Atypical miRNA expression in temporal cortex associated with dysregulation of immune, cell cycle, and other pathways in autism spectrum disorders.

BackgroundAutism spectrum disorders (ASDs) likely involve dysregulation of multiple genes related to brain function and development. Abnormalities in individual regulatory small non-coding RNA (sncRNA), including microRNA (miRNA), could have profound effects upon multiple functional pathways. We assessed whether a brain region associated with core social impairments in ASD, the superior temporal sulcus (STS), would evidence greater transcriptional dysregulation of sncRNA than adjacent, yet functionally distinct, primary auditory cortex (PAC).MethodsWe measured sncRNA expression levels in 34 samples of postmortem brain from STS and PAC to find differentially expressed sncRNA in ASD compared with control cases. For differentially expressed miRNA, we further analyzed their predicted mRNA targets and carried out functional over-representation analysis of KEGG pathways to examine their functional significance and to compare our findings to reported alterations in ASD gene expression.ResultsTwo mature miRNAs (miR-4753-5p and miR-1) were differentially expressed in ASD relative to control in STS and four (miR-664-3p, miR-4709-3p, miR-4742-3p, and miR-297) in PAC. In both regions, miRNA were functionally related to various nervous system, cell cycle, and canonical signaling pathways, including PI3K-Akt signaling, previously implicated in ASD. Immune pathways were only disrupted in STS. snoRNA and pre-miRNA were also differentially expressed in ASD brain.ConclusionsAlterations in sncRNA may underlie dysregulation of molecular pathways implicated in autism. sncRNA transcriptional abnormalities in ASD were apparent in STS and in PAC, a brain region not directly associated with core behavioral impairments. Disruption of miRNA in immune pathways, frequently implicated in ASD, was unique to STS

Single Nucleotide Polymorphisms in MicroRNA Binding Sites: Implications in Colorectal Cancer

Cancer is a complex genetic disorder, characterised by uncontrolled cell proliferation and caused by altered expression of oncogenes and tumour suppressor genes. When cell proliferation pertains to colon, it is called colorectal cancer. Most of colorectal cancer causing genes are potential targets for the miRNA (microRNA) that bind to 3′UTR (untranslated regions) of mRNA and inhibit translation. Mutations occurring in miRNA binding regions can alter the miRNA, mRNA combination, and can alter gene expression drastically. We hypothesized that 3′UTR mutation in miRNA binding site could alter the miRNA, mRNA interaction, thereby altering gene expression. Altered gene expression activity could promote tumorigenesis in colon. Therefore, we formulated a systematic in silico procedure that integrates data from various databases, followed rigorous selection criteria, and identified mutations that might alter the expression levels of cancer causing genes. Further we performed expression analysis to shed light on the potential tissues that might be affected by mutation, enrichment analysis to find the metabolic functions of the gene, and network analysis to highlight the important interactions of cancer causing genes with other genes to provide insight that complex network will be disturbed upon mutation. We provide in silico evidence for the effect of these mutations in colorectal cancer

Autism-associated miR-873 regulates ARID1B, SHANK3 and NRXN2 involved in neurodevelopment

Autism spectrum disorders (ASD) are highly heritable neurodevelopmental disorders with significant genetic heterogeneity. Noncoding microRNAs (miRNAs) are recognised as playing key roles in development of ASD albeit the function of these regulatory genes remains unclear. We previously conducted whole-exome sequencing of Australian families with ASD and identified four novel single nucleotide variations in mature miRNA sequences. A pull-down transcriptome analysis using transfected SH-SY5Y cells proposed a mechanistic model to examine changes in binding affinity associated with a unique mutation found in the conserved 'seed' region of miR-873-5p (rs777143952: T > A). Results suggested several ASD-risk genes were differentially targeted by wild-type and mutant miR-873 variants. In the current study, a dual-luciferase reporter assay confirmed miR-873 variants have a 20-30% inhibition/dysregulation effect on candidate autism risk genes ARID1B, SHANK3 and NRXN2 and also confirmed the affected expression with qPCR. In vitro mouse hippocampal neurons transfected with mutant miR-873 showed less morphological complexity and enhanced sodium currents and excitatory neurotransmission compared to cells transfected with wild-type miR-873. A second in vitro study showed CRISPR/Cas9 miR-873 disrupted SH-SY5Y neuroblastoma cells acquired a neuronal-like morphology and increased expression of ASD important genes ARID1B, SHANK3, ADNP2, ANK2 and CHD8. These results represent the first functional evidence that miR-873 regulates key neural genes involved in development and cell differentiation.We acknowledge the financial support from Australian National Health and Medical Research Council (APP1008125)

Atypical miRNA expression in temporal cortex associated with dysregulation of immune, cell cycle, and other pathways in autism spectrum disorders

BACKGROUND: Autism spectrum disorders (ASDs) likely involve dysregulation of multiple genes related to brain function and development. Abnormalities in individual regulatory small non-coding RNA (sncRNA), including microRNA (miRNA), could have profound effects upon multiple functional pathways. We assessed whether a brain region associated with core social impairments in ASD, the superior temporal sulcus (STS), would evidence greater transcriptional dysregulation of sncRNA than adjacent, yet functionally distinct, primary auditory cortex (PAC). METHODS: We measured sncRNA expression levels in 34 samples of postmortem brain from STS and PAC to find differentially expressed sncRNA in ASD compared with control cases. For differentially expressed miRNA, we further analyzed their predicted mRNA targets and carried out functional over-representation analysis of KEGG pathways to examine their functional significance and to compare our findings to reported alterations in ASD gene expression. RESULTS: Two mature miRNAs (miR-4753-5p and miR-1) were differentially expressed in ASD relative to control in STS and four (miR-664-3p, miR-4709-3p, miR-4742-3p, and miR-297) in PAC. In both regions, miRNA were functionally related to various nervous system, cell cycle, and canonical signaling pathways, including PI3K-Akt signaling, previously implicated in ASD. Immune pathways were only disrupted in STS. snoRNA and pre-miRNA were also differentially expressed in ASD brain. CONCLUSIONS: Alterations in sncRNA may underlie dysregulation of molecular pathways implicated in autism. sncRNA transcriptional abnormalities in ASD were apparent in STS and in PAC, a brain region not directly associated with core behavioral impairments. Disruption of miRNA in immune pathways, frequently implicated in ASD, was unique to STS. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13229-015-0029-9) contains supplementary material, which is available to authorized users

The non-coding genome in Autism Spectrum Disorders

Autism Spectrum Disorders (ASD) are a group of neurodevelopmental disorders (NDDs) characterized by difficulties in social interaction and communication, repetitive behavior, and restricted interests. While ASD have been proven to have a strong genetic component, current research largely focuses on coding regions of the genome. However, non-coding DNA, which makes up for ∼99% of the human genome, has recently been recognized as an important contributor to the high heritability of ASD, and novel sequencing technologies have been a milestone in opening up new directions for the study of the gene regulatory networks embedded within the non-coding regions. Here, we summarize current progress on the contribution of non-coding alterations to the pathogenesis of ASD and provide an overview of existing methods allowing for the study of their functional relevance, discussing potential ways of unraveling ASD's “missing heritability”S

Identification of two novel autism genes, TRPC4 and SCFD2, in Qatar simplex families through exome sequencing

This study investigated the genetic underpinnings of autism spectrum disorder (ASD) in a Middle Eastern cohort in Qatar using exome sequencing. The study identified six candidate autism genes in independent simplex families, including both four known and two novel autosomal dominant and autosomal recessive genes associated with ASD. The variants consisted primarily of de novo and homozygous missense and splice variants. Multiple individuals displayed more than one candidate variant, suggesting the potential involvement of digenic or oligogenic models. These variants were absent in the Genome Aggregation Database (gnomAD) and exhibited extremely low frequencies in the local control population dataset. Two novel autism genes, TRPC4 and SCFD2, were discovered in two Qatari autism individuals. Furthermore, the D651A substitution in CLCN3 and the splice acceptor variant in DHX30 were identified as likely deleterious mutations. Protein modeling was utilized to evaluate the potential impact of three missense variants in DEAF1, CLCN3, and SCFD2 on their respective structures and functions, which strongly supported the pathogenic natures of these variants. The presence of multiple de novo mutations across trios underscored the significant contribution of de novo mutations to the genetic etiology of ASD. Functional assays and further investigations are necessary to confirm the pathogenicity of the identified genes and determine their significance in ASD. Overall, this study sheds light on the genetic factors underlying ASD in Qatar and highlights the importance of considering diverse populations in ASD research

Análise de expressão de microRNA em pacientes com Transtorno do Espectro do Autismo : a relação do comportamento fenotípico com alterações epigenéticas

O Transtorno do Espectro do Autismo (TEA) é um grupo heterogêneo de desordens do desenvolvimento neurológico, caracterizado por prejuízos na comunicação e interação social e padrões repetitivos e estereotipados de comportamento. O diagnóstico clínico é avaliado apenas por entrevistas com os pais. A heterogeneidade deste transtorno é gerada por fatores genéticos e ambientais que desempenham um papel vital na predisposição dos indivíduos para o TEA com diferentes níveis de comprometimento. As alterações epigenéticas têm sido amplamente estudadas em doenças psiquiátricas. MicroRNAs (miRNAs) surgiram recentemente como reguladores epigenéticos importantes em uma variedade de processos celulares. O objetivo deste estudo foi avaliar a expressão de alguns microRNAs (miRNAs) e correlacionar com vias bioquímicas em pacientes com TEA e em modelo animal de TEA. Nos pacientes com TEA, de um total de 26 miRNAs, sete foram estatisticamente alterados em pacientes quando comparado com o grupo controle: miR34c, miR92a-2, miR145 e miR199a têm aumento na expressão em pacientes enquanto os miR27a, miR19-b e miR193a estão reprimidos nos pacientes. Este estudo ainda mostra uma correlação de expressão de 4 miRNAs, miR34c, miR145 e miR199a e miR193a, em ambos os grupos (pacientes e do modelo animal de TEA). Os principais alvos, dos miRNAs, que estão reprimidos contribuem com o fenótipo encontrado no TEA são Sirtuína 1, HDAC2 e MeCP2. Além disso, este trabalho mostra algumas semelhanças na expressão de miRNAs alterados entre as duas espécies. É importante ressaltar o papel do miR34c que está intimamente ligado ao Transtorno de Ansiedade. Isto sugere que existe uma alteração epigenética no TEA que é persistente e conservada entre espécies, induzindo, possivelmente, o comportamento ansioso nos pacientes. Além de um perfil de expressão de microRNA como um possível marcador biológico, tanto diagnóstico quanto prognóstico, este estudo mostra uma possibilidade terapêutica para o tratamento da ansiedade em pacientes com TEA.Autism Spectrum Disorder (ASD) is a heterogeneous group of neurodevelopmental disorders characterized by impairments in communication and social interaction and repetitive and stereotyped patterns of behavior. The clinical diagnosis is only assessed by interviews with parents. The heterogeneity of this disorder is generated by genetic and environmental factors that play a vital role in the predisposition of individuals to the ASD with different levels of commitment. Epigenetic changes have been widely studied in psychiatric disorders. miRNAs have recently emerged as important regulators of epigenetic a variety of cellular processes. The aim of this study was to evaluate the expression of miRNA and correlate with biochemical pathways in ASD patients and in ASD animal model. In the ASD patients, a total of 26 miRNA, seven were significantly altered when compared with the control group: miR34c, miR92a-2, miR145 and miR199a have increased expression in patients while miR27a, miR19-b miR193a are suppressed in patients. This study also shows a correlation of 4 miRNA expression, miR34c, miR145, miR199a and miR193a in both groups (patients and ASD animal model). The main targets that are repressed in these miRNAs expressed differently contribute to the phenotype found in ASD and are Sirtuin 1, HDAC2 and MeCP2. Also, this work shows some similarities in miRNAs expression changed between the two species. It is important to emphasize the role of miR34c that is closely linked to anxiety disorder. This suggests that there is an epigenetic change in ASD and is persistent and conserved among species, inducing possibly the anxious behavior in patients. Besides a microRNA expression profile as a biomarker can both diagnosis and prognosis, this study shows the therapeutic opportunity for treatment of anxiety in patients with ASD

Integrated analysis of miRNA expression in response to Salmonella Typhimurium infection in pigs

La salmonelosis es una enfermedad gastrointestinal causada por S. Typhimurium, la cual es transmitida mediante ingesta de comida de origen animal contaminada. Se considera un problema grave de salud pública, y la carne de cerdo es uno de los reservorios más importantes de la enfermedad. Como el cerdo es considerado un buen modelo animal para estudiar enfermedades humanas, la investigación sobre mecanismos moleculares involucrados en la respuesta a la infección en cerdos es necesaria para la salud humana. Los microRNAs (miRNAs) son un tipo de RNA no codificante que interfiere con la estabilidad y traducción de la proteína. Los miRNAs regulan post-transcripcionalmente procesos biológicos como aquellos causados por bacterias. Aunque la respuesta porcina a la infección por S. Typhimurium ha sido estudiada previamente, se sabe poco del papel que juegan los miRNAs en la regulación génica y expresión de proteínas durante la infección. Por tanto, el objetivo de este estudio fue la identificación los miRNAs que regulan la respuesta a la infección por S. Typhimurium en el tracto gastrointestinal y nódulo linfático mesentérico porcino, usando herramientas “ómicas” de nueva generación

Molecular and physiological roles of long 3′ UTR mRNA isoforms in neurons

The brain is an organ where the greatest proportion of genes are expressed compared to any other part of the body. To add even more complexity, gene expression in the brain is subject to various layers of regulation through RNA processing mechanisms including alternative splicing (AS) and alternative cleavage and polyadenylation (APA). These RNA processing mechanisms contribute to increased transcriptome diversity in the brain. APA often induces the synthesis of mRNA isoforms that harbor the same protein-coding sequence but different length 3′ untranslated regions (3′ UTRs) from a single gene. Alternative 3′ UTRs regulate gene expression post-transcriptionally by modulating transcript stability, translation efficiency, or subcellular localization. In Chapter 1, we reviewed all of the reported functions of 3′ UTRs in the nervous system. Despite the fact 3′ UTR is highly regarded in gene regulation, evidence of impacts of long 3′ UTR loss on in vivo animal is scarce. To study the physiological relevance of long 3′ UTR mRNA isoforms, we have driven our attention to the Calm1 gene. Calm1 is one of the three genes that encode Calmodulin which is required for proper neural development and function. In Chapter 2, we found that the expression of the long 3′ UTR mRNA isoform of Calm1 was necessary for mouse nervous system development and function. Disruption of the Calm1 long 3′ UTR isoform impaired dorsal root ganglion axon development in mouse embryos and neuronal activation upon novel environment exposure in young adult mice. Our results presented direct evidence for the physiological importance of the Calm1 long 3′ UTR mRNA isoform in vivo. To screen molecular and cellular functions of long 3′ UTRs in a fast and efficient manner, establishing an in vitro cell system is warranted. In Chapter 3, we presented mouse embryonic stem cell (mESC)-derived neurons as a suitable cell-culture system. The transcriptomic profile of the mESC-derived neurons closely resembled the profile in the mouse cerebral cortex, showing the suitability of using this system for studying long 3′ UTRs. The mESC system is amenable to genetic manipulation via CRISPR-Cas9, thus providing as good avenue for fast generation of long 3′ UTR isoform knockout lines. As a proof of principle, a workflow for the generation of Myosin phosphatase Rho interacting protein (Mprip) long 3′ UTR isoform knockout cell lines, differentiation into glutamatergic neurons, and confirmation of the long 3′ UTR expression abolishment is presented. Taking advantage of the convenient culture cell system we have established, we next aimed to explore more functions of long 3′ UTRs. A recent discovery in our lab suggested that APA and AS are closely linked RNA processing mechanisms in which long 3′ UTRs modulate upstream AS. In Chapter 4, we explored the coupling events between AS and APA in mouse neurons using Pull-a-Long-Seq (PL-Seq) pipeline, which presents a particular utility in quantifying the coordination of tandem 3′ UTR APA events with upstream cassette exon AS. PL-Seq performed on the Endonuclease V (Endov) gene reveals that expression of its long 3′ UTR in neurons is preferentially associated with an exon skipping event located far upstream of the terminal exon