Diagnostic Utility of Genome-wide DNA Methylation Analysis in Genetically Unsolved Developmental and Epileptic Encephalopathies and Refinement of a CHD2 Episignature.

Sequence-based genetic testing currently identifies causative genetic variants in ∼50% of individuals with developmental and epileptic encephalopathies (DEEs). Aberrant changes in DNA methylation are implicated in various neurodevelopmental disorders but remain unstudied in DEEs. Rare epigenetic variations ("epivariants") can drive disease by modulating gene expression at single loci, whereas genome-wide DNA methylation changes can result in distinct "episignature" biomarkers for monogenic disorders in a growing number of rare diseases. Here, we interrogate the diagnostic utility of genome-wide DNA methylation array analysis on peripheral blood samples from 516 individuals with genetically unsolved DEEs who had previously undergone extensive genetic testing. We identified rare differentially methylated regions (DMRs) and explanatory episignatures to discover causative and candidate genetic etiologies in 10 individuals. We then used long-read sequencing to identify DNA variants underlying rare DMRs, including one balanced translocation, three CG-rich repeat expansions, and two copy number variants. We also identify pathogenic sequence variants associated with episignatures; some had been missed by previous exome sequencing. Although most DEE genes lack known episignatures, the increase in diagnostic yield for DNA methylation analysis in DEEs is comparable to the added yield of genome sequencing. Finally, we refine an episignature for CHD2 using an 850K methylation array which was further refined at higher CpG resolution using bisulfite sequencing to investigate potential insights into CHD2 pathophysiology. Our study demonstrates the diagnostic yield of genome-wide DNA methylation analysis to identify causal and candidate genetic causes as ∼2% (10/516) for unsolved DEE cases.

Cation leak: a common functional defect causing HCN1 developmental and epileptic encephalopathy.

Pathogenic variants in HCN1 are an established cause of developmental and epileptic encephalopathy (DEE). To date, the stratification of patients with HCN1-DEE based on the biophysical consequence on channel function of a given variant has not been possible. Here, we analysed data from eleven patients carrying seven different de novo HCN1 pathogenic variants located in the transmembrane domains of the protein. All patients were diagnosed with severe disease including epilepsy and intellectual disability. The functional properties of the seven HCN1 pathogenic variants were assessed using two-electrode voltage-clamp recordings in Xenopus oocytes. All seven variants showed a significantly larger instantaneous current consistent with cation leak. The impact of each variant on other biophysical properties was variable, including changes in the half activation voltage and activation and deactivation kinetics. These data suggest that cation leak is an important pathogenic mechanism in HCN1-DEE. Furthermore, published mouse model and clinical case reports suggest that seizures are exacerbated by sodium channel blockers in patients with HCN1 variants that cause cation leak. Stratification of patients based on their 'cation leak' biophysical phenotype may therefore provide key information to guide clinical management of individuals with HCN1-DEE.

Rare variants in PPFIA3 cause delayed development, intellectual disability, autism, and epilepsy.

PPFIA3 encodes the Protein-Tyrosine Phosphatase, Receptor-Type, F Polypeptide-Interacting Protein Alpha-3 (PPFIA3), which is a member of the LAR protein-tyrosine phosphatase-interacting protein (liprin) family involved in synaptic vesicle transport and presynaptic active zone assembly. The protein structure and function are well conserved in both invertebrates and vertebrates, but human diseases related to PPFIA3 dysfunction are not yet known. Here, we report 14 individuals with rare mono-allelic PPFIA3 variants presenting with features including developmental delay, intellectual disability, hypotonia, autism, and epilepsy. To determine the pathogenicity of PPFIA3 variants in vivo , we generated transgenic fruit flies expressing either human PPFIA3 wildtype (WT) or variant protein using GAL4-UAS targeted gene expression systems. Ubiquitous expression with Actin-GAL4 showed that the PPFIA3 variants had variable penetrance of pupal lethality, eclosion defects, and anatomical leg defects. Neuronal expression with elav-GAL4 showed that the PPFIA3 variants had seizure-like behaviors, motor defects, and bouton loss at the 3 rd instar larval neuromuscular junction (NMJ). Altogether, in the fly overexpression assays, we found that the PPFIA3 variants in the N-terminal coiled coil domain exhibited stronger phenotypes compared to those in the C-terminal region. In the loss-of-function fly assay, we show that the homozygous loss of fly Liprin- α leads to embryonic lethality. This lethality is partially rescued by the expression of human PPFIA3 WT, suggesting human PPFIA3 protein function is partially conserved in the fly. However, the PPFIA3 variants failed to rescue lethality. Altogether, the human and fruit fly data reveal that the rare PPFIA3 variants are dominant negative loss-of-function alleles that perturb multiple developmental processes and synapse formation.

Rates of Status Epilepticus and Sudden Unexplained Death in Epilepsy in People With Genetic Developmental and Epileptic Encephalopathies.

BACKGROUND AND OBJECTIVES: The genetic developmental and epileptic encephalopathies (DEEs) comprise a large group of severe epilepsy syndromes, with a wide phenotypic spectrum. Currently, the rates of convulsive status epilepticus (CSE), nonconvulsive status epilepticus (NCSE), and sudden unexplained death in epilepsy (SUDEP) in these diseases are not well understood. We aimed to describe the proportions of patients with frequently observed genetic DEEs who developed CSE, NCSE, mortality, and SUDEP. Understanding the risks of these serious presentations in each genetic DEE will enable earlier diagnosis and appropriate management. METHODS: In this retrospective analysis of patients with a genetic DEE, we estimated the proportions with CSE, NCSE, and SUDEP and the overall and SUDEP-specific mortality rates for each genetic diagnosis. We included patients with a pathogenic variant in the genes SCN1A, SCN2A, SCN8A, SYNGAP1, NEXMIF, CHD2, PCDH19, STXBP1, GRIN2A, KCNT1, and KCNQ2 and with Angelman syndrome (AS). RESULTS: The cohort comprised 510 individuals with a genetic DEE, in whom we observed CSE in 47% and NCSE in 19%. The highest proportion of CSE occurred in patients with SCN1A-associated DEEs, including 181/203 (89%; 95% CI 84-93) patients with Dravet syndrome and 8/15 (53%; 95% CI 27-79) non-Dravet SCN1A-DEEs. CSE was also notable in patients with pathogenic variants in KCNT1 (6/10; 60%; 95% CI 26-88) and SCN2A (8/15; 53%; 95% CI 27-79). NCSE was common in patients with non-Dravet SCN1A-DEEs (8/15; 53%; 95% CI 27-79) and was notable in patients with CHD2-DEEs (6/14; 43%; 95% CI 18-71) and AS (6/19; 32%; 95% CI 13-57). There were 42/510 (8%) deaths among the cohort, producing a mortality rate of 6.1 per 1,000 person-years (95% CI 4.4-8.3). Cases of SUDEP accounted for 19/42 (48%) deaths. Four genes were associated with SUDEP: SCN1A, SCN2A, SCN8A, and STXBP1. The estimated SUDEP rate was 2.8 per 1,000 person-years (95% CI 1.6-4.3). DISCUSSION: We showed that proportions of patients with CSE, NCSE, and SUDEP differ for commonly encountered genetic DEEs. The estimates for each genetic DEE studied will inform early diagnosis and management of status epilepticus and SUDEP and inform disease-specific counseling for patients and families in this high-risk group of conditions.

Complications of Influenza A or B Virus Infection in Individuals With SCN1A-Positive Dravet Syndrome.

BACKGROUND AND OBJECTIVES: To determine the frequency and spectrum of complications of influenza infection in individuals with SCN1A-positive Dravet syndrome (SCN1A-DS). METHODS: Individuals with SCN1A-DS were identified in neurologists' care at 2 hospitals in Melbourne, Australia, with additional searches of EEG databases, the Victorian PAEDS FluCan influenza database, and the University of Melbourne Epilepsy Genetics Research Program database. Medical records were searched and families questioned to identify individuals who had an influenza infection; reported infections were confirmed by pathology report. For these individuals, we obtained baseline clinical characteristics and clinical details of the influenza infection. RESULTS: Twenty-one of 82 individuals (26%) had 24 documented influenza infections (17 influenza A and 7 influenza B) at age 0.5-25 years (median 4 years). All presented to hospital, 18/24 (75%) for status epilepticus or seizure exacerbations. Recovery was prompt in 18/24 (75%) infections, delayed but complete in 1/24 (4%) and incomplete in 5/24 (21%). One child died from influenza pneumonia, and long-term neurologic sequelae were seen with 4 infections. These individuals were poorly responsive after termination of status epilepticus. Brain imaging in 2 showed cerebral edema and 1 also having imaging features of laminar necrosis. All have ongoing neurologic deficits compared with their baseline, 1 having profound global impairment. DISCUSSION: Our data show that patients with SCN1A-DS are highly susceptible to neurologic complications during and severe sequelae after influenza infection, including moderate to severe persistent neurologic impairments and death. Safe administration of the seasonal influenza vaccine should be prioritized for this population.

Does long-term phenytoin have a place in Dravet syndrome?

Anti-seizure medications that block sodium channels are generally considered contraindicated in Dravet syndrome. There is, however, considerable debate about the sodium-channel blocker phenytoin, which is often used for status epilepticus, a frequent feature of Dravet syndrome. We describe four patients with Dravet syndrome in whom long-term phenytoin therapy reduced seizure frequency and duration. In two patients, phenytoin produced prolonged periods without status epilepticus for the first time. Attempting to wean phenytoin in all patients after 1 to 20 years of use resulted in seizure exacerbation. Reintroducing phenytoin improved seizure control, suggesting phenytoin is beneficial in some patients with Dravet syndrome.

The gain of function SCN1A disorder spectrum: novel epilepsy phenotypes and therapeutic implications.

Brain voltage-gated sodium channel NaV1.1 (SCN1A) loss-of-function variants cause the severe epilepsy Dravet syndrome, as well as milder phenotypes associated with genetic epilepsy with febrile seizures plus. Gain of function SCN1A variants are associated with familial hemiplegic migraine type 3. Novel SCN1A-related phenotypes have been described including early infantile developmental and epileptic encephalopathy with movement disorder, and more recently neonatal presentations with arthrogryposis. Here we describe the clinical, genetic and functional evaluation of affected individuals. Thirty-five patients were ascertained via an international collaborative network using a structured clinical questionnaire and from the literature. We performed whole-cell voltage-clamp electrophysiological recordings comparing sodium channels containing wild-type versus variant NaV1.1 subunits. Findings were related to Dravet syndrome and familial hemiplegic migraine type 3 variants. We identified three distinct clinical presentations differing by age at onset and presence of arthrogryposis and/or movement disorder. The most severely affected infants (n = 13) presented with congenital arthrogryposis, neonatal onset epilepsy in the first 3 days of life, tonic seizures and apnoeas, accompanied by a significant movement disorder and profound intellectual disability. Twenty-one patients presented later, between 2 weeks and 3 months of age, with a severe early infantile developmental and epileptic encephalopathy and a movement disorder. One patient presented after 3 months with developmental and epileptic encephalopathy only. Associated SCN1A variants cluster in regions of channel inactivation associated with gain of function, different to Dravet syndrome variants (odds ratio = 17.8; confidence interval = 5.4-69.3; P = 1.3 × 10-7). Functional studies of both epilepsy and familial hemiplegic migraine type 3 variants reveal alterations of gating properties in keeping with neuronal hyperexcitability. While epilepsy variants result in a moderate increase in action current amplitude consistent with mild gain of function, familial hemiplegic migraine type 3 variants induce a larger effect on gating properties, in particular the increase of persistent current, resulting in a large increase of action current amplitude, consistent with stronger gain of function. Clinically, 13 out of 16 (81%) gain of function variants were associated with a reduction in seizures in response to sodium channel blocker treatment (carbamazepine, oxcarbazepine, phenytoin, lamotrigine or lacosamide) without evidence of symptom exacerbation. Our study expands the spectrum of gain of function SCN1A-related epilepsy phenotypes, defines key clinical features, provides novel insights into the underlying disease mechanisms between SCN1A-related epilepsy and familial hemiplegic migraine type 3, and identifies sodium channel blockers as potentially efficacious therapies. Gain of function disease should be considered in early onset epilepsies with a pathogenic SCN1A variant and non-Dravet syndrome phenotype.