Altered Function of the SCN1A Voltage-gated Sodium Channel Leads to ��-Aminobutyric Acid-ergic (GABAergic) Interneuron Abnormalities

Voltage-gated sodium channels are required for the initiation and propagation of action potentials. Mutations in the neuronal voltage-gated sodium channel SCN1A are associated with a growing number of disorders including generalized epilepsy with febrile seizures plus (GEFS+),⁷ severe myoclonic epilepsy of infancy, and familial hemiplegic migraine. To gain insight into the effect of SCN1A mutations on neuronal excitability, we introduced the human GEFS+ mutation SCN1A-R1648H into the orthologous mouse gene. Scn1a^RH/RH mice homozygous for the R1648H mutation exhibit spontaneous generalized seizures and premature death between P16 and P26, whereas Scn1a^RH/+ heterozygous mice exhibit infrequent spontaneous generalized seizures, reduced threshold and accelerated propagation of febrile seizures, and decreased threshold to flurothyl-induced seizures. Inhibitory cortical interneurons from P5-P15 Scn1a^RH/+ and Scn1a^RH/RH mice demonstrated slower recovery from inactivation, greater use-dependent inactivation, and reduced action potential firing compared with wild-type cells. Excitatory cortical pyramidal neurons were mostly unaffected. These results suggest that this SCN1A mutation predominantly impairs sodium channel activity in interneurons, leading to decreased inhibition. Decreased inhibition may be a common mechanism underlying clinically distinct SCN1A-derived disorders.     

Scn1a dysfunction alters behavior but not the effect of stress on seizure response

Mutations in the voltage‐gated sodium channel gene SCN1A are responsible for a number of epilepsy disorders, including genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome. In addition, dysfunction in SCN1A is increasingly being linked to neuropsychiatric abnormalities, social deficits and cognitive disabilities. We have previously reported that mice heterozygous for the SCN1A R1648H mutation identified in a GEFS+ family have infrequent spontaneous seizures, increased susceptibility to chemically and hyperthermia‐induced generalized seizures and sleep abnormalities. In this study, we characterized the behavior of heterozygous mice expressing the SCN1A R1648H mutation (Scn1a^RH/+) and the effect of stress on spontaneous and induced seizures. We also examined the effect of the R1648H mutation on the hypothalamic–pituitary–adrenal (HPA) axis response. We confirmed our previous finding that Scn1a^RH/+ mutants are hyperactive, and also identified deficits in social behavior, spatial memory, cued fear conditioning, pre‐pulse inhibition and risk assessment. Furthermore, while exposure to a stressor did increase seizure susceptibility, the effect seen in the Scn1a^RH/+ mutants was similar to that seen in wild‐type littermates. In addition, Scn1a dysfunction does not appear to alter HPA axis function in adult animals. Our results suggest that the behavioral abnormalities associated with Scn1a dysfunction encompass a wider range of phenotypes than previously reported and factors such as stress exposure may alter disease severity in patients with SCN1A mutations.     

Variable Nav1.5 Protein Expression from the Wild-Type Allele Correlates with the Penetrance of Cardiac Conduction Disease in the Scn5a +/? Mouse Model

Background

Loss-of-function mutations in SCN5A, the gene encoding Na_v1.5 Na⁺ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a ^+/− mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A.

Methodology/Principal Findings

Based on ECG, 10-week-old Scn5a ^+/− mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS≤18 ms; QRS in wild-type littermates: 10–18 ms). Phenotypic difference persisted with aging. At 10 weeks, the Na⁺ channel blocker ajmaline prolonged QRS interval similarly in both groups of Scn5a ^+/− mice. In contrast, in old mice (>53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a ^+/− mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A–mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a ^+/− mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a ^+/− mice had similar Na_v1.5 mRNA but higher Na_v1.5 protein expression, and moderately larger I_Na current than severely affected Scn5a ^+/− mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a ^+/− mice than in mildly affected ones.

Conclusions

Scn5a ^+/− mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a ^+/− mice, phenotype severity correlates with wild-type Na_v1.5 protein expression.     

Mutations in the Scn8a DIIS4 voltage sensor reveal new distinctions among hypomorphic and null Nav1.6 sodium channels

Mutations in the voltage‐gated sodium channel gene SCN8A cause a broad range of human diseases, including epilepsy, intellectual disability, and ataxia. Here we describe three mouse lines on the C57BL/6J background with novel, overlapping mutations in the Scn8a DIIS4 voltage sensor: an in‐frame 9 bp deletion (Δ9), an in‐frame 3 bp insertion (?3) and a 35 bp deletion that results in a frameshift and the generation of a null allele (Δ35). Scn8a ^Δ9/+ and Scn8a ^?3/+ heterozygous mutants display subtle motor deficits, reduced acoustic startle response, and are resistant to induced seizures, suggesting that these mutations reduce activity of the Scn8a channel protein, Na_v1.6. Heterozygous Scn8a ^Δ35/+ mutants show no alterations in motor function or acoustic startle response, but are resistant to induced seizures. Homozygous mutants from each line exhibit premature lethality and severe motor impairments, ranging from uncoordinated gait with tremor (Δ9 and ?3) to loss of hindlimb control (Δ35). Scn8a ^Δ9/Δ9 and Scn8a ^?3/?3 homozygous mutants also exhibit impaired nerve conduction velocity, while normal nerve conduction was observed in Scn8a ^Δ35/Δ35 homozygous mice. Our results suggest that hypomorphic mutations that reduce Na_v1.6 activity will likely result in different clinical phenotypes compared to null alleles. These three mouse lines represent a valuable opportunity to examine the phenotypic impacts of hypomorphic and null Scn8a mutations without the confound of strain‐specific differences.     

Mapping genetic modifiers of survival in a mouse model of Dravet syndrome

Epilepsy is a common neurological disorder affecting approximately 1% of the population. Mutations in voltage‐gated sodium channels are responsible for several monogenic epilepsy syndromes. More than 800 mutations in the voltage‐gated sodium channel SCN1A have been reported in patients with generalized epilepsy with febrile seizures plus and Dravet syndrome. Heterozygous loss‐of‐function mutations in SCN1A result in Dravet syndrome, a severe infant‐onset epileptic encephalopathy characterized by intractable seizures, developmental delays and increased mortality. A common feature of monogenic epilepsies is variable expressivity among individuals with the same mutation, suggesting that genetic modifiers may influence clinical severity. Mice with heterozygous deletion of Scn1a (Scn1a^+/?) model a number of Dravet syndrome features, including spontaneous seizures and premature lethality. Phenotype severity in Scn1a^+/? mice is strongly dependent on strain background. On the 129S6/SvEvTac strain Scn1a^+/? mice exhibit no overt phenotype, whereas on the (C57BL/6J × 129S6/SvEvTac)F1 strain Scn1a^+/? mice exhibit spontaneous seizures and early lethality. To systematically identify loci that influence premature lethality in Scn1a^+/? mice, we performed genome scans on reciprocal backcrosses. Quantitative trait locus mapping revealed modifier loci on mouse chromosomes 5, 7, 8 and 11. RNA‐seq analysis of strain‐dependent gene expression, regulation and coding sequence variation provided a list of potential functional candidate genes at each locus. Identification of modifier genes that influence survival in Scn1a^+/? mice will improve our understanding of the pathophysiology of Dravet syndrome and may suggest novel therapeutic strategies for improved treatment of human patients.     

Generation and basic characterization of a gene-trap knockout mouse model of Scn2a with a substantial reduction of voltage-gated sodium channel Nav1.2 expression

Large-scale genetic studies revealed SCN2A as one of the most frequently mutated genes in patients with neurodevelopmental disorders. SCN2A encodes for the voltage-gated sodium channel isoform 1.2 (Na_v1.2) expressed in the neurons of the central nervous system. Homozygous knockout (null) of Scn2a in mice is perinatal lethal, whereas heterozygous knockout of Scn2a (Scn2a^+/−) results in mild behavior abnormalities. The Na_v1.2 expression level in Scn2a^+/− mice is reported to be around 50–60% of the wild-type (WT) level, which indicates that a close to 50% reduction of Na_v1.2 expression may not be sufficient to lead to major behavioral phenotypes in mice. To overcome this barrier, we characterized a novel mouse model of severe Scn2a deficiency using a targeted gene-trap knockout (gtKO) strategy. This approach produces viable homozygous mice (Scn2a^gtKO/gtKO) that can survive to adulthood, with about a quarter of Na_v1.2 expression compared to WT mice. Innate behaviors like nesting and mating were profoundly disrupted in Scn2a^gtKO/gtKO mice. Notably, Scn2a^gtKO/gtKO mice have a significantly decreased center duration compared to WT in the open field test, suggesting anxiety-like behaviors in a novel, open space. These mice also have decreased thermal and cold tolerance. Additionally, Scn2a^gtKO/gtKO mice have increased fix-pattern exploration in the novel object exploration test and a slight increase in grooming, indicating a detectable level of repetitive behaviors. They bury little to no marbles and have decreased interaction with novel objects. These Scn2a gene-trap knockout mice thus provide a unique model to study pathophysiology associated with severe Scn2a deficiency.     

Reduced Retinal Function in the Absence of Na(v)1.6

Background

Mice with a function-blocking mutation in the Scn8a gene that encodes Na_v1.6, a voltage-gated sodium channel (VGSC) isoform normally found in several types of retinal neurons, have previously been found to display a profoundly abnormal dark adapted flash electroretinogram. However the retinal function of these mice in light adapted conditions has not been studied.

Methodology/Principal Findings

In the present report we reveal that during light adaptation these animals are shown to have electroretinograms with significant decreases in the amplitude of the a- and b-waves. The percent decrease in the a- and b-waves substantially exceeds the acute effect of VGSC block by tetrodotoxin in control littermates. Intravitreal injection of CoCl₂ or CNQX to isolate the a-wave contributions of the photoreceptors in littermates revealed that at high background luminance the cone-isolated component of the a-wave is of the same amplitude as the a-wave of mutants.

Conclusions/Significance

Our results indicate that Scn8a mutant mice have reduced function in both rod and the cone retinal pathways. The extent of the reduction in the cone pathway, as quantified using the ERG b-wave, exceeds the reduction seen in control littermates after application of TTX, suggesting that a defect in cone photoreceptors contributes to the reduction. Unless the postreceptoral component of the a-wave is increased in Scn8a mutant mice, the reduction in the b-wave is larger than can be accounted for by reduced photoreceptor function alone. Our data suggests that the reduction in the light adapted ERG of Scn8a mutant mice is caused by a combination of reduced cone photoreceptor function and reduced depolarization of cone ON bipolar cells. This raises the possibility that Na_v1.6 augments signaling in cone bipolar cells.     

Scn1a missense mutation impairs GABAA receptor-mediated synaptic transmission in the rat hippocampus

Mutations of the Na_v1.1 channel subunit SCN1A have been implicated in the pathogenesis of human febrile seizures (FS). We have recently developed hyperthermia-induced seizure-susceptible (Hiss) rat, a novel rat model of FS, which carries a missense mutation (N1417H) in Scn1a[1]. Here, we conducted electrophysiological studies to clarify the influences of the Scn1a mutation on the hippocampal synaptic transmission, specifically focusing on the GABAergic system. Hippocampal slices were prepared from Hiss or F344 (control) rats and maintained in artificial cerebrospinal fluid saturated with 95% O₂ and 5% CO₂in vitro. Single neuron activity was recorded from CA1 pyramidal neurons and their responses to the test (unconditioned) or paired pulse (PP) stimulation of the Schaffer collateral/commissural fibers were evaluated. Hiss rats were first tested for pentylenetetrazole-induced seizures and confirmed to show high seizure susceptibility to the blockade of GAGA_A receptors. The Scn1a mutation in Hiss rats did not directly affect spike generation (i.e., number of evoked spikes and firing threshold) of the CA1 pyramidal neurons elicited by the Schaffer collateral/commissural stimulation. However, GABA_A receptor-mediated inhibition of pyramidal neurons by the PP stimulation was significantly disrupted in Hiss rats, yielding a significant increase in the number of PP-induced firings at PP intervals of 32-256 ms. The present study shows that the Scn1a missense mutation preferentially impairs GABA_A receptor-mediated synaptic transmission without directly altering the excitability of the pyramidal neurons in the hippocampus, which may be linked to the pathogenesis of FS.     

Genetic modifiers affecting severity of epilepsy caused by mutation of sodium channelScn2a

Mutations in the voltage-gated sodium channels SCN1A and SCN2A are responsible for several types of human epilepsy. Variable expressivity among family members is a common feature of these inherited epilepsies, suggesting that genetic modifiers may influence the clinical manifestation of epilepsy. The transgenic mouse model Scn2a^Q54 has an epilepsy phenotype as a result of a mutation in Scn2a that slows channel inactivation. The mice display progressive epilepsy that begins with short-duration partial seizures that appear to originate in the hippocampus. The partial seizures become more frequent and of longer duration with age and often induce secondary generalized seizures. Clinical severity of the Scn2a^Q54 phenotype is influenced by genetic background. Congenic C57BL/6J.Q54 mice exhibit decreased incidence of spontaneous seizures, delayed seizure onset, and longer survival in comparison with [C57BL/6J × SJL/J]F₁.Q54 mice. This observation indicates that strain SJL/J carries dominant modifier alleles at one or more loci that determine the severity of the epilepsy phenotype. Genome-wide interval mapping in an N₂ backcross revealed two modifier loci on Chromosomes 11 and 19 that influence the clinical severity of of this sodium channel-induced epilepsy. Modifier genes affecting clinical severity in the Scn2a^Q54 mouse model may contribute to the variable expressivity seen in epilepsy patients with sodium channel mutations.     

Modeling human epilepsy by TALEN targeting of mouse sodium channel Scn8a

To evaluate the efficiency of TALEN technology for introducing mutations into the mouse genome we targeted Scn8a, a member of a multigene family with nine closely related paralogs. Our goal was to generate a model of early onset epileptic encephalopathy by introduction of the Scn8a missense mutation p.Asn1768Asp. We used a pair of TALENs that were highly active in transfected cells. The targeting template for homologous recombination contained a 4 kb genomic fragment. Microinjection of TALENs with the targeting construct into the pronucleus of 350 fertilized mouse eggs generated 67 live‐born potential founders, of which 5 were heterozygous for the pathogenic mutation, a yield of 7% correctly targeted mice. Twenty‐four mice carried one or two Scn8a indels, including 12 frameshift mutations and the novel amino acid deletion p.Asn1759del. Nine off‐site mutations in the paralogs sodium channel genes Scn5a and Scn4a were identified. The data demonstrate the feasibility and efficiency of targeting members of multigene families using TALENs. The Scn8a^tm1768DMm mouse model will be useful for investigation of the pathogenesis and therapy of early onset seizure disorders. genesis 52:141–148. © 2013 The Authors genesis Published by Wiley Periodicals, Inc.     

Characterisation of new symbiotic Medicago truncatula (Gaertn.) mutants, and phenotypic or genotypic complementary information on previously described mutants

From a pool of Medicago truncatula mutants—obtained by gamma-irradiation or ethyl methanesulfonate mutagenesis—impaired in symbiosis with the N-fixing bacterium Sinorhizobium meliloti, new mutants are described and genetically analysed, and for already reported mutants, complementary data are given on their phenotypic and genetic analysis. Phenotypic data relate to nodulation and mycorrhizal phenotypes. Among the five new mutants, three were classified as [Nod⁺ Fix^– Myc⁺] and the mutations were ascribed to two loci, Mtsym20 (TRV43, TRV54) and Mtsym21 (TRV49). For the two other new mutants, one was classified as [Nod^–/+ Myc⁺] with a mutation ascribed to gene Mtsym15 (TRV48), and the other as [Nod^– Myc^-/+] with a mutation ascribed to gene Mtsym16 (TRV58). Genetic analysis of three previously described mutants has shown that [Nod^–/+ Myc⁺] TR74 mutant can be ascribed to gene Mtsym14, and that [Nod^–/+ Myc^–/+] TR89 and TRV9 mutants are ascribed to gene Mtsym2 (dmi2). Using a detailed analysis of mycorrhizal phenotype, we have observed a delayed typical arbuscular mycorrhizal formation on some mutants that present thick lens-shaped appressoria. This phenotype was called [Myc^–/+] and mutants TR25, TR26, TR89, TRV9, P1 and Y6 were reclassified as [Myc^–/+]. Mutant P1 was reclassified as [Nod^–/+] because of a late nodulation observed on roots of this mutant.     

A Heterozygous Deletion Mutation in the Cardiac Sodium Channel Gene SCN5A with Loss- and Gain-of-Function Characteristics Manifests as Isolated Conduction Disease,without Signs of Brugada or Long QT Syndrome

Background

The SCN5A gene encodes for the α-subunit of the cardiac sodium channel Na_V1.5, which is responsible for the rapid upstroke of the cardiac action potential. Mutations in this gene may lead to multiple life-threatening disorders of cardiac rhythm or are linked to structural cardiac defects. Here, we characterized a large family with a mutation in SCN5A presenting with an atrioventricular conduction disease and absence of Brugada syndrome.

Method and Results

In a large family with a high incidence of sudden cardiac deaths, a heterozygous SCN5A mutation (p.1493delK) with an autosomal dominant inheritance has been identified. Mutation carriers were devoid of any cardiac structural changes. Typical ECG findings were an increased P-wave duration, an AV-block I° and a prolonged QRS duration with an intraventricular conduction delay and no signs for Brugada syndrome. HEK293 cells transfected with 1493delK showed strongly (5-fold) reduced Na⁺ currents with altered inactivation kinetics compared to wild-type channels. Immunocytochemical staining demonstrated strongly decreased expression of SCN5A 1493delK in the sarcolemma consistent with an intracellular trafficking defect and thereby a loss-of-function. In addition, SCN5A 1493delK channels that reached cell membrane showed gain-of-function aspects (slowing of the fast inactivation, reduction in the relative fraction of channels that fast inactivate, hastening of the recovery from inactivation).

Conclusion

In a large family, congregation of a heterozygous SCN5A gene mutation (p.1493delK) predisposes for conduction slowing without evidence for Brugada syndrome due to a predominantly trafficking defect that reduces Na⁺ current and depolarization force.     

Reduced Nav1.6 Sodium Channel Activity in Mice Increases In Vivo Sensitivity to Volatile Anesthetics

Na_v1.6 is a major voltage-gated sodium channel in the central and peripheral nervous systems. Within neurons, the channel protein is concentrated at the axon initial segment and nodes of Ranvier, where it functions in initiation and propagation of action potentials. We examined the role of Na_v1.6 in general anesthesia using two mouse mutants with reduced activity of Na_v1.6, Scn8a ^medJ/medJ and Scn8a ^9J/9J. The mice were exposed to the general anesthetics isoflurane and sevoflurane in step-wise increments; the concentration required to produce loss of righting reflex, a surrogate for anesthetic-induced unconsciousness in rodents, was determined. Mice homozygous for these mutations exhibited increased sensitivity to both isoflurane and sevoflurane. The increased sensitivity was observed during induction of unconsciousness but not during the recovery phase, suggesting that the effect is not attributable to compromised systemic physiology. Electroencephalographic theta power during baseline waking was lower in mutants, suggesting decreased arousal and reduced neuronal excitability. This is the first report linking reduced activity of a specific voltage-gated sodium channel to increased sensitivity to general anesthetics in vivo.     

Three ENU-induced neurological mutations in the pore loopof sodium channel <Emphasis Type="Italic">Scn8a</Emphasis> (Na<Subscript>v</Subscript>1.6) and a genetically linkedretinal mutation, <Emphasis Type="Italic">rd13</Emphasis>

The goal of The Jackson Laboratory Neuroscience Mutagenesis Facility is to generate mouse models of human neurological disease. We describe three new models obtained from a three-generation screen for recessive mutations. Homozygous mutant mice from lines nmf2 and nmf5 exhibit hind limb paralysis and juvenile lethality. Homozygous nmf58 mice exhibit a less severe movement disorder that includes sustained dystonic postures. The mutations were mapped to the distal region of mouse Chromosome (Chr) 15. Failure to complement a mutant allele of a positional candidate gene, Scn8a, demonstrated that the mutations are new alleles of Scn8a. Missense mutations of evolutionarily conserved residues of the sodium channel were identified in the three lines, with the predicted amino acid substitutions N1370T, I1392F, and L1404H. These residues are located within the pore loop of domain 3 of sodium channel Na_v1.6. The lethal phenotypes suggest that the new alleles encode proteins with partial or complete loss of function. Several human disorders are caused by mutation in the pore loop of domain 3 of paralogous sodium channel genes. Line nmf5 contains a second, independent mutation in the rd13 locus that causes a reduction in cell number in the outer nuclear layer of the retina. rd13 was mapped to the distal 4 Mb of Chr 15. No coding or splice site mutations were detected in Pde1b, a candidate gene for rd13. The generation of three independent Scn8a mutations among 1100 tested G3 families demonstrates that the Scn8a locus is highly susceptible to ENU mutagenesis. The new alleles of Scn8a will be valuable for analysis of sodium channel physiology and disease.(David A. Buchner and Kevin L. Seburn) These authors contributed equally.     

Exaggerated Nighttime Sleep and Defective Sleep Homeostasis in a Drosophila Knock-In Model of Human Epilepsy

Despite an established link between epilepsy and sleep behavior, it remains unclear how specific epileptogenic mutations affect sleep and subsequently influence seizure susceptibility. Recently, Sun et al. (2012) created a fly knock-in model of human generalized epilepsy with febrile seizures plus (GEFS+), a wide-spectrum disorder characterized by fever-associated seizing in childhood and lifelong affliction. GEFS+ flies carry a disease-causing mutation in their voltage-gated sodium channel (VGSC) gene and display semidominant heat-induced seizing, likely due to reduced GABAergic inhibitory activity at high temperature. Here, we show that at room temperature the GEFS+ mutation dominantly modifies sleep, with mutants exhibiting rapid sleep onset at dusk and increased nighttime sleep as compared to controls. These characteristics of GEFS+ sleep were observed regardless of sex, mating status, and genetic background. GEFS+ mutant sleep phenotypes were more resistant to pharmacologic reduction of GABA transmission by carbamazepine (CBZ) than controls, and were mitigated by reducing GABA_A receptor expression specifically in wake-promoting pigment dispersing factor (PDF) neurons. These findings are consistent with increased GABAergic transmission to PDF neurons being mainly responsible for the enhanced nighttime sleep of GEFS+ mutants. Additionally, analyses under other light conditions suggested that the GEFS+ mutation led to reduced buffering of behavioral responses to light on and off stimuli, which contributed to characteristic GEFS+ sleep phenotypes. We further found that GEFS+ mutants had normal circadian rhythms in free-running dark conditions. Interestingly, the mutants lacked a homeostatic rebound following mechanical sleep deprivation, and whereas deprivation treatment increased heat-induced seizure susceptibility in control flies, it unexpectedly reduced seizure activity in GEFS+ mutants. Our study has revealed the sleep architecture of a Drosophila VGSC mutant that harbors a human GEFS+ mutation, and provided unique insight into the relationship between sleep and epilepsy.     

Induction of a Tumor-associated Activating Mutation in Protein Tyrosine Phosphatase Ptpn11 (Shp2) Enhances Mitochondrial Metabolism,Leading to Oxidative Stress and Senescence

Activating mutations in Ptpn11 (Shp2), a protein tyrosine phosphatase involved in diverse cell signaling pathways, are associated with pediatric leukemias and solid tumors. However, the pathogenic effects of these mutations have not been fully characterized. Here, we report that induction of the Ptpn11^E76K/+ mutation, the most common and active Ptpn11 mutation found in leukemias and solid tumors, in primary mouse embryonic fibroblasts resulted in proliferative arrest and premature senescence. As a result, apoptosis was markedly increased. These cellular responses were accompanied and mediated by up-regulation of p53 and p21. Moreover, intracellular levels of reactive oxygen species (ROS), byproducts of mitochondrial oxidative phosphorylation, were elevated in Ptpn11^E76K/+ cells. Since Shp2 is also distributed to the mitochondria (in addition to the cytosol), the impact of the Ptpn11^E76K/+ mutation on mitochondrial function was analyzed. These analyses revealed that oxygen consumption of Ptpn11^E76K/+ cells and the respiratory function of Ptpn11^E76K/+ mitochondria were significantly increased. Furthermore, we found that phosphorylation of mitochondrial Stat3, one of the substrates of Shp2 phosphatase, was greatly decreased in the mutant cells with the activating mutation Ptpn11^E76K/+. This study provides novel insights into the initial effects of tumor-associated Ptpn11 mutations.     

Deficiency of β Common Receptor Moderately Attenuates the Progression of Myeloproliferative Neoplasm in NrasG12D/+ Mice

Activating Ras signaling is a major driver in juvenile and the myeloproliferative variant of chronic myelomonocytic leukemia (JMML/MP-CMML). Numerous studies suggest that GM-CSF signaling plays a central role in establishing and maintaining JMML/MP-CMML phenotypes in human and mouse. However, it remains elusive how GM-CSF signaling impacts on JMML/MP-CMML initiation and progression. Here, we investigate this issue in a well characterized MP-CMML model induced by endogenous Nras^G12D/+ mutation. In this model, Nras^G12D/+ hematopoietic stem cells (HSCs) are required to initiate and maintain CMML phenotypes and serve as CMML-initiating cells. We show that the common β chain of the GM-CSF receptor (βc) is dispensable for Nras^G12D/+ HSC function; loss of βc does not affect the expansion, increased self-renewal, or myeloid differentiation bias in Nras^G12D/+ HSCs. Therefore, βc^−/− does not abrogate CMML in Nras^G12D/+ mice. However, βc deficiency indeed significantly reduces Nras^G12D/+-induced splenomegaly and spontaneous colony formation and prolongs the survival of CMML-bearing mice, suggesting that GM-CSF signaling plays an important role in promoting CMML progression. Together, our results suggest that inhibiting GM-CSF signaling in JMML/MP-CMML patients might alleviate disease symptoms but would not eradicate the disease.     

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.