Tojo Nakayama

Abstract Splice-switching antisense oligonucleotides (ASOs) could be used to treat a subset of individuals with genetic diseases 1 , but the systematic identification of such individuals remains a challenge. Here we performed whole-genome sequencing analyses to characterize genetic variation in 235 individuals (from 209 families) with ataxia-telangiectasia, a severely debilitating and life-threatening recessive genetic disorder 2,3 , yielding a complete molecular diagnosis in almost all individuals. We developed a predictive taxonomy to assess the amenability of each individual to splice-switching ASO intervention; 9% and 6% of the individuals had variants that were ‘probably’ or ‘possibly’ amenable to ASO splice modulation, respectively. Most amenable variants were in deep intronic regions that are inaccessible to exon-targeted sequencing. We developed ASOs that successfully rescued mis-splicing and ATM cellular signalling in patient fibroblasts for two recurrent variants. In a pilot clinical study, one of these ASOs was used to treat a child who had been diagnosed with ataxia-telangiectasia soon after birth, and showed good tolerability without serious adverse events for three years. Our study provides a framework for the prospective identification of individuals with genetic diseases who might benefit from a therapeutic approach involving splice-switching ASOs.

Abstract Mutations in the SCN2A gene encoding a voltage-gated sodium channel Nav1.2 are associated with epilepsies, intellectual disability, and autism. SCN2A gain-of-function mutations cause early-onset severe epilepsies, while loss-of-function mutations cause autism with milder and/or later-onset epilepsies. Here we show that both heterozygous Scn2a -knockout and knock-in mice harboring a patient-derived nonsense mutation exhibit ethosuximide-sensitive absence-like seizures associated with spike-and-wave discharges at adult stages. Unexpectedly, identical seizures are reproduced and even more prominent in mice with heterozygous Scn2a deletion specifically in dorsal-telencephalic (e.g., neocortical and hippocampal) excitatory neurons, but are undetected in mice with selective Scn2a deletion in inhibitory neurons. In adult cerebral cortex of wild-type mice, most Nav1.2 is expressed in excitatory neurons with a steady increase and redistribution from proximal (i.e., axon initial segments) to distal axons. These results indicate a pivotal role of Nav1.2 haplodeficiency in excitatory neurons in epilepsies of patients with SCN2A loss-of-function mutations.

Dravet syndrome is a severe form of epileptic encephalopathy characterized by early onset epileptic seizures followed by ataxia and cognitive decline. Approximately 80% of patients with Dravet syndrome have been associated with heterozygous mutations in SCN1A gene encoding voltage-gated sodium channel (VGSC) α(I) subunit, whereas a homozygous mutation (p.Arg125Cys) of SCN1B gene encoding VGSC β(I) subunit was recently described in a patient with Dravet syndrome. To further examine the involvement of homozygous SCN1B mutations in the etiology of Dravet syndrome, we performed mutational analyses on SCN1B in 286 patients with epileptic disorders, including 67 patients with Dravet syndrome who have been negative for SCN1A and SCN2A mutations. In the cohort, we found one additional homozygous mutation (p.Ile106Phe) in a patient with Dravet syndrome. The identified homozygous SCN1B mutations indicate that SCN1B is an etiologic candidate underlying Dravet syndrome.