Multiple molecular mechanisms for a single GABA _A mutation in epilepsy

Abstract

Objective: To understand the molecular basis and differential penetrance of febrile seizures and absence seizures in patients with the g2(R43Q) GABA A receptor mutation.Methods: Spike-and-wave discharges and thermal seizure susceptibility were measured in heterozygous GABA A g2 knock-out and GABA A g2(R43Q) knock-in mice models crossed to different mouse strains.Results: By comparing the GABA A g2 knock-out with the GABA A g2(R43Q) knock-in mouse model we show that haploinsufficiency underlies the genesis of absence seizures but cannot account for the thermal seizure susceptibility. Additionally, while the expression of the absence seizure phenotype was very sensitive to mouse background genetics, the thermal seizure phenotype was not. Conclusions:Our results show that a single gene mutation can cause distinct seizure phenotypes through independent molecular mechanisms. A lack of effect of genetic background on thermal seizure susceptibility is consistent with the higher penetrance of febrile seizures compared to absence seizures seen in family members with the mutation. These mouse studies help to provide a conceptual framework within which clinical heterogeneity seen in genetic epilepsy can be explained. Neurology Family members harboring the GABA A g2(R43Q) mutation display multiple seizure types, incomplete penetrance, and variable seizure severity, 1,2 a common feature of genetic epilepsies. 3To predict clinical outcomes for patients based on their personal genomes, it is important that we develop a conceptual framework explaining the genetic and molecular basis of heterogeneity. Animal models of epilepsy, based on human mutations, provide a means of investigating this. The knock-in mouse model based on the GABA A g2(R43Q) mutation recapitulates the 2 major phenotypes seen in family members, including febrile seizures and typical absence seizures. 4,5 However, the molecular mechanisms causing epilepsy in this model are unclear. In vitro studies suggest that the deficit could be through haploinsufficiency (loss-of-function) or a dominant impact of the mutated protein. 6 To investigate this, we compared the seizure phenotypes of heterozygous Gabrg2 knock-out mice with those of knock-in mice.Penetrance is relatively low for absence seizures in the GABA A g2(R43Q) family. 1,2 By backcrossing the GABA A g2(R43Q) knock-in mouse to strains with different seizure susceptibility, we have demonstrated that the spike-wave phenotype requires additional susceptibility alleles for full expression, 5,7 potentially explaining the low penetrance of absence in the family. In contrast, febrile seizures segregate as a more highly penetrant autosomal dominant trait, which suggests that background genetics have less impact.1,2 Here we investigate the influence of genetic background on thermal seizure and spike-wave susceptibility in the knock-in mouse model. METHODS Mice. All experiments were approved by the Animal Ethics Committee at the Florey Institute of Neuroscience and Mental Health (09-046). Genotyping of the GABA...

Three epilepsy-associated GABRG2 missense mutations at the γ+/β− interface disrupt GABAA receptor assembly and trafficking by similar mechanisms but to different extents

“…A single nucleotide substitution caused a highly conserved arginine residue located within a loop between the α-helix and the β1-sheet (the α-β1 loop) in the extracellular N terminus to be replaced by a glutamine (Figure 1A), resulting in impaired surface expression of γ2 subunits and decreased GABA A receptor currents (Bianchi et al, 2002; Kang and Macdonald, 2004; Sancar and Czajkowski, 2004; Hales et al, 2005; Eugene et al, 2007; Frugier et al, 2007). Heterozygous knock-in mice carrying this mutation displayed spontaneous spike-wave discharges and thermal-induced seizures (Tan et al, 2007; Reid et al, 2013), consistent with R82Q being an epilepsy-causing mutation. However, whether this mutation has dominant negative effects on other GABA A receptor subunits and how it affects subunit-subunit interactions is still controversial (Hales et al, 2005; Frugier et al, 2007).…”

“…Compared to a hypomorphic allele, the R82Q mutation increased seizure susceptibility indicating that it had effects in addition to haploinsufficiency (Chiu et al, 2008). A recent study also suggested that γ2 subunits haploinsufficiency could account for genesis of absence seizures in γ2 R82Q/+ knock-in mice but not the increased thermal seizure susceptibility, while the R82Q mutation increased thermal seizure susceptibility, independent of genetic background (Reid et al, 2013). …”

“…However, whether this mutation has dominant negative effects on other GABA A receptor subunits and how it affects subunit-subunit interactions is still controversial (Hales et al, 2005; Frugier et al, 2007). A recent study showed that while loss of γ2 subunit function could account for the absence seizure phenotype, the R82Q mutation might be responsible for the febrile seizure phenotype (Reid et al, 2013), further suggesting that the R82Q mutation had effects in addition to haploinsufficiency.…”

Fever-induced seizures: where are we today?

“…Primjerice, animalni model Dravet sindroma temeljen na SCN1A i SCN1B mutaciji pokazuje povećanu osjetljivost na konvulzije 45 . Nadalje, GEFK+ modeli temeljeni na SCN1B i GA-BRG2 mutacijama također pokazuju sklonost konvulzijama prilikom povećanja temperature okoliša 46 . Takva istraživanja su također pokazala o dobi ovisne učinke u pogledu konvulzija induciranim porastom temperature, modelirajući dobno ovisne manifestacije FK-a 47 .…”

Mutations in GABRB3

“…The fact that GABRB3 truncations have not been reported in the ExAC database argues for the latter hypothesis. It has also been shown that the epileptic phenotype observed in mouse models lacking one allele of a GABA A receptor subunit gene may be milder compared to missense mutation carriers, 38 although this may depend on the functional interactions of the truncated protein and the remaining WT subunits. 39 Lastly, we cannot completely exclude that the parents carry these mutations in a mosaic state, as seen in the mother of patient 9, even though this is not obvious from the blood DNA analysis.…”