Dear Dr. bao,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jun 08 2025 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.

• 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,

Jinhui Liu

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

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. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

3. Please include a separate caption for each figure in your manuscript.

Additional Editor Comments:

Authors should revise according to the suggestions of reviewers. The modifications should be marked. A point to point response letter is needed.

[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?

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: This study investigates the molecular mechanisms underlying Autism Spectrum Disorder (ASD) by exploring the interaction between CHD8—a high-risk gene in ASD—and the Notch signaling pathway. Using publicly available transcriptomic datasets, the authors performed a comprehensive bioinformatics analysis including differential expression analysis, enrichment analysis, protein-protein interaction (PPI) network construction, and regulatory network exploration via miRNAs and drug-gene interaction databases. Seven potential hub genes were identified, with IGF2 and CXCR4 highlighted as key players in ASD pathogenesis. The study is scientifically meaningful and methodologically sound, but several major issues need to be addressed before publication.

1.Please elaborate on why the Notch signaling pathway was prioritized for interaction analysis with CHD8. Citing studies that show CHD8 indirectly or directly regulates Notch components would support this choice.

2.Clarify whether adjusted p-values (FDR) were used to define DEGs. If not, please re-analyze using adjusted values to reduce false positives. Include a summary table of the top 20 DEGs with their log2 fold change, p-value, and annotation.

3. Add labels for representative genes on the volcano plots (e.g., IGF2, FN1, CXCR4). Include color scales and clustering dendrograms in the heatmaps for better interpretability.

4. In Figure 2, please provide actual gene counts or ratios involved in each GO term/pathway.

5.While several small-molecule compounds (e.g., AMD3100 and IGF-1R inhibitors) are identified, the therapeutic implications for ASD are not well explained.

6.The manuscript is generally understandable, but contains grammatical and stylistic issues. Consider professional English language editing to improve clarity and scientific tone.

Reviewer #2: Overall, this study provides valuable insights through a systematic bioinformatics analysis, identifying potential key genes associated with ASD and exploring their molecular mechanisms, making it of significant academic value. Despite some limitations in experimental validation, the innovation and proposed research directions offer valuable references for future studies. It is recommended that the authors further improve the manuscript based on the suggestions�

1.The specific processing and analysis process of the datasets GSE236993 and GSE85417 mentioned in the article is somewhat brief. It is suggested to provide a detailed description of each step of data processing in the supplementary materials to increase the transparency of the methods.

2.The methods section is not detailed enough, and the statistical analysis section lacks.

3.All the abbreviations should be explained when used the first time in the manuscript. In addition, if you can avoid any of the abbreviations, it is preferred to write only full text.

4.It will be more relevant if the authors can verify their key observations in vivo and in vitro with their own samples.

5.The discussion section is relatively brief, and it is suggested to have a more detailed discussion on the functions of each key gene, especially how they play a role in the Notch signaling pathway and neural development. In addition, further exploration should be conducted to address the limitations of current research, especially in areas where data sources and validation are insufficient, and to clearly identify which aspects need to be further supplemented in future research.

6.The authors should elaborate the difference between the current manuscript and previous similar published papers, and explain the advantages of current study.

7.Multiple grammar mistakes. English needs to be improved.

**********

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: 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.

Identification of Therapeutic Targets in Autism Spectrum Disorder through CHD8-Notch Pathway Interaction Analysis

PONE-D-25-15242R1

Dear Dr. bao,

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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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,

Jinhui Liu

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

I think this manuscript was well organized and it could be accepted.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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??>

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: (No Response)

Reviewer #2: The author has answered all the questions and this article is suitable for publication. I have no other questions

**********

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: No

**********