In pilocytic astrocytomas, which are the most common childhood brain tumours, Lambert et observed a strong positive correlation between methylation and expression, along al. En2 with a number of other key neural developmental genes []. However this methylation frequently involved the gene body rather than the promoter region. Gene body-specific methylation has previously been identified on the active X chromosome in contrast to promoter methylation with corresponding gene silencing of the inactive X chromosome []. It has been hypothesised that such gene body methylation increases levels of transcription via repression of internal gene promoters, suppressing repetitive elements and switching promoter usage within a gene [, ]. Further detailed research involving hypermethylation has been conducted in En2 conditions, such as the autism spectrum disorders. Previous work highlighted genetic linkage of in families with autistic members, along with broad similarities En2 between the neuropathology seen in knockout mice and autistic individuals, resulting in En2 it being considered as an autism susceptibility gene []. James therefore studied et al. En2 promoter region methylation in post-mortem cerebellar cortex specimens from autistic and matched unaffected individuals []. Within the context of global DNA hypermethylation in autistic samples compared with controls, they also demonstrated significant promoter En2 hypermethylation in the autistic examples compared with the control samples (23±4% versus 10±2%; p=0.005), using the McrBC-PCR assay, the same assay that we used in our methylation work. They confirmed these findings using a second assay, MSR-PCR, which demonstrated similar promoter region hypermethylation. The authors were surprised to En2 observe significantly elevated expression levels of mRNA in the autism samples, En2 alongside the promoter region hypermethylation, demonstrating a positive correlation between these variables in the case and control samples (p=0.007). They also confirmed an increased level of EN2 protein in the autism specimens via western blot, consistent with the increase in gene expression. En2 CONCLUSION
In pilocytic astrocytomas, which are the most common childhood brain tumours, Lambert et al. observed a strong positive correlation between En2 methylation and expression, along with a number of other key neural developmental genes []. However this methylation frequently involved the gene body rather than the promoter region. Gene body-specific methylation has previously been identified on the active X chromosome in contrast to promoter methylation with corresponding gene silencing of the inactive X chromosome []. It has been hypothesised that such gene body methylation increases levels of transcription via repression of internal gene promoters, suppressing repetitive elements and switching promoter usage within a gene [, ]. Further detailed research involving En2 hypermethylation has been conducted in conditions, such as the autism spectrum disorders. Previous work highlighted genetic linkage of En2 in families with autistic members, along with broad similarities between the neuropathology seen in En2 knockout mice and autistic individuals, resulting in it being considered as an autism susceptibility gene []. James et al. therefore studied En2 promoter region methylation in post-mortem cerebellar cortex specimens from autistic and matched unaffected individuals []. Within the context of global DNA hypermethylation in autistic samples compared with controls, they also demonstrated significant En2 promoter hypermethylation in the autistic examples compared with the control samples (23±4% versus 10±2%; p=0.005), using the McrBC-PCR assay, the same assay that we used in our methylation work. They confirmed these findings using a second assay, MSR-PCR, which demonstrated similar En2 promoter region hypermethylation. The authors were surprised to observe significantly elevated expression levels of En2 mRNA in the autism samples, alongside the promoter region hypermethylation, demonstrating a positive correlation between these variables in the case and control samples (p=0.007). They also confirmed an increased level of EN2 protein in the autism specimens via western blot, consistent with the increase in En2 gene expression. CONCLUSION
THESIS%20FINAL%20DRAFT%2008JUNE2015.docx