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 (ChIP- Seq) 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, a patient 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;screens 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. Disclaimer: Note that reviewers were given the following special instructions for the review of these Exceptional Unconventional Research Enabling Knowledge Accelerations (EUREKA) grant applications: The purpose of the EUREKA initiative is to foster exceptionally innovative research that, if successful, will have an unusually high impact on the areas of science that are germane to the mission of one or more of the participating NIH institutes. EUREKA is for new projects. EUREKA is not for the continuation of existing projects. EUREKA is not for support of pilot projects (i.e., projects of limited scope that are designed primarily to generate data that wll enable the PI to seek other funding opportunities). Rather, it is anticipated that EUREKA projects will begin and be completed during the funding period. Please provide an opinion and assessment of the likelihood the project will exert a sustained, powerful influence on the research field(s) involved. Significance and innovation should be the major determinants of your overall impact score. The approach should be evaluated for general feasibility. An application should score poorly if it is clear to the reviewers that the proposed methodology has no probability at all of being successful, either because it is inherently illogical or because the sae approach has already been attempted and shown not to be feasible. Remember that unavoidable risk, which is intrinsic to novel and innovative approaches, is expected for these applications and reviewers are instructed that the presence or absence of preliminary data should not be taken into account when determining the score. Applications that are good science for standard research type investigation, but not likely to exert a sustained and powerful influence on the field, should be scored down. Also note that the following critiques were prepared by the reviewers prior to the Study Section meeting and are provided in an essentially unedited form. While there is opportunity for the reviewers to update or revise their written evaluation, based upon the group's discussion, there is no guarantee that individual critiques have been updated subsequent to the discussion at the meeting. Therefore, the critiques may not fully reflect the final opinions of the individual reviewers at the close of group discussion or the final majority opinion of the group. Thus the Resume and Summary of Discussion is the final word on what the reviewers actually considered critical at the meeting.
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