PONE-D-23-41162HYPOTHESIS GENERATION FOR RARE AND UNDIAGNOSED DISEASES THROUGH CLUSTERING AND CLASSIFYING TIME-VERSIONED BIOLOGICAL ONTOLOGIESPLOS ONE

Dear Dr. Layer,

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.

ACADEMIC EDITOR:

The main comments made by the reviewers on the manuscript about the Biological Ontology Cluster Classification (BOCC) tool can be summarized as follows:

1. Figures in the manuscript are not properly labeled.

2. Inconsistency in font face and clarity between text and images.

3. Lack of clarity on the novelty of the study needs highlighting in the introduction.

4. Absence of a literature review section to establish context and credibility.

5. The manuscript seems outdated due to the limited citation of recent works; it needs updating with suggested references made by the reviewers, which I suggest you incorporate in your article, such as the introduction section or literature review section.

6. Recommendations for proofreading to correct grammatical mistakes.

7. Suggestion to compare results with state-of-the-art studies for validation.

8. Absence of a discussion on the limitations of the study; recommended for inclusion in the conclusion.

9. Overall, the paper is well-written and suitable for the journal but lacks a conclusion section to summarize findings and implications.

This summary addresses the key areas the reviewers have identified for improvement, including technical presentation, content depth, literature context, and overall structure.

Please ensure that your decision is justified on PLOS ONE’s publication criteria and not, for example, on novelty or perceived impact.

For Lab, Study and Registered Report Protocols: These article types are not expected to include results but may include pilot data. 

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We look forward to receiving your revised manuscript.

Kind regards,

Roseline Oluwaseun Ogundokun, Ph.D.

Academic Editor

PLOS ONE

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Additional Editor Comments:

The main comments made by the reviewers on the manuscript about the Biological Ontology Cluster Classification (BOCC) tool can be summarized as follows:

1. Figures in the manuscript are not properly labeled.

2. Inconsistency in font face and clarity between text and images.

3. Lack of clarity on the novelty of the study needs highlighting in the introduction.

4. Absence of a literature review section to establish context and credibility.

5. The manuscript seems outdated due to the limited citation of recent works; it needs updating with suggested references made by the reviewers, which I suggest you incorporate in your article, such as the introduction section or literature review section.

6. Recommendations for proofreading to correct grammatical mistakes.

7. Suggestion to compare results with state-of-the-art studies for validation.

8. Absence of a discussion on the limitations of the study; recommended for inclusion in the conclusion.

9. Overall, the paper is well-written and suitable for the journal but lacks a conclusion section to summarize findings and implications.

This summary addresses the key areas the reviewers have identified for improvement, including technical presentation, content depth, literature context, and overall structure.

[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

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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: Yes

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: the study presents an innovative tool named Biological Ontology Cluster Classification (BOCC). This tool is aimed at assisting in the diagnosis of rare and undiagnosed diseases by identifying potential gene-to-phenotype (g2p) associations that are not explicitly documented in the current literature. Here are my comments on the manuscript:

1. All the figures are not properly labeled.

2. the manuscript has different font face. for instance, I dont know how I will classify the text/image between line 50 and 51. Is it text or image, if it is text, while did it have different front face and if it is image while is it not label?

3. I understand that BOCC is available as both a web application and a command-line tool, making it accessible for different types of users ranging from researchers to clinicians. However, what is the novelty of this study? the author is expected to highlights the contributions of this study towards the last paragraph of the introduction section.

4. I suggest that the author should create a section to discuss the literature review of some related work done in this area so as to enhance the credibility of this study.

6. Havn't checked this manuscript thoroughly, I observed that the author makes little efforts to cite and reference 2023 work. This act makes the manuscript looks outdated. Therefore, I will suggest that the author should make use of this following searched references to updated their manuscript:

1. Zhuang, Y., Jiang, N., Xu, Y., Xiangjie, K., & Kong, X. (2022). Progressive Distributed and Parallel

Similarity Retrieval of Large CT Image Sequences in Mobile Telemedicine Networks. Wireless

communications and mobile computing, 2022. doi: 10.1155/2022/6458350

2. Lu, G., Duan, L., Meng, S., Cai, P., Ding, S.,... Wang, X. (2023). Development of a colorimetric and

turn-on fluorescent probe with large Stokes shift for H2S detection and its multiple applications

in environmental, food analysis and biological imaging. Dyes and Pigments, 220, 111687. doi:

https://doi.org/10.1016/j.dyepig.2023.111687

3. Siyu Lu, J. Y. B. Y. (2023). Analysis and Design of Surgical Instrument Localization Algorithm.

Computer Modeling in Engineering & Sciences, 137(1), 669-685. doi:

10.32604/cmes.2023.027417

4. Zhu, Y., Huang, R., Wu, Z., Song, S., Cheng, L.,... Zhu, R. (2021). Deep learning-based predictive

identification of neural stem cell differentiation. Nature Communications, 12(1), 2614. doi:

10.1038/s41467-021-22758-0

5. Chen, L., He, Y., Zhu, J., Zhao, S., Qi, S., Chen, X.,... Xie, T. (2023). The roles and mechanism of

m6A RNA methylation regulators in cancer immunity. Biomedicine & Pharmacotherapy, 163,

114839. doi: https://doi.org/10.1016/j.biopha.2023.114839

6. Huang, H., Liu, L., Wang, J., Zhou, Y., Hu, H., Ye, X.,... Tang, B. Z. (2022). Aggregation caused

quenching to aggregation induced emission transformation: a precise tuning based on BN-doped

polycyclic aromatic hydrocarbons toward subcellular organelle specific imaging. Chemical

Science, 13(11), 3129-3139. doi: 10.1039/D2SC00380E

7. Huang, H., Wu, N., Liang, Y., Peng, X., & Shu, J. (2022). SLNL: A novel method for gene selection

and phenotype classification. International Journal of Intelligent Systems, 37(9), 6283-6304. doi:

https://doi.org/10.1002/int.22844

5. I suggest that the manuscript should be thoroughly proof-read to avoid some grammar mistake.

6. I recommend that the author should compare their result with the state-of-the-art studies so as to validate the strength of the obtained result.

7. What is the limitation of this study. This can be included in the conclusion section to give room for future research.

Reviewer #2: This manuscript is well written, introducing their BOCC tool. As they write, BOCC is a series of network-science-based methodologies that identify relevant clusters from a heterogeneous network comprised of HPO, STRING, OMIM, and Orphanet. I do not have any major suggestions for how to improve this paper, and it seems suitable for this journal venue.

Reviewer #3: The research addressed the topic of discuss but there are areas to be improved. The importance of literature review cannot be overemphasized. it is of great importance as it serves the purpose of establishing the context of a research study by elucidating the existing knowledge on the subject matter. This aids researchers in situating their work within the already established body of knowledge. Secondly, it allows researchers to identify gaps within the current knowledge base, which can subsequently form the foundation for research questions or hypotheses. Thirdly, a literature review supports the justification of research methodologies or methods by showcasing the manner in which prior studies were conducted and their contributions to the field. Therefore as observed that this important section is missing in the work, I suggest that it be included and relevant work be added to improve this research. I suggest the following work:

1. Luo, Y., Chen, D., & Xing, X. (2023). Comprehensive Analyses Revealed Eight Immune Related Signatures Correlated With Aberrant Methylations as Prognosis and Diagnosis Biomarkers for Kidney Renal Papillary Cell Carcinoma. Clinical Genitourinary Cancer, 21(5), 537-545. doi: https://doi.org/10.1016/j.clgc.2023.06.011

2. Gan, Y., Xu, Y., Zhang, X., Hu, H., Xiao, W., Yu, Z.,... Zheng, S. (2023). Revisiting Supersaturation of a Biopharmaceutical Classification System IIB Drug: Evaluation via a Multi-Cup Dissolution Approach and Molecular Dynamic Simulation. Molecules , 28(19), 6962. doi: https://doi.org/10.3390/molecules28196962

3. Fan, Z., He, Y., Sun, W., Li, Z., Ye, C.,... Wang, C. (2023). Clinical characteristics, diagnosis and management of Sweet syndrome induced by azathioprine. Clinical and Experimental Medicine, 23, 3581-3587. doi: 10.1007/s10238-023-01135-9

4. Wu, J., Fang, Z., Wang, X., Zeng, W., Zhao, Y., Jiang, F.,... Li, J. (2022). SLIT2 Rare Sequencing Variants Identified in Idiopathic Hypogonadotropic Hypogonadism. Hormone Research in Paediatrics, 95(4), 384-392. doi: 10.1159/000525769

5. Gong, T., Zhang, F., Feng, L., Zhu, X., Deng, D., Ran, T.,... Ji, X. (2023). Diagnosis and surgical outcomes of coarctation of the aorta in pediatric patients: a retrospective study. Frontiers in Cardiovascular Medicine, 10. doi: 10.3389/fcvm.2023.1078038

6. Fan, Z., He, Y., Sun, W., Li, Z., Ye, C., & Wang, C. (2023). Amoxicillin-induced aseptic meningitis: clinical features, diagnosis and management. European journal of medical research, 28(1), 301. https://doi.org/10.1186/s40001-023-01251-y

7. Jin, K., Gao, Z., Jiang, X., Wang, Y., Ma, X., Li, Y.,... Ye, J. (2023). MSHF: A Multi-Source Heterogeneous Fundus (MSHF) Dataset for Image Quality Assessment. Scientific Data, 10(1), 286. doi: 10.1038/s41597-023-02188-x

The introduction should include wht the work is contribution to the body of knowledge and conclude with section of the work.

The conclusion furnishes a brief overview of the primary discoveries and outcomes of the investigation, highlighting the significant implications of the study. It permits the researcher to contemplate on the degree to which the study has accomplished its objectives and whether the research inquiries have been addressed. Implications: The conclusion provides an occasion to deliberate upon the ramifications of the research findings, encompassing their pertinence to the research domain, practical implementations, and prospective influence.

I observed that there is no conclusion. I suggest this section be included.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PONE-D-23-41162R1HYPOTHESIS GENERATION FOR RARE AND UNDIAGNOSED DISEASES THROUGH CLUSTERING AND CLASSIFYING TIME-VERSIONED BIOLOGICAL ONTOLOGIESPLOS ONE

Dear Dr. Layer,

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.

I would like to sincerely apologise for the delay you have incurred with your submission. Due to the concerns raised about the process of evaluation of this manuscript, it was considered necessary to invite additional reviewers to provide comments on your study. Although some reviewers are happy with the revised version, other reviewers have raised remaining scientific concerns about the study that need to be addressed. Please revise the manuscript to address all the reviewer's comments in a point-by-point response in order to ensure it is meeting the journal's publication criteria. Please note that the revised manuscript will need to undergo further review, we thus cannot at this point anticipate the outcome of the evaluation process.

Please submit your revised manuscript by Jun 24 2024 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,

Miquel Vall-llosera Camps

Staff Editor

PLOS ONE

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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 #3: All comments have been addressed

Reviewer #4: All comments have been addressed

Reviewer #5: (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 #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Partly

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

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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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 #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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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 #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: 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 #1: The Author have attended to all the comments raised by the reviewer. Therefore, I recommend the manuscript for publication.

Reviewer #3: (No Response)

Reviewer #4: The study is novel and Article may be accepted. This research is fascinating and incredibly promising for advancing our understanding of rare diseases and providing potential diagnoses for patients who have struggled to find answers. The approach of integrating protein-protein interactions and phenotype relationships through networks and clustering is innovative and holds great potential for uncovering latent gene-to-phenotype connections. The use of databases like STRING and HPO, combined with the development of a tool like BOCC, marks a significant step forward in addressing the diagnostic challenges posed by rare diseases. The ability to identify significant clusters that correlate with known drug interactions is particularly exciting, as it opens new avenues for targeted therapeutic interventions.

Reviewer #5: The authors introduced a clustering method that can be used to discover the potential and novel gene-phenotype relationship. It is especially important for undiagnosed patients and unknown gene-phenotype relationships. The clusters reported by the method can be a guide to shortening the search space. Although the author addressed many questions raised by the reviewers in the first review process, the experimental setup and the clinical usage and validation are still unclear to me. Moreover, the author needs to do more proofreading as numerous mistakes are observed easily.

Major comments:

1. The formatting and language are usually relatively minor during review. However, multiple reviewers have already pointed it out in the first review. Several grammatical errors and formatting errors persist in the manuscript. I think the author should take it very seriously. I still observed many formatting issues, such as a missing space before the citation. For example, in page 2, “Matchmaker Exchange[2] and MyGene2[1]”. Many issues might be easily detected by Grammarly or other tools. I will recommend really finding an English editing service for the proofreading.

2. In page 2, these two sentences: “Using the Human Phenotype Ontology, we can expand the patient’s assigned phenotypes to include all closely related phenotypes and diseases. By connecting STRING and HPO [4] with the gene-to-phenotype (g2p) connections from Orphanet[5] and OMIM[6], we can then look for indirect associations between the patient’s assigned data points.”

HPO was not introduce as abbriveation after the first appearance. And the citation for HPO should be after the first appearance.

3. Page 3: “There is a trend toward using an ensemble of methods or methods capable of using higher-order patterns and integrating multiple types of networks (Table 1 ).” Redundant space before “)”.

4. Page 10: “Fig 4. A. Procedure for training and evaluating the XGboost model. Features were generated about each cluster (identified as described in Section )”, which Section? Is the Section number missing here? In page 11: the same issue, “After performing quality control (Section ), there were on average 401 variants per patient (Fig 6.C).”

5. The author refers g2p to gene to phenotype. Does the author use Phenotype in this paper as a feature (HPO term)? We often use phenotype to refer to disorders and describe HPO terms by features.

6. The author's definition of the undiagnosed patient in CHCO and MyGene2 is unclear. The first sentence of the result section states, “We applied BOCC to 721 patients from Children’s Hospital Colorado (CHCO) with suspected genetic drivers of their diseases.” Does that mean these patients are undiagnosed?

7. Moreover, in MyGene2 section, “We found that presently 111 of these profiles contained no direct connection between any of their genes and any of their HPO terms; we assumed these cases to still be undiagnosed.”, I think it is problematic. It is dangerous to assume the patient is undiagnosed because there is no connection between HPO and the gene. I randomly checked some patients in Table S1 and Table 5. I found some of the patients are already published in the literature, and I believe they are diagnosed. The first question is the patient with ID 3071 (https://mygene2.org/MyGene2/familyprofile/3071/profile). It is the first patient in Table S1. This patient was already published in a paper with PMID (25142838) and annotated with Kabuki and KMT2D. Does the author consider this patient as diagnosed or undiagnosed? For example, patient ID 877 (https://mygene2.org/MyGene2/familyprofile/877/genetic/gene) is already published in https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5082428/. This patient was also annotated with Joubert syndrome and a likely pathogenic mutation in B9D2. Besides, patient ID 1292 has likely pathogenic mutation in TNNT3 and was diagnosed as “Arthrogryposis, Distal, Type 1A”. And the author reported “Arthrogryposis multiplex congenita” as a new relationship to TNNT3. However, we can see from the disease name linked to TNNT3, which is “Arthrogryposis, distal, type 2B2,” and the entry in omim was updated in 2019. I would say this new relationship between this HPO and TNNT3 is not surprised at all because you can see it from the disease name. Therefore, I wonder what the meaning of this prediction is.

8. My main concern is that I don’t know how I validate wheter the connection found by the cluster is correct or not. The experiement should start from validate on the existing relationship from diagnosed patients. Then we know that the relationship found in the cluster do have some meanings. Then, we might select some patients with a disease gene found after the model is built. For example, the model trained on the data from 2019, then we can check whether the model can predict the relationship between the HPO and a Gene X, which was found as a disease-causing gene in 2020. As pointed out previously, when the definition of diagnosed and undiagnosed is unclear, it is very difficult to validate whether the cluster is meaningful or not, even though the experiments reported many significant p-values from different methods.

9. In the end, how the clinicians use this tool is unclear. There are many nodes in the graph, and many clusters were reported after the HPO and genes are given as input. It will be great to show more examples and the steps how the user should use. For example, I tried several HPOs linked to Cornelia de Lange syndrome (NIPBL gene), Long eyelashes (HP:0000527). Synophrys (HP:0000664) and Highly arched eyebrow (HP:0002553). However, I only found NIPBL is the third claster. I believe it is the key features for CdLS (NIPBL). Therefore, how do I interpret the first two clusters without NIPBL? The experiment and results showed that this method is working, statistically. However, how to interpret the results and validate the clinical meaning is unclear.

10. Will the gene in the same pathway or same phenotypic series shown in the same cluster?

11. In author’s first example, Patient 1930 with VUS in NBEA and SSPO. The author stated in the introduction that with more experiment to the associated phenotype in the clusters could help us to diagnose this patient. I believe the phenotype here is the HPO term. I wonder why we can use the new gene-to-hpo to solve the case with VUS? Does it contribute to the ACMG variant classification?

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Reviewer #4: Yes: Amit Joshi

Reviewer #5: Yes: Tzung-Chien Hsieh

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HYPOTHESIS GENERATION FOR RARE AND UNDIAGNOSED DISEASES THROUGH CLUSTERING AND CLASSIFYING TIME-VERSIONED BIOLOGICAL ONTOLOGIES

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Reviewer #5: Yes: Tzung-Chien Hsieh

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