Placental DNA methylation levels at CYP2E1 and IRS2 are associated with child outcome in a prospective autism study.

DNA methylation acts at the interface of genetic and environmental factors relevant for autism spectrum disorder (ASD). Placenta, normally discarded at birth, is a potentially rich source of DNA methylation patterns predictive of ASD in the child. Here, we performed whole methylome analyses of placentas from a prospective study MARBLES (Markers of Autism Risk in Babies-Learning Early Signs) of high-risk pregnancies. A total of 400 differentially methylated regions (DMRs) discriminated placentas stored from children later diagnosed with ASD compared to typically developing controls. These ASD DMRs were significantly enriched at promoters, mapped to 596 genes functionally enriched in neuronal development, and overlapped genetic ASD risk. ASD DMRs at CYP2E1 and IRS2 reached genome-wide significance, replicated by pyrosequencing and correlated with expression differences in brain. Methylation at CYP2E1 associated with both ASD diagnosis and genotype within the DMR. In contrast, methylation at IRS2 was unaffected by within DMR genotype but modified by preconceptional maternal prenatal vitamin use. This study therefore identified two potentially useful early epigenetic markers for ASD in placenta.

Prader-Willi locus Snord116 RNA processing requires an active endogenous allele and neuron-specific splicing by Rbfox3/NeuN.

Prader-Willi syndrome (PWS), an imprinted neurodevelopmental disorder characterized by metabolic, sleep and neuropsychiatric features, is caused by the loss of paternal SNORD116, containing only non-coding RNAs (ncRNAs). The primary SNORD116 transcript is processed into small nucleolar RNAs (snoRNAs), which localize to nucleoli, and their spliced host gene 116HG, which is retained at its site of transcription. While functional complementation of the SNORD116 ncRNAs is a desirable goal for treating PWS, the mechanistic requirements of SNORD116 RNA processing are poorly understood. Here we developed and tested a novel transgenic mouse which ubiquitously expresses Snord116 on both a wild-type and a Snord116 paternal deletion (Snord116+/-) background. Interestingly, while the Snord116 transgene was ubiquitously expressed in multiple tissues, splicing of the transgene and production of snoRNAs was limited to brain tissues. Knockdown of Rbfox3, encoding neuron-specific splicing factor neuronal nuclei (NeuN) in Snord116+/--derived neurons, reduced splicing of the transgene in neurons. RNA fluorescence in situ hybridization for 116HG revealed a single significantly larger signal in transgenic mice, demonstrating colocalization of transgenic and endogenous 116HG RNAs. Similarly, significantly increased snoRNA levels were detected in transgenic neuronal nucleoli, indicating that transgenic Snord116 snoRNAs were effectively processed and localized. In contrast, neither transgenic 116HG nor snoRNAs were detectable in either non-neuronal tissues or Snord116+/- neurons. Together, these results demonstrate that exogenous expression and neuron-specific splicing of the Snord116 locus are insufficient to rescue the genetic deficiency of Snord116 paternal deletion. Elucidating the mechanisms regulating Snord116 processing and localization is essential to develop effective gene replacement therapies for PWS.

MeCP2 isoform e1 mutant mice recapitulate motor and metabolic phenotypes of Rett syndrome.

Mutations in the X-linked gene MECP2 cause the majority of Rett syndrome (RTT) cases. Two differentially spliced isoforms of exons 1 and 2 (MeCP2-e1 and MeCP2-e2) contribute to the diverse functions of MeCP2, but only mutations in exon 1, not exon 2, are observed in RTT. We previously described an isoform-specific MeCP2-e1-deficient male mouse model of a human RTT mutation that lacks MeCP2-e1 while preserving expression of MeCP2-e2. However, RTT patients are heterozygous females that exhibit delayed and progressive symptom onset beginning in late infancy, including neurologic as well as metabolic, immune, respiratory and gastrointestinal phenotypes. Consequently, we conducted a longitudinal assessment of symptom development in MeCP2-e1 mutant females and males. A delayed and progressive onset of motor impairments was observed in both female and male MeCP2-e1 mutant mice, including hind limb clasping and motor deficits in gait and balance. Because these motor impairments were significantly impacted by age-dependent increases in body weight, we also investigated metabolic phenotypes at an early stage of disease progression. Both male and female MeCP2-e1 mutants exhibited significantly increased body fat compared to sex-matched wild-type littermates prior to weight differences. Mecp2e1-/y males exhibited significant metabolic phenotypes of hypoactivity, decreased energy expenditure, increased respiratory exchange ratio, but decreased food intake compared to wild-type. Untargeted analysis of lipid metabolites demonstrated a distinguishable profile in MeCP2-e1 female mutant liver characterized by increased triglycerides. Together, these results demonstrate that MeCP2-e1 mutation in mice of both sexes recapitulates early and progressive metabolic and motor phenotypes of human RTT.

Early motor phenotype detection in a female mouse model of Rett syndrome is improved by cross-fostering.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the gene encoding methyl CpG binding protein 2 (MeCP2) that occur sporadically in 1:10,000 female births. RTT is characterized by a period of largely normal development followed by regression in language and motor skills at 6-18 months of age. Mecp2 mutant mice recapitulate many of the clinical features of RTT, but the majority of behavioral assessments have been conducted in male Mecp2 hemizygous null mice as offspring of heterozygous dams. Given that RTT patients are predominantly female, we conducted a systematic analysis of developmental milestones, sensory abilities, and motor deficits, following the longitudinal decline of function from early postnatal to adult ages in female Mecp2 heterozygotes of the conventional Bird line (Mecp2tm1.1bird-/+), as compared to their female wildtype littermate controls. Further, we assessed the impact of postnatal maternal environment on developmental milestones and behavioral phenotypes. Cross-fostering to CD1 dams accelerated several developmental milestones independent of genotype, and induced earlier onset of weight gain in adult female Mecp2tm1.1bird-/+ mice. Cross-fostering improved the sensitivity of a number of motor behaviors that resulted in observable deficits in Mecp2tm1.1bird-/+ mice at much earlier (6-7 weeks) ages than were previously reported (6-9 months). Our findings indicate that female Mecp2tm1.1bird-/+ mice recapitulate many of the motor aspects of RTT syndrome earlier than previously appreciated. In addition, rearing conditions may impact the phenotypic severity and improve the ability to detect genotype differences in female Mecp2 mutant mice.

Cognitive deficits in the Snord116 deletion mouse model for Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is an imprinted neurodevelopmental disease caused by a loss of paternal genes on chromosome 15q11-q13. It is characterized by cognitive impairments, developmental delay, sleep abnormalities, and hyperphagia often leading to obesity. Clinical research has shown that a lack of expression of SNORD116, a paternally expressed imprinted gene cluster that encodes multiple copies of a small nucleolar RNA (snoRNA) in both humans and mice, is most likely responsible for many PWS symptoms seen in humans. The majority of previous research using PWS preclinical models focused on characterization of the hyperphagic and metabolic phenotypes. However, a crucial understudied clinical phenotype is cognitive impairments and thus we investigated the learning and memory abilities using a model of PWS, with a heterozygous deletion in Snord116. We utilized the novel object recognition task, which doesn't require external motivation, or exhaustive swim training. Automated findings were further confirmed with manual scoring by a highly trained blinded investigator. We discovered deficits in Snord116+/- mutant mice in the novel object recognition, location memory and tone cue fear conditioning assays when compared to age-, sex- matched, littermate control Snord116+/+ mice. Further, we confirmed that despite physical neo-natal developmental delays, Snord116+/- mice had normal exploratory and motor abilities. These results show that the Snord116+/- deletion murine model is a valuable preclinical model for investigating learning and memory impairments in individuals with PWS without common confounding phenotypes.

Loss of MeCP2 in the rat models regression, impaired sociability and transcriptional deficits of Rett syndrome.

Mouse models of the transcriptional modulator Methyl-CpG-Binding Protein 2 (MeCP2) have advanced our understanding of Rett syndrome (RTT). RTT is a 'prototypical' neurodevelopmental disorder with many clinical features overlapping with other intellectual and developmental disabilities (IDD). Therapeutic interventions for RTT may therefore have broader applications. However, the reliance on the laboratory mouse to identify viable therapies for the human condition may present challenges in translating findings from the bench to the clinic. In addition, the need to identify outcome measures in well-chosen animal models is critical for preclinical trials. Here, we report that a novel Mecp2 rat model displays high face validity for modelling psychomotor regression of a learned skill, a deficit that has not been shown in Mecp2 mice. Juvenile play, a behavioural feature that is uniquely present in rats and not mice, is also impaired in female Mecp2 rats. Finally, we demonstrate that evaluating the molecular consequences of the loss of MeCP2 in both mouse and rat may result in higher predictive validity with respect to transcriptional changes in the human RTT brain. These data underscore the similarities and differences caused by the loss of MeCP2 among divergent rodent species which may have important implications for the treatment of individuals with disease-causing MECP2 mutations. Taken together, these findings demonstrate that the Mecp2 rat model is a complementary tool with unique features for the study of RTT and highlight the potential benefit of cross-species analyses in identifying potential disease-relevant preclinical outcome measures.

MeCP2 regulates activity-dependent transcriptional responses in olfactory sensory neurons.

During postnatal development, neuronal activity controls the remodeling of initially imprecise neuronal connections through the regulation of gene expression. MeCP2 binds to methylated DNA and modulates gene expression during neuronal development and MECP2 mutation causes the autistic disorder Rett syndrome. To investigate a role for MeCP2 in neuronal circuit refinement and to identify activity-dependent MeCP2 transcription regulations, we leveraged the precise organization and accessibility of olfactory sensory axons to manipulation of neuronal activity through odorant exposure in vivo. We demonstrate that olfactory sensory axons failed to develop complete convergence when Mecp2 is deficient in olfactory sensory neurons (OSNs) in an otherwise wild-type animal. Furthermore, we demonstrate that expression of selected adhesion genes was elevated in Mecp2-deficient glomeruli, while acute odor stimulation in control mice resulted in significantly reduced MeCP2 binding to these gene loci, correlating with increased expression. Thus, MeCP2 is required for both circuitry refinement and activity-dependent transcriptional responses in OSNs.

Mice with an isoform-ablating Mecp2 exon 1 mutation recapitulate the neurologic deficits of Rett syndrome.

Mutations in MECP2 cause the neurodevelopmental disorder Rett syndrome (RTT OMIM 312750). Alternative inclusion of MECP2/Mecp2 exon 1 with exons 3 and 4 encodes MeCP2-e1 or MeCP2-e2 protein isoforms with unique amino termini. While most MECP2 mutations are located in exons 3 and 4 thus affecting both isoforms, MECP2 exon 1 mutations but not exon 2 mutations have been identified in RTT patients, suggesting that MeCP2-e1 deficiency is sufficient to cause RTT. As expected, genetic deletion of Mecp2 exons 3 and/or 4 recapitulates RTT-like neurologic defects in mice. However, Mecp2 exon 2 knockout mice have normal neurologic function. Here, a naturally occurring MECP2 exon 1 mutation is recapitulated in a mouse model by genetic engineering. A point mutation in the translational start codon of Mecp2 exon 1, transmitted through the germline, ablates MeCP2-e1 translation while preserving MeCP2-e2 production in mouse brain. The resulting MeCP2-e1 deficient mice developed forelimb stereotypy, hindlimb clasping, excessive grooming and hypo-activity prior to death between 7 and 31 weeks. MeCP2-e1 deficient mice also exhibited abnormal anxiety, sociability and ambulation. Despite MeCP2-e1 and MeCP2-e2 sharing, 96% amino acid identity, differences were identified. A fraction of phosphorylated MeCP2-e1 differed from the bulk of MeCP2 in subnuclear localization and co-factor interaction. Furthermore, MeCP2-e1 exhibited enhanced stability compared with MeCP2-e2 in neurons. Therefore, MeCP2-e1 deficient mice implicate MeCP2-e1 as the sole contributor to RTT with non-redundant functions.

R-loop formation at Snord116 mediates topotecan inhibition of Ube3a-antisense and allele-specific chromatin decondensation.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are oppositely imprinted autism-spectrum disorders with known genetic bases, but complex epigenetic mechanisms underlie their pathogenesis. The PWS/AS locus on 15q11-q13 is regulated by an imprinting control region that is maternally methylated and silenced. The PWS imprinting control region is the promoter for a one megabase paternal transcript encoding the ubiquitous protein-coding Snrpn gene and multiple neuron-specific noncoding RNAs, including the PWS-related Snord116 repetitive locus of small nucleolar RNAs and host genes, and the antisense transcript to AS-causing ubiquitin ligase encoding Ube3a (Ube3a-ATS). Neuron-specific transcriptional progression through Ube3a-ATS correlates with paternal Ube3a silencing and chromatin decondensation. Interestingly, topoisomerase inhibitors, including topotecan, were recently identified in an unbiased drug screen for compounds that could reverse the silent paternal allele of Ube3a in neurons, but the mechanism of topotecan action on the PWS/AS locus is unknown. Here, we demonstrate that topotecan treatment stabilizes the formation of RNA:DNA hybrids (R loops) at G-skewed repeat elements within paternal Snord116, corresponding to increased chromatin decondensation and inhibition of Ube3a-ATS expression. Neural precursor cells from paternal Snord116 deletion mice exhibit increased Ube3a-ATS levels in differentiated neurons and show a reduced effect of topotecan compared with wild-type neurons. These results demonstrate that the AS candidate drug topotecan acts predominantly through stabilizing R loops and chromatin decondensation at the paternally expressed PWS Snord116 locus. Our study holds promise for targeted therapies to the Snord116 locus for both AS and PWS.

A Prader-Willi locus lncRNA cloud modulates diurnal genes and energy expenditure.

Prader-Willi syndrome (PWS), a genetic disorder of obesity, intellectual disability and sleep abnormalities, is caused by loss of non-coding RNAs on paternal chromosome 15q11-q13. The imprinted minimal PWS locus encompasses a long non-coding RNA (lncRNA) transcript processed into multiple SNORD116 small nucleolar RNAs and the spliced exons of the host gene, 116HG. However, both the molecular function and the disease relevance of the spliced lncRNA 116HG are unknown. Here, we show that 116HG forms a subnuclear RNA cloud that co-purifies with the transcriptional activator RBBP5 and active metabolic genes, remains tethered to the site of its transcription and increases in size in post-natal neurons and during sleep. Snord116del mice lacking 116HG exhibited increased energy expenditure corresponding to the dysregulation of diurnally expressed Mtor and circadian genes Clock, Cry1 and Per2. These combined genomic and metabolic analyses demonstrate that 116HG regulates the diurnal energy expenditure of the brain. These novel molecular insights into the energy imbalance in PWS should lead to improved therapies and understanding of lncRNA roles in complex neurodevelopmental and metabolic disorders.

Long-lived epigenetic interactions between perinatal PBDE exposure and Mecp2308 mutation.

The widespread use of persistent organic polybrominated diphenyl ethers (PBDEs) as commercial flame retardants has raised concern about potential long-lived effects on human health. Epigenetic mechanisms, such as DNA methylation, are responsive to environmental influences and have long-lasting consequences. Autism spectrum disorders (ASDs) have complex neurodevelopmental origins whereby both genetic and environmental factors are implicated. Rett syndrome is an X-linked ASD caused by mutations in the epigenetic factor methyl-CpG binding protein 2 (MECP2). In this study, an Mecp2 truncation mutant mouse (Mecp2(308)) with social behavioral defects was used to explore the long-lasting effects of PBDE exposure in a genetically and epigenetically susceptible model. Mecp2(308/+) dams were perinatally exposed daily to 2,2',4,4'-tetrabromodiphenyl ether 47 (BDE-47) and bred to wild-type C57BL/6J males, and the offspring of each sex and genotype were examined for developmental, behavioral and epigenetic outcomes. Perinatal BDE-47 exposure negatively impacted fertility of Mecp2(308/+) dams and preweaning weights of females. Global hypomethylation of adult brain DNA was observed specifically in female offspring perinatally exposed to BDE-47 and it coincided with reduced sociability in a genotype-independent manner. A reversing interaction of Mecp2 genotype on BDE-47 exposure was observed in a short-term memory test of social novelty that corresponded to increased Dnmt3a levels specifically in BDE-47-exposed Mecp2(308/+) offspring. In contrast, learning and long-term memory in the Morris water maze was impaired by BDE-47 exposure in female Mecp2(308/+) offspring. These results demonstrate that a genetic and environmental interaction relevant to social and cognitive behaviors shows sexual dimorphism, epigenetic dysregulation, compensatory molecular mechanisms and specific behavioral deficits.

Sex-specific single cell-level transcriptomic signatures of Rett syndrome disease progression.

Dominant X-linked diseases are uncommon due to female X chromosome inactivation (XCI). While random XCI usually protects females against X-linked mutations, Rett syndrome (RTT) is a female neurodevelopmental disorder caused by heterozygous MECP2 mutation. After 6-18 months of typical neurodevelopment, RTT girls undergo poorly understood regression. We performed longitudinal snRNA-seq on cerebral cortex in a construct-relevant Mecp2e1 mutant mouse model of RTT, revealing transcriptional effects of cell type, mosaicism, and sex on progressive disease phenotypes. Across cell types, we observed sex differences in the number of differentially expressed genes (DEGs) with 6x more DEGs in mutant females than males. Unlike males, female DEGs emerged prior to symptoms, were enriched for homeostatic gene pathways in distinct cell types over time, and correlated with disease phenotypes and human RTT cortical cell transcriptomes. Non-cell-autonomous effects were prominent and dynamic across disease progression of Mecp2e1 mutant females, indicating wild-type-expressing cells normalizing transcriptional homeostasis. These results improve understanding of RTT progression and treatment.

Integration of CTCF Loops, Methylome, and Transcriptome in Differentiating LUHMES as a Model for Imprinting Dynamics of the 15q11-q13 Locus in Human Neurons.

Human cell line models, including the neuronal precursor line LUHMES, are important for investigating developmental transcriptional dynamics within imprinted regions, particularly the 15q11-q13 Angelman (AS) and Prader-Willi (PWS) syndrome locus. AS results from loss of maternal UBE3A in neurons, where the paternal allele is silenced by a convergent antisense transcript UBE3A-ATS, a lncRNA that normally terminates at PWAR1 in non-neurons. qRTPCR analysis confirmed the exclusive and progressive increase in UBE3A-ATS in differentiating LUHMES neurons, validating their use for studying UBE3A silencing. Genome-wide transcriptome analyses revealed changes to 11,834 genes during neuronal differentiation, including the upregulation of most genes within the 15q11-q13 locus. To identify dynamic changes in chromatin loops linked to transcriptional activity, we performed a HiChIP validated by 4C, which identified two neuron-specific CTCF loops between MAGEL2-SNRPN and PWAR1-UBE3A. To determine if allele-specific differentially methylated regions (DMR) may be associated with CTCF loop anchors, whole genome long-read nanopore sequencing was performed. We identified a paternally hypomethylated DMR near the SNRPN upstream loop anchor exclusive to neurons and a paternally hypermethylated DMR near the PWAR1 CTCF anchor exclusive to undifferentiated cells, consistent with increases in neuronal transcription. Additionally, DMRs near CTCF loop anchors were observed in both cell types, indicative of allele-specific differences in chromatin loops regulating imprinted transcription. These results provide an integrated view of the 15q11-q13 epigenetic landscape during LUHMES neuronal differentiation, underscoring the complex interplay of transcription, chromatin looping, and DNA methylation. They also provide insights for future therapeutic approaches for AS and PWS.

Neuron-specific impairment of inter-chromosomal pairing and transcription in a novel model of human 15q-duplication syndrome.

Although the etiology of autism remains largely unknown, cytogenetic and genetic studies have implicated maternal copy number gains of 15q11-q13 in 1-3% of autism cases. In order to understand how maternal 15q duplication leads to dysregulation of gene expression and altered chromatin interactions, we used microcell-mediated chromosome transfer to generate a novel maternal 15q duplication model in a human neuronal cell line. Our 15q duplication neuronal model revealed that by quantitative RT-PCR, transcript levels of NDN, SNRPN, GABRB3 and CHRNA7 were reduced compared with expected levels despite having no detectable alteration in promoter DNA methylation. Since 15q11-q13 alleles have been previously shown to exhibit homologous pairing in mature human neurons, we assessed homologous pairing of 15q11-q13 by fluorescence in situ hybridization. Homologous pairing of 15q11-q13 was significantly disrupted by 15q duplication. To further understand the extent and mechanism of 15q11-q13 homologous pairing, we mapped the minimal region of homologous pairing to a ∼500 kb region at the 3' end of GABRB3 which contains multiple binding sites for chromatin regulators MeCP2 and CTCF. Both active transcription and the chromatin factors MeCP2 and CTCF are required for the homologous pairing of 15q11-q13 during neuronal maturational differentiation. These data support a model where 15q11-q13 genes are regulated epigenetically at the level of both inter- and intra-chromosomal associations and that chromosome imbalance disrupts the epigenetic regulation of genes in 15q11-q13.

15q11.2-13.3 chromatin analysis reveals epigenetic regulation of CHRNA7 with deficiencies in Rett and autism brain.

Copy number variations (CNVs) within human 15q11.2-13.3 show reduced penetrance and variable expressivity in a range of neurologic disorders. Therefore, characterizing 15q11.2-13.3 chromatin structure is important for understanding the regulation of this locus during normal neuronal development. Deletion of the Prader-Willi imprinting center (PWS-IC) within 15q11.2-13.3 disrupts long-range imprinted gene expression resulting in Prader-Willi syndrome. Previous results establish that MeCP2 binds to the PWS-IC and is required for optimal expression of distal GABRB3 and UBE3A. To examine the hypothesis that MeCP2 facilitates 15q11.2-13.3 transcription by linking the PWS-IC with distant elements, chromosome capture conformation on chip (4C) analysis was performed in human SH-SY5Y neuroblastoma cells. SH-SY5Y neurons had 2.84-fold fewer 15q11.2-13.3 PWS-IC chromatin interactions than undifferentiated SH-SY5Y neuroblasts, revealing developmental chromatin de-condensation of the locus. Out of 68 PWS-IC interactions with15q11.2-13.3 identified by 4C analysis and 62 15q11.2-13.3 MeCP2-binding sites identified by previous ChIP-chip studies, only five sites showed overlap. Remarkably, two of these overlapping PWS-IC- and MeCP2-bound sites mapped to sites flanking CHRNA7 (cholinergic receptor nicotinic alpha 7) encoding the cholinergic receptor, nicotinic, alpha 7. PWS-IC interaction with CHRNA7 in neurons was independently confirmed by fluorescent in situ hybridization analysis. Subsequent quantitative transcriptional analyses of frontal cortex from Rett syndrome and autism patients revealed significantly reduced CHRNA7 expression compared with controls. Together, these results suggest that transcription of CHRNA7 is modulated by chromatin interactions with the PWS-IC. Thus, loss of long-range chromatin interactions within 15q11.2-13.3 may contribute to multiple human neurodevelopmental disorders.

Placenta and fetal brain share a neurodevelopmental disorder DNA methylation profile in a mouse model of prenatal PCB exposure.

Polychlorinated biphenyls (PCBs) are developmental neurotoxicants implicated as environmental risk factors for neurodevelopmental disorders (NDDs). Here, we report the effects of prenatal exposure to a human-relevant mixture of PCBs on the DNA methylation profiles of mouse placenta and fetal brain. Thousands of differentially methylated regions (DMRs) distinguish placenta and fetal brain from PCB-exposed mice from sex-matched vehicle controls. In both placenta and fetal brain, PCB-associated DMRs are enriched for functions related to neurodevelopment and cellular signaling and enriched within regions of bivalent chromatin. The placenta and brain PCB DMRs overlap significantly and map to a shared subset of genes enriched for Wnt signaling, Slit/Robo signaling, and genes differentially expressed in NDD models. The consensus PCB DMRs also significantly overlap with DMRs from human NDD brain and placenta. These results demonstrate that PCB-exposed placenta contains a subset of DMRs that overlap fetal brain DMRs relevant to an NDD.

Placental methylome reveals a 22q13.33 brain regulatory gene locus associated with autism.

BACKGROUND: Autism spectrum disorder (ASD) involves complex genetics interacting with the perinatal environment, complicating the discovery of common genetic risk. The epigenetic layer of DNA methylation shows dynamic developmental changes and molecular memory of in utero experiences, particularly in placenta, a fetal tissue discarded at birth. However, current array-based methods to identify novel ASD risk genes lack coverage of the most structurally and epigenetically variable regions of the human genome. RESULTS: We use whole genome bisulfite sequencing in placenta samples from prospective ASD studies to discover a previously uncharacterized ASD risk gene, LOC105373085, renamed NHIP. Out of 134 differentially methylated regions associated with ASD in placental samples, a cluster at 22q13.33 corresponds to a 118-kb hypomethylated block that replicates in two additional cohorts. Within this locus, NHIP is functionally characterized as a nuclear peptide-encoding transcript with high expression in brain, and increased expression following neuronal differentiation or hypoxia, but decreased expression in ASD placenta and brain. NHIP overexpression increases cellular proliferation and alters expression of genes regulating synapses and neurogenesis, overlapping significantly with known ASD risk genes and NHIP-associated genes in ASD brain. A common structural variant disrupting the proximity of NHIP to a fetal brain enhancer is associated with NHIP expression and methylation levels and ASD risk, demonstrating a common genetic influence. CONCLUSIONS: Together, these results identify and initially characterize a novel environmentally responsive ASD risk gene relevant to brain development in a hitherto under-characterized region of the human genome.

MeCP2 is required for global heterochromatic and nucleolar changes during activity-dependent neuronal maturation.

Mutations in MECP2, encoding methyl CpG binding protein 2, cause the neurodevelopmental disorder Rett syndrome. MeCP2 is an abundant nuclear protein that binds to chromatin and modulates transcription in response to neuronal activity. Prior studies of MeCP2 function have focused on specific gene targets of MeCP2, but a more global role for MeCP2 in neuronal nuclear maturation has remained unexplored. MeCP2 levels increase during postnatal brain development, coinciding with dynamic changes in neuronal chromatin architecture, particularly detectable as changes in size, number, and location of nucleoli and perinucleolar heterochromatic chromocenters. To determine a potential role for MeCP2 in neuronal chromatin maturational changes, we measured nucleoli and chromocenters in developing wild-type and Mecp2-deficient mouse cortical sections, as well as mouse primary cortical neurons and a human neuronal cell line following induced maturation. Mecp2-deficient mouse neurons exhibited significant differences in nucleolar and chromocenter number and size, as more abundant, smaller nucleoli in brain and primary neurons compared to wild-type, consistent with delayed neuronal nuclear maturation in the absence of MeCP2. Primary neurons increased chromocenter size following depolarization in wild-type, but not Mecp2-deficient cultures. Wild-type MECP2e1 over-expression in human SH-SY5Y cells was sufficient to induce significantly larger nucleoli, but not a T158M mutation of the methyl-binding domain. These results suggest that, in addition to the established role of MeCP2 in transcriptional regulation of specific target genes, the global chromatin-binding function of MeCP2 is essential for activity-dependent global chromatin dynamics during postnatal neuronal maturation.

Reciprocal co-regulation of EGR2 and MECP2 is disrupted in Rett syndrome and autism.

Mutations in MECP2, encoding methyl-CpG-binding protein 2 (MeCP2), cause the neurodevelopmental disorder Rett syndrome (RTT). Although MECP2 mutations are rare in idiopathic autism, reduced MeCP2 levels are common in autism cortex. MeCP2 is critical for postnatal neuronal maturation and a modulator of activity-dependent genes such as Bdnf (brain-derived neurotropic factor) and JUNB. The activity-dependent early growth response gene 2 (EGR2), required for both early hindbrain development and mature neuronal function, has predicted binding sites in the promoters of several neurologically relevant genes including MECP2. Conversely, MeCP2 family members MBD1, MBD2 and MBD4 bind a methylated CpG island in an enhancer region located in EGR2 intron 1. This study was designed to test the hypothesis that MECP2 and EGR2 regulate each other's expression during neuronal maturation in postnatal brain development. Chromatin immunoprecipitation analysis showed EGR2 binding to the MECP2 promoter and MeCP2 binding to the enhancer region in EGR2 intron 1. Reduction in EGR2 and MeCP2 levels in cultured human neuroblastoma cells by RNA interference reciprocally reduced expression of both EGR2 and MECP2 and their protein products. Consistent with a role of MeCP2 in enhancing EGR2, Mecp2-deficient mouse cortex samples showed significantly reduced EGR2 by quantitative immunofluorescence. Furthermore, MeCP2 and EGR2 show coordinately increased levels during postnatal development of both mouse and human cortex. In contrast to age-matched Controls, RTT and autism postmortem cortex samples showed significant reduction in EGR2. Together, these data support a role of dysregulation of an activity-dependent EGR2/MeCP2 pathway in RTT and autism.

Investigation of modifier genes within copy number variations in Rett syndrome.

MECP2 mutations are responsible for two different phenotypes in females, classical Rett syndrome and the milder Zappella variant (Z-RTT). We investigated whether copy number variants (CNVs) may modulate the phenotype by comparison of array-CGH data from two discordant pairs of sisters and four additional discordant pairs of unrelated girls matched by mutation type. We also searched for potential MeCP2 targets within CNVs by chromatin immunopreceipitation microarray (ChIP-chip) analysis. We did not identify one major common gene/region, suggesting that modifiers may be complex and variable between cases. However, we detected CNVs correlating with disease severity that contain candidate modifiers. CROCC (1p36.13) is a potential MeCP2 target, in which a duplication in a Z-RTT and a deletion in a classic patient were observed. CROCC encodes a structural component of ciliary motility that is required for correct brain development. CFHR1 and CFHR3, on 1q31.3, may be involved in the regulation of complement during synapse elimination, and were found to be deleted in a Z-RTT but duplicated in two classic patients. The duplication of 10q11.22, present in two Z-RTT patients, includes GPRIN2, a regulator of neurite outgrowth and PPYR1, involved in energy homeostasis. Functional analyses are necessary to confirm candidates and to define targets for future therapies.