Although autism is a highly heritable disorder, the genetic etiology of autism remains elusive. In contrast, Rett syndrome is a phenotypically overlapping PDD primarily caused by mutations in MECP2
. The progress made in studying the function of MeCP2 and its role in Rett syndrome provides an opportunity to apply some of this knowledge to other related neurodevelopmental disorders. We previously found defective MeCP2 expression in 4/4 autism frontal cortex samples (32
). Here we expanded this analysis to frontal cortex (BA9) samples from 14 autism patients; six of these patients were represented with both frontal cortex and fusiform gyrus brain samples. Additionally, a greater number of Rett, PWS, and AS samples were included. Down syndrome, ADHD, and Fragile X samples were also included because of some degree of phenotypic overlap or comorbidity between these disorders and autism. Using a tissue microarray containing all of these samples as well as controls, we found that 79% of the autism frontal cortex samples showed significant decreases in MeCP2 total protein expression compared to age-matched controls. In general, there appeared to be good concordance in MeCP2 reduction in autism between the frontal cortex and fusiform gyrus, suggesting that MeCP2 expression defects in autism are not limited to a single brain region.
All Rett syndrome samples showed significantly reduced MeCP2. Since these experiments were performed using a C-terminal MeCP2 antibody that detects both MeCP2 protein isoforms, it is expected that RTT individuals with nonsense MECP2
mutations would show lower expression. In addition, RTT females are heterozygous and mosaic for MECP2
mutations and non-cell autonomous mechanisms result in reduced MeCP2 expression in wild-type expressing cells (35
). Reduced expression was also observed for RTT samples without MECP2
coding mutations. This might be because these samples are heterozygous for MECP2
deletions. Alternatively, these samples might harbor non-coding MECP2
mutations that affect MeCP2 expression. Promoter mutations in the MECP2
−1630 to +254 genomic region were not found in these samples, however new cis
-regulatory regions have recently been described (36
) and could be tested. The reduced MeCP2 expression in RTT samples without MECP2
mutations suggests that altered regulation of a pathway involving MeCP2 is common to all Rett cases.
PWS, AS, ADHD and Down syndrome samples also had high frequencies of MeCP2 reduction, whereas a single Fragile X brain did not. MeCP2 expression defects appeared to increase with age in the Down syndrome samples, suggesting that early changes in brain development caused by trisomy for chromosome 21 might affect MeCP2 expression in adulthood, since high MeCP2 expression is a marker of mature neurons (24
). The significantly lower MeCP2 expression in ADHD samples was surprising, but this result may be brain region-specific, and the small sample number limits further interpretation. However, given the phenotypic overlap between ADHD and autism, these results warrant further investigation with additional ADHD samples and different brain regions. Taken together, the tissue microarray data indicate that MeCP2 deficiency is frequent in autism and RTT, observed in at least two brain regions in autism, and not restricted to RTT and autism. MeCP2 expression may therefore be a marker of a shared dysregulated pathway of abnormal brain development in multiple neurodevelopmental disorders.
Reduced MeCP2 expression is hypothesized to have important functional consequences. Expression of GABRB3
were previously shown to be reduced in human MECP2
-mutant RTT brain samples, Mecp2-
null mouse brain, and human autism brain (37
). Expression of other genes regulated by MeCP2, such as BDNF
, may also be affected by reduced MeCP2 (38
). The dysregulation of MeCP2 target genes is expected to have several neurological consequences, such as defects in excitatory neurotransmission and long-term potentiation, leading to some of the cognitive and behavioral deficits in ASDs.
As a first step in finding the etiology of reduced MeCP2 in ASDs, the protein-coding region and a 1.9 kb promoter region of MECP2
were sequenced in ASD samples. As expected, coding mutations were found or confirmed in some RTT samples. One female autism sample, AUT 1638, was heterozygous for a silent polymorphism in exon 4 (c.1035 A>G). This polymorphism has been identified previously and causes a synonymous amino acid change (p.K345K) (20
promoter sequencing yielded a single sequence variant in an autistic female (AUT B5342) who was heterozygous for a novel T>C transition 5′ of the human MECP2
promoter (g.-1398 T>C). The significance of the −1398 variant remains unclear at this stage and will require further functional studies to determine if it could be a rare mutation. One conclusion drawn from these experiments is that mutations in the MECP2
−1492 to −1318 genomic region are rare in autism and are unlikely to be a major contributor to its etiology. It cannot be excluded that sequence variation in other promoter regions, introns, 3′ UTR, or distant regulatory sequences might affect MeCP2 expression. MECP2
exon 1 and promoter mutations are rare in RTT (34
), but there is some evidence for involvement of 3′ UTR variants in autism (20
). Recent data suggest that long-range regulation of MECP2
transcription is influenced by sequences up to 130 kb away from the gene itself, presumably exerting their effects through chromatin looping (36
Since coding or 5′-regulatory genetic variants did not appear to explain most cases of reduced MeCP2 expression, we hypothesized that aberrant MECP2
promoter methylation might contribute to decreased MeCP2 expression and the autism phenotype. MECP2
is an X-linked gene and is subject to X-inactivation (26
in males is expected to be unmethylated and expressed, so any promoter methylation in autism males would be aberrant. Bisulfite sequencing of the MECP2
promoter (−531 to −243) in male autism and control cerebral cortex found evidence for significantly increased promoter methylation in autism compared to controls. Although not all CpG sites or alleles were methylated in any clone or sample, the overall level of DNA methylation was statistically significantly higher in autism versus controls. One CpG site out of 15 assayed, site #3, showed significantly increased methylation in autism but not four Down syndrome males. This site is located at position −445 and is part of the functional promoter region (31
). Furthermore, ENCODE chromatin immunoprecipitation (ChIP) data show that RNA polymerase II, SP1, SP3, E2F1, and other transcription factors bind at or around CpG site #3, and this region contains a DNase I hypersensitivity site in neuronal cells, strongly suggesting functional relevance (Supplementary Figure II). Female samples were not included because evidence of bias (not the expected 50% methylation) was observed in control samples, likely due to major differences in amplification and cloning efficiency between the highly methylated inactive and unmethylated active alleles following bisulfite conversion. However, 60% of alleles were methylated at site #3 in female control samples, suggesting that methylation of this site corresponds to transcriptional silencing (data not shown).
The relatively low level of total methylation observed in autism brain samples suggests that the affected cells may only be a subpopulation of the cells from which DNA was isolated from the brain. Since LCS analysis collected data from thousands of cells per sample but bisulfite sequencing analysis was limited to 10–20 cells, the LSC data is ultimately a better reflection of actual cell populations. Despite the sampling discrepancies, the two assays showed a remarkable correlation between the percentage of methylated cells and the decrease in MeCP2hi cells, particularly for juvenile autism samples. In contrast, the adult autism samples likely had other indirect factors influencing MeCP2 expression, similar to the observation of reduced MeCP2 in adult but not juvenile Down syndrome samples (). It would be interesting to determine if changes in MECP2 promoter methylation are detectable and quantifiable in blood in young autism patients with methylation assays that reflect a larger number of cells, as this might facilitate a diagnostic tool for molecular characterization of suspected autism cases.
A mixed oligogenic and epigenetic etiology has been proposed for autism (41
). Epigenetic pathways are relevant in autism because they implicate both genetic and environmental factors. For example, dietary methyl supplementation can affect gene expression via DNA methylation (42
). The observation of monozygotic twins discordant for autism (43
) suggests epigenetic differences, as methylation changes increase over the lifespan of monozygotic twins (44
). Abnormal patterns of DNA methylation have been observed in cancer, as both whole genome hypomethylation and tumor-suppressor gene hypermethylation (45
). Interestingly, abnormal DNA methylation has also been found in a several psychiatric disorders. Promoter hypermethylation of the autism and schizophrenia candidate gene reelin (RELN)
was found in postmortem occipital and frontal cortex schizophrenia samples (46
). Interestingly, the methylation increases were not at all RELN
CpG sites analyzed, but rather only at a few key sites, similar to the increased methylation of one MECP2
CpG site in autism reported here. It seems likely that the epigenetic defects observed in psychiatric disorders are more subtle that than observed in other conditions, such as cancer.
Increased DNA methylation has been observed in alcoholism, and HERP
promoter hypermethylation in blood correlated with elevated homocysteine levels and decreased HERP
). In autism, aberrant DNA methylation was found in the gene UBE3A
, but did not correlate with reduced expression (41
). Mutations in UBE3A
cause Angelman syndrome, but UBE3A
is also an autism candidate gene based on cytogenetic, positional, epigenetic, and expression data (37
). Increased acetylated histone H3 lysine 9, which is a mark of active chromatin, correlated with decreased MeCP2 expression in autism brain (50
). These observations indicate that epigenetic pathways are relevant in multiple human neurodevelopmental disorders and open the door for research into epigenetic etiology in autism and other disorders.
What could be the cause of increased MECP2
promoter methylation in autism? One possibility is lack of complete erasure of methylation derived from X chromosome inactivation on the maternally inherited X chromosome. Alternatively, environmental factors, including in utero
or postnatal exposure to certain chemicals or pollutants, could result in de novo
). Proximal genetic variation does not appear to explain increased MECP2
promoter methylation, in contrast to Fragile X syndrome, where 5′ trinucleotide repeats influence FMR1
promoter methylation (52
). However, genetic variants in trans
could potentially cause aberrant MECP2
methylation. Since male Down syndrome samples with reduced MeCP2 expression did not show increased methylation, a simplistic explanation that abnormal brain development is the cause of aberrant MECP2
methylation is unlikely.
MeCP2 is a key epigenetic regulator which mediates the effects of DNA methylation by binding to methylated DNA and recruiting additional factors that modify chromatin (1
). In this study we show that increased MECP2
promoter methylation correlates with reduced expression of MeCP2 in autism brain. While our data suggest that aberrant methylation causes reduced MeCP2 expression, we cannot rule out the possibility that factors that cause reduced MeCP2 expression could also cause increased methylation of the MECP2
promoter. It would be interesting to determine if aberrant MECP2
promoter methylation recruits itself (MeCP2 protein) or other methyl-binding domain (MBD) proteins resulting in reduction of MeCP2 expression. Alternatively, MECP2
promoter methylation at a few key CpG sites may block specific transcription factor binding, independent of MBD protein binding. The latter case seems more likely since one specific CpG site (site #3) showed statistically significantly increased methylation in autism brain. Methylation of this site may affect several putative transcription factor binding sites (CTCF, C/EBP, CAP).
Further understanding of epigenetic pathways in autism will likely lead to a greater understanding of the complex etiology of this disorder. No molecular test currently exists for autism, and diagnosis is based solely on clinical observations. Defining epigenetic abnormalities in autism could lead to a molecular diagnostic test, such as the DNA methylation test used in Angelman and Prader-Willi syndromes (53
). Drugs that act on epigenetic pathways, such as methyltransferase or histone deacetylase inhibitors, are in development or clinical trials for the treatment of cancer (54
). By defining epigenetic defects in neurodevelopmental disorders, some of these advancements may be useful in the diagnosis and eventual treatment of autism and autism spectrum disorders.