Epigenetic Investigations of 15q11-13 Using Autism Post-Mortem Brain

Background: Autism is an increasingly common disorder of complex etiology, affected by multiple genetic and environmental influences. Epigenetic mechanisms act at the interface of genetic and environmental risk factors in autism. Methylation of CpG dinucleotides and methyl-specific binding proteins are part of an epigenetic pathway essential for parental imprinting and chromatin dynamics during normal brain development.  Autism has several phenotypic features in common with the neurodevelopmental disorders with altered epigenetic pathways. Rett syndrome (RTT) is an X-linked pervasive developmental disorder caused by mutations in MECP2, which encodes methyl-CpG-binding protein 2 (MeCP2).  Prader-Willi (PWS), Angelman (AS), and 15q duplication syndromes are imprinted disorders caused by paternal or maternal 15q11-13 deficiency or duplication, respectively. Copy number variants within 15q11-13 are also associated with a spectrum of neurodevelopmental disorders, including autism, schizophrenia, and epilepsy.  Human brain samples are required for further understanding the complex interaction between genetics and epigenetics in 15q11-13 because 1) many of the neurologically relevant genes within 15q11-13 are expressed exclusively in brain, 2) there are distinct tissue-specific patterns of DNA methylation and parental imprinting not observed in blood, and 3) a mouse model of 15q duplication syndrome shows the opposite parental inheritance pattern as the human disease.

Objectives: To perform integrated analyses of genetic, epigenetic, and environmental differences that effect transcript levels of 15q11-13 genes in autism brain.

Methods: Frozen samples of cerebrum and cerebellum from individuals with autism, 15q duplication, PWS, AS, RTT, and Down syndrome were obtained from the Autism Tissue Program, the NICHD Brain and Tissue Bank for Developmental Disorders, and Harvard Brain Tissue Resource Center.  DNA, RNA, and protein were isolated and additional tissue fixed and arrayed in tissue microarrays.  DNA methylation analyses were performed by bisulfite sequencing, and transcript levels were assessed by quantitative RT-PCR.  Immunofluorescence on tissue microarrays was quantitated by laser scanning cytometry for MeCP2 and global DNA methylation.  Fluorescence in situ hybridization was performed to investigate chromatin organization and homologous pairing of 15q11-13 alleles.

Results: Epigenetic alteration in autism human brain samples have included reduced MeCP2 due to increased MECP2 promoter methylation in males, reduced 15q11-13 GABA_A receptor subunit GABRB3 due to reduced homologous pairing and loss of biallelic expression, and altered global levels of DNA methylation.  Epigenetic differences also exist within brain samples with maternal chromosome 15 duplication syndrome, with a subset showing gene expression and DNA methylation not predicted from additional maternal copies.

Conclusions: These results suggest that multiple genetic and epigenetic alterations contribute to 15q11-13 gene dysregulation in autism.  The use of human brain samples is critical to further understanding mechanistic explanations and therapeutic solutions.