Journal of Cardiac Failure Vol. 18 No. 7 2012
Plasma Pro-B-Type Natriuretic Peptide Testing as a Screening Method for Hypertrophic Cardiomyopathy FABIO FERNANDES, MD, PhD, EDMUNDO ARTEAGA-FERNANDEZ, MD, PhD, MURILLO DE OLIVEIRA ANTUNES, MD, PAULA BUCK, PhD, JULIA DAHER CARNEIRO MARSIGLIA, MSc, AFONSO MATSUMOTO, MD, PhD, LUCIANO NASTARI, MD, PhD, JOSE EDUARDO KRIEGER, MD, PhD, ALEXANDRE COSTA PEREIRA, MD, PhD, AND CHARLES MADY, MD, PhD S~ao Paulo, Brazil
ABSTRACT Background: Clinical multistage risk assessment associated with electrocardiogram (ECG) and NTproBNP may be a feasible strategy to screen hypertrophic cardiomyopathy (HCM). We investigated the effectiveness of a screening based on ECG and NT-proBNP in first-degree relatives of patients with HCM. Methods and Results: A total of 106 first-degree relatives were included. All individuals were evaluated by echocardiography, ECG, NT-proBNP, and molecular screening (available for 65 individuals). From the 106 individuals, 36 (34%) had diagnosis confirmed by echocardiography. Using echocardiography as the gold standard, ECG criteria had a sensitivity of 0.71, 0.42, and 0.52 for the Romhilt-Estes, Sokolow-Lyon, and Cornell criteria, respectively. Mean values of NT-ProBNP were higher in affected as compared with nonaffected relatives (26.1 vs. 1290.5, P ! .001). The AUC of NT-proBNP was 0.98. Using a cutoff value of 70 pg/mL, we observed a sensitivity of 0.92 and specificity of 0.96. Using molecular genetics as the gold standard, ECG criteria had a sensitivity of 0.67, 0.37, and 0.42 for the Romhilt-Estes, SokolowLyon, and Cornell criteria, respectively. Using a cutoff value of 70 pg/mL, we observed a sensitivity of 0.83 and specificity of 0.98. Conclusion: Values of NT-proBNP above 70 pg/mL can be used to effectively select high-risk firstdegree relatives for HCM screening. (J Cardiac Fail 2012;18:564e568) Key Words: Hypertrophic cardiomyopathy, NT-proBNP, familial screening.
Undetected heart disease is the leading cause of sudden death in young individuals. In this scenario, hypertrophic cardiomyopathy is the leading diagnosis responsible for sudden death. The disease has been recognized as caused by more than 900 mutations in more than 19 genes encoding for sarcomeric, calcium handling and z-disk proteins and is inherited as an autosomal dominant trait with variable penetrance.1 Signs and symptoms of hypertrophic cardiomyopathy are not specific. The 12-lead electrocardiogram (ECG) has been classically the initial diagnostic test for the early
evaluation of a patient suspected of having hypertrophic cardiomyopathy (HCM).2 Nonetheless, the ECG is wellknown for its lack of sensitivity in this setting. On the other hand, in HCM the echocardiography (Echo) has become a more reliable diagnostic procedure, including the early detection of disease in first-degree relatives due to diastolic dysfunction.3e5 Nevertheless, it is expensive and not easily available for large pedigrees. Previous study from our group found that NT-proBNP levels were markedly elevated in patients with HCM, as compared with those in healthy individuals.6 Among HCM patients, a clear relationship between the concentrations of the peptide and the signs of left ventricular (LV) diastolic dysfunction and the severity of hypertrophy were observed. Slight elevations of NT-proBNP may reflect very early stages of diseases that precede the development of cardiac manifestations (such as measurable LV hypertrophy). An ideal biomarker for structural heart disease initial assessment would have a high sensitivity, prognostic impact, be stable, be simple to use, and be cost effective.
From the Instituto do Corac¸~ao, University of S~ao Paulo, S~ao Paulo, Brazil. Manuscript received January 13, 2012; revised manuscript received March 26, 2012; revised manuscript accepted April 4, 2012. Reprint requests: Fabio Fernandes, MD, PhD, Av. Dr. Eneas C. Aguiar, 44-Unid. Clınica de Cardiopatias Gerais, Cerqueira Cesar, 05403-900-Sao Paulo, SP, Brasil. Tel: þ55 11 3069 5057; Fax: þ55 11 3069 5346. E-mail: [email protected]
See page 568 for disclosure information. 1071-9164/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cardfail.2012.04.005
Plasma proBNP as a Screening Method for HC
The aim of this study was to evaluate the role of ECG and NT-proBNP as predictors of both structural heart disease, and a positive molecular genetic test, in first-degree relatives of patients with diagnosed HCM. Methods Population Only first-degree relatives of HCM patients were enrolled in the study. Data were obtained from 12 families with HCM followed at our tertiary referral center (Heart Institute, University of S~ao Paulo Medical School). Inclusion criteria were an established diagnosis of HCM (unexplained LV hypertrophy and an echocardiographic wall thickness $15 mm) in at least 1 first-degree relative of the studied individual. Exclusion criteria were low LV ejection fraction, systemic hypertension, proven or suspected coronary artery disease, obesity, severe mitral regurgitation, and inability to provide adequate echocardiograms.7 All patients had glomerular filtration rate higher than 60 mg/dL. The family members were studied by 12-lead electrocardiography; Echo and blood samples were obtained for NT-proBNP analysis. A total of 106 individuals were included in this study. The study protocol was approved by the scientific board of our institution, and all participants provided a written informed consent. The investigation conforms to the principles outlined in the Declaration of Helsinki. Study Groups Individuals were divided into 2 groups based on the used gold standard for the diagnosis of HCM: 1 group consisted of individuals without the disease and another group consisted of individuals with HCM diagnosed by echocardiography or by molecular genetic analysis. To reduce further variability, in the subgroup of individuals with a previously identified causative HCM mutation, we have only included individuals harboring mutations in the same gene: beta myosin heavy chain. Symptoms and functional capacity were assessed according to the New York Heart Association classification. Electrocardiography Standard 12-lead ECGs were recorded in all individuals in the supine position. The ECG voltage criteria were defined: Cornell voltage criterion: R aVL þ SV3, with 8 mm added in women, O28 mm8; Sokolow-Lyon voltage criterion: SV1 þ RV5 or RV6, whichever is larger, O35 mm9; Romhilt-Estes voltage criterion: point score, described previously, $4.10 Echocardiography All individuals were examined by an experienced echocardiographer who reported the data without knowledge of NT-proBNP or genetic tests results. The studies were performed with Acuson equipment model Sequoia 512 (Mountainview, CA) 2.5- to 3.5-MHz transducers. According to the recommendations of the American Society of Echocardiography and the Canadian consensus for diastolic dysfunction reports,11,12 the following parameters were measured in 3 cardiac cycles and averaged: M-mode. Left atrial diameter, LV diastolic internal diameter, LV systolic internal diameter, LV systolic shortening (DD%),
Fernandes et al
interventricular septal diastolic thickness, and LV posterior wall diastolic thickness. Doppler. Mitral peak early and atrial velocities, mitral peak early velocity/atrial velocity ratio, acceleration time and deceleration time of mitral peak early velocity, and isovolumetric relaxation time. Tissue Doppler Imaging. With the sample placed on the lateral border of the mitral annulus, the early longitudinal peak diastolic velocity of the annulus was obtained. The derived mitral flow velocity/mitral annulus velocity ratio was calculated with data from mitral flow and tissue Doppler. The resting LV outflow peak gradient was obtained by using a continuous wave Doppler with the cursor positioned along the LV outflow tract. NT-pro BNP Serum NT-proBNP levels were measured by immunoassay with detection by electrochemiluminescence (Roche Diagnostics, S~ao Paulo, SP, Brazil) using 20 mL of serum and polyclonal antibodies that detect epitopes in the N-terminal region (amino acids 1e76) of the proBNP (108 amino acids). Cross-reactivity with other natriuretic peptides (BNP, proANP1, CNP2) and the renin-angiotensin system were !0.001% (data from the manufacturer). The assay is fully automated using the Elecsys 2010 automated analyzer (Roche Diagnostics). For the determination of NT-proBNP, blood was collected into glass tubes without additives and centrifuged within 1 hour. In the meantime, samples were kept at room temperature. The serum was immediately frozen and stored at 20 C. The sample was thawed just before the analysis, which was carried out within 6 months of blood collection. NT-proBNP is stable for at least 1 year when frozen at 20 C.13,14 For the NT-proBNP assay the coefficient of variation was 7.0% (mean concentration 57 pg/mL) and the inter- and intra-assay variations were 4.0 and 2.6%, respectively. Concentration ranged from 5 to 35,000 pg/mL (data from the manufacturer). Statistical Analysis The diagnostic performance of proBNP for detection of HCM was evaluated by receiver operating characteristic (ROC) curves with calculation of areas under curves (AUC) of sensitivity plotted vs specificity for cutoff proBNP concentration. Sensitivity, specificity, positive and negative predictive values were calculated for each ECG diagnostic criterion and for NT-proBNP levels using a cutoff of 70 pg/mL. The cutoff value was defined based on using NT-proBNP as a continuous variable in ROC analysis. For statistical analysis we have used the statistical software SPSS version 13.0. P ! .05 was considered to be statistically significant.
Results The characteristics of the study individuals are listed in Table 1 according to the 2 used diagnostic criteria (echocardiography or molecular genetics). As observed, serum NT-proBNP levels were significantly associated with a diagnosis of HCM affected status (P ! .001). Using ROC analysis we were able to detect a high AUC for NT-proBNP levels independently of the used gold
566 Journal of Cardiac Failure Vol. 18 No. 7 July 2012 Table 1. Characteristics of Studied Individuals Echocardiography-based Diagnosis (n 5 106) Unaffected Male (%) Age (y) Septum (cm) Posterior wall (cm) Septum/posterior wall (cm) NT-proBNP (pg/mL) 5th percentile 25th percentile 75th percentile 95th percentile 99th percentile Left atrium (cm) E-wave velocity (cm/sec) A-wave velocity (cm/sec) DFT (ms) AT (ms) DT (ms) A’ velocity (mm/s) IRT (ms) E/A ratio BMI (kg/m2) Creatinine (mg/dL)
54.3 32 0.8 0.8 1.00 26.09 5 10 35 67 92 3.7 85 57 467 84 182 134 93 1.68 23.2 0.8
SD 14 0.1 0.1 0.08 20.73
0.75 22 20 138 23 49 35 24 0.60 2.8 0.1
Affected 50 34 1.9 1.0 1.89 1290.52 47 219 1337 5483 12,659 3.5 81 54 470 92 181 134 92 1.70 23.5 0.8
Molecular Genetics-based Diagnosis (n 5 65)
14 0.6 0.3 0.52 2241.25
.68 .54 !.001 !.001 !.001 !.001
57.1 32 0.9 0.8 1.00 21.78 5 9 30 47 74 3.8 88 56 471 91 184 130 95 1.8 22.8 0.8
0.83 21 19 130 23 50 57 25 0.66 2.7 0.2
SD 14 0.1 0.1 0.13 16.14
0.8 22 21 133 25 57 34 24 0.6 2.8 0.1
Affected 47.8 29 1.6 0.9 1.73 768.94 19 92 1183 1994 3549 3.4 81 55 496 87 190 129 96 1.6 23.4 0.9
12 0.5 0.2 0.58 839.72
.47 .42 !.001 .015 !.001 !.001
0.9 26 17 100 25 46 23 30 0.6 2.9 0.2
AT, acceleration time; BMI, body mass index; DFT, diastolic filling time; DT, deceleration time; IRT, isovolumetric relaxation time; SD, standard deviation. Values are presented as mean.
standard for the diagnosis of HCM in first-degree family members (Fig. 1). In Table 2, we present estimates of the sensitivity, specificity, predictive values, and likelihood ratios for the 3 possible ECG approaches for screening first-degree relatives using an echocardiographic study as the gold standard (Table 2A) and the same calculated estimative using molecular genetic test as the gold standard (Table 2B). In Table 2 we also present the same indices for a screening criterion based solely on a serum NT-proBNP level O70 pg/mL. Clearly, in either scenario, a NT-proBNP serum level
O70 pg/mL has a significantly greater diagnostic performance than any of the proposed ECG criteria for HCM diagnosis. Finally, after adjusting multivariate logistic regression models for age, sex, a NT-proBNP level O70 pg/mL, and the ECG diagnostic criteria (in 3 separate models, 1 with each of the 3 tested ECG criteria), NT-proBNP remained significantly associated with the diagnosis of HCM in screened relatives. This was true for all 3 models with different ECG criteria and for all models regardless of the used gold standard (data not shown).
Fig. 1. Receiver operating characteristic analysis of NT-proBNP levels using (A) echocardiography as the gold standard or (B) molecular genetic test as the gold standard. AUC, area under the curve.
Plasma proBNP as a Screening Method for HC
Fernandes et al
Table 2. ECG Criteria Estimative for the Diagnosis of HMC in First-degree Relatives ECG Criterion Sokolow-Lyon A: Echocardiogram as gold standard (point estimative, 95% confidence interval) Sensitivity Specificity Positive predictive value Negative predictive value Positive likelihood-ratio Negative likelihood ratio B: Molecular genetic test as gold standard (point estimative, 95% confidence interval) Sensitivity Specificity Positive predictive value Negative predictive value Positive likelihood ratio Negative likelihood ratio
NT-proBNP (O70 pg/mL)
0.42 0.91 0.68 0.78 4.89 0.64
(0.25e0.61) (0.82e0.96) (0.43e0.86) (0.67e0.86) (2.05e11.68) (0.47e0.86)
0.52 0.97 0.89 0.82 18.06 0.50
(0.33e0.69) (0.89e0.99) (0.64e0.98) (0.72e0.89) (4.42e73.83) (0.35e0.72)
0.71 0.88 0.73 0.87 6.12 0.33
(0.52e0.85) (0.79e0.95) (0.54e0.87) (0.76e0.94) (3.07e12,20) (0.19e0.57)
0.92 0.96 0.92 0.96 21.39 0.09
(0.76e0.97) (0.87e0.99) (0.76e0.98) (0.87e0.99) (7.04e64.99) (0.03e0.26)
0.37 0.93 0.70 0.76 5.16 0.68
(0.17e0.61) (0.79e0.98) (0.35e0.92) (0.62e0.87) (1.49e17.81) (0.48e0.96)
0.42 0.98 0.89 0.79 17.68 0.21
(0.21e0.66) (0.86e0.99) (0.51e0.99) (0.65e0.88) (2.38e131.58) (0.12e0.35)
0.67 0.88 0.71 0.86 5.60 0.38
(0.41e0.86) (0.74e0.96) (0.44e0.87) (0.71e0.94) (2.31e13.57) (0.20e0.73)
0.83 0.98 0.95 0.91 34.70 0.18
(0.60e0.94) (0.86e0.99) (0.73e0.99) (0.79e0.97) (4.96e242.78) (0.07e0.43)
Discussion Early identification of HCM patients will allow clinicians to implement management strategies such as risk factor modification (ie, avoid exercise) sooner. This has the potential to prevent sudden death and improve prognosis.15 BNP is produced in the ventricle as a response to ventricular overload. It was wildly described as a biomarker for disease severity in heart failure, including diastolic dysfunction.16 Several studies report the use of BNP as a biomarker for prognosis in patients with HCM. Pieroni et al17 suggests that plasmatic measure of BNP may provide a noninvasive method to evaluate hemodynamic deterioration. Other studies demonstrated that BNP measurement alone can correlate with several functional parameters.16 The focus of our work is to use the plasmatic values of NT-proBNP as a faster and cheaper biomarker for firstdegree relatives of patients with HCM. BNP was used with this purpose in cats. One study has shown that the plasmatic NT-proBNP concentration was able to discriminate normal animals from those with occult hypertrophic cardiomyopathy. Also, the values were associated with echocardiographic markers of severity.18 In this cross-sectional study, plasma NT-proBNP levels were markedly elevated in first-degree relatives of HCM patients that also had HCM, as compared with those in healthy, unaffected first-degree relatives. In addition, using different measures of diagnostic performance, we have observed that a NT-proBNP level above 70 mg/dL is a valid tool for identifying structural heart disease within the relatives’ patients with HCM. Dosing NT-proBNP may provide useful information in addition to ECG abnormalities as an independent predictor for HCM. Indeed, in our models NT-proBNP provided an independent and significantly better accuracy than any of the ECG criteria proposed for the identification of LV hypertrophy in first-degree relatives, regardless of the used gold standard for this diagnosis.
Even in the absence of clinical heart failure, diastolic dysfunction in itself is associated with increased level of BNP.16 Actually, several studies have found significant correlations between echocardiographic parameters for diastolic dysfunction and serum natriuretic levels in diagnosing diastolic heart failure. Tschope et al observed that NT-proBNP can reliably detect the presence of isolated diastolic dysfunction in symptomatic patients and is a useful tool to rule out patients with reduced exercise tolerance of noncardiac origin. In this study the reliability of NT-proBNP was similar to tissue doppler imaging indices (AUC 0.83 vs. 0.81) and improved when compared with conventional echocardiography. NT-proBNP also had the best negative predictive value of all methods (94%) and correlated strongly with indices of LV filling pressure, as determined by invasive measurements.16 BNP levels also correlate with noninvasive parameters of disease severity in children with HCM, including measures of raised LV filling pressures.19 In a screening study, there is the demand for high diagnostic sensitivity. In our study, we observed, using threshold values of 70 pg/mL, sensitivities in the range of 0.83 to 0.92, depending on the used diagnostic gold standard. Some authors suggest that, even in the primary setting, natriuretic peptides should not be used to replace cardiac imaging to confirm a final diagnosis of symptomatic heart failure. Nevertheless, natriuretic peptides could be used as a cost-effective test for ruling out the need for echocardiogram when negative.20 Also, Pagourelias et al suggest that BNP has a promising role in the exclusion of HCM in elite strength athletes and suggest that the measurement of BNP levels in a larger number of strength athletes would yield a significant difference between this group and patients with HCM.21 Comparing with BNP measurement, the Echo is more expensive, needs to be performed locally (cannot be transported to a central lab, for instance) and depend on a highly trained operator. Tissue Doppler imaging is more sensitive, but it is even more expensive because of
568 Journal of Cardiac Failure Vol. 18 No. 7 July 2012 the necessity of sophisticate equipment and a trained operator. A limitation of our study is that we selected only MYH7 gene mutations to reduce variability but one might argue that it is not possible to extend these findings to the general population of HCM. Indeed, different age-dependent penetrance and phenotype-genotype associations could potentially lead to different accuracies, and NT-proBNP cut points, in individuals harboring other HCM mutations than MYH7. Because natriuretic peptides values can range with several factors such as gender and age, we point out the necessity for the development of algorithms that take these data into account. According to Redfield et al,22 the median BNP for both women and man younger than 55 years old is between 7 and 28 pg/mL. We only have 7 patients above this age, thus we do not believe that in this specific group the age is such an important factor. Also, body mass index and kidney problems may influence NT-proBNP values. To avoid this bias, we excluded patients with kidney failure and obesity. Nonetheless, care should be exercised while one is applying this algorithm to older individuals. We expand these ideas for the scenario of HCM screening in first degree-relatives. In our study, the negative predictive value of this marker ranged from 0.91 to 0.96, dependent on the used gold standard for diagnosis, suggesting that it could improve the cost-effectiveness of screening even asymptomatic individuals with incipient diastolic dysfunction. Disclosure
Supplementary Data Supplementary data related to this article can be found online at doi:10.1016/j.cardfail.2012.04.005
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