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Division of Cardiovascular Medicine, Kanazawa University Graduate School of Medical Science, Kanazawa, JapanCenter for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USAProgram in Medical and Population Genetics, Broad Institute, Cambridge, MA, USADepartment of Medicine, Harvard Medical School, Boston, MA, USA
Division of Cardiovascular Medicine, Kanazawa University Graduate School of Medical Science, Kanazawa, JapanResearch and Education Center for Innovative and Preventive Medicine, Kanazawa University, Kanazawa, Japan
Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USAProgram in Medical and Population Genetics, Broad Institute, Cambridge, MA, USADepartment of Medicine, Harvard Medical School, Boston, MA, USACardiovascular Research Center, Massachusetts General Hospital, Boston, MA, USA
Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USAProgram in Medical and Population Genetics, Broad Institute, Cambridge, MA, USADepartment of Medicine, Harvard Medical School, Boston, MA, USACardiovascular Research Center, Massachusetts General Hospital, Boston, MA, USA
The development of candidate gene approaches to enable molecular diagnosis of hypertrophic cardiomyopathy (HCM) has required extensive and prolonged efforts. Whole exome sequencing (WES) technologies have already accelerated genetic studies of Mendelian disorders, yielding approximately 30% diagnostic success. As a result, there is great interest in extending the use of WES to any of Mendelian diseases. This study investigated the potential of WES for molecular diagnosis of HCM.
Methods
WES was performed on seven relatives from a large HCM family with a clear HCM phenotype (five clinically affected and two unaffected) in the Kanazawa University Hypertrophic Cardiomyopathy Registry. Serial bioinformatics filtering methods as well as using combined annotation dependent depletion (CADD) score and high heart expression (HHE) gene data were applied to detect the causative variant. Moreover, additional carriers of the variant were investigated in the HCM registry, and clinical characteristics harboring the variant were collected and evaluated.
Results
WES detected 60020 rare variants in the large HCM family. Of those, 3439 were missense, nonsense, splice-site, or frameshift variants. After genotype–phenotype matching, 13 putative variants remained. Using CADD score and HHE gene data, the number of candidates was reduced to one, a variant in the myosin essential light chain (MYL3, NM_000258.2:c.281G>A, p.Arg94His) that was shared by the five affected subjects. Additional screening of the HCM registry (n = 600) identified two more subjects with this variant. Serial assessments of the variant carriers revealed the following phenotypic characteristics: (1) disease-penetrance of 88%; (2) all clinically affected carriers exhibited asymmetric septal hypertrophy with a substantial maximum left ventricular wall thickness of 18 ± 3 mm without any obstruction.
Conclusions
WES combined with CADD score and HHE gene data may be useful even in HCM. Furthermore, the MYL3 Arg94His variant was associated with high disease penetrance and substantial interventricular septal hypertrophy.
Hypertrophic cardiomyopathy (HCM) is a genetically heterogeneous myocardial disorder with various morphological, functional, and clinical characteristics [
American College of Cardiology Foundation/American Heart Association Task Force on Practice G 2011 ACCF/AHA Guideline for the Diagnosis and Treatment of Hypertrophic Cardiomyopathy: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Developed in collaboration with the American Association for Thoracic Surgery, American Society of Echocardiography, American Society of Nuclear Cardiology, Heart Failure Society of America, Heart Rhythm Society, Society for Cardiovascular Angiography and Interventions, and Society of Thoracic Surgeons.
Prevalence of hypertrophic cardiomyopathy in a general population of young adults. Echocardiographic analysis of 4111 subjects in the CARDIA Study. Coronary Artery Risk Development in (Young) Adults.
]. Myosin heavy chain (MYH7, OMIM#: 160760), myosin binding protein C (MYBPC3, OMIM#: 600958), troponin T (TNNT2, OMIM#: 191045), tropomyosin (TPM1, OMIM#: 191010), and troponin I (TNNI3, OMIM#: 191044) have been reported to be the main causal genes [
A systematic review and meta-analysis of genotype–phenotype associations in patients with hypertrophic cardiomyopathy caused by sarcomeric protein mutations.
]. To date, candidate-gene approaches using traditional Sanger sequencing have enabled the identification of causative variants in approximately 50% of HCM patients [
]. However, because these techniques are onerous and time-consuming, motivating the researchers to perform target re-sequencing with next generation sequencing (NGS) as well as whole exome sequencing (WES) which is reported to have a success rate of nearly 30% in identifying disease-causing variants in patients whose causal variants have not been detected [
], it remains unclear whether WES enables comprehensive identification of causative variants in an exome-wide manner. This study investigated the effectiveness of WES with bioinformatics for molecular diagnosis for HCM.
Materials and methods
Study population
All subjects were enrolled through the Kanazawa University Hypertrophic Cardiomyopathy Registry in Kanazawa, Japan. The largest HCM family in the registry, whose causative variant had not been detected by traditional direct sequencing, was first investigated in this study. HCM was diagnosed according to the 2011 version of the guideline of the American College of Cardiology Foundation/American Heart Association [
American College of Cardiology Foundation/American Heart Association Task Force on Practice G 2011 ACCF/AHA Guideline for the Diagnosis and Treatment of Hypertrophic Cardiomyopathy: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Developed in collaboration with the American Association for Thoracic Surgery, American Society of Echocardiography, American Society of Nuclear Cardiology, Heart Failure Society of America, Heart Rhythm Society, Society for Cardiovascular Angiography and Interventions, and Society of Thoracic Surgeons.
]. In brief, subjects with a maximal left ventricular wall ≥13 mm (involving asymmetric septal hypertrophy) without extra-cardiac or metabolic findings based on echocardiography were diagnosed with HCM. Individuals were classified as HCM-affected in the case diagnosed with HCM, or HCM-unaffected in the case of either a non-HCM phenotype or known diseases such as hypertensive heart disease. Detailed clinical data, including family history, age, and symptoms at evaluation, presence of hypertension, physical examination, New York Heart Association (NYHA) classification, electrocardiogram (ECG) and echocardiographic findings were collected for each subject. Hypertrophic obstructive cardiomyopathy (HOCM) was defined as HCM with left ventricular outflow tract (LVOT) obstruction and a peak instantaneous LVOT pressure gradient of >30 mmHg. Maximum left ventricular wall thickness (MWT) was defined as the greatest thickness within the chamber. Other echocardiographic parameters were evaluated using the recommendation of the American Society of Echocardiography [
Recommendations for chamber quantification: a report from the American Society of Echocardiography's Guidelines and Standards Committee and the Chamber Quantification Writing Group, developed in conjunction with the European Association of Echocardiography, a branch of the European Society of Cardiology.
]. Hypertension was defined as when a patient's systolic blood pressure (BP) was ≥140 mmHg and/or its diastolic BP was ≥90 mmHg. Pathological Q waves were defined as follows based on previous studies: Q wave >1/4 of the ensuing R wave in depth and/or >40 ms in duration in at least two leads except aVR [
]. The Ethics Committee for Medical Research at our institution approved the study protocol, and all subjects provided written informed consent.
Exome sequencing
Genomic DNA was isolated from peripheral white blood cells of all subjects using a standard DNA extraction protocol. DNA was pooled, selected size, ligated to sequencing adapters, and amplified to enrich for targets to be sequenced by the Agilent SureSelectXT Target Enrichment System (Agilent Technologies Inc., Santa Clara, CA, USA). Exome capture was performed with the Agilent SureSelectXT Human All Exon 50 Mb Kit (Agilent Technologies Inc.). Exome enriched products were sequenced using the Illumina HiSeq 2000 by Takara Bio Inc., Shiga, Japan. One sample was sequenced per lane to obtain an average theoretical depth of 80x, using 2x100 bp sequencing.
Bioinformatics
For the samples, paired-end reads were aligned using the Burrows-Wheeler Aligner on the human reference genome build hg19 using quality score calibration, soft clipping, and adapter trimming. Following the exclusion of PCR duplicate reads using the Picard, insertions/deletions and single-nucleotide polymorphisms (SNP) were called using the Genome Analysis Toolkit (GATK) [
]. Variants (SNP/indels) were filtered on the basis of the Phred scaled genotype quality score. Re-alignment was performed and the calling algorithm merged the output of GATK Unified Genotyper. All samples were annotated using SnpEff [
A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3.
In addition to the standard variant quality controls, six independent filters were applied to facilitate detection of causal variants among the enrolled HCM families. Variants were filtered by: (1) minor allele frequency (MAF) >1% in Asian population; (2) benign, as predicted by SnpEff; (3) genotype–phenotype unmatched under the assumption of complete penetrance without phenocopies; (4) registered in the SNP Database (dbSNP137); (5) combined annotation dependent depletion (CADD) score <10; and (6) low heart expression of the genes, less than the top quartile.
The frequency filter adopted the allele frequency estimates from the Asian cohort of the 1000 Genomes Project database [
], and we used a MAF 1% as the cut-off. Genotype–phenotype matching was defined as narrowing the variants at which affected subjects had ≥1 alternative allele(s) and unaffected subjects had no alternative allele. As a functional filtering method, variants not registered in NCBI dbSNP137 were considered to be candidate variants. Prediction of in silico pathogenicity for novel missense variants was performed using the CADD prediction software (version 1.0), which objectively integrates many diverse annotations into a single measure (C-score) for each variant [
]. A variant was predicted to be pathogenic if the scaled C-score calculated by the software was above 10, a score indicative of the variant being within the top 10% of deleteriousness substitutions. Candidate variants were evaluated if the gene associated with each variant was directly involved in the myocardium or worsening cardiac function by using the high heart expression (HHE) gene data as previously reported [
]. In brief, HHE genes were defined as the top quartile [>40 reads per million mapped reads (rpm)] of 16,599 human-mouse orthologous gene expressions generated by RNA sequencing data of mice hearts at embryonic day 14.5.
After the evaluation, Sanger sequencing method was performed to confirm the putative variant identified by the bioinformatics analysis in the tested subjects and other relatives.
Additional screening for HCM registry
In addition, restriction fragment length polymorphism (RFLP) was performed on HCM probands listed in our registry to identify other subjects harboring the variant. In brief, restriction enzyme (Van91I) was added to each DNA sample and incubated for 3 h, which allowed the enzyme to cut at the recognition site (in this case, the myosin essential light chain [(MYL3, OMIM#: 160790), NM_000258.2:c.281G>A variant site]). The DNA fragments were separated by size using gel electrophoresis to allow confirmation of cutting of the sample DNAs. To investigate the ancestral origin of the variant, haplotype analysis was performed by using six microsatellite markers (short tandem repeats [(CA)n: D3S3687, D3S3678, D3S3647, D3S3582, D3S3640, D3S1568] flanking the variant gene) [
]. Clinical characteristics of variant carriers were documented for phenotype evaluation of the variant.
Results
Characteristics of HCM-F18 family members
Clinical characteristics of HCM-F18 family members are shown in Table 1, and a representative case (III:6, proband) is presented in Fig. 1. The subject was referred to our hospital because of an abnormal ECG finding detected by a local hospital. ECG showed atrial fibrillation with left ventricular hypertrophy pattern, chest X-rays displayed an increase in cardiothoracic ratio, and echocardiography revealed an apparent interventricular septal hypertrophy without LVOT obstruction. After excluding other cardiac diseases, the subject was diagnosed with HCM.
Table 1Clinical features of carriers harboring the MYL3 (Arg94His) variant in HCM-F18 and HCM-F189 families.
Fig. 1A representative case (proband) in hypertrophic cardiomyopathy (HCM)-F18 family. Clinical images of the proband (III:6 in Table 1) in HCM-F18 family are shown: (A) chest X-ray; (B) electrocardiogram; (C) echocardiography of longitudinal views. Chest X-ray showed that the cardiothoracic ratio was increased (61%); electrocardiogram showed atrial fibrillation with left ventricular hypertrophy pattern; and echocardiography presented normal ejection fraction and an apparent interventricular septal hypertrophy without left ventricular outflow tract obstruction (arrow head). Ao, aorta; LA, left atrium; LV, left ventricle; RV, right ventricle.
Although the HCM-F18 proband and her family members had been clinically observed for years, traditional Sanger sequencing failed to identify the apparent causative variants. The HCM-affected family members had a typical clinical phenotype consistent with HCM, exhibiting interventricular septal hypertrophy and asymmetric septal hypertrophy. The affected subjects comprised two males and three females with an age range at enrollment of between 24 and 74 years. The HCM-unaffected subjects were a 67-year-old female diagnosed with hypertensive heart disease and a 48-year-old female without any cardiac disease.
Exome sequencing and bioinformatics analyses
DNA samples from seven members of the HCM-F18 family, five clinically HCM-affected subjects, and two unaffected subjects were analyzed using WES (Fig. 2) followed by bioinformatics filtering methods (Table 2). The mean sequencing depth for the seven subjects was 56.1x, 60.0x, 54.8x, 58.7x, 58.2x, 63.7x, and 66.0x per base across the whole exome, for samples III:2 (unaffected), III:3 (affected), III:6 (affected), III:9 (affected), III:12 (affected), IV:7 (unaffected), and IV:11 (affected), respectively. Percentages of on-target reads were 78.6% (III:2), 78.0% (III:3), 77.3% (III:6), 79.4% (III:9), 77.2% (III:12), 78.7% (IV:7), and 77.0% (IV:11), respectively, while coverage rates of target coding lesions (20x) were 89.3% (III:2), 90.1% (III:3), 89.0% (III:6), 90.8% (III:9), 89.7% (III:12), 91.7% (IV:7), and 92.0% (IV:11), respectively.
Fig. 2Pedigrees of families: (A) hypertrophic cardiomyopathy (HCM)-F18 and (B) HCM-F189. The pedigrees of HCM families with the myosin essential light chain (MYL3) variant (Arg94His) are shown. Arrows indicate probands. Squares represent male subjects; circles represent female subjects. Diagonal lines mark deceased individuals. Solid symbols denote HCM. Open symbols represent unaffected individuals or individuals with no data available for analysis. Gray symbols represent obligate carriers. Plus “(+)” and minus “(−)” signs indicate presence or absence of the Arg94His variant confirmed by direct sequencing, respectively. Encircled numbers indicate subjects investigated by whole exome sequencing. Haplotypes (the number of [CA]n repeat) are listed for six microsatellite markers ([1] D3S3687 – [2] D3S3678 – [3] D3S3647 – [4] D3S3582 – [MYL3] – [5] D3S3640 – [6] D3S1568) from top to bottom. Boxed haplotypes demonstrate affected haplotypes. Affected haplotypes share same numbers of [CA]n repeat from [3] D3S3647 to [6] D3S1568 in both families, indicating that the MYL3 Arg94His variant originated from the same ancestor.
Subsequent to WES, bioinformatics analysis and genotype–phenotype matching following exome sequencing were performed for family HCM-F18 to identify causative variants found in all five HCM-affected subjects but not in the two unaffected subjects (Table 2). The number of aligned variants in the seven subjects with passing quality filtering was 243359. Among those, 60200 were considered to be rare variants (MAF <1%) using the Asian cohort in 1000 Genome Project [
]. Focusing on missense, nonsense, splice-site, and frameshift variants, 3439 variants were detected. Genotype–phenotype matching enabled putative variants to narrow the number to 13. After excluding variants registered in dbSNP137, six putative variants remained (Table 3). To evaluate these variants, CADD prediction software was used to calculate each scaled C-score. Five out of six variants were predicted to be damaging (scaled C-score >10), indicating that these were potentially deleterious variants. Among the candidate variants, myosin essential light chain (MYL3, c.281G>A, p.Arg94His) was the only gene that belonged to the HHE genes and was directly associated with the myocardium (ventricular muscle). Although this MYL3 Arg94His variant was not listed in the NHLBI ESP exome variant server [
]. Sanger sequencing for the 10 HCM-F18 family members validated the variant in all the affected subjects and resulted in the detection of one additional variant carrier without an apparent HCM phenotype (Fig. 2, Fig. 3).
Table 3Candidate variants after filtering methods in HCM-F18.
Gene
Function
Chr.
Exon
Position (build 37)
NM #
Amino acid
SIFT
PolyPhen-2
MutationTaster2
CADD score
HHE gene
HMGB4
High Mobility Group Box 4
1
2
34329932
145205
E47A
0.01
0.999
DC
24.5
−
HHATL
Hedgehog Acyltransferase-Like
3
9
42738359
20707
R341C
0
1
DC
18.7
−
MYL3
Myosin Light Chain 3, Ventricular, Skeletal
3
3
46902192
258
R94H
0.04
0.008
DC
16.63
+
NIPAL4
NIPA-Like Domain Containing 4
5
1
156887258
1172292
R39Q
0.25
0.002
Poly
19.5
−
PLAU
Plasminogen Activator, Urinary
10
10
75676220
1145031
R381L
0.11
0.009
Poly
9.587
−
LIPM
Lipase, Family member M
10
4
90574372
1128215
G184S
0.01
0.981
DC
35
−
The six variants in six genes filtered by bioinformatics methods are present in five HCM-affected subjects and are absent in two unaffected subjects. Of those, 5 variants had the CADD score more than 10, and MYL3 gene was the only gene that highly expressed in mice hearts.
SIFT scores (ranges from 0 to 1) were calculated by SIFT version 5.2.2, and the SIFT score less than 0.05 was considered as deleterious. PolyPhen-2 scores (ranges from 0 to 1) were calculated by PolyPhen-2 version 2.2.2, and the PolyPhen-2 score more than 0.85 was considered as probably damaging. The six variants were also evaluated by MutationTaster2 (classifying variants as “Disease causing” or “Polymorphism”).
Fig. 3NGS read alignment and the Sanger sequencing validation in the MYL3 gene at exon 3. Alignment reads are visualized by the Integrative Genomics Viewer (IGV) version 2.3. The red arrow in IGV indicates the p.Arg94His variant position in the MYL3 gene at exon 3 (5′ → 3′ complementary strand). The red arrow in Sanger sequencing data of affected subjects also shows the same variant at the same position.
Phenotype evaluation of MYL3 (c.281G>A, p.Arg94His) variant
All 600 familial or sporadic HCM probands listed in the registry were screened for the MYL3 (c.281G>A, p.Arg94His) variant by RFLP analysis using the Van91I restriction enzyme. The Arg94His variant was identified in one additional proband (HCM-F189) and Sanger sequencing confirmed that two additional subjects had the MYL3 Arg94His variant (Fig. 2). Moreover, haplotype analysis of family HCM-F18 and family HCM-F189 using six microsatellites in the neighborhood revealed that the MYL3 Arg94His variant originated from the same ancestor (Fig. 2).
The clinical characteristics of the eight MYL3 Arg94His variant carriers are summarized in Table 1. Disease-penetrance was 88%, and all clinically affected carriers exhibited asymmetrical septal hypertrophy with a substantial maximal left ventricular wall thickness of 18 ± 3 mm. Left ventricular systolic dysfunction (ejection fraction <50%) was not observed. Abnormal ECG findings were observed in seven carriers with an apparent HCM phenotype. Three subjects had left ventricular hypertrophy, two had first-degree atrioventricular block, two had bundle branch block, and one had atrial fibrillation with an enlarged left atrium. GNTW was observed in one subject, but pathological Q wave was not observed.
Clinical follow-up data were available for four of eight subjects. Although they suffered from neither cardiac death nor progression of left ventricular systolic dysfunction during >10 years follow-up, one subject (HCM-F18; III:12) received an implantable cardioverter defibrillator (ICD) because of syncope with frequent non-sustained ventricular tachycardia. Another subject (HCM-F18; III:6) died from non-cardiac causes (postoperative infection after an orthopedic surgery).
Discussion
In this study, WES was demonstrated to be an effective tool, even in HCM. Furthermore, we determined the MYL3 Arg94His variant was newly identified as a variant associated with high disease penetrance and substantial degree of interventricular septal hypertrophy for the first time. This is the first study to validate the MYL3 Arg94His as a causative variant of familial HCM using WES.
Since the application of NGS methods such as WES and target re-sequencing for clinical genetics, the number of bioinformatics approaches for detecting the causative variant in patients in cardiomyopathy has gradually increased [
]. This is because the traditional direct sequencing methods have only been able to identify approximately 50% of the causative variants in patients with cardiomyopathy [
], providing support for the notion that WES may be useful in diverse ethnic settings.
In this study, two novel filtering schemas were adopted to enable causative variants to be distinguished from thousands of other incidental variants detected via WES. The CADD C-score [
]. This means that we can obtain the comprehensive damaging scores of each candidate variant even though each annotation tool shows discordant results. For example, the MYL3 Arg94His variant was first predicted as deleterious by SIFT and disease-causing by MutationTaster2 [
], but was considered as benign by PolyPhen-2 (Table 3). However, the CADD C-score of the variant was 16.63, clearly predicting that the MYL3 Arg94His as deleterious. Furthermore, prediction of the pathogenicity of human variants with the CADD C-score is more accurate than that of any other in silico single annotation tool [
]. Actually, the usefulness of CADD C-score for detecting causative variants in patients with Tangier disease and long QT syndrome were previously reported [
], Zaidi et al. demonstrated that HHE genes had a higher frequency of protein-alternating de novo variants than genes with low heart expression (less than top quartile) in patients with congenital heart diseases [
]. In the present study, the combination of the CADD scaled C-score and the heart expression-oriented filtering schema successfully narrowed down the number of causative variants to a single variant, the MYL3 Arg94His variant, suggesting the usefulness of this schema even in heterogeneous diseases such as HCM.
Completion of the final filtering method (Table 2) indicated that MYL3 was the only gene that was directly associated with ventricular myocardium. MYL3 encodes the essential light chain, an important component of sarcomere, which wraps around the lever arm of the myosin head, and supports both the neck domain and the lever arm with myosin regulatory light chain [
]. These patients exhibited mid-cavity hypertrophy with restrictive physiology, but in none of our HCM-subjects was the feature found. Also, Andersen et al. and Kazmierczak et al. reported the MYL3 Val79Ile and Ala57Gly variants were causative in HCM [
]. In this study, all subjects with HCM harbored the MYL3 Arg94His variant. Although the Arg94His was suggested as a potentially causative variant of sporadic HCM by DNA re-sequencing array [
Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
], this is the first study to confirm and support it as a causative variant of HCM by using the co-segregation pattern in the HCM pedigrees and the unbiased method of WES. In addition, a detailed description of the clinical phenotype associated with the MYL3 Arg94His is provided. The myosin light chain belongs to the EF-hand family of calcium-binding proteins [
] also predicted this to be a damaging variant site. The MYL3 Arg94His variant is expected to cause crucial damage to EF-hand function, myosin kinetics, and its structure, and eventually hypertrophic changes in the myocardium, which may underlie the high disease penetrance and substantial interventricular septal hypertrophy observed in this study (Table 1). However, due to the small number of subjects harboring this variant, it remains unclear whether screening of families with HCM for the MYL3 Arg94His variant will be useful in patient management. Further study is needed to clarify the long-term clinical courses in HCM patients with the MYL3 Arg94His variant.
Study limitations
This study has some limitations. WES was performed with protein-coding regions only, which potentially overlooked other intergenic variants that influenced the specific phenotype. HCM is a heterogeneous disorder in terms of both clinical characteristics and age of onset, limiting the accuracy of the phenotype modeling. In this study, we could accurately find out one causative variant for HCM, but it might be controversial whether a 48-year-old woman without apparent HCM phenotype could be classified as an unaffected HCM subject. Although CADD enabled more precise prediction of in silico damaging score than other software, the optimal cut-off value for detection of deleterious variants with CADD scaled C-scores is debatable. HHE genes were generated only by known human-mouse orthologues, which might rule-out potential human-specific genes that were highly expressed in human heart. In addition, clinical information was not available for some family members, which may have affected the penetrance and clinical characteristics of HCM with MYL3 Arg94His variant.
Conclusion
Together with CADD score and HHE gene data, WES facilitated successful identification of the one causative variant, the MYL3 Arg94His, even in HCM. Furthermore, the MYL3 Arg94His variant was associated with high disease penetrance and substantial ventricular hypertrophy.
Funding
This study was supported by JSPS KAKENHI Grant Numbers 19590807, 22590808, 25460648, and 26460742. Noboru Fujino was funded by a grant for innovative research from Kanazawa University Hospital. Akihiro Nomura was also funded by a research grant from Yoshida Scholarship Foundation.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
The authors express their gratitude to Drs Christine E. Seidman, Jonathan G. Seidman, and Jason Homsy for providing heart expression RNA-sequencing data from mice. Drs Hidenobu Terai, Tomoya Kaneda, Yoshihiro Tanaka, Yoji Nagata, Toyonobu Tsuda, and Mika Mori for assistance in collecting follow-up information including clinical and echocardiographic data, and Takako Obayashi for technical assistance in collecting genetic information and laboratory experiments.
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Hypertrophic cardiomyopathy (HCM) is caused by an autosomal dominant mutation in genes that encode sarcomere proteins. The accumulated evidence indicates that mutations in 8 sarcomere protein genes definitively cause HCM: cardiac β-myosin heavy chain (MYH7), cardiac myosin binding protein-C (MYBPC3), ventricular regulatory myosin light chain (MYL2), ventricular essential myosin light chain (MYL3), cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), α-tropomyosin (TPM1), and cardiac actin (ACTC).
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