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.

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.

Generalized epilepsy with febrile seizures plus type 2 mutation W1204R alters voltage-dependent gating of Na(v)1.1 sodium channels.

Nine mutations that cause generalized epilepsy with febrile seizures plus have been identified in the SCN1A gene encoding the alpha subunit of the Na(v)1.1 voltage-gated sodium channel. The functional properties of two of these mutations (T875M and R1648H) have previously been described. T875M was shown to enhance slow inactivation, while R1648H dramatically accelerated recovery from inactivation. In this report, we have cloned, expressed and characterized the functional effects of a third generalized epilepsy with febrile seizures plus mutation, W1204R (Am J Hum Genet 68 (2001) 866). The mutation was cloned into the orthologous rat channel, rNa(v)1.1, and at the same time a single base pair insertion at base 120 in the original rNa(v)1.1 clone was corrected. The level of expression of the corrected wild-type rNa(v)1.1 was approximately 1000-fold higher than that of the original clone and comparable to that achieved with other neuronal sodium channels expressed in Xenopus oocytes. The properties of the W1204R mutant in the corrected rNa(v)1.1 were determined in the absence and presence of the beta1 subunit in Xenopus oocytes. The W1204R mutation resulted in approximately 11 mV hyperpolarized shifts in the voltage-dependence of activation and steady-state inactivation when expressed as an alpha subunit alone. When the channels were coexpressed with the beta1 subunit, the hyperpolarized shifts were still present but smaller, approximately 5 mV in magnitude. All other properties that we examined were comparable for the mutant and wild-type channels. The negative shift in activation would increase channel excitability, whereas the negative shift in inactivation would decrease excitability. The negative shifts in both properties also shifted the window current, which is the voltage region in which sodium channels can continue to open because some percentage of channels are activated and not all of the channels are inactivated. The shift in window current for the W1204R mutation could result in hyperexcitability because the neuron's potential is more likely to reach the more negative range. These results demonstrate that a third SCN1A mutation that causes generalized epilepsy with febrile seizures plus 2 alters the properties of the sodium channel in a different manner than the previous two mutations that were studied. The diversity in functional effects for these three mutations indicates that a similar clinical phenotype can result from very different underlying sodium channel abnormalities.

Identification of epilepsy genes in human and mouse.

The development of molecular markers and genomic resources has facilitated the isolation of genes responsible for rare monogenic epilepsies in human and mouse. Many of the identified genes encode ion channels or other components of neuronal signaling. The electrophysiological properties of mutant alleles indicate that neuronal hyperexcitability is one cellular mechanism underlying seizures. Genetic heterogeneity and allelic variability are hallmarks of human epilepsy. For example, mutations in three different sodium channel genes can produce the same syndrome, GEFS+, while individuals with the same allele can experience different types of seizures. Haploinsufficiency for the sodium channel SCN1A has been demonstrated by the severe infantile epilepsy and cognitive deficits in heterozygotes for de novo null mutations. Large-scale patient screening is in progress to determine whether less severe alleles of the genes responsible for monogenic epilepsy may contribute to the common types of epilepsy in the human population. The development of pharmaceuticals directed towards specific epilepsy genotypes can be anticipated, and the introduction of patient mutations into the mouse genome will provide models for testing these targeted therapies.

Functional effects of two voltage-gated sodium channel mutations that cause generalized epilepsy with febrile seizures plus type 2.

Two mutations that cause generalized epilepsy with febrile seizures plus (GEFS+) have been identified previously in the SCN1A gene encoding the alpha subunit of the Na(v)1.1 voltage-gated sodium channel (Escayg et al., 2000). Both mutations change conserved residues in putative voltage-sensing S4 segments, T875M in domain II and R1648H in domain IV. Each mutation was cloned into the orthologous rat channel rNa(v)1.1, and the properties of the mutant channels were determined in the absence and presence of the beta1 subunit in Xenopus oocytes. Neither mutation significantly altered the voltage dependence of either activation or inactivation in the presence of the beta1 subunit. The most prominent effect of the T875M mutation was to enhance slow inactivation in the presence of beta1, with small effects on the kinetics of recovery from inactivation and use-dependent activity of the channel in both the presence and absence of the beta1 subunit. The most prominent effects of the R1648H mutation were to accelerate recovery from inactivation and decrease the use dependence of channel activity with and without the beta1 subunit. The DIV mutation would cause a phenotype of sodium channel hyperexcitability, whereas the DII mutation would cause a phenotype of sodium channel hypoexcitability, suggesting that either an increase or decrease in sodium channel activity can result in seizures.

Sodium channels and neurological disease: insights from Scn8a mutations in the mouse.

The human genome contains 10 voltage-gated sodium channel genes, 7 of which are expressed in neurons of the CNS and PNS. The availability of human genome sequences and high-throughput mutation screening methods make it likely that many human disease mutations will be identified in these genes in the near future. Mutations of Scn8a in the mouse demonstrate the broad spectrum of neurological disease that can result from different alleles of the same sodium channel gene. Null mutations of Scn8a produce motor neuron failure, loss of neuromuscular transmission, and lethal paralysis. Less severe mutations result in ataxia, tremor, muscle weakness, and dystonia. The effects of Scn8a mutations on channel properties have been studied in the Xenopus oocyte expression system and in neurons isolated from the mutant mice. The Scn8a mutations provide insight into the mode of inheritance, effect on neuronal sodium currents, and role of modifier genes in sodium channel disease, highlighting the ways in which mouse models of human mutations can be used in the future to understand the pathophysiology of human disease.

A novel SCN1A mutation associated with generalized epilepsy with febrile seizures plus--and prevalence of variants in patients with epilepsy.

We recently described mutations of the neuronal sodium-channel alpha-subunit gene, SCN1A, on chromosome 2q24 in two families with generalized epilepsy with febrile seizures plus (GEFS+) type 2. To assess the contribution that SCN1A makes to other types of epilepsy, 226 patients with either juvenile myoclonic epilepsy, absence epilepsy, or febrile convulsions were screened by conformation-sensitive gel electrophoresis and manual sequencing of variants; the sample included 165 probands from multiplex families and 61 sporadic cases. The novel mutation W1204R was identified in a family with GEFS+. Seven other coding changes were observed; three of these are potential disease-causing mutations. Two common haplotypes, with frequencies of .67 and .33, were defined by five single-nucleotide polymorphisms (SNPs) spanning a 14-kb region of linkage disequilibrium. An SNP located 18 bp upstream of the splice-acceptor site for exon 3 was observed in 7 of the 226 patients but was not present in 185 controls, suggesting possible association with a disease mutation. This work has confirmed the role of SCN1A in GEFS+, by identification of a novel mutation in a previously undescribed family. Although a few candidate disease alleles were identified, the patient survey suggests that SCN1A is not a major contributor to idiopathic generalized epilepsy. The SCN1A haplotypes and SNPs identified here will be useful in future association and linkage studies.

Evolution of the ovine MHC DQA region.

Southern hybridisation was used to define an apparent gene duplication event at the ovine DQA2 locus. Approximately 500 sheep from five different breeds were genotyped at their DQA1 and DQA2 loci. A subset of these were selected for further characterisation. Southern hybridisation of TaqI digested DNA revealed no DQA1 region in some sheep. It was also noted in these DQA1 null animals the DQA2 specific probe hybridised to two bands. An EcoRV-RFLP designed to distinguish copy number confirmed this duplication of the DQA2 region. The results showed that the duplication was exclusively associated with the DQA1 null haplotype and occurred only in alleles DQA2-F, -G, -I and -J. Comparison with bovine MHC genes revealed that they also contained a DQA1 null haplotype and that this haplotype was associated with a putative DQA3 gene. The potential for an ovine DQA3 locus is discussed.

Coding and noncoding variation of the human calcium-channel beta4-subunit gene CACNB4 in patients with idiopathic generalized epilepsy and episodic ataxia.

Inactivation of the beta4 subunit of the calcium channel in the mouse neurological mutant lethargic results in a complex neurological disorder that includes absence epilepsy and ataxia. To determine the role of the calcium-channel beta4-subunit gene CACNB4 on chromosome 2q22-23 in related human disorders, we screened for mutations in small pedigrees with familial epilepsy and ataxia. The premature-termination mutation R482X was identified in a patient with juvenile myoclonic epilepsy. The R482X protein lacks the 38 C-terminal amino acids containing part of an interaction domain for the alpha1 subunit. The missense mutation C104F was identified both in a German family with generalized epilepsy and praxis-induced seizures and in a French Canadian family with episodic ataxia. These coding mutations were not detected in 255 unaffected control individuals (510 chromosomes), and they may be considered candidate disease mutations. The results of functional tests of the truncated protein R482X in Xenopus laevis oocytes demonstrated a small decrease in the fast time constant for inactivation of the cotransfected alpha1 subunit. Further studies will be required to evaluate the in vivo consequences of these mutations. We also describe eight noncoding single-nucleotide substitutions, two of which are present at polymorphic frequency, and a previously unrecognized first intron of CACNB4 that interrupts exon 1 at codon 21.

The mouse neurological mutant flailer expresses a novel hybrid gene derived by exon shuffling between Gnb5 and Myo5a.

Exon shuffling is thought to be an important mechanism for evolution of new genes. Here we show that the mouse neurological mutation flailer (flr) expresses a novel gene that combines the promoter and first two exons of guanine nucleotide binding protein beta 5 (Gnb5) with the C-terminal exons of the closely linked Myosin 5A (MyoVA) gene (Myo5a). The flailer protein, which is expressed predominantly in brain, contains the N-terminal 83 amino acids of Gnb5 fused in-frame with the C-terminal 711 amino acids of MyoVA, including the globular tail domain that binds organelles for intracellular transport. Biochemical and genetic studies indicate that the flailer protein competes with wild-type MyoVA in vivo, preventing the localization of smooth endoplasmic reticulum vesicles in the dendritic spines of cerebellar Purkinje cells. The flailer protein thus has a dominant-negative mechanism of action with a recessive mode of inheritance due to the dependence of competitive binding on the ratio between mutant and wild-type proteins. The chromosomal arrangement of Myo5a upstream of Gnb5 is consistent with non-homologous recombination as the mutational mechanism. To our knowledge, flailer is the first example of a mammalian mutation caused by germ line exon shuffling between unrelated genes.

Coding sequence, genomic organization, and conserved chromosomal localization of the mouse gene Scn11a encoding the sodium channel NaN.

Previous studies have shown that sodium channel alpha-subunit NaN is preferentially expressed in small-diameter sensory neurons of dorsal root ganglia and trigeminal ganglia. These neurons include high-threshold nociceptors that are involved in transduction of pain associated with tissue and nerve injury. In this study, we show that mouse NaN is a 1765-amino-acid peptide that is predicted to produce a current that is resistant to tetrodotoxin (TTX-R). Mouse and rat NaN are 80 and 89% identical at the nucleotide and amino acid levels, respectively. The Scn11a gene encoding this cDNA is organized into 24 exons. Unlike some alpha-subunits, Scn11a does not have an alternative exon 5 in domain I. Introns of the U2 and U12 spliceosome types are present at conserved positions relative to other members of this family. Scn11a is located on mouse chromosome 9, close to the two other TTX-R sodium channel genes, Scn5a and Scn10a. The human gene, SCN11A, was mapped to the conserved linkage group on chromosome 3p21-p24, close to human SCN5A and SCN10A. The colocalization of the three sodium channel genes supports a common lineage of the TTX-R sodium channels.

Dystonia associated with mutation of the neuronal sodium channel Scn8a and identification of the modifier locus Scnm1 on mouse chromosome 3.

The mouse mutant medJ contains a splice site mutation in the neuronal sodium channel Scn8a that results in a very low level of expression. On a C57BL/6J genetic background, medJ homozygotes exhibit progressive paralysis and juvenile lethality. The C3H genetic background has an ameliorating effect, producing viable adults with a novel dystonic phenotype. The dystonic mice exhibit movement-induced, sustained abnormal postures of the trunk and limbs. A dominant modifier locus responsible for the difference between strains was mapped to a 4.5 +/- 1.3 cM interval on mouse chromosome 3. Our findings establish a role for ion channels in dystonia and demonstrate the impact of genetic background on its severity and progression. This new model suggests that SCN8A on chromosome 12q13 and SCNM1 on chromosome 1p21-1q21 may contribute to human inherited dystonia.

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.

Association between alleles of the ovine major histocompatibility complex and resistance to footrot.

Variation in natural resistance to footrot may be genetically derived, implying that genetic markers for resistance may exist and allow selection of superior animals. In this study association between variation within the ovine MHC class II region and resistance to footrot was investigated in two trials. Half-sib progeny were subjected to a field challenge with footrot and their condition subsequently recorded. The animals were then typed at their MHC class II loci to investigate associations between inherited paternal haplotype and footrot status. In the first trial an association between MHC haplotype and footrot status was observed across all animals (P = 0.005), when the self-curing and resistant animals were combined (P = 0.002) and when the self-curing animals were excluded from the analysis (P = 0.001). No association was observed in the second trial, a result attributed to the dry weather conditions which led to poor disease transmission and unreliable disease classification.

Ion channel mutations in mouse models of inherited neurological disease.

Analysis of the molecular defects in mouse mutants can identify candidate genes for human neurological disorders. During the past 2 years, mutations in sodium channels, calcium channels and potassium channels have been identified by positional cloning of the spontaneous mouse mutants motor endplate disease, tottering, lethargic and weaver. The phenotypes of four allelic mutations identified in the sodium channel gene Scn8a range from ataxia and muscle weakness through severe dystonia and progressive paralysis, indicating that human mutations in this gene could be associated with a variety of clinical syndromes. Mutations of the calcium channel subunits beta 4 in the lethargic mouse and alpha 1A in the tottering mouse have specific effects on cerebellar function. Targeted mutation of ligand-gated ion channels has also been used to generate new models of neurological disease. We will review these recent achievements and their implications for human neurological disease. The mouse studies indicate that mutations in ion channel genes are likely to be responsible for a broad spectrum of clinical phenotypes in human neurological disorders.

Polymorphism at the ovine major histocompatibility complex class II loci.

Southern hybridization analysis of the ovine major histocompatibility complex (MHC) (MhcOvar) class II region, using sheep-specific probes for the DQA1, DQA2, DQB and DRA loci, has revealed extensive polymorphism. DQA1 and DQA2 had eight and 16 alleles respectively, DQB had six and DRA had three alleles. Little information was derived from the DRB locus owing to extensive cross-hybridization between the DRB probe and the DQB locus. Differences in allele frequency between breeds were revealed. At the DQA1 locus a null allele (DQA1-N) was observed with a frequency of between 27% and 45%, making this the most common DQA1 allele in all breeds examined. The frequency of DQA1-N homozygotes was between 11% and 18%, raising questions as to the functional significance of the DQA1 gene. Linkage analysis between the DQA1, DQA2, DQB and DRA loci did not reveal any recombination.