Ana Tahira

Co-expression network of neural-differentiation genes shows specific pattern in schizophrenia

BackgroundSchizophrenia is a neurodevelopmental disorder with genetic and environmental factors contributing to its pathogenesis, although the mechanism is unknown due to the difficulties in accessing diseased tissue during human neurodevelopment. The aim of this study was to find neuronal differentiation genes disrupted in schizophrenia and to evaluate those genes in post-mortem brain tissues from schizophrenia cases and controls.MethodsWe analyzed differentially expressed genes (DEG), copy number variation (CNV) and differential methylation in human induced pluripotent stem cells (hiPSC) derived from fibroblasts from one control and one schizophrenia patient and further differentiated into neuron (NPC). Expression of the DEG were analyzed with microarrays of post-mortem brain tissue (frontal cortex) cohort of 29 schizophrenia cases and 30 controls. A Weighted Gene Co-expression Network Analysis (WGCNA) using the DEG was used to detect clusters of co-expressed genes that werenon-conserved between adult cases and controls brain samples.ResultsWe identified methylation alterations potentially involved with neuronal differentiation in schizophrenia, which displayed an over-representation of genes related to chromatin remodeling complex (adjP = 0.04). We found 228 DEG associated with neuronal differentiation. These genes were involved with metabolic processes, signal transduction, nervous system development, regulation of neurogenesis and neuronal differentiation. Between adult brain samples from cases and controls there were 233 DEG, with only four genes overlapping with the 228 DEG, probably because we compared single cell to tissue bulks and more importantly, the cells were at different stages of development. The comparison of the co-expressed network of the 228 genes in adult brain samples between cases and controls revealed a less conserved module enriched for genes associated with oxidative stress and negative regulation of cell differentiation.ConclusionThis study supports the relevance of using cellular approaches to dissect molecular aspects of neurogenesis with impact in the schizophrenic brain. We showed that, although generated by different approaches, both sets of DEG associated to schizophrenia were involved with neocortical development. The results add to the hypothesis that critical metabolic changes may be occurring during early neurodevelopment influencing faulty development of the brain and potentially contributing to further vulnerability to the illness.

Global analysis of biogenesis, stability and sub-cellular localization of lncRNAs mapping to intragenic regions of the human genome

Long noncoding RNAs (lncRNAs) that map to intragenic regions of the human genome with the same (intronic lncRNAs) or opposite orientation (antisense lncRNAs) relative to protein-coding mRNAs have been largely dismissed from biochemical and functional characterization due to the belief that they are mRNA precursors, byproducts of RNA splicing or simply transcriptional noise. In this work, we used a custom microarray to investigate aspects of the biogenesis, processing, stability, evolutionary conservation, and cellular localization of ∼6,000 intronic lncRNAs and ∼10,000 antisense lncRNAs. Most intronic (2,903 of 3,427, 85%) and antisense lncRNAs (4,945 of 5,214, 95%) expressed in HeLa cells showed evidence of 5′ cap modification, compatible with their transcription by RNAP II. Antisense lncRNAs (median t1/2 = 3.9 h) were significantly (p < 0.0001) more stable than mRNAs (median t1/2 = 3.2 h), whereas intronic lncRNAs (median t1/2 = 2.1 h) comprised a more heterogeneous class that included both stable (t1/2 > 3 h) and unstable (t1/2 < 1 h) transcripts. Intragenic lncRNAs display evidence of evolutionary conservation, have little/no coding potential and were ubiquitously detected in the cytoplasm. Notably, a fraction of the intronic and antisense lncRNAs (13 and 15%, respectively) were expressed from loci at which the corresponding host mRNA was not detected. The abundances of a subset of intronic/antisense lncRNAs were correlated (r ≥ |0.8|) with those of genes encoding proteins involved in cell division and DNA replication. Taken together, the findings of this study contribute novel biochemical and genomic information regarding intronic and antisense lncRNAs, supporting the notion that these classes include independently transcribed RNAs with potentials for exerting regulatory functions in the cell.

Transcriptional profiling of Neurospora crassa Δmak-2 reveals that mitogen-activated protein kinase MAK-2 participates in the phosphate signaling pathway.

The filamentous fungus Neurospora crassa is an excellent model system for examining molecular responses to ambient signals in eukaryotic microorganisms. Inorganic phosphate (Pi) is an essential growth-limiting nutrient in nature and is crucial for the synthesis of nucleic acids and the flow of genetic information. The genetic and molecular mechanisms controlling the response to Pi starvation in N. crassa include at least four genes (nuc-2, preg, pogv, and nuc-1), which are involved in a hierarchical regulatory activation network. In a previous work, we identified a number of genes modulated by NUC-2 protein, including the mak-2 gene, which codes for a mitogen-activated protein kinase (MAPK), suggesting its participation in the phosphate signaling pathway. Thus, to identify other genes involved in metabolic responses to exogenous phosphate sensing and the functioning of the MAPK MAK-2, we performed microarray experiments using a mak-2 knockout strain (Δmak-2) grown under phosphate-shortage conditions by comparing its transcription profile to that of a control strain grown in low- and high-phosphate cultures. These experiments revealed 912 unique differentially expressed genes involved in a number of physiological processes related to phosphate transport, metabolism, and regulation as well as posttranslational modification of proteins, and MAPK signaling pathways. Quantitative Real-time PCR gene expression analysis of 18 selected genes, using independent RNA samples, validated our microarray results. A high Pearson correlation between microarray and quantitative Real-time PCR data was observed. The analysis of these differentially expressed genes in the Δmak-2 strain provide evidence that the mak-2 gene participates in the hierarchical phosphate-signaling pathway in N. crassa in addition to its involvement in other metabolic routes such as the isoprenylation pathway, thus revealing novel aspects of the N. crassa phosphorus-sensing network.

Sex differences in DNA methylation of the cord blood are related to sex-bias psychiatric diseases

Sex differences in the prevalence of psychiatric disorders are well documented, with exposure to stress during gestation differentially impacting females and males. We explored sex-specific DNA methylation in the cord blood of 39 females and 32 males born at term and with appropriate weight at birth regarding their potential connection to psychiatric outcomes. Mothers were interviewed to gather information about environmental factors (gestational exposure) that could interfere with the methylation profiles in the newborns. Bisulphite converted DNA was hybridized to Illumina HumanMethylation450 BeadChips. Excluding XYS probes, there were 2,332 differentially methylated CpG sites (DMSs) between sexes, which were enriched within brain modules of co-methylated CpGs during brain development and also differentially methylated in the brains of boys and girls. Genes associated with the DMSs were enriched for neurodevelopmental disorders, particularly for CpG sites found differentially methylated in brain tissue between patients with schizophrenia and controls. Moreover, the DMS had an overlap of 890 (38%) CpG sites with a cohort submitted to toxic exposition during gestation. This study supports the evidences that sex differences in DNA methylation of autosomes act as a primary driver of sex differences that are found in psychiatric outcomes.

An integrative approach to investigate the respective roles of single-nucleotide variants and copy-number variants in Attention-Deficit/Hyperactivity Disorder

Many studies have attempted to investigate the genetic susceptibility of Attention-Deficit/Hyperactivity Disorder (ADHD), but without much success. The present study aimed to analyze both single-nucleotide and copy-number variants contributing to the genetic architecture of ADHD. We generated exome data from 30 Brazilian trios with sporadic ADHD. We also analyzed a Brazilian sample of 503 children/adolescent controls from a High Risk Cohort Study for the Development of Childhood Psychiatric Disorders, and also previously published results of five CNV studies and one GWAS meta-analysis of ADHD involving children/adolescents. The results from the Brazilian trios showed that cases with de novo SNVs tend not to have de novo CNVs and vice-versa. Although the sample size is small, we could also see that various comorbidities are more frequent in cases with only inherited variants. Moreover, using only genes expressed in brain, we constructed two “in silico” protein-protein interaction networks, one with genes from any analysis, and other with genes with hits in two analyses. Topological and functional analyses of genes in this network uncovered genes related to synapse, cell adhesion, glutamatergic and serotoninergic pathways, both confirming findings of previous studies and capturing new genes and genetic variants in these pathways.

Integrative Variation Analysis Reveals that a Complex Genotype May Specify Phenotype in Siblings with Syndromic Autism Spectrum Disorder.

It has been proposed that copy number variations (CNVs) are associated with increased risk of autism spectrum disorder (ASD) and, in conjunction with other genetic changes, contribute to the heterogeneity of ASD phenotypes. Array comparative genomic hybridization (aCGH) and exome sequencing, together with systems genetics and network analyses, are being used as tools for the study of complex disorders of unknown etiology, especially those characterized by significant genetic and phenotypic heterogeneity. Therefore, to characterize the complex genotype-phenotype relationship, we performed aCGH and sequenced the exomes of two affected siblings with ASD symptoms, dysmorphic features, and intellectual disability, searching for de novo CNVs, as well as for de novo and rare inherited point variations—single nucleotide variants (SNVs) or small insertions and deletions (indels)—with probable functional impacts. With aCGH, we identified, in both siblings, a duplication in the 4p16.3 region and a deletion at 8p23.3, inherited by a paternal balanced translocation, t(4, 8) (p16; p23). Exome variant analysis found a total of 316 variants, of which 102 were shared by both siblings, 128 were in the male sibling exome data, and 86 were in the female exome data. Our integrative network analysis showed that the siblings’ shared translocation could explain their similar syndromic phenotype, including overgrowth, macrocephaly, and intellectual disability. However, exome data aggregate genes to those already connected from their translocation, which are important to the robustness of the network and contribute to the understanding of the broader spectrum of psychiatric symptoms. This study shows the importance of using an integrative approach to explore genotype-phenotype variability.

Erratum: An integrative approach to investigate the respective roles of single-nucleotide variants and copy-number variants in Attention-Deficit/Hyperactivity Disorder (Scientific Reports (2016) 6 (22851) DOI: 10.1038/srep22851)

Leandro de Araújo Lima, Ana Cecília Feio-dos-Santos, Sintia Iole Belangero, Ary Gadelha, Rodrigo Affonseca Bressan, Giovanni Abrahão Salum, Pedro Mario Pan, Tais Silveira Moriyama, Ana Soledade Graeff-Martins, Ana Carina Tamanaha, Pedro Alvarenga, Fernanda Valle Krieger, Bacy Fleitlich-Bilyk, Andrea Parolin Jackowski, Elisa Brietzke, João Ricardo Sato, Guilherme Vanoni Polanczyk, Jair de Jesus Mari, Gisele Gus Manfro, Maria Conceição do Rosário, Eurípedes Constantino Miguel, Renato David Puga, Ana Carolina Tahira, Viviane Neri Souza, Thais Chile, Gisele Rodrigues Gouveia, Sérgio Nery Simões, Xiao Chang, Renata Pellegrino, Lifeng Tian, Joseph T. Glessner, Ronaldo Fumio Hashimoto, Luis Augusto Rohde, Patrick M. A. Sleiman, Hakon Hakonarson & Helena Brentani