PONE-D-23-04707Molecular Investigation of TSHR gene in Bangladeshi Congenital Hypothyroid patientsPLOS ONE

Dear Dr. Qadri,

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.

The reviewers raised a number of important concerns, including incomplete or misleading interpretation of the data. Once I receive a revised version of your manuscript, I will send it back to at least one of the original reviewers to determine its suitability for publication in PLOS ONE.

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We look forward to receiving your revised manuscript.

Kind regards,

Dhanusha Yesudhas

Academic Editor

PLOS ONE

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Additional Editor Comments:

The manuscript require substantial modifications before considering for publication.

Please specify the region line 161, small molecule binding TM region.

The modeling of the TM region, author should consider using more than one computational tool to evaluate it, not only I-TASSER.

Since the whole work is relay on the model, the authors should have prepared the model from different tools subsequently compare and choose the most consistent one.

Table 4, write which one is acceptor and donor in proper format, the figure ligand is confusing, update it with mentioning of what are the numbers in braces. Same for table 5. It will be good to add one clear pic about the mutant position in the full length protein with more structural explanation (ref: https://www.ncbi.nlm.nih.gov/books/NBK279140/)

English editing is required.

Figure qualities need to be improved.

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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

Reviewer #2: No

Reviewer #3: Partly

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

Reviewer #1: N/A

Reviewer #2: N/A

Reviewer #3: No

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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

Reviewer #3: Yes

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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: No

Reviewer #2: No

Reviewer #3: Yes

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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: Line 68-69 needs a little bit of reconstruction. Line 78, exons should be in small letters. In line no. 81,the sentence starts with an And. Line 82, what does normally placed thyroid gland mean? Line 85, how can two molecules at the same time have the highest potency? Line 139 is a bit confusing. line 150 Magnesium chloride 's formula is wrong.

Reviewer #2: Overall, the study has several limitations and shortcomings that raise questions about its originality, accuracy, and clinical relevance.

Lack of originality: The docking analysis has already been done by another study "DOI: 10.1089/thy.2014.0119", which raises questions about the originality and novelty of this study. Additionally, the identification of the mutations and their location in the protein have already been reported in the ClinVar database "NM_000369.5(TSHR):c.1523C>T (p.Ser508Leu), further reducing the novelty of this study.

Incorrect information: The authors have incorrectly identified the location of one of the mutations (Asp727Glu) in the transmembrane region of the protein, when in fact it is located in the cytoplasmic region. This suggests a lack of attention to detail and accuracy in the study.

Small sample size: The study only includes 21 patients with dysgenesis, which is a relatively small sample size. This may limit the generalizability and statistical power of the findings.

Reviewer #3: The manuscript “Molecular Investigation of TSHR gene in Bangladeshi Congenital Hypothyroid patients” reports two mutations in the transmembrane region of TSHR gene in 21 patients. The transmembrane region of TSHR proteins (wild type and mutant) are modeled using I-TASSER. The authors did docking and molecular dynamic simulations for the two small molecule drugs with the wild type as well as mutants to understand the effect of mutations. The authors used autodock vina for docking and YASARA suits for the MD simulations.

Overall application of different computational and modeling tools looks logical for the subject of the manuscript, the obtained results lack proper scientific analysis. The work is clearly presented, however I have several significant questions. The crude values are mentioned in the text (Table 3) rather than the necessary information about the target (TPO protein) and template (TSHR protein ) alignment. As a reader, I am unable to understand the quality of the predicted model based on the information provided. The full length protein is essential to understand the function. Why is the full length of TSHR protein not modeled? The alignment information should be added and discussed. How many models were predicted by the I-TASSER tool. At least the user should discuss the top three models and explain the differences or similarities. Where all the models used to be built by the same template. What is the quality of the template structure? It is easy to get automated protein structure using multiple tools. If the target template alignment is low , it will be interesting to compare the models built by another popular tool to see the difference in the prediction.When author discusses the C-score, it will be helpful to the reader to explain what the score means.Also, whether the small molecules were built or download from the database. The reference or web link for PyRx software can be added. There is no information about the active site nor about how many docking clusters for each of the small molecules with the protein. Are there any other docking poses found if the observed author can discuss those. Adequacy of modeling and docking were not also evaluated that is specially important for modeled proteins.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PONE-D-23-04707R1Molecular Investigation of TSHR Gene in Bangladeshi Congenital Hypothyroid patientsPLOS ONE

Dear Dr. Qadri,

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 Aug 05 2023 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,

Dhanusha Yesudhas

Academic Editor

PLOS ONE

Additional Editor Comments:

The authors should pay more attention about the revised copy of the manuscript, the tracked word file is difficult to read and manage. Please make a clean copy word file highlighting the modifications.

Various part of the manuscript remains hard to read and lack of connection.

The authors mentioned about the addition of figures, but the figure remains same. Please update it accordingly.

[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 #3: (No Response)

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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 #3: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: I Don't Know

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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 #3: No

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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 #3: No

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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 #3: The writing part should be improved. Please improve the clarity and explain the necessary information in detail so that the paper explains independently. As a reader I am not convinced the author's writings are clear yet.

-What was the target template alignment percentage? Is it 30% or 70%? If the target template is very low, authors can submit distance guided protein structure https://zhanggroup.org/D-I-TASSER/. Nowadays, getting automated protein models in a few hours. Again, the authors didn’t mention the target template hence I cannot decide.

-Additional figures would have explained the target template alignment. e.g. Like the I-TASSER help page suggests https://zhanggroup.org/I-TASSER/example/ . The authors can add the figure to show the target template alignment (Page 47 for the section Prediction of 3D structure of TSHR protein and ligand selection).

- As pointed out by the other reviewer the author’s lack attention to detail. For instance in the revised manuscript the reference 8 points out to the I-Tasser reference instead of the previous high throughput study.(Introduction, Page 44, line 111).

- The author’s may submit the predicted protein and docking models in a public repository like https://figshare.com/ that might be useful to other researchers

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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 #3: No

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[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.

Molecular Investigation of TSHR Gene in Bangladeshi Congenital Hypothyroid patients

PONE-D-23-04707R2

Dear Dr. Qadri,

Thank you for submitting your work to PLOS ONE. I am happy to inform you that your paper "Molecular Investigation of TSHR Gene in Bangladeshi Congenital Hypothyroid patients" has been accepted for publication.

Regards,

Dhanusha Yesudhas

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Please look for the font changes, table alignment and figure resolutions before publication.

Reviewers' comments: