The proband was an 8-year-old male admitted to our hospital for 5 days due to severe, recurrent, and persistent chest pain. Moreover, the chest pain attacks became more frequent and his pain levels worsened, which he could not tolerate. He described the acute and repeated chest pain as aching and tightness. Additionally, the patient complained of pain spreading from the chest to the shoulders, upper abdomen, and back. In the last 2 days before hospital admission, the patient suffered severe vomiting more than five times per day. When the chest attacks occurred, he experienced palpitations and slightly reduced activity tolerance. However, he denied any syncope and shortness of breath. There was no fever, cough, or indigestion.
The initial physical examination at the emergency department revealed an acute critical illness involving severe diaphoresis, dizziness, and fatigue, with the patient presenting with a pale face and feeling restless. The heart rate was approximately 75 beats per minute, and irregular premature beats were observed. His blood pressure was 91/52 mmHg. At the same time, the breathing rate slightly increased to 25 breaths per minute. This patient had a normal nutrition status, and his response to external stimulation was also normal. No trauma injuries were found on the body surface. The respiratory movements of both lungs were symmetrical; the breath sounds of both lungs were rough, no significant wet rales were heard in the bilateral lungs, and occasional wheezing sounds were heard. The apex of the heartbeat moved to the lower left, and there was a sense of lift in the precordial area. A mildly enlarged heart boundary was identified, and the rhythm was heterogeneous with premature beats. However, the heart sound was dull, and a level I-II systolic murmur was recorded. The abdomen was soft, the liver was 2 cm below the subcostal margin and 1 cm-2 cm below the xiphoid process, the texture was medium, and the spleen was not palpable subcostally. The muscle strength and tension of the four extremities were normal. The pathological signs and meningeal irritation signs were negative.
The patient suffered influenza A infection 1 year before these chest pain attacks, and myocardial injuries had been identified by an increased level of cardiac troponin I (cTnI). The electrocardiogram (ECG) revealed multiple premature ventricular beats. After a series of myocardial protection treatments had been provided, the patient recovered and did not complain of any other cardiac-related symptoms in the year before this illness. Moreover, his parents denied any positive family history of cardiac attacks or cardiovascular, hypertension, and coronary artery diseases. The parents also denied any history of diabetes and obesity among his family members. No inherited disease had been identified in this family, including any cardiomyopathies and metabolic diseases.
Routine blood cell tests and blood gas analyses produced results that were within a standard range. In addition, hepatic and renal function tests yielded no significant findings. However, serum cardiac troponin I (2.808 microg/L, n.v. < 0.06 microg/L) and B-type natriuretic peptide (1020.24 pg/ml, n.v. < 100 pg/ml) levels were significantly elevated, demonstrating significant myocardial injury. An ECG was performed immediately, and abnormal Q waves were observed in the II, III, aVF, V5, and V6 leads (Figure 1A). At the same time, Holter scanning revealed multiple ventricular premature beats and paired atrial premature beats. Thus, ACS or viral myocarditis was primarily suspected in this patient. All infection parameters, including CRP, PCT, and ESR, were in the expected range. Additionally, antibody tests of potential viruses involved in myocarditis were assessed, including cytomegalovirus, Epstein-Barr virus, adenovirus, coxsackie virus, and herpes simplex virus, such as human parvovirus B19, and no positive viral infection results were obtained. In addition, autoimmune antibodies and rheumatic tests were performed and all the related results were negative.
Echocardiography demonstrated a slight enlargement of the left ventricle (42 mm), while the left ventricular ejection fraction dropped slightly to 46% at the time he was admitted (Figure 2A). Holter scanning revealed multiple premature ventricular beats and atrial tachycardia, while abnormal Q waves were also identified in the II, III, aVF, and V3-V6 leads (Figures 1B, C). Moreover, repeated ECG examination found an ST-segment elevation among the abovementioned leads, which strongly indicated ACS. Cardiac magnetic resonance (CMR) revealed myocardial edema in the lateral ventricular wall and apex, indicating localized myocardium injuries (Figures 2B, B'). The area of infected myocardium was consistent with the changes in ECG presentation. In addition, CT coronary artery angiography was performed to examine the morphology of the coronary arteries (Figures 2C-E). The right coronary artery was infused and presented a typical structure. Angiographic images of the left coronary artery were also obtained and no significant positive result was recorded (Figures 2F, F').
Therefore, a myocarditis attack was suspected, while ACS with non-obstructive coronary arteries could not be entirely excluded. Thus, myocardial protection treatment, including creatine phosphate and levocarnitine, and non-invasive mechanical ventilation were provided. Then, antibiotics were administered for potential infection treatment. Dexamethasone was administered to relieve edema of the myocardium. After 2 weeks of treatment and intensive care, the patient recovered from severe chest pain and did not report any discomfort of the heart. After that, the patient was discharged with strict follow-up.
Owing to the complicated symptoms and negative imaging assessments of this proband, a particular cardiomyopathy was still suspected. Therefore, whole-exome sequencing (WES) was carried out to identify any essential genetic variants. A peripheral blood sample was obtained from the patient in an ethylenediaminetetraacetic acid (EDTA) anticoagulant blood sample tube that was stored at 4 C for less than 6 h. DNA was extracted using a Blood Genome Column Medium Extraction Kit (Tiangen Biotech, Beijing, China). WES was performed using the NovaSeq 6,000 platform (Illumina, San Diego, CA, USA), and the raw data were processed using FastP to remove adapters and filter low-quality reads. Paired-end reads were aligned to the Ensembl GRCh38/hg38 reference genome using the Burrows-Wheeler Aligner. Variant annotation was performed in accordance with database-sourced minor allele frequencies (MAFs) and practical guidelines on pathogenicity issued by the American College of Medical Genetics. The annotation of MAFs was performed according to the 1,000 Genomes, dbSNP, ESP, ExAC, and Chigene inhouse MAF databases and the Provean, Sift, Polypen2_hdiv, and Polypen2_hvar databases using R software (R Foundation for Statistical Computing, Vienna, Austria). To elucidate the molecular architecture of the targeted gene, we used MutationTaster with R software to predict the pathogenicity of the targeted gene and assess the impact of the mutations on protein structure. We performed comparative modeling using SWISS-MODEL. If there was no available full-length protein crystal structure for the targeted gene, the AlphaFold protein structure database (https://alphafold.ebi.ac.uk/) tool was used to predict the protein crystal structure.
A homozygous missense variant in the DSG2 gene was identified (NM_001943, c.1592T >G; p.F531C). His biological parents were allelic carriers without any clinical manifestations (Figure 3A), and Sanger validation was performed (Figure 3B). Additionally, no other cardiovascular-related variants were retrieved between the proband and his parents. Beyond the reported DSG2 variant, there were no other potential cardiomyopathic variants, and most of them were synonymous mutations. The molecular crystal structure was built in AlphaFold (AF-Q14126-F1, Figure 3C), while the extracellular domain protein structure was used for specific site analysis (5erd.1.A, Figure 3E). In addition, the frequency of this mutation in the population according to database research is presented (Figure 3D), but only a few allele carriers were found in previous reports. This proband would be the youngest identified patient with a homozygous variant change of c.1592T > G in DSG2. This mutation site was regulated by DNA modification, which induced amino acid sequence changes, protein structure effects, and splice-site changes. According to MutationTaster analysis, the variant was considered a disease-causing mutation, and the probability value was 0.99. PolyPhen-2 analysis demonstrated a damaging change in this protein (1.00). The SIFT score predicted protein damage (0.001), and MutationAssessor indicated a high molecular function impact on the variant (FI score, 3.79; VC score, 4.80; and VS score 2.77). Then, we used SWISS-MODEL to illustrate the mutation site of p.F531C (Figure 3E). The ensemble variance of p.F531C indicated free energy changes after the amino acid change (Figure 3F).
During follow-up, the patient presented left ventricular dysfunction (LVEF = 36%) and aggressive enlargement of the left ventricle (48 mm). ECG also identified frequent premature ventricular beats (>1,000 beats per day). Based on the molecular genetic analysis of a homozygous variant of DSG2, a diagnosis of arrhythmogenic cardiomyopathy was made, as no significant involvement was found in the right ventricle. After diagnosis, metoprolol and captopril were administered to the patient, who reported no further chest pain and a reduced frequency of arrhythmia. This patient was misdiagnosed as having myocarditis at his initial admission. The results indicate that this homozygous variant of DSG2 could cause an earlier onset of an adverse cardiac event than heterozygous variants. The first attack of arrhythmogenic cardiomyopathy might result in a series of symptoms, such as ACS. Therefore, genetic screening of unexplained myocarditis is critical.