions. Our study had a 3:1 male to female ratio in the ASD and control populations. The DSM-5 states that “autism spectrum disorder is diagnosed four times more often in males than in females.”(American Psychiatric Association 2013), and most recent research suggest that it is closer to 3:1 (Loomes, Hull, and Mandy 2017). Therefore the study population is reflective of the gender ratio in ASD and provides a useful model to explore specific genetic associations. b. (2) Define severity of ASD. Answer: This was delineated in the PLOS PDF lines 145-182 as follow: The severity was defined according to the Childhood Autism Rating Scale as follow: The patients with ASD were stratified according to severity into the following sub-groups (20): mild ASD (n=39), moderate ASD (n=13) and severe ASD (n=8) according to the Childhood Autism Rating Scale (CARS) (21). The CARS is a diagnostic assessment method that rated individuals on a scale ranging from normal to severe and yields a composite score ranging from non-autistic to mildly autistic, moderately autistic, or severely autistic. The scale was used to observe and subjectively rate the following fifteen items in both ASD and case control subjects. Each of the following 15-items were scored in patients according to seven levels of severity by interview: 1. Relationship to people 2. Imitation 3. Emotional response 4. Body 5. Object use 6. Adaptation to change 7. Visual response 8. Listening response 9. Taste-smell-touch response and use 10. Fear and nervousness 11. Verbal communication 12. Non-verbal communication 13. Activity level 14. Level and consistency of intellectual response 15. General impressions c. (3) Provide required details for all used kits and reagents, such as manufacturer, city, and country. Answer: done. These were delineated in supplementary Table S1, S2 S1 Table. Instruments and Equipment used in the study. S2 Table. Chemicals and reagents for DNA extraction and polymerase chain reaction. d. (4) Experimental details on Isolation of genomic DNA and Agarose gel electrophoresis are commonly used techniques so that they should be summarized in the main text and the technical details can be better provided as supplementary. Answer: The experimental details for both isolation of genomic DNA and preparation of gels for electrophoresis are now summarized in the main manuscript with full lab protocols described in supplementary table 3. e. (5) Did the authors test data for normality? Answers: The Kolmogorov-Smirnov test was used to test normality, which showed a normal distribution, methods (statistical analysis): paragraph 270. The test for all variables was greater than 0.05 so that samples are normally distributed (Table 2). f. Did the author consider confounders “e.g., the Median age of control is lower than that of ASD, which may affect serum OXT level”. Answer. Ages were matched between the ASD and control group. There were no significant differences in age distribution between studied and control group P=0.708 (Table 2). g. (7) In table 2, authors should describe which statistical tests were used; moreover, data are presented as mean (SD) but some appear to be not normally distributed. Answer: done. Analysis of Variance (ANOVA) test were used for statistical analysis as the data were normally distributed which was ensured by doing Kolmogorov-Smirnov test (Table 2). h. (8) In addition “The ANOVA test found statistically significant difference (p=0.032) in OXT levels according to severity of ASD, as shown in Table 2”. Please, specify the difference? Answer: done. The oxytocin level was 165.02±28.03 pg/mL in mild cases, 152.78±27.86 pg/mL in moderate cases, and 129.09±11.04 pg/mL in severe cases. Furthermore, there were significant differences in oxytocin level when comparing mild and severe cases (p=0.001), moderate and severe cases (p=0.034). On other hand there were no significant differences in oxytocin level between mild and moderate cases (p=0.178) (Table 2, table footnotes). i. (9) In table 2, it seems that Oxytocin levels are lower in severe ASD (higher in mild ASD), which may be associated with older age. Answer. Done. Oxytocin level was higher in mild cases (165.02±28.03 pg/mL) than severe cases (129.09±11.04 pg/mL). However there was no significant correlation between age of the patient and oxytocin level as stated in the manuscript in lines 554 and Supplementary Figure 1 (P= 0.396, R=-0.140, R2=0.0196; Supplementary Fig 1). j. (10) It is interesting to see that healthy controls had highest OXT levels in the CC genotype, while ASD subjects had the highest OXT in the TT genotype; it would be meaningful to explain/discuss such findings. Answer. Done (Discussion, main manuscript). In stratified ASD patients, serum oxytocin levels were higher in the mild category with the polymorphism genotype TT. This suggests that patients with milder ASD symptoms and the TT genotype may have evolved an ability to upregulate OXT levels to help or try to compensate for the cerebral social response skills deficiency caused by the OXTR polymorphism. The higher amounts of OXT will help to improve facial processing and human interpersonal contact. As showed in previous study D LoParo et al 2015 showed oxytocin receptor gene rs2268491 (‘T’ allele is risk-inducing) in the meta-analysis, these SNPs were shown to be substantially related with ASD, implying that signals from these SNPs may represent a shared connection with ASD. Liu x, et al. 2010 showed two SNPs (rs2268491 and rs2254298), which were significantly associated with ASD in there study in which re 2268491 with allele C predominant. 2. Criterion 4. “Conclusions are presented in an appropriate fashion and are supported by the data.” Authors have to revise the conclusion of their study to be based on study findings (e.g., “The measurement of peripheral OXT levels and OXTR genetic alterations provide a simple dual-biomarker test to gauge social functioning in the ASD patient for diagnostic, monitoring and support purposes”; however, this study didn’t investigate monitoring and support aspects). Other suggested implications/recommendations based on study findings/conclusion should be separated from the conclusion itself. Answer: Done. The final conclusion paragraph has been amended, the implications/recommendations of the study have been presented as such from lines 485 3. Criterion 5. “The article is presented in an intelligible fashion and is written in standard English.” a. (1) Table 1: better to show ASD and controls in columns and variables as rows. Please, provide other important data, such as gender and comorbid conditions. Answer. Done. Table 1 was corrected as requested by the reviewers. b. (2) The article contain several errors related to English language, spelling, and grammar. PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. We may reject papers that do not meet these standards. If the language of a paper is difficult to understand or includes many errors, we may recommend that authors seek independent editorial help before submitting a revision. Done. 4. Criterion 6. “The research meets all applicable standards for the ethics of experimentation and research integrity.”. The authors repeat the ethics statement twice, once under study populations “Formal parental consent was obtained from families who expressed interest in the study, who were provided with consent sheets and a questionnaire to complete” and under Ethical approval “All patients were informed about the purpose of the work, and a written consent obtained from each patient.”. Authors should provide this information once. Moreover, did they obtain consent from the patients “children?”, parents, or both? Answer: In fact, written consent was taken from the father of the ASD child only who is in charge and responsible for any matter regarding the ASD patients according to the societal customs in Iraq. The ethics statement has been updated as recommended. Please refer to paragraph lines 272 Criterion 7. “The article adheres to appropriate reporting guidelines and community standards for data availability.” a. (1) In the abstract and discussion, authors seem to selectively report some findings in an inappropriate manner, such as “study characteristics in the ASD population revealed a high level of consanguinity (36.66%)” although the percentage of consanguinity was higher among control group (40%). Answer: The sample size might be behind this variation which shows that the percentage of consanguinity was higher among control group. Much larger sample sizes will facilitate a clear picture on these variations. The consanguinity findings are now fully reported in the results and discussion sections lines 282 and 403: • Results: The rate of consanguinity among the Arabic study populations was high at 36.66% and 40% in the ASD and healthy control groups (Table 1). • Discussion: Given that the consanguinity rate worldwide is 10.4% (7), the rates observed in both the ASD and control study populations are at least three fold higher. b. (2) The authors stated that “all data are fully available without restriction” but they did not provide unidentified patients’ data. Authors are required to make all data underlying the findings described fully available, without restriction, and from the time of publication. PLOS allows rare exceptions to address legal and ethical concerns, but this has to be completely explained. Please, review PLOS Data Policy. Done. All data is available as supplementary material as Supplementary S1 Appendix. 5. Data sharing statement. Answer: All the data cannot be made publically available as a results of local institution policy. However, it can be made available on a reasonable request from the corresponding author or from the department of pathology and forensic medicine (third party). Contact information of the department of pathology and forensic medicine is: Email: pathology.med@uokufa.edu.iq (email included in the cover letter) 6. Please amend either the title on the online submission form (via Edit Submission) or the title in the manuscript so that they are identical. Amended 7. PLOS PDF (phrase data not shown). Done. This phrase has been removed, and the OXT and patient age Supplementary Fig 1 is now included. Attachments Attachment Submitted filename: answer to reviewers.docx https://doi.org/10.1371/journal.pone.0265217.r002
2 Feb 2022 Decision Letter - Elsayed Abdelkreem, Editor PONE-D-21-31116R1The oxytocin receptor gene polymorphism rs2268491 and serum oxytocin alterations are indicative of autism spectrum disorder: A case-control paediatric study in Iraq with personalized medicine implications PLOS ONE Dear Dr. McAllister, 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. Besides the important reviewers' comments shown below, authors are requiried to address the following points: (1) Previous comment: Authors should clarify sampling method and justify sample size. Authors’ response: (lines 126 paragraph manuscript) literature searches were performed to assess the prevalence of autism spectrum disorder. An estimated 1 % of the world’s population has autism spectrum disorder, with recent figures of 1.5%. Since the prevalence of the disease is a relatively uncommon event at 1 out of 100 cases, a case control study was selected as the most efficient sampling method to investigate autism among the Iraqi Arab population. A study sample size of N=120 (60 ASD and 60 controls) was feasible to manage in the face of opposition to the study from local tribal and religious restrictions. Our study had a 3:1 male to female ratio in the ASD and control populations. The DSM-5 states that “autism spectrum disorder is diagnosed four times more often in males than in females.”(American Psychiatric Association 2013), and most recent research suggest that it is closer to 3:1 (Loomes, Hull, and Mandy 2017). Therefore the study population is reflective of the gender ratio in ASD and provides a useful model to explore specific genetic associations. New comment: Please, provide calculation of the sample size that would be required for this study. If the number enrolled in this study satisfy the calculated sample size, this would be perfect. If not, you can acknowledge this as one of the study limitations and provide justifications, including feasibility issues. (2) Previous comment: Did the author consider confounders “e.g., the Median age of control is lower than that of ASD, which may affect serum OXT level”. Authors response. Ages were matched between the ASD and control group. There were no significant differences in age distribution between studied and control group P=0.708 (Table 2). New comment: Thank you for your important clarification. However, as shown in Table 2, the mean age for controls is lower than that of patients, and the p-value may become significant if larger number was included. This underscores the importance of sample size calculation. (3) Previous comment: It is interesting to see that healthy controls had highest OXT levels in the CC genotype, while ASD subjects had the highest OXT in the TT genotype; it would be meaningful to explain/discuss such findings. Authors’ response. Done (Discussion, main manuscript). In stratified ASD patients, serum oxytocin levels were higher in the mild category with the polymorphism genotype TT. This suggests that patients with milder ASD symptoms and the TT genotype may have evolved an ability to upregulate OXT levels to help or try to compensate for the cerebral social response skills deficiency caused by the OXTR polymorphism. The higher amounts of OXT will help to improve facial processing and human interpersonal contact. As showed in previous study D LoParo et al 2015 showed oxytocin receptor gene rs2268491 (‘T’ allele is risk-inducing) in the meta-analysis, these SNPs were shown to be substantially related with ASD, implying that signals from these SNPs may represent a shared connection with ASD. Liu x, et al. 2010 showed two SNPs (rs2268491 and rs2254298), which were significantly associated with ASD in there study in which re 2268491 with allele C predominant. New comment: Thank you for these important discussions/thoughts . However, authors are encouraged to expand the discussion of this point in the main text (please, integrate the rest of your response, including the references, in the main text). (4) Previous comment: In the abstract and discussion, authors seem to selectively report some findings in an inappropriate manner, such as “study characteristics in the ASD population revealed a high level of consanguinity (36.66%)” although the percentage of consanguinity was higher among control group (40%). Authors’ response: The sample size might be behind this variation which shows that the percentage of consanguinity was higher among control group. Much larger sample sizes will facilitate a clear picture on these variations. The consanguinity findings are now fully reported in the results and discussion sections lines 282 and 403. •Results: The rate of consanguinity among the Arabic study populations was high at 36.66% and 40% in the ASD and healthy control groups (Table 1). •Discussion: Given that the consanguinity rate worldwide is 10.4% (7), the rates observed in both the ASD and control study populations are at least three fold higher New comment: Thank you for your important clarifications. Please, integrate your comments regarding sample size in the main text. (5)Other comments Please, provide the age of study participants in table 1 (with other characteristics) rather than table 2. It is not necessary to provide the p-value for the Kolmogorov-Smirnov test in table 2. Instead, just report this in the table footnote or results section. Authors are encouraged to acknowledge and discuss study limitations and generalizability of findings. Please submit your revised manuscript by Mar 19 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, Elsayed Abdelkreem, MD, PhD 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 #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 #2: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? 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 #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 #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 #2: Title: The oxytocin receptor gene polymorphism rs2268491 and serum oxytocin alterations are indicative of autism spectrum disorder: A case-control paediatric study in Iraq with personalized medicine implications The manuscript is precisely written and it presents an excellent point that might help in the suggestion of personalized precision intervention strategy. I recommend the acceptance but only after major revision. From my experience the number of samples is satisfactory, however, - Most recent prevalence of ASD should be cited: E.g.: Bougeard Clémence, Picarel-Blanchot Françoise, Schmid Ramona, Campbell Rosanne, Buitelaar Jan. Prevalence of Autism Spectrum Disorder and Co-morbidities in Children and Adolescents: A Systematic Literature Review, Frontiers in Psychiatry, 12, 2021 DOI=10.3389/fpsyt.2021.744709 - The significantly high oxytocin in mild ASD patients compared to moderate and severe should be explained and supported by other studies if possible. - Individuals with autism do not outgrow autism (This sentence is not clear please clarify or delete) “Ignoring early and accurate diagnosis of this disorder might lead to secondary disorders such as depression and anxiety (Reference is needed). Please see the reference below: Hollocks MJ, Lerh JW, Magiati I, Meiser-Stedman R, Brugha TS. Anxiety and depression in adults with autism spectrum disorder: a systematic review and meta-analysis. Psychol Med. 2019 Mar;49(4):559-572. doi: 10.1017/S0033291718002283. Epub 2018 Sep 4. PMID: 30178724. - “These results suggest the ROC curve could become the gold standard for the identification of parameters that are sensitive and specific enough to support ASD diagnosis”. ROC curves are already known as excellent statistical tool in the field of biomarkers, so this statement should be corrected. - The authors stated that “ within the stratified ASD population in this study, the highest OXT levels occurred in the mild subgroup, while the lowest OXT levels occurred in the severe group. This should be explained and supported at least with the fact that nasal oxytocin is recommended to decrease the severity of ASD symptoms (Support is mandatory). - The significant difference between mild, moderate and severe ASD should be clearly presented in the table not only in the text. Roc curves for mild, moderate and severe should be presented independently. - The authors mentioned that “This suggests that patients with milder ASD symptoms and the TT genotype may have evolved an ability to upregulate OXT levels to help or try to compensate for the cerebral social response skills deficiency caused by the OXTR polymorphism. The higher amounts of OXT will help to improve facial processing and human interpersonal contact” (Again support your suggestion). - Please go through the manuscript below it might help https://www.nature.com/articles/s41598-020-79109-0.pdf Horiai, M., Otsuka, A., Hidema, S. et al. Targeting oxytocin receptor (Oxtr)-expressing neurons in the lateral septum to restore social novelty in autism spectrum disorder mouse models. Sci Rep 10, 22173 (2020). https://doi.org/10.1038/s41598-020-79109-0 Gene expression analysis shows that Oxt mRNA is up-regulated in brain and bone which indicate that Oxt is adaptive and important in restoring the homeostasis of the body. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: Yes:** Afaf El-Ansary [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.0265217.r003
Revision 2
7 Feb 2022 Author Response Response to Reviewers PLOS Editor specific revisions: The authors would like to thank the PLOS editor for these helpful manuscript suggestions. • Previous comment: Authors should clarify sampling method and justify sample size. New comment to address: Please, provide calculation of the sample size that would be required for this study. If the number enrolled in this study satisfy the calculated sample size, this would be perfect. If not, you can acknowledge this as one of the study limitations and provide justifications, including feasibility issues. Response from authors: Methods, Lines (131-144) A sample size of N=36 was calculated as the minimal requirement to determine serum oxytocin alterations in the study cohort. The study variable of interest (peripheral oxytocin alterations) was identified as dichotomous (proportion). The sample size was calculated using the equation: N = 4 * Zα2 * p (1 − p)/w2. Where Zα is the confidence level, W is the width of the confidence interval and P is the study estimate of the proportion to be measured. The proportion of ASD patients who present with oxytocin alterations compared to controls was estimated at about 90% (S1 Appendix) with a selected confidence interval of ± 10 [16] N = 4 * 1.96 * 0.9 (1 − 0.9)/0.22 = 17.64 (18 cases of ASD) Minimum sample size for 1:1 case-control study cohort: N=36 Please note that a bigger sample size is ideal for estimating a single OXT SNP in a population (about N=250), this is discussed in the new study limitation section of the discussion (lines 508 paragraph). • Previous comment: Did the author consider confounders “e.g., the Median age of control is lower than that of ASD, which may affect serum OXT level”. New comment to address: Thank you for your important clarification. However, as shown in Table 2, the mean age for controls is lower than that of patients, and the p-value may become significant if larger number was included. This underscores the importance of sample size calculation Responses from authors: Thank you for raising the issue of confounders. The study is of a case control age and gender matched design which should reduce effects of these confounder variables. Although the mean age of patients with ASD is slightly higher - it is not significant (Table 1). The sample size calculations indicates that the study numbers are sufficient to investigate the variable of serum oxytocin. • Previous comment: It is interesting to see that healthy controls had highest OXT levels in the CC genotype, while ASD subjects had the highest OXT in the TT genotype; it would be meaningful to explain/discuss such findings. New comment: Thank you for these important discussions/thoughts. However, authors are encouraged to expand the discussion of this point in the main text (please, integrate the rest of your response, including the references, in the main text). Responses from authors: The discussion of this key finding of the how OXT levels can differ in relation to OXTR genotype has been reworked and expanded in relation to social functioning with references (lines 465 to 488) • New comment: Thank you for your important clarifications. Please, integrate your comments regarding sample size in the main text. Response: Done. Refer to the study limitation section of the discussion (lines 508 paragraph). • Please, provide the age of study participants in table 1 (with other characteristics) rather than table 2. Response: Done • It is not necessary to provide the p-value for the Kolmogorov-Smirnov test in table 2. Instead, just report this in the table footnote or results section. Response: Done • Authors are encouraged to acknowledge and discuss study limitations and generalizability of findings. Response: done. (Lines 507-519) ‘Ideally the conclusions presented in this study should be generalizable to the wider Iraqi Arab population. Therefore the study samples must be representative of the population and adequate in number (33). Feasibility issues in the Middle and South Euphrates population region limited recruitment of large sample sizes in the current study. Although the estimated study sample size was sufficient for the serum OXT analysis, numbers are still inadequate to generalize to the entire population. For example, consanguinity was high among the ASD populations as expected, but also the control group. The sample size might explain why the consanguinity proportions are higher among control patients. Sample size also affects the association between SNP markers and disease. Testing a single SNP marker in a 1:1 case control study requires 248 cases to achieve 80% statistical power according to the allelic genetic model, under certain assumptions (34). Insufficient study numbers are a limitation of the current investigation of SNP rs2254298. Reviewer 2: The authors would like to thank reviewer 2 for these helpful revisions. • Most recent prevalence of ASD should be cited: E.g.: Bougeard Clémence, Picarel-Blanchot Françoise, Schmid Ramona, Campbell Rosanne, Buitelaar Jan. Prevalence of Autism Spectrum Disorder and Co-morbidities in Children and Adolescents: A Systematic Literature Review, Frontiers in Psychiatry, 12, 2021 DOI=10.3389/fpsyt.2021.744709 Response: This is an excellent suggestion, we have updated our introductory prevalence of ASD literature, citing ‘recent estimates of 1.70 and 1.85% in US children aged 4 and 8 years respectively’ (lines 81-82) • The significantly high oxytocin in mild ASD patients compared to moderate and severe should be explained and supported by other studies if possible. Response: Done. Please refer to discussion lines 441- 457 • The trend for elevated OXT in this study agrees with other studies (14-16). However, the use of OXT as a biomarker of ASD is not straightforward, as these results are contradictory with those reporting lower OXT in ASD patients (10-12). However, within the stratified ASD population in this study, the highest OXT levels occurred in the mild subgroup, while the lowest OXT levels occurred in the severe group. Our study found that ASD severity categories had statistically significant (p=0.032) difference in OXT levels. This trend does agree with the previous study of ASD children in Iraq (12). Here OXT level were highest in mild autistic patients with a significant decrease (p<0.05) in moderate autistic patients, and a highly significant decrease (p<0.01) in severe autistic patients compared with control. Interestingly, this variability in OXT levels in stratified ASD patients reported both Iraqi studies may relate to a key finding of the Stanford study (27). The US investigators noted that pretreatment blood OXT concentrations also predicted response to intranasal OXT treatment(27). Those individuals with the lowest pretreatment OXT concentrations showed the greatest social improvement (27). Therefore, the ASD severe patients with lowest OXT in our study could be suitable candidates for individualized intranasal therapy with OXT to boost their social functioning. Individuals with autism do not outgrow autism (This sentence is not clear please clarify or delete) Response: Done. Sentence is updated to: ‘Autism is a lifelong neurodevelopmental disorder’. • “Ignoring early and accurate diagnosis of this disorder might lead to secondary disorders such as depression and anxiety (Reference is needed). Please see the reference below: Hollocks MJ, Lerh JW, Magiati I, Meiser-Stedman R, Brugha TS. Anxiety and depression in adults with autism spectrum disorder: a systematic review and meta-analysis. Psychol Med. 2019 Mar;49(4):559-572. doi: 10.1017/S0033291718002283. Epub 2018 Sep 4. PMID: 30178724. Response: Done. Thank you for the reference suggestion. • “These results suggest the ROC curve could become the gold standard for the identification of parameters that are sensitive and specific enough to support ASD diagnosis”. ROC curves are already known as excellent statistical tool in the field of biomarkers, so this statement should be corrected. Response: Done. ROC curves are now discussed as an existing ‘gold standard statistical tool (lines 432) • The authors stated that “within the stratified ASD population in this study, the highest OXT levels occurred in the mild subgroup, while the lowest OXT levels occurred in the severe group. This should be explained and supported at least with the fact that nasal oxytocin is recommended to decrease the severity of ASD symptoms (Support is mandatory). Response: Done. (Lines 451-456). The US investigators noted that pretreatment blood OXT concentrations also predicted response to intranasal OXT treatment (27). Those individuals with the lowest pretreatment OXT concentrations showed the greatest social improvement (27). Therefore, the ASD severe patients with lowest OXT in our study could be suitable candidates for individualized intranasal therapy with OXT to boost their social functioning. • The significant difference between mild, moderate and severe ASD should be clearly presented in the table not only in the text. Roc curves for mild, moderate and severe should be presented independently Response: Done. Figure 1 has been updated to show all ROC curves independently, including mild, moderate and sever (Fig 1C-E). • The authors mentioned that “This suggests that patients with milder ASD symptoms and the TT genotype may have evolved an ability to upregulate OXT levels to help or try to compensate for the cerebral social response skills deficiency caused by the OXTR polymorphism. The higher amounts of OXT will help to improve facial processing and human interpersonal contact” (Again support your suggestion). Please go through the manuscript below it might help https://www.nature.com/articles/s41598-020-79109-0.pdf Horiai, M., Otsuka, A., Hidema, S. et al. Targeting oxytocin receptor (Oxtr)-expressing neurons in the lateral septum to restore social novelty in autism spectrum disorder mouse models. Sci Rep 10, 22173 (2020). https://doi.org/10.1038/s41598-020-79109-0 Gene expression analysis shows that Oxt mRNA is up-regulated in brain and bone which indicate that Oxt is adaptive and important in restoring the homeostasis of the body. Response: Done. Suitable discussion material (lines 484-487) has been added from the suggested paper. ‘A recent mouse ASD model study demonstrated that targeting OXTR-expressing neurons in the lateral septum restores social skills (31). The results of the current study and previous research (27, 31) suggest that OXT is adaptive and can restore the homeostasis of the body. Attachments Attachment Submitted filename: response to reviewers 070222.docx https://doi.org/10.1371/journal.pone.0265217.r004
28 Feb 2022 Decision Letter - Elsayed Abdelkreem, Editor The oxytocin receptor gene polymorphism rs2268491 and serum oxytocin alterations are indicative of autism spectrum disorder: A case-control paediatric study in Iraq with personalized medicine implications PONE-D-21-31116R2 Dear Dr. McAllister, 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, Elsayed Abdelkreem, MD, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? 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 #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 #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 #2: Thanks for your response to my previous comments The authors answered my comments and use the provided references to modify the discussion accordingly and I think the manuscript is suitable for publication. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: Yes:** Afaf Kamal El-Din El-Ansary https://doi.org/10.1371/journal.pone.0265217.r005
Formally Accepted
7 Mar 2022 Acceptance Letter - Elsayed Abdelkreem, Editor PONE-D-21-31116R2 The oxytocin receptor gene polymorphism rs2268491 and serum oxytocin alterations are indicative of autism spectrum disorder: A case-control paediatric study in Iraq with personalized medicine implications Dear Dr. McAllister: 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. Elsayed Abdelkreem Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0265217.r006

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