This was an observational retrospective, single-centre trial. Patients were enrolled between January 2017 and January 2023 at Hunan Children’s Hospital, Changsha, China. The definitions of complex arrhythmias associated with the RYR2 gene are as follows: 1) Development of multiple types of arrhythmias. 2) The patient has RYR2 gene mutations. This study protocol was approved by the Ethics Committee of the hospital (HCHLL-2023-128). All patients’ family members were aware that their clinical data might be used for a clinical study and signed written informed consent.

Retrospective analysis of the clinical data of enrolled children was conducted, including medical history, physical examination, electrocardiogram, Holter ECG, cardiac ultrasound, and pathogenic gene analysis results. The exercise treadmill test (GET2100 exercise treadmill, United States) was performed with an improved Bruce regimen for submaximal exercise. The indications for termination of the exercise test were: 1) achieving the target heart rate [(220-age) x 85%]; 2) Malignant arrhythmias such as ventricular tachycardia appear; 3) Development of intolerable symptoms such as palpitations, dizziness, etc. Whole exome gene sequencing was performed by Guangzhou Jiajian Medical Laboratory.

The assessment of the pathogenic role of the genetic variants identified should be multiparametric. Judicious use of bioinformatic predictors, which are known for a high rate of false-positives, and in vitro studies where available, should be coupled with data derived from the families of the index case and population data. According to ACMG guidelines, we calculate the correlation using the following six scoring criteria: 1. Group frequency: total frequency of population variation. 2. Conservative analysis: the region where this mutation occurs is an important component of this protein, and the amino acid sequences of different species are highly conserved. 3. Algorithm prediction: Computer assisted analysis predicts a higher likelihood of this mutation affecting protein structure/function. 4. Public data. 5. Family analysis: parents carry RYR2 gene mutation or not. 6. Clinical notes: Based on the clinical manifestations and family analysis of the examinee, the clinical correlation is strong. The score of case 1 is PM2-P + PP3-A1+PP3-B1+PS4-M + PP2+PM6. The score of case 2 is PM2-P + PP3-A1+PP3-B2+PM1+PP2+PM6. The score of case 3 is PM2-P + PP3-A1+PP3-B1+PM1+PP2+PM6. The score of case 4 is PM2-P + PP3-A1+PP3-S + PM1+PP2+PM6. The score of case 5 is PM2-P + PP3-A1+PP3-B1+PM5+PP2+PS2. The score of case 6 is PM2-P + PP3-A1+PP3-B1-M + PM5+PP2+PS2.

All children were advised to limit their exercise and avoid emotional stimulation. They were started on β-receptor blocker therapy, initially with a dose of 0.5–1 mg/(kg·d) of propranolol, administered orally in three times, and gradually increased to a tolerable maximum dose of 2–4 mg/(kg·d). Alternatively, a starting dose of 0.5–1.0 mg/(kg·d) of metoprolol tartrate was administered orally in two times. If arrhythmias persisted despite the use of β-receptor blockers, oral flecainide (1 mg/kg, twice a day) or propafenone (5–7 mg/kg, three times a day) was added. If obvious sinus bradycardia occurred after medication treatment, implantation of a pacemaker was considered. Outpatient follow-up was conducted after discharge, with Holter ECG and echocardiography reviewed every 3–6 months to evaluate heart function and the presence of cardiovascular-related symptoms. Children over 5 years old were evaluated through exercise treadmill tests to clarify the heart rate limit for sinus tachycardia before arrhythmias occurred, with the aim of avoiding exceeding the threshold in daily life.

Among the six cases of complex arrhythmias associated with RYR2 gene mutations, there were four boys and two girls. Three children were initially misdiagnosed as “epilepsy” and treated with antiepileptic drugs in other hospitals. Two children were misdiagnosed as “myocarditis and tachycardia cardiomyopathy”, and one child was misdiagnosed as “supraventricular tachycardia”. For three children who were misdiagnosed with epilepsy at the first diagnosis, their initial symptoms were syncope and convulsions. They were admitted to neurology department for the first time, and diagnosed with epilepsy according to their clinical symptoms and therefore they were misdiagnosed. Without epileptic wave in electroencephalogram (EEG), electrocardiogram (ECG) or exercise treadmill was performed to detect that they have complex arrhythmias. Finally, genetic test was performed to detect RYR2 mutation and the diagnosis was corrected. For two children who were misdiagnosed with myocarditis and tachycardia cardiomyopathy at the first diagnosis. The onset of the disease was heart failure, with abnormal elevation of myocardial enzyme and cardiac troponin. And cardiac ultrasound showed cardiac enlargement. But later, they developed with complex arrhythmias such as atrial tachycardia and sinus node dysfunction. Finally, Genetic test was performed to confirm the diagnosis and found RYR2 gene mutation. For two children who were misdiagnosed with supraventricular tachycardia at the first diagnosis. As the electrocardiogram diagnosis was supraventricular tachycardia, she developed with syncope and Holter ECG showed sinus arrest. So genetic testing was performed to confirm the diagnosis, revealing RYR2 gene mutation. Three children developed syncope during exercise, one child during emotional arousal, and one child experienced transient loss of consciousness due to sinus bradycardia. The duration of syncope ranged from more than 10 s to a few minutes, with spontaneous recovery of consciousness. The age of initial symptoms ranged from 2.0 to 5.0 years, with an average age of 3.5 ± 0.5 years; the age at confirmed diagnosis ranged from 2.1 to 11 years, with an average age of 6.3 ± 1.4 years. The time from initial symptoms to diagnosis ranged from 0.1 to 8.4 years, with an average time of 2.7 ± 1.3 years.

Two patients were admitted, and their routine surface electrocardiogram showed atrial tachycardia; one exhibited borderline tachycardia, one had occasional premature atrial contractions, and two patients had T-wave notches in lead V₂. Six patients showed abnormalities in the Holter ECG, including four cases of atrial tachycardia, two cases of atrial flutter, one case of atrial fibrillation, three cases of sinus bradycardia and cardiac arrest (RR interval 2–4.4 s), and two cases of QT interval prolongation (QTc 476–545 m). One case developed short burst multi-source ventricular tachycardia, and three cases developed ventricular premature beats. The patients’ characteristics of electrocardiograms are shown in Figure 1 and Figure 2.

All six cases underwent whole exome gene sequencing and parental verification, revealing de novo missense mutations, with no family history of related genetic diseases in the family members. The mutation site c.12534C>A (p.N4178K) in case 2 has not been reported in clinical cases, but other pathogenic mutations p.N4178S and p.N4178Y at the same amino acid position have been reported in clinical cases. The mutation site c.536A>G (p.D179G) in case 4 has not been reported in clinical cases, but the pathogenic mutation p.G178A near this amino acid position has been reported in clinical cases. The mutation site c.41T>G (p.L14R) in case 6 has not been reported in the literature, but the region of variation is an important component of this protein. The mutation site c.12515T>A (p.F4172Y) in case 3 has not been reported in the literature. Cases 1 and 5 have been reported in clinical cases (Ohno et al., 2015; Kaneshiro et al., 2017; Kannankeril et al., 2017; Wilde et al., 2022). Based on the clinical manifestations and test results of the examinees, and according to the American College of Medical Genetics and Genomics (ACMG) mutation classification guidelines, the pathogenicity of the mutation sites in all six children is possible. The genetic examination results are shown in Table 1. The gene test had been performed in their parents and no RYR2 gene mutations were found in their parents. Their family members were unwilling to undergo the gene test. And the exome gene sequencing and mapping results, especially for the mutation sites, are shown in this paper (Figure 3; Figure 4).

All six children were strictly restricted in exercise and received treatment with antiarrhythmic drugs. After oral administration of propranolol, two children did not develop syncope. Two patients still experienced intermittent palpitations and syncope after initial oral administration of metoprolol. However, after switching to propranolol, no palpitations or syncope occurred. One patient with atrial arrhythmias and frequent sinus arrest was treated with permanent epicardial pacing (VVI, VVI means a pacing mode: single chamber ventricular pacemaker), and after oral administration of propranolol and flecainide, the arrhythmias were significantly reduced. One patient developed significant sinus bradycardia after oral administration of metoprolol, and underwent permanent endocardial pacing (VVI, VVI means a pacing mode: single chamber ventricular pacemaker). One patient still experienced short paroxysmal ventricular tachycardia after taking propranolol orally, and propafenone was added orally. All cases survived, with a follow-up period of 12–37 months, with an average time of 24.3 ± 3.7 months. Please see Table 2 for details.