SCN5A Mutation Research Articles

Novel variant of the glycerol-3-phosphate dehydrogenase-1 Like (GPD1-L) gene in Japanese Brugada syndrome patients

Abstract Background The incidence of Brugada syndrome (BrS) varies among racial groups. Several studies reported Glycerol-3-Phosphate Dehydrogenase 1-Like (GPD1-L) gene is associated with BrS. However, most of these studies were reported from Western countries, so the evidence about GPD1-L mutation is limited especially among Asian BrS patients. This study aimed to search for rare variants in GPD1-L among Japanese BrS patients and to investigate the pathogenicity. Method We performed whole-exome sequencing for patients with Brugada type 1 ECG pattern from Japanese multicenter BrS cohort consisting of SCN5A-negative BrS probands (n=288) and controls (n=372). We conducted patch-clamp study in human embryonic kidney (HEK) 293 cells cotransfected with the wild-type sodium channel (SCN5A) and wild-type or mutant GPD1-L expression plasmid. Results We identified a rare variant in GPD1-L, p.D262N (c.784g>a) in 2 of 288 BrS probands, which was not identified in 372 controls. The minor allele frequency of the variant is 0.0014% in the Genome Aggregation Database. One proband was a 49-year-old man and the other was 34-year-old man who both developed a ventricular fibrillation. ECGs of both probands showed Brugada Type 1 pattern after administration of the pilsicainide. In functional study, coexpression of D262N GPD1-L with SCN5A in HEK293 cells significantly reduced inward sodium currents compared with wild-type GPD1-L. Additionally, inward sodium currents with D262N were similar to those with A280V GPD1-L, which was associated with BrS in previous reports (Figure). Also, several pathogenicity prediction programs, such as SIFT (score: 0.031) and PolyPhen2 (score: 0.937) predicted deleterious effects of GPD1-L D262N. Conclusion We identified a rare variant in GPD1-L at the rate of 0.7% in Japanese BrS patients without SCN5A mutations. GPD1-L, p.D262N reduces inward sodium currents and may be a novel susceptible variant for BrS in the Japanese population. Funding Acknowledgement Type of funding sources: None. Figure 1. Current–voltage curve

Family screening in brugada syndrome patients

Abstract Background Brugada syndrome (BS) is a rare inherited channelopathy associated with sudden cardiac death (SCD) and family screening (FS) of index patients (pts) is recommended. However, data about pts identified through FS is lacking. Aim To compare index pts to non-index pts identified through systematic FS. Methods Single-center retrospective study of BS pts followed by the Arrhythmology Department. FS was offered to 1st degree relatives of all index pts through primary care services and a once-weekly voluntary open appointment. Genetic counselling was performed when indicated. Index and non-index pts were compared regarding baseline characteristics and events during the follow-up (syncope of probable arrhythmic origin, ventricular tachycardia/ventricular fibrillation (VT/VF) and SCD). Results We included 165 pts (61% males, mean age 47±15 years) and 72 (44%) were identified through FS. Non-index pts were diagnosed at a younger age (42±14 vs 51±14 years, p <.001), were more often female (57% vs 25%, p<.001), were diagnosed predominantly through provocative test with ajmaline/flecainide (88% vs 47%, p<.001) and had less documented spontaneous type 1 ECG pattern (17% vs 59%, p<.001). A type 2 pattern was identified in 18 (25%) non-index pts. Genetic testing was performed in 38 (53%) non-index pts: 6 had a pathogenic SCN5A mutation, 18 a likely pathogenic SCN5A mutation and 12 a mutation of uncertain significance. At diagnosis, 24 (33%) non-index pts had history of syncope, 3 (4%) had nocturnal agonal respiration and 11 (15%) had palpitations with no differences between both groups (p=.119). Non-index pts were less likely to implant a cardioverterdefibrillator (14% vs 38%, p=.001). During a median follow-up of 28 (IQR 16–41) months, 10 (6%) pts had an event - 2 (3%) in the non-index group (2 syncope) and 8 (9%) in the index group (1 syncope; 7 VT/VF) - with no significative differences between groups (p=.432). There were nocardiovascular deaths. Conclusions FS identified a considerable proportion of BS pts. Non-index pts were younger at the time of the diagnosis and had less spontaneous type 1 pattern. No differences were found in events between index and non-index pts, however, the event rate was low. Systematic FS can identify individuals at risk of SCD earlier, allowing close monitoring and, when indicated, appropriate treatment. Funding Acknowledgement Type of funding sources: None.

Transcriptional profiles of genes related to electrophysiological function in scn5a+/− murine hearts

Abstract Background The Scn5a gene encodes the major pore-forming Nav1.5 (α) subunit, of the voltage-gated Na+ channel in cardiomyocytes. The key role of Nav1.5 in action potential initiation and propagation in both atria and ventricles predisposes organisms lacking Scn5a or carrying Scn5a mutations to cardiac arrhythmogenesis. Loss-of-function Nav1.5 genetic abnormalities account for many cases of the human arrhythmic disorder Brugada Syndrome (BrS) and related conduction disorders. A murine model with a heterozygous Scn5a deletion recapitulates many electrophysiological phenotypes of BrS. Purpose This study examines relationships between its Scn5a+/− genotype, resulting transcriptional changes and the consequent phenotypic presentations of BrS. Methods Atrial and ventricular tissue samples were obtained from aged (11±3 months) wild-type (WT) and homozygous Scn5a+/− mice. Quantitative PCR was used to examine transcription of 60 genes underlying cardiac tissue excitability. Results Of selected protein-coding genes related to cardiomyocyte electrophysiological or homeostatic function, concentrations of mRNA transcribed from 15 differed significantly from WT. Despite halved apparent ventricular Scn5a transcription heterozygous deletion did not significantly downregulate its atrial expression, raising possibilities of atria-specific feedback mechanisms. Most of the remaining 14 genes whose expression differed significantly between WT and Scn5a+/− animals involved Ca2+homeostasis specifically in atrial tissue, with no overlap with any ventricular changes. All significant differences shown by the atria and ventricles were up- and downregulations, respectively. Conclusion This investigation demonstrates the value of future experiments exploring for and clarifying links between transcriptional control of Scn5a and of genes whose protein products coordinate Ca2+ regulation and examining their possible roles in BrS. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University of Surrey; Medical Research Council

Correction of the hypomorphic Gabra2 splice site variant in mouse strain C57BL/6J modifies the severity of Scn8a encephalopathy.

SummaryDe novo gain-of-function mutations of SCN8A are a significant cause of developmental and epileptic encephalopathy (DEE) (MIM: 614558). The severely affected individuals exhibit refractory seizures, developmental delay, and cognitive disabilities, often accompanied by impaired movement. Individuals with the identical SCN8A variant often differ in clinical course, suggesting a role for modifier genes in disease severity. In a previous study we demonstrated genetic linkage between a hypomorphic mutation in the Gabra2 gene and seizure severity in a mouse model of the human SCN8A pathogenic variant p.Arg1872Trp. Homozygosity for the hypomorphic Gabra2 mutation was associated with early seizure onset and shortened lifespan. We have now confirmed Gabra2 as the modifier gene using a knock-in allele that corrects the splice site variant in strain C57BL/6J. Correction of the Gabra2 variant restores transcript abundance, increases the age of seizure onset, and extends survival of the Scn8a mutant mice. GABRA2 encodes the α2 subunit of the GABAA receptor that provides inhibitory input to dendrites and the the axon initial segment of excitatory neurons. Quantitative variation in human GABAA receptor expression could contribute to variation in the severity of genetic epilepsies and suggests a potential therapeutic intervention.

Functional Analysis of SCN5A Genetic Variants Associated with Brugada Syndrome

Background: Brugada syndrome (BrS) is a rare inherited cardiac arrhythmia with increased risk of sudden cardiac death. Mutations in gene SCN5A, which encodes the α-subunit of cardiac voltage-gated sodium channel Na_V1.5, have been identified in over 20% of patients with BrS. However, only a small fraction of Na_V1.5 variants, which are associated with BrS, are characterized in electrophysiological experiments. Results: Here we explored variants V281A and L1582P, which were found in our patients with BrS, and variants F543L and K1419E, which are reportedly associated with BrS. Heterologous expression of the variants in CHO-K1 cells and the Western blot analysis demonstrated that each variant appeared at the cell surface. We further measured sodium current in the whole-cell voltage clamp configuration. Variant F543L produced robust sodium current with a hyperpolarizing shift in the voltage dependence of steady-state fast inactivation. Other variants did not produce detectable sodium currents, indicating a complete loss of function. In a recent cryoEM structure of the hNa_V1.5 channel, residues V281, K1419, and L1582 are in close contacts with residues whose mutations are reportedly associated with BrS, indicating functional importance of respective contacts. Conclusions: Our results support the notion that loss of function of Na_V1.5 or decrease of the channel activity is involved in the pathogenesis of BrS.

Mutations associated with hypokalemic periodic paralysis: from hotspot regions to complete analysis of CACNA1S and SCN4A genes.

Familial periodic paralyses (PPs) are inherited disorders of skeletal muscle characterized by recurrent episodes of flaccid muscle weakness. PPs are classified as hypokalemic (HypoPP), normokalemic (NormoPP), or hyperkalemic (HyperPP) according to the potassium level during the paralytic attacks. HypoPP is an autosomal dominant disease caused by mutations in the CACNA1S gene, encoding for Cav1.1 channel (HypoPP-1), or SCN4A gene, encoding for Nav1.4 channel (HypoPP-2). In the present study, we included 60 patients with a clinical diagnosis of HypoPP. Fifty-one (85%) patients were tested using the direct sequencing (Sanger method) of all reported HypoPP mutations in CACNA1S and SCN4A genes; the remaining 9 (15%) patients were analyzed through a next-generation sequencing (NGS) panel, including the whole CACNA1S and SCN4A genes, plus other genes rarely associated to PPs. Fifty patients resulted mutated: 38 (76%) cases showed p.R528H and p.R1239G/H CACNA1S mutations and 12 (24%) displayed p.R669H, p.R672C/H, p.R1132G/Q, and p.R1135H SCN4A mutations. Forty-one mutated cases were identified among the 51 patients managed with Sanger sequencing, while all the 9 cases directly analyzed with the NGS panel showed mutations in the hotspot regions of SCN4A and CACNA1S. Ten out of the 51 patients unresolved through the Sanger sequencing were further analyzed with the NGS panel, without the detection of any mutation. Hence, our data suggest that in HypoPP patients, the extension of genetic analysis from the hotspot regions using the Sanger method to the NGS sequencing of the entire CACNA1S and SCN4A genes does not lead to the identification of new pathological mutations.

New Challenges Resulting From the Loss of Function of Nav1.4 in Neuromuscular Diseases.

The voltage-gated sodium channel Nav1.4 is a major actor in the excitability of skeletal myofibers, driving the muscle force in response to nerve stimulation. Supporting further this key role, mutations in SCN4A, the gene encoding the pore-forming α subunit of Nav1.4, are responsible for a clinical spectrum of human diseases ranging from muscle stiffness (sodium channel myotonia, SCM) to muscle weakness. For years, only dominantly-inherited diseases resulting from Nav1.4 gain of function (GoF) were known, i.e., non-dystrophic myotonia (delayed muscle relaxation due to myofiber hyperexcitability), paramyotonia congenita and hyperkalemic or hypokalemic periodic paralyses (episodic flaccid muscle weakness due to transient myofiber hypoexcitability). These last 5 years, SCN4A mutations inducing Nav1.4 loss of function (LoF) were identified as the cause of dominantly and recessively-inherited disorders with muscle weakness: periodic paralyses with hypokalemic attacks, congenital myasthenic syndromes and congenital myopathies. We propose to name this clinical spectrum sodium channel weakness (SCW) as the mirror of SCM. Nav1.4 LoF as a cause of permanent muscle weakness was quite unexpected as the Na+ current density in the sarcolemma is large, securing the ability to generate and propagate muscle action potentials. The properties of SCN4A LoF mutations are well documented at the channel level in cellular electrophysiological studies However, much less is known about the functional consequences of Nav1.4 LoF in skeletal myofibers with no available pertinent cell or animal models. Regarding the therapeutic issues for Nav1.4 channelopathies, former efforts were aimed at developing subtype-selective Nav channel antagonists to block myofiber hyperexcitability. Non-selective, Nav channel blockers are clinically efficient in SCM and paramyotonia congenita, whereas patient education and carbonic anhydrase inhibitors are helpful to prevent attacks in periodic paralyses. Developing therapeutic tools able to counteract Nav1.4 LoF in skeletal muscles is then a new challenge in the field of Nav channelopathies. Here, we review the current knowledge regarding Nav1.4 LoF and discuss the possible therapeutic strategies to be developed in order to improve muscle force in SCW.

Clinical characteristics and risk of arrhythmic events in patients younger than 12 years diagnosed with Brugada syndrome.

Brugada syndrome (BrS) is an inheritable disease with an increased risk of sudden cardiac death. Although several score systems have been proposed, the management of children with BrS has been inconsistently described. The purpose of this study was to identify the characteristics, outcome, and risk factors associated with cardiovascular and arrhythmic events (AEs) in children younger than 12 years with BrS. In this single-center case series, all children with spontaneous or drug/fever-induced type 1 Brugada electrocardiographic (ECG) pattern and younger than 12 years at the time of diagnosis were enrolled. Forty-three patients younger than 12 years at the time of diagnosis were included. The median follow-up was 3.97 years (interquartile range 2-12 years). In terms of first-degree atrioventricular block, premature beats, nonmalignant AEs, malignant AEs, and episodes of syncope, no significant differences were observed either between patients with spontaneous and drug/fever-induced type 1 Brugada ECG pattern or between female and male patients (except a significant difference between female and male patients for first-degree atrioventricular block). A higher incidence of malignant AEs was observed in patients with syncope (3 of 8 [37.5%] vs 0 of 35 [0%]; P = .005) than in patients without syncope. SCN5A mutations were associated with a higher occurrence of malignant AEs (3 of 14 [21.4%] vs 0 of 25 [0%]; P = .04) compared with no SCN5A mutations. A spontaneous type 1 Brugada ECG pattern is not associated with a higher incidence of syncope, first-degree atrioventricular block, premature beats, nonmalignant AEs, and malignant AEs than the drug/fever-induced type 1 Brugada ECG pattern. Syncope events are correlated with an increased incidence of malignant AEs. Moreover, SCN5A mutations are associated with a higher occurrence of malignant AEs.

Nodular heterotopia: a rare finding in patients with epilepsy and SCN1A mutation.

A 25-year-old man had started febrile seizures from 6 months of age and developed important neurodevelopmental delay. At four years presented with status epilepticus and sequentially with refractory epilepsy. He presents with motor delay, behavior disorder and absent expressive language. Brain MRI showed periventricular nodular heterotopias (Figures and ). The Next Generation Sequencing (NGS) came with a rare frameshift mutation in heterozygosis for SCN1A gene. The presence of periventricular heterotopias in association with SCN1A mutation is rare¹. In literature, we [...]

Antiepleptics'drug response and SCN2A mutations in Sudanese patients with idiopathic epilepsy

Sodium channel blockers are widely used for epilepsy in Sudan however some patients don't respond to them. Since SCN2A mutations have a role in idiopathic epilepsy why does resistance develop? Aim: To detect SCN2A mutations and study their relation to Antiepileptic drug response.

Identification of a SCN5A founder mutation causing sudden death, Brugada syndrome, and conduction blocks in Southern Italy.

The genetic architecture of Brugada syndrome (BrS) is emerging as an increasingly complex area of investigation. The identification of genetically homogeneous populations can provide mechanistic insights and improve genotype-phenotype correlation. To characterize and define the clinical implications of a novel BrS founder mutation. Using a haplotype-based approach we investigated whether 2 SCN5A genetic variants could derive from founder events. Single nucleotide polymorphisms were genotyped in 201 subjects, haplotypes reconstructed, and mutational age estimated. Clinical phenotypes and historical records were collected. A SCN5A variant (c.3352C>T; p.Gln1118Ter) was identified in 3 probands with BrS originating from south Italy. The same mutation was identified in a proband from central Italy and in 1 U.S. resident subject with Italian ancestry. The 5 individuals carried a common core haplotype, whose frequency was extremely low in local noncarrier probands and in population controls (0%-6.06%). The clinical presentation included multigenerational dominant transmission of Brugada electrocardiographic pattern, high incidence of sudden cardiac death (SCD), and cardiac conduction defects (CCD). We reconstructed 7-generation pedigrees with common geographic origin. Variant's age estimates suggested that origin of the p.Gln1118Ter dates back 76 generations (95% confidence interval: 28-200). A second SCN5A variant (c.5350G>A; p.Glu1784Lys) identified in the region did not show similar founder signal. p.Gln1118Ter is a novel BrS/CCD/SCD founder mutation. We illustrate how these findings provide insights on the inheritance patterns and phenotypes associated with SCN5A mutation.

Transcriptional profiles of genes related to electrophysiological function in Scn5a+/- murine hearts.

The Scn5a gene encodes the major pore‐forming Nav1.5 (α) subunit, of the voltage‐gated Na+ channel in cardiomyocytes. The key role of Nav1.5 in action potential initiation and propagation in both atria and ventricles predisposes organisms lacking Scn5a or carrying Scn5a mutations to cardiac arrhythmogenesis. Loss‐of‐function Nav1.5 genetic abnormalities account for many cases of the human arrhythmic disorder Brugada syndrome (BrS) and related conduction disorders. A murine model with a heterozygous Scn5a deletion recapitulates many electrophysiological phenotypes of BrS. This study examines the relationships between its Scn5a +/− genotype, resulting transcriptional changes, and the consequent phenotypic presentations of BrS. Of 62 selected protein‐coding genes related to cardiomyocyte electrophysiological or homeostatic function, concentrations of mRNA transcribed from 15 differed significantly from wild type (WT). Despite halving apparent ventricular Scn5a transcription heterozygous deletion did not significantly downregulate its atrial expression, raising possibilities of atria‐specific feedback mechanisms. Most of the remaining 14 genes whose expression differed significantly between WT and Scn5a +/− animals involved Ca2+ homeostasis specifically in atrial tissue, with no overlap with any ventricular changes. All statistically significant changes in expression were upregulations in the atria and downregulations in the ventricles. This investigation demonstrates the value of future experiments exploring for and clarifying links between transcriptional control of Scn5a and of genes whose protein products coordinate Ca2+ regulation and examining their possible roles in BrS.

High FGF-21 level in a cohort of 22 patients with Dravet Syndrome-Possible relationship with the disease outcomes.

ObjectiveDravet syndrome (DS) is a severe and intractable form of epilepsy with prolonged seizures which may evolve to other seizure types and associated with mild‐to‐severe intellectual disabilities. Fibroblast growth factor 21 (FGF‐21) is a stress hormone mediating metabolic and oxidative stress and circulating level of FGF‐21 had been shown to increase in some patients with impairment of oxidative phosphorylation in muscles. In DS, FGF‐21 is of interest for further study as mitochondrial oxidative stress was identified previously in patients.MethodsPlasma FGF‐21 levels were compared between 22 DS patients and 22 normal controls, and their clinical characteristics of DS patients at the time of plasma sampling were studied retrospectively. Besides, the relationships of FGF‐21 level with intellectual development, seizure frequency, valproate treatment, and types of SCN1A mutations were analyzed. Logarithmic transformation of FGF‐21 levels was performed before comparison and statistical analysis.ResultsMean of log10 FGF‐21 level was significantly higher in DS patients when comparing with normal controls (P = .0042). Mean of log10 FGF‐21 level was significantly higher in DS patients with normal‐to‐mild ID versus mild‐to‐severe ID (P = .0193) and with valproate treatment versus without valproate treatment (P = .015). No significant difference was shown in FGF‐21 level in DS patients with missense versus truncating SCN1A variants, and no correlation could be demonstrated between seizure frequency and FGF‐21 level.SignificanceSignificantly higher level of plasma FGF‐21 was identified in DS patients. The high FGF‐21 levels were shown to be associated with developmental outcome and valproate treatment. These results support further investigation on the relationship of FGF‐21 with the clinical outcomes of DS and other related mechanism which is important for possible therapeutic development for this epileptic encephalopathy.

CHANNELOPATHIES AND RELATED DISORDERS: EP.226 Muscle fat fraction, cross-sectional area and contractility in patient with skeletal muscle sodium channel disorders

We present a cross-sectional, observational study that investigates the presence of muscle degeneration in patients with skeletal muscle sodium channel disorders (SCN4A gene mutation). This includes paramyotonia congenita (PMC), hyperkalemic periodic paralysis (HyperPP) and potassium-aggravated myotonia (PAM). Little is known about quantitative fat infiltration and contractility of patients with these disorders. So far, we have studied 13 patients (30 planned), older than 18 years and diagnosed with SCN4A mutations using dynamometry testing and quantitative (Dixon) muscle MRI.

Genetic Analysis of Children with Dravet Syndrome in a Resourcelimited Setting

Objective: To identify the common causal gene mutations in Thai children with the Dravet (DS) phenotype, using single gene analysis.Material and Methods: The study was carried out on 20 DS patients at Siriraj Hospital, Bangkok, Thailand. Sanger sequencing of the Voltage-Gated Sodium Channel Alpha Subunit 1 (SCN1A) gene was conducted in all patients. In SCN1A-negative patients, multiplex ligation-dependent probe amplification of the SCN1A gene was performed in all cases; however, direct sequencing of the Protocadherin-19 (PCDH19) gene was analyzed in girls only.Results: Fourteen (70.0%) DS patients were found to carry pathogenic SCN1A mutations, with 6 novel mutations. In SCN1A-negative patients; 1 of the 4 girl patients (25.0%) had a novel PCDH19 mutation, while none of the 6 patients had a large deletion or duplication in the SCN1A gene.Conclusion: The SCN1A gene is the most common causative mutation in Thai children with DS phenotype. This study emphasizes the benefit of Sanger sequencing of the SCN1A gene in resource-limited countries to aid in making appropriate therapeutic decisions.

Enhanced slow inactivation contributes to dysfunction of a recurrent SCN2A mutation associated with developmental and epileptic encephalopathy.

The recurrent SCN2A mutation R853Q is associated with developmental and epileptic encephalopathy with typical onset after the first months of life. Heterologously expressed R853Q channels exhibit an overall loss-of-function as a result of multiple defects in time- and voltage-dependent channel properties. A previously unrecognized enhancement of slow inactivation is conferred by the R853Q mutation and is a major driver of loss-of-function. Enhanced slow inactivation is potentiated in the canonical splice isoform of the channel and this may explain the later onset of symptoms associated with R853Q. Mutations in voltage gated sodium (NaV ) channel genes, including SCN2A (encoding NaV 1.2), are associated with diverse neurodevelopmental disorders with or without epilepsy that present clinically with varying severity, age-of-onset and pharmacoresponsiveness. We examined the functional properties of the most recurrent SCN2A mutation (R853Q) to determine whether developmentally-regulated alternative splicing impacts dysfunction severity and to investigate effects of the mutation on slow inactivation. We engineered the R853Q mutation into neonatal and adult NaV 1.2 splice isoforms. Channel constructs were heterologously co-expressed in HEK293T cells with human β1 and β2 subunits. Whole-cell patch clamp recording was used to compare time- and voltage-dependent properties of mutant and wild-type channels. The R853Q mutation exhibits an overall loss-of-function attributed to multiple functional defects including a previously undiscovered enhancement of slow inactivation. The mutation exhibited altered voltage dependence of activation and inactivation, slower recovery from inactivation and decreased channel availability during high-frequency depolarizations. More notable were effects on slow inactivation, including a 10-fold slower rate of recovery from slow inactivation exhibited by mutant channels. The impairments in fast inactivation properties were more severe in the neonatal splice isoform, whereas slow inactivation was more pronounced in the splice isoform of the channel expressed predominantly in later childhood. Enhanced later-onset slow inactivation may be a primary driver of the later onset of neurological features associated with this mutation.

Comparative analysis of the autism‑related variants between different autistic children in a family pedigree.

The present study aimed to provide evidence for the genetic heterogeneity of familial autism spectrum disorder (ASD), which might help to improve our understanding of the complex polygenic basis of this disease. Whole-exome sequencing (WES) was performed on two autistic children in a family pedigree, and reasonable conditions were set for preliminarily screening variant annotations. Sanger sequencing was used to verify the preliminarily screened variants and to determine the possible sources. In addition, autism-related genes were screened according to autism databases, and their variants were compared between two autistic children. The results showed that there were 21 genes respectively for autistic children IV2 and IV4, preliminarily screened from all variants based on the harmfulness (high) and quality (high or medium) of the variants, as well as the association between mutant genes and autism in human gene mutation database. Furthermore, candidate autism-related genes were screened according to the evidence score of >4 in the Autism KnowledgeBase (AutismKB) database or ≥3 in the AutDB database. A total of 11 and 10 candidate autism-related genes were identified in the autistic children IV2 and IV4, respectively. Candidate genes with an evidence score of >16 in AutismKB were credible autism-related genes, which included LAMC3, JMJD1C and CACNA1H in child IV2, as well as SCN1A, SETD5, CHD7 and KCNMA1 in child IV4. Other than the c.G1499A mutation of SCN1A, which is known to be associated with Dravet syndrome, the specific missense variant loci of other six highly credible putative autism-related genes were reported for the first time, to the best of the authors' knowledge, in the present study. These credible autism-related variants were inherited not only from immediate family members but also from extended family members. In summary, the present study established a reasonable and feasible method for screening credible autism-related genes from WES results, which by be worth extending into clinical practice. The different credible autism-related genes between the two autistic children indicated a complex polygenic architecture of ASD, which may assist in the early diagnosis of this disease.

B-011-05 PANORAMIC OPTICAL MAPPING AND MICRO COMPUTED TOMOGRAPHY FOR IN-DEPTH 3D CHARACTERIZATION OF THE ARRHYTHMOGENIC SUBSTRATE IN MURINE HEARTS

Optical mapping of murine hearts is currently limited to the epicardium. Action potential duration (APD) heterogeneity is a key driver for initiation and persistence of reentry. When combined with micro CT, optical mapping can provide data on anatomical alterations and structural remodeling contributing to arrhythmogenesis. In this study, we developed a 3D visualization tool combining panoramic optical mapping (POM) with a convolutional neural network automatically segmenting the heart anatomy on micro CT for improved computational modeling and arrhythmia risk stratification in mice ex vivo. We performed high resolution POM with 4 simultaneous CMOS cameras on 17 Langendorff murine hearts: 7 control, 4 transaortic constriction (TAC), 5 transgenic (TG) SCN5A mutation at lidocaine binding site F1759A with spontaneous VF and 1 post LAD ligation. Activation and APD maps were projected onto 3D reconstructed cardiac silhouettes. We measured APD dispersion and the % of ventricular tissue exhibiting dispersion > 10 ms was analyzed. We then coregistered and overlaid the functional maps onto the reconstructed structural meshes obtained with the deep learning algorithm. The mean % of ventricular tissue with APD dispersion > 10 ms was significantly different (ANOVA, p<0.0001) across control, TAC, TG and LAD mice (0.6 ± 0.4 %, 4.8 ± 0.5 %, 12.9 ± 1.4 % and 13.9 %). Myocardial thickness on the TAC micro CT depicted LV hypertrophy, correlating with increased dispersion on POM. POM merged with micro CT allows us to visualize how anatomical markers correlate with arrhythmia mechanisms and thus offers improved understanding of disease progression.

B-PO02-204 EVIDENCE OF TWIN ATRIOVENTRICULAR NODES IN A PATIENT WITH AN SCN5A MUTATION AND STRUCTURALLY NORMAL HEART

Twin atrioventricular (AV) nodes are known to exist in association with complex congenital cardiac lesions. Genetic determinants of AV conduction are poorly understood, although reports have implicated a role of both SCN5A and MYH6 genes. Existence of twin AV nodes in a patient with a structurally normal heart has not previously been reported. We describe a patient with normal cardiac structure demonstrating evidence of twin AV nodes, later found to harbor an SCN5A mutation (D1595N). N/A Evaluation of a 21 week fetus revealed tachycardia, abdominal ascites, and pericardial effusion. Maternal history was significant for pacemaker placement due to intermittent complete heart block. Fetal echocardiogram confirmed supraventricular tachycardia (SVT), which resolved with maternal flecainide therapy. Delivered at term, the infant had a wide-complex bradycardic rhythm with ventricular ectopy concerning for flecainide toxicity. Echocardiogram revealed severely depressed biventricular function with normal intracardiac anatomy and normal abdominal/atrial situs. The rhythm developed into a regular wide-complex tachycardia with no discernable P-waves. Amiodarone resulted in slowing and narrowing of the QRS and emergence of sinus rhythm. By 2 weeks of age, biventricular function normalized. Telemetry revealed sinus rhythm with two distinct QRS morphologies and axes, but with consistent PR intervals and QRS durations. With sinus slowing, accelerated junctional rhythms matching both QRS morphologies were observed suggesting the presence of 2 distinct AV nodes (as opposed to aberrant AV conduction). A single junctional morphology became dominant but with widening of the QRS at higher sinus and junctional rates. Sotalol was initiated with subsequent Holters showing a dominant QRS morphology and a second junctional QRS morphology during sinus pauses. Genetic testing revealed an SCN5A mutation (p.D1595N), later confirmed in the patient’s mother. The presence of twin AV nodes in a living patient with normal cardiac anatomy has not been previously described. Discovery of an SCN5A mutation is an interesting finding, although a mechanistic explanation for its role in AV node formation remains elusive.

Modeling NaV1.1/SCN1A sodium channel mutations in a microcircuit with realistic ion concentration dynamics suggests differential GABAergic mechanisms leading to hyperexcitability in epilepsy and hemiplegic migraine.

Loss of function mutations of SCN1A, the gene coding for the voltage-gated sodium channel NaV1.1, cause different types of epilepsy, whereas gain of function mutations cause sporadic and familial hemiplegic migraine type 3 (FHM-3). However, it is not clear yet how these opposite effects can induce paroxysmal pathological activities involving neuronal networks’ hyperexcitability that are specific of epilepsy (seizures) or migraine (cortical spreading depolarization, CSD). To better understand differential mechanisms leading to the initiation of these pathological activities, we used a two-neuron conductance-based model of interconnected GABAergic and pyramidal glutamatergic neurons, in which we incorporated ionic concentration dynamics in both neurons. We modeled FHM-3 mutations by increasing the persistent sodium current in the interneuron and epileptogenic mutations by decreasing the sodium conductance in the interneuron. Therefore, we studied both FHM-3 and epileptogenic mutations within the same framework, modifying only two parameters. In our model, the key effect of gain of function FHM-3 mutations is ion fluxes modification at each action potential (in particular the larger activation of voltage-gated potassium channels induced by the NaV1.1 gain of function), and the resulting CSD-triggering extracellular potassium accumulation, which is not caused only by modifications of firing frequency. Loss of function epileptogenic mutations, on the other hand, increase GABAergic neurons’ susceptibility to depolarization block, without major modifications of firing frequency before it. Our modeling results connect qualitatively to experimental data: potassium accumulation in the case of FHM-3 mutations and facilitated depolarization block of the GABAergic neuron in the case of epileptogenic mutations. Both these effects can lead to pyramidal neuron hyperexcitability, inducing in the migraine condition depolarization block of both the GABAergic and the pyramidal neuron. Overall, our findings suggest different mechanisms of network hyperexcitability for migraine and epileptogenic NaV1.1 mutations, implying that the modifications of firing frequency may not be the only relevant pathological mechanism.