Mitochondrial DNA Haplogroups and Variants Predispose to Chagas Disease Cardiomyopathy
, David Goudenège 2, João Paulo Silva Nunes 1,3,4,5, Pauline Andrieux 1, Barbara Maria Ianni 3, Amanda Farage Frade 3,4,5, Charles Mady 3, Ronaldo Honorato Barros Santos 6, Andreia Kuramoto 3, Samuel Steffen 6,7, Antonio Noedir Stolf 7, Pablo Pomerantzeff 8, Alfredo Inacio Fiorelli 7, Edimar Alcides Bocchi 7,9, Cristina Wide Pissetti 10, Bruno Saba 11, FabrÃcio C. Dias 12, Marcelo Ferraz Sampaio 11, Fabio Antônio Gaiotto 6,7, Ricardo Costa Fernandes Zaniratto 3, Sergio Siqueira 13, Fernando Bacal 6, André Schmidt 12, Mario Hiroyuki Hirata 14, Jorge Kalil 3,4,5, Edecio Cunha-Neto 3,4,5,17,*,‡ and Christophe Chevillard 1,*,‡add
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Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
This study by Gallardo et al examines whether the mitochondrial genome contributes to the progression/severity of Chagas disease-associated chronic cardiomyopathy via mtDNA copy number assessment and haplotyping. The authors found that mtDNA copy number was lower in heart samples from chronic cardiomyopathy patients and identified 84 mtDNA-encoded protein variants associated with chronic cardiomyopathy. Overall this is a well-designed and internally consistent study.
Additional Comments
1. Line 521 has a misspelling: perhaps change “effet” to “effect”
Author Response
Thanks to the reviewer for the comment:
This study by Gallardo et al examines whether the mitochondrial genome contributes to the progression/severity of Chagas disease-associated chronic cardiomyopathy via mtDNA copy number assessment and haplotyping. The authors found that mtDNA copy number was lower in heart samples from chronic cardiomyopathy patients and identified 84 mtDNA-encoded protein variants associated with chronic cardiomyopathy. Overall this is a well-designed and internally consistent study.
Additional Comments
Query 1: Line 521 has a misspelling: perhaps change “effet” to “effect”
Answer 1: Sentence has been corrected.
“Moreover, as no clear variant is associated to disease here, may suggested an additive effect of the mt DNA variants.”
Reviewer 2 Report
Comments and Suggestions for Authors
In this comprehensive study a correlation was found between mitochondrial DNA haplotypes and variants, and different forms of Chagas disease. The study is scientifically highly relevant, innovative, and generally well described. Some corrections and clarifications are needed.
Specific comments:
---line 60. CCC is abreviation of Chronic Chagas Cardiomyopathy.
---line 200. Many patients with moderate CCC have a normal LV ejection fraction. Which clinical criteria were used to identify the moderate CCC group?
---lines 303-304. In Figure 1A the percentage of copy number reduction seems above 50%. How to explain this discrepancy with the text?
---lines 393-395. 4 patients missing since in total 74 CCC patients.
---line 396. In table S5 106 patients. Why this discrepancy? "on"should be deleted.
Author Response
Thanks to the reviewer for the comments:
In this comprehensive study a correlation was found between mitochondrial DNA haplotypes and variants, and different forms of Chagas disease. The study is scientifically highly relevant, innovative, and generally well described. Some corrections and clarifications are needed.
Specific comments:
Query 1: line 60. CCC is abbreviation of Chronic Chagas Cardiomyopathy.
Answer 1: Sentence has been corrected.
“Cardiomyopathies are major causes of heart failure. Chagas disease (CD) is caused by the parasite Trypanosoma cruzi, and it is endemic in Central, South America. Thirty percent of the cases evolve into Chronic Chagas Cardiomyopathy (CCC) with worse prognosis as compared with other cardiomyopathies.”
Query 2: line 200. Many patients with moderate CCC have a normal LV ejection fraction. Which clinical criteria were used to identify the moderate CCC group?
Answer 2: Thanks for this query. The clinical criteria were added to the materials and methods section. Sentences had been corrected.
“Human left ventricular free wall heart tissue samples were obtained from patients with end-stage heart failure CCC at the time of heart transplantation (n = 34). These CCC patients underwent serological diagnosis of T. cruzi infection and standard electrocardiography and echocardiography, and tissues were subject to histopathological assessment (43). Moderate CCC patients are characterized by an ejection fraction ≥0.4 whereas severe CCC patients are characterized by an ejection fraction <0.4. Biopsies from controls (n = 6) were obtained from healthy hearts of organ donors having no suitable recipient. The protocol was approved by the institutional review boards of the University of São Paulo School of Medicine (n°009/2011, January 21th 2011) and INSERM (French National Institute of Health and Medical Research) (FWA00005831, March 16th 2011). Written informed consent was obtained from all patients. All experimental methods comply with the Helsinki Declaration.”
Query 3: lines 303-304. In Figure 1A the percentage of copy number reduction seems above 50%. How to explain this discrepancy with the text?
Answer 3: This discrepancy was due to typing error. Sentence has been corrected.
“The quantification of mtDNA copy number was evaluated on heart tissue samples from end stage patient heart tissues (n=33) and on healthy hearts of organ donors having no suitable recipient (n=6) (Supplementary Table S1). MtDNA copy number using (MT-ND1) was significantly lower in CCC ((MT-ND1 69%; p= 0.0034) than control samples (Figure 1A) indicating a reduction of mtDNA copy number. Mitochondrial DNA (MT-COX1) gave a similar result (37%; p= 0.0019) (Figure 1B).”
Query 4: lines 393-395. 4 patients missing since in total 74 CCC patients.
Answer 4: The whole mtDNA sequencing was performed on 74 CCC patients. Among the CCC patients, 34 patients suffered from moderate cardiomyopathy (EF≥0.4) whereas 36 patients had a severe cardiomyopathy (EF<0.4). For 4 CCC patients, the ejection fraction value is not available. Sentence has been corrected.
“Whole mtDNA sequencing was carried out on 112 samples. (Supplementary Table S5). It included 38 asymptomatic subjects and 74 CCC patients. Among the CCC patients, 34 patients suffered from moderate cardiomyopathy whereas 36 patients had a severe cardiomyopathy. For 4 CCC patients, the ejection fraction value is not available.”
Query 5: line 396. In table S5 106 patients. Why this discrepancy? "on"should be deleted.
Answer 5: Sentence has been corrected.
“The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Table S1: Heart tissue samples used for mitochondria quantification. Table S2: Blood DNAs samples used for mitochondria quantification. Table S3: Blood samples used for haplogroup analysis. Table S4: Statistics on African sub-haplogroup distribution. Table S5: Clinical information for patients whom the whole mitochondria genome has been sequenced (n=112). Table S6: Carrier analysis parformed on 12429 Variants. Table S7: Heteroplasmy level test between the three groups (ASY, moderate CCC and severe CCC) and heteroplasmy level analyses between the groups (two by two: ASY vs CCC; ASY vs Moderate CCC; ASY vs Severe CCC and Moderate CCC vs Severe CCC). Table S8: Variant annotation. Table S9: List of the variants associated to diseases.”
Supplementary table S5 was updated.
Round 2
Reviewer 2 Report
Comments and Suggestions for Authors
Thanks for appropriate response. No further comments.
