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.

Confirmation of an epilepsy modifier locus on mouse chromosome 11 and candidate gene analysis by RNA-Seq.

Epilepsy is a neurological disorder affecting approximately 1% of the worldwide population. Mutations in voltage-gated sodium channels have been identified in several monogenic epilepsy syndromes. Over 800 mutations have been identified in the voltage-gated sodium channel genes SCN1A and SCN2A in human epilepsies, including genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome. In GEFS+ families, affected members with the same mutation often display variability in clinical severity of the disease. This suggests that additional genes modify the effect of the primary mutation, resulting in the variable clinical presentation. The Scn2a(Q54) transgenic mouse model has an epilepsy phenotype that varies depending on the genetic strain background. Scn2a(Q54) mice congenic on the C57BL/6J strain exhibit delayed seizure onset and improved survival compared to (C57BL/6J × SJL/J)F1.Q54 mice. Two modifier loci of Scn2a(Q54) seizure susceptibility were mapped and designated Moe1 (modifier of epilepsy) on chromosome (chr) 11 and Moe2 on chr 19. To confirm Moe1 and refine its position, we generated interval-specific congenic lines carrying C57BL/6J-derived chr 11 alleles on the SJL/J strain and refined the map position to 89-104 Mb. We then used RNA-Seq for candidate analysis in the modifier region. C57BL/6J and SJL/J male and female brain RNAs were sequenced, revealing numerous significant transcriptome differences and coding single-nucleotide polymorphisms. Additional consideration of gene function and expression suggested several strong candidate modifier genes, including two voltage-gated calcium channel subunits, Cacna1g and Cacnb1, and the proline and acidic amino acid-rich basic leucine zipper transcription factor, Hlf.

Restoration of all dystrophin protein interactions by functional domains in trans does not rescue dystrophy.

Rescue of dystrophic skeletal muscle in mdx and utrophin/dystrophin-deficient (dko) mouse models by reintroduction of dystrophin has validated gene therapy as a potential therapeutic approach for Duchenne muscular dystrophy. However, the size of the dystrophin gene exceeds the capacity of adeno-associated viral (AAV) vectors. Dystrophin provides a mechanical link at the muscle membrane by direct binding of its amino-terminal and cysteine-rich domains to actin and a transmembrane protein complex, respectively. It has not been investigated whether restoration of these two tethering functions by two separate dystrophin molecules is sufficient to prevent dystrophic pathologies. We examine the effect of coexpression of the amino-terminal and cysteine-rich domains from separate dystrophin transgenes, Deltacys and Dp71, on the dystrophic phenotype. Expression of individual dystrophin domains from multiple vectors would effectively expand AAV capacity. Although both Deltacys and Dp71 colocalize at the membrane, there is no improvement of dystrophic pathology. The fiber-type and neuromuscular junction abnormalities of dko mice that are ameliorated by the Deltacys transgene are not further improved or disrupted by Dp71. Separate truncated dystrophins, which together restore all protein interactions and scaffolding for signaling molecules, are not sufficient to ameliorate the dystrophic phenotype and therefore dystrophin domains in trans cannot be used to increase the effective cloning capacity for AAV-mediated gene therapy.

A novel epilepsy mutation in the sodium channel SCN1A identifies a cytoplasmic domain for beta subunit interaction.

A mutation in the sodium channel SCN1A was identified in a small Italian family with dominantly inherited generalized epilepsy with febrile seizures plus (GEFS+). The mutation, D1866Y, alters an evolutionarily conserved aspartate residue in the C-terminal cytoplasmic domain of the sodium channel alpha subunit. The mutation decreased modulation of the alpha subunit by beta1, which normally causes a negative shift in the voltage dependence of inactivation in oocytes. There was less of a shift with the mutant channel, resulting in a 10 mV difference between the wild-type and mutant channels in the presence of beta1. This shift increased the magnitude of the window current, which resulted in more persistent current during a voltage ramp. Computational analysis suggests that neurons expressing the mutant channels will fire an action potential with a shorter onset delay in response to a threshold current injection, and that they will fire multiple action potentials with a shorter interspike interval at a higher input stimulus. These results suggest a causal relationship between a positive shift in the voltage dependence of sodium channel inactivation and spontaneous seizure activity. Direct interaction between the cytoplasmic C-terminal domain of the wild-type alpha subunit with the beta1 or beta3 subunit was first demonstrated by yeast two-hybrid analysis. The SCN1A peptide K1846-R1886 is sufficient for beta subunit interaction. Coimmunoprecipitation from transfected mammalian cells confirmed the interaction between the C-terminal domains of the alpha and beta1 subunits. The D1866Y mutation weakens this interaction, demonstrating a novel molecular mechanism leading to seizure susceptibility.

Sodium channels SCN1A, SCN2A and SCN3A in familial autism.

Autism is a psychiatric disorder with estimated heritability of 90%. One-third of autistic individuals experience seizures. A susceptibility locus for autism was mapped near a cluster of voltage-gated sodium channel genes on chromosome 2. Mutations in two of these genes, SCN1A and SCN2A, result in the seizure disorder GEFS+. To evaluate these sodium channel genes as candidates for the autism susceptibility locus, we screened for variation in coding exons and splice sites in 117 multiplex autism families. A total of 27 kb of coding sequence and 3 kb of intron sequence were screened. Only six families carried variants with potential effects on sodium channel function. Five coding variants and one lariat branchpoint mutation were each observed in a single family, but were not present in controls. The variant R1902C in SCN2A is located in the calmodulin binding site and was found to reduce binding affinity for calcium-bound calmodulin. R542Q in SCN1A was observed in one autism family and had previously been identified in a patient with juvenile myoclonic epilepsy. The effect of the lariat branchpoint mutation was tested in cultured lymphoblasts. Additional population studies and functional tests will be required to evaluate pathogenicity of the coding and lariat site variants. SNP density was 1/kb in the genomic sequence screened. We report 38 sodium channel SNPs that will be useful in future association and linkage studies.

A gain-of-function mutation in the sodium channel gene Scn2a results in seizures and behavioral abnormalities.

The GAL879-881QQQ mutation in the cytoplasmic S4-S5 linker of domain 2 of the rat brain IIA sodium channel (Na(v)1.2) results in slowed inactivation and increased persistent current when expressed in Xenopus oocytes. The neuron-specific enolase promoter was used to direct in vivo expression of the mutated channel in transgenic mice. Three transgenic lines exhibited seizures, and line Q54 was characterized in detail. The seizures in these mice began at two months of age and were accompanied by behavioral arrest and stereotyped repetitive behaviors. Continuous electroencephalogram monitoring detected focal seizure activity in the hippocampus, which in some instances generalized to involve the cortex. Hippocampal CA1 neurons isolated from presymptomatic Q54 mice exhibited increased persistent sodium current which may underlie hyperexcitability in the hippocampus. During the progression of the disorder there was extensive cell loss and gliosis within the hippocampus in areas CA1, CA2, CA3 and the hilus. The lifespan of Q54 mice was shortened and only 25% of the mice survived beyond six months of age. Four independent transgenic lines expressing the wild-type sodium channel were examined and did not exhibit any abnormalities. The transgenic Q54 mice provide a genetic model that will be useful for testing the effect of pharmacological intervention on progression of seizures caused by sodium channel dysfunction. The human ortholog, SCN2A, is a candidate gene for seizure disorders mapped to chromosome 2q22-24.

Intrasubthalamic nucleus metabotropic glutamate receptor activation: a behavioral, Fos immunohistochemical and [14C]2-deoxyglucose autoradiographic study.

Metabotropic glutamate receptors are a major class of excitatory amino acid receptors. Eight metabotropic glutamate receptor subtypes have been cloned, and are classified into three groups (I, II and III) based on amino acid sequence identity, effector systems and pharmacological profile. Previous results have shown that unilateral stimulation of metabotropic glutamate receptors in the subthalamic nucleus with the non-subtype-selective metabotropic glutamate receptor agonist 1S,3R-1-amino-1,3-cyclopentane dicarboxylate results in contralateral rotation in rats and Fos expression in the subthalamic nucleus. This suggests that metabotropic glutamate receptor stimulation results in altered subthalamic nucleus activity with consequent altered basal ganglia activity on the injected side. We sought to determine the metabotropic glutamate receptor subtype(s) involved and the functional neuroanatomy underlying the rotational behavior. Unilateral intrasubthalamic nucleus injection of group II or group III metabotropic glutamate receptor agonists induced contralateral rotation. In addition to producing rotation, group II and group III metabotropic glutamate receptor agonists induce toxicity in the subthalamic nucleus and overlying thalamus. Following group II or group III subthalamic nucleus metabotropic glutamate receptor stimulation, there is Fos-like immunoreactivity in the globus pallidus, subthalamic nucleus, substantia nigra pars reticulata and entopeduncular nucleus, suggesting altered activity in subthalamic nucleus target regions. However, examination of [14C]2-deoxyglucose uptake suggests that the alterations in basal ganglia activity are different following group II versus group III metabotropic glutamate receptor stimulation, suggesting that rotation is occurring via different mechanisms. It appears that stimulation of subthalamic nucleus group II metabotropic glutamate receptors induces rotation by increasing subthalamic nucleus activity. These results suggest that group II metabotropic glutamate receptor antagonists may be useful for alleviating subthalamic nucleus overactivity in Parkinson's disease.

In vitro activities of the oxazolidinone compounds linezolid (PNU-100766) and eperzolid (PNU-100592) against middle ear isolates of Streptococcus pneumoniae.

Two hundred and sixteen isolates of Streptococcus pneumoniae recovered between 1994 and 1996 from the middle ears of children with acute otitis media were tested for their susceptibility to penicillin, erythromycin, clindamycin and the oxazolidinones, linezolid (PNU-100766) and eperezolid (PNU-100592). There were 116 isolates from the Children's Hospital of Pittsburgh and 100 isolates from a national collection. Eighty percent of the local strains were susceptible to penicillin (MIC < 0.1 mg/l); 20% of the local strains and all of the national strains had reduced susceptibility to penicillin. All strains of S. pneumoniae tested had an MIC < 2.0 mg/l for both oxazolidinones. A regional difference was noted in the frequency of resistance to erythromycin with local isolates being more susceptible than isolates from the national collection. This difference was most pronounced among the high-level penicillin-resistant strains of S. pneumoniae.

The role of nigrostriatal dopamine in metabotropic glutamate agonist-induced rotation.

Metabotropic glutamate receptors are a major class of excitatory amino acid receptors. Eight metabotropic glutamate receptors subtypes have been cloned and have been classified into three groups based on their amino acid sequence homology, effector systems, and pharmacological profile. Previous results have shown that striatal group I metabotropic glutamate receptor stimulation produces vigorous contralateral rotation in intact rats, thought to be due to increased striatal dopamine release. Examination of FOS-like immunoreactivity and local cerebral glucose metabolism suggests that this occurs secondary to activation of the subthalamic nucleus. We sought to determine the contribution of dopamine by examining metabotropic glutamate receptor agonist-induced rotation in rats following acute dopamine depletion by reserpine/alpha-methyl-para-tyrosine treatment, or chronic dopamine depletion by 6-hydroxydopamine treatment. In unilateral 6-hydroxydopamine lesioned rats, the group I metabotropic glutamate receptor agonist (RS)-3,5-dihydroxyphenylglycine induced contralateral rotation with a coincident increase in striatal 3,4-dihydroxyphenylacetic acid. The rotation was attenuated by the group I antagonist 1-aminoindan-1,5-dicarboxylate. Examination of FOS-like immunoreactivity and [14C]2-deoxyglucose uptake in chronically dopamine depleted rats also revealed similar patterns to those seen previously in intact rats. However, acutely dopamine depleted rats do not exhibit metabotropic glutamate receptor agonist-induced rotation and show a different pattern of [14C]2-deoxyglucose uptake, with no increase in glucose utilization in the intermediate and deep layers of the superior colliculus. These results suggest that there are compensatory changes under conditions of chronic dopamine denervation which permit metabotropic glutamate receptor agonist-induced rotation to occur, which may include dopamine receptor supersensitivity, increased dopamine turnover, and/or changes in sensitivity of striatal group I metabotropic glutamate receptors. The group III metabotropic glutamate receptor agonist L-(+)-2-amino-4-phosphonobutyrate induced contralateral rotation in 6-hydroxydopamine lesioned rats, while it had no effect in intact rats. Additionally, examination of FOS-like immunoreactivity revealed a distinct pattern following L-(+)-2-amino-4-phosphonobutyrate administration in 6-hydroxydopamine lesioned versus intact rats. These results suggest that there is a change in the effect of striatal group III stimulation under conditions of dopamine depletion.

Calcium channel beta 4 (CACNB4): human ortholog of the mouse epilepsy gene lethargic.

The mouse neurological mutant lethargic (lh) is characterized by ataxia, focal myoclonus, and absence epilepsy due to a loss-of-function mutation in the beta4 subunit of the voltage-gated calcium channel. To evaluate the role of this channel subunit in human neurological disease, we determined the chromosomal location and intron/exon structure of the human CACNB4 gene. The 1560-bp open reading frame of the CACNB4 cDNA predicts a 58-kDa protein with an amino acid sequence that is 99% identical to the rat protein. The 13 coding exons of CACNB4 span >55 kb of genomic DNA. Human cerebellar RNA contains one major CACNB4 transcript that is 9 kb in length. Expression of CACNB4 was detected in cerebellum, kidney, testis, retina, lymphoblasts, and circulating lymphocytes. Retinal transcripts were localized by in situ hybridization to ganglion cells and the inner nuclear layer. Analysis of the GeneBridge 4 radiation hybrid mapping panel localized CACNB4 to position 791 cR on human chromosome 2, in a conserved linkage group on human 2q22-q31 and mouse chromosome 2. We localized CACNB4 to the 1.3-Mb YAC clone 952F10 in Whitehead contig WC861, along with the polymorphic markers D2S2236 and D2S2299. The chromosomal linkage of three of the four beta subunit genes to homeobox gene clusters associates the evolutionary origin of the beta gene family with the events that generated the four HOX clusters early in vertebrate evolution.

Metabotropic glutamate agonist-induced rotation: a pharmacological, FOS immunohistochemical, and [14C]-2-deoxyglucose autoradiographic study.

Metabotropic glutamate receptors (mGluRs) are a major class of excitatory amino acid receptors. Eight mGluR subtypes, coupled to a variety of effector systems, have been cloned. These receptors have been classified into three groups based on amino acid sequence homology, effector systems, and pharmacological profile. Group I mGluRs increase phosphoinositide turnover, whereas groups II and III mGluRs are negatively coupled to adenylyl cyclase. The striatum possesses a high density of mGluR binding sites, and several mGluR mRNAs and proteins are expressed by striatal neurons. In rats, unilateral striatal injection of the nonsubtype selective mGluR agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD) results in contralateral rotation with delayed onset, thought to be secondary to an increase in dopamine release. We sought to determine the mGluR subtype(s) involved, the modulation of the rotation by other basal ganglia neurotransmitter systems, and the functional anatomy underlying the rotational behavior. The group I mGluR agonist 3,5-dihydroxyphenylglycine (DHPG) induced contralateral rotation in a dose-dependent manner, whereas group II and group III agonists were ineffective. Rotation induced by DHPG or 1S,3R-ACPD was attenuated by group I antagonists, but not by group II or group III antagonists. This suggests that the rotation is mediated by group I mGluRs. Rotation induced by DHPG or 1S,3R-ACPD was attenuated by pretreatment with antagonists at muscarinic cholinergic, adenosine A2, dopamine D2, or dopamine D1 receptors. Examination of FOS-like immunoreactivity after group I and group II mGluR agonist administration suggests increased activity in the striatopallidal pathway. However, [14C]-2-deoxyglucose uptake studies indicate increased activity in nuclei of the striatopallidal (indirect) pathway, particularly in the subthalamic nucleus, only after group I mGluR activation.

Emotional development in infancy: theoretical models and nursing implications.

TOPIC: Theories of emotional development in infancy that are relevant to clinical practice. PURPOSE: A discussion of the complementary theories such as Emde's (1989) psychoanalytically based developmental model of early socioemotional reorganizations and Stern's (1985a) model of the developing self, with the goal of presenting an integrated view of key socioemotional processes and their developmental foundations during infancy. Concepts such as "emotional availability" and "affect attunement" are examined as vehicles for early socioemotional organization and adaptation. SOURCES: Relevant literature from nursing, child psychiatry, and developmental psychology. CONCLUSIONS: Implications are drawn from clinical assessment of high-risk caregiver-infant relationships and early causal pathways for later socioemotional dysfunction. Nurses should focus on the importance of assessing interpersonal variables and their ongoing contribution to internalizing patterns of dysfunctional behavior in children.

Nurse to nurse referral: the role of the child psychiatric nurse consultant.

Nurse to nurse referral is a process by which nurses can use various areas of expert knowledge within their profession. Nurse to nurse referral is examined here with specific reference to the role of the child psychiatric clinical nurse specialist as consultant to the pediatric nurse practitioner. Current research and Chisholm's guidelines for the nurse consultant role are reviewed. With a focus maintained on the ambulatory care area, the referral and consultation process in such settings is discussed. Parameters for using nurse to nurse referral between pediatric nurse practitioners and child psychiatric clinical specialists are outlined and recommendations given for implementing the process. The conclusion is reached that this type of referral process includes a number of components that must be examined, if it is to continue to develop and flourish.

Adenosine A2 receptor-mediated modulation of contralateral rotation induced by metabotropic glutamate receptor activation.

Systemic pretreatment with the adenosine receptor antagonist theophylline significantly decreases contralateral rotation induced by unilateral intrastriatal 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD). Intrastriatal or intrasubthalamic nucleus coadministration of theophylline and 1S,3R-ACPD significantly decreases contralateral rotation suggesting that metabotropic glutamate (mGlu) receptors and adenosine receptors interact locally. These appear to be adenosine A2 receptor effects as the adenosine A2 receptor antagonist 8-(3-chlorostyryl)caffeine (CSC) also decreases contralateral rotation induced by unilateral intrastriatal and intrasubthalamic nucleus administration of 1S,3R-ACPD, while the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) has no effect. Pretreatment with the adenosine A2 receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido adenosine hydrochloride (CGS 21680) potentiates contralateral rotation induced by unilateral striatal 1S,3R-ACPD, whereas pretreatment with the adenosine A1 receptor agonist N6-cyclopentyl-adenosine (CPA) has no effect. These results suggest that mGlu receptor effects may be due, in part, to modulation of adenosine action.