Andrew Escayg

Mutations of SCN1A, encoding a neuronal sodium channel, in two families with GEFS+2

Generalized epilepsy with febrile seizures plus type 2 (GEFS+2, MIM 604233) is an autosomal dominant disorder characterized by febrile seizures in children and afebrile seizures in adults. We describe here two mutations of the gene encoding the neuronal voltage-gated sodium channel (SCN1A), Thr875Met and Arg1648His, that co-segregate with the disorder in two families with GEFS+ linked to chromosome 2q. These mutations identify a new disease gene for human inherited epilepsy.

Coding and Noncoding Variation of the Human Calcium-Channel β4-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.

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.

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 27u2009kb of coding sequence and 3u2009kb 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 Nav1.1 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 neurons 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.

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 Xenopusoocyte 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.

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.