Hypertrophic cardiomyopathy (HC) is a hereditary heterogeneous cardiovascular disorder. Existing data have been of predominantly Caucasian samples, and a large study is needed in Chinese population. The present study was intended to explore the genetic basis and clinical characteristics correlated with different genotypes in a large cohort of Chinese patients. Direct gene sequencing of β-myosin heavy chain ( MYH7 ), myosin binding protein-C ( MYBPC3 ), and cardiac troponin T ( TNNT2 ) was performed in 136 unrelated Chinese HC patients. Clinical evaluations were conducted. In total, 32 mutations were identified in 36 patients (27%), including 10 novel ones. Distribution of mutations was 56% ( MYBPC3 ), 31% ( MYH7 ), and 13% ( TNNT2 ), respectively. Double mutations were identified in 3% patients. The occurrence of HC-associated sarcomeric mutations was associated with an earlier age of onset, increased left ventricular hypertrophy, a higher incidence of syncope, previous family history, and sudden cardiac death. No statistical difference was identified in patients carrying MYBPC3 and MYH7 mutations with regard to clinical characteristics and outcomes. Patients with double mutations were associated with malignant progression in the study. In conclusion, MYBPC3 is the most predominant gene in HC. Multiple mutations are common in MYH7 , MYBPC3 , and TNNT2 . The present study suggests a large diversity of HC and a prognostic role of genotype.
Hypertrophic cardiomyopathy (HC) is an autosomal-dominant cardiovascular disorder characterized by asymmetric left ventricular hypertrophy with a high risk of sudden cardiac death (SCD) in youth. Up to the present, more than 900 mutations have been identified in 13 genes encoding both sarcomere and sarcomere-related proteins. β-myosin heavy chain ( MYH7 ), myosin binding protein-C ( MYBPC3 ), and cardiac troponin T ( TNNT2 ) are the most important causative genes accounting for approximately 30% to 50%, 20%, and 20% of familial HC, respectively. Existing data have come largely from Caucasian samples. A large study is expected in Chinese population because of ethnic genetic heterogeneity. The objective of the present study was to explore the genetic basis and clinical characteristics correlated with different genotypes in a large cohort of Chinese patients.
Methods
One hundred thirty-six unrelated Chinese HC patients (94 men and 42 women) were recruited from 2002 to 2010. Informed written consent was obtained from all study participants under the approval of the ethics committee of Peking University People’s Hospital. The diagnosis of HC was based on the presence of hypertrophied left ventricle on echocardiography (left ventricular wall thickness ≥13 mm) in the absence of other cardiac or systemic diseases. Another 100 healthy gender- and-age-matched subjects (200 alleles) with normal electrocardiography (ECG) and echocardiography findings were selected as the control group.
Genomic DNA of peripheral blood was harvested from all subjects and their family members. The sequence of all coding sequences (exon 2-39 of MYH7 , exon 1-35 of MYBPC3 , exon 2-17 of TNNT2 ) for selected genes was studied by polymerase chain reaction (PCR), and direct sequencing. PCR primers were provided by reference ( http://genetics.med.harvard.edu/∼seidman/cg3 ) or redesigned with Primer 3 software according to reference sequence from GenBank ( MYH7 : NM_000257.2 ; MYBPC3 : NM_000256.3 ; TNNT2 : NM_001001430.01 ) to amplify the coding sequence and intron-exon junctions. PCR products were purified by vacuum pump MultiScreen PCR Plate (Millipore, Bedford, Massachusetts). Direct sequencing was conducted with BigDye Terminator DNA sequencing kit (version 3.1) and 3730XL DNA Analyzer (Applied Biosystems, Carlsbad, California). All experimental procedures were performed in accordance to standard protocols. Nomenclature of mutations followed the recommendations of Human Genome Variation Society (2007). To exclude polymorphism from mutations, 100 healthy Han volunteers were selected as control subjects, and the results were compared with information of databases from National Center for Biotechnology Information.
Clinical evaluations included family and personal history, physical examinations, 12-lead electrocardiogram, conventional M-mode, and 2-dimensional and Doppler echocardiography. Twenty-four-hour ambulatory electrocardiogram and magnetic resonance imaging were performed if needed. Follow-up studies included New York Heart Association (NYHA) functional classification, treatment, and adverse event with regard to HC-related death, heart failure requiring hospitalization, and stroke caused by atrial fibrillation. The information was obtained by telephone contact or standard questionnaires. SCD was defined as sudden and unexpected death within 1 hour of the onset of symptoms with a relatively stable clinical course in the past.
Statistical analysis was performed with software SPSS 13.0 (SPSS Inc., Chicago, Illinois). Mean value for clinical parameters was expressed as mean ± SD. Difference between groups was assessed with chi-square analysis for categorical variables and t test and nonparametric test for normally distributed continuous data and abnormally distributed continuous variables respectively. A p value <0.05 was considered statistically significant.
Results
The clinical characteristics of all patients were summarized in Table 1 . Overall, 32 mutations in the selected genes were identified in 36 patients leading to genetic diagnosis of 27% patients ( Table 2 ). Nearly 1/3 of mutations (10 mutations) were novel, and 7 mutations were first published by our center in a previous study. None of the mutations were found in 200 control alleles.
| Clinical Phenotype | Patients (n = 136) |
|---|---|
| Male/female | 94/42 |
| Mean onset age (yrs) | 45.7 ± 17.6 |
| Syncope | 15% (21/136) |
| Family history of HC | 20% (27/136) |
| Family history of SCD | 10% (13/136) |
| Maximal LVWT (mm) | 18.6 ± 4.7 |
| LVOTO | 30% (38/136) |
| LVEF (%) | 68.2 ± 9.0 |
| Disease-Causing Gene | Site of Mutation | DNA Sequence Change | Amino Acid Change | Type of Mutation | Frequency | Domain |
|---|---|---|---|---|---|---|
| MYH7 | Exon2 | c.183C>T | p.Ala 26 Val | Missense | 1 | Actin-binding |
| Intron4 | c.452-7T>C ∗ | Splice site | 1 | S1 domain | ||
| Exon4 | c.533C>T ‡ | p.Arg 143Trp | Missense | 1 | ATP-binding | |
| Exon15 | c.1874A>G ∗ | p.Ile590Val | Missense | 1 | Actin-binding | |
| Exon17 | c.2094G>A | p.Arg663His | Missense | 1 | S1 domain | |
| Exon18 | c.2262G>A | p.Arg719Gln | Missense | 1 | S1 domain | |
| Exon19 | c.2297C>T ∗ | p.Pro731Ser | Missense | 1 | S1 domain | |
| Exon19 | c.2313T>C ‡ | p.Ile736Thr | Missense | 3 | S1 domain | |
| Exon22 | c. 2876G>A | p.Glu924Lys | Missense | 1 | S2 domain | |
| Exon26 | c.3753A>T ∗ | p.Lys1216Met | Missense | 2 | Rod domain | |
| MYBPC3 | Intron4 | c.560+5 G>A ∗ | Splice site | 1 | Splice site | |
| Intron9 | c.907-10 C>G ∗ | Splice site | 1 | Splice site | ||
| Exon 9 | c.958deleteG † | p.Lys301LysfsX49 | Frameshift | 1 | Frameshift | |
| Exon12 | c.1055G>A ∗ | p.Glu334Lys | Missense | 2 | MYBPC3 motif | |
| Exon12 | c.1093_1102dupCGGCA † | p.Met348ThrfsX4 | Frameshift | 1 | Frameshift | |
| Exon16 | c.1432deleteC | p.Pro459ProfsX7 | Frameshift | 1 | Frameshift | |
| Exon16 | c.1442C>T † | p.Gln463X | Nonsense | 1 | C3 | |
| Exon17 | c.1559C>T † | p.Arg502Trp | Missense | 1 | C3 | |
| Exon17 | c.1574 G>A | p.Gly507Arg | Missense | 1 | C3 | |
| Exon17 | c.1622G>A † | p.Gly523Arg | Missense | 1 | C3 | |
| Exon21 | c.2058 G>C ‡ | p.Arg668Pro | Missense | 1 | C5 | |
| Intron23 | c.2204-3 C>G ∗ | Splice site | 1 | Splice site | ||
| Exon25 | c.2495_2497delete AAG | p.Lys814Del | Frameshift | 1 | Frameshift | |
| Exon25 | c.2559G>T ∗ | p.Arg835Leu | Missense | 1 | C6 | |
| Exon25 | c.2594T>C † | p.Tyr847His | Missense | 1 | C6 | |
| Intron26 | c.2792+12 C>T † | Splice site | 3 | Splice site | ||
| Exon31 | c.3422deleteC ∗ | p.His1123ThrfsX66 | Frameshift | 1 | Frameshift | |
| Exon32 | c.3679delete C | p.Pro1208ProfsX29 | Frameshift | 2 | Frameshift | |
| TNNT2 | EXON 9 | c.373C>T | p.Arg92Trp | Missense | 2 | T1 domain |
| EXON 9 | c.379C>T | p.Arg94Cys | Missense | 1 | T1 domain | |
| EXON 10 | c.435G>A † | p.Glu128Lys | Missense | 1 | T1 domain | |
| EXON 10 | c.947G>A | p.Arg286H | Missense | 1 | T2 domain |
∗ Novel mutations identified in current study.
† First published by our center in the previous study.
Screening of the whole coding region of MYH7 uncovered 10 mutations in 13 patients, including 4 novel mutations (c.452-7T>C, I590V, P731S, K1216M). Nine mutations led to a change of a single amino acid, and 1 was located in splice region. All mutations were private except for mutation MYH7 -A26V. Analysis of MYBPC3 revealed 18 mutations, including 7 missense mutations, 6 frameshift, 4 splice, and 1 nonsense mutations. Among them, 6 mutations (c.560+5 G>A, c.907-10 C>G,E334K, c.2204-3 C>G, R835L, p.His1123ThrfsX6) were first to be reported. Four missense mutations were identified in TNNT2 . In this cohort, 4 patients (3%) were reported to have complex genetic status with 2 compound heterozygotes in MYBPC3 ( MYBPC3 -G523R and MYBPC3 -Y847H; MYBPC3 -R502W and MYBPC3 -c.2792+12 C>T) and 2 double heterozygotes ( MYH7 -A26V and MYBPC3 -E334K; MYBPC3 – c.2792+12 C>T and TNNT2 -E128K).
Overall, mutational screening yielded a genetic diagnosis of 36 patients (27%) in the 136 unrelated HC patients. Among them, 16 (12%) patients had mutations of MYBPC3 , 12 (9%) patients had mutations of MYH7 , and 4 (3%) patients had mutations of TNNT2 . Double mutations were identified in 4 (3%) patients.
Patients with mutations in selected genes were associated with an earlier age of onset (36.1 ± 17.7 vs 49.2 ± 16.3 years, p = 0.001), a higher incidence of syncope (31% vs 10%, p = 0.003), and more pronounced left ventricular hypertrophy (21.7 ± 5.6 vs 17.4 ± 3.8 mm, p = 0.001) compared with those without mutations. No difference existed between genotyped patients and those without mutations in proportion of obstructive-HC and incidence of ventricular tachycardia, ventricular fibrillation, frequent ventricular premature contraction, and atrial fibrillation. No statistical difference was identified in any variable between patients carrying mutations of MYH7 and MYBPC3 with regard to symptoms, echocardiography, and ECG characteristics ( Table 3 ). Patients with double mutations had an early age of onset. Three-quarters of patients developed HC before age 35 years. The mean maximum left ventricular wall thickness was 29.0 ± 9.5 mm ( Table 4 ).
| Clinical Phenotype | Genotype Negative (n = 100) | Genotype Positive (N = 36) | p Value (Positive Genotype vs Negative Genotype) | p Value ( MYH7 vs MYBPC3 ) | |||
|---|---|---|---|---|---|---|---|
| MYH7 (n = 12) | MYBPC3 (n = 16) | TNNT2 (n = 4) | All (n = 36) | ||||
| Male/female | 68/32 | 9/3 | 12/4 | 2/2 | 26/10 | NS | NS |
| Mean age of onset (yrs) | 49.2 ± 16.3 | 38.7 ± 17.2 | 39.3 ± 16.0 | 32.3 ± 25.0 | 36.1 ± 17.7 | 0.001 | NS |
| Age of onset ≤35 yrs | 22% (22/100) | 42% (5/12) | 38% (6/16) | 50% (2/4) | 44% (16/36) | 0.01 | NS |
| Syncope | 10% (10/100) | 33% (4/12) | 31% (5/16) | 25% (1/4) | 31% (11/36) | 0.003 | NS |
| Family history of HC | 13% (13/100) | 50% (6/12) | 25% (4/16) | 50% (2/4) | 39% (14/36) | 0.001 | NS |
| Family history of SCD | 3% (3/100) | 25% (3/12) | 25% (4/16) | 50% (2/4) | 28% (10/36) | 0.001 | NS |
| Maximal LVWT (mm) | 17.4 ± 3.8 | 21.4 ± 3.7 | 20.4 ± 5.1 | 20.6 ± 4.6 | 21.7 ± 5.6 | 0.001 | NS |
| Severe hypertrophy ≥20mm | 20% (20/100) | 75% (9/12) | 50% (8/16) | 50% (2/4) | 64% (23/36) | 0.001 | NS |
| Apical hypertrophy | 22% (22/100) | 0% (0/12) | 13% (2/16) | 25% (1/4) | 8% (3/36) | NS | NS |
| LVEF (%) | 69.0 ± 7.7 | 68.2 ± 12.5 | 66.0 ± 8.9 | 62.8 ± 5.3 | 65.8 ± 11.8 | NS | NS |
| LVOTO | 27% (27/100) | 42% (5/12) | 25% (4/16) | 0% (0/4) | 31% (11/36) | NS | NS |
| ELVH | 40% (40/100) | 50% (6/12) | 38% (6/16) | 50% (2/4) | 47% (17/36) | NS | NS |
| AF | 16% (16/100) | 8% (1/12) | 6% (1/16) | 0% (0/4) | 8% (3/36) | NS | NS |
| VT/VF/FPVC | 7% (7/100) | 25% (3/12) | 13% (2/16) | 0% (0/4) | 14% (5/36) | NS | NS |
| Known prognosis | 88/100 | 11/12 | 12/16 | 3/4 | 29/36 | — | — |
| NYHA at latest evaluation | 0.004 | NS | |||||
| I/II | 94% (83/88) | 73% (8/11) | 75% (9/12) | 100% (3/3) | 72% (21/29) | ||
| III/IV | 6% (5/88) | 27% (3/11) | 25% (3/12) | 0% (0/3) | 28% (8/29) | ||
| Alcohol septal ablation | 1% (1/88) | 9% (1/11) | 8% (1/12) | 0% (0/3) | 7% (2/29) | NS | NS |
| SCD | 0% (0/88) | 0% (0/11) | 8% (1/12) | 33% (1/3) | 10% (3/29) | 0.017 | NS |
| Heart failure | 2% (2/88) | 9% (1/11) | 17% (2/12) | 0% (0/3) | 14% (4/29) | NS | NS |
| Stroke caused by AF | 1% (1/88) | 9% (1/11) | 8% (1/12) | 0% (0/3) | 10% (3/29) | NS | NS |
| Combined adverse event | 34% (3/88) | 18% (2/11) | 33% (4/12) | 33% (1/3) | 35% (10/29) | 0.001 | NS |
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