Juvenile Myoclonic Epilepsy (JME) is among the most common types of syndromes of Idiopathic Generalized Epilepsy (IGE) and has a complex genetic inheritance. Although several putative JME susceptibility genes have been identified, the functions of these genes in the brain and their roles in the pathogenesis of seizures are unknown. Linkage and association analysis led us to the bromodomain-containing gene BRD2. BRD2 is a member of BET subfamily of double bromodomain-containing genes implicated in regulating gene expression. We have shown that BRD2 is expressed in developing and adult brain, and recently, that Brd2-null embryos die at mid-gestation with central nervous system malformations. Further, our preliminary data show that heterozygous Brd2 mice exhibit a reduction in the number of GABAergic interneurons in the frontal cortex and, strikingly, heterozygous Brd2 mice exhibit an enhanced sensitivity to chemically-induced seizures. Our working hypothesis is that mis-expression of BRD2 contributes to seizure susceptibility. We have also discovered that intron 2 of BRD2 contains a highly conserved, alternatively spliced exon, which introduces a premature termination codon and we have identified polymorphisms in the lengths of CA-repeats in intron 2 that are associated with JME. Variation in the length of CA-repeats has been shown to affect alternative splicing events in other experimental models. We now wish to test the hypothesis that the polymorphisms in intron 2, in particular variants in CA repeat-length that we have identified in patient DNA that associate with JME, affect splicing of the alternative exon and the levels of the corresponding transcripts, and hence, function of BRD2 products. In Aim 1, we will initially examine the effect of variable CA-repeat lengths on the relative levels of the two alternatively spliced BRD2 transcripts by transient expression of 'mini-gene'constructs containing [exon 2]-[intron 2]-[exon3] of human BRD2 with varying CA-repeat lengths and measuring the ratio of the resulting two transcripts. In Aim 2, we will begin to elucidate the functional significance of the alternatively spliced transcripts by determining whether the alternative transcripts are translated and if so, whether the proteins made affect homodimerization or sub-cellular localization. PUBLIC HEALTH RELEVANCE: Juvenile Myoclonic Epilepsy (JME) is among the most common types of syndromes of Idiopathic Generalized Epilepsy (IGE) and is characterized by adolescent onset and life-long affliction, requiring continual medication to suppress seizures. The proposed experiments will demonstrate whether the JME- associated variants in intron 2 of BRD2 play a direct role in the frequency of alternative splicing, and further, will provide insight into the possible physiological significance of the alternative transcripts, outcomes that are highly relevant to the stated goals of the RFA and the R03 funding mechanism.
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