Dear Dr. Shehata.

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 findings of the manuscript provide valuable insights on   molecular basis of Epidermolysis Bullosa (EB) from Middle eastern families. The study  reports novel and known pathogenic mutations in three genes (COL7A1 , COL17A1 , LAMB3 )  from  12 families affected with EB by employing exome sequencing. The manuscript is well structured technically sound. However the manuscript requires minor reversions for improvements of quality and better comprehension before acceptance for publication. The following points needs to be considered:

1. As the study reports families from Saudi Arabia, Yemen and Syria, so the title referring ‘ Saudi Arabian families’ is misleading. The use of  term ‘Middle eastern’ would have been more appropriate.
2. The quality of pedigrees in  figure 1 needs considerable  improvements for clarity.
3.  A  flow chart  showing  exome sequencing pipeline will be useful.
4. More Information on inclusion  and diagnostic criteria, details on clinical phenotypes should be added.
5. Some gene mutation nomenclature  should be checked especially the variant described at protein level. Please fellow the guideline set by Human Genome Variation Society (HGVS).
6. Functional consequences of splice site and misssense variant should be  discussed in discussion.
7. There are grammatical and typological  errors in different sections of the manuscript which needs to corrected

Furthermore, the reviewers’ feedback provide extensive reversion points to consider that  will be useful in improving the quality of the manuscript.

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We look forward to receiving your revised manuscript.

Kind regards,

Rabia Habib, 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?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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Reviewer #1:  This manuscript will make a nice contribution to the field of human genetics. But needs some improvements and minor revisions before formal acceptance for publication in PlosOne. Authors should considering following points during minor revisions:

1. Authors must provide the transcript IDs of different genes in which mutations have been identified.

2. In the abstract there are empty parentheses "()".

3. Quality of all the figures needs improvement.

4. A figure detailing the flow chart of exome sequencing should be made for clear understanding.

5. There are some splice variants, these splice variants should be analyzed using bioinformatics softwares like NetGene2 etc. for assessing the effect of these variants. And graphical representation in the form of a a figure showing intron inclusion in the final protein or exon skipping due to aberrant splicing should be given.

6. Carefully check the grammar and other typographical mistakes before resubmission.

Reviewer #2:  The study presents significant findings on novel and recurrent mutations in EB among Saudi families, contributing valuable data on a region with limited molecular epidemiological studies. The manuscript is well-structured and follows a logical flow, but some sections require refinement for clarity and conciseness.

Specific Comments:

Title & Abstract

- The title is appropriate but could be more specific by highlighting "molecular findings" or "genetic profiling."

- The abstract summarizes key findings well, but some sentences are wordy. Consider shortening complex phrases for better readability.

- The Results section should clearly differentiate between novel and recurrent mutations in the abstract.

- The conclusion should explicitly state the clinical significance of the study.

Introduction

- The introduction provides good background information, but the prevalence statistics on EB in Saudi Arabia should be expanded with references.

- The final paragraph should clearly define the research gap and justify why studying Saudi families is important.

Methodology

- Recruitment Criteria: The inclusion and exclusion criteria should be more explicit, especially concerning how patients were diagnosed and classified.

- Computational Analysis: Provide references for the specific thresholds used in pathogenicity predictions (e.g., CADD score cutoff).

Results

- Clearly separate findings related to novel versus known variants to emphasize new contributions.

- Consider reorganizing the section to first present clinical findings, then genetic results, and finally computational predictions.

Discussion

- Comparisons with global EB cases are insightful, but more emphasis should be placed on how Saudi genetic variants differ from other populations.

- Implications for genetic counseling in consanguineous populations should be elaborated on.

Conclusion

- The conclusion should be more concise and explicitly highlight future research directions.

- Mention potential applications of findings, such as early genetic screening programs.

Figures & Tables

- Figure 1 (Pedigrees): Improve clarity by ensuring consistent labeling across all families.

- Figure 2 (Clinical Presentation): Enhance image descriptions to clarify key features of EB.

- Tables 3 & 4: Consider moving some detailed variant data to supplementary material to improve readability.

Reviewer #3:  The authors have done a good work on the EB families from the middle east. But I have some suggestions and changes for the integrity of the manuscript, I trust in the authors’ capabilities to address all the comments.

# This study includes families from three ethnic origins i.e., Saudi Arabia, Syria and Yemen, so how the authors focus in the title only on the Saudi Arabian families?

# “This study intends to shed some light on recurrent as well as novel genetic abnormalities that cause EB in the Western region of Saudi Arabia” is a poor presentation please rephrase this sentence e.g., “This study intends to highlight the recurrent as well as novel genetic abnormalities that cause EB in the Western region of Saudi Arabia”.

# “Twelve EB families in Saudi Arabia were recruited from clinical settings” this sentence is ambiguous to understand please try to make its sense clearer. What do you mean by clinical settings here?

# In line 43 (abstract), please correct “classify disease subtypes precisely” as “classified the disease subtypes precisely”.

# In line 44 “to identify EB-causing variants” as “to identify EB-causing gene variants”.

# In line 47, the authors state that “We identified 11 pathogenic variants”. Were all the variants pathogenic, confirmed by the pathogenicity prediction tools? Otherwise rephrase this sentence and use correct terms regarding classification of the identified variants.

# Use correct nomenclature regarding the identified frameshift, nonsense, and missense variants nonsense (c.1633C>T, c.1837C>T, c.2005C>T, and c.5888G>A), missense (c.4448G>A, and c.8245G>A). Please follow the variant nomenclature guidelines, how to write a variant that has not been verified through functional studies in the model organisms.

# The sentences “The COL7A1 variants included frameshift (c.5924_5927del and c.6268_6269del,), nonsense (c.1633C>T, c.1837C>T, c.2005C>T, and c.5888G>A), missense (c.4448G>A, and c.8245G>A), and splice site (c.6751-1G>A). Additionally, splice site variants were detected in the COL17A1 () and LAMB3 genes (c.8305-1G>A and c.1977-1G>A, respectively)” contains many mistakes please correct.

# In line 53, “predicted These variants” correct it as “predicted these variants”.

# In line 53, the variants are either pathogenic or likely pathogenic but not highly pathogenic, so please correct.

# In lines 54, 55, the authors state that Protein deficiency occurs due to frameshift and truncating mutations, and splice site changes impairing RNA processing. In connection with the above sentence please mention, “what are the consequences of missense variants?”

# The pedigrees are unreadable and illegible. In some of the pedigrees the lines (horizontal/vertical) are missing, in such situations it is very difficult to reach a conclusion about. To make it more clear and easier for the readers it is advised to write the gene name and variant in the left upper side of the corresponding pedigree. It will be also very helpful to write genotype below each individual in the pedigree. Please follow this article DOI: 10.1007/s10528-025-11087-2 as a guide.

# Add OMIM IDs with the gene and disease names.

# Write p.Glu1975fs and p.Pro2090fs frameshift variants in their full form. Such as, p.(Glu1975GlyfsTer29) and p.(Pro2090TrpfsTer8). Also please use uniform nomenclature for c.5924_5927delAACG, c.5924_5927del and other variants like c.1633C>T (p. Gln545Ter), c.5888G>A (p. Trp1963X), c.2005C>T (p.Arg669*), c.1837C>T (p.Arg613*) etc., throughout the manuscript.

# In line 140, it will be better to use GRCh37/hg19 in place of only hg19, because it has already been used in line 211.

# In line 147, add the reference for ACMG guidelines (DOI: 10.1038/gim.2015.30).

# The COL17A1 nonsense variant c.1394G>A; p.(Trp465X) is not mentioned in the text on page 6 under the heading “Genetic Analysis”.

# Write the gene names in italics throughout the manuscript, e.g., in lines 247 etc.

# The “in text-citations” format is not correct, e.g., line 70-- (1) (2) (3), line 93-- (12) (13), line 97-- (14) (12). Write in a correct format such as (1-3), (12, 13), (12, 14).

# In lines 150, 151… segregation of the variant with disease in other affected family members. It will be more appropriate to state “segregation of the variant with disease in other family members”, because the variant may segregate randomly in any of the siblings in heterozygous/homozygous form.

# Use proper reference/URL with the softwares or tools used in the text, e.g., SnapGene version 6.0.2, CADD, FATHMM and SpliceAI etc.

# In line 179, replace “variant forms” with “mutant forms”.

# In line 187, the authors have simply mentioned that “sixteen patients from 12 different families were clinically diagnosed in the clinic” please mention the name of the clinic/s in order to increase credibility and trustworthiness of the research work.

# In line 189, please write the families numbers (11 8 and 12) in a sequence such as 8, 11, and 12. Here it is difficult to confirm that which family belongs to Syria and which one is of Yemeni origin?

# In line 191, the authors have mentioned that “Pedigree analysis (Fig 1) suggested an

autosomal recessive mode of inheritance” but family 12 has autosomal dominant inheritance pattern (have not mentioned).

# In line 194, replace (Fig 2) with Figure 2.

# In table 1, correct the font style in column 7.

# In table 2, correct the gene symbol COL17A as COL17A1.

# For each variant classification based on the ACMG/AMP criteria, write the evidences such as very strong (PVS1), Strong (PS1-4), moderate (PM1-6) or supporting (PP1-5) to be pathogenic or likely pathogenic.

# Correct the heading of table 4, these are the “details of mutations identified in EB patients” add at the end “so far”.

# Correct the sentence “The current study sheds light on the genetic makeup of EB in Saudi patients by detecting causative variants in the COL7A1, COL17A1, and LAMB3 genes” here not the genetic make up of EB but the genetic makeup of Saudi EB patients. So, correct the above sentence. Also replace “sheds light on” with “highlights”.

# It will be more suitable to use “variant” in place of “mutation”.

# No description about the families, consanguineous or nonconsanguineous and how many individuals are present each family etc, which is mandatory to give the detailed description of each family to the readers.

# In lines 360-364 “A total of ten mutations were identified in COL7A1, including three missense mutations (c.4448G>A and c.8245G>A), three frameshift mutations (c.5924_5927delAACG, c.6268_6269del, c.1837C>T), two splice site mutations (c.8305-1G>A, c.6751-1G>A), and two truncating mutations (c.1633C>T, c.2005C>T and c.5888G>A)”. This statement is incorrect because:

Missense: 2 --------(c.4448G>A and c.8245G>A)

Frameshift: 2-------(c.5924_5927del, c.6268_6269del)

Nonsense/truncating: 4------- c.1633C>T; p.(Gln545Ter), c.1837C>T; p.(Arg613Ter), c.2005C>T; p.(Arg669Ter), c.5888G>A; p.(Trp1963Ter).

Splice site: 2--------(c.6751-1G>A; p.?, c.8305-1G>A ;p.?), so correct the above statement.

# The discussion is about the comparison of your results as in this case “the patients’ clinical phenotypes, number of patients in the families, their gender, geography and the identified variants in each family” are logically compared with the previous literature. But here in this manuscript this critical point seems to be missing. The authors are advised to compare their results with those present in table 4.

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Reviewer #1: Yes:  Muhammad Ajmal

Reviewer #2: No

Reviewer #3: Yes:  Dr. Sher Alam Khan

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Identifying Novel Genetic Variants in Epidermolysis Bullosa Among Middle Eastern Arab Families: Insights from Whole Exome Sequencing and Computational Analysis

PONE-D-24-59641R1

Dear Dr. Shehata,

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.

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.

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Kind regards,

Rabia Habib, Ph.D

Academic Editor

PLOS ONE

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