Genetic and molecular network of the calcium/calmodulin-dependent serine protein kinase CASK

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Next-generation sequencing is a powerful approach for unravelling the molecular basis of Mendelian disorders. Recently, we and others identified mutations in the X-linked CASK (calcium/calmodulin-dependent serine protein kinase) gene in females and males with intellectual disability with or without additional clinical features. In synapses of the central nervous system, CASK contributes to the formation of the presynaptic specialization, and to postsynaptic delivery of NMDA receptors. CASK is also redistributed to the neuronal nucleus where it binds the transcription factor Tbr1 and activates the target genes Reelin and NR2B (encoding a regulatory subunit of NMDA receptors). As NMDA receptors function as switches for memory formation by gating synaptic plasticity, the contribution of CASK to expression and transport of NMDA receptors strongly suggests a critical role of CASK in synaptic plasticity, learning and memory. Our current understanding indicates that a complex genetic and molecular synaptic network determines the phenotypic outcome of individuals with a CASK mutation. Phenotypic variability suggests that there are a number of modifier genes which affect CASK-related pathways. On the protein and cellular level, CASK acts at three different locations in neurons in different protein complexes, and it is currently unclear which of these complexes is relevant for disease. Our goals are to1) establish a genotype-phenotype correlation in males with a CASK mutation by analyzing CASK transcripts and protein in patient-derived cells.2) identify modifier genes responsible for differences in phenotypic expression of females with a heterozygous deleterious CASK mutation. We will use an extreme phenotype study design by defining individuals who are at both ends of a phenotype distribution. Whole exome sequencing on individuals of both groups will help to identify rare sequence variants enriched in one group.3) characterize the effect of CASK missense mutations (p.Y268H, p.L354P, p.P396S, p.Y723C and p.W914R) on transcriptional regulation of specific target genes of the CASK-Tbr1 transcriptional activator complex. We will carry out dual luciferase reporter assays in primary cortical neurons derived from Cask knockout mice expressing wild-type or mutant CASK. 4) analyze the effect of CASK missense mutations on specific protein-protein interactions and on intracellular trafficking of neurotransmitter receptors. CASK wildtype and mutants will be expressed in neurons derived from Cask-deficient mice, and the effect of CASK mutations on synaptogenesis and spinogenesis will be analyzed by immunocytochemistry and confocal microscopy. We will also determine the cell surface level of NMDA receptors by using live-cell surface immunostaining and biotinylation assays.5) study the in vivo effects of a selected CASK mutation in a Cask knock-in mouse, which will be analyzed with respect to synapse formation, neuronal morphology, and behavior.