Tuberous sclerosis complex (TSC) is a genetic disorder that arises from mutations in either the TSC1 or TSC2 gene. These mutations cause regions of abnormal tissue development (lesions) that can occur in a range of organs. Most TSC patients have brain lesions called "tubers" that are associated with a number of neurological disorders. Up to 90% of TSC patients suffer from epileptic seizures, and many do not respond to conventional medications. TSC seizures are usually associated with tubers, although not all tubers cause seizures. It is unknown why some tubers cause seizures (epileptogenic) while others do not (non-epileptogenic). TSC patients with seizures that do not respond to medications may undergo surgery to remove epileptogenic tubers. Our group has pioneered diagnostic imaging methods that assist in the identification of epileptogenic tubers. TSC patients from worldwide referrals come to our institution for diagnostic imaging and treatment, allowing us to assemble an unparalleled repository of brain tissue that was resected to treat intractable epilepsy. We propose to test a novel hypothesis for why some tubers are epileptogenic while others are not by analyzing epileptogenic and nonepileptogenic tissue in our existing biorepository with new technology to understand how genes in the brain are regulated.

The cell has a number of ways to turn genes on and off. One mechanism requires the chemical modification of DNA called "methylation." This type of modification is not permanent like gene mutations, and methylation of DNA may change during development and aging. Abnormal DNA methylation has been reported in a number of neurological disorders, including autism and Alzheimer's disease, as well as in cancer. In the past 3 years, a newer form of methylation, called 5-hydroxymethylcytosine (5hmC), was found to be particularly important in the brain. The role of 5hmC in neurological disorders is just starting to be uncovered, and the role of 5hmC in epilepsy and TSC is completely unknown. However, it is known that genes regulated by 5hmC include those involved in establishing the signaling connections within the brain (synapses). Synapse development can influence seizures; therefore, we hypothesize that abnormal 5hmC is disrupting the regulation of these genes, leading to epileptic seizures. We will test this hypothesis by comparing 5hmC in epileptogenic tubers to non-epileptogenic tubers. To perform this experiment, we will use state-of-the-art genomics technology that can "read" the sequence of DNA in a sample. Using chemical methods, we will isolate fragments of DNA from brain tissue, selecting DNA having 5hmC modifications, and then we will determine their sequence. We will compare these regions between epileptogenic and non-epileptogenic tubers. 5hmC changes associated with genes that are already known to confer risk of epilepsy (often due to mutation or dysregulation) will be further investigated. For these genes we will use the same genomics technology to assess the abundance of the gene product (messenger RNA) to quantify the "activity" of each gene. We will again compare these results between epileptogenic and nonepileptogenic tubers. The results are expected to identify genes with abnormal 5hmC that are contributing to the seizures and may lead to the development of new medicines for epilepsy in TSC. Therefore, the outcomes of this research have a direct impact on TSC patient care.