A Novel Nonsense Pathogenic TTN Variant Identified in a Patient with Severe Dilated Cardiomyopathy

Round 1

Reviewer 1 Report

In the manuscript 'A novel nonsense pathogenic TTN variant identified in a patient with severe hypokinetic dilated cardiomyopathy, submitted by C. Micolonghi et al. to Current Issues in Molecular Biology, the authors identied a novel truncating TTN variant by next generation sequencing. 

The information that TTN truncatin mutations can cause DCM is not really novel. However, small papers like this one might be suitable to improve the genetic counseling of families with similar mutations. Therefore, this manuscript can be published but needs several changes. Especially relevant citations are missing for some parts:

1.) Please indicate the specific TTN mutation also in the abstract with cDNA and protein nomenclature.

2.) Abstract: Sudden cardiac death and not sudden death.

3.) Line 24: are localized instead of "fall"

4.) Plage 2, Line 55-59: Please include relevant citations for each gene:

TTN: Gerull, Brenda, et al. "Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cardiomyopathy." Nature genetics 30.2 (2002): 201-204.

LMNA: Keller, Hans, et al. "Novel c. 367_369del LMNA mutation manifesting as severe arrhythmias, dilated cardiomyopathy, and myopathy." heart & lung 41.4 (2012): 382-386.

MYH7:De Frutos, Fernando, et al. "Natural history of MYH7-related dilated cardiomyopathy." Journal of the American College of Cardiology 80.15 (2022): 1447-1461.

FLNC: Begay, Rene L., et al. "FLNC gene splice mutations cause dilated cardiomyopathy." JACC: Basic to Translational Science 1.5 (2016): 344-359.

BAG3: Arimura, Takuro, et al. "Dilated cardiomyopathy‐associated BAG3 mutations impair Z‐disc assembly and enhance sensitivity to apoptosis in cardiomyocytes." Human mutation 32.12 (2011): 1481-1491.

TNNT2: Hershberger, Ray E., et al. "Clinical and functional characterization of TNNT2 mutations identified in patients with dilated cardiomyopathy." Circulation: Cardiovascular Genetics 2.4 (2009): 306-313.

RBM20: Gaertner, Anna, et al. "Cardiomyopathy‐associated mutations in the RS domain affect nuclear localization of RBM20." Human Mutation 41.11 (2020): 1931-1943.

SCN5A: McNair, William P., et al. "SCN5A mutation associated with dilated cardiomyopathy, conduction disorder, and arrhythmia." Circulation 110.15 (2004): 2163-2167.

DES: Brodehl, Andreas, et al. "Functional characterization of the novel DES mutation p. L136P associated with dilated cardiomyopathy reveals a dominant filament assembly defect." Journal of molecular and cellular cardiology 91 (2016): 207-214.

PLN: Schmitt, Joachim P., et al. "Dilated cardiomyopathy and heart failure caused by a mutation in phospholamban." Science 299.5611 (2003): 1410-1413.

TNNC1: Pinto, Jose Renato, et al. "Functional characterization of TNNC1 rare variants identified in dilated cardiomyopathy." Journal of Biological Chemistry 286.39 (2011): 34404-34412.

5.) The authors should also indicate in the introduction, that some rare cases with digenetic mutations (in two different cardiomyopathy genes) are known. See for example:

Gaertner, Anna, et al. "The combined human genotype of truncating TTN and RBM20 mutations is associated with severe and early onset of dilated cardiomyopathy." Genes 12.6 (2021): 883.

6.) Please indicate in Figure 1B also the codons and the encoded amino acids.

Good luck with the revision!

Author Response

Reviewer 1

In the manuscript 'A novel nonsense pathogenic TTN variant identified in a patient with severe hypokinetic dilated cardiomyopathy, submitted by C. Micolonghi et al. to Current Issues in Molecular Biology, the authors identified a novel truncating TTN variant by next generation sequencing. 

The information that TTN truncatin mutations can cause DCM is not really novel. However, small papers like this one might be suitable to improve the genetic counseling of families with similar mutations. Therefore, this manuscript can be published but needs several changes. Especially relevant citations are missing for some parts:

We thank the Reviewer for her/his general comments on our work. The replies to the specific comments follow.

1.) Please indicate the specific TTN mutation also in the abstract with cDNA and protein nomenclature.

We added the TTN variant with cDNA and protein nomenclature in the abstract.

2.) Abstract: Sudden cardiac death and not sudden death.

As suggested, we replaced it.

3.) Line 24: are localized instead of "fall"

Done

4.) Page 2, Line 55-59: Please include relevant citations for each gene:

TTN: Gerull, Brenda, et al. "Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cardiomyopathy." Nature genetics 30.2 (2002): 201-204.

LMNA: Keller, Hans, et al. "Novel c. 367_369del LMNA mutation manifesting as severe arrhythmias, dilated cardiomyopathy, and myopathy." heart & lung 41.4 (2012): 382-386.

MYH7:De Frutos, Fernando, et al. "Natural history of MYH7-related dilated cardiomyopathy." Journal of the American College of Cardiology 80.15 (2022): 1447-1461.

FLNC: Begay, Rene L., et al. "FLNC gene splice mutations cause dilated cardiomyopathy." JACC: Basic to Translational Science 1.5 (2016): 344-359.

BAG3: Arimura, Takuro, et al. "Dilated cardiomyopathy‐associated BAG3 mutations impair Z‐disc assembly and enhance sensitivity to apoptosis in cardiomyocytes." Human mutation 32.12 (2011): 1481-1491.

TNNT2: Hershberger, Ray E., et al. "Clinical and functional characterization of TNNT2 mutations identified in patients with dilated cardiomyopathy." Circulation: Cardiovascular Genetics 2.4 (2009): 306-313.

RBM20: Gaertner, Anna, et al. "Cardiomyopathy‐associated mutations in the RS domain affect nuclear localization of RBM20." Human Mutation 41.11 (2020): 1931-1943.

SCN5A: McNair, William P., et al. "SCN5A mutation associated with dilated cardiomyopathy, conduction disorder, and arrhythmia." Circulation 110.15 (2004): 2163-2167.

DES: Brodehl, Andreas, et al. "Functional characterization of the novel DES mutation p. L136P associated with dilated cardiomyopathy reveals a dominant filament assembly defect." Journal of molecular and cellular cardiology 91 (2016): 207-214.

PLN: Schmitt, Joachim P., et al. "Dilated cardiomyopathy and heart failure caused by a mutation in phospholamban." Science 299.5611 (2003): 1410-1413.

TNNC1: Pinto, Jose Renato, et al. "Functional characterization of TNNC1 rare variants identified in dilated cardiomyopathy." Journal of Biological Chemistry 286.39 (2011): 34404-34412.

5.) The authors should also indicate in the introduction, that some rare cases with digenetic mutations (in two different cardiomyopathy genes) are known. See for example:

Gaertner, Anna, et al. "The combined human genotype of truncating TTN and RBM20 mutations is associated with severe and early onset of dilated cardiomyopathy." Genes 12.6 (2021): 883.

We included the suggested references in the revised version of the manuscript (see new refs: 4-16).

6.) Please indicate in Figure 1B also the codons and the encoded amino acids.

We modified the figure 1B by following the Reviewer suggestion.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Micolonghi et al. have written a nice case report which highlights the interplay between genetic- and environmental factors on disease expression and progression and the utility of performing genetic screening in patients with DCM/HF even when other potentially causative insults are identified. The authors also highlight that TTNtv seems to be especially susceptiple to these "second hits" as previously reported by in manuscripts in NEJM, Circulation and JACC (PMID:36154167, PMID: 29773157, PMID: 30987448 and PMID: 27355546). The manuscript is well-written and while not novel is a fine addition to the litterature as a case report.

I don't have any major concerns with the manuscript, but some general suggestions include:

1. The use of the term hypokinetic when describing the phenotype might cause some confusion,. This is due to the milder phenotype of DCM being called hypokinetic non-dilated cardiomyopathy. I believe just referring to the phenotype as "severe dilated cardiomyopathy" conveys the same message.

2. Please provide information on genetic testing in family members. Even if none was performed please state this and give reasons for not performing this analyses (e.g., genetic testing was not performed in the affected brother or father since...)

3. I propose focusing the discussion on the observed clinical phenotype which mimics acquired DCM. In particular the sections on the molecular effects of variants in the M-band and the impact of the location of TTNtv on the observed phenotype (while nice reviews of the litterature) are not really relevant to the study and are not supported by data from this specific study. In fact, most previous research suggests that age at diagnosis and the observed clinical phenotype are not influenced by the specific location of the TTNtv as long as it is in a high PSI exon (PMID: 33106378  & PMID: 32964742). 

4. In relation to this I suggest including other papers which describe the clinical phenotype of this genetic DCM subtype and in particular the seminal papers by Ware which describe the impact of second hits (PubMed ID's given in the first section).

5. Consider if you want to refer to the patient as a patient or proband and stick with it.

 

Specific minor comments:

1. Line 60: Change gene to genes.

2. Line 62: delete protein or write The titin protein.

3. Line 73-74: please clarify what you mean here. I think you refer to the difference between the two transcripts being the exons spliced in in the I-band, but not entirely clear as stated now.

4. Line 136: Can you please also provide the BSA-indexed LVEDD.

5. Line 158: You say that the LA was normal... Judging from the figure 2 panel B it does seem quite large (by my unscientific and perhaps inaccurate eye-balling). This would also fit with most prior reports on the atrial cardiomyopathy observed in TTNtv. Can you provide left atrial volumes?.

6. Line 209-211: In my mind TTNtv is not associated with a particularily severe DCM phenotype. Rather, while patients often present with severe systolic dysfunction it is associated with good reversibility and equivalent or favourable outcomes (PMID: 33106378,  PMID: 32964742, PMID: 29073955, PMID: 29377983 & PMID: 27813223)

7. Line 260: I'm not sure I would say that this case report provides new insight into the proteomic basis of DCM.

 

Author Response

Reviewer 2

Micolonghi et al. have written a nice case report which highlights the interplay between genetic- and environmental factors on disease expression and progression and the utility of performing genetic screening in patients with DCM/HF even when other potentially causative insults are identified. The authors also highlight that TTNtv seems to be especially susceptiple to these "second hits" as previously reported by in manuscripts in NEJM, Circulation and JACC (PMID:36154167, PMID: 29773157, PMID: 30987448 and PMID: 27355546). The manuscript is well-written and while not novel is a fine addition to the litterature as a case report.

We wish to thank the Reviewer for her/his general positive comments on our work.

I don't have any major concerns with the manuscript, but some general suggestions include:

1. The use of the term hypokinetic when describing the phenotype might cause some confusion,. This is due to the milder phenotype of DCM being called hypokinetic non-dilated cardiomyopathy. I believe just referring to the phenotype as "severe dilated cardiomyopathy" conveys the same message.

We deleted the word "hypokinetic" as suggested by the Reviewer.

2. Please provide information on genetic testing in family members. Even if none was performed please state this and give reasons for not performing this analyses (e.g., genetic testing was not performed in the affected brother or father since...)

The Fig 2 of the original manuscript contained the information on the genetic testing in the family members. To better clarify this issue, we specified in the revised text the segregation analysis performed in the family (see page 4, lines 26-29).

3. I propose focusing the discussion on the observed clinical phenotype which mimics acquired DCM. In particular the sections on the molecular effects of variants in the M-band and the impact of the location of TTNtv on the observed phenotype (while nice reviews of the literature) are not really relevant to the study and are not supported by data from this specific study. In fact, most previous research suggests that age at diagnosis and the observed clinical phenotype are not influenced by the specific location of the TTNtv as long as it is in a high PSI exon (PMID: 33106378  & PMID: 32964742). 

We agree that the originality of the current case report mainly relies on the clinical phenotype which mimics acquired DCM but has also a genetic cause. To follow your suggestion, we modified the discussion in the revised manuscript and quoted the references you indicated (see page 5, third paragraph of the revised discussion, new ref: 31-35).

4. In relation to this I suggest including other papers which describe the clinical phenotype of this genetic DCM subtype and in particular the seminal papers by Ware which describe the impact of second hits (PubMed ID's given in the first section).

We agree with your suggestion. In fact, we focused the discussion on the importance of the genetic predisposition to hereditary cardiomyopathy in patients with acquired risk factors, highlighting the relevance of the interaction between genetic and environmental factors.

5. Consider if you want to refer to the patient as a patient or proband and stick with it.

 We have corrected it.

Specific minor comments:

1. Line 60: Change gene to genes. Done
2. Line 62: delete protein or write The titin protein. Done
3. Line 73-74: please clarify what you mean here. I think you refer to the difference between the two transcripts being the exons spliced in in the I-band, but not entirely clear as stated now.

We clarified it. (see page 2, line 29-32)

4. Line 136: Can you please also provide the BSA-indexed LVEDD.

We added the requested information in the revised manuscript (page 3, case presentation paragraph, line 14)

5. Line 158: You say that the LA was normal... Judging from the figure 2 panel B it does seem quite large (by my unscientific and perhaps inaccurate eye-balling). This would also fit with most prior reports on the atrial cardiomyopathy observed in TTNtv. Can you provide left atrial volumes?.

We added the requested information in the revised manuscript. (page 3, case presentation paragraph, line 31)

6. Line 209-211: In my mind TTNtv is not associated with a particularily severe DCM phenotype. Rather, while patients often present with severe systolic dysfunction it is associated with good reversibility and equivalent or favourable outcomes (PMID: 33106378,  PMID: 32964742, PMID: 29073955, PMID: 29377983 & PMID: 27813223)

We agree with your point and modify the discussion accordingly. (see third paragraph of the discussion, page 6)

7. Line 260: I'm not sure I would say that this case report provides new insight into the proteomic basis of DCM.

We agree with your comment and we do not mention the “proteomic basis” in the revised manuscript.

Reviewer 3 Report

cimb-2274131, A novel nonsense pathogenic TTN variant identified in a patient with severe hypokinetic dilated cardiomyopathy by Caterina Micolonghi et al. The authors aimed in their report to describe the role of the TTN gene in thedevelopment of dilated cardiomyopathy, which may have important implications for the diagnosis, management, and treatment of dilated cardiomyopathy in patients with TTN truncations. Their case report supports the pathogenicity of a novel TTN variant that can contribute to the dilated cardiomyopathy phenotype in a variable manner, along with other genetic and environmental factors. They concluded that geneticcounseling and comprehensive genetic testing can help determine the specific implications of this mutation in each affected individual. 

 

Comments 

Abstract 

- The authors should aim to provide a clear and concise statement of their research objective in the abstract.

- Page 1, line 26: the reviewer suggests rephrasing “She was affected by hypertension and ….” With “She had a history of hypertension…”

- Page 1, line 23: “ACMG”. Can the authors spell out the abbreviation.

 

Case presentation 

- Page 3, line 131: “BNP”. Can the authors spell out the abbreviation.

- Page 4, line 154: “NYHA class I-II”. Can the authors spell out the abbreviation.

Author Response

Reviewer 3

cimb-2274131, A novel nonsense pathogenic TTN variant identified in a patient with severe hypokinetic dilated cardiomyopathy by Caterina Micolonghi et al. The authors aimed in their report to describe the role of the TTN gene in thedevelopment of dilated cardiomyopathy, which may have important implications for the diagnosis, management, and treatment of dilated cardiomyopathy in patients with TTN truncations. Their case report supports the pathogenicity of a novel TTN variant that can contribute to the dilated cardiomyopathy phenotype in a variable manner, along with other genetic and environmental factors. They concluded that geneticcounseling and comprehensive genetic testing can help determine the specific implications of this mutation in each affected individual. 

 

Comments 

Abstract 

- The authors should aim to provide a clear and concise statement of their research objective in the abstract

We thank the Reviewer for her/his comments. We modified the abstract focusing on the aim of the study.

- Page 1, line 26: the reviewer suggests rephrasing “She was affected by hypertension and ….” With “She had a history of hypertension…”

- Page 1, line 23: “ACMG”. Can the authors spell out the abbreviation.

We corrected it.

 

Case presentation 

- Page 3, line 131: “BNP”. Can the authors spell out the abbreviation.

- Page 4, line 154: “NYHA class I-II”. Can the authors spell out the abbreviation.

 Done