CURE Discovery: Using Patient-Specific Cardiac Cells to Predict SUDEP Risk

CURE-funded researchers are using a novel technique to discover ways to predict patients at an increased risk of Sudden Unexpected Death in Epilepsy (SUDEP). Dr. Lori Isom, her team, and co-investigator Dr. Jack Parent at the University of Michigan are transforming skin cells from patients with developmental and epileptic encephalopathy (DEE) syndromes into induced pluripotent stem cells (iPSCs). The team then generates cardiac cells from the iPSCs which retain the patients’ exact genetic information. These unique, patient-specific cardiac cells are used as models to understand if DEE-associated genes play a role in causing heart abnormalities which may lead to SUDEP. The team also hopes to develop measurable indicators, known as biomarkers, of SUDEP risk.

Severe DEE syndromes, such as Dravet syndrome, are associated with a high incidence of SUDEP. It is estimated that up to 20% of patients with Dravet syndrome die from SUDEP.1 There is still much to be understood about the mechanisms of SUDEP and how to predict who is at risk for it.

Dravet syndrome and other DEEs are often associated with variants in genes, such as SCN1A, SCN1B, and SCN8A. These genes provide instructions to make sodium ion channels, which are very important proteins that help brain cells transmit electrical signals. The same genes are also expressed in the heart; thus, the team hypothesizes that any variants in these genes that disrupt electrical signaling in the brain would affect normal electrical function of the heart as well. In support of this hypothesis, the investigators’ previous work in mouse models of Dravet syndrome and DEEs showed that these mice exhibited irregular heartbeat, which in some cases preceded SUDEP-like events.2-4

In this CURE-funded project, the investigators expanded upon their previous work by testing their hypothesis in heart muscle cells called cardiac myocytes, generated in the laboratory from skin cells of patients with Dravet syndrome or other DEEs using iPSC technology. This Nobel Prize-winning technology involves obtaining skin or blood cells from patients and converting them to iPSCs. These are stem cells that can be converted into almost any specialized cell type in the body, such as heart, muscle, pancreatic, or neuronal cells. The cells are patient-specific, meaning they retain the unique genetic make-up of the patient they originated from, allowing investigators to study cell types which would otherwise be very difficult or impossible to obtain from a living patient.

Dr. Isom, Dr. Parent, and their colleagues previously used iPSC technology to generate heart muscle cells from four patients with variants in the SCN1A gene and found increased sodium currents and spontaneous contraction rates in these cells, suggesting cardiac electrical dysfunction.5 Cardiac abnormalities were subsequently found in the patient with the highest increase in sodium current.5 These data suggest that iPSC-cardiac cells may be useful models for identifying and developing biomarkers, such as increased sodium current, as indicators of SUDEP risk.

The investigators used the same technique to study variants in the SCN1B and SCN8A genes. The team observed that iPSC-cardiac myocytes derived from a patient with SCN1B Dravet syndrome had increased sodium currents similar to those seen in iPSC-cardiac myocytes from the patient with SCN1A Dravet syndrome, suggesting that variants in these two different genes could cause heart abnormalities through similar mechanisms. Preliminary data in iPSC-cardiac myocytes from patients with DEE caused by variants in SCN8A, suggest that these cells have altered beating rates but no change in sodium current, which is aligned with their observations in a mouse model with a variant in SCN8A.

Taken together, these results reveal mechanisms by which different epilepsy-related genes can affect heart function and SUDEP. Future research will investigate the impact of variants of a specific non-ion channel gene to see if it causes altered cardiac beating. Patient-specific iPSC cardiac myocytes are a very useful model to study SUDEP mechanisms and could be developed as diagnostic biomarkers to identify SUDEP risk in patients.

1 Cooper MS et al. Mortality in Dravet Syndrome. Epilepsy Res. 2016 Dec; 128:43-47.
2 Auerbach DS et al. Altered Cardiac Electrophysiology and SUDEP in a Model of Dravet Syndrome. PLoS One. 2013;8(10).
3 Lopez-Santiago LF et al. Sodium channel Scn1b null mice exhibit prolonged QT and RR intervals. J Mol Cell Cardiol. 2007;43(5):636-47.
4 Frasier CR et al. Cardiac arrhythmia in a mouse model of SCN8A Epileptic Encephalopathy. Proc Natl Acad Sci U S A. 2016; in press.
5 Frasier CR et al. Channelopathy as a SUDEP Biomarker in Dravet Syndrome Patient Derived Cardiac Myocytes. Stem Cell Reports. 2018 Sep 11;11(3):626-634.

Schizophrenia is a Later-Onset Feature of PCDH19 Girls Clustering Epilepsy

Objective: To investigate the occurrence of psychosis and serious behavioral problems in females with protocadherin 19 gene (PCDH19) pathogenic variants.

Methods: This study evaluated whether psychosis and serious behavioral problems had occurred in 60 females (age 2-75 years) with PCDH19 pathogenic variants belonging to 35 families. Patients were identified from epilepsy genetics databases in Australia, New Zealand, the United States, and Canada. Neurologic and psychiatric disorders were diagnosed using standard methods.

Results: Eight of 60 females (13%) from 7 families developed a psychotic disorder: schizophrenia (6), schizoaffective disorder (1), or an unspecified psychotic disorder (1). Median age at onset of psychotic symptoms was 21 years (range 11-28 years). In our cohort of 39 females aged 11 years or older, 8 (21%) developed a psychotic disorder. Seven had ongoing seizures at onset of psychosis, with 2 continuing to have seizures when psychosis recurred. Psychotic disorders occurred in the setting of mild (4), moderate (2), or severe (1) intellectual disability, or normal intellect (1). Preexisting behavioral problems occurred in 4 patients, and autism spectrum disorder in 3. Two additional females (3%) had psychotic features with other conditions: an adolescent had recurrent episodes of postictal psychosis, and a 75-year-old woman had major depression with psychotic features. A further 3 adolescents (5%) with moderate to severe intellectual disability had onset of severe behavioral disturbance, or significant worsening.

Significance: Researchers identified that psychotic disorders, including schizophrenia, are a later-onset manifestation of PCDH19 Girls Clustering Epilepsy. Affected girls and women should be carefully monitored for later-onset psychiatric disorders.

Latest Genetic Sequencing Techniques Reveal New Disease Mutations Associated With Epilepsy and Dravet Syndrome

Next-generation sequencing techniques have revealed that genetic mutations in the KCND3 gene may be responsible for more types of epilepsy than previously thought, and new candidate genes associated with Dravet syndrome have been identified, a new study reports.

The study, “Gene mutational analysis in a cohort of Chinese children with unexplained epilepsy: identification of a new KCND3 phenotype and novel genes causing Dravet syndrome,” was published in the journal Seizure.

Dante Labs Unveils 1,000 Patient Epilepsy Whole Genome Study to Advance Research and Therapies for Patients With Epilepsy

Today, Dante Labs launched its Global Epilepsy Whole Genome Study, which will sequence 1,000 patients diagnosed with epilepsy over the next nine months, further advancing the research and development of gene therapies for epilepsy.

The study was first advocated by Epilepsy Awareness Day at Disneyland (EADDL), which will also lead the selection of epilepsy patients for this project.

“Brad and Candy Levy at the Epilepsy Awareness Day at Disneyland have done an amazing job supporting epilepsy patients and their families across the United States and the world,” said Dante Labs CEO Andrea Riposati. “We are excited to dedicate resources to the study of epilepsy.”

Pediatric Epilepsy Genomic Results Should Be Reinterpreted Regularly

In a new report reinterpreting clinical genomic epilepsy test results, about a third of patients had variants reclassified. This led to a clinically significant change in the interpretation in a third of that cohort.

Investigators from the University of Texas Southwestern Medical Center retrospectively reviewed 309 pediatric epilepsy screenings in order to examine the value of reinterpreting previously reported genomic test results. The patients underwent genomic epilepsy testing at a single tertiary care pediatric health care facility between July 2012 and August 2015. The investigators wondered how often genomic test result interpretations change, and so the reinterpretation took place in May 2017.

Genomic testing is used to judge several pediatric neurological diseases, the study authors said. While recent studies have focused on the discovery and identification of new disease relationships using the genomic data, only a few studies have looked into the scope of re-analysis to include all the gene variants of previously reported conditions.

Park said that physicians should consider asking laboratories to reinterpret previously reported genetic test results in a few scenarios, such as:

  • when a period of 13 years has elapsed from the initially reported results;
  • when they are considering making changes to a patient’s medication or other therapies;
  • when they are considering ordering further genetic testing.

A Two-Hit Story: Genetic Mutations Increase the Risk of a ‘Second Hit’ to Developing Severe Epilepsy

SCN1A (NaV1.1 sodium channel) mutations cause Dravet syndrome (DS) and GEFS+ (which is in general milder), and are risk factors in other epilepsies. Phenotypic variability limits precision medicine in epilepsy, and it is important to identify factors that set phenotype severity and their mechanisms. It is not yet clear whether SCN1A mutations are necessary for the development of severe phenotypes or just for promoting seizures. A relevant example is the pleiotropic R1648H mutation that can cause either mild GEFS+ or severe DS.

Researchers used a R1648H knock-in mouse model (Scn1aRH/+) with mild/asymptomatic phenotype to dissociate the effects of seizures and of the mutation per se. The induction of short repeated seizures, at the age of disease onset for Scn1a mouse models (P21), had no effect in WT mice, but transformed the mild/asymptomatic phenotype of Scn1aRH/+ mice into a severe DS-like phenotype, including frequent spontaneous seizures and cognitive/behavioral deficits. In these mice, we found no major modifications in cytoarchitecture or neuronal death, but increased excitability of hippocampal granule cells, consistent with a pathological remodeling.

Therefore, this study claims to demonstrate for their model that an SCN1A mutation is a prerequisite for a long term deleterious effect of seizures on the brain, indicating a clear interaction between seizures and the mutation for the development of a severe phenotype generated by pathological remodeling. Applied to humans, this result suggests that genetic alterations, even if mild per se, may increase the risk of second hits to develop severe phenotypes.

Ultra-Rare Genetic Variation in the Epilepsies: a Whole-Exome Sequencing Study of 17,606 Individuals

Sequencing-based studies have identified novel risk genes for rare, severe epilepsies and revealed a role of rare deleterious variation in common epilepsies. To identify the shared and distinct ultra-rare genetic risk factors for rare and common epilepsies, researchers performed a whole-exome sequencing (WES) analysis of 9,170 epilepsy-affected individuals and 8,364 controls of European ancestry.

The study focused on three phenotypic groups; the rare but severe developmental and epileptic encephalopathies (DEE), and the commoner phenotypes of genetic generalized epilepsy (GGE) and non-acquired focal epilepsy (NAFE).

Researchers observed that compared to controls, individuals with any type of epilepsy carried an excess of ultra-rare, deleterious variants in constrained genes and in genes previously associated with epilepsy, with the strongest enrichment seen in DEE and the least in NAFE. Moreover, they found that inhibitory GABAA receptor genes were enriched for missense variants across all three classes of epilepsy, while no enrichment was seen in excitatory receptor genes. The larger gene groups for the GABAergic pathway or cation channels also showed a significant mutational burden in DEE and GGE.

Although no single gene surpassed exome-wide significance among individuals with GGE or NAFE, highly constrained genes and genes encoding ion channels were among the top associations, including CACNA1G, EEF1A2, and GABRG2for GGE and LGI1, TRIM3, and GABRG2 for NAFE.

Researchers state their study confirms a convergence in the genetics of common and rare epilepsies associated with ultra-rare coding variation and highlights a ubiquitous role for GABAergic inhibition in epilepsy etiology in the largest epilepsy WES study to date.

Diagnostic Yield of Genetic Tests in Epilepsy: A Meta-Analysis and Cost-Effectiveness Study

Article featuring the work of CURE Grantee Dr. Annapurna Poduri, Supported by the Isaiah Stone Foundation Award

Objective: To compare the cost-effectiveness of genetic testing strategies in patients with epilepsy of unknown etiology.

Methods: This meta-analysis and cost-effectiveness study compared strategies involving 3 genetic tests: chromosomal microarray (CMA), epilepsy panel (EP) with deletion/duplication testing, and whole-exome sequencing (WES) in a cost-effectiveness model, using “no genetic testing” as a point of comparison.

Results: Twenty studies provided information on the diagnostic yield of CMA (8 studies), EP (9 studies), and WES (6 studies). The diagnostic yield was highest for WES: 0.45 (95% confidence interval [CI]: 0.33–0.57) (0.32 [95% CI: 0.22–0.44] adjusting for potential publication bias), followed by EP: 0.23 (95% CI: 0.18–0.29), and CMA: 0.08 (95% CI: 0.06–0.12). The most cost-effective test was WES with an incremental cost-effectiveness ratio (ICER) of $15,000/diagnosis. However, after adjusting for potential publication bias, the most cost-effective test was EP (ICER: $15,848/diagnosis) followed by WES (ICER: $34,500/diagnosis). Among combination strategies, the most cost-effective strategy was WES, then if nondiagnostic, EP, then if nondiagnostic, CMA (ICER: $15,336/diagnosis), although adjusting for potential publication bias, the most cost-effective strategy was EP ± CMA ± WES (ICER: $18,385/diagnosis). While the cost-effectiveness of individual tests and testing strategies overlapped, CMA was consistently less cost-effective than WES and EP.

Conclusion: Whole-exome sequencing and epilepsy panel are the most cost-effective genetic tests for epilepsy. Our analyses support, for a broad population of patients with unexplained epilepsy, starting with these tests. Although less expensive, chromosomal microarray has lower yield, and its use as the first-tier test is thus not supported from a cost-effectiveness perspective.

ILAE Consortium Identifies Novel Epilepsy Genes

A genome-wide analysis of nearly 45,000 people has identified 16 regions of DNA associated with epilepsy, 11 of which are newly identified.

The International League Against Epilepsy (ILAE) Consortium on Complex Epilepsies did the analysis, which involved DNA from 15,212 people with epilepsy and 29,677 people without the condition. It is the largest study of its kind. The analysis was published in the Dec 10, 2018 issue of Nature Communications.

Most of these identified genes are associated with generalized epilepsy. The genes have diverse biological functions, including coding for ion-channel subunits, transcription factors and a vitamin B6 metabolism enzyme.

Compared with focal epilepsies, generalized epilepsies appear to have a stronger heritable component. However, fewer single genes have been implicated in generalized epilepsies.

Epilepsy Research Findings: December 2018

Exciting epilepsy research discoveries include two groundbreaking studies. Dr. Steven Petrou created “minibrains” using stem cells to better understand how neurons behave in children with epilepsy. Dr. Harald Sontheimer discovered the previously unknown function of perineuronal nets, which may lead to new treatments for acquired epilepsy. Both Dr. Petrou and Dr. Sontheimer are CURE grantees, and we’re thrilled to see these innovations from them beyond the work they do with us!

In diagnostic news regarding children with epilepsy, scientists are calling for parents to have their children’s genes reviewed at least every two years. This is to ensure their diagnoses and treatments are based on the latest discoveries.

Summaries of all highlighted studies follow below. I’ve organized the findings into four categories: Treatment Advances, Diagnostic Advances, Research Discoveries, and Also Notable.

Treatment Advances

Diacomit Add-On Therapy More Effective in Children with Dravet Syndrome Who Carry Pathogenic SCN1A Mutations, Study Shows

Diacomit (stiripentol) add-on therapy is more effective in children with Dravet syndrome who have pathogenic (disease-causing) SCN1A mutations than in those with variants of unknown significance and benign SCN1A mutations, a study has found.

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GW Pharmaceuticals Announces Second Positive Phase 3 Pivotal Trial for EPIDIOLEX® (Cannabidiol) Oral Solution CV in Patients with Dravet Syndrome

GW Pharmaceuticals announces positive top-line results of the second randomized, double-blind, placebo-controlled Phase 3 clinical trial of EPIDIOLEX® (cannabidiol or CBD) CV in the treatment of seizures associated with Dravet syndrome, a rare and severe form of childhood-onset epilepsy.

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Aquestive Therapeutics Announces FDA Approval for SYMPAZAN™ (clobazam) Oral Film

The FDA approved SYMPAZAN™ (clobazam) oral film for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome (LGS) in patients 2 years of age or older. SYMPAZAN is the first and only oral film FDA-approved to treat seizures associated with LGS. Previously, clobazam was marketed as ONFI® and offered in two formulations – either tablet or oral suspension.

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Diagnostic Advances

Reanalyzing Gene Tests Prompts New Diagnoses in Kids

A new study from UT Southwestern quantifies for the first time how quickly rapid advancements in genomics may benefit patients. Research published in JAMA Pediatrics includes a five-year review of more than 300 epilepsy cases showing nearly a third of children had a change in diagnosis based on new data.

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Research Discoveries

Could Lab-Grown Human Minibrains Help Treat Alzheimer’s and Epilepsy?

Featuring the work of CURE Grantee Dr. Steven Petrou

Florey Institute Director Dr. Steven Petrou leads research creating organoids to mimic the behavior of children’s brains with rare, debilitating forms of epilepsy. Replicating the way neurons behave in children with epilepsy using stem cells in a dish allowed the researchers to tailor a treatment; Petrou is on the verge of announcing a clinical trial of a gene therapy to treat one variant of the disorder.

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Scientists Solve Century-Old Neuroscience Mystery; Answers May Lead to Epilepsy Treatment

Featuring the work of CURE Grantee Dr. Harald Sontheimer

A research team led by Dr. Harald Sontheimer determined that perineuronal nets, whose function was previously unknown, modulate electrical impulses in the brain. Seizures can occur if the nets are dissolved. This discovery may lead to a potential treatment for acquired epilepsy.

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Epidemiology of Status Epilepticus in Adults: A Population-Based Study on Incidence, Causes, and Outcomes

The first population-based study using the International League Against Epilepsy 2015 definition and classification of status epilepticus found an increase of incidence of 10% compared to previous definitions. The study also provides epidemiologic evidence that different patterns of status evolution and level of consciousness have strong prognostic implications.

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Can Genetic Therapy Help Kids with Angelman Syndrome Overcome Seizures?

Scientists at the UNC School of Medicine found evidence that genetic therapy may prevent the enhanced seizure susceptibility common in children with Angelman Syndrome. The research marks the first time scientists reduced seizure susceptibility in mice by activating a dormant copy of the UBE3A gene, so it could replace the faulty mutant version.

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Also Notable

Parents and Researchers Work to Find Cause of Neonatal Epilepsy

Three US families aim to help researchers develop better treatments for neonatal-onset epilepsy with a US-wide study called Early Recognition of Genetic Epilepsy in Neonates (ERGENT). This study provides free-of-charge genetic testing to babies who have features suggestive of a genetically-caused epilepsy.

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Alzheimer’s and Epilepsy: Intimate Connections

Like people with Alzheimer’s disease, people with epilepsy can experience memory loss or confusion. As part of an aura, they may hear or see things that aren’t there. When older adults display these symptoms, they may be misdiagnosed with Alzheimer’s disease, when in fact they are having (or just had) a seizure.

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