Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

This manuscript, entitled “Evolutionary Restructuring and Systematic Review of the NBPF Gene Family: Comparative Genomics, Functional Divergence, and Disease-Linked Pathways, represents a substantial and ambitious submission that integrates comparative genomics, phylogenetics, and disease association literature for one of the most complex duplicated gene families in the human genome. While the scope, data compilation, and overall scholarship are substantial, the manuscript in its current form does not yet meet the acceptance threshold for the Journal of Developmental Biology. It would almost certainly require a significant revision following peer review.

The most significant issue concerns the use of the PRISMA-S methodology. Although the manuscript explicitly states that PRISMA-S was applied, the reporting does not yet meet PRISMA-S standards. The search strategy lacks sufficient transparency, including complete database-specific search strings, explicit inclusion and exclusion criteria, language restrictions, and justification for including Google Scholar alongside curated biomedical databases. In addition, no formal PRISMA-S checklist is provided. Without these elements, reviewers are likely to conclude that the work does not qualify as a fully compliant systematic review, even though the literature coverage itself is extensive.

A second primary concern is over-interpretation of disease associations. Throughout the neurological, oncological, skeletal, metabolic, and rare disease sections, the manuscript frequently implies mechanistic or causal roles for NBPF genes where the underlying evidence is largely correlational, based on copy-number variation, expression changes, methylation patterns, or mutation frequency. Statements suggesting that Olduvai copy number “promotes accelerated neurogenesis”, that NBPF1 functions as a “metabolic switch”, or that specific NBPF genes act as “driver genes” in cancer go beyond what the cited evidence can support. Reviewers are likely to require systematic softening of this language and the explicit introduction of a limitations paragraph clarifying that most associations are indirect and that functional validation is absent for the majority of NBPF family members.

The phylogenetic analysis, while technically rich, also requires more apparent methodological justification. Multiple tree-building approaches are used across genomic DNA, cDNA, CDS, and protein sequences, including UPGMA and neighbour-joining methods, sometimes without bootstrap support. Although the authors acknowledge these limitations in places, the rationale for method selection and the interpretive boundaries of each tree type are not consolidated. In particular, the inferred placement of the pseudogene NBPF2P is explicitly described as speculative, yet is still discussed structurally. Reviewers will likely request a more explicit statement that phylogenetic conclusions are robust primarily at the cluster or lineage level rather than at fine-scale branching resolution.

Another issue relates to the breadth of the disease-association review. The manuscript covers an extensive range of pathologies, from neurodevelopmental disorders and cancers to diabetes, cardiac syndromes, and congenital malformations. While this breadth is impressive, it lacks a clear hierarchy of evidentiary strength. Without explicit weighting or categorization of associations based on the type and quality of evidence, reviewers may view the disease sections as descriptive catalogs rather than a critically structured synthesis. Reframing the disease tables as evidence-mapping tools rather than functional summaries would significantly improve scientific rigor.

The title and abstract also slightly overstate novelty. Claims regarding being the “first integrative synthesis” or the “first phylogenetic reconstruction including all NBPF members” are likely defensible but should be carefully qualified. Adding phrases such as “to our knowledge” and specifying reliance on current public annotations would reduce the risk of reviewer pushback without diminishing the manuscript’s contribution.

There are also minor editorial issues that, while unlikely to cause rejection on their own, should be addressed during revision. These include the visual density of phylogenetic figures, occasional inconsistency in gene name formatting, and the inclusion of preprints without clear labeling as non–peer-reviewed sources.

In summary, this manuscript is not acceptable in its current form. The primary barriers to acceptance are incomplete PRISMA-S compliance, over-interpretation of association data, and insufficient methodological consolidation in the phylogenetic analysis. We can correct these issues without altering the core scope or datasets. With a focused major revision that addresses the transparency of methodology, causal restraint, and evidentiary hierarchy, the manuscript would be well-positioned for acceptance after a single revision cycle.

Comments on the Quality of English Language

Professional editing recommended

Author Response

Dear Reviewer, Thank you for giving us the opportunity to submit a revised draft of our manuscript. We would like to thank you for your time and effort in providing detailed and insightful comments.  We have addressed all the concerns raised, and the changes have been incorporated into the manuscript. The revisions are highlighted in the document.  Below, we provide a point-by-point response to your comments.  Best regards,

 

Coment: The most significant issue concerns the use of the PRISMA-S methodology. Although the manuscript explicitly states that PRISMA-S was applied, the reporting does not yet meet PRISMA-S standards. The search strategy lacks sufficient transparency, including complete database-specific search strings, explicit inclusion and exclusion criteria, language restrictions, and justification for including Google Scholar alongside curated biomedical databases. In addition, no formal PRISMA-S checklist is provided. Without these elements, reviewers are likely to conclude that the work does not qualify as a fully compliant systematic review, even though the literature coverage itself is extensive.

Response: In this new version, we have included all changes proposed and attached a PRISMA-S checklist within the new submission.

 

Coment: A second primary concern is over-interpretation of disease associations. Throughout the neurological, oncological, skeletal, metabolic, and rare disease sections, the manuscript frequently implies mechanistic or causal roles for NBPF genes where the underlying evidence is largely correlational, based on copy-number variation, expression changes, methylation patterns, or mutation frequency. Statements suggesting that Olduvai copy number “promotes accelerated neurogenesis”, that NBPF1 functions as a “metabolic switch”, or that specific NBPF genes act as “driver genes” in cancer go beyond what the cited evidence can support. Reviewers are likely to require systematic softening of this language and the explicit introduction of a limitations paragraph clarifying that most associations are indirect and that functional validation is absent for the majority of NBPF family members.

Response: We softened our interpretation of the available data, but we wanted to show each author’s findings and hypothesis which could lead to feel like we are over-interpretating data when we are only reporting the article’s author’s conclusions. Now the article includes a limitations paragraph in lines 573-579.

Coment: The phylogenetic analysis, while technically rich, also requires more apparent methodological justification. Multiple tree-building approaches are used across genomic DNA, cDNA, CDS, and protein sequences, including UPGMA and neighbour-joining methods, sometimes without bootstrap support. Although the authors acknowledge these limitations in places, the rationale for method selection and the interpretive boundaries of each tree type are not consolidated. In particular, the inferred placement of the pseudogene NBPF2P is explicitly described as speculative, yet is still discussed structurally. Reviewers will likely request a more explicit statement that phylogenetic conclusions are robust primarily at the cluster or lineage level rather than at fine-scale branching resolution.

Response: We addressed the comment expanding the material and methods section to include further explanations on method selection and data curation and analysis. We also included a paragraph saying that phylogenetic conclusions are robust primarily at the cluster or lineage level rather than at fine-scale branching resolution as proposed by the reviewer in lines 313-316.

NBPF2P is a really convoluted pseudogene, it appears to be a segmental duplication of a gene-specific region of NBPF3. Which makes him impossible to analyze using neighbour joining methods since it does not have a shared region with all other NBPF genes. That is why we briefly discuss its apparition. Also, it does not have any disease or phenotype related to it.

Coment: Another issue relates to the breadth of the disease-association review. The manuscript covers an extensive range of pathologies, from neurodevelopmental disorders and cancers to diabetes, cardiac syndromes, and congenital malformations. While this breadth is impressive, it lacks a clear hierarchy of evidentiary strength. Without explicit weighting or categorization of associations based on the type and quality of evidence, reviewers may view the disease sections as descriptive catalogs rather than a critically structured synthesis. Reframing the disease tables as evidence-mapping tools rather than functional summaries would significantly improve scientific rigor.

Response: We thank the reviewer for this constructive observation. In response, we have revised Tables 2 and 3 to provide a clearer and more structured synthesis of the disease associations. Specifically, we have added two new columns—Type of Evidence and Strength of Association—to better reflect the quality and hierarchy of the available data. These additions allow readers to easily distinguish between associations supported by strong genetic or functional evidence and those based on preliminary or observational findings.

Coment: The title and abstract also slightly overstate novelty. Claims regarding being the “first integrative synthesis” or the “first phylogenetic reconstruction including all NBPF members” are likely defensible but should be carefully qualified. Adding phrases such as “to our knowledge” and specifying reliance on current public annotations would reduce the risk of reviewer pushback without diminishing the manuscript’s contribution.

Response:  We fully agree that claims of novelty should be carefully framed. However, after reconsidering the title, we believe that “Evolutionary Restructuring and Systematic Review of the NBPF Gene Family: Comparative Genomics, Functional Divergence, and Disease-Linked Pathways” accurately reflects the content and scope of the manuscript without overstating its originality. The term “systematic review” already conveys methodological rigor rather than novelty, and “evolutionary restructuring” refers to the comparative and phylogenetic analyses performed. To address the reviewer’s concern, we have nonetheless softened the wording in the abstract and main text, adding expressions such as “to our knowledge” and specifying that the conclusions are based on currently available public genome annotations, ensuring a balanced and accurate representation of the work.

Coment: There are also minor editorial issues that, while unlikely to cause rejection on their own, should be addressed during revision. These include the visual density of phylogenetic figures, occasional inconsistency in gene name formatting, and the inclusion of preprints without clear labeling as non–peer-reviewed sources.

Response: We improved the quality of some of the figures, corrected formatting inconsistencies and labeled preprints as non-peer reviewed sources (lines 363-366).

Author Response File: Author Response.pdf

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The authors have adequately addressed the suggestions I've made, and the manuscript appears to me to have improved. I have no further comments to add.

Author Response

We are grateful for the constructive comments and that the reviewer is satisfied with the revised manuscript.

Round 2

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

After reading the updated article in its entirety, it is clear that the authors have addressed most of the substantive reviewer's concerns. The work is now internally consistent, methodologically clear, and much more scientifically rigorous than prior versions. The scope and goals are clearly stated; the PRISMA-S framework is clearly explained and used consistently; and the phylogenetic analyses can now be repeated, with clear reasons given for choosing the method (UPGMA vs. neighbour-joining), the rooting strategy, the bootstrapping interpretation, and how to deal with pseudogenes and NOTCH2NL fusion contexts. Claims that were previously exaggerated have been largely moderated with appropriate cautionary language, especially in the disease-association sections, where the authors now distinguish genetic association from observational evidence and functional/mechanistic support and clearly indicate preliminary or limited findings. The discussion properly combines evolutionary, structural, and pathological aspects without mixing up CNV-level Olduvai effects with single-gene causation. The limitations section also properly notes that it is based on case reports, cohort heterogeneity, and incomplete functional validation. The remaining issues are relatively minor and do not detract from the main contribution. There are still some grammatical errors and stylistic redundancies, some repetition across disease subsections that could be lightly condensed, and a few references that rely on preprints or database annotations that would benefit from clearer contextual framing. However, these are more editorial than scientific problems. In general, the manuscript now gives a full, fair, and methodologically sound synthesis of the NBPF gene family. It also provides a plausible, well-supported evolutionary model based on PDE4DIP-derived duplications and a useful reference framework for future functional studies. The work is ready for publication with just a few minor changes, and I recommend it be accepted with only a few small changes.

Author Response

We sincerely thank the reviewer for this positive and constructive assessment. We have addressed the remaining minor comments by correcting grammatical and stylistic issues, lightly reducing redundancy in some disease-related sections, and clarifying the context of references based on preprints or database annotations. These small editorial changes further improve the clarity of the manuscript without affecting its scientific content. We appreciate the reviewer’s recommendation and thoughtful guidance throughout the review process.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript "Evolutionary Restructuring and Systematic Review of the NBPF Gene Family: Comparative Genomics, Functional Divergence, and Disease-Linked Pathways" presents a comprehensive attempt to elucidate the evolutionary history of the NBPF gene family. It focuses on the Olduvai (DUF1220) domains, their phylogenetic relationships, and their links to neurological and cancer-related diseases. As a peer-reviewed version (v1), it builds upon previous research by incorporating comparative genomics using Ensembl data, phylogenetic reconstructions utilizing tools such as ClustalW and Muscle, and a systematic review of illness links employing the PRISMA-S technique. Adding Pan troglodytes orthologs as comparators strengthens the evolutionary view. The proposed phylogenetic tree (Figure 5) provides a new insight into how gene duplication events may have originated with PDE4DIP (myomegalin). The discussion about domain subgroups (CON and HLS) and their relationship to human-specific adaptations, including brain size and cognitive impairments, aligns well with existing knowledge. This could be particularly useful for subjects such as developmental biology and neurogenetics.

The document, on the other hand, has numerous typos, formatting issues, and flaws from the OCR process that make it difficult to read and appear unprofessional. Gene names are written in different ways (for example, "NBFF," "NBBPF," "NBPT," and "NBPFF" instead of the usual "NBPF"), and dates are mixed up (for example, "Dec. 201.2. 13," "1. Dec. 201. 201. 13," which could be meant to be "J. Dev. Biol. 2025"). There are repeating numbers on every page (for example, "99"s on page 4, "181"s on page 9, and "201"s on page 11), which are probably caused by scanning problems. These numbers make it hard to read the content and signal that the document needs to be resubmitted cleanly. There are a lot of grammatical problems, like awkward phrasing ("the group made up of NBPFs and NBPF74 had a common ancestor similar to NBPF4") and sentences that are too short or missing words (like "The next division is visible in the form of two branches, one containing NBBPF7. NBBPF28..."). The techniques section mentions outdated alignment tools (ClustalW from 1994) without explaining why they are superior to newer ones, such as MAFFT or IQ-TREE. It also discusses bootstrap values for trees, but doesn't provide them in the text or pictures. The systematic review states that it follows PRISMA-S; however, it lacks a clear flow diagram or explicit search strings, which could make it challenging to reproduce.

While the evolutionary story is interesting from a scientific point of view—suggesting that NBPF2S is primordial and tying expansions to chromosomal areas like 1q21—some of the statements require verification or revision. For example, the manuscript doesn't talk about discoveries from 2024 to 2025, including how NBPF14 plays a role in NOTCH2NLB orchestration for neural progenitor proliferation (Science Advances, 2025) or how the Olduvai domain downregulates mitochondrial pathways in brain neoteny (bioRxiv, 2024). Table 2 lists disease associations (like neuroblastoma, autism, and Brugada syndrome), but they are only briefly discussed without going into detail about how copy number variation (CNV) dosage effects play a role in Olduvai-linked diseases like schizophrenia and myalgic encephalomyelitis. The examination of the protein tree in Section 3.3.4 is repetitive and unclear, and it doesn't quantitatively consider functional divergence (for example, by using dN/dS ratios). It is said that pseudogenes like NBPF3SP come from "unknown origins." However, the possible retrotransposition or segmental duplication processes identified in the literature have not been explored.

The authors need to make significant changes to the paper before it can be published. First, they need to carefully proofread and reformat the entire document to eliminate mistakes and artifacts, ensure consistency in names, and verify the quality of figures (for example, clarify that the node numbers in Figure 5 are not bootstrap values). Utilize modern tools to enhance phylogenetic algorithms and verify trees against newly generated genomic assemblies (e.g., utilizing long-read sequencing findings from bioRxiv 2025 preprints). Add a comprehensive PRISMA flow, clear search criteria, and references from after 2023 to the systematic review to include new connections like Olduvai-mitochondrial interactions or NBPF diversity in non-human apes. Make talks stronger by incorporating numbers into evolutionary rates, discussing trade-offs (such as brain evolution vs. disease susceptibility), and suggesting ways to test these ideas, like CRISPR-based domain editing. Raw alignment files and the entire tree data should be included in the supplementary materials. Since the publication focuses primarily on developmental biology, it emphasizes how it affects uniquely human qualities. The manuscript could be suitable for resubmission if these changes are implemented,

 

Author Response

The document, on the other hand, has numerous typos, formatting issues, and flaws from the OCR process that make it difficult to read and appear unprofessional. Gene names are written in different ways (for example, "NBFF," "NBBPF," "NBPT," and "NBPFF" instead of the usual "NBPF"), and dates are mixed up (for example, "Dec. 201.2. 13," "1. Dec. 201. 201. 13," which could be meant to be "J. Dev. Biol. 2025"). There are repeating numbers on every page (for example, "99"s on page 4, "181"s on page 9, and "201"s on page 11), which are probably caused by scanning problems. These numbers make it hard to read the content and signal that the document needs to be resubmitted cleanly. There are a lot of grammatical problems, like awkward phrasing ("the group made up of NBPFs and NBPF74 had a common ancestor similar to NBPF4") and sentences that are too short or missing words (like "The next division is visible in the form of two branches, one containing NBBPF7. NBBPF28...").

The formatting and grammatical errors have been corrected, but we could not find the repeated numbers that are mentioned. We think that the download could have corrupted the file that was reviewed, but we are open to trying to solve this problem if it continues to happen.

 

The techniques section mentions outdated alignment tools (ClustalW from 1994) without explaining why they are superior to newer ones, such as MAFFT or IQ-TREE. It also discusses bootstrap values for trees, but doesn't provide them in the text or pictures. The systematic review states that it follows PRISMA-S; however, it lacks a clear flow diagram or explicit search strings, which could make it challenging to reproduce.

We heard your advice on newer alignment tools and used them to generate new phylogenetic trees, including bootstrap values in all that were compatible for bootstrap analysis (lines 94-116 and figures 3, 4, 5, 6, 7, S.3 and S.4).

We also updated the PRISMA-S section explaining the search strings and improving the flow diagram for an easier reproduction (lines 130-132 and figure 2).

While the evolutionary story is interesting from a scientific point of view—suggesting that NBPF2S is primordial and tying expansions to chromosomal areas like 1q21—some of the statements require verification or revision. For example, the manuscript doesn't talk about discoveries from 2024 to 2025, including how NBPF14 plays a role in NOTCH2NLB orchestration for neural progenitor proliferation (Science Advances, 2025) or how the Olduvai domain downregulates mitochondrial pathways in brain neoteny (bioRxiv, 2024).

The suggestions of some NBPF members about being primordial are based on current results and the revision of articles from other authors, the same is true for the 1q21 region who happens to be a hotspot for microduplications and microdeletions, explaining the apparition of copy number variants (CNVs) which the bibliography agrees that are extremely important in the evolution of the NBPF family. With all this said, I agree with you with the need for verification of some of these statements involving the NBPF family due to them being mostly theoretical and I hope that future studies can settle these theories.

We must thank you for noticing that the discoveries from 2024 to 2025 were not included. We do not know what could have happened, but we revised the search strings and finally incorporated 13 new articles, including the ones you suggested.

Table 2 lists disease associations (like neuroblastoma, autism, and Brugada syndrome), but they are only briefly discussed without going into detail about how copy number variation (CNV) dosage effects play a role in Olduvai-linked diseases like schizophrenia and myalgic encephalomyelitis. The examination of the protein tree in Section 3.3.4 is repetitive and unclear, and it doesn't quantitatively consider functional divergence (for example, by using dN/dS ratios). It is said that pseudogenes like NBPF3SP come from "unknown origins." However, the possible retrotransposition or segmental duplication processes identified in the literature have not been explored.

We have improved the CNV dosage effects sections (lines 553-582) and explanations on why Olduvai-linked diseases exist instead of diseases linked to a specific NBPF gene (lines 56-61).

The repetitiveness of the protein tree (now figure 7) is due to multiple transcripts of a few genes and single transcripts of most of its components. The bibliography showed us that NBPF genes are, mostly, under heavy evolutionary pressure, meaning that there are little changes in the sequences and most of them are synonymous or disease linked. And looking at the tree is worth noticing that most transcripts are dispersed and do not correlate with the associations they had in the genomic DNA, cDNA and CDS trees; giving the feeling that they are organized randomly except for NBPF4 and NBPF6. Also, as we explain with the Olduvai-linked diseases (lines 56-61), the functionality of each NBPF gene could depend only on the number of CON1 and HLS triplets copies of its pro-protein before they are cut by the enzyme furin.

The authors need to make significant changes to the paper before it can be published. First, they need to carefully proofread and reformat the entire document to eliminate mistakes and artifacts, ensure consistency in names, and verify the quality of figures (for example, clarify that the node numbers in Figure 5 are not bootstrap values). Utilize modern tools to enhance phylogenetic algorithms and verify trees against newly generated genomic assemblies (e.g., utilizing long-read sequencing findings from bioRxiv 2025 preprints).

We already answered these commentaries in the sections before.

Add a comprehensive PRISMA flow, clear search criteria, and references from after 2023 to the systematic review to include new connections like Olduvai-mitochondrial interactions or NBPF diversity in non-human apes.

We have improved the PRISMA flow diagrams and specified our search strings (lines 130-132 and figure 2) and we included the mitochondrial interactions of the group (lines 286-292) and an explanation of NBPF diversity and evolution in mammals and specifically in non-human primates (lines 589-612).

Make talks stronger by incorporating numbers into evolutionary rates, discussing trade-offs (such as brain evolution vs. disease susceptibility), and suggesting ways to test these ideas, like CRISPR-based domain editing.

We include a discussion about the evolutionary trade-off of total copies of the Oduvai domain (lines 575-582) and suggestions for future studies (lines 635-646).

Raw alignment files and the entire tree data should be included in the supplementary materials. Since the publication focuses primarily on developmental biology, it emphasizes how it affects uniquely human qualities. The manuscript could be suitable for resubmission if these changes are implemented,

All data employed in the analysis and additional materials will be available as soon as we upload the answers to the reviewer’s commentaries, thank you for your suggestions.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The topic addressed in this manuscript is timely and of significant interest. The study provides a systematic overview of the NBPF gene family, which is potentially highly relevant in neurobiology and human disease. The manuscript offers a valuable integrative synthesis of the NBPF family, linking its evolutionary origin, gene expression patterns, and disease associations, thereby reinforcing its biological and clinical significance.

However, the manuscript lacks a more detailed background on the evolutionary importance of DUF1220/Olduvai domains and the clinical relevance of the NBPF gene family. These aspects should be further developed in the Introduction.

Below are specific suggestions that could help improve the manuscript:

Introduction

• Include at least one figure illustrating the gene structure, protein domains, and overall organisation of the NBPF genes considered in this study (e.g., HLS, DUF1220).

Results

• In several sections, the Results include interpretative and speculative comments that would be more appropriately placed in the Discussion section. A clearer separation between results and their interpretation is recommended.
• Table 1: Please indicate the reference genome build or sequence release used.
• Tables 2 and 3: For each gene listed, add a final column with 1–2 relevant references supporting the gene–disease association reported.

Conclusion
The Conclusion is brief and should be expanded to highlight:

• The evolutionary implications of NBPF family expansion in primates
• Emerging clinical connections (e.g., neurodevelopment, cancer, rare diseases)
• Future research directions (e.g., functional validation, model organism studies)

Additional suggestions

• Improve the graphical quality of Supplementary Figure S1.
• Gene names should be italicised throughout the manuscript according to standard gene nomenclature (e.g., NBPF1, NBPF26).
• A careful proofreading is recommended to correct some typographical errors found throughout the text.

 

Author Response

However, the manuscript lacks a more detailed background on the evolutionary importance of DUF1220/Olduvai domains and the clinical relevance of the NBPF gene family. These aspects should be further developed in the Introduction.

We added an explanation of the Olduvai domain structure with an illustration of a NBPF gene structure (lines 43-48 and figure 1) and an explanation to why this domain has a clinical relevance (lines 56-61). The evolutionary relevance of the domain is briefly explained in the introduction due to its function is still unknown, but we develop an explanation of NBPF evolution in mammals and specifically in non-human primates in the conclusion (lines 589-612).

 

Below are specific suggestions that could help improve the manuscript:

Introduction

Include at least one figure illustrating the gene structure, protein domains, and overall organisation of the NBPF genes considered in this study (e.g., HLS, DUF1220).

We included a figure illustrating the general organization of a NBPF gene and the internal organization of subdomains of the Olduvai domain (figure 1).

Results

• In several sections, the Results include interpretative and speculative comments that would be more appropriately placed in the Discussion section. A clearer separation between results and their interpretation is recommended.
• Table 1: Please indicate the reference genome build or sequence release used.
• Tables 2 and 3: For each gene listed, add a final column with 1–2 relevant references supporting the gene–disease association reported.

The speculative and interpretative comments were cut out of the results, leaving only objective descriptions of the results provided with minor incisions of explaining why there are differences between each other (lines 156-256 and figures 3, 4, 5, 6, 7, S.3 and S.4).

A reference genome build was included in the table 1 and a reference column was added to the tables 2 and 3.

 

Conclusion
The Conclusion is brief and should be expanded to highlight:

• The evolutionary implications of NBPF family expansion in primates
• Emerging clinical connections (e.g., neurodevelopment, cancer, rare diseases)
• Future research directions (e.g., functional validation, model organism studies)

The updated conclusion expands on the commentaries given, including explanations for the evolutionary implications of NBPF family expansion in primates (lines589-612), emerging clinical connections and future research connections (614-653)

Additional suggestions

• Improve the graphical quality of Supplementary Figure S1.
• Gene names should be italicised throughout the manuscript according to standard gene nomenclature (e.g., NBPF1, NBPF26).
• A careful proofreading is recommended to correct some typographical errors found throughout the text.

We improved the quality of our figures and corrected the grammatical, typographical and nomenclature errors found in the text, thank you for your suggestions.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Reviewer’s Report

Title: Evolutionary Restructuring and Systematic Review of the NBPF Gene Family: Comparative Genomics, Functional Divergence, and Disease-Linked Pathways

General Assessment

This manuscript presents a comprehensive systematic review and comparative genomic analysis of the Neuroblastoma Breakpoint Family (NBPF) genes, focusing on their evolution, duplication patterns, phylogenetic classification, and association with human diseases. The study employs the PRISMA-S methodology, multi-level sequence alignments (cDNA, CDS, protein, and genomic DNA), and novel phylogenetic trees that integrate NOTCH2NL gene fusion relationships.

The manuscript presents a comprehensive synthesis of a poorly understood yet biologically and medically significant gene family. It offers the first integrative phylogenetic reconstruction including all NBPF members, coupled with disease correlations. The paper is well-researched, data-driven, and relevant to the fields of developmental genomics, neurogenetics, and evolutionary biology. However, several issues, mainly in structure, focus, and analytical depth, should be addressed before publication.

Major Comments

1. Scope and Focus:

• The manuscript simultaneously covers evolutionary, genomic, and clinical aspects, resulting in uneven emphasis. The phylogenetic sections are strong, but disease summaries rely on secondary literature without analytical synthesis; recommendation: Consolidate disease associations into summarized tables and emphasize mechanistic or evolutionary explanations.

2. Phylogenetic Methods and Interpretation:

• The phylogenetic analysis uses MAFFT, UPGMA, and Neighbor-Joining methods. Clarify justification for tree rooting, substitution models (JTT, Jukes-Cantor), and bootstrap thresholds for reproducibility.

3. Integration of NOTCH2NL Fusion Genes:

• The inclusion of NBPF–NOTCH2NL fusion events is novel, but their interpretation is limited. Recommendation: Expand on functional coevolution and expression regulation, referencing cortical progenitor studies involving NOTCH2NLB.

4. Evolutionary Hypothesis:

• The proposed model of NBPF evolution, stemming from PDE4DIP duplication and the formation of the 'Ancestral LINE-less Group', is compelling. Recommendation: Support with cross-species synteny or supplementary comparative figures.

5. Clinical Relevance:

• Disease sections mix confirmed and speculative associations. Recommendation: Clearly mark confirmed vs. candidate links in Tables 2–3 for transparency.

6. Writing and Structure:

• Sections 3.1.4–3.2.3 and the Discussion contain redundancy. Recommendation: Streamline repetitive tree comparisons and focus on evolutionary interpretation.

Minor Comments

• Italicize all gene symbols (e.g., NBPF1, NBPF14, NOTCH2NL) consistently throughout the text.
• Ensure figures include scale bars, clear legends, and consistent formatting.
• Correct typographical and spelling errors (e.g., 'pathologDies', 'aggrupation').
• Standardize reference formatting with consistent DOI inclusion.
• Explicitly reference supplementary data (Figures S1–S4, Table S1) in the main text.
• Enhance abstract clarity by highlighting novel contributions (e.g., NBPF26's ancestral role).
• Conclude with suggestions for future experimental validation (e.g., CRISPR domain studies).

Overall Recommendation

Major Revision – The manuscript makes a significant contribution to NBPF evolutionary genomics but requires refinement in terms of structure, methodological justification, and more precise differentiation between descriptive and interpretive findings.

Author Response

Comments 

Reply

Major Comments

Scope and Focus:

The manuscript simultaneously covers evolutionary, genomic, and clinical aspects, resulting in uneven emphasis. The phylogenetic sections are strong, but disease summaries rely on secondary literature without analytical synthesis; recommendation: Consolidate disease associations into summarized tables and emphasize mechanistic or evolutionary explanations.

Since most of the NBPF genes are not well studied and most of the studies showing the relation of an NBPF gene to a disease is based on expression changes or mutations make it difficult to associate one gene with one disease. This is especially true in the case of oncological development, which is a very complex process and multiple driver genes are needed.

The most studied NBPF gene is NBPF1, which is not completely understood, and its protein has been recently associated with other proteins like Chibby, but its function is still unknown. Trying to define mechanistic interactions of NBPF genes with the current information is daring because there are not sufficient studies to confirm how their proteins interact with other proteins or their functions.

Finally, we provide evolutionary explanation of the most known part of the NBPF genes, the Olduvai domain. But these studies are predominantly focused on brain development and even in those studies, it cannot be proved the mechanistic association of these domains to any other protein or if they function as transcription factors. The only evolutionary explanation that we can provide is that there may exist a dosage-dependent genomic trade-off of the subdomains of the Olduvai domain.

 

Phylogenetic Methods and Interpretation:

The phylogenetic analysis uses MAFFT, UPGMA, and Neighbor-Joining methods. Clarify justification for tree rooting, substitution models (JTT, Jukes-Cantor), and bootstrap thresholds for reproducibility.

The rooting of each tree is explained earlier in the introduction in the case of the trees rooted with PDE4DIP, and the trees rooted with the NBPF4-NBPF6 group are explained in the lines 241-245.

We have now defined the substitution models in lines 116-122 and the bootstrap thresholds employed in lines 109-110.

 

Integration of NOTCH2NL Fusion Genes:

The inclusion of NBPF–NOTCH2NL fusion events is novel, but their interpretation is limited. Recommendation: Expand on functional coevolution and expression regulation, referencing cortical progenitor studies involving NOTCH2NLB.

The limited interpretation of the NBPF-NOTCH2NL gene fusion is because of the limited comprehension on both NBPF and NOTCH2NL genes. Not knowing the exact function of either genes or the regulatory relationship between them. The only described NBPF-NOTCH2NL interaction is between NBPF14 and NOTCH2NLB, and in the study of Eşiyok et al (2025) they used microinjections of plasmids of both genes in embryonic mouse neocortex and chimpanzee cerebral organoids. This means that this study only covers the functional co-expression of both genes but cannot deduce any expression regulation on any of these genes.

Also, neither NBPF14 nor NOTCH2NLB are described as transcription factors or have a clear influence on each other, so the only safe thing to say about them is that their co-expression amplifies neuron development and maintenance of the cortical progenitors and that they are spatially close.

Further studies will clarify the relation of NBPF and NOTCH2NL genes, but now it is unclear.

 

Evolutionary Hypothesis:

The proposed model of NBPF evolution, stemming from PDE4DIP duplication and the formation of the 'Ancestral LINE-less Group', is compelling. Recommendation: Support with cross-species synteny or supplementary comparative figures.

It would be a great idea for a new study, as it would allow the study of the complete evolution of the gene group within the primate group. But these genes are not very well mapped within the chromosomes and even in chimpanzee there are some genes that are believed to be NBPF genes (like ENSPTRG00000001028) but it is not manually confirmed. The same is true for most primates, so it will be a very interesting future study that will help reaffirm the proposed model.

Clinical Relevance:

Disease sections mix confirmed and speculative associations. Recommendation: Clearly mark confirmed vs. candidate links in Tables 2–3 for transparency.

As said in answers before, the NBPF family is poorly understood, being a family of genes that show altered in many diseases but its function in the development of the disease is unknown. Depending on the author, some articles give a more in-depth analysis of the possible consequences of the alterations detected in NBPF genes, while other authors do not explain in such depth even if it is one of their most altered genes due to not being well studied. In the case of oncological development, it is even more controversial due to the complex development of the oncological processes, needing multiple driver genes to evade cell death and generate a tumor.

Writing and Structure:

Sections 3.1.4–3.2.3 and the Discussion contain redundancy. Recommendation: Streamline repetitive tree comparisons and focus on evolutionary interpretation.

The sections included in the results and discussion contain redundancy due to one being a descriptive analysis of each individual result and the other being the synthesis and clarification of all results. Giving a clearer understanding of why each result is important by itself and discussing the relation between results, focusing the writing on a conclusion of our analysis.

Minor Comments

Italicize all gene symbols (e.g., NBPF1, NBPF14, NOTCH2NL) consistently throughout the text.

We realized these corrections

Correct typographical and spelling errors (e.g., 'pathologDies', 'aggrupation').

We realized these corrections

Standardize reference formatting with consistent DOI inclusion.

We realized these corrections

 

Explicitly reference supplementary data (Figures S1–S4, Table S1) in the main text.

Figures S. 1 and S. 2 are non-edited versions of figures 4 and 6 which contain a reference to their non-edited versions on the supplementary materials. Meanwhile Figures S.3 and S.4 are referenced in the text in the lines 235, 518 and 532.

Table S. 1 is referenced in the text in line 152.

Enhance abstract clarity by highlighting novel contributions (e.g., NBPF26's ancestral role).

The hypothesis of the segmental duplication of PDE4DIP and the ancestral role of NBPF26 in the evolution of the NBPF family is already referenced in the abstract (lines 22-27).

Conclude with suggestions for future experimental validation (e.g., CRISPR domain studies).

We included CRISPR domain-based studies in the last correction in the conclusions in lines 641-652

Ensure figures include scale bars, clear legends, and consistent formatting.

We are new to the MAFFT software and its tree visualizer does not let us import the scale to the final image, we have tried different configurations to try to include it in the final trees but we have not been able to achieve. We have included the evolutionary distance in figures 3, 5 and 7 to facilitate the interpretation of the obtained trees. But we will gladly accept recommendations or guides to include all possible information in our figures.

 

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

Reviewer Comments to the Authors

Overall Assessment:
This manuscript offers a systematic and comprehensive analysis of the Neuroblastoma Breakpoint Family (NBPF) genes, including evolutionary reconstruction, domain analysis, and disease association. The topic is timely and relevant, but clarity, structure, and figure descriptions need improvement to meet publication standards.

Major Comments

1. Clarify whether the focus is on evolutionary reconstruction or disease association, As the current balance is uneven.
2. Explain why using MAFFT v7 with UPGMA/Neighbor-Joining is preferred over maximum-likelihood or Bayesian methods.
3. Strengthen the PRISMA section by clarifying inclusion and exclusion criteria and adding Table S1 with PubMed IDs.
4. Figures 3–7 require detailed legends (e.g., bootstrap support, color coding, scale bars).
5. The discussion includes exaggerated claims (e.g., ongoing human evolution assertions) that need to be toned down.
6. Improve English grammar and eliminate redundancies like repeated ‘being theorized’ and ‘it has been proved’.
7. Support the novelty claim (“first phylogenetic tree including all members”) by comparing it with existing literature.
8. Make sure all references follow MDPI style and eliminate duplicates (e.g., references 33 and 35).

Minor Comments

• Clarify in the abstract that this study is a systematic review and comparative genomic analysis.
• Make sure all supplementary materials (Table S1, Figures S1–S4) are included and properly labeled.
• Standardize terminology for diseases by consistently using “oncological processes.” Revise abbreviation usage and define all abbreviations upon first mention.
• Ensure consistent resolution and scaling in Figures 3–7.
• Improve reference formatting to include complete DOIs and consistent punctuation.

Comments on the Quality of English Language

Needs professional editing

Author Response

Major Comments

1. Clarify whether the focus is on evolutionary reconstruction or disease association, As the current balance is uneven.

The main objective of the study is to propose a new hypothesis of the NBPF gene family evolution in human and study and group all diseases in which NBPF genes have been associated, including all the data available to support those claims. In this work we tried to link this family evolution with some of the diseases in which they have been associated, unifying the works theme and showing the importance of this gene family and its alterations.

1. Explain why using MAFFT v7 with UPGMA/Neighbor-Joining is preferred over maximum-likelihood or Bayesian methods.

We appreciate this valuable comment. We selected MAFFT v7 using the UPGMA/Neighbor-Joining approach because our primary objective was to obtain a general overview of the evolutionary relationships among the 23 NBPF genes, rather than to reconstruct deep phylogenetic relationships or infer substitution models. The NBPF gene family is characterized by extensive internal duplications, repetitive sequences, and high sequence similarity, which can lead to model overfitting and instability when using maximum likelihood or Bayesian methods.

MAFFT combined with UPGMA/NJ provides a computationally efficient and robust alignment framework that performs well for closely related paralogs and large datasets with high redundancy, allowing us to visualize clustering patterns and divergence trends across genomic, cDNA, CDS, and protein sequences.

Maximum-likelihood methods are great for phylogeny reconstruction in distant models (same gene but in different species including not closely related species), but it tries to find an ancestral sequence to root its trees. Since the NBPF family is theorized to be a product of segmental duplications of PDE4DIP, some members differ in their original duplication, and some pseudogenes are placed in unusual branches due to less evolutionary restrictions of the nature of pseudogenes (like NBPF25P, NBPF7P and NBPF17P). On the other hand, Neighbor-joining methods calculate distances between sequences instead of rooting them to a single ancestral sequence and have better Bootstrap method support than most of the other methods, having statistically stronger results.

Bayesian methods can get stuck in local maximums and the NBPF family has extremely conserved regions, so this methodology could result in less reliable trees than UPGMA methods, which recalculates the distances between nodes after each iteration.

Nevertheless, we acknowledge the importance of more complex phylogenetic inference methods and plan to explore maximum likelihood or Bayesian analyses in future work focused on finer evolutionary resolution.

1. Strengthen the PRISMA section by clarifying inclusion and exclusion criteria and adding Table S1 with PubMed IDs.

It is described in lines 133-150 and the PRISMA figure and table S1 have been corrected.

1. Figures 3–7 require detailed legends (e.g., bootstrap support, color coding, scale bars).

In the figure foots there is a description of each figure, including bootstrap numbers and absolute distance between nodes, not needing additional scales.

1. The discussion includes exaggerated claims (e.g., ongoing human evolution assertions) that need to be toned down.

All claims were based on hypothesis of other authors and included explanations of why some results support those claims, but we have changed them (lines 579-581)

1. Improve English grammar and eliminate redundancies like repeated ‘being theorized’ and ‘it has been proved’.

All redundancies corrected and the grammar was revised by an expert in the English language.

1. Support the novelty claim (“first phylogenetic tree including all members”) by comparing it with existing literature.

Now mentioned in lines 75-80.

1. Make sure all references follow MDPI style and eliminate duplicates (e.g., references 33 and 35).

All references are in APA7 (one of the supported MDPI style references) and all duplications and errors have been corrected.

Minor Comments

• Clarify in the abstract that this study is a systematic review and comparative genomic analysis.

Now included in lines 25-26

• Make sure all supplementary materials (Table S1, Figures S1–S4) are included and properly labeled.

All included in the same document labeled as “Suplementary”

• Standardize terminology for diseases by consistently using “oncological processes.” Revise abbreviation usage and define all abbreviations upon first mention.

The terminology has been standardized, and the abbreviations are defined upon first mention

• Ensure consistent resolution and scaling in Figures 3–7.

Improved the resolution of the figures

• Improve reference formatting to include complete DOIs and consistent punctuation.

Fixed