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Objective

Genetic epilepsies and many other human genetic diseases display phenotypic heterogeneity, often for unknown reasons. Disease severity associated with nonsense mutations is dependent partially on mutation gene location and resulting efficiency of nonsense-mediated mRNA decay (NMD) to eliminate potentially toxic proteins. Nonsense mutations in the last exon do not activate NMD, thus producing truncated proteins. We compared the protein metabolism and the impact on channel biogenesis, function, and cellular homeostasis of truncated γ2 subunits produced by GABRG2 nonsense mutations associated with epilepsy of different severities and by a nonsense mutation in the last exon unassociated with epilepsy.

Methods

γ-Aminobutyric acid type A receptor subunits were coexpressed in non-neuronal cells and neurons. NMD was studied using minigenes that support NMD. Protein degradation rates were determined using 35S radiolabeling pulse chase. Channel function was determined by whole cell recordings, and subunits trafficking and cellular toxicity were determined using flow cytometry, immunoblotting, and immunohistochemistry.

Results

Although all GABRG2 nonsense mutations resulted in loss of γ2 subunit surface expression, the truncated subunits had different degradation rates and stabilities, suppression of wild-type subunit biogenesis and function, amounts of conjugation with polyubiquitin, and endoplasmic reticulum stress levels.

Interpretation

We compared molecular phenotypes of GABRG2 nonsense mutations. The findings suggest that despite the common loss of mutant allele function, each mutation produced different intracellular levels of trafficking-deficient subunits. The concentration-dependent suppression of wild-type channel function and cellular disturbance resulting from differences in mutant subunit metabolism may contribute to associated epilepsy severities and by implication to phenotypic heterogeneity in many inherited human diseases. Ann Neurol 2013;74:547–559