Objective Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the most severe developmental and epileptic encephalopathies. We delineate the genetic causes and genotype–phenotype correlations of a large EIMFS cohort. Methods Phenotypic and molecular data were analyzed on patients recruited through an international collaborative study. Results We ascertained 135 patients from 128 unrelated families. Ninety‐three of 135 (69%) had causative variants (42/55 previously reported) across 23 genes, including 9 novel EIMFS genes: de novo dominant GABRA1, GABRB1, ATP1A3; X‐linked CDKL5, PIGA; and recessive ITPA, AIMP1, KARS, WWOX. The most frequently implicated genes were KCNT1 (36/135, 27%) and SCN2A (10/135, 7%). Mosaicism occurred in 2 probands (SCN2A, GABRB3) and 3 unaffected mothers (KCNT1). Median age at seizure onset was 4 weeks, with earlier onset in the SCN2A, KCNQ2, and BRAT1 groups. Epileptic spasms occurred in 22% patients. A total of 127 patients had severe to profound developmental impairment. All but 7 patients had ongoing seizures. Additional features included microcephaly, movement disorders, spasticity, and scoliosis. Mortality occurred in 33% at median age 2 years 7 months. Interpretation We identified a genetic cause in 69% of patients with EIMFS. We highlight the genetic heterogeneity of EIMFS with 9 newly implicated genes, bringing the total number to 33. Mosaicism was observed in probands and parents, carrying critical implications for recurrence risk. EIMFS pathophysiology involves diverse molecular processes from gene and protein regulation to ion channel function and solute trafficking. ANN NEUROL 2019;86:821–831

Summary Objective To characterize the phenotypic spectrum associated with GNAO1 variants and establish genotype‐protein structure‐phenotype relationships. Methods We evaluated the phenotypes of 14 patients with GNAO1 variants, analyzed their variants for potential pathogenicity, and mapped them, along with those in the literature, on a three‐dimensional structural protein model. Results The 14 patients in our cohort, including one sibling pair, had 13 distinct, heterozygous GNAO1 variants classified as pathogenic or likely pathogenic. We attributed the same variant in two siblings to parental mosaicism. Patients initially presented with seizures beginning in the first 3 months of life (8/14), developmental delay (4/14), hypotonia (1/14), or movement disorder (1/14). All patients had hypotonia and developmental delay ranging from mild to severe. Nine had epilepsy, and nine had movement disorders, including dystonia, ataxia, chorea, and dyskinesia. The 13 GNAO1 variants in our patients are predicted to result in amino acid substitutions or deletions in the GNAO1 guanosine triphosphate (GTP)‐binding region, analogous to those in previous publications. Patients with variants affecting amino acids 207‐221 had only movement disorder and hypotonia. Patients with variants affecting the C‐terminal region had the mildest phenotypes. Significance GNAO1 encephalopathy most frequently presents with seizures beginning in the first 3 months of life. Concurrent movement disorders are also a prominent feature in the spectrum of GNAO1 encephalopathy. All variants affected the GTP‐binding domain of GNAO1, highlighting the importance of this region for G‐protein signaling and neurodevelopment.

Objective We evaluated the yield of systematic analysis and/or reanalysis of whole exome sequencing (WES) data from a cohort of well‐phenotyped pediatric patients with epilepsy and suspected but previously undetermined genetic etiology. Methods We identified and phenotyped 125 participants with pediatric epilepsy. Etiology was unexplained at the time of enrollment despite clinical testing, which included chromosomal microarray (57 patients), epilepsy gene panel (n = 48), both (n = 28), or WES (n = 8). Clinical epilepsy diagnoses included developmental and epileptic encephalopathy (DEE), febrile infection‐related epilepsy syndrome, Rasmussen encephalitis, and other focal and generalized epilepsies. We analyzed WES data and compared the yield in participants with and without prior clinical genetic testing. Results Overall, we identified pathogenic or likely pathogenic variants in 40% (50/125) of our study participants. Nine patients with DEE had genetic variants in recently published genes that had not been recognized as epilepsy‐related at the time of clinical testing (FGF12, GABBR1, GABBR2, ITPA, KAT6A, PTPN23, RHOBTB2, SATB2), and eight patients had genetic variants in candidate epilepsy genes (CAMTA1, FAT3, GABRA6, HUWE1, PTCHD1). Ninety participants had concomitant or subsequent clinical genetic testing, which was ultimately explanatory for 26% (23/90). Of the 67 participants whose molecular diagnoses were "unsolved" through clinical genetic testing, we identified pathogenic or likely pathogenic variants in 17 (25%). Significance Our data argue for early consideration of WES with iterative reanalysis for patients with epilepsy, particularly those with DEE or epilepsy with intellectual disability. Rigorous analysis of WES data of well‐phenotyped patients with epilepsy leads to a broader understanding of gene‐specific phenotypic spectra as well as candidate disease gene identification. We illustrate the dynamic nature of genetic diagnosis over time, with analysis and in some cases reanalysis of exome data leading to the identification of disease‐associated variants among participants with previously nondiagnostic results from a variety of clinical testing strategies.

BackgroundPseudohypoparathyroidism (PHP) is caused by (epi)genetic defects in the imprinted GNAS cluster. Current classification of PHP patients is hampered by clinical and molecular diagnostic overlaps. The European Consortium for the study of PHP designed a genome-wide methylation study to improve molecular diagnosis.MethodsThe HumanMethylation 450K BeadChip was used to analyze genome-wide methylation in 24 PHP patients with parathyroid hormone resistance and 20 age- and gender-matched controls. Patients were previously diagnosed with GNAS-specific differentially methylated regions (DMRs) and include 6 patients with known STX16 deletion (PHPΔstx16) and 18 without deletion (PHPneg).ResultsThe array demonstrated that PHP patients do not show DNA methylation differences at the whole-genome level. Unsupervised clustering of GNAS-specific DMRs divides PHPΔstx16 versus PHPneg patients. Interestingly, in contrast to the notion that all PHP patients share methylation defects in the A/B DMR while only PHPΔstx16 patients have normal NESP, GNAS-AS1 and XL methylation, we found a novel DMR (named GNAS-AS2) in the GNAS-AS1 region that is significantly different in both PHPΔstx16 and PHPneg, as validated by Sequenom EpiTYPER in a larger PHP cohort. The analysis of 58 DMRs revealed that 8/18 PHPneg and 1/6 PHPΔstx16 patients have multi-locus methylation defects. Validation was performed for FANCC and SVOPL DMRs.ConclusionsThis is the first genome-wide methylation study for PHP patients that confirmed that GNAS is the most significant DMR, and the presence of STX16 deletion divides PHP patients in two groups. Moreover, a novel GNAS-AS2 DMR affects all PHP patients, and PHP patients seem sensitive to multi-locus methylation defects.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0175-8) contains supplementary material, which is available to authorized users.

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Background Sudden Unexpected Death in Pediatrics (SUDP) is a tragic event, likely caused by the complex interaction of multiple factors. The presence of hippocampal abnormalities in many children with SUDP suggests that epilepsy‐related mechanisms may contribute to death, similar to Sudden Unexplained Death in Epilepsy. Because of known associations between the genes SCN1A and SCN5A and sudden death, and shared mechanisms and patterns of expression in genes encoding many voltage‐gated sodium channels (VGSCs), we hypothesized that individuals dying from SUDP have pathogenic variants across the entire family of cardiac arrhythmia‐ and epilepsy‐associated VGSC genes. Methods To address this hypothesis, we evaluated whole‐exome sequencing data from infants and children with SUDP for variants in VGSC genes, reviewed the literature for all SUDP‐associated variants in VGSCs, applied a novel paralog analysis to all variants, and evaluated all variants according to American College of Medical Genetics and Genomics (ACMG) guidelines. Results In our cohort of 73 cases of SUDP, we assessed 11 variants as pathogenic in SCN1A, SCN1B, and SCN10A , genes with long‐standing disease associations, and in SCN3A, SCN4A, and SCN9A , VGSC gene paralogs with more recent disease associations. From the literature, we identified 82 VGSC variants in SUDP cases. Pathogenic variants clustered at conserved amino acid sites intolerant to variation across the VGSC genes, which is unlikely to occur in the general population ( p < .0001). For 54% of variants previously reported in literature, we identified conflicting evidence regarding pathogenicity when applying ACMG criteria and modern population data. Conclusion We report variants in several VGSC genes in cases with SUDP, involving both arrhythmia‐ and epilepsy‐associated genes. Accurate variant assessment as well as future studies are essential for an improved understanding of the contribution of sodium channel‐related variants to SUDP.

Early infantile epileptic encephalopathy (EIEE) is a severe disorder associated with epilepsy, developmental delay and intellectual disability, and in some cases premature mortality. We report the case of a female infant with EIEE and strikingly suppressed respiratory dysfunction that led to death. Postmortem research evaluation revealed hypoplasia of the arcuate nucleus of the medulla, a candidate region for respiratory regulation. Genetic evaluation revealed heterozygous variants in the related genes NRXN1 (c.2686C>T, p.Arg896Trp) and NRXN2 (c.3176G>A, p.Arg1059Gln), one inherited from the mother with family history of sudden infant death syndrome (SIDS) and one from the father with family history of febrile seizures. Although there are no previous reports with the digenic combination of NRXN1 and NRXN2 variants, patients with biallelic loss of NRXN1 in humans and double neurexin 1α/2α knockout mice have severe breathing abnormalities, corresponding to the respiratory phenotype of our patient. These observations and the known interaction between the NRXN1 and NRXN2 proteins lead us to hypothesize that digenic variants in NRXN1 and NRXN2 contributed to the phenotype of EIEE, arcuate nucleus hypoplasia, respiratory failure, and death.

Neural tube defects (NTDs), affecting 1-2 per 1000 pregnancies, are severe congenital malformations that arise from the failure of neurulation during early embryonic development. The methylation hypothesis suggests that folate prevents NTDs by stimulating cellular methylation reactions. Folate is central to the one-carbon metabolism that produces pyrimidines and purines for DNA synthesis and for the generation of the methyldonor S-adenosyl-methionine. This review focuses on the relation between the folate-mediated one-carbon metabolism, DNA methylation and NTDs. Studies will be discussed that investigated global or locus-specific DNA methylation differences in patients with NTDs. Folate deficiency may increase NTD risk by decreasing DNA methylation, but to date, human studies vary widely in study design in terms of analyzing different clinical subtypes of NTDs, using different methylation quantification assays and using DNA isolated from diverse types of tissues. Some studies have focused mainly on global DNA methylation differences while others have quantified specific methylation differences for imprinted genes, transposable elements and DNA repair enzymes. Findings of global DNA hypomethylation and LINE-1 hypomethylation suggest that epigenetic alterations may disrupt neural tube closure. However, current research does not support a linear relation between red blood cell folate concentration and DNA methylation. Further studies are required to better understand the interaction between folate, DNA methylation changes and NTDs.