The Association of BRD2 with Juvenile Myoclonic Epilepsy Abstract Idiopathic generalized epilepsy (IGE) constitutes a variety of epileptic disorders in humans, the most common being juvenile myoclonic epilepsy (JME).
There is strong evidence that JME can be caused by a faulty genetic variation in the human gene bromodomain-containing protein 2 (BRD2). For this reason, many studies on BRD2 homologues have been conducted to determine the function of this protein. Studies on human BRD2 have revealed that certain polymorphisms in non-coding regions near the promoter are associated with JME.Knock-out experiments in mice reveal that the Brd2 deficient mice are smaller, display embryonic lethality, have slower cell proliferation in mouse embryonic fibroblasts and display defects during neurogenesis. Furthermore, studies in zebrafish confirm that Brd2 has a role in the development of vertebrates. Introduction Juvenile myoclonic epilepsy (JME) is a form of idiopathic generalized epilepsy (IGE). JME is characterized by monoclonic seizures, which consists of small jerks of the arms, shoulders and sometimes legs.
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It accounts for 26% of all IGEs and begins by late childhood or early adolescence1.Many forms of IGEs have been linked to a single genetic mutation that follows Mendelian Inheritance; however it appears that there is a more complex mechanism that causes JME2,3. Another insight on the causation of this disorder is that there are many genes associated with JME, in particular the EJM1 locus on chromosome 6, as shown by Greenberg and collegues by linkage analysis4. EJM1 was later found to include bromodomain-containing protein 2 (BRD2)1,3,5. BRD2 has been characterized as a transcriptional activator of proteins involved in the cell cycle6.This has generated an interest in determining the function of BRD2 in embryogenesis. Researchers studying orthologs of BRD2 have discovered a role in brain development7,8,9 and this knowledge might help contribute to the diagnosis and treatment of JME.
JME JME is most commonly characterized by myoclonic seizures; however some individuals who have this disorder may also experience tonic-clonic seizures (extensive jerks of all limbs) and absence seizures (where responsiveness and awareness is disabled for a few seconds). Individuals who have two or more of any of these 3 types of seizures may have JME.Other characteristics of JME is that seizures often occur in the morning and that occur more often in females than in males10. Individuals with this disorder will most likely continue to have seizures if not otherwise treated with divalproex sodium. In a study conducted on individuals under 19 with JME, researchers using magnetic resonance imaging found aberrations in the corpus callosum, frontal lobe, hippocampus and prefrontal area of the brain11. The researchers in this study concluded that minimal discrepancies are present during the formation of neural networking in the developing brain of patients with JME11.The mode of JME causation is not completely known. However, it is established that JME arises by genetic inheritance, where a family history of epilepsy is common.
Thus far, five genes have been associated with JME: GABRA1, CLCN2, GABRD, EFHC1 and BRD2. All five of these proteins, even though are not within the same family, have evidence that associates them to improper neural networking. Both GABRA1 and GABRD are GABA receptors that when mutated, as in some individuals with JME, are desensitized to GABA (an inhibitory neurotransmitter) leading to increased neuronal excitability12,13.CLCN2 is also linked to GABA: it codes for a chloride channel present in areas of the brain that is sensitive to GABA and leads to increased neuronal excitability14. EFHC1 mutations was found to cause reduced neuronal cell death during development of mouse embryos15, where apoptosis is a common event during development.
The fact that EFHC1partakes in inducing neuronal apoptosis may mean it is a part of the ras signalling pathway; thus sharing a common feature with BRD2. BRD2: gene and proteinBRD2 is a gene that encodes a transcription factor that is a member of the BET subfamily whose members contain two bromodomains and an extra terminal domain at their carboxyl termini. The bromodomains allow BRD2 to function as a chromatin-remodelling protein as shown by Kanno and colleagues who proposed that BRD2 interacts with acetylated histones16 for the transcriptional activation of cell-cycle genes. BRD2 is known to regulate the expression of genes involved in the cell cycle, such as cyclin D1, cyclin A and cyclin E, in response to ras signalling6.
The ras signalling pathway is activated to ultimately induce cell processes such as apoptosis and cell proliferation (which causes the cells to progress through the cell cycle). Cyclins are also involved in the cell cycle by controlling the progression through each phase of the cell cycle. Studies on the function of BRD2 have linked this protein to the involvement of cell proliferation within the developing embryo and it can be considered as part of the mitogen-activated protein (MAP) kinase class within the ras signal pathway6,9.Furthermore, analysis of the BRD2 gene sequence has revealed that there are many alternatively spliced mRNAs, most whose function are not known17. By analyzing these alternate transcripts to determine their function, it is possible to associate them with JME; thus further connecting BRD2 as a potential causation of this disorder. Few studies were performed on the BRD2 gene itself; however those that did occur found evidence linking to JME.It was first confirmed by Pal and colleagues that BRD2 variation due to single nucleotide polymorphisms (SNPs) in non-coding regions of the gene accounts for the association of BRD2 to JME3.
This study sequenced the exons in BRD2 of patients with JME and found no obvious mutations in the coding region. They did find, however, variations in SNPs in the promoter region that was characteristic of JME individuals. These findings led to the speculation that BRD2 may have a role in brain development10 and subsequent homolog studies have since arisen. Another study analyzed SNP variations in BRD2 of numerous nationalities.Cavalleri et al reported that their studies revealed similar results as in the Pal et al study conducted in 2003 for English and Irish descent, however not other nationalities, such as Australian and South Indian1. The actual role in SNP variation in causing JME is unknown. It is possible that proteins may interact with particular SNP sequences as part of a developmental pathway that results in a proper neural network10.
An additional study further examined the SNPs within BRD2 to find that particular SNP variations have increased sensitivity to flashing lights which resulted in seizures5.A seizure occurring in response to flashing light that is due to visual sensitivity of the brain is called a photoparoxysmal response (PPR). PPR typically occurs in childhood epilepsies, such as JME, thus contributing to the idea that BRD2 is associated with this disorder. Role of BRD2 in other vertebrates Researchers have examined both mouse and zebrafish BRD2 homologs thus far. Two knock-out studies concentrating on the mouse ortholog, Brd2, concluded that Brd2 is required for embryogenesis8,9. Both studies generated Brd2 -/- mutants and observed neural tube defects in developing embryos8,9.In the Shang et al study, Brd2 -/-was found to be lethal, had slow cell proliferation mouse embryonic fibroblasts (MEFs) and that Brd2 deficient embryos were smaller than wild-type (see Figure 1)9.
The fact that Brd2 deficient mice expire shows that Brd2 is a critical transcription factor that functions during embryogenesis. Also, the fact that MEFs proliferate slower than wild-type in vitro is consistent with the concept that human BRD2 regulates genes involved in the cell cycle in response to ras signalling, which is responsible for cell proliferation9.In the Gyuris et al study, the observed neural tube closure defects is in correlation with the expression of Brd2 in the neural tube, which is a structure that forms the nervous system (see Figure 2)8. They also connected that the unfused neural tube led to the disruption of proper hindbrain formation. The association of Brd2 with the developing brain further shows evidence in this protein as a causitive mediator of JME.
Lastly, in a study conducted to examine the expression pattern of BRD2 during development, DiBeneditto et al used zebrafish as an experimental model.The two paralogs that exist in this model, brd2a and brd2b, were found to be expressed during central nervous system formation (most prominent in the head region in later stages; see Figure 3) and during morphogenesis 7. Their studies led to their proposed idea that brd2 paralogs may be maternal effect genes and zygotic genes with role in oogenesis, embryogenesis and neurogenesis7. Aside from expression profiling and knockdown studies, the next step is to perform an ectopic expression study or perhaps an over-expression study to help determine the function of BRD2.