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Abstract
Objectives This study aimed to describe the genetic and clinical characteristics of paediatric cardiomyopathy in a cohort of Chinese patients.
Methods We retrospectively reviewed the clinical history and mutation spectrum of 75 unrelated Chinese paediatric patients who were diagnosed with cardiomyopathy and referred to our hospital between January 2016 and December 2022.
Results Seventy-five children with cardiomyopathy were enrolled, including 32 (42.7%) boys and 43 (57.3%) girls. Dilated cardiomyopathy was the most prevalent cardiomyopathy (61.3%) in the patients, followed by hypertrophic cardiomyopathy (17.3%), ventricular non-compaction (14.7%), restrictive cardiomyopathy (5.3%) and arrhythmogenic right ventricular cardiomyopathy (1.3%). Whole-exome sequencing and targeted next-generation sequencing identified 34 pathogenic/likely pathogenic variants and 1 copy number variant in 14 genes related to cardiomyopathy in 30 children, accounting for 40% of all patients. TNNC1 p.Asp65Asn and MYH7 p.Glu500Lys have not been reported previously. The follow-up time ranged from 2 months to 6 years. Twenty-two children died (mortality rate 29%).
Conclusions Comprehensive genetic testing was associated with a 40% yield of causal genetic mutations in Chinese cardiomyopathy cases. We found diversity in the mutation profile in different patients, which suggests that the mutational background of cardiomyopathy in China is heterogeneous, and the findings may be helpful to those counselling patients and families.
- Cardiology
- Genetics
Data availability statement
Data are available in a public, open access repository. The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA006972) that are publicly accessible at https://bigd.big.ac.cn/gsa-human/browse/HRA006972.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
Existing study on spectrum of genetic causes and clinical manifestations of paediatric cardiomyopathy is limited to white population.
WHAT THIS STUDY ADDS
The diagnostic yield of genetic testing in restrictive cardiomyopathy and hypertrophic cardiomyopathy was higher than that in dilated cardiomyopathy and ventricular non-compaction.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
It provides a basis for genetic counselling in paediatric cardiomyopathy in China.
Introduction
Paediatric cardiomyopathy is a rare disease with an annual incidence of 1.1–1.5/100 000,1 which is the primary indication for heart transplantation during childhood, particularly among children >1 year of age.2 The Precise Diagnosis and Treatment Cooperation Group of the Cardiology Group of the Pediatric Branch of the Chinese Medical Association investigated 1823 hospitalised children with cardiomyopathy in 16 hospitals in China from 2006 to 2016.3 This study showed that paediatric cardiomyopathy accounted for 0.1% of hospitalised children during this period. Dilated cardiomyopathy (DCM) is the most common cardiomyopathy in children (approximately 50%), followed by hypertrophic cardiomyopathy (HCM, approximately 35%–50%), ventricular non-compaction (VNC, approximately 5%), restrictive cardiomyopathy (RCM, <5%) and arrhythmogenic right ventricular cardiomyopathy (ARVC), which is extremely rare.4 With the development of second-generation sequencing technology in recent years, diagnosing diseases at the genetic level and analysing the relationship of genetic variants with clinical phenotypes have become a research hotspot. In a study of 66 patients with serious paediatric cardiomyopathy, 39% were found to have pathogenic variants.2 In this study, we investigated cardiomyopathy-related genes in 75 unrelated paediatric patients with cardiomyopathy in our hospital. We also discussed the role of genetic variants in the diagnosis, prognosis and treatment of paediatric cardiomyopathy.
Materials and methods
Patients and ethical approval
Seventy-five paediatric patients with cardiomyopathy admitted to the Department of Cardiology of Henan Children’s Hospital from January 2016 to December 2022 were recruited. Clinical data of the 75 patients, including sex, age of onset, family history, echocardiography, treatment and follow-up were collected. The relationship between the genotype and clinical phenotype was examined, followed by an assessment of the prognosis based on genetic variants.
Inclusion criteria were patients who met the classification and diagnostic criteria for cardiomyopathy in children in the 2019 ‘Classification and Diagnosis of Childhood Cardiomyopathy: AHA Scientific Statement’.4 Exclusion criteria were as follows: ischaemic cardiomyopathy, hypertensive cardiomyopathy, valvular heart disease, pre-excitation-induced dilated cardiomyopathy, myocardial lesions caused by pulmonary hypertension and congenital heart disease, cardiac enlargement caused by infection, myocarditis, or Kawasaki disease, tachycardia, bradycardia and pacemaker-induced cardiomyopathy.
Whole-exome sequencing
Whole-exome sequencing was performed in 66 patients. Genomic DNA was fragmented to an average size of approximately 200 base pairs (bp) using the Biorupter UCD-200 (Diagenode, Belgium). The DNA fragments were end-repaired and an A base was added to the 3’end. Fragments with an average size of 320 bp were collected on XP beads and then captured by hybridisation to IDT’s xGen Exome Research Panel (Integrated DNA Technologies, San Diego, CA, USA) according to the manufacturer’s protocol. The hybridised products were then eluted and collected for PCR amplification and purification. Finally, genomic DNA was sequenced using the Novaseq6000 platform (Illumina, San Diego, CA, USA) to produce 150 bp paired-end reads. Raw image files were processed using CASAVA v1.82 for base calling and generating raw data. The sequencing reads were aligned to the human reference genome (hg19/GRCh37) using the Burrows-Wheeler Aligner tool.
Targeted next-generation sequencing
Targeted next-generation sequencing was performed in nine patients. Candidate genes reported to be causative of inherited cardiomyopathy, according to Online Mendelian Inheritance in Man (http://omim.org) and PubMed literature retrieval,5 were selected for panel design. The primers of overlapping amplicons covering the coding sequence region and flanking sequences (padding +25 base pairs) of each targeted gene were automatically generated using Ion AmpliSeq designer software.
Samples comprising 1–3 µg of genomic DNA were fragmented to an average size of 150 bp using the S220 Focused-ultrasonicator (Covaris, MA, USA). The library was constructed using the Ion AmpliSeq Inherited Cardiomyopathy Panel kit and the Ion AmpliSeqTM Library kit (Thermo Fisher Scientific, USA). All coding sequence regions and flanking sequences (padding +25 base pairs) of candidate genes were amplified. The PCR products were purified using SPRI beads (Beckman Coulter) according to the manufacturer’s protocol. The enrichment libraries were sequenced on the Novaseq6000 platform to generate paired-end reads of 150 bp. Raw image files were processed using CASAVA v1.82 for base calling and generating raw data. The sequencing reads were aligned to the human reference genome (hg19/GRCh37) using the Burrows-Wheeler Aligner tool.
Data analysis
Variant annotation databases mainly included human population databases (such as gnomAD, the 1000 Genome Project and dbSNP), disease and phenotype databases (such as OMIM, ClinVar, HGMD and HPO) and prediction algorithms (such as SIFT, polyphen2 and MutationTaster), which are used to analyse the pathogenic potential of variants. According to the American College of Medical Genetics and Genomics (ACMG) guidelines for interpretation of genetic variants, the variants were classified into five categories, including ‘pathogenic’, ‘likely pathogenic’, ‘uncertain significance’, ‘likely benign’ and ‘benign’. Considering ACMG category, evidence of pathogenicity, as well as clinical synopsis and inheritance model of the associated disease, variants with minor allele frequencies <1% in exonic region or with splicing impact were implemented for deep interpretation.
Patient and public involvement
This survey involved doctors collecting clinical and genetic data from patients, implementing treatment and family members cooperating with treatment and follow-up work. Laboratory personnel were responsible for interpreting the experimental data.
Results
Clinical phenotypic characteristics
Basic characteristics
A total of 75 cases with cardiomyopathy were studied, including 32 boys and 43 girls. The median age of onset was 16 months (1–156 months). Of all patients, three had been diagnosed with cardiomyopathy in other hospitals, and the rest were first diagnosed in our hospital. Most of the patients (71/75) had no familial history of cardiomyopathy. Only four families presented additional members affected by disease: the fathers of two patients had abnormal cardiac ultrasound results (cases 2 and 7), one patient’s mother had an abnormal cardiac ultrasound (case 22) and one patient’s brother died suddenly (case 19).
Types of cardiomyopathy
Among the 75 patients, 46 (61.3%) were diagnosed with DCM (44 primary cases and 2 secondary cases) and 13 (17.3%) were diagnosed with HCM (9 primary cases and 4 secondary cases), including a case of HCM combined with atrial septal defect. Eleven cases were diagnosed with VNC (14.7%), including 10 cases with left VNC and 1 case with right VNC. Furthermore, 6 cases of VNC were combined with DCM, and 2 were combined with congenital heart disease (patent ductus arteriosus and atrial septal defect). In addition, there were 4 cases diagnosed with RCM including 1 with phenylketonuria, and 1 case diagnosed with ARVC (figure 1A).
Number of cardiomyopathy. (A) Types of cardiomyopathy. (B) Mutation-positive cases of cardiomyopathy.
Electrocardiographic findings
Of the 75 index patients, the highest frequency of electrocardiographic findings were T wave changes or ST-T changes (60/75), followed by sinus tachycardia (27/75), ventricular premature beats (6/75), left axis deviation (5/75), PR interval prolongation (4/75), intraventricular block (3/75), intermittent pre-excitation (2/75), corrected QT interval prolongation (2/75), abnormal Q waves (2/75), atrial premature beats and short array atrial tachycardia (1/75), polymorphic ventricular premature beats (1/75) and left anterior branch block (1/75). Electrocardiographic findings in each kind of cardiomyopathy were presented in table 1.
Electrocardiographic findings in each kind of cardiomyopathy
Characteristics of pathogenic genetic variants
The identified genetic variants were classified using ACMG mutation standards and guidelines. Among the 75 patients with cardiomyopathy, 34 ‘pathogenic’ or ‘likely pathogenic’ variants (online supplemental table 1) and 1 copy number variant were identified in 30 cases (40%) in 14 genes. Four of these cases have been previously published6–8 .The majority of variants were missense (26/34), followed by frameshift (4/34). According to the available description of these genes in the literature, 25 (83.3%) patients had variants with autosomal dominant inheritance, and 5 (16.7%) had variants with autosomal recessive inheritance.
Supplemental material
Of the 46 patients with DCM, 10 variants of TNNT2, TNNI3, CDH2, TPM1, DES, GLB1 and FHL2 were detected in 10 patients (21.7%) (figure 1B), including 9 patients with primary DCM and 1 patient with secondary DCM. TNNT2 was the most frequently mutation gene (4/9) in patients with primary DCM. One case of secondary DCM was caused by GM1 ganglioside storage disease due to a heterozygous variant in GLB1 and a copy number variant containing exon 3 to exon 8 in GLB1 (seq[GRCh37]dup(3)(3p22.3)chr3:g.33093249_33114205dup), which was consistent with the clinical phenotype of low muscle tone, poor growth and stunted development.
Of the 13 patients with HCM, 11 variants in MYH7, GAA, MYL2, TPM1 and ACTC1 were identified in 11 patients with HCM (84.6%) (figure 1B), including 7 patients with primary HCM and 4 patients with secondary HCM. MYH7 was the most frequently mutation gene (4/7) in patients with primary HCM. One variant in MYH7 (p.Glu500Lys) had not been previously reported, and was classified as possibly pathogenic. Furthermore, compound heterozygous variants in GAA were detected in all of the 4 patients with secondary HCM.
Four variants in RBM20, TNNC1 and TNNT2 were detected in 4 of the 11 patients with VNC (36.4%) (figure 1B). Among them, two variants in RBM20 have been reported in our previous studies, which caused left VNC combined with DCM.9
Variants in TNNI3 were detected in all four patients with RCM (100%)(figure 1B), including a splice site variant and three missense variants. Three de novo variants were identified in three patients without family history. A variant c.433C>T (p.Arg145Trp) in case 2 was inherited from the patient’s father. Ultrasound results showed that slight thickening of the father’s left ventricular wall, indicating a high penetrance of variants in TNNI3.
Furthermore, we found a frame shift variant c.2554del (p.Glu852fs) in PKP2 in a patient with ARVC (figure 1B), which was classified as pathogenic variant according to ACMG guidelines without parents’ genetic information.
Treatment
Management of the DCM cases was based on inotropic agents (mostly digitalis), diuretics, β-receptor blockers and angiotensin inhibitors. Infants with an unknown first history were administered low-dose immunoglobulin therapy. None of the four patients with secondary HCM (glycogen accumulation disease type II) were treated with glucosidase alfa owing to the family’s economic situation. Another small infant with HCM was untreated, and the remaining eight patients with HCM were all administered β-blockers. The treatment of VNC with heart failure is similar to DCM. The treatment of RCM targeted diastolic heart failure, and this included low doses of diuretics, vasodilators and angiotensin-converting enzyme inhibitors. One patient with DCM and multiple premature ventricular contractions was administered oral mexiletine and metoprolol, and one patient with ARVC and polymorphic premature beats was administered metoprolol.
Follow-up
The patients with cardiomyopathy were followed up for different durations, ranging from 2 months to 6 years, and the overall mortality rate was 29.3% (22/75). Among the 46 patients with DCM, 4 patients were lost to follow-up. Three patients underwent heart transplantation, including a patient with causative variant (case 13). 13 patients died (2 with secondary DCM and 1 after transplantation), including 3 patients with causative variants (case 7, case 8 and case 9). In this study, 41% of the patients with DCM had a ≥50% left ventricular ejection fraction after treatment. Of the 4 patients with variants in TNNT2, 3 had a fluctuating left ventricular ejection fraction, ranging from 18% to 36% at the initial diagnosis. They were followed up for periods ranging from 7 months to 6 years, and all of them currently have a normal heart size with an ejection fraction ≥52%. One patient with DCM had premature ventricular contractions that were reduced after oral administration of drugs.
None of the four patients with HCM and GAA variants survived. Among the four patients with VNC who were positive for genetic variants, two patients with RBM20 variants died and one patient with TNNT2 variant was lost to follow-up. The other patient with TNNC1 variant was followed up to 3 years old, and the degree of cardiac function and non-compaction was similar to that at the initial diagnosis. One patient with RCM died, one patient was lost to follow-up, and the status quo was maintained in the remaining two patients following treatment with oral drugs.
One patient with ARVC and polymorphic premature beats did not improve much after oral medication.
Discussion
With the development of genetic screening technology, an increasing number of children with cardiomyopathy are eligible for the detection of pathogenic gene variants. The present study showed causative variants in 40% of children with cardiomyopathy (25% were pathogenic and 15% were likely pathogenic), and 56% were de novo. In this study, autosomal dominant inheritance was most common (83.3%). The overall prognosis of primary DCM in children is poor, and this prognosis is mainly related to the age at diagnosis and the degree of heart failure. According to previous studies, the predictors of recovery of cardiac function included younger age, female sex and a smaller left ventricular end-diastolic Z score on an echocardiogram.10 In this study, 41% of the patients with DCM had a normalisation of function after treatment and 32.6% of the patients died or undergo transplantation, which is consistent with the literature.1
Approximately 40%–60% of HCM patients with HCM have multiple independent pathogenic variants in sarcomere protein genes.11 Among patients with positive genetic tests, most pathogenic variants occurred in MYH7 and MYBPC3. In addition, 5%–10% of HCM cases are caused by non-sarcomere gene variants associated with neuromuscular diseases, metabolic disorders or genetic syndromes.12 The total positive rate of genetic variants detected in children with HCM was 60%–80%.13 14 In this study, variants in MYH7, GAA, MYL2, TPM1 and ACTC1 were detected, with a positive rate of approximately 85%. Variants of MYH7 were the most frequently mutation genes in paediatric patients with primary HCM, which is consistent with the literature.13 However, we have not found pathogenic/likely pathogenic variants in MYBPC3 in our cohort. The four patients with variants in GAA causing glycogen accumulation disease type II in this study all died before the age of 1.5 years, while the remaining children with HCM are still alive. Patients with variants in GAA demonstrate a more serious influence of sudden cardiac death than those without.
The prevalence of VNC in the general population ranges from 0.05% to 0.24% but can be as high as 22% to 30% in families.15 None of the 11 patients with VNC in this study had a family history and four patients (36%) were positive for disease-causing variants. Two patients with NVC and DCM carried variants in RBM20. One patient had a de novo variant in TNNC1 (p.Asp65Asn), which was classified as likely pathogenic according to the ACMG guidelines, and has not been reported previously. The other patient carried a variant in TNNT2 (p.Arg131Trp), which was inherited from the patient’s mother. This variant has been previously reported as pathogenic variant for DCM and may have contributed to the new clinical phenotype in this patient. The echocardiogram of the affected child’s mother was normal, indicating that this variant is incomplete penetrance. There have been studies of RBM20 mutation carriers with a mean age at transplantation of 28.5 years at transplantation. The two patients in this study died at approximately 13 years of age, which is lower than the age at transplantation reported before.1
RCM accounts for only 3%–5% of paediatric cardiomyopathy and is clinically characterised by diastolic dysfunction with restricted ventricular filling and atrial enlargement. The pathogenesis of RCM remains unclear, and 30% of cases have a family history of this disease. In recent years, variants in the cardiac sarcomere protein genes (MYH17, TNNT2 and TNNI3) and in EDS have been found in children with sporadic RCM. All four patients with RCM in this study had TNNI3 variants. RCM has the worst prognosis among the types of cardiomyopathy, with a mean survival time of only 2 years after its diagnosis.
ARVC mostly shows autosomal dominant inheritance, and it has been reported that 30% to 70% of children with ARVC harbour variants of desmosomal protein genes.16 ARVC is characterised by the progressive replacement of the right ventricular myocardium by fibrotic fatty tissue, and it often manifests clinically as right ventricular enlargement, arrhythmias and sudden death. The diagnosis of ARVC in children is difficult because the myocardial changes during childhood are not specific.17 Therefore, genetic information and the clinical phenotype in children are necessary to make an accurate diagnosis of ARVC.
The clinical phenotypes of inherited cardiomyopathy in children, as well as the causative genes, can overlap with each other.18–20 In this study, TNNI3 variants presented with DCM or RCM. Similarly, TNNI2 variants can manifest as DCM or VNC, while TPM1 variants can manifest as DCM or HCM.
Additionally, this was a single-centre study. Furthermore, not all patients with cardiomyopathy in our hospital were genetically examined. Despite these limitations, the results of this study may be helpful to those counselling patients and families.
In conclusion, cardiomyopathy in children is predominantly sporadic, has a complex aetiology and is highly heterogeneous with diverse clinical phenotypes, which pose challenges for the precise diagnosis. Nevertheless, genetic testing should be performed in children with cardiomyopathy of unknown aetiology and suboptimal treatment outcomes, especially secondary cardiomyopathy, to improve the chance of early diagnosis and successful treatment.
Data availability statement
Data are available in a public, open access repository. The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA006972) that are publicly accessible at https://bigd.big.ac.cn/gsa-human/browse/HRA006972.
Ethics statements
Patient consent for publication
Ethics approval
This study involves human participants and was approved by the Medical Ethics Review Board of Zhengzhou Children’s Hospital (Approval No.: 2023 K-041). Participants gave informed consent to participate in the study before taking part.
Acknowledgments
We thank the patients, their families and their clinicians for the participation in this study.
References
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Footnotes
QS and JG contributed equally.
ZX and FW contributed equally.
Contributors QS, FW and ZX contributed to initiation and conception of the protocol. QS, FW, ZX, JG, YZ, RZ, KH, YC and CH were responsible for the experimental design, data collection and manuscript writing. QS, FW, ZX, JG, YZ, RZ, KH, YC and CH contributed to the final approval of the manuscript.
Funding This research was supported by Beijing Municipal Natural Science Foundation (7232053).
Competing interests None declared.
Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.