Title: Brain Natriuretic Peptide is not predictive of Dilated Cardiomyopathy in Becker and Duchenne Muscular Dystrophy patients and carriers.

Transcription

1 Author's response to reviews Title: Brain Natriuretic Peptide is not predictive of Dilated Cardiomyopathy in Becker and Duchenne Muscular Dystrophy patients and carriers. Authors: Steven Schade van Westrum Lukas Dekker Rob de Haan Erik Endert Ieke Ginjaar Marianne de Visser Anneke van der Kooi Version: 2 Date: 26 May 2013 Author's response to reviews: see over

2 Dear Dr. Brannagan, First of all we would like to thank all the reviewers for their effort and their questions in order to strengthen our manuscript: Brain natriuretic Peptide in Becker and Duchenne muscular dystrophy patients and carriers Herewith we would like to reply to the questions raised. Reviewer 1, DM Sproule There is a spelling error of page 7, "Body Surface Area (BSA) AND" We corrected the error. 1. I am very confused, frankly, about your patient population. You describe a large cohort of boys with dystrophinopathy, as well as maternal "carriers". I found the use of steroids and, particularly, ACEI to be extraordinarily low in comparison with my own clinical practice. I would like the authors to comment on the role of steroid use and cardiomyopathy management that is followed, and whether the low rates of use reflects cessation upon loss of ambulation or other factors (or poor adherence to international standards of care). I think this has a major effect on the external validity of the work as applied to DMD/BMD populations outside of the Netherlands. The use of corticosteroids in our cohort is low because the use of corticosteroids in the management of Duchenne patients is accepted only relatively recently as a proven treatment option in the Netherlands. Since this cohort was formed between 2003 and 2005, only some patients were using it at the time. The same holds true for the use of ACE-inhibitors. The landmark article on perindopril of Duboc et al. was published in (Duboc et al. J Am Col Cardiol Mar;45(6):855 7). Although we agree with the reviewer that this population is probably not completely comparable to DMD/BMD populations outside the Netherlands, we do not think this affects our aim tot test whether NT-proBNP can distinguish patients with Duchenne or Becker muscular dystrophy and carriers of a dystrophin mutation with a dilated cardiomyopathy from those without. 2. As noted in the discussion, symptoms of heart failure are limited in non-ambulatory patients, who often are incapable of sufficient exertion to generate dyspnea. I would appreciate a more extensive discussion and defense of "symptoms of heart failure" being incorporated as a criteria for heart failure - this may lead to tremendous under-recognition of incidence - This is a major, stressed difference of the paper compared to related works. I am less convinced than the authors that this is, in fact, a strength of the work. A major concern that I have is that the strict definition of cardiomyopathy creates an unbelievably low incidence rate in the study population - is this what drives the poor correlation of BNP and DCM? - My gut feelings on the numbers are that the BNP number is more accurate; this is the critical issue in the literature, and a more extensive discussion of the rationale for the chosen study criteria is warranted. A thorough and convincing argument as such will immensely strengthen the work. - You mention that the ROC curve demonstrates that there is no superior "cut

3 point" to improve the AUC - it is clear from the image. But what range was examined? Is there an argument that could be made to extend the range? We investigated chronic heart failure and cardiomyopathy separately. We see the reviewer s point. However, we adhered to the standard for dilated cardiomyopathy, viz. the criteria of the World Health Organisation/International Society and Federation of Cardiology Task Force, which does not include symptoms of heart failure as a criterion. In addition we used the criteria for chronic heart failure of the European Society of Cardiology, to evaluate whether patients had symptoms related to heart failure. We found CHF in 9% of the total cohort and DCM in 17%. Not only did we look at strictly defined DCM, but we additionally analysed the association between an elevated NT-proBNP and the separate parameters of DCM, viz. an enlarged LVEDD, a decreased FSI or global left ventricle hypokinesia at 2D echocardiography to avoid underrecognition. No correlation between BNP and these parameters was found either. In the ROC-curve we tested all possible cut-off values which might occur in this group. In this full range the ROC-analysis did not show a discriminatory threshold. 3. Here is my question regarding the external validity of the work: What I care about in clinical practice is the early recognition of DCM and cardiomyopathy in my dystrophinopathy patients who do not have well established cardiac disease. Is this test actually useless in such a population? Any discussion making an argument for or against the utility of this test should reflect back to this question. I am not convinced that the discussion, or methodology, properly does so. We agree with the reviewer that early recognition of DCM and cardiomyopathy in dystrophinopathy patients is the ultimate goal of any clinician. In this study we examined whether NT-proBNP distinguishes patients with dilated cardiomyopathy from those without, and demonstrated that it does not. Given the relatively high sensitivity of NT-proBNP for DCM it might be considered as a screening tool but compared to the non-invasive and relatively cheap echocardiogram, NTproBNP does not outperform the echocardiogram as a screening tool. Therefore we think this test is not useful in this population to screen for DCM. We added this to the discussion accordingly (page 16, line 8 to 16) - Discretionary Revisions 1. Obesity - the use of BMI is invalid for the assessment of obesity in a disease marked by muscle atrophy. It has been well demonstrated that such measures markedly understate obesity rates in DMD, SMA and other such populations. I would suggest either clarifying the limitations of the approach or qualifying your conclusions with relation to this (secondary) argument. On the point of the BMI we agree with the reviewer and changed the text (page 14, line 10). Reviewer 2, Mazen Dimachkie The authors do not comment on the specificity of BNP in the Duchenne subgroup. The authors found that the sensitivity and specificity of NT-proBNP for the presence of DCM in the whole cohort were 85% and 23%, respectively. Others have found comparable results in that the sensitivity and specificity of NT-proBNP in primary care adult patients with suspected heart failure, for the

4 detection of LVSD were 0.97 and 0.46 respectively (J Card Fail Jun;11(5 Suppl):S15-20). The authors state in the discussion the overall diagnostic accuracy of the NTproBNP test to screen for dilated cardiomyopathy was poor. The authors should be more cautious and make the distinction between a screening test and a diagnostic test. While it is true that NTproBNP accuracy was low as a diagnostic test, the value of a screening test is in its high sensitivity at the cost of losing specificity as a trade-off for its ease of use and therefore a decrease in accuracy in comparison to the standard criterion. The authors do not comment on whether or not NTproBNP qualifies as a screening test for DCM. We thank the reviewer for this comment. We agree that a diagnostic test, which fails to perform as a diagnostic tool, might be useful as a screening tool when the sensitivity is high. In our opinion, NT-proBNP measurement does not outperform the echocardiogram as a screening tool in comparison to the non-invasive and relatively cheap echocardiogram. We added this to the discussion (page 16, line 8 to 16). Another issue is that while BNP is a good marker for acute decompensation of congestive heart failure, its level significantly decreases after treatment. Although as the authors state The level of NT-proBNP was significantly higher in those receiving medication compared to those without (800 vs 590 pmol/l, p < 0.001), treatment would have blunted any increase in BNP. In the discussion, the authors indicate it is unlikely the treatment would have interfered with correlating BNP with DCM since BNP levels were higher in the treated group. Could it be that the lack of correlation is due to this blunting effect of treatment on BNP? Would the results have been any different if treated cases were excluded from analysis? We agree that treatment might have effect on the level of BNP and therefore affects the power to diagnose DCM. Therefore we added treatment in the logistic regression analysis for the whole group as well in the separate sub-groups. In all these models (dichotomous NTproBNP, continuous NT-proBNP, in specific subgroups or in the group as a whole) the odds for NT-proBNP were not significant with wide 95% confidence intervals. We conclude that treatment did not make a difference. Moreover NT-proBNP levels were significantly higher in treated versus untreated; exclusion of treated cases will therefore not have effect. In 16% the diagnosis was established based on pedigree or linkage analysis in carriers. Was this only for mothers of affected DMD / BMD boys? Only for a proportion (16%) of the DMD and BMD carriers pedigree or linkage analysis was used. We changed the text to clarify this (page 6, line 11-13). There is no discussion of study limitations: 1. In the results it is odd that CHF was less common in Duchenne (6%) than in BMD (14%) or carriers (8%) while these numbers for DCM were 16%, 32% and 11%, respectively. How do the authors explain these findings? We investigated chronic heart failure and cardiomyopathy separately. We adhered to the standard for dilated cardiomyopathy, viz. the criteria of the World Health Organisation/International Society and Federation of Cardiology Task Force, which does not include symptoms of heart failure as a criterion. In addition we used the criteria for chronic

5 heart failure of the European Society of Cardiology, to evaluate whether patients had symptoms related to heart failure. Some patients did not have symptoms of chronic heart failure (probably due to their inactivity due to muscle weakness), but nevertheless had dilated cardiomyopathy, based on the abovementioned criteria of the World Health Organisation/International Society and Federation of Cardiology Task Force, which does not include symptoms of heart failure as a criterion. 2. Why did the authors use a stricter and older definition of DCM (ref 14) rather than that used in more recent publications (ref 2 and 7)? If the authors had used another definition of DCM (echocardiographic evidence of a dilated left ventricle and ejection fraction < 45%), how different would the results have been? The definition of Nigro et al. was not based on internationally accepted criteria (ref 2). We used the criteria for dilated cardiomyopathy of the World Health Organisation/ International Society as has also been used by Grain et al. (ref 7). We apologize for the confusion. 3. As in Table 1, there was more hypertension in the BMD than in the DMD boys. Did these differences contribute to the higher proportion of DCM in BMD? It might well be true that hypertension contributes to some extent to the presence of DCM. In this study however we focussed on the question whether NT-proBNP distinguishes patient with dilated cardiomyopathy from those without. 4. There also was a higher proportion of elevated blood pressure in functionally symptomatic DMD carriers (70%) than in the asymptomatic carriers (42%), and this could have impacted the frequency of DCM. See previous question. 5. In Table 2, the authors present LVF dysfunction as less than good based on Global Left Ventricular Function (LVF) was judged as good, fair, or poor by an experienced cardiologist. This introduces significant subjectivity. Was intra-observer reliability testing conducted? One experienced cardiologist evaluated all echocardiograms, no formal intra-observer testing was conducted for this study. It has been proven that qualitative judgement of left ventricular function can reliably be used in clinical decision taking. (Heart 1997, 78 (3): Silcocks; Thorac Cardiovasc Surg 1993, 41(1):54-8). Though the Brooke scale may be good to describe the state of arm function in Duchenne, it has low discriminant powers, large floor effects and lacks responsiveness (Arch Phys Med Rehabil Dec;93(12): e1.). Perhaps the Motor Functional Scale would have been a more robust measure. We agree that the MFM could have been used with more discriminate power, less floor effects, and more responsiveness in studies with a different study goal. We used the Brooke scale in our cross sectional study not only to describe the state of arm, but also of leg function. It was only used to discriminate between patients/carriers with and without functional impairment.

6 It is unclear what the paragraph on diastolic dysfunction under results adds to the findings. We agree with the reviewer and omitted the data on the diastolic function. In the introduction the DCM fractional shortening is listed < 25%, and the authors used the fractional shortening < 28%. Why is this difference? This is indeed confusing and therefore we omitted this from the Introduction section. In the methods, the authors do not define the abbreviations FSI and E/A. We corrected this. In results, the authors need to clarify Of the carrier group 93% DMD and 95% BMD carriers were functionally asymptomatic due to skeletal muscle weakness. Do they mean due to lack of skeletal muscle weakness? The reviewer is correct. Reviewer 3, Elena Pegoraro Major Compulsory Revisions (.) The main weakness is to have clustered together DMD, BMD and DMD/BMD carriers in a single dystrophinopathy group and to have conducted some, even if not all, analysis in the entire group instead of in the single subset of patients. This approach, even if justifiable by the low number of DCM patient in each subset to allow meaningful conclusions, may be misleading. Even if the entire cohort carries a DMD gene mutation, the consequences of the mutation at the biochemical level are dramatically different in the three subsets of patients: complete lack of dystrophin in DMD, abnormal but partially functional dystrophin in BMD, and variable amount of normal/partially functional dystrophin in carriers. As a consequence the impact of the DMD gene mutation on cardiac myocytes susceptibility to produce NTproBNP is a priori very different and conclusions may likely be different in the diverse subsets of patients. It is true that the different subpopulations have different risk of developing DCM and therefore the incidence of DCM varies. That is why we did analyse the subgroups separately (page 11, line 6 and 15). Only in DMD patients with DCM the median NT-proBNP level was higher than in DMD patients without DCM. It was impossible to correct for disease duration, any weakness or complaints of heart failure with a logistic regression analysis since the model did not fit. Moreover, the carriers subset of patients was, on average, older than DMD and BMD. In older patients many potentially confounding variables for the interpretation of plasma N- terminal probnp should be considered (i.e. haematocrit, CRP; FT3, renal failure, etc) leading to increased levels of NT-proBNP independent of overt cardiovascular disease.

7 All the carriers examined were in good health. It may be true that there are potentially confounding variables for the interpretation of NT-pro BNP levels, but we feel it is unlikely that this has influenced the results of our study. The analysis is very thoroughly and accurate but only separate subsets of patients need to be analysed. We followed the suggestion of the reviewer. Minor Essential Revisions Given this, there are also additional minor points to be addressed: - In the Methods section, patients inclusion criteria, the authors stated that to achieve a definite diagnosis would be necessary a DMD gene mutation or, only for DMD, an absence of dystrophin on immunohistochemical studies. In the Results section 13% of DMD and BMD diagnosis was established by immunohistochemical or biochemical investigations. How many BMD patients without DMD gene mutations were included? We agree that the information could have been more specific. We changed the text accordingly (page 10, line 5). In 72% of BMD patients a mutation was found with DNAanalysis. - Results section:...bmd carriers were functionally asymptomatic due to skeletal muscle weakness omit due to skeletal muscle weakness ; We agree and omit that part of the sentence (page 10, line 13) - Please omit p value when not significant. We changed the text accordingly. - Reference 1: Kichmann et al, 2005 and not 2004; Our apologies for this mistake, we corrected it. - Quality of figures may be improved. We are sorry but did not manage to improve the quality of the figures before the deadline but will do so when the manuscript is accepted for publication. - Few typos need to be corrected. We corrected the typo.

8 Reviewer 4: Jacinda Sampson Reviewer's report: 1. Discretionary Revisions : suggest inclusion of CHF into title of article, or more concrete regarding conclusions, such as " BNP is not predictive of CHF or cardiomyopathy in DMD, BMD or DMD/or BMD carriers. We thank the reviewer for the suggestion and changed the title accordingly 2. Discretionary Revisions : minor spelling punctuation errors. We corrected the errors.