Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain

Abstract

Autism is currently considered a multigene disorder with epigenetic influences. To investigate the contribution of DNA methylation to autism spectrum disorders, we have recently completed large-scale methylation profiling by CpG island microarray analysis of lymphoblastoid cell lines derived from monozygotic twins discordant for diagnosis of autism and their nonautistic siblings. Methylation profiling revealed many candidate genes differentially methylated between discordant MZ twins as well as between both twins and nonautistic siblings. Bioinformatics analysis of the differentially methylated genes demonstrated enrichment for high-level functions including gene transcription, nervous system development, cell death/survival, and other biological processes implicated in autism. The methylation status of 2 of these candidate genes, BCL-2 and retinoic acid-related orphan receptor alpha (RORA), was further confirmed by bisulfite sequencing and methylation-specific PCR, respectively. Immunohistochemical analyses of tissue arrays containing slices of the cerebellum and frontal cortex of autistic and age- and sex-matched control subjects revealed decreased expression of RORA and BCL-2 proteins in the autistic brain. Our data thus confirm the role of epigenetic regulation of gene expression via differential DNA methylation in idiopathic autism, and furthermore link molecular changes in a peripheral cell model with brain pathobiology in autism.

Figure 1

Overlap of dysregulated genes identified from CpG island microarray and global gene expression profiling previously performed using the same samples. A) Scatter plot shows inverse correlation of log2 ratios of gene expression and methylation of genes with differentially methylated CpG islands directly overlapping with its 5′ end. Inset: plot of log2 ratios of genes with differentially methylated CpG islands located either upstream or downstream of the CpG island. Interestingly, the majority of genes identified from these analyses were hypermethylated in the twins relative to their respective nonautistic siblings, with a corresponding decrease in gene expression. B) Network analysis of 25 genes (circumscribed in blue) that were both differentially methylated and expressed. The network was generated using Pathway Studio 5 network prediction software and identified common biological themes, including apoptosis, cellular differentiation, and inflammation. The analysis also revealed neurologically relevant functions and disorders including synaptic regulation, development, and mental deficiency.

Figure 2

A) Bisulfite sequencing of the BCL-2 P1 promoter region. B). Bisulfite sequencing was performed across 38 CpG sites from genomic DNA isolated from LCLs of discordant MZ twins. Autistic co-twins are designated by A_ preceding the blood identifier number (e.g., A_809); undiagnosed co-twins are designated by M_ preceding the blood number (e.g., M_810). Control (C) individuals include unaffected siblings (e.g., C_813), one normal monozygotic twin pair (C_2744 and C_2745). A pair of siblings, one autistic (A_2020) and one unaffected (C_2019), was also included in this analysis. Inset: average percentage methylation with sd across all 38 CpG sites for each group of samples.

Figure 3

A) Representative MSP image of the upstream CpG island region of RORA in LCL from autistic (A_737 and A_2020) and unaffected control (C_735 and C_2019) siblings. LCLs were treated with DMSO (vehicle control) or 5 uM of 5-Aza-2-deoxycytidine for 48 h. Genomic DNA was isolated and bisulfite modified. B) Modified DNA was used for PCR containing primers specific for unmethylated (U) and methylated (M) CpG sites of RORA.

Figure 4

qRT-PCR of BCL-2 and RORA transcripts from LCLs of 3 pairs of discordant twins and respective sibling controls for 2 pairs of twins. A) Percentage of gene expression of autistic co-twin (#_A). B) RNA isolated from LCLs following 48 h treatment with 5 μM 5-Aza-2-deoxycytidine was also analyzed by qRT-PCR. Results are average ± se fold change in expression following treatment.

Figure 5

Immunohistochemical staining for RORA protein in the cerebellum. Immunohistochemistry for RORA protein was performed using a standard avidin-biotin complex method of detection, as described in Materials and Methods. Representative images (×20) were taken from the autism tissue array (prepared by Dr. Charles Eberhart, Johns Hopkins University, Baltimore, MD, USA), which contained tissue cores from 5 autistic brains (A, C, E, G, I) and age-matched controls (B, D, F, H, J). Samples E, F, G, H were from female donors. It should be noted that not all autistic samples exhibit reduced RORA staining. This is expected because we observed that only the autistic phenotype associated with severe language impairment exhibited reduced RORA expression . Arrows indicate Purkinje cells, which are the largest cells in the sections.

Figure 6

Immunohistochemical staining for BCL-2 protein in the cerebellum. Immunohistochemistry for BCL-2 protein was performed as described in Materials and Methods for RORA protein with the substitution of a primary antibody for BCL-2 instead of for RORA. In addition, the tissue was counterstained with hematoxylin to reveal the nuclei. Tissue samples from 5 autistic brains (A, C, E, G, I) and age-matched controls (B, D, F, H, J); samples E, F, G, H were from female donors. Images were taken at ×20. Arrows indicate Purkinje cells.

Figure 7

Higher magnification (×40) images of immunohistochemical staining for RORA and BCL-2 on cerebellar sections from autistic and age-matched control subjects. ML, molecular layer; PL, Purkinje layer; GL, granule layer. Arrows indicate Purkinje cells.

Figure 8

RORA protein also appears to be reduced in the frontal cortex (BA9) in the majority of tissue sections from age- and sex-matched autistic and control subjects. This tissue array, obtained through the Autism Tissue Program, was prepared as described by Nagarajan et al. in the laboratory of Dr. Janine LaSalle (University of California, Davis, CA, USA). Immunohistochemical staining for RORA was accomplished as in Fig. 5.

Figure 9

Genes, cellular processes (yellow rectangles), disorders (purple), metabolites (green), and functional complexes (orange) associated with RORA by pathway analyses using Pathway Studio. Genes highlighted in blue were found to be differentially expressed between autistic individuals with severe language impairment and nonautistic controls in our recent study profiling gene expression in several autistic subtypes ; genes highlighted in yellow were associated with autism in other studies.