The clinical characteristics of 46 (female-26 and male-20) pediatric patients with DCM onset are presented in Table 1 and Table 2. Almost all the patients were presented with a common respiratory syndrome like shortness of breath and cough. Notably, heart rhythm disturbances (1 atrial tachycardia, 2 premature ventricular contractions with low voltage, 1 transient junctional rhythm, 7 premature ventricular contractions, 2 left anterior fascicular blocks, 1 left bundle branch block, 3 atrial premature beats) were observed in 17 patients. It is important to note that 67.4% of patients (31/46) manifested the disease before 1 year of age with a median age of diagnosis of 6.5 months, and 84.8% (39/46) cases were diagnosed before the child’s third birthday. At presentation, the mean LVEF was 28.5%, and the mean LVEDD Z-score was 7.02. The mean serum N-terminal pro-brain natriuretic peptide (NT-pro BNP) level was 14406pg/ml, much higher than the reference range (Table 3). During follow-up of median 12.5 months, 54.3% of them (25/46) were recovered from DCM and their LVEF was rescued to normal range. Because of the severity of DCM, 10 out of 46 patients died, and the average death timing range was 1-36 months (median 7 months) after the initial diagnosis. Importantly, 90% of deaths occurred within the first year after diagnosis, and it was found to be a critical time period reflected by the significant morbidity and mortality. Genetic analysis of DCM showed dramatically high heterogeneity in these patients, while only 3 patients had a familial history of DCM. No significant sex difference was observed in LVEF, LVEDD Z-scores, age of onset, serum NT-proBNP level at diagnosis, and in follow-up time, the outcome of death or recovery (all P>0.05).
Genetic Characteristics of Mutation-Positive Group
Based on the sequencing results, the cohort was divided into two groups: the mutation-positive (MP) group (16/46) and the mutation-negative (MN) group (30/46), as shown in Table 3. Considering the stringent selection criteria and reclassification of DCM according to the ACMG guidelines, the genetic analysis suggested that these 16 patients could be classified as either ‘pathogenic’ or ‘likely pathogenic’ mutant carriers. All carriers were identified with only one pathogenic or likely pathogenic mutation in DCM-associated genes (Figure 1). We identified 10 genes with disease causing heterozygous mutations, including those with integral sarcomere functions like titin (TTN) [(OMIM *604145), (n=6, 37.5%)], myosin heavy chain 7 (MYH7) [(OMIM *613426) (n=1, 6.25%)], troponin T2 (TNNT2) [(OMIM *601494) (n=1, 6.25%)], Nexilin (NEXN) [(OMIM* 613122) (n=1, 6.25%)], troponin I3 (TNNI3) [(OMIM*613286) (n=1, 6.25%)], cytoskeletal structure-related genes like filamin-C (FLNC) [(OMIM*617047) (n=1, 6.25%)], vinculin (VCL) [(OMIM*611407) (n=1, 6.25%)], and other genes like RNA binding motif Protein 20 (RBM20) [(OMIM*613172) (n=2, 12.5%)], NK2 homeobox 5 (NKX2-5) [(OMIM:108900) (n=1, 6.25%)] and PR domain containing 16 (PRDM16) [(OMIM*615373) (n=1, 6.25%)]. Table 4 summarizes the main clinical features and the details of identified variants in DCM patients. Excluding 3 patients with familial mutations in TTN, MYH7 and NEXN genes, 5 de novo variants were identified in NKX2-5, TNNI3, PRDM16, and RBM20 (n=2) genes. Among the identified 16 gene mutations (missense-5, non-sense-5, frameshift-4 and splice site-2), 2 same mutations were identified in a pair of monozygotic (MZ) twin patients, and 10 (62.5%) other mutations were novel.,
In total, we identified 6 different (5 novels) TTN truncation variants, including 3 nonsense, 1 frameshift, and 2 splice-site variants, in 13% of patients (Table 4). Furthermore, we mapped the identified variants to protein domains of TTN gene (Figure 3a), which showed consistent results with previous reports. Notably, 4 variants were in I-band, and the remaining 2 in A-band and M-band regions, respectively.
Genotype and Clinical Phenotype Analyses
The phenotypic severity of these patients at presentation was measured by LVEDD Z-score, LVEF, and serum proBNP level. There were no significant differences in distribution based on sex (P=0.202), age (P=0.23), LVEF (P=0.935), LVEDD Z-score (P=0.42), and serum proBNP level (P=0.115) between MP and MN groups. The MP group probands exhibited lower phenotypic severity (echocardiographic parameters) than MN group DCM probands, with the same LVEF of 28.5% and LVEDD Z-score of 6.4 versus 7.08, respectively. Also, we observed a trend that the onset age (median 9.5 months) in MP group was slightly higher than that in the MN group (median 6 months). Nine MP patients developed DCM before 12 months of age, and the age distribution was between 1 month and 13 years.
Despite the observation of better echocardiographic parameters and seemingly better phenotypes at presentation in MP group patients, there was no significant difference in the outcome (the number of deceased patients, P=0.145) and LVEF recovery patients, P=0.665) between the two groups during the follow-up of median time of 12.5 months (P=0.972). Survival curves free from death and recovery also showed similar results (Figure 3). 37.5% (6/16) of MP DCM patients eventually suffered from heart failure related death compared to only 13% (4/30) of the MN group. Although no significant differences in survival free of death (P= 0.093) were documented, however, there was a trend toward a worse prognosis in our patients at advanced ages (Figure 2a) when compared to the MN patients. The outcome of cardiac death occurred in patients with TTN truncating mutants (c.50065C>T, c.98421_98422insGG, c.37454-2A>T), and NKX2-5 (c.242delA), TNNT2 (c.422G>A), TNNI3 (c.544G>A) mutations. Notably, 5 out of 6 disease causing mutations belong to sarcomeric genes (TTN=3, MYH7,TNNT2, TNNI3).
In this study, a significant proportion of the patients exhibited marked improvement and better prognostic outcomes in response to heart failure treatment. In both groups, almost 50% of pediatric patients could recover (LVEF above 50%). There was a trend toward better recovery in MN patients at advanced ages (Figure 2b).
TTN Truncation Variants in Pediatric DCM Patients
Here, we describe the clinical and genetic characteristics of 4 probands with 6 TTN mutations (as summarized in Table 4).
P2: A 12 years-old girl presented with dyspnea and fatigue. Her chest x-ray revealed enlarged heart size, and echocardiography showed LVEDD value 5.86cm and LVEF of 27%. Her brother died of severe congenital heart disease at <1 year age. Her LVEF was recovered to 58% after treatment. A novel ‘likely pathogenic’ nonsense heterozygous truncation mutation (c.43298T>G, p.Leu14433*) in TTN gene was confirmed in the proband and her mother by genetic analysis (Figure 4b). But her mother had no symptoms.
P4: A 12 years-old boy with DCM performed a cardiology outpatient clinical evaluation due to the sudden onset of shortened breath with abdominal pain, cough, chest tightness, and fatigue. The proband performed an electrocardiogram (ECG) showing the left anterior fascicular block. Also, he exhibited severely elevated plasmatic transaminases (AST = 275U/L, ALT = 881U/L). The abdominal CT elsewhere revealed ascites, pleural effusion. Remarkably, the plasmatic NT-proBNP level was also drastically high (960.4pg/ml) (normal values less than 125pg/ml at the proband’s age). He also performed an electrocardiogram of the left anterior fascicular block and echocardiography showing LVEF of 20%, LVEDD of 7.29cm. After receiving the regimen of spironolactone 40mg/day, captopril 8.3mg/day, furosemide 40mg/day, digoxin 0.25mg/day, potassium chloride sustained release tablets 6000mg/day for 13 days, a repeated echocardiography showed a more severe dilated left ventricle (7.45cm) and a suspicion for left ventricular apex noncompaction. Three months after diagnosis of DCM, he was hospitalized again and died of ventricular tachycardia at a young age. NGS and Sanger sequencing confirmed the ‘pathogenic’ TTN (c.50065C>T, p.Arg16689*) variant in the proband and his mother (Figure 4b). His mother had no symptoms, and she refused to perform echocardiography. For the first time, a pathogenic variant in TTN was associated with the DCM phenotype.
P8: A 10 years-old girl, who suffered from DCM for about 3 years and showed no response to heart failure medication, died of heart failure and pneumonia. At the onset, she had an increased serum phosphocreatine kinase (up to 2900u/L) level, and her ECG showed premature ventricular contractions. We identified a novel ‘likely pathogenic’ frameshift mutation in TTN (c.98421_98422insGG, p.I30808Pfs * 7) by NGS analysis in the proband and her father. Her father didn’t show any symptoms. Involvement of skeletal myopathy was suspected in this patient because of increased phosphocreatine kinase level. The skeletal myopathy was then identified by muscle biopsy (failing to record the pathologic result). This suggests that this variant can cause disease in both heart and skeletal muscles.
P13: A girl diagnosed with DCM at the age of 3 months had an autosomal dominant family history of DCM. Her maternal grandfather (diagnosed at 36 and died at 52 years), the brother of the grandfather (diagnosed at 42 and died at 46 years), and their sister (diagnosed at 44 and died at 52 years) died of DCM. The proband presented with shortness of breath and atrial tachycardia. She was given a treatment of dopamine cardiotonic, furosemide, spironolactone, amiodarone, and captopril. Unfortunately, 6 months later Her mother felt shortness of breath after activity and performed echocardiography elsewhere, exhibiting LVEF=29% and LVEDD=6.8cm. After 3 years of follow-up, the proband could recover her LVEF to 62% with no signs of arrhythmia. But her mother still receives drug treatment and has not been recovered yet. By trio WES method, we identified a novel truncating ‘likely pathogenic’ mutation in TTN gene (c.105541A> T, p.K35181X) in the proband and her mother, but not in her father and 8-years old brother (Figure 3), who both had normal echocardiography parameters.
RBM20 Gene Mutation in Pediatric DCM
P6 and P7, monozygotic (MZ) twin brothers (a kinship coefficient of 0.4901), were diagnosed with DCM with LVEF 29% and 31%, respectively at the age of 12 years (the interval time of onset was 9 months). They both felt nausea, chest tightness, and shortness of breath. Both ECG showed ventricular premature beats. The MZ twin was administered with standard heart failure medication. After seven years of follow-up, the LVEF of the two patients were 56% and 55%, respectively, and remain asymptomatic. By WES screening, a ‘pathogenic’ de novo mutation c.2746G>A (p.E916K) in RBM20 gene was identified. The missense mutation was absent from controls (1000G, ESP, and ExAC-ALL), and was predicted to be ‘pathogenic’ by PolyPhen2, SIFT databases. The amino acid p.E916 is highly conserved across vertebrate species (Figure 5C). We confirmed this missense mutation by Sanger sequencing in the twin patients but not in their parents (Figure 5B).