Genetically Confirmed Familial Case of Nonsyndromic Cardiac Progeria Caused by the LMNA p.Asp300Asn Variant with Presumed Gonadal Mosaicism: Phenotypic Comparison with Previously Reported Patients

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In your manuscript you describe a familial form of nonsyndromic cardiac progeria based on the Asp300Asn LMNA variant whereby you emphasize that it is the first genetically confirmed familial case for this variant and that evidence suggests gonadal mosaicism as the mechanism of inheritance. In addition, you provide an overview of the phenotype of all reported carriers of this specific LMNA variant highlighting the variability in age of onset, type and severity of cardiovascular manifestations and other LMNA related manifestations. You conclude appropriately the importance of genetic evaluation in patients with early-onset cardiac atherosclerotic disease in the absence of cardiovascular risk factors.

Your manuscript is relevant as it adds to the growing literature on LMNA variants that are associated with another phenotype than the classical ones.  In addition, the overview of all patients with the Asp30Asn variant is valuable as it is increasingly important to obtain better insight in the variability of the natural history of patients with a same genetic variant. I would therefore emphasize much more on the phenotypic comparison with the previously reported patients as well as with other LMNA variants that are also related to nonsyndromic cardiac progeria.

General comments:

1. The title is somewhat ‘pretentious’:
   • It may be the first genotypically confirmed family with nonsyndromic cardiac progeria with the specific LMNA Asp300Asn variant, but it is not the first reported familial case of nonsyndromic cardiac progeria caused by an LMNA variant (see for instance JCDD 2024.11,86, PMID: 38535109). Moreover, only the death causes of the brother and sister of the index patient were mentioned, but I presume that they have not been physically examined so that a systemic progeroid phenotype cannot be ruled out and as such not clear whether they fit the ‘nonsyndromic’ cardiac progeria phenotype. In addition 3 of the 4 publications reporting patients with the specific LMNA Asp300Asn variant (as reported in the manuscript) also describe a familial burden in line with the LMNA phenotype (albeit not genetically confirmed).
   • The title mentions 'confirmed' gonadal mosaicism. This is somewhat misleading as somatic mosaicism in one of the parents has not been ruled out.
2. Spelling errors, absence of punctuation and the use of the English language should be addressed throughout the entire manuscript.
3. Sloppy mistakes (such as duplicated references) should be addressed. 

 

Specific comments:

1. Regarding the Case Presentation:
   • Indicate that the cause of death of the siblings can be associated with a LMNA phenotype and add references.
   • Any additional information on the siblings (medical history) would be useful to add.
   • Were there any indications whether the proband has osteoporosis or liver steatosis?
   • In the text it is mentioned that WES was done, but be more specific: was it a gene panel based on WES or an HPO analysis or was it open WES?
   • Sanger sequencing was used as a technique to confirm the variant in the proband and to test family members. Have you considered to perform ultra-deep NGS (as this technique might detect low-level mosaicism in blood where Sanger sequencing is ineffective, see AJMG 2019.179(6), PMID: 30912254)?
   • You indicate that the LMNA variant results in a missense substitution of the highly conserved amino acid residue. It would be more useful to add the ACMG criteria so that it is clear how pathogenicity has been classified.
   • It is mentioned that two unaffected daughters and two unaffected nieces are carriers of the LMNA variant. No ages are mentioned. They probably are young which can explain why they have not yet developed a phenotype.
   • Table 1 can be much improved by taking out the cardiovascular manifestations and surgical interventions and focusing more on the systemic progeroid features of all reported patients and maybe include the brother and sister in the table. In addition the row with the genetic results can be erased as this is already mentioned in the title of the table.
   • I would suggest to present the cardiovascular manifestations and surgical interventions in a figure (with a line per patient over time and bullets in color for the different type of manifestations/interventions) to better show the chronological order of cardiovascular manifestations/interventions.
2. Regarding the Discussion:
   • Please provide a reference why alterations at that specific site is strongly associated with pathogenic cardiac and progeroid phenotypes.
   • I miss a broader context and discussion regarding patients with nonsyndromic (familial) cardiac progeria with other variants in LMNA, not located in that same region.
   • As for the gonadal mosaicism, I miss the context of previously described mosaicism in LMNA.

Comments on the Quality of English Language

Use of the English language is poor. Sentences throughout the text can be improved. It could be useful to ask a native speaker to go through the entire text.

 

Author Response

General comments:

Comment 1 The title is somewhat ‘pretentious’

Response 1 Thank you for pointing this out. We agree with this comment. The title has been revised to avoid the potentially misleading term “confirmed” gonadal mosaicism. We also acknowledge that, although our family may represent the first genotypically confirmed case of nonsyndromic cardiac progeria with the specific LMNA p.Asp300Asn variant, it is not the first reported familial case of nonsyndromic cardiac progeria caused by an LMNA variant. Furthermore, as the brother and sister of the proband were not examined clinically, a systemic progeroid phenotype cannot be entirely excluded.

Comment  2 Spelling errors, absence of punctuation and the use of the English language should be addressed throughout the entire manuscript.

Response 2 Thank you for pointing this out. We agree with this comment. All spelling, punctuation, and language errors have been carefully corrected throughout the entire manuscript.

Comment  3 Sloppy mistakes (such as duplicated references) should be addressed. 

Response 3 Thank you for pointing this out. We agree with this comment. All duplicated references have been corrected in the revised manuscript.

 

Specific comments:

Comment 1.

Indicate that the cause of death of the siblings can be associated with a LMNA phenotype and add references.

Any additional information on the siblings (medical history) would be useful to add.

Response 1 Thank you for pointing this out. We absolutely agree with this comment.  Unfortunately, detailed clinical information about the deceased siblings is not available. However, the early cardiovascular deaths of the proband’s brother and sister, together with the identification of the LMNA p.Asp300Asn variant in their children (the proband’s nieces), suggest that the siblings may also have carried the same variant. This raises the possibility that their deaths could be associated with an LMNA-related cardiac phenotype.

Comment 2. Were there any indications whether the proband has osteoporosis or liver steatosis?

Response  2. Thank you for pointing this out. We absolutely agree with this comment. The proband underwent comprehensive clinical and instrumental evaluation, and no signs of osteoporosis or liver steatosis were identified. These details have been added to the revised manuscript on page 3, lines 22–24.

Comment 3. In the text it is mentioned that WES was done, but be more specific: was it a gene panel based on WES or an HPO analysis or was it open WES?

Response 3. Thank you for pointing this out. We absolutely agree with this comment.  We appreciate the reviewer’s comment. We confirm that a comprehensive whole-exome sequencing (WES) analysis was performed, rather than a targeted gene panel or HPO-based virtual analysis. The methodology and analytical workflow are described in detail in the Materials and Methods section.

Comment 4. Sanger sequencing was used as a technique to confirm the variant in the proband and to test family members. Have you considered to perform ultra-deep NGS (as this technique might detect low-level mosaicism in blood where Sanger sequencing is ineffective, see AJMG 2019.179(6), PMID: 30912254)?

Response 4. Thank you for pointing this out. We absolutely agree with this comment. No, ultra-deep NGS was not performed. Sanger sequencing was used to confirm the variant in the proband and to determine the genetic status of her family members. Unfortunately, we were unable to obtain additional tissue samples (such as buccal epithelial cells or sperm) for analysis; therefore, even ultra-deep sequencing would not be sufficient to exclude the possibility of somatic mosaicism. We thank the reviewer for this valuable comment, and this clarification has been added to the Discussion section of the revised manuscript.

Comment 5. You indicate that the LMNA variant results in a missense substitution of the highly conserved amino acid residue. It would be more useful to add the ACMG criteria so that it is clear how pathogenicity has been classified.

Response 5. Thank you for pointing this out. We absolutely agree with this comment. We thank the reviewer for this valuable suggestion. The ACMG/AMP criteria used to classify the pathogenicity of the LMNA variant have been added to the revised manuscript on page 4, lines 10–11.

Comment 6. It is mentioned that two unaffected daughters and two unaffected nieces are carriers of the LMNA variant. No ages are mentioned. They probably are young which can explain why they have not yet developed a phenotype.

Response 6. Thank you for pointing this out. We absolutely agree with this comment. The ages of the proband’s daughters and nieces are indicated in the text and figure and have been clarified in the revised version. They are indeed young, which likely explains the absence of phenotypic manifestations at this stage.

Comment 7. Table 1 can be much improved by taking out the cardiovascular manifestations and surgical interventions and focusing more on the systemic progeroid features of all reported patients and maybe include the brother and sister in the table. In addition the row with the genetic results can be erased as this is already mentioned in the title of the table.

Response 7. Thank you for pointing this out. We absolutely agree with this comment. Table 1 has been revised according to the reviewer’s recommendations, with a focus on systemic progeroid features and removal of the cardiovascular manifestations, surgical interventions, and the row containing genetic results. However, the proband’s siblings were not included in the table, as detailed clinical information about them is not available.

Comment 8. I would suggest to present the cardiovascular manifestations and surgical interventions in a figure (with a line per patient over time and bullets in color for the different type of manifestations/interventions) to better show the chronological order of cardiovascular manifestations/interventions.

Response 8. Thank you for pointing this out. We absolutely agree with this comment. In accordance with the reviewer’s comment, we have prepared a figure  (Figure 2 in manuscript)  illustrating the timeline of disease onset and surgical interventions for each patient to better demonstrate the chronological order of cardiovascular manifestations and interventions.

Comment 9. Please provide a reference why alterations at that specific site is strongly associated with pathogenic cardiac and progeroid phenotypes.

Response 9. Thank you for pointing this out. We absolutely agree with this comment. We have revised the text on page 9, lines 15–24, to include a reference supporting the association between alterations at this specific site and pathogenic cardiac and progeroid phenotypes. This information is also summarized in Table 1, which presents previously reported patients carrying the same LMNA p.Asp300Asn variant and their corresponding clinical features.

Comment 10. I miss a broader context and discussion regarding patients with nonsyndromic (familial) cardiac progeria with other variants in LMNA, not located in that same region.

Response 10. Thank you for pointing this out. We absolutely agree with this comment. We have expanded the discussion to provide a broader context regarding patients with nonsyndromic (familial) cardiac progeria caused by other LMNA variants located outside this region. These additions have been incorporated on page 10, lines 1–10.

Comment 11. As for the gonadal mosaicism, I miss the context of previously described mosaicism in LMNA.

Response 11. Thank you for pointing this out. We absolutely agree with this comment. We have revised the Discussion section to include additional context on previously described cases of mosaicism involving the LMNA gene. These updates have been incorporated on page 10, lines 21–25, and page 11, lines 1–4.

 

 

 

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors describe a familial case of cardiovascular disease without systemic progeroid features caused by the heterozygous missense variant p.(Asp300Asn) in the LMNA gene. Comparison with previously reported cases in literature, the authors underline the clinical heterogeneity associated with this variant ranging from isolated cardiovascular involvement to syndromic progeroid manifestations with metabolic and skeletal abnormalities.

This is an interesting and valuable manuscript of clinical heterogeneity of the LMNA variants. The manuscript is concise and well-written.

I have only a few minor suggestions for clarification:

• It would be helpful to improve the layout of the Table 1 for better readability.
• Proband and his relatives had a history of non syndromic cardiovascular disease. Could authors provide a proband’s photo? Moreover, the p.(Asp300Asn) variant has ben associated to Hepatic Steatosis (Mahdi et al.,). Have the authors excluded this phenotype in the proband? I report that the reference Mahdi et al., has been included twice in the References section ([5] and [10]).
• The novelty of this familial case is the gonadal mosaiciscm as the mechanism of inheritance of the LMNA variant, although the limited demonstration. Non-paternity-event was also excluded. I think proof that in fact proband is the biological daughter of her father is key. Could authors provide further info about these data?
• According to HGVS nomenclature the format to descrive a predicted aminoacid change into protein based only on DNA level data is p.(Asp300Asn).

Author Response

Comment 1 It would be helpful to improve the layout of the Table 1 for better readability.

Response 1 Thank you for pointing this out. We agree with this comment. The layout of the table has been revised accordingly to enhance clarity and visual organization.

Comment 2. Proband and his relatives had a history of non syndromic cardiovascular disease. Could authors provide a proband’s photo? Moreover, the p.(Asp300Asn) variant has ben associated to Hepatic Steatosis (Mahdi et al.,). Have the authors excluded this phenotype in the proband? I report that the reference Mahdi et al., has been included twice in the References section ([5] and [10]).

Thank you for pointing this out. We agree with this comment. Unfortunately, it is not possible to provide a photograph of the patient. The proband underwent a comprehensive clinical and instrumental evaluation, and hepatic steatosis was excluded. This information has been clarified in the revised version of the manuscript (page 3, lines 22–24).

Comment 3 The novelty of this familial case is the gonadal mosaiciscm as the mechanism of inheritance of the LMNA variant, although the limited demonstration. Non-paternity-event was also excluded. I think proof that in fact proband is the biological daughter of her father is key. Could authors provide further info about these data? 

Response 3 Thank you for pointing this out. We agree with this comment. Confirmation of the proband’s biological relationship to her father has been obtained, and the supporting documentation is provided in the attached file.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Nuzhnaya et al. present a case report of familial nonsyndromic cardiac progeria associated with the LMNA NM_170707.4:c.898G>A mutation. The authors describe a 40-year-old woman with a long history of valvular and coronary artery disease. Whole exome sequencing revealed a variant in the LMNA gene (NM_170707.4: c.898G>A), which results in an Asp300Asn substitution. Sanger sequencing revealed a heterozygous state of NM_170707.4(LMNA):c.898G>A in asymptomatic carrier relatives: two daughters and two nieces. The other three siblings did not show LMNA gene mutations. The proband’s brother died from a pulmonary embolism at age 32, her sister died from coronary artery disease at age 53, and her father underwent coronary artery bypass grafting at age 63.

Several authors have previously described premature aging disorder with severe cardiac manifestations.

The novel findings of this study show familial accumulation and suggest gonadal mosaicism.

 The LMNA gene, which encodes Lamin A/C, causes a diverse array of phenotypes and variable skeletal muscle involvement, though it does not affect the coronary arteries. Less commonly, LMNA mutations cause progeroid syndromes, one of which is characterized by early-onset coronary artery disease. All reported studies identified a heterozygous LMNA:c.898G>A mutation and various cardiac manifestations ranging from aortic stenosis to cardiac arrhythmias.

This report expands understanding of the LMNA p.Asp300Asn variant by illustrating its potential to cause nonsyndromic cardiac progeria without systemic features. The authors emphasize that early genetic diagnosis is crucial for the effective management and counseling of affected families.

This is a well-designed case report overall. To improve the manuscript, I have several suggestions:

1. The authors should briefly discuss other possible genetic mechanisms. The proband may also have a de novo mutation of the LMNA gene; thus, mixed mechanisms may have been at play.
2. It would be worthwhile to investigate histone methylation and acetylation status in asymptomatic carrier relatives.
3. The male offspring were unavailable for investigation. Do the authors suppose there to be any sex-specific effects on gene expression and organic manifestations?Some misspellings should be revised throughout of the manuscript, like: L52: “with burnbed familly history”.

 

Author Response

Comment 1. The authors should briefly discuss other possible genetic mechanisms. The proband may also have a de novo mutation of the LMNA gene; thus, mixed mechanisms may have been at play.

Response 1 Thank you for pointing this out. We agree with this comment. Therefore, the presence of the LMNA variant not only in the proband’s daughters but also in her nieces supports gonadal mosaicism as the most plausible mechanism of inheritance, rather than a purely de novo event, although mixed mechanisms cannot be entirely excluded.

Comment 2 It would be worthwhile to investigate histone methylation and acetylation status in asymptomatic carrier relatives.

Response 2 Thank you for pointing this out. We agree with this comment. We also appreciate the suggestion to investigate histone methylation and acetylation status in asymptomatic carrier relatives. Unfortunately, we currently lack the technical resources to perform such analyses; however, this represents an interesting direction for future research.

Comment 3 

The male offspring were unavailable for investigation. Do the authors suppose there to be any sex-specific effects on gene expression and organic manifestations? Some misspellings should be revised throughout of the manuscript, like: L52: “with burnbed familly history”.

Response 3 Thank you for pointing this out. We agree with this comment. Regarding the comment on potential sex-specific effects, we thank the reviewer for raising this point. We do not suspect any sex-related differences in LMNA gene expression or phenotypic manifestations within this family, and such effects have not been reported in the literature. Finally, all typographical errors noted by the reviewer have been corrected in the revised manuscript.

Comment 4 According to HGVS nomenclature the format to descrive a predicted aminoacid change into protein based only on DNA level data is p.(Asp300Asn).

Response 4 Thank you for pointing this out. In our manuscript, we indicate the variant as NM_170707.4(LMNA):c.898G>A (p.Asp300Asn) in accordance with the HGVS nomenclature.