A Family with a Single LMNA Mutation Illustrates Diversity in Cardiac Phenotypes Associated with Laminopathic Progeroid Syndromes

Round 1

Reviewer 1 Report

Authors reported a family showing a progeroid syndrome and cardiac alterations such as aortic stenosis. Affected relatives carry a deleterious mutation in the LMNA gene, most plausible cause of the phenotype. Some points to clarify:

1.- please, genes (LMNA) should be written in Italic format.

2.- please add data concerning the variant classified as pathogenic (following ACMG recommendations).

3.- Please include data concerning genetic analysis (whole exome, panel...). Which genes were analyzed?

4.- Any other rare variant in any gene currently associated with any of symptom identified in the family? 

Author Response

1. Please, genes (LMNA) should be written in Italic format.

Thank you for recalling us. Changes have been done accordingly from title to the end of manuscript.

2. Please add data concerning the variant classified as pathogenic (following ACMG recommendations).

According to the last update of the ACGM recommendations on the classification of genomic variants, our variant is considered as likely pathogenic (Masson et al. Human Genomics (2022) 16:31):

• 3 PM: absent in database (PM2), affect a functional domain (PM1), and affect the same position as another pathogenic variant already described –PM5).
• 2 PP: segregation (PP1) and bioinformatics arguments (PP3, even if discordant).

We have added these precisions from lines 76 to 84 as follows, and modified variant class in the whole test.

Before:

Lines 75-78 - Currently, this point substitution is not indexed in ClinVar or GnomAD population databases. Another missense variant substituting the Asp for a His at the same position was identified in a family presenting with an atypical progeroid syndrome associating generalized lipodystrophy, diabetes mellitus and dyslipidemia

After:

Line 76-79: Currently, this point substitution is located in the coil1b functional domain of the pro-tein's central rod. Bioinformatics tools produce conflicting predictions. It is not indexed in ClinVar or GnomAD population databases. It is found in all affected individuals in the family. It is absent in unaffected individuals.

Lines 82-84: According to the last update of the ACGM recommendations on the classification of genomic variants, our variant is considered as likely pathogenic.

3. Please include data concerning genetic analysis (whole exome, panel...). Which genes were analyzed?

Initially, line 81 states that the genetic test has been done using Sanger analysis. And that LMNA was the only gene screened. To fully take into account the reviewers’ questions, we decided to precise that the Sanger method was done as usual with screening of exonic and intronic regions. New statements have been included as follows:

Before:

Line 81 - All genetic analyses were performed by direct Sanger sequencing

After:

Lines 87-88: All genetic analyses were performed by direct Sanger sequencing of exonic and flanking regions

4. Any other rare variant in any gene currently associated with any of symptom identified in the family?

As said earlier, we decided from the start to screen only the LMNA gene, using Sanger analysis, because of the very highly suggestive of a laminopathy, phenotype of our patients. Therefore, no data was available for any other gene.

Reviewer 2 Report

In the manuscript 'A Family with a Single LMNA mutation illustrates diversity in cardiac phenotypes associated with Laminopathic progeroid syndromes' submittes by Giuet-Valard and coworkers to Cardiogenetics, the authors identified a novel LMNA variant in a three generation family.

In general this manuscript is interesting but needs several changes and extensions:

1.) Please write all human gene names in the complete manuscript in Italics.

2.) A material and methods section is missing. This is strange. How was genetic analysis been done? How was clinical analysis done? 

3.) Please write all numbers between 1-12 without units as words in the complete manuscript. (e.g. Line 34 abstract)

4.) Please add OMIM identifiers, when you describe an associated disease (e.g. line 59-62).

5.) What is a broad genes --> line 47? Please rephrase this sentence?

6.) Please improve Figure 2 and seperate the Figure legends from the figure. Increase the size. The writing within the figures can not be read. 

7.) Please prepare a figure with the genetic data to this family as a seperate figure. Sanger sequencing data for each patient should be shown in a relevant region of the LMNA gene.

8.) Could you explain the disease classification of this variant according the ACMG guidelines (Richards et al. 2015)?

9.) Please increase the size of Table 1. It is a little bit small.

10.) COuld you please compare your clinical phenotype with other reports describing LMNA variants in close proximity to this variant.

11.) Do you have any functional data at the molecular or cellular level about this variant?

12.) Which other variants are present in your patients? Was a NGS analysis performed? If not, please consider to do this? If it was performed, please list all variants found by this NGS analysis and explain their impact on the described phentype or why you think that they are not relevant?

In total, this manuscripts needs several changes and extension before it can be reviewed a second time. I suggest a major revision.

Author Response

1. Please write all human gene names in the complete manuscript in Italics.

This has been done.

2. A material and methods section is missing. This is strange. How was genetic analysis been done? How was clinical analysis done?

We presented our paper as a case report. This is the reason why there was no structured material and methods sections, as recommended by the journal’s author guidelines. However, to take into account the reviewer’s comment, we decided to add more information as follows:

Before:

Line 81 - All genetic analyses were per-formed by direct Sanger sequencing

After:

Lines 87-88: All genetic analyses were performed by direct Sanger sequencing of exonic and flanking regions

3. Please write all numbers between 1-12 without units as words in the complete manuscript. (e.g. Line 34 abstract)

Thank you for recalling us. According to suggestions, modifications have been done, at lines: 34, 36, 55, 111, 113, 114, 179, 187, 226 and 232.

4. Please add OMIM identifiers, when you describe an associated disease (e.g. line 59-62).

Thank you for recalling us. OMIM modifiers have been added and generate modifications highlighted from line 60 to 65.

5. What is a broad genes --> line 47? Please rephrase this sentence?

After consultation with the authors, we recognize that using “broad” could be misleading and decided to delete it.

Before:

Line 47- The LMNA is a broad gene consisting of approximately 24kb and 12 exons

After:

Line 47 - LMNA gene consists of approximately 24kb and 12 exons

6. Please improve Figure 2 and seperate the Figure legends from the figure. Increase the size. The writing within the figures can not be read.

To improve readability, all suggested modifications have been done.

7. Please prepare a figure with the genetic data to this family as a separate figure. Sanger sequencing data for each patient should be shown in a relevant region of the LMNA gene.

Thank you for this suggestion. We added Sanger sequencing data to figure1.

8. Could you explain the disease classification of this variant according the ACMG guidelines (Richards et al. 2015)?

According to the last update of the ACGM recommendations on the classification of genomic variants, our variant is considered as likely pathogenic (Masson et al. Human Genomics (2022) 16:31).

• 3 PM: absent in database (PM2), affect a functional domain (PM1), and affect the same position as another pathogenic variant already described –PM5).
• 2 PP: segregation (PP1) and bioinformatics arguments (PP3, even if discordant).

We have given precisions from lines 76 to 84. Modifications have been done as follow:

Before:

Lines 75-78 - Currently, this point substitution is not indexed in ClinVar or GnomAD population databases. Another missense variant substituting the Asp for a His at the same position was identified in a family presenting with an atypical progeroid syndrome associating generalized lipodystrophy, diabetes mellitus and dyslipidemia

After:

Line 76-79: Currently, this point substitution is located in the coil1b functional domain of the pro-tein's central rod. Bioinformatics tools produce conflicting predictions. It is not indexed in ClinVar or GnomAD population databases. It is found in all affected individuals in the family, and absent in unaffected individuals.

Lines 82-84: According to the last update of the ACGM recommendations on the classification of genomic variants, our variant is considered as likely pathogenic.

Variant class has been also changed all along the manuscript, accordingly.

9. Please increase the size of Table 1. It is a little bit small.

Change has been done.

10. Could you please compare your clinical phenotype with other reports describing LMNA variants in close proximity to this variant.

LMNA is a polymorphic gene (1795 variants in ClinVar, 07/16/2023). To our knowledge, genomic DNA variants impacted proximate AA are Variants of Unknown Significance or variants reported with conflicting interpretations. They are associated with Charcot-Marie-Tooth type2, a neurological disorder characterized by muscle weakness and atrophy starting during the second decade of life.

A previously published family with the LMNA p.Asp136His missense variant presented an atypical progeroid syndrome combining generalized lipodystrophy, diabetes mellitus and dyslipidemia. Noticeably, none of our patients had diabetes mellitus.

Take into account the reviewer’s remark, we added a new sentence as follows:

Lines 229-231: But unlike the previous family described, which has a variant at the same position, none of our patients had diabetes mellitus.

We also added this information in Table 1.

11.) Do you have any functional data at the molecular or cellular level about this variant?

Thank you again for this relevant question. So far there was no available functional analysis of our variant, which prompted us to perform in-silico analysis, and to change the manuscript in the Introduction and in the Case Presentation sections as follows:

Lines 90-91: Finally, we investigated the pathogenic effect of this variant using “Waggawagga” bioinformatics prediction software.

Lines 272-286: Functional analysis studies may be complex to perform due to pleiotropism of Lamin-A. To our knowledge, there are very few and discordant in silico and in vitro data. An experimental study aimed at systematically identifying disruptions to peptide interactions generated by deleterious lamin A variants showed that the Asp136 substitution with His amino acid would not affect any of the interactions tested [23]. On another side, using a machine learning approach for in silico prediction provided evidence of a biochemical and pathogenic effect of the p.Asp136Glu LMNA variant [24]. Asp, His and Glu are 3 polar and charged amino acids whereas Val is non-polar hydrophobic AA. It probably the reason why, using bioinformatics prediction tool, we find that changing the Asp in Val to position 136 abolishes the interaction with the Arg133 residue, whereas other interactions are intact. The absence of interaction would weaken the bond between the laminating monomers and the tetramer assembly.

12.) Which other variants are present in your patients? Was a NGS analysis performed? If not, please consider to do this? If it was performed, please list all variants found by this NGS analysis and explain their impact on the described phentype or why you think that they are not relevant?

As explained in the report, the phenotype of our index case was very suggestive of a laminopathy, so that we used a very targeted approach of the LMNA gene using the Sanger analysis. This approach was all the more legitimate since family screening revealed that non-affected relatives did not carry the variant. Moreover, according to the recent ACMG classification guidelines our variant is not a predisposing but a likely pathogenic variant of a disease causing gene segregating in an autosomal dominant pattern. Therefore, extending the screening by NGS should not be informative in terms of identification of Mendelian causing gene.

Round 2

Reviewer 1 Report

No comments

Reviewer 2 Report

The authors have addressed my concerns in a convining way.