-->PONE-D-25-67348-->-->Human α-Galactosidase A is Stimulated by Folic Acid Supplementation – Possible Implications in Fabry Disease Management-->-->PLOS One

Dear Dr. Junaid,

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 current study is interesting and contribute largely in inventing new treatment for Fabry Disease, however, the methods and results are not sufficient to support the conclusion.  Three experts raised several issues to be clarified.  Please revise extensively to draw the conclusion.-->-->

Please submit your revised manuscript by Mar 25 2026 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Tatsuo Shimosawa, M.D., Ph.D.

Academic Editor

PLOS One

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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: Yes

Reviewer #3: Partly

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

Reviewer #1: I Don't Know

Reviewer #2: I Don't Know

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: No

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: No

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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 manuscript describes a possible interesting effect of folic acid on alpha-galactosidase A expression and activity, which could be helpful for the treatment of Fabry Disease.

Numerous experiments were performed to support the conclusions, however some of them are not methodologically precise or not enough described, therefore these aspects have to be clarified, emended or at least some methodological deficiency has to be mentioned as a limitation.

In particular:

There is no mention to the fact that N-Acetyl-galactosamine was added as a reagent in the activity tests performed in all cell lysates. This compound is used to exclude the contribution of the alpha-gal isoform B (or alpha-N-Acetylgalactosaminidase Ec 3.2.1.49 encoded by NAGA) to the enzymatic activity, since the two enzyme are homoulogus in their sequence and share the same substrate. Ideally, lysate activity assays should be repeated in the presence of N-Acetyl-galactosamine, however, since the experiments on the purified alpha-Gal A activity also show an effect of folic acid, it is assumable that the effect on the cell lysates could also be genuinely due to folic acid action on the correct isoform, but the limitation of the assays performed in cell lysate should be mentioned.

As a consequence, the sentence in page 10 line 205, in which author says that data obtained in lymphoblasts are "highly specific", should be avoided because the effect of the most important isoform of the enzyme was not excluded, according to the draft.

Authors also state that they have performed similar experiments in other lysosomal enzyme with no effect on folic acid. These experiments are not included in the manuscript, presumibly to simplify the reading and center the attention on the relevant results, but these experiments could be included as supplementary figures for transparency.

Statistical analysis was performed and it was indicated in the text which are the significant values, however not all the graphs include significant data representation and, more importantly, there is no mention in the whole manuscript on which tests were applied to analyze statistical significance, therefore statistical analysis accuracy cannot be reviewed.

It should also be clearly indicated in the text, which region of the enzyme or which exons are amplified in the m-RNA expression analysis and confirm that the primers are specific for GLA expressed isoform.

Methylation analysis of GLA could also be included as supplementary figure.

The figure relative to Western Blot data includes bands intensity quantification, but panel C is not mentioned in the caption. According to Western blot data, the quantification was performed on the bases of the relative expression of GLA compared to house keeping gene GAPDH, however it cannot be excluded that level of expression of GAPDH are also affected by folic acid, therefore quantification of additional house keeping genes or quantification of GLA relative to total protein load could have been performed.

It should be made more clear in the discussion that the presented experiments appoint to a double mechanism of folic acid to stimulate alpha-Galactosidase A production at the expression level and also determine a possible direct interaction with the protein which apparently does not involve the active site of the protein.

Other important limitations of the manuscript are correctly described by the authors (luck of in vivo effect, luck of data to support a clear mechanism such as the identification of the binding site for folic acid or the mechanism that support the increase in GLA m-RNA production).

Reviewer #2: I read with interest the article Human α-Galactosidase A is Stimulated by Folic Acid Supplementation – Possible Implications in Fabry Disease Management by Sabiha Khatoon.

The study reports the effect of a FA supplementation in augmenting AGLA enzymatic activity in-vivo and in a patients with Fabry disease.

Even if the study represents a news and an innovative trial to find further material for FD treatment , I believe that the sample results scarcely representative. The application of the FA supplementation in a single cell-line in a single patients (besides with a late-onset phenotype) represents an important limitation. It could be more interesting if the authors investigate also patients with missense mutation and classic phenotype.

Moreover, the results should better specify the exact percentage or n.of fold of increased AGAL level (compared to the 1.2 fold, 3% of DGJ).

Reviewer #3: General Comments

This study analyses the effect of folic acid supplementation on GLA/α-galactosidase A expression and enzymatic activity, and explores whether FA may enhance DGJ (migalastat)-mediated rescue in a Fabry disease lymphoblastoid cell line. Although the topic is innovative and potentially translationally relevant, several methodological issues should be addressed.

Specific revision comments

1) In Introduction (line 57–60), this paragraph is misleading; FD classification should follow clinical history, GLA gene analysis, α-Gal A assay, and blood accumulation of LysoGb3. (reference: Int. J. Mol. Sci. 2018, 19, 3726; doi:10.3390/ijms19123726). Please discuss this point, adding the reference.

2) In Introduction (also in Abstract), the aims and type of study should be clearly stated at the end of this section (e.g., experimental design, models used, and primary endpoints).

3) In Introduction (line 93–104), the paragraph summarizes the results of the study; therefore, it is out of context and should be erased or moved to the Results/Discussion section.

4) In Methods, AGLA activity is quantified by 4-MU fluorescence after stopping the reaction at pH 11. Since FA is a chromophore and can alter optical readouts and/or quench/enhance fluorescence depending on conditions and may also affect micro-pH or ionic strength, please provide more details regarding this issue.

5) In Methods, the term “Quantitative RT-PCR” should be better defined, specifying quantification method (e.g., ΔΔCt), amplification chemistry, primer efficiencies, reference gene strategy, and whether the plotted metric is truly “fold change” (including how normalization was performed).

6) In Statistics, this section is not sufficient. Authors state: “Each experiment was performed in triplicates. Values are presented as means ± SEM.” However, multiple figure captions say values are medians of three independent experiments with SEM bars. Additionally: Are the “triplicates” technical replicates within one culture, or independent biological replicates? Why are medians used with SEM ? What statistical tests were used, given multiple FA concentrations compared to baseline? Please clarify n, define what is meant by “independent experiments,” adopt consistent summary statistics, and report the full statistical workflow.

7) In Results, Authors state several lysosomal enzymes are unaffected, referring to Table 1. However, Table 1 is only a list of enzymes, not the quantitative activities, variability, or statistical comparisons. Please provide enzyme activity values (or normalized activities) across FA doses with statistics, at least for the most relevant comparator enzymes.

8) In Results, please report disease-relevant substrates, such as Gb3 and/or lyso-Gb3, to support biological relevance beyond enzyme activity.

9) In Discussion, only one FD lymphoblastoid line is tested (male with the Asn215Ser variant and mild manifestations). Considering the wide spectrum of FD, these results cannot be generalized to all FD populations. Please acknowledge this limitation, nuancing the discussion and clearly delimiting the scope of inference.

10) In Conclusion (also in Abstract), Authors state no methylation differences in one CpG island. However, this is insufficient to support conclusions about epigenetic mechanisms. Please nuance the conclusions based on the actual scope of methylation analysis (single region interrogated) and avoid broader mechanistic claims unless additional data are provided.

11) In Competing Interest Statement, Authors state “no competing interest to declare” but also state “Patent Applied For” with a USPTO application number. In addition, the submission form text indicates “no specific funding,” while the Acknowledgments say the study “was supported by funds” from a state office. Please verify disclosures.

12) Figures, the quality should be improved.

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Reviewer #1: Yes: Saida Ortolano

Reviewer #2: No

Reviewer #3: No

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-->PONE-D-25-67348R1-->-->Human α-galactosidase A is stimulated by folic acid supplementation – possible implications in Fabry disease management-->-->PLOS One

Dear Dr. Junaid,

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 04 2026 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 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.

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As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only the individual author can complete the verification step; PLOS staff cannot verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

Tatsuo Shimosawa, M.D., Ph.D.

Academic Editor

PLOS One

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If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

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Additional Editor Comments:

Almost all the concerns were clarified, however, one expert has comments to be answered.

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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: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: 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 #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

-->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 #2: Yes

Reviewer #3: No

**********

-->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 #2: Yes

Reviewer #3: No

**********

-->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: I believe that the comments from the reviewers were well addressed and the manuscript improved considerably is quality, however in the statistical analysis author say that they have performed a student t-test, while the comparison of more than two conditions should be done by ANOVA, parametrics or non parametric test, depending on the case. This change won't change the general finding and will be more accurate.

Reviewer #2: the revised form of the article has reached almost of reviewer' suggestions and comments and is now suitable for publication

Reviewer #3: The authors have satisfactorily addressed all the concerns I had with the previous submission and made the necessary changes in the manuscript.

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Reviewer #1: Yes: Saida Ortolano

Reviewer #2: No

Reviewer #3: No

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-->

Human α-galactosidase A is stimulated by folic acid supplementation – possible implications in Fabry disease management

PONE-D-25-67348R2

Dear Dr. Junaid,

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,

Tatsuo Shimosawa, M.D., Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

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

**********

-->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

**********

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

Reviewer #1: Yes

**********

-->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

**********

-->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

**********

-->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: Authors applied all the suggested improvements to the submitted article. The statistical analysis was correctly modified.

**********

-->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 #1: Yes: Saida Ortolano

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