De novo and rare inherited mutations implicate the transcriptional coregulator TCF20/SPBP in autism spectrum disorder

BACKGROUND: Autism spectrum disorders (ASDs) are common and have a strong genetic basis, yet the cause of ∼70-80% ASDs remains unknown. By clinical cytogenetic testing, we identified a family in which two brothers had ASD, mild intellectual disability and a chromosome 22 pericentric inversion, not detected in either parent, indicating de novo mutation with parental germinal mosaicism. We hypothesised that the rearrangement was causative of their ASD and localised the chromosome 22 breakpoints. METHODS: The rearrangement was characterised using fluorescence in situ hybridisation, Southern blotting, inverse PCR and dideoxy-sequencing. Open reading frames and intron/exon boundaries of the two physically disrupted genes identified, TCF20 and TNRC6B, were sequenced in 342 families (260 multiplex and 82 simplex) ascertained by the International Molecular Genetic Study of Autism Consortium (IMGSAC). RESULTS: IMGSAC family screening identified a de novo missense mutation of TCF20 in a single case and significant association of a different missense mutation of TCF20 with ASD in three further families. Through exome sequencing in another project, we independently identified a de novo frameshifting mutation of TCF20 in a woman with ASD and moderate intellectual disability. We did not identify a significant association of TNRC6B mutations with ASD. CONCLUSIONS: TCF20 encodes a transcriptional coregulator (also termed SPBP) that is structurally and functionally related to RAI1, the critical dosage-sensitive protein implicated in the behavioural phenotypes of the Smith-Magenis and Potocki-Lupski 17p11.2 deletion/duplication syndromes, in which ASD is frequently diagnosed. This study provides the first evidence that mutations in TCF20 are also associated with ASD

Identification of Neural Outgrowth Genes using Genome-Wide RNAi

While genetic screens have identified many genes essential for neurite outgrowth, they have been limited in their ability to identify neural genes that also have earlier critical roles in the gastrula, or neural genes for which maternally contributed RNA compensates for gene mutations in the zygote. To address this, we developed methods to screen the Drosophila genome using RNA-interference (RNAi) on primary neural cells and present the results of the first full-genome RNAi screen in neurons. We used live-cell imaging and quantitative image analysis to characterize the morphological phenotypes of fluorescently labelled primary neurons and glia in response to RNAi-mediated gene knockdown. From the full genome screen, we focused our analysis on 104 evolutionarily conserved genes that when downregulated by RNAi, have morphological defects such as reduced axon extension, excessive branching, loss of fasciculation, and blebbing. To assist in the phenotypic analysis of the large data sets, we generated image analysis algorithms that could assess the statistical significance of the mutant phenotypes. The algorithms were essential for the analysis of the thousands of images generated by the screening process and will become a valuable tool for future genome-wide screens in primary neurons. Our analysis revealed unexpected, essential roles in neurite outgrowth for genes representing a wide range of functional categories including signalling molecules, enzymes, channels, receptors, and cytoskeletal proteins. We also found that genes known to be involved in protein and vesicle trafficking showed similar RNAi phenotypes. We confirmed phenotypes of the protein trafficking genes Sec61alpha and Ran GTPase using Drosophila embryo and mouse embryonic cerebral cortical neurons, respectively. Collectively, our results showed that RNAi phenotypes in primary neural culture can parallel in vivo phenotypes, and the screening technique can be used to identify many new genes that have important functions in the nervous system

Self-oligomerization regulates stability of survival motor neuron protein isoforms by sequestering an SCF^Slmb degron

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers

Conserved Genes Act as Modifiers of Invertebrate SMN Loss of Function Defects

Spinal Muscular Atrophy (SMA) is caused by diminished function of the Survival of Motor Neuron (SMN) protein, but the molecular pathways critical for SMA pathology remain elusive. We have used genetic approaches in invertebrate models to identify conserved SMN loss of function modifier genes. Drosophila melanogaster and Caenorhabditis elegans each have a single gene encoding a protein orthologous to human SMN; diminished function of these invertebrate genes causes lethality and neuromuscular defects. To find genes that modulate SMN function defects across species, two approaches were used. First, a genome-wide RNAi screen for C. elegans SMN modifier genes was undertaken, yielding four genes. Second, we tested the conservation of modifier gene function across species; genes identified in one invertebrate model were tested for function in the other invertebrate model. Drosophila orthologs of two genes, which were identified originally in C. elegans, modified Drosophila SMN loss of function defects. C. elegans orthologs of twelve genes, which were originally identified in a previous Drosophila screen, modified C. elegans SMN loss of function defects. Bioinformatic analysis of the conserved, cross-species, modifier genes suggests that conserved cellular pathways, specifically endocytosis and mRNA regulation, act as critical genetic modifiers of SMN loss of function defects across species

Alternative splicing and transcriptome profiling of experimental autoimmune encephalomyelitis using genome-wide exon arrays

BACKGROUND: Multiple Sclerosis (MS) is a chronic inflammatory disease causing demyelination and nerve loss in the central nervous system. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS that is widely used to investigate complex pathogenic mechanisms. Transcriptional control through isoform selection and mRNA levels determines pathway activation and ultimately susceptibility to disease. METHODOLOGY/PRINCIPAL FINDINGS: We have studied the role of alternative splicing and differential expression in lymph node cells from EAE-susceptible Dark Agouti (DA) and EAE-resistant Piebald Virol Glaxo.AV1 (PVG) inbred rat strains using Affymetrix Gene Chip Rat Exon 1.0 ST Arrays. Comparing the two strains, we identified 11 differentially spliced and 206 differentially expressed genes at day 7 post-immunization, as well as 9 differentially spliced and 144 differentially expressed genes upon autoantigen re-stimulation. Functional clustering and pathway analysis implicate genes for glycosylation, lymphocyte activation, potassium channel activity and cellular differentiation in EAE susceptibility. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that alternative splicing occurs during complex disease and may govern EAE susceptibility. Additionally, transcriptome analysis not only identified previously defined EAE pathways regulating the immune system, but also novel mechanisms. Furthermore, several identified genes overlap known quantitative trait loci, providing novel causative candidate targets governing EAE

Fibroblasts from patients with major depressive disorder show distinct transcriptional response to metabolic stressors

Major depressive disorder (MDD) is increasingly viewed as interplay of environmental stressors and genetic predisposition, and recent data suggest that the disease affects not only the brain, but the entire body. As a result, we aimed at determining whether patients with major depression have aberrant molecular responses to stress in peripheral tissues. We examined the effects of two metabolic stressors, galactose (GAL) or reduced lipids (RL), on the transcriptome and miRNome of human fibroblasts from 16 pairs of patients with MDD and matched healthy controls (CNTR). Our results demonstrate that both MDD and CNTR fibroblasts had a robust molecular response to GAL and RL challenges. Most importantly, a significant part (messenger RNAs (mRNAs): 26-33%; microRNAs (miRNAs): 81-90%) of the molecular response was only observed in MDD, but not in CNTR fibroblasts. The applied metabolic challenges uncovered mRNA and miRNA signatures, identifying responses to each stressor characteristic for the MDD fibroblasts. The distinct responses of MDD fibroblasts to GAL and RL revealed an aberrant engagement of molecular pathways, such as apoptosis, regulation of cell cycle, cell migration, metabolic control and energy production. In conclusion, the metabolic challenges evoked by GAL or RL in dermal fibroblasts exposed adaptive dysfunctions on mRNA and miRNA levels that are characteristic for MDD. This finding underscores the need to challenge biological systems to bring out disease-specific deficits, which otherwise might remain hidden under resting conditions

Physiological suppression of microRNA-silencing pathway by HIV-1 during virus replication

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