Hyperglycosylation and Reduced GABA Currents of Mutated GABRB3 Polypeptide in Remitting Childhood Absence Epilepsy

Abstract

Childhood absence epilepsy (CAE) accounts for 10% to 12% of epilepsy in children under 16 years of age. We screened for mutations in the GABA(A) receptor (GABAR) beta 3 subunit gene (GABRB3) in 48 probands and families with remitting CAE. We found that four out of 48 families (8%) had mutations in GABRB3. One heterozygous missense mutation (P11S) in exon 1a segregated with four CAE-affected persons in one multiplex, two-generation Mexican family. P11S was also found in a singleton from Mexico. Another heterozygous missense mutation (S15F) was present in a singleton from Honduras. An exon 2 heterozygous missense mutation (G32R) was present in two CAE-affected persons and two persons affected with EEG-recorded spike and/or sharp wave in a two-generation Honduran family. All mutations were absent in 630 controls. We studied functions and possible pathogenicity by expressing mutations in HeLa cells with the use of Western blots and an in vitro translation and translocation system. Expression levels did not differ from those of controls, but all mutations showed hyperglycosylation in the in vitro translation and translocation system with canine microsomes. Functional analysis of human GABA(A) receptors (alpha 1 beta 3-v2 gamma 2S, alpha 1 beta 3-v2[P11S]gamma 2S, alpha 1 beta 3-v2[S15F]gamma 2S, and alpha 1 beta 3-v2[G32R]gamma 2S) transiently expressed in HEK293T cells with the use of rapid agonist application showed that each amino acid transversion in the beta 3-v2 subunit (P11S, S15F, and G32R) reduced GABA-evoked current density from whole cells. Mutated beta 3 subunit protein could thus cause absence seizures through a gain in glycosylation of mutated exon 1a and exon 2, affecting maturation and trafficking of GABAR from endoplasmic reticulum to cell surface and resulting in reduced GABA-evoked currents.

GABRB3 mutations: a new and emerging cause of early infantile epileptic encephalopathy

“…A single nucleotide polymorphism at the promoter region of GABRB3 (T>C substitution in position −897, numbering with respect to the initiator methionine of exon 1a), which leads to reduction of the promoter's transcriptional ability, has been associated with childhood absence epilepsy in previous studies 12. Other researchers have reported that a rare GABRB3 single nucleotide polymorphism (c.31C>T; p.Pro11Ser)15 and other variants (c.44C>T; p.Ser15Phe, and c.94G>A; p.Gly32Arg) (NM_021912.4) are linked with childhood absence epilepsy phenotypes associated with eyelid myoclonus and generalized tonic–clonic seizures 6, 11. Probands had typical generalized 3Hz spike and wave discharges during childhood, with clinical symptoms later remitting, without neurological sequelae.…”

“…Recent studies suggest that GABRB3 mutations cause attenuated chloride currents and channel activity impairment through subunit hyperglycosylation 11. The mechanisms through which GABRR3 mutations affect neuronal networks giving rise to epilepsy and neurodevelopmental delay are yet to be fully elucidated.…”

“…Reduced GABRB3 expression has been postulated in the pathogenesis of absence seizures, abnormal sensory processing, and other neurodevelopmental disorder phenotypes such as Angelman syndrome, autism spectrum disorders, and intellectual disability 8, 9, 10. Furthermore, single nucleotide polymorphisms and missense mutations in GABRB3 have previously been implicated in childhood absence epilepsy 11, 12…”

Glycosylation of β2 Subunits Regulates GABAA Receptor Biogenesis and Channel Gating

“…A mutation, G32R, that is one amino acid N-terminal to the first glycosylation site of GABA A receptor ␤3 subunits and is associated with childhood absence epilepsy has been demonstrated to reduce peak current amplitudes of ␣1␤3(G32R)␥2S receptor channels (48). The G32R mutation may affect glycosylation efficiency of ␤3 N33 and in turn potentially could affect channel function.…”

GABRB3 Mutation, G32R, Associated with Childhood Absence Epilepsy Alters α1β3γ2L γ-Aminobutyric Acid Type A (GABAA) Receptor Expression and Channel Gating

“…Three separate CAE-associated mutations were recently identified in GABA A receptor ␤3 subunits: GABRB3(P11S), GABRB3(S15F), and GABRB3(G32R) (15). Mutant subunitcontaining receptors exhibited reduced current density.…”