Negative for SCN1A. Today the first major paper by the EuroEPINOMICS-RES consortium was published in the American Journal of Human Genetics online. As you might recall from some of our previous posts, RES has worked on gene identification in patients with Dravet Syndrome negative for SCN1A using trio exome sequencing. A significant fraction of patients turned out to be positive for SCN1A with mutations initially missed using conventional sequencing techniques. However, there was also a second gene that we discovered in an initial cohort of patients with SCN1A-negative Dravet Syndrome. This gene was CHD2. While working on the functional studies in zebrafish, CHD2 was also discovered as a novel gene for epileptic encephalopathies by both Carvill and collaborators and the Epi4K consortium. These parallel discoveries clearly highlight the relevance of this gene in human epilepsy and suggest that CHD2 mutations might be more common than mutations in many of the other candidate genes discovered in the last 12 months. In addition, when looking closer, the phenotype of the patients was not exactly Dravet Syndrome, but might represent a novel fever-related epileptic encephalopathy.
Dravet Syndrome. Dravet Syndrome is a one of the more common epileptic encephalopathies, and mutations or deletions in SCN1A can be identified in the vast majority of patients. In addition, in up to one third of patients with typical Dravet Syndrome initially negative for SCN1A, mutations can subsequently be identified through gene panel or exome sequencing, suggesting that Dravet Syndrome is a genetically homogeneous disorder. Nevertheless, up to 20 percent of patients are negative for SCN1A mutations, particularly if the phenotype is slightly expanded. Some female Dravet patients have mutations in PCDH19, even though seizure onset is later. For the RES project, we included patients with Dravet Syndrome and borderline Dravet Syndrome to search for novel genes. We performed trio-based exome sequencing in 9 patient-proband trios with SCN1A negative fever-associated epileptic encephalopathies. This is how we stumbled upon two patients with mutations in CHD2, a gene that I personally did not believe in for almost an entire year.
CHD2. In “The Five Temptations of a CEO” motivational author Patrick Lencioni tells us about the three most powerful words that a project leader can say. These words are “I WAS WRONG”. I had written off CHD2 as a genetic mirage, a chance finding due to genomic noise with little chance of validation in other cohorts. In retrospect, I am thankful for the perseverance of everybody else, particularly the Antwerp group, as I was incorrect on both accounts. CHD2 is actually a highly mutation-resistant gene, and we identified a third patient with a de novo CHD2 mutation after screening a cohort of 150 patients with fever-associated epileptic encephalopathies. Likewise, this gene was found by three additional exome studies, including the gene panel study by Carvill and collaborators and the publication of the Epi4K paper. In addition, a CHD2-positive patient had been described by Rauch and collaborators in a study on non-syndromic intellectual disability, a finding that was hidden in the Supplement of their Lancet paper. CHD2 stands for chromodomain helicase DNA binding protein 2. This suggests that the role of this gene is in the nucleus close to the DNA, but does not tell us anymore regarding a possible function of this gene in the context of epilepsy. Taken together, these findings suggest that CHD2 is a recurrent cause of epileptic encephalopathy. And of course there are at least five individuals with well-described epilepsy identified with a small deletion spanning CHD2.
The CHD2 phenotype. When we reviewed the phenotypes of the three patients with CHD2 mutations, we were surprised by the relatively late onset, which started between 14 months and 3 years. Patients had one or more febrile seizures, which were then soon followed by other seizure types including predominant generalized tonic-clonic seizures and absence seizures. In addition, all patients had myoclonic seizures, but not the typical alternating hemiclonic seizures seen in patients with typical Dravet Syndrome. All three patients had frequent generalized EEG discharges. We felt that for a typical Myoclonic Astatic Epilepsy, the generalized tonic-clonic seizures were too prominent, and for typical Dravet Syndrome, the onset was too late. Seizures in the three patients that we reported were fever-sensitive.
Danio rerio. In order to further assess the role of CHD2 in epilepsy, we investigated the role of this gene using a zebrafish model system. For us as a consortium, this was the first time that we reached out to a group working on a zebrafish model, and the Leuven group worked tirelessly to learn as much about this gene in an epilepsy animal model as possible in a very short time frame. In brief, CHD2 was knocked down using morpholino antisense oligonucleotides. In animals with reduced CHD2 expression, twitching movements were identified, and these movements were found to represent seizures when some of the larvae were measured electrographically. These experiments suggest that the human phenotype can be modeled in zebrafish to a certain extent. This experimental platform may eventually be used for other candidate genes, as it allows for a medium-throughput screen with a reliable phenotypic readout. Given the plethora of gene findings that emerge from large-scale genetic studies, this is the model system that we have been waiting for.
How to assess epileptic activity in zebrafish larvae (video above). On the left panel, movements of a control zebrafish larva are shown; on the right, PTZ is added. PTZ is a proconvulsant that leads to increased movements of the larva, which is a correlate of epileptic seizures. These seizures can then be characterized more thoroughly using field recordings. Several larvae can be imaged in parallel, and the movement can be analyzed in a computerized fashion. This allows researchers to use zebrafish larvae as a medium throughput screening system for both gene knock-downs and pharmacological tests.
Future directions. CHD2 encephalopathy is an emerging epilepsy syndrome with a phenotypic spectrum between Dravet Syndrome and Myoclonic Astatic Epilepsy. It will be interesting to learn more about the CHD2-related phenotypes including the natural course of the epilepsy and response to antiepileptic drugs. In addition, as the function of CHD2 in the context of epilepsy is unknown, this gene may hint at novel pathogenic mechanisms in human epilepsy that may be used for novel treatment options. Finally, as CHD2 encephalopathy has been successfully modeled in zebrafish, this system may be used for high-throughput drug screening.