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Cited by 531Autism Spectrum Disorder ResearchGenetics and Neurodevelopmental DisordersNeuroinflammation and Neurodegeneration Mechanisms

Transcriptome analysis reveals dysregulation of innate immune response genes and neuronal activity-dependent genes in autism

Open access
Gupta, Simone, Ellis, Shannon E., Ashar, Foram N., Moes, Anna, Bader, Joel S., Zhan, Jianan, West, Andrew B., Arking, Dan E.·2014·Nature Communications
DOI 10.1038/ncomms6748PMC PMC4270294
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Abstract

Recent studies of genomic variation associated with autism have suggested the existence of extreme heterogeneity. Large-scale transcriptomics should complement these results to identify core molecular pathways underlying autism. Here we report results from a large-scale RNA sequencing effort, utilizing region-matched autism and control brains to identify neuronal and microglial genes robustly dysregulated in autism cortical brain. Remarkably, we note that a gene expression module corresponding to M2-activation states in microglia is negatively correlated with a differentially expressed neuronal module, implicating dysregulated microglial responses in concert with altered neuronal activity-dependent genes in autism brains. These observations provide pathways and candidate genes that highlight the interplay between innate immunity and neuronal activity in the aetiology of autism.

AI snapshot

Written from the paper’s full text.

TL;DR

Large-scale RNA sequencing of autism and control cortical brain tissue found two transcriptome-wide significant differentially expressed genes and a co-expression module enriched for M2-microglial and type I interferon immune genes. The M2 microglial module was strongly negatively correlated with a differentially expressed neuronal module, suggesting interplay between innate immunity and neuronal activity-dependent genes in autism.

Key findings

  • In 104 cortical samples (57 controls, 47 autism), 13,262 genes were tested and two transcriptome-wide significant differentially expressed genes were identified: MAL (P=2.16 × 10−7) and C11orf30/EMSY (P=3.29 × 10−7).
  • WGCNA identified 12 co-expressed modules; mod5 was differentially co-expressed (P=9.64 × 10−4), comprised 759 genes, and was enriched for M2-microglial cell states (P=1.22 × 10−39) and “Type I Interferon pathway” (P=1.19 × 10−20).
  • The M2 microglial module mod5 was strongly negatively correlated with the neuronal module mod1 (r=−0.92); mod1 was downregulated in autism and mod6 was upregulated in autism, both nominally differentially co-expressed (P<0.005).
  • Genes with autism genetic association were not enriched for altered expression overall; mod2 was enriched for common (P=2.49 × 10−6) and rare autism variants (P=4.29 × 10−4) but was comparably expressed between cases and controls.

Methods

  • Post-mortem cerebral cortex samples from Brodmann areas BA19, BA10, and BA44 were sequenced: 62, 14, and 28 samples respectively, totaling 57 controls (40 unique individuals) and 47 autism cases (32 unique individuals).
  • RNA-Seq libraries were prepared from total RNA, sequenced on Illumina HiSeq 2000, mapped with Tophat2, and gene counts normalized with EDASeq/CQN; differential expression used linear mixed regression with collection site, age, sex, brain region, and independent surrogate variables as fixed effects.
  • Co-expression analysis used WGCNA on an artefact-corrected data set of 13,443 genes; module associations were tested with linear mixed regression, and enrichment was assessed with hypergeometric tests, GO/MSigDB gene sets, permutation thresholds, and Bonferroni correction.

Limitations

  • Stated: the genetic gene lists used are “not comprehensive and only reflect the current understanding of the genetic basis of autism”; the youngest individual was 2 years of age, so mutations important in fetal development may not be captured in the current study.
  • Stated: no gene-set enrichment with FWER ≤0.05 was observed for the top single-gene differentially expressed genes, and exon-level analysis of 21,310 exons found no differences after multiple-testing correction.
  • Evident from the text: the study is cross-sectional post-mortem transcriptomics, so it cannot establish causality or directionality between immune and neuronal changes; some cortical regions had relatively small sample numbers (BA10 n=14).

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/ncomms6748

Cites

58 of 58 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 41 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Autism brains are specifically enriched for 'immune response' genes.

    “Using co-expression network analysis, we demonstrate that autism brains are specifically enriched for ‘activated’ M2 microglial and ‘immune response’ genes.”
    Quote in the paperSubstantial claim
  • Findingown data

    The M2 microglial module is strongly negatively correlated with one of two differentially expressed neuronal modules.

    “Remarkably, we note that a gene expression module corresponding to M2-activation states in microglia is negatively correlated with a differentially expressed neuronal module”
    • Also stated in: § Results · boosted
      “there is a strong negative correlation between two differentially co-expressed modules, mod5 (activated M2-state microglia genes) and mod1 (synaptic transmission genes; r=−0.92”
    Quote in the paperSubstantial claim
  • Findingown data

    There are clear differences in the transcriptome between control and ASD cortical brains.

    “We find clear differences in the transcriptome between control and ASD cortical brains.”
    Quote in the paperSubstantial claim
  • Findingown data

    The most significant differentially expressed gene was MAL (P=2.16 × 10−7).

    “The most significant differentially expressed gene was Myelin And Lymphocyte Protein (MAL; P=2.16 × 10−7”
    Quote in the paperNot a substantial claim
  • Findingown data

    Two transcriptome-wide significant differentially expressed genes associated with autism were identified.

    “two transcriptome-wide significant differentially expressed genes associated with autism were identified”
    Quote in the paperSubstantial claim
  • May combine two claims. 13
  • Classified as probably not a substantial claim. 58

  • May combine two claims.

    Findingown data

    The genetic signal is stronger in the non-differentially expressed module (mod2), with a twofold enrichment for FMRP targets compared with the differentially expressed mod6, whereas no enrichment was observed for mod1.

    “we again find evidence that the genetic signal is stronger in the non-differentially expressed module (mod2), with a twofold enrichment for FMRP targets compared with the differentially expressed mod6, whereas no enrichment was observed for mod1.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Mod5 comprised 759 genes.

    “Mod5 comprised 759 genes”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    5,075 genes were assigned into the predefined mod0, which is reserved for non-module genes.

    “However, 5,075 genes were assigned into the predefined mod0, which is reserved for non-module genes.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Findingown data

    The co-expression analysis on 13,443 genes from the AC data identified 12 modules.

    “The co-expression analysis on 13,443 genes from the AC data identified 12 modules, with each module being represented by its first principal component or eigengene (for example, ME1) for each sample.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The number of genes differentially expressed at P<0.01 was 749.

    “The number of genes differentially expressed at P<0.05, P<0.01, P<0.001 and P<0.0001 were 1,964, 749, 185 and 50, respectively.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The number of genes differentially expressed at P<0.001 was 185.

    “The number of genes differentially expressed at P<0.05, P<0.01, P<0.001 and P<0.0001 were 1,964, 749, 185 and 50, respectively.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The number of genes differentially expressed at P<0.005 was 494.

    “Four lists of differentially expressed genes at P values <0.001,<0.005,<0.01 and<0.05, with 185, 494, 749 and 1,964 genes, respectively, were generated.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The number of genes differentially expressed at P<0.0001 was 50.

    “The number of genes differentially expressed at P<0.05, P<0.01, P<0.001 and P<0.0001 were 1,964, 749, 185 and 50, respectively.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The number of genes differentially expressed at P<0.05 was 1,964.

    “The number of genes differentially expressed at P<0.05, P<0.01, P<0.001 and P<0.0001 were 1,964, 749, 185 and 50, respectively.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Most of the 13,443 genes were clustered into mutually exclusive co-expressing modules.

    “Most of the 13,443 genes were clustered into mutually exclusive co-expressing modules.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Voineagu et al. identified a set of co-expressed neuronal genes enriched for known autism susceptibility genes.

    “Indeed, Voineagu et al. recently utilized co-expressed gene networks from RNA-Sequencing (RNA-Seq) carried out in 19 autism brains and 17 controls to identify a set of co-expressed neuronal genes enriched for known autism susceptibility genes as well as a set of co-expressed genes enriched for both immune genes and glial markers10.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Voineagu, I. et al. Transcriptomic analysis of autistic brain reveals convergent molecular pathology. Nature 474, 380–384 (2011). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Voineagu et al. identified a set of co-expressed genes enriched for both immune genes and glial markers.

    “Indeed, Voineagu et al. recently utilized co-expressed gene networks from RNA-Sequencing (RNA-Seq) carried out in 19 autism brains and 17 controls to identify a set of co-expressed neuronal genes enriched for known autism susceptibility genes as well as a set of co-expressed genes enriched for both immune genes and glial markers10.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Voineagu, I. et al. Transcriptomic analysis of autistic brain reveals convergent molecular pathology. Nature 474, 380–384 (2011). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    A major mode of action for common genetic variation is through altered gene expression.

    “For common genetic variation, altered gene expression is a major mode of action2021.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [2021]

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    Premisecitation

    Autism is characterized by extreme phenotypic heterogeneity and involvement of hundreds of loci.

    “This slow progress can be attributed, in part, to both the extreme phenotypic heterogeneity and involvement of hundreds of loci in the disorder45.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [45] Hansen, K. D., Irizarry, R. A. & Wu, Z. Removing technical variability in RNA-seq data using conditional quantile normalization. Biostatistics 13, 204–216 (2012). source

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    Premisecitation

    Previous genome-wide association studies have linked C11orf30 to inflammatory and malignant diseases.

    “previous genome-wide association studies have linked C11orf30 to inflammatory and malignant diseases1819.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1819]

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    Premisecitation

    Voineagu et al. previously reported a co-expression module dysregulated in autism brains, termed asdM16, enriched in astrocytes and microglia-expressed genes.

    “Voineagu et al. previously reported a co-expression module dysregulated in autism brains, termed asdM16, enriched in astrocytes and microglia-expressed genes10.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [10] Voineagu, I. et al. Transcriptomic analysis of autistic brain reveals convergent molecular pathology. Nature 474, 380–384 (2011). source

      Not checked in the free run.

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    Premisecitation

    Key regulatory genes may not harbour common inherited variation because of natural selection.

    “suggesting that key regulatory genes may not harbour common inherited variation because of natural selection34.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] Mäkinen, V.-P. et al. Integrative genomics reveals novel molecular pathways and gene networks for coronary artery disease. PLoS Genet. 10, e1004502 (2014). source

      Not checked in the free run.

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    Premisecitation

    M2 microglial cells promote myelination.

    “M2 microglial cells also secrete brain-derived neurotrophic factor (BDNF), increase the production of neural progenitor cells and promote myelination272829.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites 272829

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    Premisecitation

    A recent study of gene networks in coronary artery disease has shown that genes at the centre of the networks, referred to as 'key drivers', were largely not genome-wide association study signal genes.

    “a recent study of gene networks in coronary artery disease has shown that genes at the centre of the networks, referred to as ‘key drivers’, were largely not genome-wide association study signal genes”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [34] Mäkinen, V.-P. et al. Integrative genomics reveals novel molecular pathways and gene networks for coronary artery disease. PLoS Genet. 10, e1004502 (2014). source

      Not checked in the free run.

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    Premisecitation

    M2 microglial cells increase the production of neural progenitor cells.

    “M2 microglial cells also secrete brain-derived neurotrophic factor (BDNF), increase the production of neural progenitor cells and promote myelination272829.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites 272829

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Type I Interferon responses in the brain are classically attributed to viral infections that can produce M1 activation states in microglia.

    “Type I Interferon responses in the brain are classically attributed to viral infections that can produce M1 activation states in microglia26.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [26] Delhaye, S. et al. Neurons produce type I interferon during viral encephalitis. Proc. Natl Acad. Sci. USA 103, 7835–7840 (2006). source

      Not checked in the free run.

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    Premisecitation

    Iossifov and colleagues reported an enrichment of ASD RDNVs in FMRP targets.

    “Similarly, Iossifov and colleagues reported an enrichment of ASD RDNVs in FMRP targets5.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [5] Iossifov, I. et al. De novo gene disruptions in children on the autistic spectrum. Neuron 74, 285–299 (2012). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    In this study, we provide transcriptomic evidence for type I interferon and M2-activation state abnormalities in autism.

    “In this study, we provide transcriptomic evidence for type I interferon and M2-activation state abnormalities in autism”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    This process may drive changes in neural progenitor cell proliferation and connectivity with resultant altered activity-dependent neural expression profiles in post-natal development.

    “This process may drive changes in neural progenitor cell proliferation and connectivity with resultant altered activity-dependent neural expression profiles in post-natal development3839.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [3839]

      Not checked in the free run.

    • Rests on an assumption: M2 responses are responsible for mediating anti-inflammatory remediation responses to damage caused by viral infections.

  • Classified as probably not a substantial claim.

    Premisecitation

    Direct evidence for the role of Fragile X mental retardation protein (FMRP) in autism has been provided by Darnell et al.

    “Direct evidence for the role of Fragile X mental retardation protein (FMRP) in autism has been provided by Darnell et al.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [35] Darnell, J. C. et al. FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell 146, 247–261 (2011). source

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    Premisecitation

    Microglia have been identified as cells capable of restoring neural function in the ASD-model MECP2 knockout mice.

    “Recently, microglia have been identified as cells capable of restoring neural function in the ASD-model MECP2 knockout mice37.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [37] Derecki, N. C. et al. Wild-type microglia arrest pathology in a mouse model of Rett syndrome. Nature 484, 105–109 (2012). source

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    Premisecitation

    Many of the protein-interacting partners of FMRP harbour ASD-associated common variants.

    “These authors reported that many of the protein-interacting partners of FMRP harbour ASD-associated common variants35.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [35] Darnell, J. C. et al. FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell 146, 247–261 (2011). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Brain-critical exons have high gene expression in brain and low rare burden of rare mutations, and have been proposed to represent autism candidate genes.

    “which have high gene expression in brain and low rare burden of rare mutations, and have been proposed to represent autism candidate genes25”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [25] Uddin, M. et al. Brain-expressed exons under purifying selection are enriched for de novo mutations in autism spectrum disorder. Nataure Genet. 46, 742–747 (2014). source

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    Premisecitation

    Steinberg et al. organized the FMRP target genes into distinct temporally expressed subpopulations affected by different classes of genetic variation associated with ASD.

    “Recently, Steinberg et al. organized the FMRP target genes into distinct temporally expressed subpopulations affected by different classes of genetic variation associated with ASD36.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [36] Steinberg, J. & Webber, C. The roles of FMRP-regulated genes in autism spectrum disorder: single- and multiple-hit genetic etiologies. Am. J. Hum. Genet. 93, 825–839 (2013). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    MAL and other myelination genes have previously been reported to show altered expression in patients with psychiatric disorders.

    “MAL, along with other myelination genes, has previously been reported to show altered expression in patients with psychiatric disorders17.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [17] Esparza-Gordillo, J. et al. A common variant on chromosome 11q13 is associated with atopic dermatitis. Nat. Genet. 41, 596–601 (2009). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    M2 microglial cells secrete brain-derived neurotrophic factor (BDNF).

    “M2 microglial cells also secrete brain-derived neurotrophic factor (BDNF), increase the production of neural progenitor cells and promote myelination272829.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites 272829

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Previous studies have identified an enrichment of genetic association signals in genes selectively expressed in neurons.

    “Previous studies have identified an enrichment of genetic association signals in genes selectively expressed in neurons10253233.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites 10253233

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  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Quality control measures during library amplification included PCR from reactions with no template, from libraries made with no ligase, and from libraries with no adaptor oligonucleotides; in these cases, the library failed to amplify.

    “Quality control measures during library amplification included PCR from reactions with no template, from libraries made with no ligase (hence no adaptors) and finally from libraries with no adaptor oligonucleotides included in the ligase reaction. In these cases, the library failed to amplify”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The I-exomes list comprises 59 genes disrupted by de novo nonsense, frameshift or splice-site point mutations in autism probands from Iossifov et al.

    “(vi) Steinberg et al.36 compiled a list of genes disrupted by de novo nonsense, frameshift or splice-site point mutations in autism probands that were obtained from Iossifov et al.5 (59 genes; referred to as ‘I-exomes’)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [5] Iossifov, I. et al. De novo gene disruptions in children on the autistic spectrum. Neuron 74, 285–299 (2012). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The T-BCAs list comprises 32 genes disrupted by breakpoints of balanced chromosomal abnormalities observed in individuals with ASD, obtained from Talkowski et al.

    “(vii) A list of genes disrupted by breakpoints of balanced chromosomal abnormalities (BCAs) observed in individuals with ASD was obtained from Talkowski et al.56 (32 genes; referred to as ‘T-BCAs’).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [56] Talkowski, M. E. et al. Sequencing chromosomal abnormalities reveals neurodevelopmental loci that confer risk across diagnostic boundaries. Cell 149, 525–537 (2012). source

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    Premisecitation

    The ASD SFARI 2014 CV list comprises 197 genes, a subset of the ASD SFARI 2014 list after removing 896 genes with rare de novo variants from four whole-exome sequencing publications.

    “(iii) 197 genes (ASD SFARI 2014 CV) are a subset of the ASD SFARI 2014 after removing the 896 genes with rare de novo variant from the four whole-exome sequencing publications9101112”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites 9101112

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    Study design and conductown data

    ISVs were generated while protecting for case-control status.

    “ISVs were generated while protecting for case–control status”
    Quote in the paperSubstantial claim

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    Study design and conductown data

    The permutation threshold, P<2.0 × 10−3, was determined by permuting case-control labels (n=100 permutations) for the eigengene values and re-running the regression analysis.

    “The permutation threshold, P<2.0 × 10−3, was determined by permuting case–control labels (n=100 permutations) for the eigengene values and re-running the regression analysis.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Permutation testing was used to estimate the threshold for transcriptome-wide significant differential expression.

    “Permutation testing was used to estimate the threshold for transcriptome-wide significant differential expression”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The ASD [Pinto] list comprises 124 genes implicated in ASD and ID, updated from a list by Betancur in 2011.

    “(iv) Pinto et al.33 compiled a list of 124 genes (ASD [Pinto]) that have been implicated in ASD and was updated from a list provided by Betancur in 2011 (ref. 55). All of the 124 genes have also been implicated in ID.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [33] Pinto, D. et al. Convergence of genes and cellular pathways dysregulated in autism spectrum disorders. Am. J. Hum. Genet. 94, 677–694 (2014). source

      Not checked in the free run.

    • cites [55] Betancur, C. Etiological heterogeneity in autism spectrum disorders: more than 100 genetic and genomic disorders and still counting. Brain Res. 1380, 42–77 (2011). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    To account for false positives in gene list enrichment, the 0.05 FWER was calculated as described above.

    “To account for false positives, the 0.05 FWER was calculated as described above.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Steinberg et al. demonstrated that the 832 FMRP-interacting partners, particularly in two modules (FMRP 1 and FMRP 2), exhibit differential temporal expression: FMRP 1 genes tend to be upregulated during fetal development, whereas FMRP 2 genes are generally upregulated in adolescence and adulthood.

    “In additionally, Steinberg et al.36 demonstrated that the 832 FMRP-interacting partners, particularly in two modules (represented as FMRP 1 and FMRP 2), exhibit differential temporal expression: genes in FMRP 1 tend to be specifically upregulated during fetal development, whereas genes in FMRP 2 were generally upregulated in adolescence and adulthood.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [36] Steinberg, J. & Webber, C. The roles of FMRP-regulated genes in autism spectrum disorder: single- and multiple-hit genetic etiologies. Am. J. Hum. Genet. 93, 825–839 (2013). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductown data

    To account for false positives in GO enrichment, 12 random modules of the same size were generated 2,000 times, and the 0.05 FWER was calculated.

    “To account for false positives, 12 random modules of the same size were generated 2,000 times, the hypergeometic test was carried out, and the 0.05 FWER was calculated.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductcitation

    We assessed summarized values on a per-gene basis, removing gene estimates for samples whose gene expression values were more than three standard deviations from the mean expression of each gene, as these outliers are artefactual in origin.

    “We assessed summarized values on a per-gene basis, removing gene estimates for samples whose gene expression values were more than three standard deviations (s.d.) from the mean expression of each gene (per-gene outlier), as these outliers are artefactual in origin16.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [16] Ellis, S. E. et al. RNA-Seq optimization with eQTL gold standards. BMC Genomics 14, 892 (2013). source

      Not checked in the free run.

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    Study design and conductown data

    We assessed the possibility of confounding in the expression of the two differentially expressed genes by investigating the expression stratified by sample collection site.

    “We assessed the possibility of confounding in the expression of the two differentially expressed genes by investigating the expression stratified by sample collection site”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The module preservation statistic Zsummary was used to assess the overlap in network modules obtained from the autism case and control brain data sets.

    “We utilized the module preservation statistic Zsummary50, described in the ‘modulePreservation’ R function implemented in WGCNA, to assess the overlap in network modules obtained from the autism case and control brain data sets.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Six samples with low gene coverage were dropped from all downstream analyses, resulting in 104 samples.

    “Six samples with low gene coverage (>20% of the 48,260 genes had zero coverage) were dropped from all downstream analyses, resulting in 104 samples.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    The model included a random intercept term to account for the correlation of gene expression estimated from multiple brain regions obtained from the same individual.

    “the model included a random intercept term to account for the correlation of gene expression estimated from multiple brain regions obtained from the same individual.”
    Quote in the paperSubstantial claim

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    Study design and conductnone given

    The single-gene differential expression analysis used independent surrogate variables (ISVs) to correct for both technical and biological confounders in the expression data.

    “For the single-gene differential expression analysis, we used the ISVs to correct for both technical and biological confounders in the expression data.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The ASD SFARI 2014 list comprises 235 genes from the SFARI AutDB database.

    “(ii) 235 genes (ASD SFARI 2014) from the SFARI AutDB database22 (accessed on July 2014).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22] Abrahams, B. S. et al. SFARI Gene 2.0: a community-driven knowledgebase for the autism spectrum disorders (ASDs). Mol. Autism 4, 36 (2013). source

      Not checked in the free run.

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    Study design and conductown data

    There was no significant difference in cause of death between the two groups.

    “there was no significant difference in cause of death between the two groups.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Each sequencing run included samples randomized by sex, collection site and case-control status.

    “Each sequencing run included samples randomized by sex, collection site and case–control status.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The SON-exomes list comprises 65 genes disrupted by de novo nonsense, frameshift or splice-site point mutations in autism probands from three other studies (Sanders et al., O'Roak et al., Neale et al.).

    “and three other recent studies by Sanders et al.8, O’Roak et al.7 and Neale et al.23 (65 genes combined from all three; referred to as ‘SON-exomes’);”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [8] Sanders, S. J. et al. De novo mutations revealed by whole-exome sequencing are strongly associated with autism. Nature 485, 237–241 (2012). source

      Not checked in the free run.

    • cites [7] O’Roak, B. J. et al. Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations. Nature 485, 246–250 (2012). source

      Not checked in the free run.

    • cites [23] Neale, B. M. et al. Patterns and rates of exonic de novo mutations in autism spectrum disorders. Nature 485, 242–245 (2012). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    The multiple test correction threshold using the Bonferroni method was 4.0 × 10−3.

    “The multiple test correction threshold using the Bonferroni method was 4.0 × 10−3.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The average age at time of death of the control and autism individuals was similar.

    “The average age at time of death of the 40 control and 32 autism individuals was similar (cases median age=20 years, controls median age=17 years)”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductreasoning

    To account for false positives, 100 sets of balanced permutations were generated, and the 5th best P value was set as the threshold for a true discovery, giving a 0.05 FWER.

    “We then extract the best P value for each permutation, ranked them and set the 5th best P value as the threshold for a true discovery. This gives us a 0.05 FWER.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Voineagu et al. identified two co-expression gene modules dysregulated in post-mortem ASD brains: asdM12 (neuronal, enriched for ASD associated genes) and asdM16 (enriched with astrocyte and activated microglial markers, immune response, but no enrichment for ASD associated genes).

    “Voineagu et al. identified two co-expression gene modules that were dysregulated in post-mortem ASD brains, asdM12 (a neuronal module, enriched for ASD associated genes) and asdM16 (enriched with astrocyte, activated microglial markers, with functional annotation immune response, but no enrichment for ASD associated genes)10.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Voineagu, I. et al. Transcriptomic analysis of autistic brain reveals convergent molecular pathology. Nature 474, 380–384 (2011). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductown data

    A multivariate linear regression model was used to correct the gene expression estimates of sequencing artefacts, collection site, sex, age and brain region, yielding an artefact corrected (AC) data set.

    “We used a multivariate linear regression model to correct the gene expression estimates of sequencing artefacts (SAs), collection site (CS), sex (S), age (A) and brain region (BR), yielding an artefact corrected (AC) data set.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    FMRP and its interacting partners are implicated in translational regulation of synaptic proteins and are enriched with diverse classes of ASD variants.

    “FMRP and its interacting partners (FMRP interacting) implicated with translational regulation of synaptic proteins35 and shown to be enriched with diverse class of ASD variants57.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [35] Darnell, J. C. et al. FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell 146, 247–261 (2011). source

      Not checked in the free run.

    • cites [57] Nagata, T. et al. Profiling of genes associated with transcriptional responses in mouse hippocampus after transient forebrain ischemia using high-density oligonucleotide DNA array. Brain Res. Mol. Brain Res. 121, 1–11 (2004). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The robustness of correlations between modules was assessed using bootstrap with replacement analysis.

    “The Pearson’s correlation between modules is shown in Supplementary Data 14, and the robustness of correlations was assessed using bootstrap with replacement analysis.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Uddin et al. identified 3,955 exons mapping to 1,744 genes with high expression in the brain and a low burden of rare mutations, designated as 'brain-critical exons'.

    “Finally, Uddin et al. identified 3,955 exons mapping to 1,744 genes with high expression in the brain and a low burden of rare mutations, and designate these as ‘brain-critical exons’25.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [25] Uddin, M. et al. Brain-expressed exons under purifying selection are enriched for de novo mutations in autism spectrum disorder. Nataure Genet. 46, 742–747 (2014). source

      Not checked in the free run.

61 of 61

  • The paper relies on this without support.

    M2 responses are responsible for mediating anti-inflammatory remediation responses to damage caused by viral infections.

    “In opposition to M1-activated microglia, M2 responses are responsible for mediating anti-inflammatory remediation responses to damage caused by viral infections.”

  • The paper relies on this without support.

    M2 activation state responses have not previously been attributed to the pathogenesis of autism.

    “To our knowledge, M2 activation state responses have not previously been attributed to the pathogenesis of autism.”
2 of 2

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • The finding of immune gene upregulation and synaptic gene downregulation in idiopathic ASD was replicated in a larger ASD sample.

    “Upregulation of immune genes and downregulation of synaptic genes was observed in postmortem brain from idiopathic ASD (Voineagu et al., 2011), and this finding has been replicated in a larger ASD sample (Gupta et al., 2014).”

    From Getting to the cores of autism (2019) · cites this paper as Gupta et al., 2014

  • Compared with a prior RNA-seq study of occipital cortex, there is a weak but significant correlation in fold changes, mostly due to upregulated genes (P = 0.038).

    “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).”

    From Genome-wide changes in lncRNA, splicing, and regional gene expression patterns in autism (2016) · cites this paper as [4]

  • Upregulated modules are enriched in genes associated with inflammatory pathways and glial function.

    “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).”

    From Genome-wide changes in lncRNA, splicing, and regional gene expression patterns in autism (2016) · cites this paper as [4]

  • Samples from ref. 4 have lower RNA integrity, lower sequencing depth, greater 5′−3′ bias, and greater variability across quality-control metrics than samples in this study.

    “Compared to this study, samples from ref. 4 were prepared by poly(A) selection RNA-seq, exhibit lower RNA integrity number (RIN, median 4.8 versus 7.3), have lower median sequencing depth (11 million versus 40 million), exhibit greater 5′−3′ bias, and have generally greater variability across all QC metrics.”

    From Genome-wide changes in lncRNA, splicing, and regional gene expression patterns in autism (2016) · cites this paper as [4]

  • ASD-associated modules are enriched with modules from ref. 4.

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

    From Genome-wide changes in lncRNA, splicing, and regional gene expression patterns in autism (2016) · cites this paper as [4]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Module-preservation thresholds overlap

    Minor

    As written, any Zsummary value below 2 satisfies both thresholds, assigning it both no evidence and weak-to-moderate evidence for module preservation.

1 of 1

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Related

Authors
Gupta, S., Ellis, S. E., Ashar, F. N., Moes, A., Bader, J. S., Zhan, J., West, A. B., & Arking, D. E.
Affiliations
  • Johns Hopkins University (US)
  • University of Alabama at Birmingham (US)
Corresponding author
  • West, Andrew B.
  • Arking, Dan E.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2014-12-10
Volume
5
Issue
1
Pages
5748-5748
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/ncomms6748
PMC
PMC4270294
OpenAlex
W2076040334
Cited by
504
References
59
Citations by year
2026: 23 · 2025: 23 · 2024: 39 · 2023: 32 · 2022: 55 · 2021: 46 · 2020: 56 · 2019: 52 · 2018: 42 · 2017: 50 · 2016: 59 · 2015: 27
FWCI
25.29
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
Funders
  • Simons Foundation (137603)
  • Simons Foundation Autism Research Initiative
  • National Institute of Mental Health (R24MH068855, R01MH100027, MH 068855, R01MH081754)
  • National Institute of General Medical Sciences (T32GM007814)
Topics
  • Life Sciences › Neuroscience › Cognitive Neuroscience › Autism Spectrum Disorder Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetics and Neurodevelopmental Disorders
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
Keywords
autism spectrum disorder, transcriptome, microglia, innate immune response, gene modules, autism heterogeneity, candidate genes

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-06.

Statistics and arithmetic are recomputed, not judged.

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