PAX 2 Mutation in an Indian Family with Renal Coloboma Syndrome

Round 1

Reviewer 1 Report

In the present paper, Digvijay K and co-authors describe a novel heterozygous mutation in the PAX2 gene associated with Renal Coloboma Syndrome together with 3 new polymorphisms.

The following comments are for the authors’ consideration:

ü  It would be interesting to sequence PAX2 in the healthy father (I.2) and sister (II.4) to evaluate if they are carriers of the PAX2 c.617-19 T>A c.617-70 C>A, c.617-41 T>A variations detected. If this is the case, these polymorphisms were inherited form the father and it could be that their presence worsened the disease phenotype.  Interestingly, affected siblings appear to have a more severe clinical presentation and progression than the mother.  Otherwise, the above polymorphism are on the same allele as the maternal inherited c.352C>G mutation.

ü  The authors should specify if there are malformations in the auditory system of the patients II:2 and II:5.

ü  Chromatograms in figure 1 and 3 should be of better quality.

ü  The authors should add a table with the primers used to sequence PAX2 exon and intron-exon boundaries. Please specify the sequencer used, as well.

ü  Figure 4 shows only the c.617-19 T>A variation and not c.617-70 C>A, c.617-41 T>A ones as specified at page 5, lines 141-144. Please correct the figure accordingly.

ü  Two sentences at page 1 lines 38-40 are in contrast: 1- “It is caused by mutations in the PAX2 gene located on chromosome 38 10q24.3-10q25”. 2- “In 50% of RCS cases, mutations can be found in the PAX2 transcription 39 factor gene”.  Which of the two is true? Please correct accordingly.

Author Response

Dear Reviewer,

We would like to express our sincere gratitude for the time and effort you put into reviewing our manuscript. Your constructive feedback and insightful comments were immensely valuable in improving the quality of our work.

We are pleased to inform you that we have carefully considered each of your suggestions and made the necessary changes to our manuscript. Your feedback helped us to clarify several points, improve the organization of our results, and address potential weaknesses in our methodology.

Once again, thank you for your thoughtful review and for your contributions to the advancement of our field. We appreciate your time and effort, and we hope that our revised manuscript meets your expectations.

Sincerely,

Kumar Digvijay

Answers to the reviewer:

1. It would be interesting to sequence PAX2 in the healthy father (I.2) and sister (II.4) to evaluate if they are carriers of the PAX2 c.617-19 T>A c.617-70 C>A, c.617-41 T>A variations detected. If this is the case, these polymorphisms were inherited form the father and it could be that their presence worsened the disease phenotype.  Interestingly, affected siblings appear to have a more severe clinical presentation and progression than the mother.  Otherwise, the above polymorphism are on the same allele as the maternal inherited c.352C>G mutation.

Answer: The father and sister of the affected individual have not been diagnosed with any renal, ocular, or auditory problems. Therefore, it is possible that they are not carriers of the PAX2 c.617-19 T>A c.617-70 C>A, c.617-41 T>A variations detected in the affected individual.

Also, as this was a diagnostic test, we were unable to sequence the father and sister at the time of the study. We will keep the suggestion in mind for future studies and if we have the opportunity to obtain samples from the father and sister, we will evaluate if they are carriers of the PAX2 c.617-19 T>A, c.617-70 C>A, and c.617-41 T>A variations detected. It is possible that these polymorphisms were inherited from the father and could have contributed to the more severe clinical presentation and progression seen in the affected siblings compared to the mother. Additionally, it is important to note that these polymorphisms are on the same allele as the maternal inherited c.352C>G mutation, which further supports the pathogenicity of this mutation.

2. The authors should specify if there are malformations in the auditory system of the patients II:2 and II:5.

Answer: We appreciate the reviewer's suggestion to provide information on the auditory system of the patients. We do have an audiogram report for the younger brother (II:5) which indicates conductive hearing loss in the right ear and sensoneural hearing loss in the left ear. We have included this information in the revised version of the manuscript.

3. Chromatograms in figure 1 and 3 should be of better quality.

Answer: Thank you for your comment. We agree that the quality of the chromatograms in Figures 1 and 3 could be improved. We have regenerated higher quality chromatograms. The updated figures have been included in the revised manuscript.

4. The authors should add a table with the primers used to sequence PAX2 exon and intron-exon boundaries. Please specify the sequencer used, as well.

Answer: Thank you for the suggestion. We added a table with the primers used to sequence PAX2 exon and intron-exon boundaries in the methods section of our manuscript. We also specified the sequencer used for the sequencing.

5. Figure 4 shows only the c.617-19 T>A variation and not c.617-70 C>A, c.617-41 T>A ones as specified at page 5, lines 141-144. Please correct the figure accordingly.

Answer: Thank you for bringing this to our attention. We apologize for the error in Figure 4. We  revised the figure to include all three variations (c.617-19 T>A, c.617-70 C>A, c.617-41 T>A) and ensure that it accurately reflects the information presented in the manuscript.

6. Two sentences at page 1 lines 38-40 are in contrast: 1- “It is caused by mutations in the PAX2 gene located on chromosome 38 10q24.3-10q25”. 2- “In 50% of RCS cases, mutations can be found in the PAX2 transcription 39 factor gene”.  Which of the two is true? Please correct accordingly.

Answer: I apologize for the inconsistency in the sentences. The correct information is that mutations in the PAX2 gene located on chromosome 10q24.3-10q25 are responsible for the majority of cases of renal coloboma syndrome (RCS), while mutations in the PAX2 transcription factor gene can be found in around 50% of RCS cases. Thank you for bringing this to our attention.

Author Response File: Author Response.docx

Reviewer 2 Report

Report of a novel heterozygous variant in PAX2 in two affected individuals with RCS.

Strengths:

1. Detected in 2 affected individuals of the kindred

2. Occular phenotype shown with case

3. Novel Sequence variant predicted to be pathogenic by software

Weakness:

1. Does not show segregation within the kindred. Would be much sounder if authors could show that the variant is not present in the father (Unaffected).

2. Does not provide allele frequency/healthy control chromosomes from a population with comparable ancestry. It is possible that an innocuous variant may not be present in genomic databases if the population from which it is derived is not well represented in these datasets.

3. Should indicate the reference sequence that is being used when describing the sequence variant position.

4. Labeling on the figures is too small to be legible.

5. If segregation in the kindred and evaluation of a healthy ancestral cohort is not possible the conclusions regarding the pathogenicity of the variant should be softened. It is possible that this variant is pathogenic, but certainly not definitive.

Author Response

Dear Reviewers,

We would like to express our sincere gratitude for the time and effort you put into reviewing our manuscript. Your constructive feedback and insightful comments were immensely valuable in improving the quality of our work.

We are pleased to inform you that we have carefully considered each of your suggestions and made the necessary changes to our manuscript. Your feedback helped us to clarify several points, improve the organization of our results, and address potential weaknesses in our methodology.

Once again, thank you for your thoughtful review and for your contributions to the advancement of our field. We appreciate your time and effort, and we hope that our revised manuscript meets your expectations.

Sincerely,

Kumar Digvijay

Answers to the reviewer:

1. Does not show segregation within the kindred. Would be much sounder if authors could show that the variant is not present in the father (Unaffected).

Answer: The father does not have any renal, ocular, or auditory problems and has a normal creatinine level of 0.58 mg/dl. Similarly, the elder sister does not have any renal, visual, or auditory complaints and has a normal creatinine level of 0.60 mg/dl. It would have been useful if the variant could have been checked for the father to ascertain if the variant is present in him as well. However, as a prospective donor, the father may not have been willing to undergo genetic testing. Nonetheless, the absence of the variant in the father and the elder sister further adds to the complexity of the genetic inheritance of the disease.

Moreover, it is important to note that these polymorphisms are on the same allele as the maternal inherited c.352C>G mutation, which further supports the pathogenicity of this mutation.

2. Does not provide allele frequency/healthy control chromosomes from a population with comparable ancestry. It is possible that an innocuous variant may not be present in genomic databases if the population from which it is derived is not well represented in these datasets.

Answer: Although we did not obtain allele frequencies from healthy control individuals with comparable ancestry, it is important to note that an innocuous variant may not be present in genomic databases if the population from which it is derived is not well represented in these datasets. Future studies including larger cohorts of individuals with diverse ancestry will be needed to confirm the pathogenicity of the identified variant and assess its prevalence in different populations.

3. Should indicate the reference sequence that is being used when describing the sequence variant position.

Answers: We added a table with the primers used to sequence PAX2 exon and intron-exon boundaries in the Methods section of our manuscript. We also specified the sequencer used for the sequencing.

4. Labeling on the figures is too small to be legible.

Answer: We apologize for any inconvenience caused by the small size of the labeling, and we have taken your feedback into consideration.

We have corrected the labeling on the figures to ensure that it is now legible. We hope that this addresses your concerns and improves the overall clarity of the article.

5. If segregation in the kindred and evaluation of a healthy ancestral cohort is not possible the conclusions regarding the pathogenicity of the variant should be softened. It is possible that this variant is pathogenic, but certainly not definitive.

Answer: Due to the unavailability of a healthy ancestral cohort and the inability to perform segregation analysis, the conclusions regarding the pathogenicity of the identified variant should be interpreted with caution. It is possible that the variant is pathogenic, but the lack of definitive evidence warrants further investigation.

Round 2

Reviewer 1 Report

1-    It would be interesting to sequence PAX2 in the healthy father (I.2) and sister (II.4) to evaluate if they are carriers of the PAX2 c.617-19 T>A c.617-70 C>A, c.617-41 T>A variations detected. If this is the case, these polymorphisms were inherited form the father and it could be that their presence worsened the disease phenotype.  Interestingly, affected siblings appear to have a more severe clinical presentation and progression than the mother.  Otherwise, the above polymorphism are on the same allele as the maternal inherited c.352C>G mutation.

Answer: The father and sister of the affected individual have not been diagnosed with any renal, ocular, or auditory problems. Therefore, it is possible that they are not carriers of the PAX2 c.617-19 T>A c.617-70 C>A, c.617-41 T>A variations detected in the affected individual. Also, as this was a diagnostic test, we were unable to sequence the father and sister at the time of the study. We will keep the suggestion in mind for future studies and if we have the opportunity to obtain samples from the father and sister, we will evaluate if they are carriers of the PAX2 c.617-19 T>A, c.617-70 C>A, and c.617-41 T>A variations detected. It is possible that these polymorphisms were inherited from the father and could have contributed to the more severe clinical presentation and progression seen in the affected siblings compared to the mother. Additionally, it is important to note that these polymorphisms are on the same allele as the maternal inherited c.352C>G mutation, which further supports the pathogenicity of this mutation

R2: It is not clear how the authors can conclude from the ion torrent analysis that the polymorphisms detected are on the same allele as the maternal inherited c.352C>G mutation. If this were the case, there would be no doubt that they were inherited by the mother together with the c.352C>G mutation.

5.     Figure 4 shows only the c.617-19 T>A variation and not c.617-70 C>A, c.617-41 T>A ones as specified at page 5, lines 141-144. Please correct the figure accordingly.

Answer: Thank you for bringing this to our attention. We apologize for the error in Figure 4. We revised the figure to include all three variations (c.617-19 T>A, c.617-70 C>A, c.617-41 T>A) and ensure that it accurately reflects the information presented in the manuscript.

R2: New figure 5 (old figure 4) does not yet include all three variations

Author Response

Dear Reviewer,

We would like to express our sincere gratitude for the time and effort you put into reviewing our manuscript. Your constructive feedback and insightful comments were immensely valuable in improving the quality of our work.

We are pleased to inform you that we have carefully considered each of your suggestions and made the necessary changes to our manuscript. Your feedback helped us to clarify several points, improve the organization of our results, and address potential weaknesses in our methodology.

Once again, thank you for your thoughtful review and for your contributions to the advancement of our field. We appreciate your time and effort, and we hope that our revised manuscript meets your expectations.

Sincerely,

Kumar Digvijay

Answers to the reviewer:

1. It is not clear how the authors can conclude from the ion torrent analysis that the polymorphisms detected are on the same allele as the maternal inherited c.352C>G mutation. If this were the case, there would be no doubt that they were inherited by the mother together with the c.352C>G mutation.

Answer: I want to apologize for the misrepresentation of the sequencer that was used in our genetic analysis study. It has come to our attention that there was a miscommunication from the technician of the company where we sent our samples, which resulted in incorrect information.

We understand the importance of accurate and transparent reporting of the methodology and equipment used in scientific research, and we regret any confusion or inconvenience that this may have caused. We would like to clarify that the correct sequencer used in our study was the 3500xl genetic analyzer.

The 3500xl genetic analyzer is a reliable instrument used in the field of genetic analysis. However, it is not possible to conclusively determine whether the polymorphisms detected are on the same allele as the maternal inherited c.352C>G mutation solely based on the type of sequencer used.

Based on the information provided, it is possible that the father and sister of the affected individual are not carriers of the PAX2 c.617-19 T>A, c.617-70 C>A, c.617-41 T>A variations detected in the affected individual.

Also, as this was a diagnostic test, we were unable to sequence the father and sister at the time of the study.

2. New figure 5 (old figure 4) does not yet include all three variations

Answer: We apologize for the error in Figure 5. We revised the figure to include all three variations (c.617-19 T>A, c.617-70 C>A, c.617-41 T>A) and ensure that it accurately reflects the information presented in the manuscript.

Round 3

Reviewer 1 Report

The paper is now suitable for publication