-->PONE-D-26-20888-->-->Advancing mental health and well-being of Nigerian children through public health screening for Fragile X disorders (CHAMP-FX): a prospective multicentre screening study with longitudinal follow-up-->-->PLOS One

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“Randi Hagerman (RH) reports research grants from Zynerba/Harmony for a cannabidiol (CBD) trial in Fragile X syndrome, and from Shionogi & Co., Ltd. for a zatolmilast study, awarded to UC Davis Medical Center.

RH also serves on advisory boards for Shionogi and Harmony Biosciences and is a member of the Clinical Trials Committee of the National Fragile X Foundation.

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-->Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.-->

Reviewer #1: Yes

Reviewer #2: Partly

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-->3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.-->

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

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(Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: Reviewer comments

06/06/2026

ID: PONE-D-26-20888

Title: Advancing mental health and well-being of Nigerian children through public health screening for Fragile X disorders (CHAMP-FX): a prospective multicentre screening study with longitudinal follow-up

Dear Editor and Authors,

Thank you for giving me the opportunity to read the present work and evaluate the study. I found the protocol quite interesting to read regarding this prospective study in Nigeria advancing pediatric neurodegenerative disease evaluation in the African continent. I believe the present work is quite strong, rigorous, and provides a strong foundation for this multi centred study around Nigeria. In this ongoing study I wish the present authors the best of luck.

This manuscript addresses an important gap in Fragile X diagnosis and public-health genetics in Nigeria and has several strengths, including a multicentre design, planned DBS-based molecular screening, and integration of counselling/referral pathways. The methods used in the study are also described in sufficient detail and quite rigorously which aligns with PLOS One’s goals of reproducible and transparent research.

Despite this I have a few comments that I believe the author should address to make this work a publishable protocol. Kindly find my comments below.

Major Comments

- Please include the word “Protocol” in your title as per PLOS One recommendations.

-Line 170, please ensure the formula is displayed correctly, as it seems to have lost correct display. I would also recommend saying that the sample size “was” calculated instead of “will” be calculated.

- Line 201 please elaborate what the validated workflow is. This section (200 DNA extraction and quality assessment) needs to be more thorough for a protocol.

-The use of STROBE is mentioned in the appendix. Please ensure that you also mention that findings will be reported as per STROBE in the methods section.

- The phrase “where feasible” is too weak for transfer of identifiable or genetic data. The protocol should state the actual minimum required security practices.

- The discussion says the study will generate “robust data,” but given purposive sampling and n = 102, “foundational” or “pilot” data would be more accurate.

- The sample-size calculation is a major weakness. The authors use P = 0.023, then apply a finite population correction using records from a single centre: 12 autism cases every 5 months at NAUTH, estimated as 28.9 per year. This produces an adjusted minimum sample size of 15.7, after which the study selects 17 participants per zone, totalling 102. The value P = 0.023 appears to relate to autism prevalence rather than the expected prevalence of FMR1 expansion among the study’s target population. If the primary outcome is Fragile X full mutation/premutation positivity, the sample-size calculation should be based on the expected FMR1 positivity rate or on desired precision around that estimate. The finite population correction is not appropriate as presented. The denominator is based only on NAUTH autism clinic records, not on the six-site target population and not on all included diagnoses, namely ID, ASD, and GDD. With n = 102, if the expected FMR1 expansion rate is low, the number of positive cases may be very small. The planned association testing and possible logistic regression may therefore be infeasible. The statistical plan says logistic regression “may be explored for correlates of screen positivity if event counts permit,” but this needs a clearer decision rule and a more cautious framing. The authors should provide either a precision-based calculation for prevalence/proportion estimation (which would lead to an inclusion population much higher than 102) or explicitly describe this as a feasibility/pilot screening study rather than a robust prevalence study. The present proposal, regardless, will provide an idea regarding the prevalence of Fragile X in Nigeria.

Minor Comments

-Line 115 “In” should not be capitalized.

-Line 123 please expand CHAMP-FX upon first use, even if the expansion is there in the abstract.

- I recommend moving the “objectives” section to be part of the material and methods (specifically moving the main and specific objectives bullets) while adding a more narrative and small paragraph at the end of the introduction to briefly explore the objectives. This is just for ease of readership and to highlight the specific objectives as part of the methods.

-Line 168 please consider changing the last sentence to: “See Supplementary Material S1 for the Informed Consent Form.”

- Line 185 and 186 please be consistent with capitalization.

- Line 193 seems to have language structural issues. Please fix.

- Line 196 it would be a good idea to specify the temperature of the refrigeration.

- Line 197 please specify what you mean by courier service in the context of Nigeria. Do you mean it will be shipped by mail or special delivery? Etc.

- You can use the abbreviation NTC for No-template control in line 213 as this may be more familiar to some readers.

- Line 215 please expand the word UCD.

- Line 216 a full stop is needed before this sentence “PCR…”.

- Line 240 expand upon first use.

- Line 241 “;” can be replaced with “.”.

-Line 244 is missing a full stop.

- Line 246 needs to be replaced to the following: “using Stata (16.0; StataCorp LLC, College Station, TX, US)”.

-Line 247 please cite the declaration of Helsinki.

- The consent for publication declaration is unnecessary.

- DSM-5 and other diagnostic criteria are mentioned in the consent form. It would be a good idea to mention them as inclusion criteria in the main manuscript.

- I would encourage the authors to be more detailed with the laboratory workflow.

Additionally, I recommend that the authors produce a graphical abstract outlining the workflow of the proposed study. This is not necessary per se; however, I believe it would strengthen the work and allow for a better graphical abstract representation of how the overall study will be conducted. Whether the authors decide to produce or not produce a graphical abstract does not personally affect my recommendation for publication.

Once again thank you for giving me the opportunity to read the present work. I recommend that the present work is published following these changes, and wish the authors the best of luck in conducting this study.

I acknowledge that I do not have any conflicts of interest. The present peer review is of my own expertise and artificial intelligence was not consulted.

Reviewer #2: Thank you for the opportunity to review this interesting paper. This manuscript presents a multicentre protocol for screening Fragile X disorders among Nigerian children with neurodevelopmental conditions, addressing an important evidence gap in sub-Saharan Africa. The manuscript is generally well structured but requires major revision to improve clarity, writing quality, and methodological presentation before publication.

In general, the manuscript should maintain a consistent protocol-writing style. The text occasionally shifts between protocol, grant, implementation, and advocacy language, particularly in the abstract, discussion, conclusion, follow-up, and timeline sections.

Under introduction

Maintain consistent terminology for Fragile X conditions throughout the manuscript. Standardize the use of terms such as Fragile X syndrome, Fragile X disorders, and FMR1-related disorders. Additionally, revise the sentence beginning "Despite global advances…" for grammar, style, and capitalization, removing the unnecessary capitalized "In."

Under objectives

Objective number 4, which stated, (To develop a context-appropriate model for integrating Fragile X screening into paediatric/developmental health services in Nigeria.)

I have noticed that the methods do not describe how the integration model will be developed. Every study objective should correspond to a defined methodology. Therefore, I suggest to describe the methodology for developing the model (e.g., stakeholder consultation, implementation evaluation, consensus methods) or revise the objective to reflect feasibility assessment.

Under methodology

Study Design: the statement: “This approach enables the study to track outcomes over time, assess intervention impacts…”The term “intervention impacts” may be misleading because this is primarily an observational screening protocol rather than an intervention trial. Therefore, replace with “assess longitudinal screening outcomes, referral processes, and implementation feasibility.”

Study Population: Children aged 1–18 years attending paediatric neurology/paediatric clinics. Briefly state how autism spectrum disorder, intellectual disability, and global developmental delay diagnoses are verified.

Sample Size: Sample size derived from records at a single hospital. Please justify the use of patient records from one coordinating centre to estimate the sample size for a multicentre national study. Discuss any limitations of this approach and add a brief rationale explaining why local records were used and acknowledge the absence of national prevalence data.

Sampling Technique: “This pragmatic sample is intended to estimate prevalence…” Clarify that prevalence estimates apply to the selected high-risk clinical cohort rather than the general paediatric population.

Statistical Analysis: “Descriptive statistics will be done…” Use standard statistical terminology throughout.Replace “will be done” with “will be performed” and “frequencies/percentages will be calculated.”

Statistical Analysis: “Logistic regression may be explored…”Clarify under what circumstances regression modelling will be performed (e.g., sufficient number of positive cases).

Result disclosure, counselling, and follow-up

In the manuscript, it is mentioned that targeted therapy will be offered using metformin for participants diagnosed with FXS. As written, this section could be interpreted as moving from screening into therapeutic intervention without adequate explanation. The wording should be revised carefully to avoid ambiguity and to maintain a protocol-appropriate scope. Therefore, I suggest to clearly distinguishing:

•what is part of the research protocol,

•what is part of post-test counselling,

•what constitutes standard clinical referral,

•and what, if any, treatment is outside the study but may be discussed or offered in routine care.

Under discussion

The discussion should focus on the anticipated contribution of the protocol rather than expected outcomes. Since this is a protocol paper, statements regarding improved clinical outcomes, national impact, or service transformation should be presented as potential benefits rather than established or expected results.

This is where the manuscript shifts most from a protocol to advocacy.

Examples include statements such as:

•the study "will substantially improve care"

•the project "will transform diagnosis and management"

•the programme "will improve the mental health and well-being of Nigerian children"

•statements implying that the screening programme will be successfully scaled nationally.

It is important to keep in mind that the discussion should acknowledge limitations more directly. These likely include:

•relatively small sample size,

•purposive rather than population-based recruitment,

•recruitment from selected health facilities,

•limited generalisability to all Nigerian children,

•possible underrepresentation of children outside formal care pathways,

•and feasibility challenges for counselling and follow-up.

Under Conclusion

The Conclusion reads more like the conclusion of a completed study than a study protocol. Rewrite with more focus on emphasize the anticipated contribution of the proposed study rather than implying demonstrated effectiveness or future implementation success.

Under Appendix

Ensure all appendices and supplementary materials are cited at their first mention in the manuscript. Several supporting documents (e.g., data collection proforma, examination checklists, workflow flowchart, SOP, and project timeline) are not consistently referenced.For example, CHAMP-FX Workflow Flowchart, It is listed at the end of the PDF but not cited in the manuscript. Please cite it in the Laboratory Workflow section

(S1 Appendix)

The statement : “Fragile X syndrome is a genetic condition caused by changes in the FMR1 gene on the X chromosome.” This is accurate, but may be difficult for some participants. Consider a plainer explanation such as “Fragile X syndrome is an inherited condition caused by a change in a gene.”

The word “premutation” may not be understandable to non-specialists. It should either be explained in plain language or omitted from the consent form if not essential for participant understanding.

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Reviewer #1: No

Reviewer #2: No

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Attachment

Submitted filename: comments for PLOS ONE 2026.docx

Advancing Mental Health and Well-being of

Nigerian Children Through Public Health Screening for Fragile X Disorders (CHAMP-FX): Protocol of a prospective Multicentre Screening Study with Longitudinal Follow-up

PONE-D-26-20888R1

Dear Dr. Mbachu,

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.

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Kind regards,

David Chibuike Ikwuka, Ph.D.

Academic Editor

PLOS One

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