Investigation of epigenetic regulatory networks associated with autism spectrum disorder (ASD) by integrated global LINE-1 methylation and gene expression profiling analyses.

BACKGROUND:The exact cause and mechanisms underlying the pathobiology of autism spectrum disorder (ASD) remain unclear. Dysregulation of long interspersed element-1 (LINE-1) has been reported in the brains of ASD-like mutant mice and ASD brain tissues. However, the role and methylation of LINE-1 in individuals with ASD remain unclear. In this study, we aimed to investigate whether LINE-1 insertion is associated with differentially expressed genes (DEGs) and to assess LINE-1 methylation in ASD. METHODS:To identify DEGs associated with LINE-1 in ASD, we reanalyzed previously published transcriptome profiles and overlapped them with the list of LINE-1-containing genes from the TranspoGene database. An Ingenuity Pathway Analysis (IPA) of DEGs associated with LINE-1 insertion was conducted. DNA methylation of LINE-1 was assessed via combined bisulfite restriction analysis (COBRA) of lymphoblastoid cell lines from ASD individuals and unaffected individuals, and the methylation levels were correlated with the expression levels of LINE-1 and two LINE-1-inserted DEGs, C1orf27 and ARMC8. RESULTS:We found that LINE-1 insertion was significantly associated with DEGs in ASD. The IPA showed that LINE-1-inserted DEGs were associated with ASD-related mechanisms, including sex hormone receptor signaling and axon guidance signaling. Moreover, we observed that the LINE-1 methylation level was significantly reduced in lymphoblastoid cell lines from ASD individuals with severe language impairment and was inversely correlated with the transcript level. The methylation level of LINE-1 was also correlated with the expression of the LINE-1-inserted DEG C1orf27 but not ARMC8. CONCLUSIONS:In ASD individuals with severe language impairment, LINE-1 methylation was reduced and correlated with the expression levels of LINE-1 and the LINE-1-inserted DEG C1orf27. Our findings highlight the association of LINE-1 with DEGs in ASD blood samples and warrant further investigation. The molecular mechanisms of LINE-1 and the effects of its methylation in ASD pathobiology deserve further study

Integrated genome-wide Alu methylation and transcriptome profiling analyses reveal novel epigenetic regulatory networks associated with autism spectrum disorder

Abstract Background Alu elements are a group of repetitive elements that can influence gene expression through CpG residues and transcription factor binding. Altered gene expression and methylation profiles have been reported in various tissues and cell lines from individuals with autism spectrum disorder (ASD). However, the role of Alu elements in ASD remains unclear. We thus investigated whether Alu elements are associated with altered gene expression profiles in ASD. Methods We obtained five blood-based gene expression profiles from the Gene Expression Omnibus database and human Alu-inserted gene lists from the TranspoGene database. Differentially expressed genes (DEGs) in ASD were identified from each study and overlapped with the human Alu-inserted genes. The biological functions and networks of Alu-inserted DEGs were then predicted by Ingenuity Pathway Analysis (IPA). A combined bisulfite restriction analysis of lymphoblastoid cell lines (LCLs) derived from 36 ASD and 20 sex- and age-matched unaffected individuals was performed to assess the global DNA methylation levels within Alu elements, and the Alu expression levels were determined by quantitative RT-PCR. Results In ASD blood or blood-derived cells, 320 Alu-inserted genes were reproducibly differentially expressed. Biological function and pathway analysis showed that these genes were significantly associated with neurodevelopmental disorders and neurological functions involved in ASD etiology. Interestingly, estrogen receptor and androgen signaling pathways implicated in the sex bias of ASD, as well as IL-6 signaling and neuroinflammation signaling pathways, were also highlighted. Alu methylation was not significantly different between the ASD and sex- and age-matched control groups. However, significantly altered Alu methylation patterns were observed in ASD cases sub-grouped based on Autism Diagnostic Interview-Revised scores compared with matched controls. Quantitative RT-PCR analysis of Alu expression also showed significant differences between ASD subgroups. Interestingly, Alu expression was correlated with methylation status in one phenotypic ASD subgroup. Conclusion Alu methylation and expression were altered in LCLs from ASD subgroups. Our findings highlight the association of Alu elements with gene dysregulation in ASD blood samples and warrant further investigation. Moreover, the classification of ASD individuals into subgroups based on phenotypes may be beneficial and could provide insights into the still unknown etiology and the underlying mechanisms of ASD

Additional file 6: of Integrated genome-wide Alu methylation and transcriptome profiling analyses reveal novel epigenetic regulatory networks associated with autism spectrum disorder

Correlation analysis between AluS methylation and expression level of ASD subgroup S. (PDF 333 kb

Additional file 3: of Integrated genome-wide Alu methylation and transcriptome profiling analyses reveal novel epigenetic regulatory networks associated with autism spectrum disorder

List of the overlapping genes. (DOC 118 kb

Correlation analysis of LINE-1 methylation and expression in ASD individuals with severe language deficits and sex-/age-matched controls.

<p>The X-axis represents the percentage of LINE-1 methylation. The Y-axis represents the log₂ fold change of LINE-1 expression against <i>GAPDH</i>. R² and P-values were calculated via Spearman’s correlation method.</p

Developmental and neurological diseases significantly associated with dysregulated genes with LINE-1 insertion predicted via IPA.

<p>Developmental and neurological diseases significantly associated with dysregulated genes with LINE-1 insertion predicted via IPA.</p

Additional file 1: of Integrated genome-wide Alu methylation and transcriptome profiling analyses reveal novel epigenetic regulatory networks associated with autism spectrum disorder

Demographic information of LCLs used in this study. (DOC 73 kb

Additional file 2: of Integrated genome-wide Alu methylation and transcriptome profiling analyses reveal novel epigenetic regulatory networks associated with autism spectrum disorder

Association analysis between the DEGs in ASD from GSE15402 and the human Alu-inserted genes lists when ASD individuals were sub-grouped based on ADI-R scores. The comparisons were performed for five types of Alu insertions, including exonized, exonic, intronic, promoter and all types. The Fisher’s exact test P value and number of DEGs are shown. (DOC 46 kb

Association analyses between the differentially expressed genes in ASD and the human LINE-1-inserted gene lists.

<p>Association analyses between the differentially expressed genes in ASD and the human LINE-1-inserted gene lists.</p

Canonical pathways significantly associated with dysregulated genes with LINE-1 insertion predicted by the Ingenuity Pathway Analysis (IPA).

<p>Canonical pathways significantly associated with dysregulated genes with LINE-1 insertion predicted by the Ingenuity Pathway Analysis (IPA).</p