Echocardiographic characterization of hypertrophic cardiomyopathy in Chinese patients with myosin-binding protein C3 mutations
- Authors:
- Published online on: January 25, 2017 https://doi.org/10.3892/etm.2017.4089
- Pages: 995-1002
Abstract
Introduction
Hypertrophic cardiomyopathy (HCM) is a common autosomal dominant cardiac disease characterized by asymmetric left ventricular (LV) hypertrophy (LVH) with heterogeneous morphologic, functional and clinical features (1). Since the pathogenic missense mutation in the β-myosin heavy chain 7 (MYH7 R403Q) was revealed two decades ago, over 1,400 gene mutations have been identified in ≥11 putative HCM-susceptibility genes (2–4), which predominantly encode for proteins of the thick filament of the cardiac sarcomere. Cardiac myosin-binding protein C3 (MyBPC3) and myosin heavy chain 7 (MYH7) are, by far, the most common HCM-associated genes with an estimated prevalence of 25–35% for each gene (5). MyBPC3 is a key constituent of the thick filaments localized to doublets in the C-zone of the A-band of the sarcomere. By binding to myosin, titin and actin, MyBPC3 contributes to the structural integrity of the cardiac sarcomere and regulates cardiac muscle contractility (6).
Although the mechanisms underlying the correlations between disease-causing genetic mutations and characteristic pathological and morphologic (or phenotypic) features of HCM are unclear, preliminary studies suggest that particular gene abnormalities are associated with specific clinical phenotypes, such as degree of hypertrophy, risk of sudden death, onset of disease and disease penetrance in families (7–9). Mutations in the MyBPC3 gene were found to be responsible for 15–20% of cases of familial HCM, and were generally associated with mild and age-related penetrance disease (10). However, the prevalence of MyBPC3 mutations and the clinical characteristics associated in the Chinese HCM population remain unclear.
The present study is, to the best of our knowledge, the first to identify the prevalence of MyBPC3 gene mutations and the associated characteristic echocardiographic phenotypes in Chinese patients with HCM. To identify these clinical characteristics, cutting-edge echocardiographic techniques, such as 2-dimensional echocardiography (2DE), Tissue Doppler imaging (TDI) and real-time 3-dimensional echocardiography (RT-3DE), were employed.
Materials and methods
Study population
A total of 48 Chinese adult patients (age, 48±12 years) with familial or sporadic HCM were enrolled in the study after informed written consent was obtained. All patients were in sinus rhythm at the time of enrollment and were diagnosed clinically by echocardiography between December 2008 and February 2010 at the General People's Liberation Army Hospital in Beijing, China. A total of 37 patients had a family history of HCM while the other 11 patients did not have confirmed relatives with HCM. The diagnosis of HCM was based on 2DE showing an unexplained maximum left ventricular wall thickness (MLVWT) of ≥15 mm (11) in the absence of other cardiac or systemic disease capable of producing the observed magnitude of hypertrophy (such as systemic hypertension, aortic stenosis and amyloidosis). The family members of index patients with positive MyBPC3 mutations were divided into the following two groups according to whether the patients and their family members met the criteria for diagnosis of HCM from the results of routine echocardiography, as described by McKenna et al (12): Mutation carriers in the LVH group (G+/LVH+; n=18), and mutation carriers not in the LVH group (G+/LVH-; n=23). The control group consisted of 30 healthy age- and gender-matched subjects without cardiological disorders.
Genetic study
Venous blood (3 ml) was collected from each subject for MyBPC3 mutation screening. Systematic family screenings of genotyped patients with HCM were performed after identifying MyBPC3 mutations. DNA was extracted from whole blood and stored at −70°C. Genomic primer pairs were designed to amplify the whole coding exons of MyBPC3 (35 exons) using Primer BLAST (www.ncbi.nlm.nih.gov/tools/primer-blast/) and Primer Premier 5 software (PREMIER Biosoft, Palo Alto, CA, USA). Nested polymerase chain reaction (PCR) was used to amplify the extracted DNA (Table I). Briefly, for the initial PCR, 0.1 µg DNA and primers were used in a 20-µl reaction: 2.5 µl 10X buffer, 0.5 µl 2.5 mM dNTPs, 0.4 µl of each primer forward (10 µM) and reverse (10 µM) and 2.5 µl of Taq (TaKaRa) (5 U/µl). The target gene was amplified according to the following parameters: Denaturation at 95°C for 5 min, 45 cycles of denaturation at 95°C for 25 sec, annealing at 50°C for 60 sec and extension at 72°C for 2 min, and a final extension of 5 min at 72°C. The second round of PCR was performed using the first-round PCR products in a 20-µl reaction: 2.5 µl 10X buffer, 0.5 µl 2.5 mM dNTPs, 0.5 µl of each primer forward (10 µM) and reverse (10 µM) and 2.5 µl of Taq (TaKaRa) (5 U/µl). The target gene was amplified according to the following parameters: denaturation at 95°C for 3 min, 30 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 45 sec and extension at 72°C for 45 sec, and a final extension of 2 min at 72°C. Following PCR amplification, PCR products were analyzed by direct sequencing on an ABI PRISM 3130 DNA Analyzer with BigDye Terminator chemistry (version 3.1; Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The fluorophore used was SYBR green (Applied Biosystems; Thermo Fisher Scientific, Inc.). The 2−ΔΔcq method was used to quantify the results (13). All results were repeated four independent experiments. The relative level of mRNA was calculated by the comparative CT method with GAPDH mRNA as the housekeeping gene.
For every sequence variant detected, a cohort of 200 ethnically matched control subjects were screened by using the same methodology. Conservation of residues was determined by Homologene (www.ncbi.nlm.nih.gov/homologene) by multiple alignment of orthologues in various species, including Homo sapiens, Pan troglodytes, Canis lupus, Bos taurus, Mus musculus, Rattus norvegicus, Gallus gallus and Danio rerio. A variant was considered a mutation if it showed co-segregation with affected members in the family, was not present in the 200 healthy adult controls, and if the mutated residue was conserved among species.
Clinical evaluation
Index patients and their relatives were evaluated for family history, age, gender, body surface area and clinical symptoms. They also underwent 12-lead electrocardiography, 2DE, TDI, and 3DE.
2-DE
2DE images were acquired with a commercially available ultrasound system (Acuson Sequoia 512; Siemens AG, Munich, Germany) equipped with a 4V1c transducer (1–4 MHz). LV end-diastolic diameter (LVEDD) and LV end-systolic diameter (LVESD) were measured using M-mode and 2D images obtained from parasternal long-axis views. The severity and distribution of LVH was assessed in the parasternal short-axis plane at the mitral valve and papillary muscle levels. MLVWT was regarded as the greatest thickness at any site in the LV wall (14). Patients were classified as having obstructive HCM when the LV outflow tract gradient was ≥30 mmHg (15). The mitral early (E-wave) and late (A-wave) filling velocities ratio (E/A), and the E-wave velocity deceleration time (DT) were measured by pulsed-wave Doppler. TDI was performed by placing the sample volume at the side of the basal septal and lateral wall in the apical 4-chamber view. The mitral annular systolic (Sa), and early (Ea) and late (Aa) diastolic velocities were recorded at late expiration in the pulsed-wave Doppler mode. Diastolic function was subsequently graded 0 to 3, as previously described (16): Grade 0 (normal relaxation): DT >140 msec, E/A ratio >1 and septal E/Ea ratio <8; Grade 1 (impaired relaxation): DT >140 msec, E/A ratio <0.75 and E/Ea ratio <8; Grade 2 (pseudo-normal filling): DT >140 msec, 1 <E/A ratio <1.5 and E/Ea ratio >15, and systolic/diastolic pulmonary vein ratio <1; Grade 3 (restrictive filling): E/A ratio ≥1.5, DT <140 msec, E/Ea ratio >15, and systolic/diastolic pulmonary vein ratio <1.
3-DE
3DE images were obtained with a commercially available ultrasound system (Acuson SC2000; Siemens AG) equipped with a 4Z1c transducer (2–4 MHz). This system has 15–17 cm detectable depth, 90×90° scanning angle, and >20 frames/sec volume rate. Electrocardiogram (ECG) images were obtained in the left lateral decubitus position. The 3D data sets were analyzed offline using the Left Ventricular Analysis System (Siemens Medical Solutions, Inc., Malvern, PA, USA). Various sequences of the endocardium were automatically signed and manually revised. The LV was automatically divided into 17 segments. Diastolic dyssynchrony index (DDI), as a mechanical dyssynchrony parameter used in the software, was derived from the standard deviation of the regional end-diastolic peak volume times of the 17-segment model and represented the mechanical dyssynchrony of a ventricle. End-diastolic synchrony with the DDI index was evaluated using the following equation:
DDI=1N–1∑i=1N(ti–t¯)Where ti represents regional relaxation times, and t represents the mean regional relaxation times.
The images were stored by adopting the P-P interval in the ECG to acquire a harmonized standard. Then, the sampling points for every segment were automatically located in the maximum volume and the DDI was automatically calculated by the software.
Statistical analysis
Data were expressed as mean ± standard deviation, data was analyzed using the Chi-square test for qualitative data and one-way analysis of variance or Wilcoxon rank sum test for quantitative data, followed by Student-Newman-Keuls multiple range test for multiple comparisons, and variables were analyzed by post-hoc tests between the control group and the G+/LVH+ or G+/LVH- groups. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using the SPSS version 16 for Windows (SPSS, Inc., Chicago, IL, USA).
Results
Genetic study
Nine mutations in the MyBPC3 gene were identified in 7/48 cases (15%), with two patients having digenic mutations in the same gene. Specifically, there are 7 missense mutations (c.2541C>G/p.Y847X, c.2526C>G/p.Y842X, c.2527G>T/p.Y843X, c.706A>G/p.S236G, c.772G>A/p.E258K, c.2971G>A/p.V991M and c.3272A>G/p.D1091G), and 2 synonymous mutations (c.2118T>C/p.G706G and c.2025G>A/p.G675). Among them, four mutations were novel while the other five mutations had been previously published (Table II). In addition, all the index patients with these novel mutations have a familial HCM history. Systematic genetic screenings and echocardiographic characterization of these patients showed that 18 subjects had MyBPC3 mutations and HCM phenotype (G+/LVH+ group), and the other 23 subjects had MyBPC3 mutations only (G+/LVH- group) (Table III). All mutations located in exon regions were concentrated in 5 myosin domains, C1, C5, C6, C8 and C9 (Fig. 1).
Clinical characteristics
The clinical variables of subjects enrolled in this study at the baseline are displayed in Table IV. There was no significant difference in blood pressure or heart rate among the three groups. However, by comparing with the other two groups, the G+/LVH+ population was significantly older (P<0.0001) and had more males than females. All patients were symptomatic at the first presentation; 90% of them had mild symptoms (functional New York Heart Association [NYHA] class). Patients with HCM were managed primarily through medication (β-blockers and calcium channel blockers). Diastolic function was normal (grade 0) in all controls and all G+/LVH- subjects. The majority of G+/LVH+ subjects had grades 1 to 2 diastolic dysfunction and asymmetrical septal hypertrophy (14/18).
Echocardiographic features
The results of the echocardiographic analysis are displayed in Table V. As expected, maximal LV wall thickness and interventricular septal thickness (IVS) were higher in the G+/LVH+ subjects compared with control (P<0.05) and G+/LVH-subjects. In addition, the left atrial dimension was significantly higher in the G+/LVH+ subjects compared with the control. There were no significant differences in LVEDD, LVESD and LV ejection fraction among the three groups.
In the G+/LVH+ group, velocity parameters including septal Sa, Ea and Aa, and lateral Sa and Ea velocities were significantly lower compared with their counterparts in the control group (P<0.05). However, there was no significant difference in lateral Aa velocity between the G+/LVH+ and control groups. In the G+/LVH- group, only the septal Ea peak velocity (rather than septal Sa and Aa, and lateral Sa, Ea and Aa velocities) was significantly lower (P<0.0001) compared with the control group (Fig. 2). Fig. 2 shows the comparison between the G+/LVH- or G+/LVH+ group and the control. There was no significant difference in septal Sa and Aa, and lateral Sa, Ea and Aa velocities between the G+/LVH- and control groups. The DT was significantly increased in the G+/LVH+ and G+/LVH-groups compared with the control group (P<0.0001), although there was no significant difference in E/A ratios among the three groups.
According to the 3DE images, the 17 segmental curves, divided according to the recommendations of the Joint Committee of the American Heart Association (17), were smooth and regular for the controls, whereas the curves for HCM subjects were dyssynchronous (Fig. 3). A significant increase in DDI was found in the G+/LVH+ group (P<0.0001). However, no difference was observed between controls and the G+/LVH- group. This implies that the diastolic dyssynchrony may be a predominant cause of the diastolic dysfunction in patients with HCM.
Discussion
HCM is a primary myocardial disorder marked by genotypic and phenotypic heterogeneity (18). More than 200 different gene mutations have been reported for HCM, and the disease is typically caused by single heterozygous mutations in the gene encoding sarcomere proteins. Mutations in MyBPC3 are the most common type found in patients with HCM. Identification of specific MyBPC3 mutations may give clinicians insight into the genetic course of the disease. Various reports have indicated that MyBPC3 mutations are present in 14.5–26% of patients with HCM [18.2% at the Mayo Clinic, 14.5% at Harvard Medical School, 26% in France, 18% in Germany, 21.7% in Sweden and 24% in Finland (19)]. However, the prevalence of MyBPC3 mutations in Chinese patients with HCM has, to the best of our knowledge, not been investigated. The present study revealed that there is a 15% prevalence of MyBPC3 mutations in Chinese patients with HCM, which is within the range reported above. In addition, double MyBPC3 mutations were identified in two families, which accounts for 3% of the multiple mutation rate, similar to as previously described (19,20). These data indicate that MyBPC3 mutations may be the most common genetic cause of HCM in patients in China.
MyBPC3 is a structural and regulatory protein found in the sarcomere. The C0 and C1 domains at the N-terminus are primarily responsible for sarcomere regulation and include a Pro-Ala-rich region and a myosin binding domain, which is a target of post-translational modifications. The C5-C10 domains at the C-terminus participate in binding myosin and titin, and are necessary for MyBPC3 stability and sarcomere organization (21,22). The novel mutations identified in the present study are located at C5, C8 and C9, suggesting that these mutations may affect myocardial function through changing MyBPC3 protein stability and sarcomere structure.
Diastolic dysfunction has been documented as the early sign of HCM preceding LVH in human and animal models harboring HCM disease causing genetic mutations (23–26). Previous studies have demonstrated that nonhypertrophied regions of LV may contribute to diastolic dysfunction in patients with HCM (27). Diastolic dysfunction was observed in all patients with HCM with MyBPC3 gene mutations in the present study, along with significantly increased septal and lateral E/Ea ratio and significantly increased DDI values. E/Ea ratio reflects the filling pressure in patients with HCM by TDI, and the higher ratio reflects a higher severity of symptoms (28). The DDI value is a mechanical dyssynchrony parameter in 3DE; the higher the value, the more severe the diastolic dyssynchrony (29). Furthermore, the present study showed that regional remodeling of LV may be independent and occur prior to global LVH. Increased septal E/Ea ratio was observed in the G+/LVH- group in the absence of lateral E/Ea ratio change, and increased IVS and LPW ratio in G+/LVH- group was observed, which suggests local remodeling, consistent with previous studies which showed histological abnormalities in the absence of significant LVH (30). In addition, no significant change in DDI value was observed in the G+/LVH- group, indicating that regional myocardial function abnormalities were not serious enough to affect global LV diastolic function. These findings suggest that regional hypertrophic remodeling proceeds myocardial dysfunction in patients with MyBPC3 mutations. However, detailed prospective studies are required to better understand the chronology of the development of HCM caused by MyBPC3 mutations.
In conclusion, the present study revealed a 15% prevalence of MyBPC3 gene mutations in the Chinese HCM population. MyBPC3 gene mutation caused regional LV hypertrophic remodeling first and further proceeded to global hypertrophic remodeling and myocardial diastolic dysfunction. Combined applications of 2DE, TDI and 3DE are a feasible way to detect early myocardial remodeling and myocardial dysfunction in patients with genetic predisposition.
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