Genome-wide changes in lncRNA, splicing, and regional gene expression patterns in autism

In the paper

“Our analysis reveals ASD-associated dysregulation of primate-specific long noncoding RNAs (lncRNAs), downregulation of the alternative splicing of activity-dependent neuron-specific exons, and attenuation of normal differences in gene expression between the frontal and temporal lobes.”

In the paper

“Our analysis reveals ASD-associated dysregulation of primate-specific long noncoding RNAs (lncRNAs), downregulation of the alternative splicing of activity-dependent neuron-specific exons, and attenuation of normal differences in gene expression between the frontal and temporal lobes.”

In the paper

“Our analysis reveals ASD-associated dysregulation of primate-specific long noncoding RNAs (lncRNAs), downregulation of the alternative splicing of activity-dependent neuron-specific exons, and attenuation of normal differences in gene expression between the frontal and temporal lobes.”

In the paper

“We performed a classification analysis to confirm that gene expression in ASD could separate samples by disease status (Extended Data Fig. 2a) and confirmed the technical quality of our data with qRT–PCR (Extended Data Fig. 2b, c).”

In the paper

“Therefore, we next investigated whether the shared splicing signature in ASD could be explained by perturbations in splicing factors known to be important in nervous system function8,14 (Extended Data Fig. 4j), and found high correlations between splicing factor expression and differential splicing in the cortex (Fig. 1f) but not the cerebellum (Fig. 1g).”

In the paper

“We next evaluated enrichment of the gene sets for pathways and cell types (Extended Data Fig. 2d, e), and found that the downregulated set was enriched in genes expressed in neurons and involved in neuronal pathways, including PVALB and SYT2, which are highly expressed in interneurons; by contrast, the upregulated gene set was enriched in genes expressed in microglia and astrocytes8.”

In the paper

“As a group, these lncRNAs are enriched in the brain relative to other tissues (Extended Data Fig. 2l, m) and most that have been evaluated across species exhibit primate-specific expression patterns in the brain11, which we confirm for several transcripts (Supplementary Information, Extended Data Fig. 3a–h).”

In the paper

“The lack of significant DGE in the cerebellum is explained by the fact that changes in expression were consistently stronger in the cortex than in the cerebellum (Extended Data Fig. 2h), which suggests that the cortex is more selectively vulnerable to these transcriptomic alterations.”

In the paper

“We found a significant differential splicing signal over background in the cortex (1,127 differential splicing events in 833 genes; Methods), but not in the cerebellum (P distributions in Extended Data Fig. 4b, c).”

In the paper

“We found a significant differential splicing signal over background in the cortex (1,127 differential splicing events in 833 genes; Methods), but not in the cerebellum (P distributions in Extended Data Fig. 4b, c).”

In the paper

“Pathway and cell-type analysis showed that the ACP set is enriched in Wnt signalling, calcium binding, and neuronal genes (Extended Data Fig. 6i, j, Supplementary Information).”

In the paper

“We further demonstrate that a genetically defined subtype of ASD, chromosome 15q11.2–13.1 duplication syndrome (dup15q), shares the core transcriptomic signature observed in idiopathic ASD.”

In the paper

“Co-expression network analysis reveals that individuals with ASD show age-related changes in the trajectory of microglial and synaptic function over the first two decades, and suggests that genetic risk for ASD may influence changes in regional cortical gene expression.”

In the paper

“Indeed, differential splicing events were significantly enriched in those previously shown to be regulated by neuronal activity21 (Fig. 1h).”

What the sources say

• Not checked yetcites [21] Maze, I. et al. Critical role of histone turnover in neuronal transcription and plasticity. Neuron 87, 77–94 (2015) source

In the paper

“It also confirms that downregulated modules are enriched in synaptic function and neuronal genes, that upregulated modules are enriched in genes associated with inflammatory pathways and glial function4,8, and that microglial and synaptic modules exhibit significant anticorrelation (Fig. 4c).”

In the paper

“We first validated differential gene expression (DGE) between samples of cortex from control individuals and those with ASD (ASD cortex) by comparing gene expression with that of different individuals from those previously profiled by microarray8, and found strong concordance (R2 = 0.60; Fig. 1a, Extended Data Fig. 1i).”

What the sources say

• Not checked yetcites [8] Voineagu, I. et al. Transcriptomic analysis of autistic brain reveals convergent molecular pathology. Nature 474, 380–384 (2011) source

In the paper

“Cell-type specific enrichment and pathway analysis of alternative splicing demonstrated that most differential splicing events involve exclusion of neuron-specific exons17 (Fig. 1e, Extended Data Fig. 4i).”

What the sources say

• Not checked yetcites [17] Zhang, Y. et al. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J. Neurosci. 34, 11929–11947 (2014) source

In the paper

“Furthermore, the downregulated modules CTX.M10 and CTX.M16 are enriched in genes previously related to neuronal firing rate, consistent with the overlap of dysregulated splicing with events regulated by neuronal activity (Extended Data Fig. 9b and Fig. 1h).”

In the paper

“Although there was no significant DGE in the cerebellum (FDR < 0.05, P distributions in Fig. 1b), similar to observations in a smaller cohort8, there was a replication signal in the cerebellum and overall concordance between ASD-related fold changes in the cortex and cerebellum (Extended Data Fig. 2f–h).”

What the sources say

• Not checked yetcites [8] Voineagu, I. et al. Transcriptomic analysis of autistic brain reveals convergent molecular pathology. Nature 474, 380–384 (2011) source

In the paper

“As a group, these lncRNAs are enriched in the brain relative to other tissues (Extended Data Fig. 2l, m) and most that have been evaluated across species exhibit primate-specific expression patterns in the brain11, which we confirm for several transcripts (Supplementary Information, Extended Data Fig. 3a–h).”

What the sources say

• Not checked yetcites [11] Necsulea, A. et al. The evolution of lncRNA repertoires and expression patterns in tetrapods. Nature 505, 635–640 (2014) source

In the paper

“We identified a remarkable difference in CTX.M19 and CTX.M20 during the first two decades of life (Fig. 4g, additional age trajectories in Extended Data Fig. 9f) that is most consistent with an evolving process during early brain development that stabilizes starting in late childhood and early adolescence.”

In the paper

“Despite significant technical differences that reduce power to detect DGE, and profiling of different brain regions in that study, there was a weak but significant correlation in fold changes, which was due mostly to upregulated genes in both studies (P = 0.038, Extended Data Fig. 2i, j).”

What the sources say

• Not checked yetcites [4] Gupta, S. et al. Transcriptome analysis reveals dysregulation of innate immune response genes and neuronal activity-dependent genes in autism. Nat. Commun. 5, 5748 (2014) source

In the paper

“However, other phenotypes with larger, better-powered genome-wide association studies (GWAS) also demonstrate enrichment (Extended Data Fig. 9h–i).”

In the paper

“Therefore, we next investigated whether the shared splicing signature in ASD could be explained by perturbations in splicing factors known to be important in nervous system function8,14 (Extended Data Fig. 4j), and found high correlations between splicing factor expression and differential splicing in the cortex (Fig. 1f) but not the cerebellum (Fig. 1g).”

In the paper

“When we compared the first principal component across samples for protein coding DGE, lncRNA DGE and differential splicing, we found remarkably high correlations (R2 > 0.8), indicating that molecular convergence is likely to be a unitary phenomenon across multiple levels of transcriptome regulation in ASD (Fig. 1i).”

In the paper

“We next explored lncRNAs, most of which have little functional annotation, and identified 60 lncRNAs in the DGE set (FDR < 0.05, Extended Data Fig. 2k).”

In the paper

“We confirmed that a significant proportion of ACP genes are regulated by SOX5 by overexpressing it in human neural progenitors. SOX5 induced synaptic genes and repressed cell proliferation (Fig. 2e), and predicted SOX5 targets exhibited net down-regulation, consistent with the repressive function of SOX5 (Fig. 2f, Extended Data Fig. 6l, m).”

In the paper

“It also confirms that downregulated modules are enriched in synaptic function and neuronal genes, that upregulated modules are enriched in genes associated with inflammatory pathways and glial function4,8, and that microglial and synaptic modules exhibit significant anticorrelation (Fig. 4c).”

In the paper

“Moreover, 20 of these lncRNAs have been shown to interact with microRNA (miRNA)–protein complexes, and 9 with the fragile X mental retardation protein (FMRP), whose mRNA targets are enriched in ASD risk genes9,10.”

In the paper

“Compared to control cortex, 584 genes showed increased expression and 558 showed decreased expression in ASD cortex (Fig. 1b; Benjamini–Hochberg FDR < 0.05, linear mixed effects model; see Methods).”

In the paper

“Remarkably, the upregulated module CTX.M20 was not found in previous analyses, overlaps significantly with the ACP set (FDR < 0.05, Extended Data Fig. 9a), and contains genes implicated in development and regulation of cell differentiation (Fig. 4f).”

In the paper

“One module, CTX.M24, exhibited significant enrichment for rare mutations found in ASD, while rare de novo mutations associated with intellectual disability were most strongly enriched in CTX.M22 (FDR < 0.05, Extended Data Fig. 9a).”

In the paper

“Moreover, 20 of these lncRNAs have been shown to interact with microRNA (miRNA)–protein complexes, and 9 with the fragile X mental retardation protein (FMRP), whose mRNA targets are enriched in ASD risk genes9,10.”

In the paper

“Compared to control cortex, 584 genes showed increased expression and 558 showed decreased expression in ASD cortex (Fig. 1b; Benjamini–Hochberg FDR < 0.05, linear mixed effects model; see Methods).”

In the paper

“It also confirms that downregulated modules are enriched in synaptic function and neuronal genes, that upregulated modules are enriched in genes associated with inflammatory pathways and glial function4,8, and that microglial and synaptic modules exhibit significant anticorrelation (Fig. 4c).”

What the sources say

• Not checked yetcites [4] Gupta, S. et al. Transcriptome analysis reveals dysregulation of innate immune response genes and neuronal activity-dependent genes in autism. Nat. Commun. 5, 5748 (2014) source
• Not checked yetcites [8] Voineagu, I. et al. Transcriptomic analysis of autistic brain reveals convergent molecular pathology. Nature 474, 380–384 (2011) source

In the paper

“We highlight two primate-specific lncRNAs, LINC00693 and LINC00689. Both interact with miRNA processing complexes and are typically downregulated during development12, but are upregulated in ASD cortex (Fig. 1c, d, Extended Data Fig. 2n).”

In the paper

“We next evaluated enrichment of the gene sets for pathways and cell types (Extended Data Fig. 2d, e), and found that the downregulated set was enriched in genes expressed in neurons and involved in neuronal pathways, including PVALB and SYT2, which are highly expressed in interneurons; by contrast, the upregulated gene set was enriched in genes expressed in microglia and astrocytes8.”

What the sources say

• Not checked yetcites [8] Voineagu, I. et al. Transcriptomic analysis of autistic brain reveals convergent molecular pathology. Nature 474, 380–384 (2011) source

In the paper

“Notably, the differential splicing molecular signature is not driven by DGE (Extended Data Fig. 4h), consistent with the observation that splicing alterations are related to common disease risk independently of gene expression changes16.”

In the paper

“Remarkably, the upregulated module CTX.M20 was not found in previous analyses, overlaps significantly with the ACP set (FDR < 0.05, Extended Data Fig. 9a), and contains genes implicated in development and regulation of cell differentiation (Fig. 4f).”

In the paper

“By contrast, enrichment for ASD-associated common variation was observed in CTX. M20 (FDR < 0.1, Extended Data Fig. 9h–1, Methods).”

In the paper

“Remarkably, genome-wide (trans) DGE and differential splicing patterns were highly concordant between dup15q and ASD (Fig. 3b, c, Extended Data Fig. 7c–e).”

In the paper

“Remarkably, the upregulated module CTX.M20 was not found in previous analyses, overlaps significantly with the ACP set (FDR < 0.05, Extended Data Fig. 9a), and contains genes implicated in development and regulation of cell differentiation (Fig. 4f).”

In the paper

“One module, CTX.M24, exhibited significant enrichment for rare mutations found in ASD, while rare de novo mutations associated with intellectual disability were most strongly enriched in CTX.M22 (FDR < 0.05, Extended Data Fig. 9a).”

In the paper

“Examples of specific lncRNA transcripts that show primate-specific (in human and macaque, or only in human, but not in mouse) expression.”

In the paper

“demonstrating three major clusters”

In the paper

“Enrichment statistics for transcription factor motifs found to be significantly enriched in the ACP set”

In the paper

“Lines above the plot indicate pairwise significance with a one-sided Wilcoxon rank-sum test between brain and the other tissues.”

What the sources say

• Not checked yetcites [12] Jaffe, A. E. et al. Developmental regulation of human cortex transcription and its clinical relevance at single base resolution. Nat. Neurosci. 18, 154–161 (2015) source

In the paper

“Comparison of fold changes between frontal cortex (FC) and temporal cortex (TC) for all samples, demonstrating similar changes in both regions.”

In the paper

“Differential splicing analysis identifies events independent of DGE signal.”

In the paper

“Temporal trajectories for four module eigengenes (CTX.M4, CTX.M9, CTX.M10, CTX.M16) associated with ASD”

In the paper

“Similar to l, except for embryonic stem cells and stem-cell-derived cell types.”

In the paper

“Samples from individuals with diagnoses confirmed by dup15q mutations, confirmed by Autism Diagnostic Interview-Revised (ADI-R), and supported by clinical records are all significantly different from controls by two-sided pairwise Wilcoxon rank sum tests.”

In the paper

“Notably, these genes are not in the DGE set, but are detected in the differential alternative splicing set owing to altered transcript structure.”

In the paper

“Grey bars with labels signify three ASD-associated modules.”

In the paper

“Independent replication of ASD versus control DGE fold changes between previously evaluated and new ASD samples in cortex by RNA-seq using samples from ref. 8”

What the sources say

• Not checked yetcites [8] Voineagu, I. et al. Transcriptomic analysis of autistic brain reveals convergent molecular pathology. Nature 474, 380–384 (2011) source

In the paper

“Enrichment of ASD-associated modules with that from ref. 4.”

What the sources say

• Not checked yetcites [4] Gupta, S. et al. Transcriptome analysis reveals dysregulation of innate immune response genes and neuronal activity-dependent genes in autism. Nat. Commun. 5, 5748 (2014) source

In the paper

“The first principal component of the cortex DGE set is primarily associated with diagnosis, and not with other factors.”

In the paper

“Independent replication analysis of ASD versus control DGE fold changes between previously evaluated and new ASD samples from cerebellum by microarray and RNA-seq using samples from ref. 8”

What the sources say

• Not checked yetcites [8] Voineagu, I. et al. Transcriptomic analysis of autistic brain reveals convergent molecular pathology. Nature 474, 380–384 (2011) source

In the paper

“Autism spectrum disorder (ASD) involves substantial genetic contributions.”

What the sources say

• Not checked yetcites [1] Gaugler, T. et al. Most genetic risk for autism resides with common variation. Nat. Genet. 46, 881–885 (2014) source
• Not checked yetcites [2] Gratten, J., Visscher, P. M., Mowry, B. J. & Wray, N. R. Interpreting the role of de novo protein-coding mutations in neuropsychiatric disease. Nat. Genet. 45, 234–238 (2013) source
• Not checked yetcites [3] de la Torre-Ubieta, L., Won, H., Stein, J. L. & Geschwind, D. H. Advancing the understanding of autism disease mechanisms through genetics. Nat. Med. 22, 345–361 (2016) source

In the paper

“Our data suggest that SOX5, a transcription factor involved in neuron fate specification, contributes to this reduction in regional differences.”

In the paper

“These data show that dysregulation of lncRNAs, many of which are brain-enriched, primate-specific, and predicted to affect protein expression through miRNA or FMRP interactions, is an integral component of the transcriptomic signature of ASD.”

In the paper

“The lack of significant DGE in the cerebellum is explained by the fact that changes in expression were consistently stronger in the cortex than in the cerebellum (Extended Data Fig. 2h), which suggests that the cortex is more selectively vulnerable to these transcriptomic alterations.”

In the paper

“Previous experimental perturbation of three splicing factors, Rbfox1 (ref. 18), SRRM4 (ref. 19), and PTBP1 (ref. 20), shows strong overlap with the differential splicing changes found in ASD cortex, further supporting these predicted relationships (Fig. 1h, Extended Data Fig. 5b).”

What the sources say

• Not checked yetcites [18] Lovci, M. T. et al. Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges. Nat. Struct. Mol. Biol. 20, 1434–1442 (2013) source
• Not checked yetcites [19] Raj, B. et al. A global regulatory mechanism for activating an exon network required for neurogenesis. Mol. Cell 56, 90–103 (2014) source
• Not checked yetcites [20] Gueroussov, S. et al. An alternative splicing event amplifies evolutionary differences between vertebrates. Science 349, 868–873 (2015) source

In the paper

“When we compared the first principal component across samples for protein coding DGE, lncRNA DGE and differential splicing, we found remarkably high correlations (R2 > 0.8), indicating that molecular convergence is likely to be a unitary phenomenon across multiple levels of transcriptome regulation in ASD (Fig. 1i).”

In the paper

“We identified a remarkable difference in CTX.M19 and CTX.M20 during the first two decades of life (Fig. 4g, additional age trajectories in Extended Data Fig. 9f) that is most consistent with an evolving process during early brain development that stabilizes starting in late childhood and early adolescence.”

In the paper

“Our findings illustrate how diverse genetic perturbations can lead to phenotypic convergence at multiple biological levels in a complex neuropsychiatric disorder.”

In the paper

“As CTX.M20 is enriched for the ACP gene set, this suggests a potential link between polygenic risk and regional attenuation of gene expression in ASD.”

In the paper

“Parsimony suggests that the highly overlapping expression pattern shared by individuals with dup15q and the majority of those with idiopathic ASD represents an evolving adaptive or maladaptive response to a primary insult rather than a secondary environmental hit.”

In the paper

“These findings support the prediction that attenuated patterning of the transcription factor SOX5 between cortical regions contributes to direct alterations in patterning of SOX5 targets.”

In the paper

“We performed a classification analysis to confirm that gene expression in ASD could separate samples by disease status (Extended Data Fig. 2a) and confirmed the technical quality of our data with qRT–PCR (Extended Data Fig. 2b, c).”

In the paper

“Co-expression network analysis reveals that individuals with ASD show age-related changes in the trajectory of microglial and synaptic function over the first two decades, and suggests that genetic risk for ASD may influence changes in regional cortical gene expression.”

In the paper

“The absence of neuronal splicing factor DGE or correlation with splicing changes in the cerebellum is consistent with the absence of a differential splicing signal in the cerebellum and suggests that these splicing factors contribute to cortex-biased differential splicing.”

In the paper

“Comparison of DGE fold changes demonstrating that using different control samples (control samples used in the idiopathic analysis, column 2 of Extended Data Fig. 7b) for the dup15q cortex analysis yields similar findings.”

In the paper

“This corresponds to the period of synapse elimination and stabilization after birth in humans28,29, which may have significant implications for intervention.”

What the sources say

• Not checked yetcites [28] Huttenlocher, P. R. Morphometric study of human cerebral cortex development. Neuropsychologia 28, 517–527 (1990) source
• Not checked yetcites [29] Khundrakpam, B. S., Lewis, J. D., Zhao, L., Chouinard-Decorte, F. & Evans, A. C. Brain connectivity in normally developing children and adolescents. Neuroimage 134, 192–203 (2016) source

In the paper

“Our analyses also reveal primate-specific lncRNAs that are probably relevant to understanding human higher cognition11,30.”

What the sources say

• Not checked yetcites [11] Necsulea, A. et al. The evolution of lncRNA repertoires and expression patterns in tetrapods. Nature 505, 635–640 (2014) source
• Not checked yetcites [30] Zhang, Y. E., Landback, P., Vibranovski, M. D. & Long, M. Accelerated recruitment of new brain development genes into the human genome. PLoS Biol. 9, e1001179 (2011) source

In the paper

“We interpret these data to suggest that aberrant microglia–neuron interactions reflect an early alteration in developmental trajectory that becomes more evident in late childhood.”

In the paper

“Co-expression network analysis demonstrated that the moderate agreement is largely driven by concordance in upregulation of microglial genes in both studies”

What the sources say

• Not checked yetcites [4] Gupta, S. et al. Transcriptome analysis reveals dysregulation of innate immune response genes and neuronal activity-dependent genes in autism. Nat. Commun. 5, 5748 (2014) source

In the paper

“demonstrating that confounders do not drive clustering of about two-thirds of samples.”

In the paper

“Although we observe no significant association of the ASD-associated transcriptome signature with either clinical or technical confounders, some of the changes are likely to represent consequences or compensatory responses, rather than causal factors.”