Abstracts

Rationale: Juvenile Myoclonic Epilepsies are a heterogeneous disorder affecting over 8.5 million people worldwide. Although JMEs have been hypothesized to have a genetic basis, specific genes have not been found for >90% of cases. We use large multiplex, multigeneration families to identify variants of candidate JME genes and follow NHGRI and ACMG guidelines for implicating and assigning disease causality to sequence variants.Methods: Genome-wide genotyping with 5185 SNPs of 37 members in a 3-generation family, was followed by genetic linkage and haplotype analyses. Whole exomes of four JME affected individuals and 2 married-in controls were sequenced (WES). After ANNOVAR program identified a candidate variant, Sanger sequencing confirmed co-segregation of the variant with all affected members. LightCycler real time PCR then scanned for additional variants in 336 JME (177 Hispanic from Honduras and Mexico, 65 Caucasian from Los Angeles, and 94 Japanese from Japan) and 1011 ethnically-matched controls. Functional analyses including quantitation of mitotic index, cell cycle exit and neuronal migration after ex vivo electroporation in mouse developing neocortex, assessed pathogenicity of variants.Results: Linkage analysis revealed a JME locus in 6p11-12 and fine mapping with additional SNPs and microsatellites refined the location to 6p12. WES identified Intestinal Cell Kinase (ICK) c.914A>C (p.K305T) as the candidate variant in 6p12·2 and Sanger sequencing confirmed co-segregation of c.914A>C with all nine affected members across three generations. We found three more ICK missense mutations – c.304A>C (p.I102L), c.658A>G (p.K220E) and c.1843G>A (p.A615T) in three other Hispanic and Japanese families and a Japanese singleton, and one nonsense mutation (c.1894C>T (p.R632X)) in another Japanese singleton. Because ICK encodes serine/threonine kinase involved in apoptosis, cell proliferation and cell-cycle regulation, we proved pathogenicity of ICK variants by demonstrating enhanced apoptosis and impaired radial migration of neuronal progenitor cells due to decreased mitosis and cell cycle exit. ICK was observed on ependymal cells lining walls of lateral ventricles, choroid plexus, CA1 pyramidal neurons, neocortical cells, and Purkinje cells of the cerebellum. Using EGFP, EGFP-tagged wildtype and mutant ICK proteins in neural progenitor cells in the mouse neocortex, we showed impaired radial migration, [Ld1] supporting a dominant-negative effect of the mutant proteins on ICK function. [Ld1]This has been explain 3 lines before.Conclusions: Intestinal Cell Kinase (ICK) is implicated as disease causing in JME using NHGRI and ACMG guidelines that included genetic, and functional evidence. Funding. NIH R01NS055057, VACO Merit Review, FRS-FNRS (T.0009.13), University of Liege, Center for Inherited Disease Research (CIDR)