The role of neuronal GABA(A) receptor subunit mutations in idiopathic generalized epilepsies.

Rare GABA(A) receptor gamma2 and alpha1 subunit mutations of pathogenic effect have been described segregating in families with "monogenic" epilepsies. We now report globally on the genetic variation contained within all 16 neuronal GABA(A) receptor subunit genes from the one patient cohort. The cohort consists of GEFS(+), FS, and IGE subgroups as either sporadic cases or index cases from small families, with one index case from one large IGE family. The rarity of mutations and coding variation in general across all of the subunits suggests a low tolerance for mutations affecting GABA mediated neuronal inhibition. Characterization of the broader channelopathy load associated with susceptibility to these common epilepsies mostly with complex genetics will need to be expanded beyond the family of GABA(A) receptor subunits to all families of neuronal ion channels and their interacting molecules by systematic mutation detection associated with functional investigation of their naturally occurring genetic variations.

Generalized epilepsy with febrile seizures plus-associated sodium channel beta1 subunit mutations severely reduce beta subunit-mediated modulation of sodium channel function.

Two novel mutations (R85C and R85H) on the extracellular immunoglobulin-like domain of the sodium channel beta1 subunit have been identified in individuals from two families with generalized epilepsy with febrile seizures plus (GEFS+). The functional consequences of these two mutations were determined by co-expression of the human brain NaV1.2 alpha subunit with wild type or mutant beta1 subunits in human embryonic kidney (HEK)-293T cells. Patch clamp studies confirmed the regulatory role of beta1 in that relative to NaV1.2 alone the NaV1.2+beta1 currents had right-shifted voltage dependence of activation, fast and slow inactivation and reduced use dependence. In addition, the NaV1.2+beta1 current entered fast inactivation slightly faster than NaV1.2 channels alone. The beta1(R85C) subunit appears to be a complete loss of function in that none of the modulating effects of the wild type beta1 were observed when it was co-expressed with NaV1.2. Interestingly, the beta1(R85H) subunit also failed to modulate fast kinetics, however, it shifted the voltage dependence of steady state slow inactivation in the same way as the wild type beta1 subunit. Immunohistochemical studies revealed cell surface expression of the wild type beta1 subunit and undetectable levels of cell surface expression for both mutants. The functional studies suggest association of the beta1(R85H) subunit with the alpha subunit where its influence is limited to modulating steady state slow inactivation. In summary, the mutant beta1 subunits essentially fail to modulate alpha subunits which could increase neuronal excitability and underlie GEFS+ pathogenesis.

NEDD4-2 as a potential candidate susceptibility gene for epileptic photosensitivity.

Photosensitive seizures occur most commonly in childhood and adolescence, usually as a manifestation of complex idiopathic generalized epilepsies (IGEs). Molecular mechanisms underlying this condition are yet to be determined because no susceptibility genes have been identified. The NEDD4-2 (Neuronally Expressed Developmentally Downregulated 4) gene encodes a ubiquitin protein ligase proposed to regulate cell surface levels of several ion channels, receptors and transporters involved in regulating neuronal excitability, including voltage-gated sodium channels (VGSCs), the most clinically relevant of the epilepsy genes. The regulation of NEDD4-2 in vivo involves complex interactions with accessory proteins in a cell type specific manner. We screened NEDD4-2 for mutations in a cohort of 253 families with IGEs. We identified three NEDD4-2 missense changes in highly conserved residues; S233L, E271A and H515P in families with photosensitive generalized epilepsy. The NEDD4-2 variants were as effective as wild-type NEDD4-2 in downregulating the VGSC subtype Na(v)1.2 when assessed in the Xenopus oocyte heterologous expression system showing that the direct interaction with the ion channel was not altered by these variants. These data raise the possibility that photosensitive epilepsy may arise from defective interaction of NEDD4-2 with as yet unidentified accessory or target proteins.

Severe myoclonic epilepsy of infancy (Dravet syndrome): recognition and diagnosis in adults.

Establishing an etiologic diagnosis in adults with refractory epilepsy and intellectual disability is challenging. We analyzed the phenotype of 14 adults with severe myoclonic epilepsy of infancy. This phenotype comprised heterogeneous seizure types with nocturnal generalized tonic-clonic seizures predominating, mild to severe intellectual disability, and variable motor abnormalities. The diagnosis was suggested by a characteristic evolution of clinical findings in the first years of life. Ten had mutations in SCN1A and one in GABRG2.

A new molecular mechanism for severe myoclonic epilepsy of infancy: exonic deletions in SCN1A.

We examined cases of severe myoclonic epilepsy of infancy (SMEI) for exon deletions or duplications within the sodium channel SCN1A gene by multiplex ligation-dependent probe amplification. Two of 13 patients (15%) who fulfilled the strict clinical definition of SMEI but without SCN1A coding or splicing mutations had exonic deletions of SCN1A.

LGI1 mutations in temporal lobe epilepsies.

BACKGROUND AND OBJECTIVES: A number of familial temporal lobe epilepsies (TLE) have been recently recognized. Mutations in LGI1 (leucine-rich, glioma-inactivated 1 gene) have been found in a few families with the syndrome of autosomal dominant partial epilepsy with auditory features (ADPEAF). The authors aimed to determine the spectrum of TLE phenotypes with LGI1 mutations, to study the frequency of mutations in ADPEAF, and to examine the role of LGI1 paralogs in ADPEAF without LGI1 mutations. METHODS: The authors performed a clinical and molecular analysis on 75 pedigrees comprising 54 with a variety of familial epilepsies associated with TLE and 21 sporadic TLE cases. All were studied for mutations in LGI1. ADPEAF families negative for LGI1 mutations were screened for mutations in LGI2, LGI3, and LGI4. RESULTS: Four families had ADPEAF, 22 had mesial TLE, 11 had TLE with febrile seizures, two had TLE with developmental abnormalities, and 15 had various other TLE syndromes. LGI1 mutations were found in two of four ADPEAF families, but in none of the other 50 families nor in the 21 individuals with sporadic TLE. The mutations were novel missense mutations in exons 1 (c.124T-->G; C42G) and 8 (c.1418C-->T; S473L). No mutations in LGI2, LGI3, or LGI4 were found in the other two ADPEAF families. CONCLUSION: In TLE, mutations in LGI1 are specific for ADPEAF but do not occur in all families. ADPEAF is genetically heterogeneous, but mutations in LGI2, LGI3, or LGI4 did not account for families without LGI1 mutations.

Sodium channel alpha1-subunit mutations in severe myoclonic epilepsy of infancy and infantile spasms.

BACKGROUND: Mutations in SCN1A, the gene encoding the alpha1 subunit of the sodium channel, have been found in severe myoclonic epilepsy of infancy (SMEI) and generalized epilepsy with febrile seizures plus (GEFS+). Mutations in SMEI include missense, nonsense, and frameshift mutations more commonly arising de novo in affected patients. This finding is difficult to reconcile with the family history of GEFS+ in a significant proportion of patients with SMEI. Infantile spasms (IS), or West syndrome, is a severe epileptic encephalopathy that is usually symptomatic. In some cases, no etiology is found and there is a family history of epilepsy. METHOD: The authors screened SCN1A in 24 patients with SMEI and 23 with IS. RESULTS: Mutations were found in 8 of 24 (33%) SMEI patients, a frequency much lower than initial reports from Europe and Japan. One mutation near the carboxy terminus was identified in an IS patient. A family history of seizures was found in 17 of 24 patients with SMEI. CONCLUSIONS: The rate of SCN1A mutations in this cohort of SMEI patients suggests that other factors may be important in SMEI. Less severe mutations associated with GEFS+ could interact with other loci to cause SMEI in cases with a family history of GEFS+. This study extends the phenotypic heterogeneity of mutations in SCN1A to include IS.

Mutant GABA(A) receptor gamma2-subunit in childhood absence epilepsy and febrile seizures.

Epilepsies affect at least 2% of the population at some time in life, and many forms have genetic determinants. We have found a mutation in a gene encoding a GABA(A) receptor subunit in a large family with epilepsy. The two main phenotypes were childhood absence epilepsy (CAE) and febrile seizures (FS). There is a recognized genetic relationship between FS and CAE, yet the two syndromes have different ages of onset, and the physiology of absences and convulsions is distinct. This suggests the mutation has age-dependent effects on different neuronal networks that influence the expression of these clinically distinct, but genetically related, epilepsy phenotypes. We found that the mutation in GABRG2 (encoding the gamma2-subunit) abolished in vitro sensitivity to diazepam, raising the possibility that endozepines do in fact exist and have a physiological role in preventing seizures.

Mutations at G:C base pairs predominate after replication of peroxyl radical-damaged pSP189 plasmids in human cells.

The mutagenicity of peroxyl radicals, important participants in lipid peroxidation cascades, was investigated using a plasmid-based mutational assay system. Double-stranded pSP189 plasmids were incubated with a range of concentrations of the water-soluble peroxyl radical generator 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH). Following replication in human Ad293 cells, the plasmids were screened for supF mutations in indicator bacteria. Exposure to peroxyl radicals caused strand nicking and a decrease in transfection efficiency, which was accompanied by a significant increase in supF mutants. Each of these effects was abolished in the presence of the water-soluble vitamin E analogue Trolox. Automated sequencing of 76 AAPH-induced mutant plasmids revealed that substitutions at G:C base pairs were the most common changes, accounting for 85.5% of all identified mutations. Of these, most comprised G:C-->T:A transversions (53.5%), with lesser contributions by G:C-->A:T transitions (23.9%) and G:C-->C:G transversions (22.5%). Collectively, these data confirm our previous findings concerning the spectrum of mutations produced upon bacterial replication of peroxyl radical-damaged phage DNA and extend them by showing that such damage has mutagenic consequences during replication in more complex eukaryotic systems.

Formation of novel C1-oxidised abasic sites in alkylperoxyl radical-damaged plasmid DNA.

We have recently shown that peroxyl radicals react with DNA to form alkali-labile sites. To further characterise these lesions, we studied their susceptibility to digestion by repair endonucleases that recognise different types of abasic sites. We found that peroxyl radical-damaged pSP189 plasmids were resistant to cleavage by T4 endonuclease V, an enzyme that incises DNA at "regular" and C4-oxidised abasic residues. In contrast, the DNA was digested by exonuclease III, an enzyme that recognises "regular" and C1-oxidised abasic sites. The presence of Trolox during exposure to peroxyl radicals reduced subsequent DNA cleavage by exonuclease III, while prior incubation of damaged plasmids with methoxyamine potentiated digestion by this enzyme. These findings suggest that peroxyl radical-induced DNA damage involves the generation of novel C1-oxidised deoxyribose residues.

Role of G-->T transversions in the mutagenicity of alkylperoxyl radicals: induction of alkali-labile sites in bacteriophage M13mp19.

The mutagenicity of peroxyl radicals, ubiquitous products of lipid peroxidation, was assessed using an in vitro M13 forward mutational assay. Single-stranded M13mp19 plasmids were incubated with a range of concentrations of the azo initiator 2,2'-azobis(2-amidinopropane) hydrochloride, and then transfected into competent, SOS-induced Escherichia coli JM105 cells. Incubation with peroxyl radicals produced a concentration-dependent decrease in phage survival, with a 500 microM concentration of the azo initiator reducing the transfection efficiency by more than 90% while inducing a corresponding 6-fold increase in lacZ alpha mutation frequencies. Peroxyl radical-induced mutagenesis was completely prevented by the peroxyl radical scavenger Trolox. Automated DNA sequence analysis of the lacZ alpha gene of 100 peroxyl radical-induced mutants revealed that the most frequent sequence changes were base pair substitutions (92/95), with G-->T transversions predominating (73/92). Alkaline treatment prior to transfection diminished the mutagenicity of damaged plasmids to a level resembling that of unmodified DNA. While abasic sites might account for the sensitivity to alkaline cleavage, the possibility that unidentified nonabasic alkaline-labile lesions also contribute to peroxyl radical mutagenesis cannot be excluded. Collectively, these findings raise the possibility that DNA damage caused by a major class of endogenous radicals contributes to one of the most common spontaneous mutational events, the G-->T transversion.

Genotoxicity of acyl glucuronide metabolites formed from clofibric acid and gemfibrozil: a novel role for phase-II-mediated bioactivation in the hepatocarcinogenicity of the parent aglycones?

Glucuronides formed from carboxylate-containing xenobiotics are more chemically reactive than most Phase II conjugates. However, while they have been shown to form protein adducts, their reactions with DNA have received little attention. We thus used the M13 forward mutational assay to assess the genotoxicity of acyl glucuronides formed from two widely used fibrate hypolipidemics, clofibric acid and gemfibrozil. Single-stranded M13mp19 bacteriophage DNA was incubated in pH 7.4 buffer for 16 h in the presence of 0, 1, 2.5, and 5 mM concentrations of each glucuronide as well as the respective aglycones. The modified DNA was then transfected into SOS-induced competent Escherichia coli JM105 cells and the transfection efficiency was determined after phage growth overnight at 37 degrees C. Significantly, both acyl glucuronides, but not the aglycones, caused a concentration-dependent decrease in the transfection efficiency of the DNA, with a greater than 80% decrease in phage survival produced by the 5 mM concentrations of the glucuronides. No increase in lacZa mutations accompanied the loss of phage survival. We propose that these genotoxic effects involve reactions with nucleophilic centers in DNA via a Schiff base mechanism that is analogous to the glycosylation of DNA by endogenous sugars. Since strand nicking is known to accompany such damage, we also analyzed glucuronide-treated pSP189 plasmids for strand breakages via agarose gel electrophoresis. Both clofibric acid and gemfibrozil glucuronides produced significant concentration-related strand nicking and exhibited over 10-fold greater reactivity than the endogenous glycosylating agent, glucose 6-phosphate. On the basis of these findings, the possibility that this novel bioactivation route participates in the carcinogenicity of the fibrate hypolipidemics deserves investigation.

Resistance vessel gene expression of nerve growth factor is elevated in young spontaneously hypertensive rats.

OBJECTIVE: In this study we determined whether the enhanced production of nerve growth factor (NGF) and the associated hypernoradrenergic innervation of the vasculature of the spontaneously hypertensive rat (SHR) was associated with an increased gene expression of messenger (m)RNA encoding for nerve growth factor. DESIGN: It has been shown previously that the hypernoradrenergic innervation of the SHR occurs early, as does the enhanced expression for NGF. In this study we analysed the content of NGF mRNA in blood vessels from young SHR and normotensive Wistar-Kyoto (WKY) rats. METHODS: Total RNA was isolated from mesenteric arteries from 2-, 10- and 43-day-old SHR and WKY rats and RNA was also isolated from caudal arteries from 43-day-old rats. The RNA was subjected to Northern transfer or slot blots and the content of NGF mRNA measured after hybridization with a 32P-labelled complementary (c)DNA probe for NGF. RESULTS: Slot blot analysis indicated a larger concentration of NGF mRNA in mesenteric and caudal arteries from SHR than for tissues from WKY rats. CONCLUSIONS: In this genetic model of hypertension the results indicate an association between an enhanced level of NGF mRNA and the appearance of vascular hypernoradrenergic hypertension.

Nerve growth factor mRNA content parallels altered sympathetic innervation in the spontaneously hypertensive rat.

1. In order to explore the mechanisms responsible for the hypernoradrenergic innervation of the vasculature in the spontaneously hypertensive rat (SHR) the tissue content of nerve growth factor messenger ribonucleic acid (NGFmRNA) was examined. 2. The concentration of NGFmRNA was markedly elevated in mesenteric veins obtained from SHR when compared with the contents of NGFmRNA in veins from Wistar Kyoto rats (WKY). 3. The NGFmRNA content of kidneys was greater in SHR when compared with the levels present in WKY rats for 10- and 43-day-old animals. 4. In contrast to the pattern observed for veins and kidneys, the NGFmRNA content of SHR hearts was smaller than those present in hearts from WKY rats for 2, 10 and 43-day-old animals. 5. The results demonstrate that tissues with enhanced innervation (the kidney and mesenteric vasculature) in SHR are associated with an enhanced expression of NGFmRNA. In contrast, the heart, which does not display an enhanced sympathetic innervation in the SHR, does not have an increased expression of NGFmRNA. 6. It is suggested that in the SHR there is a tight relationship between hypernoradrenergic innervation in the vasculature and gene expression for NGFmRNA.