Epilepsy is one of the most common neurological disorders. It is a complex and heterogeneous disease which makes it difficult to precisely diagnose and provide effective treatment. A major and underexplored cause of complex disorders such as epilepsy could be mutations in gene regulatory elements. For example, disruption of these elements and subsequently the gene regulatory networks that are involved in brain development can lead to epilepsy subtypes such as infantile spasms (IS). However, the regulatory elements of brain expressed genes involved in IS are unknown. Using chromatin immunoprecipitation followed by deep sequencing (ChIPSeq) with active enhancer chromatin marks (H3K4me, H3K27ac, p300), we will identify potential enhancers in the mouse embryonic day 16.5 (E16.5) developing forebrain. In order to determine which genes physically interact with these potential enhancers, we will carry out chromatin interaction analysis followed by paired-end tag sequencing (ChIA-PET) on E16. 5 mouse forebrains. Candidate enhancers of genes associated with IS will be tested for forebrain enhancer expression using zebrafish and mouse transgenic enhancer assays. IS patients from two different cohorts will be screened for coding and copy number variant (CNV) mutations. Potential forebrain enhancers that are found within IS-associated CNVs will be assayed for their enhancer activity in mice. IS patients without IS-associated CNVs and coding mutations will be screened for mutations in our characterized enhancers. Potential causative enhancer mutations will be functionally assessed for their enhancer expression in mice compared to the wild type allele and for differential binding affinity to transcription factors. Combined, these results will generate a regulatory landscape of the developing mouse forebrain, identify and functionally characterize potential IS-associated gene regulatory elements, screen IS patients for mutations in these elements and provide novel functional noncoding DNA sequences for the genetic diagnosis of epilepsy. In addition, this study will serve as a model for the functional characterization of gene regulatory elements involved in other complex human diseases.
Epilepsy is a complex and heterogeneous disease which makes it difficult to precisely diagnose and provide an effective treatment. A major cause of complex diseases, such as epilepsy, could be due to mutations in gene regulatory elements that instruct genes when, where and at what levels to turn on or off. Here, we will identify and characterize gene regulatory elements that could be associated with epilepsy and screen epilepsy patients for mutations in them, thus improving the genetic diagnosis of epilepsy.
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