Pathophysiology and treatment of GABRG2 nonsense epilepsy mutations

Vanderbilt University, Nashville TN

Investigators

Linked publications & trials

Abstract

DESCRIPTION (provided by applicant): Epilepsy affects more than 0.5% of the population worldwide. Genetic factors play an important role in many of the epilepsy syndromes. GABAA receptors (GABAARs) are the major inhibitory receptors in the brain and mutations in GABAAR subunit genes (GABRs) coding for the 32, 11 and 4 GABAAR subunits are associated with idiopathic generalized epilepsy syndromes (IGEs). To understand the bases for IGEs associated with GABR mutations, it is necessary to determine the errors in receptor biogenesis and function that are produced by the mutations. Many of them are single nucleotide missense mutations, but recently nonsense mutations that introduce a premature translation-termination codon (PTC) and mutations in splice donor sites have been reported. PTCs might produce nonsense mediated mRNA decay (NMD) if the mutation is not in the last exon or produce a truncated subunit if it is in the last exon that could have dominant negative effects on wt subunits. Splice donor site mutations produce mutant subunits by: (1) exon skipping, (2) use of a cryptic splice site within the downstream intron, or (3) intron inclusion if the intron is small. It is possible that all three mechanisms would generate a PTC, thus triggering NMD. There are several approaches that can be taken to treat patients with nonsense epilepsy mutations. If the mutation produces no functional protein and therefore is haplo-insufficient, the subunit could be over expressed in neurons to overcome the loss of subunit or if the PTC is in frame, a read through strategy could be applied to read through the PTC and express a full length functional subunit. If the mutation produces a truncated, nonfunctional dominant negative protein, the subunit could be over expressed in neurons to overcome the loss of subunit or a read through strategy could be applied to read through the PTC and express a full length functional subunit. The goals of this proposal are to characterize the altered receptor biogenesis and function produced by GABRG2 epilepsy PTC and splice donor site mutations and to develop strategies to overcome the resultant deficits as potential therapies for the associated epilepsies. The hypotheses of the proposal are: 1) The GABRG2(IVS6+2T`G) mutation leads to aberrant mRNA splicing that creates a PTC and activates NMD and produces a stable, nonfunctional, truncated dominant negative protein whose effects can be reversed by siRNA knock down and/or GABRG2 over expression; 2) The GABRG2(Q1X) mutation activates NMD to degrade the mutant mRNA, producing loss of function that can be reversed by PTC read through and/or GABRG2 over expression; and 3) The GABRG2(Q351X) mutation produces a stable, nonfunctional, truncated dominant negative protein that can be reversed by PTC read through and/or GABRG2 over expression. The specific aims of the proposal are: 1) To determine the aberrant mRNA splicing pattern produced by the GABRG2(IVS6+2T`G) mutation and how the mutant translation products affect g2 subunit expression and GABAAR function and to evaluate potential treatment strategies; 2) To determine the impairment of g2 subunit biogenesis produced by the GABRG2(Q1X) mutation and to evaluate potential treatment strategies, and 3) To characterize GABRG2(Q351X) knock in mice and evaluate PTC read through and/or GABRG2 over expression as potential treatment strategies.