An Unusual Presentation of Novel Missense Variant in PAX6 Gene: NM_000280.4:c.341A>G, p.(Asn114Ser)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors, this is an interesting case study on novel PAX6 gene missense mutation first time identified and yet to be characterized fully. Though I support this novel study, however I would not suggest this manuscript in current form for publication. The major issues with this manuscript are I have summarized in the word document attached. However, I believe that with those issues addressed by your team, this manuscript can hold a great strength for publication in future.  

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Author Response

This is an interesting study on novel PAX6 gene missense mutation (based on my PubMed search), first time identified in one and only patient, however yet to be characterized fully. Though I support this novel case study, however I would not suggest this manuscript in current form for publication. The major issues are:

Q1.1 The manuscript is not well-structured and organized. Lack of additional background information (different variants of PAX6 gene identified so far), introduction, poor results description and discussions.

A1.1 Thank you for your comment. We have restructured the manuscript by adding substantial sections in the text and expanding the introduction section with the background information. We have also extended Discussion section with the comparison of 2 cases: one ours and similar from cited paper.

 

Q1.2. Figures are not well organized and represented, and within figures no labeling or markings done.

A1.2 We have changes the figure 2 according to your suggestions.

 

Q1.3. There are no tables for results description.

A1.3 We have added 2 tables for clinical description of the patient as well as comparative table in the Discussion section.

 

Q1.4. Methods sections are lacking.

A1.4 We have added the Materials and Methods section in the manuscript.

 

Q1.5. Sample size (just one patient) is major drawback of this manuscript. I believe with a greater number of samples/patients, this study can get more strength of publishing in near future.

A1.5. We should mention that phenotypes which is described here is thought to be extremely rare. In our the largest cohort of nearly 300 probands with PAX6-associated ocular diseases, this is the only one with such distinguished clinical picture. In our opinion, this case is worth to be published as a separate case report. We have found a single report in available literature with similar phenotypic presentations which we discussed in the Discussion section.

 

Q1.6. Is there any information/background that support that this substitution (Asn114Ser) in Pax6 protein or any other unrelated protein can lead to gain-offunction mutation?

A1.6. GoF effect of the described substitution is our hypothesis supported by several levels of evidence. Bioinformatic analysis of the impact of this substitution on the protein structure predicts this variant to be stabilizing. Mouse models also suggest that increased dosage of PAX6 leads to severe ocular abnormalities. We have added these references to the discussion section to support our hypothesis.

 

Q1.7. References lacking at multiple places.

A1.7. Thank you. We have added references to the text were applicable.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors of this study linked an N114S mutation in PAX6 to an ocular phenotype. They provide fairly convincing arguments for this mutation to be causative of the observed clinical phenotypes, but do not prove this point. Nevertheless, the study is useful as a starting point for further exploration of other variants behaving in these way and future functional-genetic studies in the animal model systems. I have the following concerns 

Major:

- Was any exome of whole-genome sequencing performed on the patient?

- What was the clinical rationale to sequence PAX6 or were multiple other genes sequenced?

- Line 109: Can you explain the significance of the phrase: "one allele out of a total of 251,484 alleles"? Does this suggest a population frequency of the allele?

- What is the level of conservation of the mutated residue across evolution?

- I would also like to request that the authors list a few studies where PAX6 mutations caused phenotypes in a heterozygous state. 

- Materials & Methods seem absent. Is it by design of the manuscript type or was it omitted for another reason? Even bioinformatics studies need such descriptions.

Minor:

- Line 13: The phrase "Despite not directly contacting DNA" does not seem to modify any meaningful noun.

- Line 51: RCMG needs to be defined.

- Lines 205-206: "Our findings are similar to that described by Sharan et al. " does not carry much meaning. Can you specify the precise similarities or describe exactly what that study did?

 

Author Response

The authors of this study linked an N114S mutation in PAX6 to an ocular phenotype. They provide fairly convincing arguments for this mutation to be causative of the observed clinical phenotypes, but do not prove this point. Nevertheless, the study is useful as a starting point for further exploration of other variants behaving in these way and future functional-genetic studies in the animal model systems. I have the following concerns

 

Major:

 

Q2.1. - Was any exome of whole-genome sequencing performed on the patient?

A2.1. No, the patient was initially referred to the analysis of PAX6 nucleotide variants based on our expertise in involvement of PAX6 in ocular development and associated pathology.

 

Q2.2. - What was the clinical rationale to sequence PAX6 or were multiple other genes sequenced?

A2.2. – Since the PAX6 is a major regulator of eye development, this clinical case is initially thought to be linked to PAX6 nucleotide variant, specifically, subtle iris malformations accompanied by posterior segment involvement usually pointed to the PAX6 causative role in the development of such complex eye phenotype.

 

Q2.3. - Line 109: Can you explain the significance of the phrase: "one allele out of a total of 251,484 alleles"? Does this suggest a population frequency of the allele?

A2.3. You are right. This phrase refers to the population allelic frequency of the variant. This is not contradictory to the fact of variable phenotypic presentation of PAX6 missense variants observed in other studies (e.g. Sharan et al., 2008; cited upon num. [26]) where other nonsynonymous substitution also demonstrated variable expressivity within one large family.

 

Q2.4. - What is the level of conservation of the mutated residue across evolution?

A2.4. The PhyloP score of this position is 7.92828 which is shown in Fig. 2a under the sequence alignment. We have updated the Figure 2 as well as its legend to highlight this fact.

 

Q2.5. - I would also like to request that the authors list a few studies where PAX6 mutations caused phenotypes in a heterozygous state.

A2.5. PAX6-associated disorders exhibit autosomal dominant mode of inheritance and heterozygous state of a pathogenic variant is sufficient to phenotype development. We have supported this fact with several references.

 

Q2.6. - - Materials & Methods seem absent. Is it by design of the manuscript type or was it omitted for another reason? Even bioinformatics studies need such descriptions.

A2.6. – Thank you for your comment. We have significantly restructured the manuscript and added Materials and Methods section with detailed description of methods used.

 

Minor:

 

Q2.7. -  Line 13: The phrase "Despite not directly contacting DNA" does not seem to modify any meaningful noun.

A2.7. -  We have changed the text accordingly.

 

Q2.8. -  Line 51: RCMG needs to be defined.

A2.8. -  We have changed the text accordingly.

 

Q2.9. -  Lines 205-206: "Our findings are similar to that described by Sharan et al. " does not carry much meaning. Can you specify the precise similarities or describe exactly what that study did?

A2.9. -  We have added the comparative table 2 with similarities between two patients: one ours and second from the Sharan et al. study.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I am really thankful to the authors for taking time and efforts and I appreciate them for addressing my initial comments. Overall, they have made valuable revision, now able to get published. Some changes still need to be done for finalizing it before. The issues are:

1.    Figures still needs improvement in term of sizes and labeling. They still not organized well. If possible, add some labels in figure 1 to describe the phenotype on eye images.

2.    Same goes for tables. If they are adjusted well with proper font size.

Author Response

I am really thankful to the authors for taking time and efforts and I appreciate them for addressing my initial comments. Overall, they have made valuable revision, now able to get published. Some changes still need to be done for finalizing it before. The issues are:

1. Figures still needs improvement in term of sizes and labeling. They still not organized well. If possible, add some labels in figure 1 to describe the phenotype on eye images.
2. Same goes for tables. If they are adjusted well with proper font size.

Reply: 
Thank you for your comment. We have significantly improved sizes and labeling in Fig. 1. In the current form, it seems to be much clearer. 

We have also reformatted Tables 1 and 2 by changing rows and columns. Now they have much better readability and clarity. The font size corresponds to the Journal's requirements. We think it could be changed upon proofreading in case of necessity. 

Reviewer 2 Report

Comments and Suggestions for Authors

My comments were addressed to a sufficient degree.

Author Response

Thank you very much for your time and help with the improvement of our manuscript.