Genetic studies have identified many risk loci for autism spectrum disorder (ASD) although causal factors in the majority of cases are still unknown. Currently, known ASD risk genes are all protein-coding genes; however, the vast majority of transcripts in humans are non-coding RNAs (ncRNAs) which do not encode proteins. Recently, long non-coding RNAs (lncRNAs) were shown to be highly expressed in the human brain and crucial for normal brain development. We have constructed a computational pipeline for the integration of various genomic datasets to identify lncRNAs associated with ASD. This pipeline utilizes differential gene expression patterns in affected tissues in conjunction with gene co-expression networks in tissue-matched non-affected samples. We analyzed RNA-seq data from the cortical brain tissues from ASD cases and controls to identify lncRNAs differentially expressed in ASD. We derived a gene co-expression network from an independent human brain developmental transcriptome and detected a convergence of the differentially expressed lncRNAs and known ASD risk genes into specific co-expression modules. Co-expression network analysis facilitates the discovery of associations between previously uncharacterized lncRNAs with known ASD risk genes, affected molecular pathways and at-risk developmental time points. In addition, we show that some of these lncRNAs have a high degree of overlap with major CNVs detected in ASD genetic studies. By utilizing this integrative approach comprised of differential expression analysis in affected tissues and connectivity metrics from a developmental co-expression network, we have prioritized a set of candidate ASD-associated lncRNAs. The identification of lncRNAs as novel ASD susceptibility genes could help explain the genetic pathogenesis of ASD.

Smith-Magenis Syndrome is a contiguous gene syndrome in which the dosage sensitive gene has been identified: the Retinoic Acid Induced 1 (RAI1). Little is known about the function of human RAI1.

Microarray gene expression data are accumulating in public databases. The expression profiles contain valuable information for understanding human gene expression patterns. However, the effective use of public microarray data requires integrating the expression profiles from heterogeneous sources.

In utero exposure to maternal immune activation (MIA) is an environmental risk factor for neurodevelopmental and neuropsychiatric disorders. Animal models provide an opportunity to identify mechanisms driving neuropathology associated with MIA. We performed time-course transcriptional profiling of mouse cortical development following induced MIA via poly(I:C) injection at E12.5. MIA-driven transcriptional changes were validated via protein analysis, and parallel perturbations to cortical neuroanatomy were identified via imaging. MIA-induced acute upregulation of genes associated with hypoxia, immune signaling, and angiogenesis, by 6 hr following exposure. This acute response was followed by changes in proliferation, neuronal and glial specification, and cortical lamination that emerged at E14.5 and peaked at E17.5. Decreased numbers of proliferative cells in germinal zones and alterations in neuronal and glial populations were identified in the MIA-exposed cortex. Overall, paired transcriptomic and neuroanatomical characterization revealed a sequence of perturbations to corticogenesis driven by mid-gestational MIA.

Copy number variations (CNV) within the genome are extremely abundant. In this closeup, Canales and Walz discuss how CNV are associated with normal variation, genomic disorders, genome evolution, adaptive traits and how the use of a novel screen described by Ermakova et al in this issue that is designed to identify human diseaserelevant phenotypes associated with CNV in the mouse can help elucidating susceptibility or predisposition to diseases loci.

Smith-Magenis Syndrome (SMS) is a complex genomic disorder mostly caused by the haploinsufficiency of the Retinoic Acid Induced 1 gene (RAI1), located in the chromosomal region 17p11.2. In a subset of SMS patients, heterozygous mutations in RAI1 are found. Here we investigate the molecular properties of these mutated forms and their relationship with the resulting phenotype. We compared the clinical phenotype of SMS patients carrying a mutation in RAI1 coding region either in the N-terminal or the C-terminal half of the protein and no significant differences were found. In order to study the molecular mechanism related to these two groups of RAI1 mutations first we analyzed those mutations that result in the truncated protein corresponding to the N-terminal half of RAI1 finding that they have cytoplasmic localization (in contrast to full length RAI1) and no ability to activate the transcription through an endogenous target: the BDNF enhancer. Similar results were found in lymphoblastoid cells derived from a SMS patient carrying RAI1 c.3103insC, where both mutant and wild type products of RAI1 were detected. The wild type form of RAI1 was found in the chromatin bound and nuclear matrix subcellular fractions while the mutant product was mainly cytoplasmic. In addition, missense mutations at the C-terminal half of RAI1 presented a correct nuclear localization but no activation of the endogenous target. Our results showed for the first time a correlation between RAI1 mutations and abnormal protein function plus they suggest that a reduction of total RAI1 transcription factor activity is at the heart of the SMS clinical presentation.

Williams-Beuren Syndrome (WBS) is a genetic disorder associated with multisystemic abnormalities, including craniofacial dysmorphology and cognitive defects. It is caused by a hemizygous microdeletion involving up to 28 genes in chromosome 7q11.23. Genotype/phenotype analysis of atypical microdeletions implicates two evolutionary-related transcription factors, GTF2I and GTF2IRD1, as prime candidates for the cause of the facial dysmorphology.

Genes with multiple co-active promoters appear common in brain, yet little is known about functional requirements for these potentially redundant genomic regulatory elements. SCN1A, which encodes the NaV1.1 sodium channel alpha subunit, is one such gene with two co-active promoters. Mutations in SCN1A are associated with epilepsy, including Dravet syndrome (DS). The majority of DS patients harbor coding mutations causing SCN1A haploinsufficiency; however, putative causal non-coding promoter mutations have been identified.

Enhancers are cis-regulatory elements that play critical regulatory roles in modulating developmental transcription programs and driving cell-type-specific and context-dependent gene expression in the brain. The development of massively parallel reporter assays (MPRAs) has enabled high-throughput functional screening of candidate DNA sequences for enhancer activity. Tissue-specific screening of in vivo enhancer function at scale has the potential to greatly expand our understanding of the role of non-coding sequences in development, evolution, and disease. Here, we adapted a self-transcribing regulatory element MPRA strategy for delivery to early postnatal mouse brain via recombinant adeno-associated virus (rAAV). We identified and validated putative enhancers capable of driving reporter gene expression in mouse forebrain, including regulatory elements within an intronic CACNA1C linkage disequilibrium block associated with risk in neuropsychiatric disorder genetic studies. Paired screening and single enhancer in vivo functional testing, as we show here, represents a powerful approach towards characterizing regulatory activity of enhancers and understanding how enhancer sequences organize gene expression in the brain.

Mutations in Transient Receptor Potential Channel 6 (TRPC6) gene are associated with autosomal dominant focal and segmental glomerulosclerosis (FSGS). The majority of the identified mutations affect the ion channel function. Since calcium channels are promising candidate drug targets, there is an an urgent need for a mouse model to assess new therapeutic drugs and to help delineate the pathogenic process leading to FSGS. We have previously reported the generation of three independent transgenic mouse lines carrying different Trpc6 mutations that display a glomerular disease comparable to the phenotype presented by individuals with FSGS. However, the utility of these models for drug testing is dampened by the late-onset of the presentation and the mild phenotypic manifestations.

Mutations in the TRPC6 calcium channel (Transient receptor potential channel 6) gene have been associated with familiar forms of Focal and Segmental Glomerulosclerosis (FSGS) affecting children and adults. In addition, acquired glomerular diseases are associated with increased expression levels of TRPC6. However, the exact role of TRPC6 in the pathogenesis of FSGS remains to be elucidated. In this work we describe the generation and phenotypic characterization of three different transgenic mouse lines with podocyte-specific overexpression of the wild type or any of two mutant forms of Trpc6 (P111Q and E896K) previously related to FSGS. Consistent with the human phenotype a non-nephrotic range of albuminuria was detectable in almost all transgenic lines. The histological analysis demonstrated that the transgenic mice developed a kidney disease similar to human FSGS. Differences of 2-3 folds in the presence of glomerular lesions were found between the non transgenic and transgenic mice expressing Trpc6 in its wild type or mutant forms specifically in podocytes. Electron microscopy of glomerulus from transgenic mice showed extensive podocyte foot process effacement. We conclude that overexpression of Trpc6 (wild type or mutated) in podocytes is sufficient to cause a kidney disease consistent with FSGS. Our results contribute to reinforce the central role of podocytes in the etiology of FSGS. These mice constitute an important new model in which to study future therapies and outcomes of this complex disease.

Autism Spectrum Disorder (ASD) is the umbrella term for a group of neurodevelopmental disorders convergent on behavioral phenotypes. While many genes have been implicated in the disorder, the predominant focus of previous research has been on protein coding genes. This leaves a vast number of long non-coding RNAs (lncRNAs) not characterized for their role in the disorder although lncRNAs have been shown to play important roles in development and are highly represented in the brain. Studies have also shown lncRNAs to be differentially expressed in ASD affected brains. However, there has yet to be an enrichment analysis of the shared ontologies and pathways of known ASD genes and lncRNAs in normal brain development.

Cell-adhesion molecules of the immunoglobulin superfamily play critical roles in brain development, as well as in maintaining synaptic plasticity, the dysfunction of which is known to cause cognitive impairment. Recently dysfunction of KIRREL3, a synaptic molecule of the immunoglobulin superfamily, has been implicated in several neurodevelopmental conditions including intellectual disability, autism spectrum disorder, and in the neurocognitive delay associated with Jacobsen syndrome. However, the molecular mechanisms of its physiological actions remain largely unknown. Using a yeast two-hybrid screen, we found that the KIRREL3 extracellular domain interacts with brain expressed proteins MAP1B and MYO16 and its intracellular domain can potentially interact with ATP1B1, UFC1, and SHMT2. The interactions were confirmed by co-immunoprecipitation and colocalization analyses of proteins expressed in human embryonic kidney cells, mouse neuronal cells, and rat primary neuronal cells. Furthermore, we show KIRREL3 colocalization with the marker for the Golgi apparatus and synaptic vesicles. Previously, we have shown that KIRREL3 interacts with the X-linked intellectual disability associated synaptic scaffolding protein CASK through its cytoplasmic domain. In addition, we found a genomic deletion encompassing MAP1B in one patient with intellectual disability, microcephaly and seizures and deletions encompassing MYO16 in two unrelated patients with intellectual disability, autism and microcephaly. MAP1B has been previously implicated in synaptogenesis and is involved in the development of the actin-based membrane skeleton. MYO16 is expressed in hippocampal neurons and also indirectly affects actin cytoskeleton through its interaction with WAVE1 complex. We speculate KIRREL3 interacting proteins are potential candidates for intellectual disability and autism spectrum disorder. Moreover, our findings provide further insight into understanding the molecular mechanisms underlying the physiological action of KIRREL3 and its role in neurodevelopment.

Intellectual disability (ID) is a common developmental disability observed in 1 to 3% of the human population. A possible role for the Angiotensin II type 2 receptor (AGTR2) in brain function, affecting learning, memory, and behavior, has been suggested in humans and rodents. Mice lacking the Agtr2 gene (Agtr2(-/y)) showed significant impairment in their spatial memory and exhibited abnormal dendritic spine morphology. To identify Agtr2 influenced genes and pathways, we performed whole genome microarray analysis on RNA isolated from brains of Agtr2(-/y) and control male mice at embryonic day 15 (E15) and postnatal day one (P1). The gene expression profiles of the Agtr2(-/y) brain samples were significantly different when compared to profiles of the age-matched control brains. We identified 62 differently expressed genes (p< or =0.005) at E15 and in P1 brains of the Agtr2(-/y) mice. We verified the differential expression of several of these genes in brain samples using quantitative RT-PCR. Differentially expressed genes encode molecules involved in multiple cellular processes including microtubule functions associated with dendritic spine morphology. This study provides insight into Agtr2 influenced candidate genes and suggests that expression dysregulation of these genes may modulate Agtr2 actions in the brain that influences learning and memory.

Cell-adhesion molecules play critical roles in brain development, as well as maintaining synaptic structure, function, and plasticity. Here we have found the disruption of two genes encoding putative cell-adhesion molecules, CDH15 (cadherin superfamily) and KIRREL3 (immunoglobulin superfamily), by a chromosomal translocation t(11;16) in a female patient with intellectual disability (ID). We screened coding regions of these two genes in a cohort of patients with ID and controls and identified four nonsynonymous CDH15 variants and three nonsynonymous KIRREL3 variants that appear rare and unique to ID. These variations altered highly conserved residues and were absent in more than 600 unrelated patients with ID and 800 control individuals. Furthermore, in vivo expression studies showed that three of the CDH15 variations adversely altered its ability to mediate cell-cell adhesion. We also show that in neuronal cells, human KIRREL3 colocalizes and interacts with the synaptic scaffolding protein, CASK, recently implicated in X-linked brain malformation and ID. Taken together, our data suggest that alterations in CDH15 and KIRREL3, either alone or in combination with other factors, could play a role in phenotypic expression of ID in some patients.

We have identified three truncating, two splice-site, and three missense variants at conserved amino acids in the CUL4B gene on Xq24 in 8 of 250 families with X-linked mental retardation (XLMR). During affected subjects' adolescence, a syndrome emerged with delayed puberty, hypogonadism, relative macrocephaly, moderate short stature, central obesity, unprovoked aggressive outbursts, fine intention tremor, pes cavus, and abnormalities of the toes. This syndrome was first described by Cazebas et al., in a family that was included in our study and that carried a CUL4B missense variant. CUL4B is a ubiquitin E3 ligase subunit implicated in the regulation of several biological processes, and CUL4B is the first XLMR gene that encodes an E3 ubiquitin ligase. The relatively high frequency of CUL4B mutations in this series indicates that it is one of the most commonly mutated genes underlying XLMR and suggests that its introduction into clinical diagnostics should be a high priority.

Deletions and mutations involving the Retinoic Acid Induced 1 (RAI1) gene at 17p11.2 cause Smith-Magenis syndrome (SMS). Here we report a patient with autism as the main clinical presentation, with some SMS-like features and a rare de novo RAI1 gene mutation, c.3440G > A (p.R1147Q). We functionally characterized the RAI1 p.R1147Q mutant protein. The mutation, located near the nuclear localization signal, had no effect on the subcellular localization of the mutant protein. However, similar to previously reported RAI1 missense mutations in SMS patients, the RAI1 p.R1147Q mutant protein showed a significant deficiency in activating in vivo transcription of a reporter gene driven by a BDNF (brain-derived neurotrophic factor) intronic enhancer. In addition, expression of other genes associated with neurobehavioral abnormalities and/or neurodevelopmental disorders were found to be altered in this patient. These results suggest a likely contribution of RAI1, either alone or in combination of other factors, to social behavior and reinforce the RAI1 gene as a candidate gene in patients with autistic manifestations or social behavioral abnormalities.

Dendritic spine morphology and dendritic arborization are key determinants of neuronal connectivity and play critical roles in learning, memory and behavior function. Recently, defects of ZBTB20, a BTB and zinc finger domain containing transcriptional repressor, have been implicated in a wide range of neurodevelopmental disorders, including intellectual disability and autism. Here we show distinct effects of expression of two major isoforms, long and short, of ZBTB20, and its neurodevelopmental disorder-linked variants, on dendritic architecture of cultured rat cortical pyramidal neurons. The N-terminal of ZBTB20 showed a role in regulating dendritic spine morphology. Two ZBTB20 single nucleotide variants, located at the N-terminal and central regions of the protein and potentially conferring autism risk, altered dendritic spine morphology. In contrast, a single nucleotide variant identified in patients with intellectual disability and located at the C-terminus of ZBTB20 affected dendritic arborization and dendritic length but had no effect on dendritic spine morphology. Furthermore, truncation of the extreme C-terminus of ZBTB20 caused spine and dendritic morphological changes that were similar but distinct from those caused by the C-terminal variant. Taken together, our study suggests ZBTB20's role in dendritic and synaptic structure and provide possible mechanisms of its effect in neurodevelopmental disorders.