Abstracts

Rationale: Epilepsy of infancy with migrating focal seizures (EIMFS) is a severe infantile-onset pharmacoresistant epilepsy syndrome.1 Mutations in a number of genes, notably KCNT1, have been previously implicated.2,3 The potassium-chloride co-transporter KCC2, encoded by SLC12A5, plays a fundamental role in fast synaptic inhibition by maintaining a hyperpolarizing gradient for chloride ions. 4 Acquired KCC2 dysfunction has been implicated in temporal lobe epilepsy and heterozygous SLC12A5 polymorphisms were recently associated with febrile seizures and idiopathic generalised epilepsy. 5-7 We present the first report of mutations in SLC12A5 as a monogenic cause of human epilepsy.Methods: Family 1 is a consanguineous Asian family and Family 2 is a Caucasian non-consanguineous family, both with two children affected by EIMFS. Homozygosity mapping (Illumina cytoSNP-12) was performed in Family 2 and whole exome sequencing (HiSeq sequencing system, Illumina) in both families. Protein homology modelling of KCC2 was undertaken. HEK-293 cells were transiently transfected with equivalent amounts of GFP, the human glycine receptor (GlyR) α2 subunit and WT or mutant KCC2 and voltage-clamp recording performed following puff application of glycine. Myc-tagged WT and mutant KCC2 constructs were generated for immunoblotting and biotinylation studies. For confocal microscopy, a construct FLAG-tagged at the second extracellular loop was utilised. TALEN-mediated genome editing generated a double KCC2a-KCC2b knockout zebrafish model.Results: Seven regions of homozygosity were identified in Family 2 with SLC12A5 located in a region on chromosome 20. WES revealed compound heterozygous missense variants in Family 1 (c.1277T>C, L426P and c.1652G>A, G551D) and a homozygous missense variant in Family 2(c.932T>A, L311H). These were confirmed by direct Sanger sequencing and showed appropriate familial segregation. All mutations are highly conserved, predicted to be deleterious and are absent in control populations. Protein homology modelling predicts damaging effects on the structure-function of KCC2. Mutant KCC2 exhibits a depolarised chloride reversal potential (ECl-) and delayed recovery from chloride load compared to WT KCC2. Immunoblotting and surface protein biotinylation studies demonstrate reduced total and cell surface expression levels of mutant KCC2 and decreased protein glycosylation, indicating impaired post-translational processing. Confocal microscopy confirms decreased cell surface expression in mutant versus WT KCC2. The double KCC2a-KCC2b knockout demonstrates abnormal jerky movements on tactile response testing at 2 days post-fertilisation.Conclusions: Decreased cell surface expression of KCC2, abnormal post-translational modification and transporter dysfunction result in a loss of function with reduced chloride extrusion. This is likely to impair normal synaptic inhibition and promote neuronal excitability in this epileptic encephalopathy. Elucidation of novel disease mechanisms is vital to the development of targeted therapies in these devastating early onset epilepsies.