15q11-13 GABA_A receptor genes are normally biallelically expressed in brain yet are subject to epigenetic dysregulation in autism-spectrum disorders

Introduction

Human chromosome 15q11-13 is a complex locus containing imprinted genes involved in two neurological disorders, Prader-Willi syndrome (PWS) and Angelman syndrome (AS). Prader-Willi syndrome, a disorder characterized by hyperphagia leading to obesity with mild to moderate mental retardation, results from paternal deficiency of 15q11-13. Angelman syndrome, characterized by severe mental retardation with a lack of speech and excessive inappropriate laughter, is caused by maternal 15q11-13 deficiency (1). The majority of PWS and AS cases (~ 70%) result from de novo deletions spanning the 4 MB region of 15q11-13. Uniparental disomy (UPD) of the maternal chromosome is another common cause (~25% of cases) of PWS, while paternal UPD is an infrequent cause of AS. Mutations within the maternally expressed imprinted gene UBE3A, are sufficient to cause AS (2, 3), and occur in ~10% of cases (1).

Autism is a complex neurodevelopmental disorder characterized by impairments in social interaction, restricted and stereotyped behaviors, and deficits in language and communication (4). Although there is a strong genetic component to autism, few candidate genes have been identified (5). Chromosome 15q11-13 is one of the genetic loci implicated in autism, as maternal duplications of this region remain one of the most common cytogenetic abnormalities found in cases of idiopathic autism (6). Additional evidence from multiple linkage and association studies suggests 15q11-13 genes are involved in autism (7–10).

Rett syndrome (RTT) is a severe neurodevelopmental disorder that primarily affects females and is caused by mutations in the X-linked gene MECP2, the methyl CpG binding protein 2 (11). RTT is characterized by normal early postnatal development followed by an abrupt loss of developmental milestones around 6–18 months of age leading to absence of speech, severe mental retardation, and loss of purposeful hand movements (12). Like autism, RTT is categorized as a pervasive developmental disorder (PDD) and is the only one of the five PDDs with a known genetic cause (4). In a recent molecular study of post-mortem human brain, both RTT and autism samples exhibited expression defects in two 15q11-13 genes, UBE3A and GABRB3, suggesting overlapping pathways are dysregulated in these disorders (13).

Chromosome 15q11-13 contains a cluster of three GABA_A receptor subunit genes, GABRB3, GABRA5, and GABRG3, encoding receptor subunits for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). The mouse orthologues, contained within a syntenic block on chromosome 7qB5, are biallelically expressed in brain (14–16), however, conflicting data exist on the imprinting status of the human 15q11-13 GABA_A receptor subunit genes (17–21). An early study of GABRB3 expression in human hydatidiform mole and normal villi tissues suggested preferential maternal gene expression (20). Gabriel et al (21) later detected 15q11-13 GABA_A receptor expression in multiple human-mouse somatic-cell hybrid lines containing a maternal or paternal human chromosome 15, suggesting that these genes are non-imprinted. Meguro et al (17) performed microcell-mediated cell transfer to create mouse A9 hybrids harboring either a normal maternal or paternal human chromosome 15. The human paternally expressed imprinted genes retained normal imprinting in the A9 hybrids, however, the GABA_A receptor genes were only expressed from the paternal chromosome. Bittel et al (18, 19) demonstrated evidence in support of paternal expression bias of human GABA_A receptor genes with microarray analyses. Both studies, comparing deletion and UPD lymphoblastoid cell lines from PWS and AS patients, found consistent paternal bias of GABRB3 and GABRA5 gene expression.

Additional suggestive evidence for imprinting of human 15q11-13 GABA_A receptors comes from allele-specific replication timing differences of genomic sequences near the GABA_A receptor genes (22), as replication timing differences between parental homologues is a characteristic of imprinted genes (23). Both parental chromosomal contributions are required for proper replication timing of the 15q11-13 GABA_A receptor genes, suggesting trans interactions between parental homologs (24). Furthermore, homologous association of 15q11-13 domains occurs in normal lymphocytes, yet is deficient in PWS and AS samples, suggesting that the trans interaction involves the association of oppositely imprinted homologues (25). Homologous association of 15q11-13 GABRB3 alleles occurs in mature neurons within brain, and is deficient in Rett syndrome and autism (26).

Since the 15q11-13 GABA_A receptor genes are implicated in multiple human disorders, including autism, definitively determining the imprinting status of these genes in human brain is critical to understanding their potential role in the pathogenesis of neurodevelopmental disorders. In this study, single nucleotide polymorphisms (SNPs) within the coding region of GABRB3, GABRA5, and GABRG3 were used to investigate allelic expression of transcripts from cerebral cortex. Since several imprinted genes, including UBE3A, exhibit neuron-specific imprinting (16, 27–29), this imprinting study was performed using post-mortem human brain tissue. The human 15q11-13 GABA_A receptor subunit genes were determined to have equal biallelic expression in all control samples, demonstrating that these genes are not imprinted in normal frontal cortex. In contrast, allelic expression analysis revealed that four idiopathic autism samples and one Rett syndrome sample exhibited monoallelic or highly skewed allelic expression of one or more GABA_A receptor gene. Furthermore, quantitative analysis of GABRB3 transcript in PWS and AS brain samples showed evidence for a paternal expression bias when a biparental 15q11-13 contribution was lacking. These results explain prior observations of paternal bias of GABRB3 expression, and suggest that epigenetic abnormalities in 15q11-13 that serve to dysregulate GABRB3 expression may be relatively common in autistic individuals without detectable cytogenetic alterations.

Results

Discussion

Because human chromosome 15q11-13 is implicated in multiple neurodevelopmental disorders, it is critical to understand how genes within this locus are regulated and expressed. In this study, we have resolved the dispute over imprinting of the 15q11-13 GABA_A receptor cluster by showing that these genes are biallelically expressed in human frontal cortex yet are aberrantly expressed in multiple neurodevelopmental disorders. Our data also provide evidence for homologous trans effects on expression levels of GABRB3, as samples with a single parental contribution show sub-optimal expression. We have identified both trans and gender effects on brain-specific methylation of an intronic sequence of GABRB3 that serves as a binding site for MeCP2. Finally, we have provided supporting evidence that MeCP2 is a positive regulator of GABRB3 expression and propose a novel mechanistic role for MeCP2 as an activator of gene expression.

While imprinting is well characterized for some 15q11-13 genes, such as SNRPN (36) and UBE3A (27, 28), the imprinting status of the GABA_A receptor cluster has been disputed (18–21). We directly analyzed allelic expression of 15q11-13 GABA_A receptor subunit genes in human brain and report that GABRB3, GABRA5, and GABRG3 are normally biallelically expressed in frontal cortex, supporting previous findings in mouse (14, 15). Interestingly, we detected a paternal expression bias of GABRB3 in brain samples with single 15q11-13 parental contributions, as previously observed in human PWS and AS lymphoblastoid cell lines (18, 19) and microcell-mediated chromosome transfer (17). We hypothesize that GABRB3 is expressed more efficiently from the paternal chromosome than the maternal chromosome in isolation because the paternal chromosome, containing many paternally expressed imprinted genes, inherently has a more active chromatin configuration than the maternal chromosome. No evidence for a paternal bias was observed in 7 different heterozygous control samples with equal biallelic GABRB3 expression (Table 2), suggesting that the maternal GABRB3 expression defect is rescued by complementation with a paternal 15q11-13 allele.

Despite having biparental 15q11-13 contributions, one RTT and four autism brain samples exhibited loss of equal biallelic expression of one or more of the 15q11-13 GABA_A receptor subunit genes (Table 3). Although loss of biallelic expression was observed for all three of the 15q11-13 GABA_A receptor subunit genes, GABRB3 was the most highly expressed and is the most relevant to autism-spectrum disorders. Autism samples with loss of biallelic expression of any one of the 15q11-13 GABA_A receptor subunit genes had significantly reduced GABRB3 protein levels compared to samples with biallelic expression. Furthermore, one sample, AUT 797, heterozygous for all three GABA_A receptor genes, showed loss of biallelic expression for both GABRB3 and GABRG3 (Figure 1). These observations suggest that the defects in autism samples exist throughout the 15q11-13 GABA_A receptor cluster and may be due to epigenetic dysregulation of one parental allele. Parental DNA samples were not available for the post-mortem brain samples, therefore the parental origin of the expressed allele is currently unknown.

We expected GABRB3 transcript to be significantly reduced in samples with loss of biallelic expression, however, transcript levels correlated to the reduced protein in only two of four autism samples with loss of biallelic expression. This discrepancy may be partially explained by the complex genetic backgrounds of autism samples. No mutations were detected within the coding sequence of GABRB3 in autism samples, however variations in regulatory sequences may dramatically impact transcription and stability of transcripts. Interestingly, a GABRB3 promoter polymorphism was recently reported to impair transcriptional activity in a luciferase assay (37), however this polymorphism was found frequently within our samples and could not explain differences in GABRB3 expression (data not shown). Alternatively, individual differences in post-transcriptional regulation of GABRB3 within the autism samples may explain discrepancies between transcript and protein. Additionally, GABA_A receptor subunit genes have been shown to exhibit complex coordinate regulation (38), thus a compensation mechanism may account for the transcriptional differences observed in some autism brain samples.

Consistent with actively expressed genes, the promoter of GABRB3 was hypomethylated, however a nearby intronic region was more highly methylated in human brain than in blood (Figure 5C). Our quantitative real-time RT-PCR data demonstrate that cerebral cortex has high levels of GABRB3 expression (Figure 3A) while lymphoblastoid cells have barely detectable GABRB3 expression levels less than 1/1000 of the brain transcript levels (data not shown). DNA methylation within intronic or intergenic regions has been previously described to confer a positive influence on gene expression (39, 40), therefore we suggest that intronic methylation of GABRB3 is positively associated with expression. While PWS and AS brain samples did not reveal parent-of-origin specific methylation patterns at GABRB3 and GABRG3, AS deletion samples and PWS female samples had significantly reduced levels of GABRB3 intron methylation. The hypomethylation of both AS and PWS samples suggests that trans effects between parental alleles serve to regulate proper methylation levels in this locus in cortex. The gender difference in PWS samples for GABRB3 was unexpected, however, Liljelund et al (41), also found gender and parent-of-origin effects on Gabrb3 protein expression in mice heterozygous for a small Gabrb3 deletion that includes a syntenic region for the intron 3 methylation sites. Control and autism brain samples did not exhibit gender effects on methylation and autism samples with loss of biallelic expression did not have methylation defects within the assayed regions of GABRB3 or GABRG3 (Figure 5). Perhaps the significantly reduced MeCP2 expression observed in the autism samples with loss of biallelic expression (30, 42) could be the major epigenetic defect causing dysregulated GABRB3 expression. In addition, methylation defects may be found in distal regions within or near GABRB3 using high-throughput techniques in autism samples with loss of biallelic 15q11-13 GABA_A receptor expression in future studies.

We have previously described a developmentally regulated process of homologous pairing of human 15q11-13 GABRB3 alleles in normal brain that is deficient in PWS, AS, RTT, and autism samples (26). Deficiencies in neuronal trans interactions of 15q11-13 domains have been directly linked to MeCP2 by ablation of MeCP2 binding in SH-SY5Y neuroblastoma cells that significantly reduces pairing of GABRB3 alleles (26). Although MeCP2 mutation or deficiency is not sufficient to cause loss of biallelic expression of 15q11-13 GABA_A receptor genes in all RTT and autism samples, our results suggest that trans interactions between 15q11-13 parental homologues contribute to the proper normal biallelic gene expression within the GABA_A receptor domain. The unexpected significant reduction of SNRPN expression in AS deletion samples (Figure 3C) further suggests that trans interactions are important for obtaining optimal gene expression levels within 15q11-13. Our data show that monoallelic expression of GABRB3 does not occur in normal brain, which suggests that biallelic expression of GABRB3 is actively maintained. We hypothesize that the proximity of the 15q11-13 GABA receptor cluster to imprinted genes requires an active process in which both GABRB3 alleles physically associate in order to be expressed optimally from both alleles.

RTT samples with biallelic expression also have significantly reduced GABRB3 expression (Figure 3A), implicating a positive role for MeCP2 in regulation of GABRB3 expression by binding to methylated CpG sites in both cis and trans. The model shown in Figure 6 illustrates two possibilities to explain reduced biallelic and monoallelic expression of GABRB3 in RTT brain. In normal brain, MeCP2 binding to intron 3 methylated sites serves to positively regulate GABRB3 expression by bringing both parental alleles to a nuclear location with optimal transcriptional machinery, such as a transcription factory (43). In cases of reduced biallelic expression, 15q11-13 homologues are not paired due to deficiencies in MeCP2, and neither parental chromosome is optimally transcribed. Similarly, Mecp2-deficient mice also exhibit a significant two-fold reduction in Gabrb3 despite normal biallelic expression (13). In autism and RTT samples with monoallelic expression, pairing is disrupted, but an additional epigenetic or genetic defect prohibits transcription of one allele (Figure 6). Although we speculate that the maternal allele is silent, due to evidence for a paternal bias in expression (Figure 3A), the parental origin of alleles in the autism and RTT post-mortem brain samples is currently unknown.

In this report we show by ChIP and bisulfite sequencing that MeCP2 binds to intronic methylated CpG sequences within GABRB3 in differentiated human SH-SH5Y cells (Figure 4). Although traditionally viewed as a transcriptional repressor (44, 45), MeCP2 has recently been shown indirectly to positively regulate the target gene Bdnf (46) and genome-wide expression profiling analyses have demonstrated many potentially down-regulated genes with MeCP2 deficiency (47–49). In our model (Figure 6), we suggest that binding of MeCP2 to brain-specific methylated CpG sites within GABRB3 positively influences expression by promoting trans interactions between homologues and by positively organizing chromatin in cis. Horike et al (34) previously described a role for MeCP2 as an organizer of chromatin loops that silence gene expression. We hypothesize that by binding to multiple sites in cis within the GABA_A receptor cluster, MeCP2 promotes formation of chromatin structures that are favorable for active transcription, as illustrated by proximity to an active transcription factory in the model in Figure 6.

GABA is the major inhibitory neurotransmitter in the brain and plays an important role in brain function. Partial disruption of GABAergic neurons in the cortex of mice causes seizures, anxiety, and impaired social behavior, consistent with GABA signaling defects contributing to the etiology of autism-spectrum disorders (50). Unlike GABRB3, GABRA5 and GABRG3 proteins do not primarily localize in cerebral cortex (51–53), consistent with our expression data (Supplementary Figure 1). Deletion of both Gabra5 and Gabrg3 does not cause an apparent phenotype in mice (54, 55), however, Gabrb3-deficient mice exhibit phenotypes such as seizures, sleep abnormalities, and stereotyped behaviors consistent with characteristics of AS, RTT, and autism (55).

Genetic and phenotypic overlap in RTT, autism, and Angelman syndrome suggests that overlapping molecular pathways are dysregulated in these similar neurodevelopmental disorders. Although rare, MECP2 mutations have been reported in individuals clinically diagnosed with AS or autism (56, 57), and MECP2 mutations combined with rearrangements in 15q11-13 have been reported in individuals with RTT (58). In this study we show that GABRB3, a 15q11-13 gene, is positively regulated by MeCP2 and significantly reduced in both RTT and AS. Clinical observations of EEG abnormalities typical of AS in RTT patients support the involvement of GABRB3 in the shared epileptic phenotypes in these disorders (59, 60). Maternal duplications of 15q11-13, including GABRB3, occur in a small percent of autistic individuals, however it is unclear how this duplication affects GABRB3 expression in brain. Although increased copy number is predicted to increase expression, our data demonstrate complex trans regulation of GABRB3 in human brain suggesting that deviations from the normal biparental chromosomal contribution may negatively impact GABRB3 expression. Our current study also provides evidence for complex epigenetic dysregulation of the 15q11-13 GABA_A receptor cluster in idiopathic autism samples with GABRB3 protein defects, further suggesting shared molecular pathways are dysregulated in related neurodevelopmental disorders.

Materials and Methods