International Meeting for Autism Research: Genetic and epigenetic analysis of SHANK3 in humans and mice

Genetic and epigenetic analysis of SHANK3 in humans and mice

Friday, May 13, 2011: 5:15 PM
Douglas Pavilion A (Manchester Grand Hyatt)
4:45 PM
Y. H. Jiang, Pediatrics, Duke University, Durham, NC

SHANK3 is a synaptic scaffolding protein enriched in the postsynaptic density of excitatory synapses. Point mutations in the SHANK3 gene have been identified in individuals with autism spectrum disorder (ASD) and these mutations are isoform specific.  SHANK3 lies also within the critical region of chromosome 22q13.3 microdeletion syndrome (Phelan-McDermid syndrome), which includes ASD as a major clinical feature. SHANK3 protein has 5 conserved domains that interact with diverse of synaptic proteins at postsynaptic density. SHNAK3 gene displayed tissue specific DNA methylation for its 5 CpG islands in different brain regions. 


To test a hypothesis that epimutation of DNA methylation of SHANK3 in brains contribute to the susceptibility of idiopathic ASD cases.  To understand the role of SHANK3 protein in synaptic function and model the pathogenesis of ASD caused by SHANK3 deficiency in mice.


We carried out extensive DNA methylation profiling for SHANK3 CpG islands using pyrosequencing and bisulfite genomic sequencing methods. We also performed RNA expression analysis using real time RT-PCR analysis in postmortem ASD brain tissues. We used mouse embryonic stem cell gene targeting and chromosomal engineering approach to generated Shank3 mutant mice by deleting a different set of Shank3 coding exons. We analyzed Shank3 mutant mice by biochemical, morphological, neurophysiological, and neurobehavioral methods.


We have discovered both human and mouse SHANK3 gene have a complex transcriptional pattern with multiple promoters and extensive alternative splicing of coding exons.   Through analysis of a large set of ASD brain tissues and controls, we discovered ASD brain tissues have significant increase DNA methylation (epimutation) in selective promoter bound intragenic CpG islands of SHANK3. We further showed that increased DNA methylation of CpG islands is associated with aberrant SHANK3 isoform specific expression and alternative splicing of coding exons. The epimuations of SHANK3 is associated with repressed chromatin conformation of SHANK3. These data  support a causative association between epimutation of SHANK3 and the pathogenesis of ASD in these cases. We produced three different lines of Shank3 mutant mice by disrupting a different set of Shank3 coding exon. Shank3 mutant mice lacking the major isoforms display impaired social behaviors, abnormal communication patterns, repetitive behaviors, and deficient cognitive abilities-reminiscent of the core features of ASD in humans.  Shank3 isoform-specific mutant mice have reduced levels of the synaptic proteins at the postsynaptic density, and attenuated response of activity-dependent distribution of the AMPA receptor. The ultrastructure of synapses in Shank3 mutant mice is normal but the maturation of dendritic spine in cultured neurons is impaired. Although synaptic transmission is normal in CA1 hippocampus, long-term potentiation is deficient in Shank3e4-9 mice. 


We discovered that epimutation of SHANK3 is by far the most significant molecular defect found in brain tissues of idiopathic ASD. The results from Shank3 mutant mice support the hypothesis that loss of major Shank3 isoforms produces synaptic dysfunctions that lead to behavioral abnormalities that have many similarities to ASD in humans.  The data from epigenetic and genetic analysis Shank3 support a much broad role of SHANK3 in the pathogenesis of ASD.

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