Association of nonsense mutation in GABRG2 with abnormal trafficking of GABA_A receptors in severe epilepsy

Summary

Mutations in GABRG2, which encodes the γ2 subunit of GABA_A receptors, can cause both genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome. Most GABRG2 truncating mutations associated with Dravet syndrome result in premature termination codons (PTCs) and are stably translated into mutant proteins with potential dominant-negative effects. This study involved search for mutations in candidate genes for Dravet syndrome, namely SCN1A, 2A, 1B, 2B, GABRA1, B2, and G2. A heterozygous nonsense mutation (c.118C>T, p.Q40X) in GABRG2 was identified in dizygotic twin girls with Dravet syndrome and their apparently healthy father. Electrophysiological studies with the reconstituted GABA_A receptors in HEK cells showed reduced GABA-induced currents when mutated γ2 DNA was cotransfected with wild-type α1 and β2 subunits. In this case, immunohistochemistry using antibodies to the α1 and γ2 subunits of GABA_A receptor showed granular staining in the soma. In addition, microinjection of mutated γ2 subunit cDNA into HEK cells severely inhibited intracellular trafficking of GABA_A receptor subunits α1 and β2, and retention of these proteins in the endoplasmic reticulum. The mutated γ2 subunit-expressing neurons also showed impaired axonal transport of the α1 and β2 subunits. Our findings suggested that different phenotypes of epilepsy, e.g., GEFS+ and Dravet syndrome (which share similar abnormalities in causative genes) are likely due to impaired axonal transport associated with the dominant-negative effects of GABRG2.

Introduction

Epilepsy is associated with various gene mutations. However, the exact molecular mechanisms underlying the pleomorphic phenotypes of this disease remain unclear. There are two major epilepsy phenotypes associated with mutations in the GABA_A receptor. The first is genetic epilepsy with febrile seizures plus (GEFS+). Individuals with GEFS+ have a missense mutation in the gene encoding the γ2 subunit of the GABA_A receptor, GABRG2 (Baulac et al., 2001). The second phenotype is Dravet syndrome, which is a malignant epilepsy condition characterized by refractory seizures and psychomotor developmental arrest.

Compared with Dravet syndrome, GEFS+ is relatively benign. However, these seemingly different syndromes are considered part of a single disease spectrum as mutations detected in both syndromes (Mulley et al., 2005, Singh et al., 2001) lie on the same genes, namely GABRG2 (Baulac et al., 2001, Harkin et al., 2002), the gene encoding the α1 subunit of the neuronal voltage-gated sodium channel, SCN1A (Abou-Khalil et al., 2001, Claes et al., 2001, Claes et al., 2003, Escayg et al., 2000, Escayg et al., 2001, Fujiwara et al., 2003, Fukuma et al., 2004, Gennaro et al., 2003, Kimura et al., 2005, Nabbout et al., 2003, Ohmori et al., 2002, Sugawara et al., 2001, Sugawara et al., 2002, Wallace et al., 2003), and the gene encoding the α2 subunit of the neuronal voltage-gated sodium channel, SCN2A (Shi et al., 2009, Kamiya et al., 2004, Sugawara et al., 2001). Most of the mutations initially identified in Dravet syndrome were truncation mutations (Claes et al., 2001, Sugawara et al., 2002). On the other hand, mutations identified in GEFS+ were exclusively missense mutations (Abou-Khalil et al., 2001, Escayg et al., 2000, Sugawara et al., 2001). Based on these studies and the more malignant nature of Dravet syndrome, it has been since postulated that mutations found in Dravet syndrome are associated with the more severe phenotypes due to a more significant genetic loss compared to the usually milder effects of missense mutations. Intriguingly, subsequent analysis revealed that missense mutations are also associated with Dravet syndrome (Fujiwara et al., 2003, Fukuma et al., 2004, Gennaro et al., 2003, Ohmori et al., 2002).

Since most of the nonsense mutations are located in the 5′ end of SCN1A, the mutant transcripts go through a nonsense-mediated decay (NMD) pathway (Holbrook et al., 2004). Thus, NMD processing underlies the distinct phenotypes resulting from truncation mutations and, further, may explain the variety of phenotypes associated with different mutations in the same gene. However, the evidence that GEFS+ and Dravet syndrome occupy the same spectrum of disorders or are allelic variants is not compelling.

In this study, we identified a nonsense mutation (c.118C>T, p.Q40X) in the GABRG2 gene in individuals with Dravet syndrome. To specify the molecular mechanisms underlying the phenotypes of GEFS+ and Dravet syndrome resulting from allelic GABRG2 truncation mutations, we focused on intracellular trafficking since several mutants are retained in the endoplasmic reticulum (ER) (Hirose, 2006, Gallagher et al., 2005, Hales et al., 2005, Harkin et al., 2002, Kang and Macdonald, 2004, Macdonald et al., 2004).