Clinical Spectrum of SCN5A Channelopathy in Children with Primary Electrical Disease and Structurally Normal Hearts

Sodium voltage-gated channel α subunit 5 (SCN5A)-mutations may cause an array of arrhythmogenic syndromes most frequently as an autosomal dominant trait, with incomplete penetrance, variable expressivity and male predominance. In the present study, we retrospectively describe a group of Mexican patients with SCN5A-disease causing variants in whom the onset of symptoms occurred in the pediatric age range. The study included 17 patients with clinical diagnosis of primary electrical disease, at least one SCN5A pathogenic or likely pathogenic mutation and age of onset <18 years, and all available first- and second-degree relatives. Fifteen patients (88.2%) were male, and sixteen independent variants were found (twelve missense, three truncating and one complex inframe deletion/insertion). The frequency of compound heterozygosity was remarkably high (3/17, 17.6%), with early childhood onset and severe disease. Overall, 70.6% of pediatric patients presented with overlap syndrome, 11.8% with isolated sick sinus syndrome, 11.8% with isolated Brugada syndrome (BrS) and 5.9% with isolated type 3 long QT syndrome (LQTS). A total of 24/45 SCN5A mutation carriers were affected (overall penetrance 53.3%), and penetrance was higher in males (63.3%, 19 affected/30 mutation carriers) than in females (33.3%, 5 affected/15 carriers). In conclusion, pediatric patients with SCNA-disease causing variants presented mainly as overlap syndrome, with predominant loss-of-function phenotypes of sick sinus syndrome (SSS), progressive cardiac conduction disease (PCCD) and ventricular arrhythmias.


Introduction
The function of the cardiac voltage-gated sodium channel α subunit protein (Na V 1.5) is crucial because it initiates the cardiac action potential by generating the inward sodium current (I Na ), which mediates cardiomyocyte excitability and impulse conduction in the myocardium and specialized conduction system. This channel also generates the late sodium current (I NaL ), which plays a role in repolarization and refractoriness [1]. The α subunit of Na V 1.5 is a 220 kDa transmembrane protein encoded by the sodium voltage-gated channel α subunit (SCN5A) gene, consisting of a cytoplasmic N-terminal domain, four homologous transmembrane domains (TMD, DI-DIV) and a cytoplasmic C-terminal domain. Each TMD has six transmembrane α-helix segments (S1-S6) connected by cytoplasmic linkers [2]. Although sodium channel α-subunits are traditionally believed to form functional monomers, some SCN5A mutation studies in patients with inherited arrhythmias indicate that oligomerization of the sodium channel α-subunits may occur, and recent experiments suggested that sodium channel α-subunits physically interact, assemble, function and gate as a dimer [3].
SCN5A-mutations may cause several arrhythmogenic phenotypes most frequently inherited in an autosomal dominant fashion [4,5]. These mutations cause an array of arrhythmogenic syndromes including dilated, arrhythmogenic or non-compaction cardiomyopathy, or arrhythmogenic syndromes with minimal or no structural defects such as sick sinus syndrome (SSS), progressive cardiac conduction disease (PCCD), type 1 Brugada syndrome (BrS), familial atrial fibrillation, idiopathic ventricular tachycardia (VT), multifocal ectopic Purkinje-related premature contractions (MEPPC), and type 3 long QT syndrome (LQTS) [6]. Characteristically, SCN5A disease-causing mutations show incomplete penetrance, variable expressivity and male predominance for reasons that are not fully understood, which include both genetic background and environmental factors [7].
Early age of onset is associated with severity in many genetic diseases. Although there are many isolated case-reports of SCN5A-channelopathy in children in the medical literature [8][9][10][11][12][13][14], few studies have analyzed SCN5A-channelopathy cohorts where onset of symptoms occurs in pediatric patients. A large prospective multi-center pediatric cohort of SCN5A mutation-positive neonates reported that 67.9% were asymptomatic at diagnosis, while age <1 year at diagnosis, compound heterozygous mutations, and mutations with both gain-and loss-of-function were identified as independent risk factors for cardiac events [15]. In the present study, we retrospectively describe a group of Mexican patients with SCN5A-disease causing variants in whom the onset of symptoms occurred in the pediatric age range, attending the Ignacio Chávez National Institute of Cardiology over a 15 year period. This study provides insight, which can potentially be of aid for diagnosis, phenotypic characterization and to define therapeutic approaches in pediatric patients with SCN5A-disease causing variants.

Subjects
The study included a group of pediatric patients (age of onset <18 years) recruited from the Department of Cardiac Electrophysiology at the National Institute of Cardiology "Ignacio Chavez" in Mexico City over a 15-year period (2005 to 2020). All patients had a clinical diagnosis of primary electrical disease (SSS, PCCD, BrS, VT, LQTS) and a sodium channel disease-causing variant. All available first-and second-degree relatives were also included in the study. Index cases underwent routine clinical evaluation, including interrogation of family and medical history, physical examination, electrocardiogram (standard and high precordial leads), 24-h Holter, stress test, and had a yearly clinical examination for follow-up. Structural heart disease was ruled out by trans-thoracic echocardiography and/or magnetic resonance imaging. Informed consent was provided by participants or by their parents or legal guardian. The study was approved by the Ethics Committees of the National Institute of Cardiology Ignacio Chávez and the National Institute of Genomic Medicine in Mexico City.
The arrhythmogenic phenotypes considered were the following: SSS, sinus bradycardia, sinus pause or arrest with or without escape rhythm, sinoatrial exit block, tachy-brady syndrome, atrial fibrillation with slow ventricular response in the absence of AV node blocking agents, chronotropic incompetence [16]; PCCD: finding of a major conduction defect on the electrocardiogram (ECG) such as complete right bundle branch block, complete left bundle branch block, left anterior fascicular block / hemiblock or left posterior hemiblock, prolonged PR interval or complete AV block with broad QRS complexes [17]; BrS, appearance of a type one ST-segment elevation (coved type) in more than one right precordial lead (V 1 to V 3 ), in the presence or absence of a sodium channel blocker, and one of the following: documented ventricular fibrillation, ventricular tachycardia or selfterminating polymorphic ventricular tachycardia, a family history of sudden cardiac death (SCD, <45 years), coved type ECGs in family members, electrophysiological inducibility, syncope, or nocturnal agonal breathing [18]; LQTS was diagnosed when considered as high probability according the criteria of Schwartz [19]. Patients with more than one of these arrhythmogenic phenotypes were considered as SCN5A overlap syndrome cases.
All novel or very low frequency variants (minor allele frequency <0.0005) affecting the amino acid sequence of arrhythmogenic channelopathy-related genes were classified according to the American College of Medical Genetics and Genomics criteria as benign, likely benign, of unknown clinical significance (VUS), likely pathogenic (LP) or pathogenic. Patients with at least one pathogenic or likely pathogenic SCN5A variant and their available relatives were included in the analysis. Amino acid numbering was made according to transcription variant NM_198056 (http://www.ncbi.nlm.nih.gov/, accessed on 8 September 2021).

Statistical Analysis
The numerical variables are expressed as mean ± SD or median and interquartile range (IQR) as appropriate, and categorical variables as percentages. Overall penetrance for arrhythmogenic syndromes was estimated including all available individuals from families in which at least one first-degree relative was screened, as the ratio between number of SCN5A mutation carriers with arrhythmogenic phenotypes (affected) and the total number of carriers (affected and non-affected). Missense variants found in compound heterozygosity in probands, with no symptomatic heterozygous carriers in the family, were excluded from the overall penetrance estimation. Differences in SCN5A mutation penetrance between males and females were compared using the Chi-squared test.

SCN5A Mutations
A total of 17 apparently unrelated patients with at least one pathogenic or likely pathogenic SCN5A variant were included in the study, along with a total of 42 firstand second-degree relatives. Table 1 summarizes the characteristics of all pathogenic or likely pathogenic SCN5A variants identified in the probands [21][22][23][24][25][26][27][28][29]. A total of sixteen independent variants were identified, twelve were missense, while four were inferred as null (three truncating frameshift and one a complex inframe multiple amino acid (aa) deletion/insertion occurring in a non-repetitive aa sequence in DIII/S5 (p.Trp1345-Ser1349delinsPhe). All but three of the missense variants (p.Arg811Cys, p.Pro1730Leu and p.Ala1778Asp) had at least one functional study reporting loss and/or gain of function. Thirteen patients were heterozygous for missense, and one was heterozygous for a frameshift truncating mutation. Notably, three patients (17.6%) were compound heterozygous for an inferred null mutation and a missense SCN5A variant. Most missense mutations were located within the DIV domain (6/12, 50%), four (33.3%) affected the pore forming region (three in DIV and one in DII), while two affected voltage sensor domains (S4) in DII and DIV. No other pathogenic or likely pathogenic variants were found among 174 cardiogenes in this group of patients.
Three apparently unrelated patients were heterozygous for the p.Ser1710Leu mutation, each with a different phenotype: overlap syndrome (SSS and PCCD), isolated SSS and isolated BrS. Two apparently unrelated patients were p.Arg893His heterozygous, one with overlap and the other with isolated BrS. In addition, two apparently unrelated children were heterozygous for the p.Thr1708Asn mutation, both with overlap syndrome and schoolaged at the onset of symptoms. On further interrogation, family members were able to identify that both index cases were in fact second cousins once removed.

Syncope, Seizures, Non-Fatal Cardiac Arrest (NFCA) and Sudden Cardiac Death (SCD)
Syncope was reported in ten (58.8%) patients, and seizures in four (23.5%). The frequency of non-fatal cardiac arrest was 29.4% (5/17), and three of these five patients suffered SCD afterwards. The overall frequency of sudden cardiac death was also 29.4%, occurring before age 12 in 4 patients with overlap syndrome, and at age 17 in an isolated BrS patient.

ECG and Holter Findings
Twelve patients had at least one type of supraventricular tachycardia (SVT). The most prevalent was atrial flutter (8/17, 47.1%), followed by atrial fibrillation (35.3%), cavotricuspid isthmus-dependent atrial flutter (17.6%) and atrial tachycardia (11.8%). Moreover, twelve patients had one or more documented ventricular tachycardia events; the most prevalent was sustained monomorphic ventricular tachycardia (58.8%), followed by ventricular fibrillation (17.6%), polymorphic ventricular tachycardia (11.8%); and torsade de points (5.9%); ten had atrio-ventricular block (58.8%) and five had documented atrial standstill (29.4%). Finally, 15/17 patients (76.5%) showed a prolonged QTc interval (QTc max >460 ms), which was likely secondary to intraventricular conduction defect or AV block in 13, and only two patients had a normal maximum QTc interval (<440 ms). penetrance and variable expressivity. Different phenotypes were observed both for the same mutation in different cases (cases 1, 2 and 3 for p.Ser1710Leu; cases 6 and 7 for p.Thr1708Asn), and within the same family (pedigrees for cases 6, 7 and 9). Case 14 was the only isolated LQTS case, she and her father were heterozygous for the p.Arg1644His mutation; however, her father's ECG showed a normal QTc interval (432 ms) with bradycardia (49 bpm). From a total of 45 SCN5A mutation carriers, 24 were affected with an arrhythmogenic phenotype (overall penetrance 53.3%), and penetrance was higher in males (63.3%, 19 affected/30 carriers) than in females (33.3%, five affected/15 carriers; p = 0.057).    Figure 4 shows the pedigrees of the three index cases (17.6%) found to be compound heterozygous for SCN5A mutations, with an onset of symptoms in early childhood (younger than age 4 years), overlap syndrome and prolonged QTc intervals. Case 8 (p.Trp1345_Ser1349delinsPhe/ p.Pro1730Leu) was a severely affected six year-old girl who showed episodes of palpitations, fatigue and documented bradycardia since age three years, and was diagnosed with SSS, PCCD, VT, type 1 BrS and showed a prolonged QTc interval (495ms). During hospital stay she suffered various arrhythmic events including atrial flutter, atrial standstill, sustained ventricular tachycardia, ventricular fibrillation, and notably, bidirectional ventricular tachycardia and two cardiac arrest episodes with successful reanimation after cardio-pulmonary resuscitation. Both parents were asymptomatic, however a p.Trp1345_Ser1349delinsPhe heterozygous paternal first cousin showed a type 1 BrS ECG pattern. In addition, four asymptomatic relatives (II-7, III-5, III-6 and IV-3) carried the p.Pro1730Leu variant.

Compound Heterozygosity for SCN5A Mutations
Case 10 (p.Arg34fs*60/p.Arg1195His) was a 22 month-old male toddler [27], who was admitted to the emergency room with febrile seizures and syncope and was diagnosed with SSS and VT, and a prolonged QTc interval (maximum QTc = 519 ms) with no apparent intraventricular conduction defect. The boy showed signs of severe neurological injury and died shortly after being admitted. He inherited the p.Arg1195His variant from his father, the p.Arg34fs*60 variant from his mother, and both parents were asymptomatic. Penetrance of individual mutations was estimated when five or more heterozygous carriers were found. A total of 14 individuals of a single kindred carried the p.Thr1708Asn mutation (eight male and six female); penetrance for arrhythmogenic phenotypes was 6/8 (75.0%) in males (three with overlap syndrome, two with BrS and one with sudden cardiac death at age 6). No arrhythmic phenotypes were observed in available female carriers, but 3/4 (75%) showed minor ECG abnormalities such as AV block (PR < 200 ms) and/or prolonged QTc interval (>470 ms, range 478-488 ms). Moreover, a total of seven individuals from a single kindred carried the p.Leu1821fs*10 mutation. Four were female (two with PCCD and two asymptomatic) and three were male (one with PCCD, one with overlap syndrome and one asymptomatic). Finally, five individuals from three different kindreds were heterozygous for the p.Ser1710Leu mutation, an asymptomatic female and four males (one asymptomatic, one each with overlap syndrome, PCCD and BrS). Figure 4 shows the pedigrees of the three index cases (17.6%) found to be compound heterozygous for SCN5A mutations, with an onset of symptoms in early childhood (younger than age 4 years), overlap syndrome and prolonged QTc intervals. Case 8 (p.Trp1345_Ser1349delinsPhe/ p.Pro1730Leu) was a severely affected six year-old girl who showed episodes of palpitations, fatigue and documented bradycardia since age three years, and was diagnosed with SSS, PCCD, VT, type 1 BrS and showed a prolonged QTc interval (495ms). During hospital stay she suffered various arrhythmic events including atrial flutter, atrial standstill, sustained ventricular tachycardia, ventricular fibrillation, and notably, bidirectional ventricular tachycardia and two cardiac arrest episodes with successful reanimation after cardio-pulmonary resuscitation. Both parents were asymptomatic, however a p.Trp1345_Ser1349delinsPhe heterozygous paternal first cousin showed a type 1 BrS ECG pattern. In addition, four asymptomatic relatives (II-7, III-5, III-6 and IV-3) carried the p.Pro1730Leu variant.

Compound Heterozygosity for SCN5A Mutations
Genes 2022, 13, x FOR PEER REVIEW 10 of Finally, case 13 (p.Asp1741Glyfs*48/p.Val240Met) presented with syncope at age 4 year and was diagnosed with SSS, PCCD and recurrent monomorphic VT. The QT interval o ECG was prolonged (QTc = 630 ms). The mother (II-9) and two siblings (III-1 and IIIhad the Val240Met variant and were asymptomatic, while the father wa p.Asp1741Glyfs*48 heterozygous and was also asymptomatic.  Case 10 (p.Arg34fs*60/p.Arg1195His) was a 22 month-old male toddler [27], who was admitted to the emergency room with febrile seizures and syncope and was diagnosed with SSS and VT, and a prolonged QTc interval (maximum QTc = 519 ms) with no apparent intraventricular conduction defect. The boy showed signs of severe neurological injury and died shortly after being admitted. He inherited the p.Arg1195His variant from his father, the p.Arg34fs*60 variant from his mother, and both parents were asymptomatic. Finally, case 13 (p.Asp1741Glyfs*48/p.Val240Met) presented with syncope at age 4 years, and was diagnosed with SSS, PCCD and recurrent monomorphic VT. The QT interval on ECG was prolonged (QTc = 630 ms). The mother (II-9) and two siblings (III-1 and III-2) had the Val240Met variant and were asymptomatic, while the father was p.Asp1741Glyfs*48 heterozygous and was also asymptomatic.

Discussion
The field of cardiovascular genetics is rapidly evolving and has greatly contributed to the understanding of primary electrical heart disease. However, this field faces an enormous challenge when it comes to the integration of genetics, functional studies, diagnosis, prognosis, and management [30]. The growing list of SCN5A channelopathy and overlap syndrome reports in the medical literature confirms that incomplete penetrance, male predominance and variable expressivity are characteristic of the disease, attributed to both genetic and non-genetic factors [7]. In the present study, all families with more than one SCN5A mutation carrier showed this variable expressivity, involving differences in age of onset (childhood-adulthood), arrhythmogenic phenotypes and disease severity. Similarly, apparently unrelated patients sharing the same mutation (Cases 1, 2 and 3 for p.Ser1710Leu) also showed variable expressivity regarding arrhythmogenic phenotypes. As expected, overall penetrance and penetrance of individual mutations was higher in male than in female individuals.
To our knowledge only one prospective study of children with SCN5A mutations included 442 neonates, where most showed no ECG alterations at birth (44.3%) and the most frequent arrhythmogenic phenotype was isolated PCCD (25.6%), followed by overlap syndrome (15%), LQTS (10.6%) and BrS (1.8%) [15]. The present study includes only pediatric patients who were symptomatic and required specialized medical assistance at the Ignacio Chávez National Institute of Cardiology. In agreement with the study in neonates, isolated LQTS and isolated BrS were the least frequent phenotypes (5.9% each), however among the symptomatic children of the present study, the most frequent phenotype was overlap syndrome (70.6%).
The high frequency of children with compound heterozygous SCN5A mutations in our group of symptomatic children (17.6%) is noteworthy. Compound heterozygosity is infrequent in SCN5A channelopathies, reported only in 13/2111 (0.62%) of BrS patients [31], and in 0.7% of neonates with SCN5A mutations [15]. Moreover, among ten individual SCN5A compound heterozygous case reports [12,13,27,[32][33][34][35][36][37][38], only two cases reported adult age of onset [35,36], and 6/8 childhood onset cases had a truncating mutation combined with a missense mutation [12,27,[32][33][34]. All three of our compound heterozygous cases (cases 8, 10 and 13) carried an inferred null and a missense mutation, had a severe overlap syndrome with early childhood onset (<4 years), and NFCA events occurred in two of these cases. Notably, case 8 had documented polymorphic ventricular tachycardia and bidirectional ventricular tachycardia, considered a calcium-handling arrhythmia. To our knowledge, there is only one report of a SCN5A mutation (p.T1857I) in a family with multiple sudden cardiac deaths where afflicted probands presented with atrial and ventricular arrhythmias including bidirectional VT [39]. Functional studies in heterologous cells and the use of O'Hara Rudy and Grandi models [40,41] suggested the mutation caused right-shifted voltage-dependence of both activation (gain of function, GOF) and inactivation (loss of function, LOF), with a net gain-of-function in the Na V 1.5 gating, an increased window current and resultant ventricular tissue after depolarization.
Among the genetic modifiers contributing to variable expressivity and incomplete penetrance, studies in heterologous cells have shown common SCN5A polymorphisms p.H558R and p.del1077Q not only affect electrophysiological properties of Na V 1.5, but may also modulate the effects of co-existing disease-causing mutations [42][43][44]. In this regard, a functional study in HEK293 cells showed that the p.Thr1708Asn mutation decreased the peak I Na current and increased the late I NaL current, decreasing the peak I Na current even further when found in cis with the p.H558R polymorphism [25]. Segregation analysis of the p.Thr1708Asn kindred (Figure 3) showed that both variants were in fact on the same allele, as two of the p.Thr1708Asn carriers (IV-13 and IV-14) were p.558R homozygous (data not shown), compatible with the severe phenotype observed in several affected males. Unfortunately, the number of individuals with SCN5A mutations was insufficient to assess the possible association of common polymorphisms with clinical severity in our series.
The consequences of most of the mutations identified in this group of patients have been studied in heterologous ion channel expression systems [16][17][18][19][20][21][22][23][24]. However, because of the many consequences of mutations on electrophysiological phenotypes (early and late inward Na currents, activation or inactivation properties, interactions with other proteins, response to adrenergic or cholinergic stimuli, response to anti-arrhythmic drugs, etc.), and because different functional phenotypes often coexist, labeling variants as GOF and/or LOF may be an oversimplification. Particularly, because it has been proposed that Na V 1.5 channels assemble as dimers [3], compound heterozygous mutations may require the biophysical characterization of both mutations separately and in coexpression. In this regard, previous functional studies of the mutations of case 10 showed the frame-shifted and prematurely truncated peptide SCN5A-Arg34fs*60 produced no current, while SCN5A-Arg1195His had a normal peak and late current but abnormal voltage-dependent gating parameters. Interestingly, co-expression of both variants led to a significant increase in late I NaL current [27]. Functional studies of SCN5A variants found in case 8 (p.Trp1345_Ser1349delinsPhe/p.Pro1730Leu) and in case 13 (p.Asp1741Glyfs*48/ p.Val240Met), both individually and in co-expression, are pending, as are functional studies of three of the missense variants here identified (p.Arg811Cys; p.Pro1730Leu, p.Ala1778Asp). While these models have a number of shortcomings, as they do not closely reproduce human heart physiology and clinical manifestations [45,46], functional and pharmacological studies in induced pluripotent cell (iPSC)-derived cardiomyocytes have overcome several of these shortcomings and have become a promising option for precision medicine [47]. These functional studies, together with careful and detailed clinical characterization of patients and apparently asymptomatic mutation carriers, are crucial to understand the complexities of SCN5A channelopathies.
In conclusion, SCN5A-disease associated phenotypes occurring in the pediatric age were characterized mainly by overlap syndrome, sick sinus syndrome, cardiac conduction disease and ventricular tachycardia. It is important to recognize these phenotypes as potential markers of sodium channel disease, and to avoid the use of sodium channel blockers which can exacerbate the phenotype or even cause irreversible ventricular arrhythmias in the setting of pathogenic variants affecting SCN5A.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study or their parents or legal guardians. Data Availability Statement: Data available on request from the corresponding authors, due to restrictions on patient privacy as established in the informed consent.