Epigenetic clock analysis and increased plasminogen activator inhibitor-1 in high-functioning autism spectrum disorder

Satoshi Okazaki; Ryo Kimura; Ikuo Otsuka; Yasuko Funabiki; Toshiya Murai; Akitoyo Hishimoto

doi:10.1371/journal.pone.0263478

Peer Review History

Original SubmissionOctober 21, 2021
20 Dec 2021 Decision Letter - Tadafumi Kato, Editor PONE-D-21-33732Epigenetic clock analysis in high-functioning autism spectrum disorderPLOS ONE Dear Dr. Hishimoto, 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. Two reviewers assessed the paper, and gave important suggestions.The authors are requested to revise the manuscript considering all the points raised by the referees.Please submit your revised manuscript by Feb 03 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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Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: No Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In this manuscript, the authors investigated epigenetic age acceleration associated with autism spectrum disorder (ASD) using two independent datasets from blood samples consisting of adult patients with high-functioning ASD and controls. As results, they could not find significant difference in epigenetic age acceleration between ASD and controls in both cohorts. On the other hand, they found a significant increase of DNA methylation-based plasminogen activator inhibitor-1 (DNAmPAI-1) level in individuals with ASD compared to controls in the 1st cohort but not in the 2nd cohort. Finally, they performed a meta-analysis of DNAmPAI-1, and confirmed the significant increase of DNAmPAI-1 in ASD. Furthermore, they discussed the association PAI-1 and ASD. The content of this manuscript was not suitable for the title and objective. If they focus the association PAI-1 and ASD, they should change the title and objective of this manuscript. If they focus epigenetic age acceleration associated with ASD, they should perform meta-analysis of epigenetic clock analysis and discuss the differences between the previous report which found the epigenetic age acceleration of ASD (McEwen LM, et al., 2020) and their study. In addition, they should consider other factors affected the alteration of DNA methylation such as medical condition (ie. metabolic problems), prescription, real smoking condition and so on. Reviewer #2: The authors investigated whether biological aging is observed in adult high-functioning ASD individuals based on age prediction from genome-wide DNA methylation profiles using DNAs extracted from whole blood. The participants in the two independent cohorts (1st: 38 ASD cases vs. 31 controls; 2nd: 6 vs. 10) were Japanese, non-smokers, recruited from a single university hospital, had no comorbidity, no psychotropic medication in at least three months prior to blood collection, and were phenotyped appropriately on the standardized assessment tools. The controls were recruited from the same site and were matched for age, sex, and IQ. The methylation analysis were also performed using the well-documented method, and the statistics were processed appropriately. As a result, they observed a non-significant trend in GrimAge acceleration and a significant difference in DNAmPAI-1 levels between ASD and control groups in the 1st cohort but not in the 2nd cohort. The meta-analysis showed significantly increased DNAmPAI-1 levels in individuals with ASD compared to controls. Although this study has some limitations such as small sample size and lack of plasma PAI-1 levels, to the best of my knowledge, it is the first report of epigenetic clock analysis of whole blood DNA in ASD. 1, P3 L12 "HorvahAge" -> "HorvathAge" 2, P16 L5 The fact that there are no racial differences in the samples is also a strength, so it would be good to describe it in the method. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: Yes:** Takafumi Shimada [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.0263478.r001
Revision 1
7 Jan 2022 Author Response We greatly appreciate the review of our paper entitled “Epigenetic clock analysis and increased plasminogen activator inhibitor-1 in high-functioning autism spectrum disorder”: Manuscript ID PONE-D-21-33732_R1”. The comments from the reviewer were helpful to improve and polish our paper. We have revised our paper according to the comments and marked added texts made in the revised manuscript in red for better readability. Point by point replies are below for the reviewers. Reviewer #1: In this manuscript, the authors investigated epigenetic age acceleration associated with autism spectrum disorder (ASD) using two independent datasets from blood samples consisting of adult patients with high-functioning ASD and controls. As results, they could not find significant difference in epigenetic age acceleration between ASD and controls in both cohorts. On the other hand, they found a significant increase of DNA methylation-based plasminogen activator inhibitor-1 (DNAmPAI-1) level in individuals with ASD compared to controls in the 1st cohort but not in the 2nd cohort. Finally, they performed a meta-analysis of DNAmPAI-1, and confirmed the significant increase of DNAmPAI-1 in ASD. Furthermore, they discussed the association PAI-1 and ASD. The content of this manuscript was not suitable for the title and objective. If they focus the association PAI-1 and ASD, they should change the title and objective of this manuscript. Response: Thank you for your valuable comment. We revised the title as follows: - Epigenetic clock analysis and increased plasminogen activator inhibitor-1 in high-functioning autism spectrum disorder (Page 1) If they focus epigenetic age acceleration associated with ASD, they should perform meta-analysis of epigenetic clock analysis and discuss the differences between the previous report which found the epigenetic age acceleration of ASD (McEwen LM, et al., 2020) and their study. Response: Thank you for your important pointing. As you indicated, McEwen LM, et al previously reported that epigenetic age is accelerated in children with ASD. Our results appear to be inconsistent with theirs. They used the Pediatric-Buccal-Epigenetic clock based on buccal epithelium cells. In contrast, we used other epigenetic clocks based on blood samples. The differences between our and their findings may be due to difference in the type of tissues used for the development of each epigenetic clock. We revised the Discussion as follows: - McEwen LM, et al previously reported that epigenetic age is accelerated in children with ASD [13], which is inconsistent with our present findings. They used the Pediatric-Buccal-Epigenetic clock based on buccal epithelium cells. In contrast, we used other epigenetic clocks based on blood samples. The differences between our and their findings may be due to difference in the type of tissues used for the development of each epigenetic clock. (Page 14) - Finally, only blood samples were analyzed. Future studies using other tissue and cell types, such as neuronal/glial cells and buccal epithelium cells, are required. (Page 16) In addition, they should consider other factors affected the alteration of DNA methylation such as medical condition (ie. metabolic problems), prescription, real smoking condition and so on. Response: Thank you for your valuable comment. We additionally performed correlation analysis between epigenetic age accelerations/PAI-1 levels and IQ, ADOS, and ASSQ-R scores in individuals with ASD. We found that GrimAge acceleration was significantly positively correlated with ADOS total in both the 1st cohort (rho = 0.415, p = 0.0131) and the 2nd cohort (rho = 0.886, p = 0.0333), but did not show the Bonferroni correction level (corrected significance was defined as p-value < 0.05/5 = 0.01) in either cohort. We have revised the manuscript as follows: - The relationship between continuous variables was analyzed with Spearman’s rank correlation coefficient. (Page 9) - We additionally performed correlation analysis between epigenetic age accelerations/PAI-1 levels and IQ, ADOS, and ASSQ-R scores in individuals with ASD. We found that GrimAge acceleration was significantly positively correlated with ADOS total in both the 1st cohort (rho = 0.415, p = 0.0131) and the 2nd cohort (rho = 0.886, p = 0.0333), but did not show the Bonferroni correction level (corrected significance was defined as p-value < 0.05/5 = 0.01) in either cohort (S4 Fig). (Page 13) - We additionally observed a modest significant correlation between GrimAge acceleration and ADOS total in both the 1st and 2nd cohorts; however, these results did not show Bonferroni correction level. Therefore, it was not possible to draw a definitive conclusion. Further studies with a larger sample size are required. (Page 14) - S4 Fig. Relationship between epigenetic clock acceleration/PAI-1 and IQ, ADOS, and ASSQ-R scores in individuals with ASD. Scatter plots show epigenetic clock acceleration/PAI-1 vs. IQ, ADOS, and ASSQ-R scores. The relationship was analyzed with Spearman’s rank correlation coefficient. ADOS, the Autism Diagnostic Observation Schedule; ASD, autism spectrum disorder; ASSQ-R, the high-functioning Autism Spectrum Screening Questionnaire; CTL, control; DNAmTL, DNA methylation-based telomere length; IQ, intelligence quotient; PAI-1, plasminogen activator inhibitor-1. (Page 21) Response: Regarding other medical conditions, our samples have no real smoking history and no use psychotropic medication. We revised the Methods as follows. Unfortunately, we don't have any information about other diseases or metabolic problems including body mass index. We added the limitation in the Discussion as follows. - We excluded participants with an IQ < 80, additional psychiatric/neurologic disorders, other medical disorders, history of cigarette smoking, or use of psychotropic medication for at least three months before the blood collection in both ASD and control groups. (Page 7) - Fourth, information about other diseases or metabolic problems such as body mass index was lacking. (Page 16) Reviewer #2: The authors investigated whether biological aging is observed in adult high-functioning ASD individuals based on age prediction from genome-wide DNA methylation profiles using DNAs extracted from whole blood. The participants in the two independent cohorts (1st: 38 ASD cases vs. 31 controls; 2nd: 6 vs. 10) were Japanese, non-smokers, recruited from a single university hospital, had no comorbidity, no psychotropic medication in at least three months prior to blood collection, and were phenotyped appropriately on the standardized assessment tools. The controls were recruited from the same site and were matched for age, sex, and IQ. The methylation analysis were also performed using the well-documented method, and the statistics were processed appropriately. As a result, they observed a non-significant trend in GrimAge acceleration and a significant difference in DNAmPAI-1 levels between ASD and control groups in the 1st cohort but not in the 2nd cohort. The meta-analysis showed significantly increased DNAmPAI-1 levels in individuals with ASD compared to controls. Although this study has some limitations such as small sample size and lack of plasma PAI-1 levels, to the best of my knowledge, it is the first report of epigenetic clock analysis of whole blood DNA in ASD. 1, P3 L12 "HorvahAge" -> "HorvathAge" Response: Thank you for your comment. We revised the Abstract as follows: - ... HorvathAge, ... (Page 2) 2, P16 L5 The fact that there are no racial differences in the samples is also a strength, so it would be good to describe it in the method. Response: Thank you for your valuable comment. We revised the Methods as follows: - All participants were of Japanese descent and there are no racial differences in the samples. (Page 7) Journal Requirements: 2. You indicated that you had ethical approval for your study. In your Methods section, please ensure you have also stated whether you obtained consent from parents or　guardians of the minors included in the study or whether the research ethics committee or IRB specifically waived the need for their consent. Response: Our cohorts consist of adult patients with high-functioning ASD and controls. We revised the Methods as follows: - Briefly, we used two independent cohorts consisting of adult patients with high-functioning ASD and controls; the 1st cohort comprised 38 ASD cases and 31 controls, and the 2nd cohort was six ASD cases and 10 controls. (Page 7) Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0263478.r002
20 Jan 2022 Decision Letter - Tadafumi Kato, Editor Epigenetic clock analysis and increased plasminogen activator inhibitor-1 in high-functioning autism spectrum disorder PONE-D-21-33732R1 Dear Dr. Hishimoto, 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, Tadafumi Kato 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 #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: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: (No Response) ****** 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: (No Response) ****** 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: (No Response) ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) Reviewer #2: (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: No Reviewer #2: Yes:** Takafumi Shimada https://doi.org/10.1371/journal.pone.0263478.r003
Formally Accepted
24 Jan 2022 Acceptance Letter - Tadafumi Kato, Editor PONE-D-21-33732R1 Epigenetic clock analysis and increased plasminogen activator inhibitor-1 in high-functioning autism spectrum disorder Dear Dr. Hishimoto: 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. Tadafumi Kato Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0263478.r004

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