Global Methylation Profiling of Lymphoblastoid Cell Lines Reveals Epigenetic Contributions to Autism Spectrum Disorders

Background: Epigenetic mechanisms have been suggested as contributing factors in higher-order regulation of aberrant gene expression in autism spectrum disorders (ASD). One such mechanism, DNA methylation, involves the addition of a methyl group onto the C5 position of CpG dinucleotides. An increase of methylated CpG sites, particularly in the promoter region, is associated with transcriptional repression by either disruption of transcriptional machinery binding or recruitment of repressive methyl-binding proteins.

Objectives: The current study utilizes two complementary genomic approaches to globally identify genes regulated by methylation in lymphoblastoid cell lines (LCL) derived from monozygotic twins (MZ) discordant in diagnosis of autism and nonautistic sibling controls. The use of discordant twins and unaffected siblings enabled identification of epigenetic differences between genetically identical twins, as well as differences significant across all autistic individuals compared to nonautistic controls.

Methods: Two distinct forms of DNA microarray analyses were conducted on LCL from 3 pairs of discordant monozygotic twins and nonautistic sibling controls. Firstly, a methylated CpG island recovery assay (MIRA) was used to enrich the methylated fraction of genomic DNA. Enriched fractions were hybridized onto CpG island microarrays to identify genes with differentially methylated CpG island regions. Secondly, gene expression microarrays were utilized to identify methylation-sensitive genes exhibiting differential response between discordant MZ twins to treatment with a global methylation inhibitor, 5-Aza-2-deoxycytidine. Datasets from these analyses were analyzed using Ingenuity Pathway Analysis and Pathway Studio 5 software. The methylation status of candidate genes identified from both analyses was confirmed using bisulfite sequencing or methylation-specific PCR.

Results: Methylation profiling revealed many genes differentially methylated between discordant MZ twins and between twins and unaffected siblings. Bioinformatics analysis of the differentially methylated genes demonstrated enrichment for high level functions including gene transcription, inflammation, nervous system development, and cell death/survival. The overlap of genes identified in both analyses provided a stringent method of prioritizing ASD candidate genes. One hypermethylated candidate gene, BCL-2, was chosen for further study because of its anti-apoptotic functions, role in proper neurodevelopment, and reported protein decrease in autistic brain. Another gene, retinoic acid receptor (RAR)-related orphan receptor, RORA, also identified as hypermethylated in autistic individuals was chosen for its roles in Purkinje neuron differentiation, regulation of circadian rhythm, and protection against oxidative stress. In autistic individuals, there was a 2.5-fold and 2-fold decrease in gene expression for BCL-2 and RORA, respectively. Reactivation of gene expression was observed following treatment with the methylation inhibitor, 5-Aza-2-deoxycytidine, as confirmed by qRT-PCR. An increase of methylated CpG residues within CpG island and promoter regions of BCL-2 and RORA was demonstrated by bisulfite sequencing and methylation-specific PCR in genomic DNA of LCL from autistic individuals.

Conclusions: Global methylation profiling of lymphoblastoid cell lines from autistic individuals highlight the role of epigenetic regulation in idiopathic autism and reveals potential cellular pathways through which this regulation may occur. In addition, a number of differentially methylated genes identified from these studies corroborate aberrant gene expression previously characterized in ASD.

This study was supported by Autism Speaks, Grant #2381.