The GABRG2 nonsense mutation, Q40X, associated with Dravet syndrome activated NMD and generated a truncated subunit that was partially rescued by aminoglycoside-induced stop codon read-through

Abstract

The GABRG2 nonsense mutation, Q40X, is associated with the severe epilepsy syndrome, Dravet syndrome, and is predicted to generate a premature translation-termination codon (PTC) in the GABAA receptor γ2 subunit mRNA in a position that codes for the first amino acid of the mutant subunit. We determined the effects of the mutation on γ2 subunit mRNA and protein synthesis and degradation, as well as on α1β2γ2 GABAA receptor assembly, trafficking and surface expression in HEK cells. Using bacterial artificial chromosome (BAC) constructs, we found that γ2(Q40X) subunit mRNA was degraded by nonsense mediated mRNA decay (NMD). Undegraded mutant mRNA was translated to a truncated peptide, likely the signal peptide, which was cleaved further. We also found that mutant γ2(Q40X) subunits did not assemble into functional receptors, thus decreasing GABA-evoked current amplitudes. The GABRG2(Q40X) mutation is one of several epilepsy-associated nonsense mutations that have the potential to be rescued by reading through the PTC, thus restoring full-length protein translation. As a first approach, we investigated use of the aminoglycoside, gentamicin, to rescue translation of intact mutant subunits by inducing mRNA read-through. In the presence of gentamicin, synthesis of full length γ2 subunits was partially restored, and surface biotinylation and whole cell recording experiments suggested that rescued γ2 subunits could corporate into functional, surface GABAA receptors, indicating a possible direction for future therapy.

Ionotropic GABA and Glutamate Receptor Mutations and Human Neurologic Diseases

“…Premature termination codons can result in the production of truncated proteins or truncated mRNAs that are degraded prior to translation. These effects appear to occur with a1 (S326fs), g2(Q40X), g2(Q429X), as well as a splice site mutation in g2 at the boundary of intron6/exon6 (Kananura et al, 2002;Hirose 2006;Maljevic et al, 2006;Sun et al, 2008;Huang et al, 2012;Tian and Macdonald, 2012), resulting in seizure disorders, including DS, GEFS1, FS, and CAE. Promoter mutations (GABRB3 haplotype) result in CAE following impairment of transcription, which might reduce surface expression of this subunit (Urak et al, 2006).…”

“…The same three diseases can result from mutations in the Kir6.2 inwardly rectifying potassium channel gene KCNJ11 (Aittoniemi et al, 2009). In the central nervous system (CNS), mutations in both SCN1A and GABA A receptor gene GABRG2 are associated with Dravet syndrome (Huang et al, 2012). Another epilepsy (early infantile epileptic encephalopathy) can be categorized into multiple subtypes, many being associated with a different channelopathy in a different gene (KCNQ2, SCN2A, SCN8A, and KCNT1) (Kim 2014).…”

When proteins start to make sense: fine-tuning of aminoglycosides for PTC suppression therapy

“…These encouraging results were further exploited for the establishment of several clinical trials in DMD patients 22 altogether with massive experiments performed in various in-vitro , ex-vivo and in-vivo systems on DMD 23, 24 CF 18 , Rett syndrome 25, 26 , HS 27 , nephrogenic diabetes insipidus 28 , nephropathic cystinosis 29 , retinitis pigmentosa 30 , ataxia-telegiectasia 31 , spinal muscular atrophy (SMA) 32 , severe epilepsy syndrome 33 and several genetically induced cancer types 34–36 . The resulting data supported the previous findings and highlighted AGs as possible candidates for PTC suppression therapy.…”

Molecular Pathogenic Basis forGABRG2Mutations Associated With a Spectrum of Epilepsy Syndromes, From Generalized Absence Epilepsy to Dravet Syndrome

“…Q40X is associated with twin sisters with Dravet syndrome 37;38,39 . R136X is associated with GEFS+ and other extended phenotypes like eye myoclonia and autistic features 40 .…”