De Novo Loss-of-Function Mutations in CHD2 Cause a Fever-Sensitive Myoclonic Epileptic Encephalopathy Sharing Features with Dravet Syndrome

Dravet syndrome is a severe epilepsy syndrome characterized by infantile onset of therapy-resistant, fever-sensitive seizures followed by cognitive decline. Mutations in SCN1A explain about 75% of cases with Dravet syndrome; 90% of these mutations arise de novo. We studied a cohort of nine Dravet-syndrome-affected individuals without an SCN1A mutation (these included some atypical cases with onset at up to 2 years of age) by using whole-exome sequencing in proband-parent trios. In two individuals, we identified a de novo loss-of-function mutation in CHD2 (encoding chromodomain helicase DNA binding protein 2). A third CHD2 mutation was identified in an epileptic proband of a second (stage 2) cohort. All three individuals with a CHD2 mutation had intellectual disability and fever-sensitive generalized seizures, as well as prominent myoclonic seizures starting in the second year of life or later. To explore the functional relevance of CHD2 haploinsufficiency in an in vivo model system, we knocked down chd2 in zebrafish by using targeted morpholino antisense oligomers. chd2-knockdown larvae exhibited altered locomotor activity, and the epileptic nature of this seizure-like behavior was confirmed by field-potential recordings that revealed epileptiform discharges similar to seizures in affected persons. Both altered locomotor activity and epileptiform discharges were absent in appropriate control larvae. Our study provides evidence that de novo loss-of-function mutations in CHD2 are a cause of epileptic encephalopathy with generalized seizures.     

A putative disease-associated haplotype within the SCN1A gene in Dravet syndrome

Dravet syndrome (DS), previously known as severe myoclonic epilepsy of infancy, is one of the most severe forms of childhood epilepsy. DS is caused by a mutation in the neuronal voltage-gated sodium-channel alpha-subunit gene (SCN1A). However, 25–30% of patients with DS are negative for the SCN1A mutation screening, suggesting that other molecular mechanisms may account for these disorders. Recently, the first case of DS caused by a mutation in the neuronal voltage-gated sodium-channel beta-subunit gene (SCN1B) was also reported. In this report we aim to make the molecular analysis of the SCN1A and SCN1B genes in two Tunisian patients affected with DS. The SCN1A and SCN1B genes were tested for mutations by direct sequencing. No mutation was revealed in the SCN1A and SCN1B genes by sequencing analyses. On the other hand, 11 known single nucleotide polymorphisms were identified in the SCN1A gene and composed a putative disease-associated haplotype in patients with DS phenotype. One of the two patients with putative disease-associated haplotype in SCN1A had also one known single nucleotide polymorphism in the SCN1B gene. The sequencing analyses of the SCN1A gene revealed the presence of a putative disease-associated haplotype in two patients affected with Dravet syndrome.     

Altered Cardiac Electrophysiology and SUDEP in a Model of Dravet Syndrome

Objective

Dravet syndrome is a severe form of intractable pediatric epilepsy with a high incidence of SUDEP: Sudden Unexpected Death in epilepsy. Cardiac arrhythmias are a proposed cause for some cases of SUDEP, yet the susceptibility and potential mechanism of arrhythmogenesis in Dravet syndrome remain unknown. The majority of Dravet syndrome patients have de novo mutations in SCN1A, resulting in haploinsufficiency. We propose that, in addition to neuronal hyperexcitability, SCN1A haploinsufficiency alters cardiac electrical function and produces arrhythmias, providing a potential mechanism for SUDEP.

Methods

Postnatal day 15-21 heterozygous SCN1A-R1407X knock-in mice, expressing a human Dravet syndrome mutation, were used to investigate a possible cardiac phenotype. A combination of single cell electrophysiology and in vivo electrocardiogram (ECG) recordings were performed.

Results

We observed a 2-fold increase in both transient and persistent Na⁺ current density in isolated Dravet syndrome ventricular myocytes that resulted from increased activity of a tetrodotoxin-resistant Na⁺ current, likely Na_v1.5. Dravet syndrome myocytes exhibited increased excitability, action potential duration prolongation, and triggered activity. Continuous radiotelemetric ECG recordings showed QT prolongation, ventricular ectopic foci, idioventricular rhythms, beat-to-beat variability, ventricular fibrillation, and focal bradycardia. Spontaneous deaths were recorded in 2 DS mice, and a third became moribund and required euthanasia.

Interpretation

These data from single cell and whole animal experiments suggest that altered cardiac electrical function in Dravet syndrome may contribute to the susceptibility for arrhythmogenesis and SUDEP. These mechanistic insights may lead to critical risk assessment and intervention in human patients.     

Identification of SCN1A and PCDH19 Mutations in Chinese Children with Dravet Syndrome

Background

Dravet syndrome is a severe form of epilepsy. Majority of patients have a mutation in SCN1A gene, which encodes a voltage-gated sodium channel. A recent study has demonstrated that 16% of SCN1A-negative patients have a mutation in PCDH19, the gene encoding protocadherin-19. Mutations in other genes account for only a very small proportion of families. TSPYL4 is a novel candidate gene within the locus 6q16.3-q22.31 identified by linkage study.

Objective

The present study examined the mutations in epileptic Chinese children with emphasis on Dravet syndrome.

Methods

A hundred children with severe epilepsy were divided into Dravet syndrome and non-Dravet syndrome groups and screened for SCN1A mutations by direct sequencing. SCN1A-negative Dravet syndrome patients and patients with phenotypes resembling Dravet syndrome were checked for PCDH19 and TSPYL4 mutations.

Results

Eighteen patients (9 males, 9 females) were diagnosed to have Dravet syndrome. Among them, 83% (15/18) had SCN1A mutations including truncating (7), splice site (2) and missense mutations (6). The truncating/splice site mutations were associated with moderate to severe degree of intellectual disability (p<0.05). During the progression of disease, 73% (11/15) had features fitting into the diagnostic criteria of autism spectrum disorder and 53% (8/15) had history of vaccination-induced seizures. A novel PCDH19 p.D377N mutation was identified in one SCN1A-negative female patient with Dravet syndrome and a known PCDH19 p.N340S mutation in a female non-Dravet syndrome patient. The former also inherited a TSPYL4 p.G60R variant.

Conclusion

A high percentage of SCN1A mutations was identified in our Chinese cohort of Dravet syndrome patients but none in the rest of patients. We demonstrated that truncating/splice site mutations were linked to moderate to severe intellectual disability in these patients. A de novo PCDH19 missense mutation together with an inherited TSPYL4 missense variant were identified in a patient with Dravet syndrome.     

Proliferation of embryonic cardiomyocytes in zebrafish requires the sodium channel scn5Lab

In mice, homozygous deletion of the cardiac sodium channel Scn5a results in defects in cardiac morphology and embryonic death before robust sodium current can be detected. In zebrafish, morpholino knockdown of cardiac sodium channel orthologs scn5Laa and scn5Lab perturbs specification of precardiac mesoderm and inhibits growth of the embryonic heart. It is not known which developmental processes are perturbed by sodium channel knockdown and whether reduced cell number is from impaired migration of cardiac progenitors into the heart, impaired myocyte proliferation, or both. We found that embryos deficient in scn5Lab displayed defects in primary cardiogenesis specific to loss of nkx2.5, but not nkx2.7. We generated kaede reporter fish and demonstrated that embryos treated with anti‐scn5Lab morpholino showed normal secondary differentiation of cardiomyocytes at the arterial pole between 30 and 48 h post‐fertilization. However, while proliferating myocytes were readily detected at 48 hpf in wild type embryos, there were no BrdU‐positive cardiomyocytes in embryos subjected to anti‐scn5Lab treatment. Proliferating myocytes were present in embryos injected with anti‐tnnt2 morpholino to phenocopy the silent heart mutation, and absent in embryos injected with anti‐tnnt2 and anti‐scn5Lab morpholinos, indicating cardiac contraction is not required for the loss of proliferation. These data demonstrate that the role of scn5Lab in later heart growth does not involve contribution of the secondary heart field, but rather proliferation of cardiomyocytes, and appears unrelated to the role of the channel in cardiac electrogenesis. genesis 51:562–574. © 2013 Wiley Periodicals, Inc.     

Sporadic Infantile Epileptic Encephalopathy Caused by Mutations in PCDH19 Resembles Dravet Syndrome but Mainly Affects Females

Dravet syndrome (DS) is a genetically determined epileptic encephalopathy mainly caused by de novo mutations in the SCN1A gene. Since 2003, we have performed molecular analyses in a large series of patients with DS, 27% of whom were negative for mutations or rearrangements in SCN1A. In order to identify new genes responsible for the disorder in the SCN1A-negative patients, 41 probands were screened for micro-rearrangements with Illumina high-density SNP microarrays. A hemizygous deletion on chromosome Xq22.1, encompassing the PCDH19 gene, was found in one male patient. To confirm that PCDH19 is responsible for a Dravet-like syndrome, we sequenced its coding region in 73 additional SCN1A-negative patients. Nine different point mutations (four missense and five truncating mutations) were identified in 11 unrelated female patients. In addition, we demonstrated that the fibroblasts of our male patient were mosaic for the PCDH19 deletion. Patients with PCDH19 and SCN1A mutations had very similar clinical features including the association of early febrile and afebrile seizures, seizures occurring in clusters, developmental and language delays, behavioural disturbances, and cognitive regression. There were, however, slight but constant differences in the evolution of the patients, including fewer polymorphic seizures (in particular rare myoclonic jerks and atypical absences) in those with PCDH19 mutations. These results suggest that PCDH19 plays a major role in epileptic encephalopathies, with a clinical spectrum overlapping that of DS. This disorder mainly affects females. The identification of an affected mosaic male strongly supports the hypothesis that cellular interference is the pathogenic mechanism.     

Prevalence of SCN1A-Related Dravet Syndrome among Children Reported with Seizures following Vaccination: A Population-Based Ten-Year Cohort Study

Objectives

To determine the prevalence of Dravet syndrome, an epileptic encephalopathy caused by SCN1A-mutations, often with seizure onset after vaccination, among infants reported with seizures following vaccination. To determine differences in characteristics of reported seizures after vaccination in children with and without SCN1A-related Dravet syndrome.

Methods

Data were reviewed of 1,269 children with seizures following immunization in the first two years of life, reported to the safety surveillance system of the Dutch national immunization program between 1 January 1997 and 31 December 2006. Selective, prospective follow-up was performed of children with clinical characteristics compatible with a diagnosis of Dravet syndrome.

Results

In 21.9% (n = 279) of children, a diagnosis of Dravet syndrome could not be excluded based on available clinical data (median age at follow-up 16 months). Additional follow-up data were obtained in 83.9% (n = 234) of these children (median age 8.5 years).15 (1.2% of 1,269; 95%CI:0.6 to 1.8%) children were diagnosed with SCN1A-related Dravet syndrome. Of all reported seizures following vaccinations in the first year of life, 2.5% (95%CI:1.3 to 3.6%) were due to SCN1A-related Dravet syndrome, as were 5.9% of reported seizures (95%CI:3.1 to 8.7%) after 2^nd or 3^rd DTP-IPV-Hib vaccination.Seizures in children with SCN1A-related Dravet syndrome occurred more often with a body temperature below 38.5°C (57.9% vs. 32.6%, p = 0.020) and reoccurred more often after following vaccinations (26.7% vs. 4.0%, p = 0.003), than in children without a diagnosis of SCN1A-related Dravet Syndrome.

Conclusions

Although Dravet syndrome is a rare genetic epilepsy syndrome, 2.5% of reported seizures following vaccinations in the first year of life in our cohort occurred in children with this disorder. Knowledge on the specific characteristics of vaccination-related seizures in this syndrome might promote early diagnosis and indirectly, public faith in vaccination safety.     

Loss of zebrafish lgi1b leads to hydrocephalus and sensitization to pentylenetetrazol induced seizure-like behavior

Mutations in the LGI1 gene predispose to a hereditary epilepsy syndrome and is the first gene associated with this disease which does not encode an ion channel protein. In zebrafish, there are two paralogs of the LGI1 gene, lgi1a and lgi1b. Knockdown of lgi1a results in a seizure-like hyperactivity phenotype with associated developmental abnormalities characterized by cellular loss in the eyes and brain. We have now generated knockdown morphants for the lgi1b gene which also show developmental abnormalities but do not show a seizure-like behavior. Instead, the most striking phenotype involves significant enlargement of the ventricles (hydrocephalus). As shown for the lgi1a morphants, however, lgi1b morphants are also sensitized to PTZ-induced hyperactivity. The different phenotypes between the two lgi1 morphants support a subfunctionalization model for the two paralogs.     

A reverse genetic screen in the zebrafish identifies crb2b as a regulator of the glomerular filtration barrier

The glomerular filtration barrier is necessary for the selective passage of low molecular weight waste products and the retention of blood plasma proteins. Damage to the filter results in proteinuria. The filtration barrier is the major pathogenic site in almost all glomerular diseases and its study is therefore of clinical significance. We have taken advantage of the zebrafish pronephros as a system for studying glomerular filtration. In order to identify new regulators of filtration barrier assembly, we have performed a reverse genetic screen in the zebrafish testing a group of genes which are enriched in their expression within the mammalian glomerulus. In this novel screen, we have coupled gene knockdown using morpholinos with a physiological glomerular dye filtration assay to test for selective glomerular permeability in living zebrafish larvae. Screening 20 genes resulted in the identification of ralgps1, rapgef2, rabgef1, and crb2b. The crumbs (crb) genes encode a family of evolutionarily conserved proteins important for apical-basal polarity within epithelia. The crb2b gene is expressed in zebrafish podocytes. Electron microscopic analysis of crb2b morphants reveals a gross disorganization of podocyte foot process architecture and loss of slit diaphragms while overall polarity is maintained. Nephrin, a major component of the slit diaphragm, is apically mis-localized in podocytes from crb2b morphants suggesting that crb2b is required for the proper protein trafficking of Nephrin. This report is the first to show a role for crb function in podocyte differentiation. Furthermore, these results suggest a novel link between epithelial polarization and the maintenance of a functional filtration barrier.     

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system^1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 μl during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds⁴. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish^5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.     

Molecular cloning and knockdown of galactocerebrosidase in zebrafish: New insights into the pathogenesis of Krabbe's disease

The lysosomal hydrolase galactocerebrosidase (GALC) catalyzes the removal of galactose from galactosylceramide and from other sphingolipids. GALC deficiency is responsible for globoid cell leukodystrophy (GLD), or Krabbe's disease, an early lethal inherited neurodegenerative disorder characterized by the accumulation of the neurotoxic metabolite psychosine in the central nervous system (CNS). The poor outcome of current clinical treatments calls for novel model systems to investigate the biological impact of GALC down-regulation and for the search of novel therapeutic strategies in GLD. Zebrafish (Danio rerio) represents an attractive vertebrate model for human diseases. Here, lysosomal GALC activity was demonstrated in the brain of zebrafish adults and embryos. Accordingly, we identified two GALC co-orthologs (named galca and galcb) dynamically co-expressed in CNS during zebrafish development. Both genes encode for lysosomal enzymes endowed with GALC activity. Single down-regulation of galca or galcb by specific antisense morpholino oligonucleotides results in a partial decrease of GALC activity in zebrafish embryos that was abrogated in double galca/galcb morphants. However, no psychosine accumulation was observed in galca/galcb double morphants. Nevertheless, double galca/galcb knockdown caused reduction and partial disorganization of the expression of the early neuronal marker neuroD and an increase of apoptotic events during CNS development. These observations provide new insights into the pathogenesis of GLD, indicating that GALC loss-of-function may have pathological consequences in developing CNS independent of psychosine accumulation. Also, they underscore the potentiality of the zebrafish system in studying the pathogenesis of lysosomal neurodegenerative diseases, including GLD.     

Neph3 associates with regulation of glomerular and neural development in zebrafish

Neph3 (filtrin) is a membrane protein expressed in the glomerular epithelial cells (podocytes), but its role in the glomerulus is still largely unknown. To characterize the function of Neph3 in the glomerulus, we employed the zebrafish as a model system. Here we show that the expression of neph3 in pronephros starts before the onset of nephrin and podocin expression, peaks when the nephron primordium differentiates into glomerulus and tubulus, and is then downregulated upon glomerular maturation. By histology, we found that neph3 is specifically expressed in pronephric podocytes at 36 hpf. Furthermore, disruption of neph3 expression by antisense morpholino oligonucleotides results in distorted body curvature and transient pericardial edema, the latter likely reflecting perturbation of glomerular osmoregulatory function. Histological analysis of neph3 morphants reveals altered glomerular morphology and dilated pronephric tubules. The phenotype of neph3 morphants, curved body and pericardial edema, is rescued by wild-type zebrafish neph3 mRNA. In addition to glomerulus, neph3 is highly expressed in the developing brain and specific regions of mature midbrain and hindbrain. In line with this, neph3 morphants show aberrant brain morphology. Collectively, the expression of neph3 in glomerulus and brain together with the morphant phenotype imply that neph3 is a pleiotropic gene active during distinct stages of tissue differentiation and associates directly in the regulation of both glomerular and neural development.     

Zebrafish scube1 (Signal Peptide-CUB (Complement Protein C1r/C1s,Uegf, and Bmp1)-EGF (Epidermal Growth Factor) Domain-containing Protein 1) Is Involved in Primitive Hematopoiesis

scube1 (signal peptide-CUB (complement protein C1r/C1s, Uegf, and Bmp1)-EGF domain-containing protein 1), the founding member of a novel secreted and cell surface SCUBE protein family, is expressed predominantly in various developing tissues in mice. However, its function in primitive hematopoiesis remains unknown. In this study, we identified and characterized zebrafish scube1 and analyzed its function by injecting antisense morpholino-oligonucleotide into embryos. Whole-mount in situ hybridization revealed that zebrafish scube1 mRNA is maternally expressed and widely distributed during early embryonic development. Knockdown of scube1 by morpholino-oligonucleotide down-regulated the expression of marker genes associated with early primitive hematopoietic precursors (scl) and erythroid (gata1 and hbbe1), as well as early (pu.1) and late (mpo and l-plastin) myelomonocytic lineages. However, the expression of an early endothelial marker fli1a and vascular morphogenesis appeared normal in scube1 morphants. Overexpression of bone morphogenetic protein (bmp) rescued the expression of scl in the posterior lateral mesoderm during early primitive hematopoiesis in scube1 morphants. Biochemical and molecular analysis revealed that Scube1 could be a BMP co-receptor to augment BMP signaling. Our results suggest that scube1 is critical for and functions at the top of the regulatory hierarchy of primitive hematopoiesis by modulating BMP activity during zebrafish embryogenesis.     

Aromatic L-Amino Acid Decarboxylase (AADC) Is Crucial for Brain Development and Motor Functions

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare pediatric neuro-metabolic disease in children. Due to the lack of an animal model, its pathogenetic mechanism is poorly understood. To study the role of AADC in brain development, a zebrafish model of AADC deficiency was generated. We identified an aadc gene homolog, dopa decarboxylase (ddc), in the zebrafish genome. Whole-mount in situ hybridization analysis showed that the ddc gene is expressed in the epiphysis, locus caeruleus, diencephalic catecholaminergic clusters, and raphe nuclei of 36-h post-fertilization (hpf) zebrafish embryos. Inhibition of Ddc by AADC inhibitor NSD-1015 or anti-sense morpholino oligonucleotides (MO) reduced brain volume and body length. We observed increased brain cell apoptosis and loss of dipencephalic catecholaminergic cluster neurons in ddc morphants (ddc MO-injected embryos). Seizure-like activity was also detected in ddc morphants in a dose-dependent manner. ddc morphants had less sensitive touch response and impaired swimming activity that could be rescued by injection of ddc plasmids. In addition, eye movement was also significantly impaired in ddc morphants. Collectively, loss of Ddc appears to result in similar phenotypes as that of ADCC deficiency, thus zebrafish could be a good model for investigating pathogenetic mechanisms of AADC deficiency in children.