#These authors contributed equally to this work
Cardiomyopathies are structural and functional heart muscle disorders that affect ventricular systolic function, diastolic function, or both. The incidence of cardiomyopathy is approximately one per 100,000 in children less than 18 years old [
Generally, the most common cause of childhood cardiomyopathy is idiopathic [
As more than 60 genes are associated with cardiomyopathy, multi-gene testing using next-generation sequencing techniques, such as panel-gene testing, is an appropriate diagnostic method. However, panel-gene testing cannot identify genetic causes outside the list of genes in the designed panel. Moreover, the time, cost, and effectiveness of a genomic study must be considered; whole exome sequencing (WES) is considered one of the fastest diagnostic tools in cardiomyopathy [
Therefore, the aim of the present study was to identify variants by performing WES and additional mitochondrial DNA (mtDNA) sequencing in patients with infantile-onset cardiomyopathy. By assessing the pathogenicity of the variant and its existence in the parents, we investigated the association of the identified variant with the disease in detail.
WES was conducted in patients with cardiomyopathy diagnosed at Asan Medical Center Children's Hospital, Seoul, Korea between August 2018 and June 2020. Among them, eight patients, including four with congenital cardiomyopathy suspected at prenatal examination, were infantile-onset and included in this study. Detailed demographics and clinical characteristics of the patients were reviewed, including age, initial presentations, gender, family history, laboratory results, radiological results, and genetic test results.
Informed consent was obtained from the parents of the patients for the genetic test. This study was approved by the Institutional Review Board for Human Research of Asan Medical Center (IRB numbers 2018-0574 and 2018-0180).
Dilated cardiomyopathy (DCMP): Left ventricular fractional shortening (FS) or ejection fraction (EF) > 2 SD below the normal mean for age and Left ventricular end-diastolic dimension (LVEDD) or volume > 2 SD above the normal mean for body surface area [ Left ventricular non-compaction (LVNC): Prominent LV trabeculations with deep recesses communicating with the LV cavity and a thin, compacted epicardial layer [ Hypertrophic cardiomyopathy (HCMP): Left ventricular posterior wall thickness at end diastole >2 SD above the normal mean for body surface area [
WES was performed using genomic DNA isolated from patient’s whole blood or a buccal swab sample. All exons of all human genes (approximately 22,000) were captured using a Twist Human Core Exome Kit (Twist Bioscience, San Francisco, CA, USA). The captured genomic regions were sequenced using a NovaSeq 6000 platform (Illumina, San Diego, CA, USA). Raw genome sequencing data analysis included alignment to the reference sequence (NCBI genome assembly GRCh37; accessed February 2009). The mean depth of coverage was 100-fold with 99.2% higher coverage than 10-fold. The pathogenicity of each variant according to the American College of Medical Genetics (ACMG) guidelines [
Genomic DNA was isolated from the peripheral blood using a PUREGENE DNA isolation kit (Gentra, Minneapolis, MN, USA). The amplifications by PCR were performed in 30 cycles. After verifying that the single-specific PCR product was amplified, DNA sequencing was performed using the same primers used in PCR and BigDye Terminatore V3.1 Cycle Sequencing Ready reaction kit (Applied Biosystems, Foster city, CA, USA) according to the manufacturer’s instructions. Electrophoresis and analysis of the reaction mixtures were conducted with an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
The patient’s clinical data are summarized in
Pt ID | Type of CMP | Sex | Onset age (day) | Initial presentation | Admission duration (days) | Coexisting symptom | Family history | EF (%) | FS (%) | LV mass index (g/m2) | LVOTO | Mechanical circulatory support devices | Mortality |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | DCMP | F | 77 | Poor oral intake | 28 | – | – | 32 | 15 | 103 | – | LVAD | NA |
2 | DCMP | M | 0 | Prenatal | 58 | – | Paternal cousin with myocarditis | 21 | 9 | 95 | – | – | – |
3 | LVNC | F | 0 | Prenatal | 7 | Large VSD | Paternal AR | 22 | 9 | 75 | – | – | Expired |
4 | LVNC | F | 63 | Murmur | 11 | – | 22 | 10 | 119 | – | – | – | |
5 | HCMP | M | 0 | Prenatal | 48 | Dysmorphic face, polydactyly, cystic hygroma | – | 93 | 62 | 22 | o | – | Expired |
6 | HCMP | M | 271 | Murmur | 0 | Dysmorphic face | – | 86 | 53 | 71 | o | – | – |
7 | HCMP | F | 0 | Prenatal | 87 | TGA, VSD, CoA | – | 26 | 11 | 150 | o | ECMO | Expired |
8 | HCMP | F | 94 | Lethargy | 52 | Stroke, lactic acidosis | – | 90 | 58 | 170 | o | – | – |
The presenting signs in those diagnosed later were heart murmur (two patients), lethargy (one patient), and poor oral intake (one patient). All patients were hospitalized in the intensive care unit, and their median duration of admission was 48 (IQR 19.5–55) days. Two patients (25%) were provided mechanical circulatory support; three (37.5%) died at 48 [27.5–67.5] days of life.
Five patients had coexisting symptoms. Structural abnormalities in the heart were found in two patients—patient No. 3 had a large ventricular septal defect (VSD) with a diameter of 12 mm and patient No. 7 had transposition of great arteries, VSD, and coarctation of aorta. Multiple extracardiac anomalies with dysmorphic face were found in two patients—patient No. 5 had macrocephaly, cystic hygroma, epicanthal fold, proptosis, low set ears, micrognathia, widely spaced nipples, and undescended left testis and patient No. 6 had fragile hair, low set ears, and a webbed neck. Patient No. 8 showed symptoms of metabolic diseases, such as hemorrhagic stroke and severe lactic acidosis [>15 mmol/L (ref. 0.56–1.390)]. The median (IQR, reference range) of EF and FS for the two patients with DCMP was 27% (24–29%, ref. > 50) and 12.5% (11–14%, ref. > 28), respectively; the median (IQR, reference range) LV mass index for four patients with HCMP was 110 g/m2 (59–155 g/m2, ref. >95), and all four had a degree of left ventricular outflow tract obstruction (LVOTO).
The echocardiography images representing each phenotype are summarized in
WES identified a genetic variation in seven out of eight patients. A detailed analysis of the genetic variation is summarized in
Pt ID | Gene name | Position | Nucleotide change | Protein change | GenBank number | Predicted pathogenicity* | Bayesian probability | Genetic origin | |
---|---|---|---|---|---|---|---|---|---|
1 | chr15:63349193 | NM_001330351.1 | VUS | 0.812 | -# | REVEL: 0.84 | |||
2 | chr14:23893263 | NM_000257.3 | VUS | 0.675 | Paternal | REVEL: 0.83 | |||
3 | chr16:15812228 | NM_001040114.1 | VUS | 0.812 | Paternal | REVEL: 0.82 | |||
4 | chr20:30419659 | NM_033118.3 | VUS | 0.499 | Maternal | ADA: 0.99, RF: 0.94 | |||
5 | chr11:534288 | c.35G > A | p.Gly12Asp | NM_001130442.2 | Pathogenic | 0.999 | REVEL: 0.78 | ||
6 | chr7:140477806 | c.1502A > T | p.Glu501Val | NM_004333.4 | Pathogenic | 0.999 | REVEL: 0.99 | ||
7 | chr14:23898513 | c.1182C > A | p.Asp394Glu | NM_000257.3 | Likely pathogenic | 0.949 | Paternal | REVEL: 0.59 | |
8 | m.3946G > A | p.Glu214Lys | NC_012920.1 | Pathogenic |
Notes: #Parent test was not performed
*As presented in the consensus statement of the ACMG (American College of Medical Genetics) [
†
Bold: novel variant
In the two patients with DCMP, genetic variations were identified in the genes
In the two patients with LVNC, the
In the four patients with HCMP, the genetic variations were identified in the genes
The clinical courses of the subjects are summarized in
Pt ID | Current age (10/2020) | Height | Weight | Development | Cardiovascular medication | Cardiovascular disease course | Other medical conditions |
---|---|---|---|---|---|---|---|
1 | HF managed for 28 days (including 20 days of ICU stay) then discharged and lost to follow-up | ||||||
2 | 6 m | 75–85 p | 10–25 p | Normal | 1 vasodilator, 3 diuretics, 1 inotropic | EF 31%, RV function recovered and considering reducing LV loading by PAB | – |
3 | HF aggravation caused cardiogenic shock and death despite maximum dose of inotropic at 7 days old | ||||||
4 | 2 y 11 m | 90–95 p | 50–75 p | Normal | 2 vasodilators, 1 diuretic | EF recovered up to 47% | – |
5 | LVH progressed rapidly and the patient failed to recover from cardiogenic shock and LVOTO and died at 48 days old | ||||||
6 | 1 y 1 m | 50–75 p | 95–97 p | Global developmental delay | 1 vasodilator | Stationary | AED with infantile spasms at 1 year old, keeping stationary supravalvar PS 2.3 m/s |
7 | Thirteen days after birth, arch repair, atrial septectomy, PAB, and PDA division were conducted, but ECMO weaning failure and cardiogenic shock was irreversible; the patient died at 87 days old | ||||||
8 | 7 m | <3 p | <3 p | Global developmental delay | 1 vasodilator, 3 diuretics | Stationary | After diagnosis of MELAS, taking AED with multivitamins and coenzyme therapy |
Abbreviations: EF, ejection fraction; HF, heart failure; ICU, intensive care unit; MVR, mitral valve replacement; RV, right ventricle; LV, left ventricle; PAB, pulmonary artery banding; LVH, left ventricular hypertrophy; LVOTO, left ventricle outflow tract obstruction; AED, anti-epileptic drug; PS, pulmonary stenosis; PDA, patent ductus arteriosus; ECMO, extracorporeal membrane oxygenation; MELAS, mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes
Among the two patients with DCMP, patient No. 1 was lost from follow-up and patient No. 2 had a persistently low cardiac output of 31% in EF but had normal growth and development until 6 months of age.
Among the two patients with LVNC, patient No. 3 died 1 week after birth due to progressive heart failure, and patient No. 4 is now recovering heart function to EF 47%, showing normal development and growth until 35 months of age.
Among the four patients with HCMP, patient No. 5 with Costello syndrome had progressive LVOTO and died due to cardiogenic shock at 48 days of life and patient No. 7 had an open-heart operation and died from failure of extracorporeal membrane oxygenation weaning at 86 days of life. Patient No. 6 with CFC has a similar level of hypertrophy and LVOTO until 13 months of age and is currently taking an anti-epileptic drug and undergoing rehabilitation for epilepsy and global development delay (case 17 in a previous study by Lee et al. [
In this study, all patients with infantile-onset cardiomyopathy were identified based on variants associated with cardiomyopathy via WES and additional mtDNA sequencing. Kindel et al. [
In general, the phenotype suggested by a genetic defect identified in each of the patients was consistent with that of the affected patient. Genetic variants in patients with DCMP and HCMP in this study have been found in similar phenotypes in previous studies [
In this study, one patient (patient No. 8) was diagnosed with MELAS through whole mtDNA sequencing, although WES did not find pathogenic variants in her nuclear DNA. Cardiomyopathy is one of the most characteristic features of mitochondrial disease, and her high blood lactic acid level was an additional pathognomonic finding to suspect a mitochondrial disease [
Importantly, one of the greatest challenges in the interpretation of genetic testing results in cardiomyopathy is that many parents share the variant found in their affected child but they do not have any subjective, relevant symptoms [
As subclinical cardiomyopathy is likely to progress gradually, a single evaluation is not sufficient to determine whether the asymptomatic carrier is not affected by cardiomyopathy; thus, the identified variant is not the causative variant. Rather, assessing their cardiac function on a regular basis for the long-term should be considered. There are no defined guidelines for these genotype-positive/phenotype-negative patients. However, those parameters would be helpful to predict the cardiomyopathy in the early stage, such as left ventricular global longitudinal strain, peak left atrial longitudinal strain that can be measured by cardiac magnetic resonance imaging, or three-dimensional echocardiography [
As an increasing number of subjects become involved in genomic studies such as WES, a major concern is the interpretation of variants identified in each subject. Importantly, according to the ACMG guidelines, the variants in the genes associated with cardiomyopathy should be considered to inform the subject regardless of the primary medical condition for which the genomic study was required [
There are several limitations to this study. Due to the small number of patients, it was impossible to perform subgroup analysis among the patients with infantile cardiomyopathy. In addition, as mentioned earlier, the interpretation of the pathogenicity of each variant was limited by the observation of the same variant in asymptomatic family members. Due to the variable and mostly short-term follow-up periods, it was difficult to describe the long-term outcome of infantile cardiomyopathy in association with the genetic findings, which must be re-evaluated in a larger cohort with longer-term evaluation. Lastly, multiple variants in different genes can be found in a single patient [
Genomic studies, such as WES, can identify a genetic variant in high proportions of patients with infantile-onset cardiomyopathy. However, due to a wide range of penetrance and expressivity, the interpretation of each variant’s pathogenicity is limited in most cases.