Supplementary MaterialsSupp info. characterized by ID and adjustable dysmorphic features such as for example hypotonia and constipation. mutations primarily happen mutations are detected typically in females. Nevertheless, affected men have already been reported, plus they usually screen a more serious phenotype than their feminine counterparts. The under-representation of men among mutation-bearing people is probably because of decreased male viability or lethality (Moog et al., 2015; Najm et al., 2008). was identified at first through yeast two-hybrid screening for intracellular molecules getting together with neurexins, extremely polymorphic cell surface area proteins involved in the formation of synaptic junctions (Hata, Butz, & Sdhof, 1996). CASK is a member of a super-family of proteins known as MAGUKs (Membrane-Associated GUanylate Kinases), multi-domain proteins characterized by the presence of a common set of structural domains that bind both polypeptide and nucleotide ligands (Kim, 1995). CASK is composed of 3 MAGUK-specific protein Rabbit polyclonal to Caldesmon domains: one PDZ (PSD-95, Discs-large, ZO-1), one SH3 (homology 3), and a GUK (GUanylate Kinase) domain. In addition, MK-2206 2HCl ic50 CASK contains a calcium/calmodulin-dependent protein kinase-like domain (CaMK-like) at MK-2206 2HCl ic50 its N-terminus (Baines, 1996; Hata et al., 1996), followed by two L27 (LIN-2 and LIN-7 interaction) domains (Lee, Fan, Makarova, Straight, & Margolis, 2002). In neurons, CASK is involved in the regulation of several processes, and each of its domains has specific binding partners with which it forms protein complexes with discrete molecular functions. For instance, at pre-synaptic sites, CASK forms a complex with MALS/Mint-1/liprin through its CaMK and L27A domains. This complex is involved in the organization of synaptic vesicle pools, thus regulating neurotransmitter release (Olsen, Moore, Nicoll, & Bredt, 2006). Through its PDZ and SH3 domains, CASK interacts with and regulates the synaptic targeting of neurexin-1 and ion channels in a CDK5-dependent fashion (Hsueh, 2009). At post-synaptic termini, CASK binds to syndecan-2 via its PDZ domain and contributes to the regulation of axon branching and dendritic outgrowth (Cohen et al., 1998; Ethell & Yamaguchi, 1999). In a SUMOylation-dependent manner, CASK connects plasma membrane proteins such as syndecan-2 to the actin cytoskeleton via protein 4.1 and spectrin, thus stabilizing dendritic spine morphology (Chao, Hong, Huang, Lin, & Hsueh, 2008). Thus, the known versatility of CASK is likely reflected in the variable clinical presentations associated with different gene mutations (Kuo, Hong, Chien, & Hsueh, 2010; Moog et al., 2011, 2015; Najm et al., 2008). In this study, we report the identification of previously undescribed mutations in four female subjects by applying WES to a cohort of undiagnosed microcephalic individuals. Additionally, we have developed a physiologically relevant zebrafish model of depletion, and employ complementation (Davis, Frangakis, & Katsanis, 2014; Niederriter et al., 2013) to assess variant pathogenicity. As proof of concept, we investigated the effect of three variants by focusing on MC and cerebellar phenotypic readouts analogous to those commonly observed in patients with MICPCH. MATERIALS AND METHODS Library preparation, exome enrichment and massive parallel sequencing Genomic DNA was extracted from whole blood according to standard procedures, quantified using the Qubit? dsDNA Broad Range Assay kit on a Qubit? Fluorometer (Thermo Fisher Scientific Inc.), and fragmented using MK-2206 2HCl ic50 a Diagenode Bioruptor?. Sequencing libraries were prepared using the TruSeq? DNA Sample Preparation kit (Illumina) following a gel-free of charge Low Throughput process. The SeqCap EZ Exome Enrichment v.3.0 package (Roche) was used for exome enrichment according to producers specs. Sheared DNA, full-genomic and exome-enriched library quality was assessed with either Agilent DNA 1000 or DNA Large Sensitivity chips operate on an Agilent 2100 Bioanalyzer device. The samples had been after that sequenced on an Illumina HiSeq2500 machine in fast mode utilizing a paired-end 2100 bp process. Sequence reads had been aligned to the human being genome reference sequence (Genome Assembly GRCh37/hg19) with the Burrows-Wheeler Aligner (BWA v. 0.7.8). SAMtools (v. 0.1.19) was used for SAM to BAM file conversion, sorting and indexing alignments. Picard equipment (v. 1.118) were used to compute quality metrics and tag PCR-generated duplicates. The Genome Evaluation Toolkit (GATK v. 3.2.2) program was used to execute community realignment around indels and foundation quality rating recalibration. SNPs and little indels were known as using GATK HaplotypeCaller.