-->PONE-D-25-57492-->-->Distinct Gut and Oral Microbiome Patterns Associated with Dyslexia in a Family-Based Cohort-->-->PLOS One

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Academic Editor

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Additional Editor Comments:

General comments:

Kindly, remove the repetitive speech in the manuscript about the sample size and the limitations. Only include it in the limitation and conclusion section.

Abstract:

Kindly, rewrite the abstract making it structured.

The aim of the study needs to be clarified in the abstract

Introduction and rational

Some issues needs clarification in the introduction and rational section

Why the authors compared the dyslexia with the CAS?- Kindly clarify your rational

Methods:

Why the number of saliva samples is different from fecal samples? Have some patients refused to give fecal samples? Were some samples spoilt?- Kindly, clarify these points in the methods section

Did you get the saliva and fecal samples from the same patient?

The authors mentioned the categorization of patients including undetermined phenotyping and neurotypical. Do the authors mean that 18 individual have symptoms and the others did not have? Could you clarify this point more for the reader in the manuscript?

The definitions of phenotype, neurotypical and alpha and beta diversity is better to be included to make it easier for the reader to catch up the meanings.

The results:

The results contain parts that looks like discussion. The standard is to display the results with a short comment about the trend if needed to express the meaning of the results. After that, you can discuss the results in details and compare with others using supporting references. Therefore, kindly, rewrite the result section based on these recommendations and omit the repeated parts.

The authors mentioned the mechanistic pathway results. Kindly, mention the importance of this mechanistic pathway briefly in the introduction section and add a sentence that reveals that you will assess it in the corresponding part in the methods section.

Limitation and conclusion:

Thanks for including the limitations and discussing them explicitly.

Add the inter-sex variability and ethnic variability to the limitations

Make the conclusion more concise and avoid repetition. Although the study is novel and important, Avoid speech that is overestimating of the results importance.

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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: Partly

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: 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: No

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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: Yes

Reviewer #2: 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: Major Comments

1. Clarify the Study Design and Cohort Composition

The abstract mentions a “family-based cohort,” but the relationships between dyslexia, CAS, and neurotypical participants are not explicitly stated.

Suggestion: Specify how many individuals belonged to each group and whether participants with dyslexia and CAS were from the same families or separate families.

2. Distinguish Between Dyslexia and CAS Groups More Clearly

The study groups appear uneven (fecal n=19, saliva n=29), making it unclear how many samples belonged to each diagnostic category.

Suggestion: Provide a brief breakdown (e.g., “X dyslexia, Y CAS, Z neurotypical”).

3. Interpretation of Findings Needs More Caution

The enrichment of Alistipes A 871400 communis is interesting, but the text risks implying a causal or mechanistic link.

Suggestion: Reinforce that the associations are correlative and the sample size is small.

4. More Detail About the Analytical Methods

Many tools and databases are listed, but the abstract does not explain why multiple databases were used or whether consistency across them was evaluated.

Suggestion: Briefly mention whether the enrichment or diversity patterns were robust across databases.

5. Clarify the Saliva vs. Fecal Microbiome Findings

The abstract notes differences in fecal microbiome profiles but is vague regarding saliva findings.

Suggestion: Clarify whether saliva microbiomes showed group-level differences or not.

Minor Comments

6. Precision in Terminology

“Participants with CAS histories” is unclear—does this mean persistent CAS, resolved CAS, or a history of speech delay?

Suggestion: Define the diagnostic category more precisely.

7. Improve Flow and Readability

The abstract is dense with methods, abbreviations, and technical references.

Suggestion: Streamline by focusing on the most important methodological points and moving details (e.g., specific database names) to methods in the full paper.

8. Grammar / Wording Adjustments

“remain underexplored” → “remains underexplored” (subject: microbiome).

“patterns compared to neurotypical family members and participants with CAS histories” — the comparison could be clearer if rephrased as two explicit comparisons.

9. Consider Briefly Describing Functional Results

PICRUSt2 analysis is mentioned, but no functional findings are described.

Suggestion: Add 1–2 words on whether functional predictions aligned with taxonomic differences.

Reviewer #2: Thank you for the opportunity to review this manuscript.

I have reviewed the manuscript entitled “Distinct Gut and Oral Microbiome Patterns Associated with Dyslexia in a Family-Based Cohort.” This is an exploratory study that addresses an interesting question using paired fecal and saliva 16S rRNA (V4) data. The overall concept is timely, and the hypothesis-generating framing is appropriate. However, for the work to be publishable as a technically robust exploratory report, several sensitivity analyses and methodological clarifications are needed primarily because (i) there appears to be substantial imbalance in age and collection site across comparison groups, and (ii) the family-based sampling introduces non-independence that must be accounted for in inferential analyses.

In my view, the key requirements are:

1. Explicitly addressing age/site confounding;

2. Accounting for clustering at the family/household level;

3. Tempering species-level interpretations from 16S V4 and clarifying classifier training;

4. Expanding reporting on negative controls/contamination and QC;

5. Ensuring metadata availability is sufficient for reproducibility; and

6. Aligning the strength of conclusions with the exploratory design and inference limits (including PICRUSt-based functional predictions).

The study is thoughtfully designed in several respects, including the paired gut/oral sampling, the focus on a family-based cohort, and the use of multiple reference databases. With the revisions below, the manuscript would be substantially stronger in technical validity and interpretability.

Major comments

1) Age and site imbalance (key confounding)

Microbiome composition is strongly influenced by age and by environmental/site factors. The current group structure appears meaningfully imbalanced by age and collection site, and these variables could plausibly explain a non-trivial portion of the observed separation. Please add sensitivity analyses that directly test robustness to these factors.

At minimum, I recommend considering:

• Stratified analyses, e.g., adult-only and child-only subsets where feasible;

• Models that include age and site as covariates (even if power is limited, reporting effect direction and magnitude is informative); and/or

• An approach to reduce imbalance, such as matched subsets or excluding the most extreme strata.

If these analyses are not feasible, the manuscript should substantially soften phenotype-linked interpretations and emphasize that findings are cohort-specific, exploratory signals.

2) Family-based clustering (non-independence)

Because participants are related and may share household/environment, observations are not independent. For PERMANOVA and differential abundance analyses, the family/household structure should be incorporated explicitly for example:

• Restricted permutations/stratified permutations for PERMANOVA; and

• Where feasible, mixed-effects or otherwise cluster-robust approaches for differential abundance.

If mixed-effects modeling is not feasible due to sample size, a practical alternative is to report sensitivity checks such as leave-one-family-out analyses (or excluding influential family clusters) to demonstrate that key signals are not driven by a small number of families. In all cases, please include a clear statement describing pseudo-replication risk and how it was mitigated.

3) Species-level reporting from 16S V4 and database dependence

Species-level assignments from 16S V4 can be unstable and highly database-dependent. Since the manuscript already indicates differences across reference databases, I recommend making genus-level (or higher) interpretations the primary biological narrative, and clearly labeling any species-level findings as exploratory.

Please also clarify classifier training for each database, including whether reference sequences were trimmed to the exact amplicon region used in this study. If species-level highlights are retained, avoid diagnostic or causal phrasing, and consider explicitly noting that confirmation would require shotgun metagenomics and/or targeted qPCR in future work.

4) Negative controls and contamination/QC reporting

Given the small cohort and multi-step wet-lab pipeline, transparent reporting of extraction blanks, PCR negatives, and contamination screening is essential. Please add a dedicated Methods paragraph specifying:

• Which negative controls were included (and at what stages);

• QC thresholds used; and

• How potential contaminants were assessed and handled.

If negative controls were not included, this should be stated prominently, and low-abundance taxa findings should be interpreted conservatively.

5) Data availability and reproducible metadata

Sharing reads and code is a strong step. To fully support reproducibility, please ensure that a de-identified metadata table required to reproduce the primary analyses is accessible, including at minimum: age, sex, collection site, phenotype variables, and an anonymized family/household ID. If some metadata must remain controlled, please describe a formal access pathway with clear criteria and governance.

6) Conclusions and strength of language

The Discussion should consistently match claim strength to the exploratory design and to the limitations imposed by confounding and clustering. Please avoid wording that implies diagnostic utility, therapeutic targeting, or causal directionality. For PICRUSt-based functional predictions, emphasize that these represent inferred functional potential rather than measured function, and add brief interpretive cautions (and any quality/coverage indicators you can report).

Minor comments

1. Please correct minor typographical issues and use consistent statistical notation (e.g., “q = …”). Ensure consistent formatting for taxonomic names (italics for genus/species) and consistent naming of software/tools.

2. Please explicitly state which feature table level was used for alpha diversity, beta diversity, and PERMANOVA (ASV-level vs taxonomically collapsed tables), as this choice can affect results.

Overall assessment

Overall, this work has promise as a hypothesis-generating contribution. Addressing the confounding structure, family clustering, and reporting clarifications above would substantially improve technical soundness and better align the presented evidence with the manuscript’s conclusions.

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Reviewer #1: Yes: ahmed alshewered

Reviewer #2: No

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Distinct gut and oral microbiome patterns associated with dyslexia in a family-based cohort

PONE-D-25-57492R1

Dear Dr. Wright,

Dear Authors

I am delighted to inform you that your article is provisionally accepted for publication.  However, minor corrections need to be done before publication.

-The title identifies the work as a preliminary exploratory study.Add this sentence to the title: "a preliminary exploratory study".

-The Abstract and Conclusion explicitly state that the findings are hypothesis-generating. Add this sentence to the abstract" "This preliminary exploratory study should be considered hypothesis-generating."

-The limitations section clearly emphasizes the unresolved issues of family clustering, site confounding, and lack of contamination controls. Add this sentence to the discussion: "Given the potential confounding by collection site, family clustering, and the absence of laboratory negative controls, these findings require validation in larger independent cohorts."

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. For questions related to billing, please contact billing support.

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,

Shimaa Mohammad Yousof, Msc, M.D., Ph.D

Academic Editor

PLOS One

Additional Editor Comments

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)

Reviewer #4: All comments have been addressed

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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

Reviewer #4: Yes

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

Reviewer #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: Yes

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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: Thank you for the invitation to review the revision.

The authors have addressed several reporting and transparency concerns; however, important methodological limitations remain, particularly unresolved confounding by site/phenotype, unaccounted family-level clustering, absence of negative controls, and limited reproducibility of metadata. These should be explicitly reflected in the Results, Discussion, Limitations, Abstract, and Data Availability Statement before further consideration.

1. The authors state they examined age and collection site, and acknowledge geography as a meaningful source of variation, but they do not clearly report adjusted models, matched analyses, or stratified sensitivity analyses. Since phenotype and site appear to overlap strongly, this remains a major interpretive limitation.

2. Family/household clustering remains insufficiently handled. The original concern was non-independence due to related participants and shared household/environment. The authors acknowledge this and explain that mixed-effects modelling was not feasible. Still, they also did not provide restricted PERMANOVA, leave-one-family-out analyses, or cluster-robust sensitivity checks. This is probably the most important unresolved methodological issue.

3. Authors now state that no extraction blanks or PCR-negative controls were included. This is transparent, but it means formal contaminant detection could not be performed. For a small microbiome study, this substantially weakens confidence in low-abundance and species-level findings.

4. Data availability may still be incomplete. Authors say reads and scripts are available, but the data availability statement indicates that deidentified demographic information “can be requested,” rather than being fully publicly available. This may conflict with PLOS ONE’s preference for complete public availability unless an ethics-based restriction is justified.

5. Some wording still risks overstatement. Phrases such as “distinct faecal and saliva microbiomes” and “key taxa associated with dyslexia” are acceptable only if consistently framed as exploratory, cohort-specific, and non-causal. I would recommend replacing “key taxa” with “candidate taxa” or “taxa showing exploratory associations.”

Reviewer #4: Wright and colleagues investigated fecal and salivary microbiome diversity, composition, and functional potential using 16S rRNA gene amplicon sequencing to explore potential developmental and etiological links in childhood apraxia of speech (CAS) and dyslexia. The manuscript reports that individuals with dyslexia exhibit distinct fecal microbiome diversity patterns compared to those with CAS, along with dyslexia-associated functional pathways in the fecal microbiome, whereas no functional differences were observed in the salivary microbiome. In addition to genetic predisposition studies, this work provides an interesting perspective suggesting that microbial communities along the oral–gut–brain axis may be associated with dyslexia and CAS, potentially influencing neurodevelopment and neural activity.

However, as this is an exploratory study, the sample size is limited, which may lead to overinterpretation and should caution against inferring causality.

The manuscript appropriately acknowledges and addresses several key methodological concerns:

• The cohort composition has been clarified by explicitly describing family relationships, and the issue of non-independence due to family-based clustering has been acknowledged, with an emphasis on the need for replication in independent cohorts.

• The authors recognize that causal inference is not possible given the limited sample size and appropriately highlight the need for larger cohorts and more comprehensive analytical approaches in future studies.

• To avoid overinterpretation, the results have been appropriately reframed as correlational, and functional findings are clearly presented as predictive rather than causal.

Overall, the authors have thoughtfully addressed the major and minor concerns raised, improving the clarity, rigor, and interpretability of the manuscript while appropriately maintaining an exploratory and hypothesis-generating framework.

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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.

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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: Yes: Sonu Bhaskar

Reviewer #4: No

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