Amy L. Schneider

DNA methylation signatures ("episignatures") can be used as biomarkers of genetic aberrations, clinical phenotypes, and environmental exposures in rare diseases. Episignatures are utilized in molecular diagnostics and can clarify variants of uncertain significance. A growing number of disease genes, including epilepsy genes, exhibit robust and reproducible episignatures. However, whether SCN1A, the most prominent epilepsy gene, has one or more episignatures has not yet been determined. We generated genome-wide DNA methylation data and performed episignature analysis on 64 individuals with Dravet syndrome due to pathogenic loss-of-function (LOF) variants in SCN1A and seven individuals with early infantile SCN1A developmental and epileptic encephalopathy due to pathogenic gain-of-function (GOF) variants in SCN1A, relative to a large reference database of controls and rare disease episignature-positive cohorts. We analyzed all samples with LOF variants together and performed separate analyses for missense, nonsense, and GOF variant cohorts. A reproducible blood-derived episignature was not evident in any of the cohorts using current analytical approaches and reference data.

Objective: Dravet syndrome (DS) is a developmental and epileptic encephalopathy. Diagnosis is clinical, but ~90% of patients have pathogenic variants in SCN1A. ATP6V0C has recently been proposed as a novel candidate gene for epilepsy, with or without developmental delay. Here we describe two adult patients with a clinical diagnosis of DS associated with ATP6V0C variants. Methods: Patients with developmental and epileptic encephalopathies were evaluated by physicians who are experts in DS, and their clinical diagnosis was correlated with genetic findings. A subgroup of those patients with DS but without known genetic causes were evaluated through gene panels, whole exome sequencing, and chromosome microarray. Phenotype was determined by pediatric and adult chart reviews, interviews, and physical examinations. Results: Of 753 patients with DS, two unrelated individuals with classic features of DS during childhood and adulthood were identified with heterozygous de novo missense variants in ATP6V0C (c.319G > C, p.(Gly107Arg) and c.284C > T, p.(Ala95Val), respectively). Both variants were absent in normal populations and computational prediction algorithms suggested deleterious effects on protein structure and/or function. No disease-causing variants in other genes previously associated with DS were found. Conclusions: Here we describe two adult patients with Dravet-like syndrome and pathogenic/likely pathogenic variants in ATP6V0C. We propose that abnormal ATP6V0C function can, at the severe end of the clinical spectrum, be associated with Dravet-like phenotype. This is relevant, as these patients would not qualify for disease-modifying antisense nucleotide or gene therapies targeting SCN1A.

Objective: To understand the etiological landscape and phenotypic differences between 2 developmental and epileptic encephalopathy (DEE) syndromes: DEE with spike-wave activation in sleep (DEE-SWAS) and epileptic encephalopathy with spike-wave activation in sleep (EE-SWAS). Methods: All patients fulfilled International League Against Epilepsy (ILAE) DEE-SWAS or EE-SWAS criteria with a Core cohort (n = 91) drawn from our Epilepsy Genetics research program, together with 10 etiologically solved patients referred by collaborators in the Expanded cohort (n = 101). Detailed phenotyping and analysis of molecular genetic results were performed. We compared the phenotypic features of individuals with DEE-SWAS and EE-SWAS. Brain-specific gene co-expression analysis was performed for D/EE-SWAS genes. Results: We identified the etiology in 42/91 (46%) patients in our Core cohort, including 29/44 (66%) with DEE-SWAS and 13/47 (28%) with EE-SWAS. A genetic etiology was identified in 31/91 (34%). D/EE-SWAS genes were highly co-expressed in brain, highlighting the importance of channelopathies and transcriptional regulators. Structural etiologies were found in 12/91 (13%) individuals. We identified 10 novel D/EE-SWAS genes with a range of functions: ATP1A2, CACNA1A, FOXP1, GRIN1, KCNMA1, KCNQ3, PPFIA3, PUF60, SETD1B, and ZBTB18, and 2 novel copy number variants, 17p11.2 duplication and 5q22 deletion. Although developmental regression patterns were similar in both syndromes, DEE-SWAS was associated with a longer duration of epilepsy and poorer intellectual outcome than EE-SWAS. Conclusions: DEE-SWAS and EE-SWAS have highly heterogeneous genetic and structural etiologies. Phenotypic analysis highlights valuable clinical differences between DEE-SWAS and EE-SWAS which inform clinical care and prognostic counseling. Our etiological findings pave the way for the development of precision therapies. ANN NEUROL 2024;96:932-943.

Sequence-based genetic testing identifies causative 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. We interrogate the diagnostic utility of genome-wide DNA methylation array analysis on peripheral blood samples from 582 individuals with genetically unsolved DEEs. We identify rare differentially methylated regions (DMRs) and explanatory episignatures to uncover causative and candidate genetic etiologies in 12 individuals. Using long-read sequencing, we identify DNA variants underlying rare DMRs, including one balanced translocation, three CG-rich repeat expansions, and four copy number variants. We also identify pathogenic variants associated with episignatures. Finally, we refine the CHD2 episignature using an 850 K methylation array and 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 variants as 2% (12/582) for unsolved DEE cases.

Objective: SCN1A variants are associated with epilepsy syndromes ranging from mild genetic epilepsy with febrile seizures plus (GEFS+) to severe Dravet syndrome (DS). Many variants are de novo, making early phenotype prediction difficult, and genotype-phenotype associations remain poorly understood. Methods: We assessed data from a retrospective cohort of 1018 individuals with SCN1A-related epilepsies. We explored relationships between variant characteristics (position, in silico prediction scores: Combined Annotation Dependent Depletion (CADD), Rare Exome Variant Ensemble Learner (REVEL), SCN1A genetic score), seizure characteristics, and epilepsy phenotype. Results: DS had earlier seizure onset than other GEFS+ phenotypes (5.3 vs. 12.0 months, p < .001). In silico variant scores were higher in DS versus GEFS+ (p < .001). Patients with missense variants in functionally important regions (conserved N-terminus, S4-S6) exhibited earlier seizure onset (6.0 vs. 7.0 months, p = .003) and were more likely to have DS (280/340); those with missense variants in nonconserved regions had later onset (10.0 vs. 7.0 months, p = .036) and were more likely to have GEFS+ (15/29, χ2 = 19.16, p < .001). A minority of protein-truncating variants were associated with GEFS+ (10/393) and more likely to be located in the proximal first and last exon coding regions than elsewhere in the gene (9.7% vs. 1.0%, p < .001). Carriers of the same missense variant exhibited less variability in age at seizure onset compared with carriers of different missense variants for both DS (1.9 vs. 2.9 months, p = .001) and GEFS+ (8.0 vs. 11.0 months, p = .043). Status epilepticus as presenting seizure type is a highly specific (95.2%) but nonsensitive (32.7%) feature of DS. Conclusions: Understanding genotype-phenotype associations in SCN1A-related epilepsies is critical for early diagnosis and management. We demonstrate an earlier disease onset in patients with missense variants in important functional regions, the occurrence of GEFS+ truncating variants, and the value of in silico prediction scores. Status epilepticus as initial seizure type is a highly specific, but not sensitive, early feature of DS.