Alpha1 Subunit Research Articles

Na/K-ATPase amplification of oxidant stress; a universal but unrecognized clinical target?

The Na/K-ATPase has a signaling function which appears to be separate from its ion pumping function. This signaling function refers to the transduction of conformational changes in the Na/K-ATPase alpha1 subunit into activating Src’s tyrosine kinase activity, triggering a cascade which generates reactive oxygen species (ROS), modulates other signaling pathways, and causes many physiological and pathophysiological effects. We have recently observed that ROS themselves as well as cardiotonic steroids can actually initiate the signal by directly inducing conformational changes in alpha1. It therefore appears that the Na/K-ATPase signal cascade can serve as a feed forward amplification for ROS with circulating cardiotonic steroids setting the gain. Work in both cellular and animal models of disease suggest that this amplification process is activated in conditions characterized by oxidant stress ranging from cancer to obesity/metabolic syndrome and may serve as a potential clinical target for interventions.

Calcium Current Properties and Leak Conductance in Mouse Muscle Fibers Overexpressing a Type 1 Hypokalemic Periodic Paralysis Mutant L-Type Calcium Channel

Missense mutations in the gene encoding the alpha1 subunit of the L-type calcium channel CaV1.1 induce type 1 Hypokalemic Periodic Paralysis (HypoPP1). These mutations mainly occur at arginine residues in the fourth transmembrane segment of voltage-sensor domains. Very few studies have investigated the acute effects of these mutations on channel function and muscle membrane electrical properties because of the difficulty to express Cav1.1 in heterologous systems. In the present study we successfully transferred by electroporation the genes encoding the turboGFP-tagged human wildtype (WT) and R1239H HypoPP1 mutant Cav1.1 into hind limb mouse muscles. The expression profile of the two channels showed a regular striated pattern indicative of the localization of the channels in the t-tubule membrane. Measurement of the L-type current using the silicone-clamp technique showed that the maximal conductance and the voltage-dependence of the Cav1.1 channel were significantly reduced and shifted towards negative potentials respectively in fibers expressing R1239H Cav1.1 as compared to fibers expressing WT Cav1.1. Applying voltage ramps from a holding potential of 0, −20, −40 or −60 mV to −120 mV in the presence of an external low-chloride, sodium-free and potassium-free solution revealed a significant higher leak conductance measured between −80 and −120 mV in fibers expressing R1239H Cav1.1. Acidification of the external solution significantly increased the leak inward current, leak conductance and the fluorescence of an internally loaded pH indicator in fibers expressing R1239H. These data suggest that an elevated leak inward current, likely carrying protons, flows at resting membrane potentials in fibers expressing R1239H Cav1.1 and that acidification of the external medium following muscle exercise could contribute to favor the onset of muscle paralysis in HypoPP1.

Ethanol-induced GABAA receptor alpha4 subunit plasticity involves phosphorylation and neuroactive steroids.

GABAA receptors containing α4 subunits are widely implicated in acute ethanol sensitivity, and their spatial and temporal regulation prominently contributes to ethanol-induced neuroplasticity in hippocampus and cortex. However, it is unknown if α4-containing GABAA receptors in the thalamus, an area of high α4 expression, display similar regulatory patterns following ethanol administration, and if so, by which molecular mechanisms. In the current study, thalamic GABAA receptor α4 subunit levels were increased following a 6-week-, but not a 2-week chronic ethanol diet. Following acute high-dose ethanol administration, thalamic GABAA receptor α4 subunit levels were regulated in a temporal fashion, as a decrease was observed at 2h followed by a delayed transient increase. PKCγ and PKCδ levels paralleled α4 temporal expression patterns following ethanol exposure. Initial decreases in α4 subunit expression were associated with reduced serine phosphorylation. Delayed increases in expression were not associated with a change in phosphorylation state, but were prevented by inhibiting neuroactive steroid production with the 5α-reductase inhibitor finasteride. Overall, these studies indicate that thalamic GABAA receptor α4 subunit expression following acute and chronic ethanol administration exhibits similar regulatory patterns as other regions and that transient expression patterns following acute exposure in vivo are likely dependent on both subunit phosphorylation state and neuroactive steroids.

Ethanol-induced GABAA receptor alpha4 subunit plasticity involves phosphorylation and neuroactive steroids

Ethanol-induced GABAA receptor alpha4 subunit plasticity involves phosphorylation and neuroactive steroids

The Role of the Paratrigeminal Nucleus in Vagal Afferent Evoked Respiratory Reflexes: A Neuroanatomical and Functional Study in Guinea Pigs.

The respiratory tree receives sensory innervation from the jugular and nodose vagal sensory ganglia. Neurons of these ganglia are derived from embryologically distinct origins and as such demonstrate differing molecular, neurochemical and physiological phenotypes. Furthermore, whereas nodose afferent neurons project to the nucleus of the solitary tract (nTS), recent neuroanatomical studies in rats suggest that jugular neurons have their central terminations in the paratrigeminal nucleus (Pa5). In the present study we confirm that guinea pigs demonstrate a comparable distinction between the brainstem terminations of nodose and jugular ganglia afferents. Thus, microinjection of fluorescently conjugated cholera toxin B (CT-B) neural tracers into the caudal nTS and Pa5 resulted in highly specific retrograde labeling of neurons in the nodose and jugular ganglia, respectively. Whereas, nodose neurons more often expressed 160 KD neurofilament proteins and the alpha3 subunit of Na+/K+ ATPase, significantly more jugular neurons expressed the neuropeptides substance P (SP) and, especially, Calcitonin Gene-Related Peptide (CGRP). Indeed, terminal fibers in the Pa5 compared to the nTS were characterized by their significantly greater expression of CGRP, further supporting the notion that jugular afferents project to trigeminal-related brainstem regions. Electrical stimulation of the guinea pig larynx following selective surgical denervation of the nodose afferent innervation to the larynx (leaving intact the jugular innervation) resulted in stimulus dependent respiratory slowing and eventual apnea. This jugular ganglia neuron mediated response was unaffected by bilateral microinjections of the GABAA agonist muscimol into the nTS, but was abolished by muscimol injected into the Pa5. Taken together these data confirm that jugular and nodose vagal ganglia afferent neurons innervate distinct central circuits and support the notion that multiple peripheral and central pathways mediate sensory responses associated with airway irritations.

Upregulation of Nicotinic Acetylcholine Receptor alph4+beta2 through a Ligand-Independent PI3Kbeta Mechanism That Is Enhanced by TNFalpha and the Jak2/p38Mapk Pathways.

High affinity nicotine-binding sites in the mammalian brain are neuronal nicotinic acetylcholine receptors (nAChR) assembled from at least alpha4 and beta2 subunits into pentameric ion channels. When exposed to ligands such as nicotine, these receptors respond by undergoing upregulation, a correlate of nicotine addiction. Upregulation can be measured using HEK293 (293) cells that stably express alpha4 and beta2 subunits using quantification of [3H]epibatidine ([3H]Eb) binding to measure mature receptors. Treatment of these cells with choline also produces upregulation through a hemicholinium3 (HC3)-sensitive (choline kinase) and an HC3-insensitive pathway which are both independent of the mechanism used by nicotine for upregulation. In both cases, upregulation is significantly enhanced by the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) which signals through its receptor Tnfr1 to activate p38Mapk. Here we report that the inhibition of class1 phosphoinositide 3-kinases isoform PI3Kbeta using the selective antagonist PI828 is alone sufficient to produce upregulation and enhance both nicotine and choline HC3-sensitive mediated upregulation. Further, these processes are impacted upon by an AG-490 sensitive Jak2-associated pathway. Both PI3Kbeta (negative) and Jak2 (positive) modulation of upregulation converge through p38Mapk and both overlap with TNFalpha enhancement of this process. Upregulation through the PI3Kbeta pathway did not require Akt. Collectively these findings support upregulation of endogenous alpha4beta2 as a balance among cellular signaling networks that are highly responsive to multiple environmental, inflammatory and metabolic agents. The findings also suggest how illness and metabolic stress could alter the expression of this important nicotinic receptor and novel avenues to intercede in modifying its expression.

Genetic Evidence for Possible Involvement of the Calcium Channel Gene CACNA1A in Autism Pathogenesis in Chinese Han Population.

Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders. Recent studies suggested that calcium channel genes might be involved in the genetic etiology of ASD. CACNA1A, encoding an alpha-1 subunit of voltage-gated calcium channel, has been reported to play an important role in neural development. Previous study detected that a single nucleotide polymorphism (SNP) in CACNA1A confers risk to ASD in Central European population. However, the genetic relationship between autism and CACNA1A in Chinese Han population remains unclear. To explore the association of CACNA1A with autism, we performed a family-based association study. First, we carried out a family-based association test between twelve tagged SNPs and autism in 239 trios. To further confirm the association, the sample size was expanded to 553 trios by recruiting 314 additional trios. In a total of 553 trios, we identified association of rs7249246 and rs12609735 with autism though this would not survive after Bonferroni correction. Our findings suggest that CACNA1A might play a role in the etiology of autism.

Chronic Intermittent Ethanol Regulates Hippocampal GABA(A) Receptor Delta Subunit Gene Expression.

Chronic ethanol consumption causes structural and functional reorganization in the hippocampus and induces alterations in the gene expression of gamma-aminobutyric acid type A receptors (GABAARs). Distinct forced intermittent exposure models have been used previously to investigate changes in GABAAR expression, with contrasting results. Here, we used repeated cycles of a Chronic Intermittent Ethanol paradigm to examine the relationship between voluntary, dependence-associated ethanol consumption, and GABAAR gene expression in mouse hippocampus. Adult male C57BL/6J mice were exposed to four 16-h ethanol vapor (or air) cycles in inhalation chambers alternated with limited-access two-bottle choice between ethanol (15%) and water consumption. The mice exposed to ethanol vapor showed significant increases in ethanol consumption compared to their air-matched controls. GABAAR alpha4 and delta subunit gene expression were measured by qRT-PCR at different stages. There were significant changes in GABAAR delta subunit transcript levels at different time points in ethanol-vapor exposed mice, while the alpha4 subunit levels remained unchanged. Correlated concurrent blood ethanol concentrations suggested that GABAAR delta subunit mRNA levels fluctuate depending on ethanol intoxication, dependence, and withdrawal state. Using a vapor-based Chronic Intermittent Ethanol procedure with combined two-bottle choice consumption, we corroborated previous evidences showing that discontinuous ethanol exposure affects GABAAR delta subunit expression but we did not observe changes in alpha4 subunit. These findings indicate that hippocampal GABAAR delta subunit expression changes transiently over the course of a Chronic Intermittent Ethanol paradigm associated with voluntary intake, in response to ethanol-mediated disturbance of GABAergic neurotransmission.

Learning-Induced Gene Expression in the Hippocampus Reveals a Role of Neuron -Astrocyte Metabolic Coupling in Long Term Memory.

We examined the expression of genes related to brain energy metabolism and particularly those encoding glia (astrocyte)-specific functions in the dorsal hippocampus subsequent to learning. Context-dependent avoidance behavior was tested in mice using the step-through Inhibitory Avoidance (IA) paradigm. Animals were sacrificed 3, 9, 24, or 72 hours after training or 3 hours after retention testing. The quantitative determination of mRNA levels revealed learning-induced changes in the expression of genes thought to be involved in astrocyte-neuron metabolic coupling in a time dependent manner. Twenty four hours following IA training, an enhanced gene expression was seen, particularly for genes encoding monocarboxylate transporters 1 and 4 (MCT1, MCT4), alpha2 subunit of the Na/K-ATPase and glucose transporter type 1. To assess the functional role for one of these genes in learning, we studied MCT1 deficient mice and found that they exhibit impaired memory in the inhibitory avoidance task. Together, these observations indicate that neuron-glia metabolic coupling undergoes metabolic adaptations following learning as indicated by the change in expression of key metabolic genes.

T-type Calcium Channel Regulation of Neural Tube Closure and EphrinA/EPHA Expression.

A major class of human birth defects arise from aberrations during neural tube closure (NTC). We report on a NTC signaling pathway requiring T-type calcium channels (TTCCs) that is conserved between primitive chordates (Ciona) and Xenopus. With loss of TTCCs, there is a failure to seal the anterior neural folds. Accompanying loss of TTCCs is an upregulation of EphrinA effectors. Ephrin signaling is known to be important in NTC, and ephrins can affect both cell adhesion and repulsion. In Ciona, ephrinA-d expression is downregulated at the end of neurulation, whereas, with loss of TTCC, ephrinA-d remains elevated. Accordingly, overexpression of ephrinA-d phenocopied TTCC loss of function, while overexpression of a dominant-negative Ephrin receptor was able to rescue NTC in a Ciona TTCC mutant. We hypothesize that signaling through TTCCs is necessary for proper anterior NTC through downregulation of ephrins, and possibly elimination of a repulsive signal.

AB026. SCN1A mutational analysis in 20 Vietnamese children with Dravet syndrome

Background Dravet syndrome is one of the most catastrophic types of epilepsy in infants. It is found that 70-80% of cases of Dravet syndrome are caused by mutations in SCN1A, the gene encoding alpha-1 subunit of the sodium channel. Mutations of the SCN1A gene have an autosomal dominant inheritance pattern. To date, over 1,000 SCN1A mutations have been reported all over the world, however, no SCN1A mutation studies have been performed in the Vietnamese population, and genetic characteristics of Vietnamese Dravet patients are not yet clear. In this study, we analyzed SCN1A gene in 20 Vietnamese patients with clinical features of Dravet syndrome at Children’s Hospital 2, Ho Chi Minh City, Vietnam.

GABAergic control of the activity of the central nucleus of the amygdala in low- and high-anxiety rats

The aim of this study was to examine the role of GABAergic neurotransmission in amygdala nuclei in low- (LR) and high-anxiety (HR) rats after repeated corticosterone administration and acute injection of the benzodiazepine midazolam. The animals were divided into LR and HR groups based on the duration of their conditioned freezing in a contextual fear test (CFT). Repeated daily administration of corticosterone (20 mg/kg s.c.) for 21 injections increased anxiety-like behavior in the open field and reduced body weight in both the LR and HR groups. These effects of corticosterone administration were more pronounced in the HR group. Moreover, in the HR group, chronic corticosterone administration increased the duration of freezing in the CFT test compared with the appropriate control group and treated LR rats. The behavioral effects in HR rats were accompanied by an increase in the expression of c-Fos in the lateral (LA) and central (CeA) nuclei of the amygdala and by a decrease in GABA-A alpha-2 subunit density in the CeA. Acute midazolam administration significantly attenuated the neophobia and conditioned fear responses, decreased c-Fos expression in the LA and CeA, and increased alpha-2 subunit density in the CeA only in the HR group. These studies have shown that HR rats are more susceptible to the anxiogenic effects of chronic corticosterone administration, which are associated with the attenuation of GABAergic control over the amygdala output that controls emotional responses. The current data may increase understanding of the neurobiological mechanisms responsible for individual differences in the psychopathological processes induced by repeated administration of high doses of glucocorticoids or by elevated levels of these hormones, which are associated with chronic stress and affective pathology.

Role of gabra2, GABAA receptor alpha-2 subunit, in CNS development

gabra2 gene codes for the alpha-2 subunit of the GABAA receptor, one of the ionotropic receptors which has been related to anxiety, depression and other behavioural disorders, including drug dependence and schizophrenia. GABAergic signalling also plays a role during development, by promoting neural stem cell maintenance and renewal. To investigate the role of gabra2 in CNS development, gabra2 deficient zebrafish were generated. The pattern of proliferation during the embryonic development was disrupted in morphant embryos, which also displayed an increase in the number of apoptotic nuclei mainly at the mid- and hindbrain regions. The expression of several genes (notch1, pax2, fgf8 and wnt1) known to contribute to the development of the central nervous system was also affected in gabra2 morpholino-injected embryos, although no changes were found for pax6a and shh a expression. The transcriptional activity of neuroD (a proneural gene involved in early neuronal determination) was down-regulated in gabra2 deficient embryos, and the expression pattern of gad1b (GABA-synthesising enzyme GAD67) was clearly reduced in injected fish. I propose that gabra2 might be interacting with those signalling pathways that regulate proliferation, differentiation and neurogenesis during the embryonic development; thus, gabra2 might be playing a role in the differentiation of the mesencephalon and cerebellum. Given that changes in GABAergic circuits during development have been related to several psychiatric disorders, such as autism and schizophrenia, this work might be helpful to understand the role of neurotransmitter systems during CNS development and to assess the developmental effects of several GABAergic drugs.

The combined use of miRNAs and mRNAs as biomarkers for the diagnosis of papillary thyroid carcinoma.

Thyroid carcinoma (TC) is the most common malignancy of the endocrine system, and papillary thyroid carcinoma(PTC) accounts for the largest proportion of cases with TC. Although histology is considered the gold standard in the diagnosis of PTC, the sensitivity and specificity of this method is low. Therefore, developing novel diagnostic and prognostic biomarkers for PTC is essential. MicroRNAs (miRNAs or miRs) and their target RNAs play critical roles in tumorigenesis and tumor progression. Thus, the characteristic miRNA and mRNA expression profiles may function as diagnostic biomarkers for tumors, making it possible to predict the tumor stage and the prognosis of patients. In the present study, we detected miRNAs and mRNAs which can function as novel biomarkers for the diagnosis of PTC. The sensitivity of the diagnostic tests was evaluated by receiver operating characteristic curve analysis. Pearson's correlation analysis was used to determine the correlation between mRNAs and miRNAs, and cancer types. We found that the area under the curve(AUC) values of 8miRNAs (miR-106a, miR-15a, miR-30a, miR-30b, miR-20a, miR-20b, miR-30d and miR-30e) and 8mRNAs [axis inhibition protein2(AXIN2), integrin, alpha3 (antigen CD49C, alpha3 subunit of VLA-3 receptor)(ITGA3), tumor protein p53 inducible nuclear protein(TP53INP)1, TP53INP2, B-cell CLL/lymphoma2(BCL2), phosphatase and tensin homolog(PTEN), FOS and K(lysine) acetyltransferase2B(KAT2B)] were >0.90. The combination of miR-15a and AXIN2 significantly improved the diagnostic accuracy. Therefore, our data indicate that the differential expression of miRNAs combined with that of their target mRNAs may serve as a powerful biomarker for distinguishing PTC from benign tissues.

Subacute encephalitis in a child seropositive for alpha-3 subunit of neuronal nicotinic acetylcholine receptors antibody

A previously healthy and developmentally normal 5-year-old boy from Saudi Arabia presented with a 3 mo history of abnormal behavior in the form of hyperactivity, decreased social engagement, episodic aggressiveness, decreased cognitive performance in school, inappropriate laughter and talking to himself, insomnia, tics and other stereotyped behaviors. His clinical neurological examination did not reveal any focal neurological deficits, but a prolonged electroencephalogram showed non-specific slowing over the right hemisphere. A diagnosis of encephalopathy was entertained, and he underwent testing for infectious, toxic, metabolic and mitochondrial disorders. Magnetic resonance imaging of the brain and cerebrospinal fluid studies were all normal. However, a comprehensive evaluation of paraneoplastic antibodies from cerebrospinal fluid demonstrated the presence of antibodies against the alpha-3 subunit of the neuronal nicotinic acetylcholine receptor, a finding also documented by tests from two different laboratories. An extensive diagnostic investigation that included a fludeoxyglucose-positron emission tomography scan for malignancies showed negative results. The patient was treated with plasmapheresis treatments (five ex- changes) that resulted in a significant improvement in his clinical picture. Following his initial treatments, the patient continued to receive monthly infusions of intravenous immunoglobulin. An evaluation at 5.5 mo after initiation of first treatment revealed a considerable improvement of his behavior and language abilities. The patient returned to his school where he has performed satisfactorily. To our knowledge, this is the first report of a child who developed sub-acute cognitive and neurobehavioral re- gression in association with the presence of a serum antibody to a neuronal ganglionic nicotinic acetylcholine receptor and who improved after immunotherapy.

Sexual maturation and changes in water and salt transport components in the kidney and intestine of three-spined stickleback (Gasterosteus aculeatus L.)

Mature three-spined stickleback males use spiggin threads secreted from their kidney to glue together nest material. This requires strongly hypertrophied renal proximal tubular cells, which compromises renal osmoregulatory function during the breeding period. Experimental evidence suggests that the intestine takes over hypotonic fluid secretion at that stage but the mechanism is unexplored. To unravel the molecular mechanism we analyzed and compared transcript levels of several membrane proteins involved in water and salt transport in intestinal and renal tissues, in non-mature males (NM), mature males (MM), and mature females (MF). Aquaporin paralogs aqp1a, -3a, -8aa, -8ab, -10a, and -10b, two Na+,K+-ATPase alpha-1 subunit isoforms (nka547, nka976), Na+,K+,2Cl−-, and Na+,Cl−-cotransporters (nkcc1a, nkcc2, ncc), the cystic fibrosis transmembrane conductance regulator (cftr) and two claudin isoforms (cldn2, cldn15a) were expressed in the intestine and kidney in all groups. There were no differences in aqp and cldn expression between intestines of NM and MM; nkcc2 was lower and nka levels tended to be higher in intestines of MM than in NM. In the kidney, aqp1 and aqp8ab levels were lower in MM than in NM, whereas aqp3a, nkcc1a, cldn15a, and spiggin were markedly elevated. This was accompanied by marked hypertrophy of kidney tubules in MM. The data support an altered kidney function in terms of water handling in mature males, whereas there was no support for modified trans-epithelial water permeability or salt-secretory activity in the intestine of mature males. Salt-absorptive activity in the intestine may, however, be down-regulated during male maturation.

497. HSV Vector-Mediated Expression of a Novel Drug Activated Pain Signalling Therapeutic Abrogates Pain

Gene therapy approaches to treat chronic pain have been limited by short-term duration, the inability to turn off therapeutic gene expression to avoid unwanted side-effects or tolerance. To overcome this, we employed replication-defective herpes simplex virus (rdHSV) vectors expressing the glycine receptor alpha-1 (GlyRa1) subunit to silence pain-signaling neurons in a variety of pain models upon the addition of glyine. However, the therapeutic window was small since centrally occurring GlyR in the spinal cord were activated at higher glycine doses. While this was encouraging, the application of GlyRa1 as an effective therapeutic requires further modifications to avoid off-target effects. It was clear that the most effective use of this method would require targeted GlyRa1 expression in specific neuronal subpopulations to avoid silencing of non-nociceptive neurons, limiting receptor expression to neurons involved in chronic pain signaling. Treatment specificity may be achieved through the use of a GlyRa1 that is altered in a manner to be activated by a specific drug that does not cause activation of the normal receptor. Others have identified a mutant form of the receptor (GlyRa1F207A/A288G or G2M) that no longer responds to glycine but rather responds to a novel ligand, ivermectin, an FDA-approved anti-helmenthes drug that binds to the mutant receptor in the nM range while mM quantities of ivermectin are needed to activate the wt-GlyRa1.We engineered G2M rdHSV vectors under the control of the CMV promoter and only upon addition of 100μM Ivermectin could we silence thermal pain while the wt-GlyRa1 was only activated by 100mM Glycine. We tested the effective dose range in a rat model of post-herpetic neuralgia (PHN) that elicits both thermal and mechanical pain behaviors that last out to 9 weeks. We showed that 100μM-10nM Ivermectin was able to block both mechanical and thermal pain, while only 100mM Glycine was able to abrogate those same behaviors, over a 10,000-fold difference. To further reduce off target effects, we engineered rdHSV vectors expressing G2M using neuronal fiber-specific promoters whose transgene expression will be limited to either large As-fibers (NF200) or C-fibers (TRPV1). Studies in rats showed that TRPV1p driven G2M dramatically reduced thermal pain as expected with this C-fiber specific promoter only upon Ivermectin addition to a greater magnitude than that observed with CMV. This activity could be reversed specifically using resiniferatoxin (RTx), a capsaicin analogue that caused C-fiber retraction. NF200 driven G2M failed to block thermal pain, though mildly effected mechanical pain only after RTx treatment. We are currently testing the transcriptionally targeted G2M vectors in the rat PHN pain model. Together, these results demonstrate the power of employing a therapeutic product that responds preferentially to Ivermectin rather than glycine, thereby providing the only receptor in vector-infected animals that is activated by relatively low doses of this well-tolerated drug that can be administered systemically, orally or even topically.

PP01.10 – 3014: GABRA1 neonatal encephalopathy: a clinical, genetic and pathological study

Objectives To describe a novel phenotype of neonatal encephalopathy and infantile refractory epilepsy and their genetic and pathological underpinnings. Methods After unremarkable gestation and delivery, a male newborn presented with profound lethargy immediately after birth. Initial physical exam was otherwise normal with no microcephaly or dysmorphic features. He remained in a state of continuous behavioural sleep – arguably a coma state starting at birth – in the ensuing weeks. EEG was initially normal. Brain MRI, metabolic screen and array CGH were normal. By age 2 months, developmental arrest, hypotonic tetraparesis and head growth deceleration were evident and remained unchanged thereafter. Around that age, seizures were first noticed and included daily focal clonic, myoclonic and tonic seizures that proved resistant to multiple AEDs. EEG revealed multifocal epileptiform discharges and diffuse slowing of background activity. Ictal recordings suggested a migrating pattern of the seizures. Throughout patient's life, eye opening was only noted at the time of tonic seizures. He died at age 16 months from respiratory complications. Results Brain pathology showed diffuse hypomyelination and mild gliotic changes. The mamillary bodies were conspicuously absent. Microscopically, there were no cytoarchitectural abnormalities but the neocortex showed abundance of type II Alzheimer astrocytes. Whole exome sequencing revealed a heterozygous c.1303C> G missense mutation in the GABRA1A gene, producing a p.L267V substitution. This de novo variant was confirmed by PCR and Sanger sequencing and is predicted to alter the assembly and functional properties of the GABA receptor. Conclusions Mutations in GABRA1, encoding the GABA-A receptor alpha-1 subunit, have been associated with generalized epilepsies and more recently childhood epileptic encephalopathies. The present observation expands the spectrum of GABRA1-linked phenotypes to include a severe neonatal-onset encephalopathy featuring a continuous sleep state, infantile refractory epilepsy and distinctive neuropathological abnormalities.

The incidence of SCN1A‐related Dravet syndrome in Denmark is 1:22,000: A population‐based study from 2004 to 2009

Dravet syndrome is a severe infantile-onset epileptic encephalopathy associated with mutations in the sodium channel alpha-1 subunit gene SCN1A. We aimed to describe the incidence of Dravet syndrome in the Danish population. Based on a 6-year birth cohort from 2004 to 2009, we propose an incidence of 1:22,000, which is higher than what has been established earlier. We identified 17 cases with SCN1A mutation-positive Dravet syndrome. Fifteen patients were found, by conventional Sanger sequencing. Two additional patients with clinical Dravet syndrome, but without a detectable SCN1A mutation by Sanger sequencing, were diagnosed with a SCN1A mutation after using a targeted next-generation sequencing gene panel.

Analysis of CHRNA7 rare variants in autism spectrum disorder susceptibility.

Chromosome 15q13.3 recurrent microdeletions are causally associated with a wide range of phenotypes, including autism spectrum disorder (ASD), seizures, intellectual disability, and other psychiatric conditions. Whether the reciprocal microduplication is pathogenic is less certain. CHRNA7, encoding for the alpha7 subunit of the neuronal nicotinic acetylcholine receptor, is considered the likely culprit gene in mediating neurological phenotypes in 15q13.3 deletion cases. To assess if CHRNA7 rare variants confer risk to ASD, we performed copy number variant analysis and Sanger sequencing of the CHRNA7 coding sequence in a sample of 135 ASD cases. Sequence variation in this gene remains largely unexplored, given the existence of a fusion gene, CHRFAM7A, which includes a nearly identical partial duplication of CHRNA7. Hence, attempts to sequence coding exons must distinguish between CHRNA7 and CHRFAM7A, making next-generation sequencing approaches unreliable for this purpose. A CHRNA7 microduplication was detected in a patient with autism and moderate cognitive impairment; while no rare damaging variants were identified in the coding region, we detected rare variants in the promoter region, previously described to functionally reduce transcription. This study represents the first sequence variant analysis of CHRNA7 in a sample of idiopathic autism.