Functional and Genetic Analyses Unveil the Implication of CDC27 in Hemifacial Microsomia
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript describes a complex study for the identification of candidate causative genes and gene variationsin the etiology of Goldenhar syndrome spectrum. The careful and well planned methods are updated and informative. Most figures and tables are inforamtive.
Some remarks : 1./ It is not clear, which disease entry is the target of the investigations. The title speaks about 'hemifacial microsomia ', but the introduction mentions MIM number incorrectly 164210, which is 'craniofacial microsomia '. Hemifacial microsomia has the MIM number 141400. (Page 1, line 35.) The precise name of disease is imortant already in the title of manuscript, because Goldenhar syndrome is a spectrum, also referred as oculoauriculovertebral spectrum (OAVS) . 2./ One gene variation as etiology is known in craniofacial microsomia, namely SF3B2 (MIM number 605591) , it should be mentioned. 3./ In the demographic part of the patients (2.1. chapter) genders, ages of children are missing, and the side of unilateral malformations (right or left side) should be given, as well. 4./ The text writes on page 3, lines 90 - 91: ...analysis across various families... A more precise description is needed . 5./ The authors found CDC27 expression in eye, trunk and heart tissues, which was not present in a later pahase of development. How do they explain this expression difference? This fact seems to be important, because several organs, not only mandibulofacial regions may be involved in the malformation phenotype in patients. 6./ Fig. 4, page 6 line 184: "in the majority...", a correct number has to be given. 7./ Page 7, lines 191, 195 - 196 : Sanger chromatograms and black boxes are not clearly seen, they are too small. 8./ Page 16 line 521: ...showed no phenotype... what does it mean ? Perhaaps 'pathologic phenotype ' is the correct formulation. 9./ The mnnuscript is too long, several parts of the text should concentrate on e new findings. If so, the reading of the complex text of results and methods could be easier and more interesting. 10./ A list of abbreviations would be also useful.
The manuscript is recommended for publication after some minor corrections.
Author Response
Comments and Suggestions for Authors
The manuscript describes a complex study for the identification of candidate causative genes and gene variations in the etiology of Goldenhar syndrome spectrum. The careful and well-planned methods are updated and informative. Most figures and tables are informative.
Response: I am really appreciated for you to review the current study. All suggestions have been carefully reviewed and our responses to each comment were attached.
Some remarks:
1. It is not clear, which disease entry is the target of the investigations. The title speaks about ‘hemifacial microsomia’, but the introduction mentions MIM number incorrectly 164210, which is ‘craniofacial microsomia’. Hemifacial microsomia has the MIM number 141400. (Page 1, line 35.) The precise name of disease is important already in the title of manuscript, because Goldenhar syndrome is a spectrum, also referred as oculoauriculovertebral spectrum (OAVS).
Response: Thank you for your valuable feedback. The MIM number 141400 denotes “hemifacial microsomia with radial defects.” Nevertheless, probands in our current study did not exhibit radial defects. Conversely, the MIM number 164210 corresponds to “craniofacial microsomia-1,” with alternative designations such as “hemifacial microsomia,” “Goldenhar syndrome,” and “oculoauriculovertebral spectrum (OAVS).” To mitigate potential confusion, we will remove the MIM number from the “Introduction” section in the revised manuscript.
2. One gene variation as etiology is known in craniofacial microsomia, namely SF3B2 (MIM number 605591), it should be mentioned.
Response: Thank you for your suggestion. SF3B2 has been confirmed as a crucial pathogenic gene in CFM, and we have cited this in the “Introduction” section (Reference 6). To highlight the significance of this gene’s pathogenicity, we will present the important results related to SF3B2 in the revised manuscript (Page 2, Line 48 to 50).
3. In the demographic part of the patients (2.1. chapter) genders, ages of children are missing, and the side of unilateral malformations (right or left side) should be given, as well.
Response: Thanks for the suggestion. We have added this information to 2.1. chapter in our revised manuscript.
4. The text writes on page 3, lines 90 - 91: ...analysis across various families... A more precise description is needed.
Response: Thank you for your suggestion. Our intention here is to convey that there are at least two or more probands from distinct families exhibiting a novel mutation in a specific gene. We added a more precise description in the revised manuscript.
5. The authors found CDC27 expression in eye, trunk and heart tissues, which was not present in a later phase of development. How do they explain this expression difference? This fact seems to be important, because several organs, not only mandibulofacial regions may be involved in the malformation phenotype in patients.
Response: During the initial stage of zebrafish embryonic development, we observed significant expression of cdc27 primarily in the head, eyes, and trunk. Subsequently, its expression became more localized to the pharyngeal arch region. The cdc27-/- zebrafish embryos exhibited progressive development of craniofacial abnormalities, including reduced head and eye size, diminutive jaw, and spinal deformities, beginning from 2 dpf. These phenotypic traits closely resemble those observed in individuals with HFM. While the upregulation of cdc27 expression in the head, eyes, trunk, and other regions was not as pronounced beyond the second day, its regulatory influence might persist in these areas. We hypothesize that while the cdc27 expression in the head, eyes, trunk, and other regions diminishes after the second day, its regulatory impact continues in these areas.
6. Fig. 4, page 6 line 184: “in the majority...”, a correct number has to be given.
Response: Thanks for the suggestion. In over 90% of the gRNA injected embryos, a notable reduction in the overall size of craniofacial structures, along with spine malformation and cardiac edema, was evident compared to control siblings. We have made an update in the revised manuscript.
7. Page 7, lines 191, 195-196: Sanger chromatograms and black boxes are not clearly seen, they are too small.
Response: The clarity of the Sanger chromatograms in the manuscript is compromised due to the inability to display the original TIFI format images clearly. Upon acceptance and official publication of our article, we commit to providing high-definition original images to the journal. Additionally, we have added relevant sequence information in the figure legends.
8. Page 16 line 521: ...showed no phenotype... what does it mean? Perhaps ‘pathologic phenotype’ is the correct formulation.
Response: Thanks for your comment. Our expression is inaccurate; what we intended to convey here was “pathological phenotype”.
9. The manuscript is too long; several parts of the text should concentrate on e new findings. If so, the reading of the complex text of results and methods could be easier and more interesting.
Response: Thank you for your comment. Despite our efforts to condense the article, it encompasses patient clinical data, gene screening, zebrafish phenotype validation, and rescue experiments, resulting in a lengthy manuscript. In the revised version, we aimed to streamline certain sections to enhance readability and emphasize key points.
10. A list of abbreviations would be also useful.
Response: Thank you for the suggestion. An abbreviation list was added to the revised manuscript at the end of the main text before the “Reference” section.
The manuscript is recommended for publication after some minor corrections.
Response: Thank you again for all your work and support.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe article "Functional and Genetic Analyses Unveil the Implication of CDC27 in Hemifacial Microsomia" provides a comprehensive investigation into the role of CDC27 in the development of hemifacial microsomia (HFM), a rare congenital disorder characterized by asymmetry of the face due to underdevelopment of the facial structures. The study employs a multidisciplinary approach, combining whole-exome sequencing (WES) of affected individuals with experimental validation in zebrafish models to identify CDC27 as a novel candidate gene associated with HFM.
**Strengths:**
1. **Innovative Methodology:** The use of WES to identify novel non-synonymous de novo variants in CDC27 among HFM patients represents a significant advancement in understanding the genetic basis of HFM. The combination of predictive structural analysis, in situ hybridization, and CRISPR/Cas9 gene editing in zebrafish to model the disease phenotype and validate the gene's role is methodologically sound and innovative.
2. **Comprehensive Analysis:** The article presents a thorough analysis of the CDC27 variants, including their effects on protein structure and function, interaction with other proteins, and impact on cell proliferation and apoptosis. This holistic approach provides valuable insights into the pathogenic mechanisms underlying HFM.
3. **Clear Presentation of Results:** The findings are clearly presented, with well-organized sections and informative figures that illustrate the research outcomes effectively. The statistical analysis is robust, lending credibility to the conclusions drawn.
4. **Potential Clinical Implications:** Identifying CDC27 as a potential pathogenic gene for HFM opens new avenues for genetic counseling, early diagnosis, and possibly targeted therapeutic interventions, offering hope for affected individuals and their families.
**Weaknesses:**
1. **Limited Sample Size:** The study is based on a relatively small number of HFM cases. While the identification of CDC27 variants in these cases is compelling, a larger cohort would strengthen the evidence for CDC27's role in HFM and help identify other potentially involved genes.
2. **Lack of Functional Studies in Mammalian Models:** Although the zebrafish model provides valuable insights, the absence of functional validation in mammalian models, particularly in models that more closely resemble human facial development, is a limitation. Such studies could further elucidate the role of CDC27 in craniofacial development and its relevance to human HFM.
3. **Need for Further Research on Therapeutic Implications:** While the study highlights the potential clinical implications of CDC27 in HFM, it stops short of exploring therapeutic interventions. Further research is needed to translate these findings into practical treatments or preventive strategies for HFM.
4. **Consideration of Environmental Factors:** The article focuses primarily on the genetic aspects of HFM without extensive consideration of environmental factors that may interact with genetic predispositions to influence the development of the condition.
In conclusion, this article makes a significant contribution to the genetic understanding of HFM, identifying CDC27 as a key player in its pathogenesis. The findings set the stage for further research on the mechanisms of HFM development and open potential pathways for future therapeutic interventions. However, additional studies with larger cohorts and in mammalian models are necessary to fully understand the implications of CDC27 mutations in HFM and to explore their therapeutic potential.
Author Response
Comments and Suggestions for Authors
The article "Functional and Genetic Analyses Unveil the Implication of CDC27 in Hemifacial Microsomia" provides a comprehensive investigation into the role of CDC27 in the development of hemifacial microsomia (HFM), a rare congenital disorder characterized by asymmetry of the face due to underdevelopment of the facial structures. The study employs a multidisciplinary approach, combining whole-exome sequencing (WES) of affected individuals with experimental validation in zebrafish models to identify CDC27 as a novel candidate gene associated with HFM.
Response: I am really appreciated for you to review the current study. All suggestions and comments have been carefully reviewed and our responses to each comment were attached.
**Strengths: **
- **Innovative Methodology: ** The use of WES to identify novel non-synonymous de novo variants in CDC27 among HFM patients represents a significant advancement in understanding the genetic basis of HFM. The combination of predictive structural analysis, in situ hybridization, and CRISPR/Cas9 gene editing in zebrafish to model the disease phenotype and validate the gene's role is methodologically sound and innovative.
Response: Thanks for your comment.
- **Comprehensive Analysis: ** The article presents a thorough analysis of the CDC27 variants, including their effects on protein structure and function, interaction with other proteins, and impact on cell proliferation and apoptosis. This holistic approach provides valuable insights into the pathogenic mechanisms underlying HFM.
Response: Thank you for your comment.
- **Clear Presentation of Results: ** The findings are clearly presented, with well-organized sections and informative figures that illustrate the research outcomes effectively. The statistical analysis is robust, lending credibility to the conclusions drawn.
Response: Thank you for your recognition of the “Results” section.
- **Potential Clinical Implications: ** Identifying CDC27 as a potential pathogenic gene for HFM opens new avenues for genetic counseling, early diagnosis, and possibly targeted therapeutic interventions, offering hope for affected individuals and their families.
Response: Thank you for your review. We aspire to delve deeper into the clinical applications of CDC27 in our further study.
**Weaknesses: **
- **Limited Sample Size: ** The study is based on a relatively small number of HFM cases. While the identification of CDC27 variants in these cases is compelling, a larger cohort would strengthen the evidence for CDC27's role in HFM and help identify other potentially involved genes.
Response: Thank you for your suggestion. In our further study, we aim to conduct additional analyses of the CDC27 gene in a larger cohort of HFM patients.
- **Lack of Functional Studies in Mammalian Models: ** Although the zebrafish model provides valuable insights, the absence of functional validation in mammalian models, particularly in models that more closely resemble human facial development, is a limitation. Such studies could further elucidate the role of CDC27 in craniofacial development and its relevance to human HFM.
Response: Thanks for your suggestion. We have initiated knockout experiments targeting the cdc27 gene in mice. We look forward to the results which could further elucidate the role of CDC27 in HFM.
- **Need for Further Research on Therapeutic Implications: ** While the study highlights the potential clinical implications of CDC27 in HFM, it stops short of exploring therapeutic interventions. Further research is needed to translate these findings into practical treatments or preventive strategies for HFM.
Response: Currently, investigations into HFM and the cdc27 gene primarily reside in the animal model phase. While various methodologies have been explored to alleviate cdc27-/- zebrafish phenotypes, further endeavors are essential for rescuing phenotypes in HFM patients. Future prospects may involve integrating prenatal screenings to identify suspected pathogenic CDC27 mutations and executing gene therapy on fetuses harboring such mutations.
- **Consideration of Environmental Factors: ** The article focuses primarily on the genetic aspects of HFM without extensive consideration of environmental factors that may interact with genetic predispositions to influence the development of the condition.
Response: Thanks for your comment. Other researchers of our team have focused on the environmental factors of HFM.
In conclusion, this article makes a significant contribution to the genetic understanding of HFM, identifying CDC27 as a key player in its pathogenesis. The findings set the stage for further research on the mechanisms of HFM development and open potential pathways for future therapeutic interventions. However, additional studies with larger cohorts and in mammalian models are necessary to fully understand the implications of CDC27 mutations in HFM and to explore their therapeutic potential.
Response: Thank you again for all your work and support. In the further study, we will undertake the detection of variant sites within the CDC27 gene across a larger sample size of HFM patients, and subsequently observe alterations in phenotypes associated with HFM through cdc27 knockout and rescue experiments in mammals.
Reviewer 3 Report
Comments and Suggestions for AuthorsI would like to thank the authors for their submission and allowing me to review their work.
This is an interesting study on an important topic. However, I would be grateful if you could add further explanations and changes on the following points:
1) RESULTS: Page 2, line 75
What was the mean age (± standard deviation) of the probands?
2) RESULTS: Page 2, line 78
What hearing classification did you use to classify the different degrees of hearing loss?
3) RESULTS: Page 2, line 78
What was the mean hearing threshold (± standard deviation) of the probands?
4) RESULTS: Page 2, line 78
Did the probands use a hearing device (e.g. a bone anchored implant)?
5) RESULTS: Page 2, line 78
Had the probands undergone any vestibular examination? Unilateral hearing loss may also be associated with unilateral vestibular hypofunction.
6) RESULTS: Page 2, line 78
Was a high resolution computed tomography (CT) scan performed to better study the ear with atresia?
7) DISCUSSION: Page 16, line 523
A detailed section on the limitations of the study should be added.
8) DISCUSSION: Page 16, line 523
What are the future prospects of this study?
Comments on the Quality of English Languagegood
Author Response
Comments and Suggestions for Authors
I would like to thank the authors for their submission and allowing me to review their work.
This is an interesting study on an important topic. However, I would be grateful if you could add further explanations and changes on the following points:
Response: I am really appreciated for you to review the current study. All suggestions have been carefully reviewed and our responses to each comment were attached.
1) RESULTS: Page 2, line 75
What was the mean age (± standard deviation) of the probands?
Response: Thanks for the question. The mean age of the participants is 6.9 years with a standard deviation of 0.9. We have added the age range in the revised manuscript.
2) RESULTS: Page 2, line 78
What hearing classification did you use to classify the different degrees of hearing loss?
Response: The hearing classification employed in our study follows the guidelines outlined in the World Hearing Report 1997, reflecting the timeframe during which participants were enrolled, from January 2017 to December 2019.
3) RESULTS: Page 2, line 78
What was the mean hearing threshold (± standard deviation) of the probands?
Response: The mean hearing threshold of the probands was 80 ± 5 dBHL.
4) RESULTS: Page 2, line 78
Did the probands use a hearing device (e.g. a bone anchored implant)?
Response: In the current study, two probands, Proband 2 and Proband 10, were fitted with bone conduction hearing devices (soft band, MED-EL, Innsbruck, Austria).
5) RESULTS: Page 2, line 78
Had the probands undergone any vestibular examination? Unilateral hearing loss may also be associated with unilateral vestibular hypofunction.
Response: We totally agreed that unilateral hearing loss may also be associated with unilateral vestibular hypofunction. No probands in the current study underwent vestibular examination since none complained of dizziness.
6) RESULTS: Page 2, line 78
Was a high-resolution computed tomography (CT) scan performed to better study the ear with atresia?
Response: Temporal bone CT scans were exclusively performed on probands manifesting stenosis of the external auditory canal, with the specific objective of identifying the presence of external auditory canal cholesteatoma. Those presenting with atresia of the external auditory canal did not undergo temporal bone CT scans following physical examination.
7) DISCUSSION: Page 16, line 523
A detailed section on the limitations of the study should be added.
Response: Thanks for the suggestion. We have added a detailed section on the limitations of the current study in the revised manuscript.
The current study has several limitations. Firstly, it relies on a relatively small cohort of HFM cases. Although the discovery of CDC27 variants in these cases is compelling, a larger sample size would strengthen the evidence supporting the involvement of CDC27 in HFM and facilitate the identification of other potentially implicated genes. Additionally, while the zebrafish model provides valuable insights, the lack of functional validation in mammalian models, particularly those closely resembling human facial development, is a limitation. Lastly, although the study underscores the potential clinical implications of CDC27 in HFM, it does not explore therapeutic interventions. Further research is necessary to translate these findings into actionable treatments or preventive measures for HFM.
8) DISCUSSION: Page 16, line 523
What are the future prospects of this study?
Response: Thanks for the question. In the further study, we will undertake the detection of variant sites within the CDC27 gene across a larger sample size of HFM patients, and subsequently observe alterations in phenotypes associated with HFM through cdc27 knockout and rescue experiments in mammals. We also added these to the revised manuscript.
