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

下一代测序是揭示孟德尔疾病分子基础的有力方法。最近，我们和其他人在有或没有其他临床特征的智力残疾的女性和男性中发现了x连锁CASK（钙/钙调素依赖性丝氨酸蛋白激酶）基因突变。在中枢神经系统的突触中，CASK有助于突触前特化的形成，以及突触后NMDA受体的传递。CASK也被重新分配到神经元核，在那里它结合转录因子Tbr1并激活靶基因Reelin和NR2B（编码NMDA受体的调节亚基）。由于NMDA受体通过控制突触可塑性作为记忆形成的开关，CASK对NMDA受体的表达和转运的贡献强烈表明CASK在突触可塑性、学习和记忆中起着关键作用。我们目前的理解表明，一个复杂的遗传和分子突触网络决定了CASK突变个体的表型结果。表型变异性表明，有许多修饰基因影响cask相关途径。在蛋白质和细胞水平上，CASK作用于神经元中不同蛋白复合物的三个不同位置，目前尚不清楚这些复合物中哪一个与疾病相关。我们的目标是1)通过分析患者来源细胞中的CASK转录物和蛋白质，在CASK突变的男性中建立基因型-表型相关性。2)鉴定导致杂合CASK有害突变雌性表型表达差异的修饰基因。我们将通过定义处于表型分布两端的个体来使用极端表型研究设计。对两组个体的全外显子组测序将有助于确定在一组中富集的罕见序列变异。3)表征CASK错义突变（p.Y268H、p.L354P、p.P396S、p.Y723C和p.W914R）对CASK- tbr1转录激活因子复合物特异性靶基因转录调控的影响。我们将在表达野生型或突变型Cask的Cask基因敲除小鼠的原代皮质神经元中进行双荧光素酶报告基因检测。4)分析CASK错义突变对特异性蛋白-蛋白相互作用和神经递质受体细胞内转运的影响。CASK野生型和突变型将在来自CASK缺陷小鼠的神经元中表达，并通过免疫细胞化学和共聚焦显微镜分析CASK突变对突触发生和脊髓发生的影响。我们还将通过使用活细胞表面免疫染色和生物素化测定来确定NMDA受体的细胞表面水平。5)研究CASK突变在CASK敲入小鼠体内的影响，并从突触形成、神经元形态和行为方面进行分析。

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.