Background: CaM (calmodulin) mutations are associated with congenital arrhythmia susceptibility (calmodulinopathy) and are most often de novo. In this report, we sought to broaden the genotype-phenotype spectrum of calmodulinopathies with 2 novel calmodulin mutations and to investigate mosaicism in 2 affected families. Methods: CaM mutations were identified in 4 independent cases by DNA sequencing. Biochemical and electrophysiological studies were performed to determine functional consequences of each mutation. Results: Genetic studies identified 2 novel CaM variants ( CALM3 -E141K in 2 cases; CALM1 -E141V) and one previously reported CaM pathogenic variant ( CALM3 -D130G) among 4 probands with shared clinical features of prolonged QTc interval (range 505–725 ms) and documented ventricular arrhythmia. A fatal outcome occurred for 2 of the cases. The parents of all probands were asymptomatic with normal QTc duration. However, 2 of the families had multiple affected offspring or multiple occurrences of intrauterine fetal demise. The mother from the family with recurrent intrauterine fetal demise exhibited the CALM3 -E141K mutant allele in 25% of next-generation sequencing reads indicating somatic mosaicism, whereas CALM3 -D130G was present in 6% of captured molecules of the paternal DNA sample, also indicating mosaicism. Two novel mutations (E141K and E141V) impaired Ca 2+ binding affinity to the C-domain of CaM. Human-induced pluripotent stem cell-derived cardiomyocytes overexpressing mutant or wild-type CaM showed that both mutants impaired Ca 2+ -dependent inactivation of L-type Ca 2+ channels and prolonged action potential duration. Conclusions: We report 2 families with somatic mosaicism associated with arrhythmogenic calmodulinopathy, and demonstrate dysregulation of L-type Ca 2+ channels by 2 novel CaM mutations affecting the same residue. Parental mosaicism should be suspected in families with unexplained fetal arrhythmia or fetal demise combined with a documented CaM mutation.

Developmental and epileptic encephalopathies (DEEs) describe a subset of neurodevelopmental disorders categorized by refractory epilepsy that is often associated with intellectual disability and autism spectrum disorder. The majority of DEEs are now known to have a genetic basis with de novo coding variants accounting for the majority of cases. More recently, a small number of individuals have been identified with intronic <i>SCN1A</i> variants that result in alternative splicing events that lead to ectopic inclusion of poison exons (PEs). PEs are short highly conserved exons that contain a premature truncation codon, and when spliced into the transcript, lead to premature truncation and subsequent degradation by nonsense-mediated decay. The reason for the inclusion/exclusion of these PEs is not entirely clear, but research suggests an autoregulatory role in gene expression and protein abundance. This is seen in proteins such as RNA-binding proteins and serine/arginine-rich proteins. Recent studies have focused on targeting these PEs as a method for therapeutic intervention. Targeting PEs using antisense oligonucleotides (ASOs) has shown to be effective in modulating alternative splicing events by decreasing the amount of transcripts harboring PEs, thus increasing the abundance of full-length transcripts and thereby the amount of protein in haploinsufficient genes implicated in DEE. In the age of personalized medicine, cellular and animal models of the genetic epilepsies have become essential in developing and testing novel precision therapeutics, including PE-targeting ASOs in a subset of DEEs.

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Biallelic pathogenic variants in SZT2 result in a neurodevelopmental disorder with shared features, including early-onset epilepsy, developmental delay, macrocephaly, and corpus callosum abnormalities. SZT2 is as a critical scaffolding protein in the amino acid sensing arm of the mTOR signaling pathway. Due to its large size (3432 amino acids), lack of crystal structure, and absence of functional domains, it is difficult to determine the pathogenicity of SZT2 missense and in-frame deletions. We report a cohort of twelve individuals with biallelic SZT2 variants and phenotypes consistent with SZT2-related neurodevelopmental disorder. The majority of this cohort contained one or more SZT2 variants of uncertain significance (VUS). We developed a novel individualized platform to functionally characterize SZT2 VUSs. We identified a recurrent in-frame deletion (SZT2 p.Val1984del) which was determined to be a loss-of-function variant and therefore likely pathogenic. Haplotype analysis determined this single in-frame deletion is a founder variant in those of Ashkenazi Jewish ancestry. Overall, we present a FACS-based rapid assay to distinguish pathogenic variants from VUSs in SZT2, using an approach that is widely applicable to other mTORopathies including the most common causes of the focal genetic epilepsies, DEPDC5, TSC1/2, MTOR and NPRL2/3.

Purpose: Sub-Saharan Africa bears the highest burden of epilepsy worldwide. A presumed proportion is genetic, but this aetiology is buried under the burden of infections and perinatal insults, in a setting of limited awareness and few options for testing. Children with developmental and epileptic encephalopathies (DEEs), are most severely affected by this diagnostic gap in Africa, as the rate of actionable findings is highest in DEE-associated genes. Methods: We tested 235 genetically naive South African children diagnosed with/possible DEE, using gene panels, exome sequencing and chromosomal microarray. Statistical comparison of electroclinical features in children with and without candidate variants was performed to identify characteristics most likely predictive of a positive genetic finding. Results: Of 41/235 children with likely/pathogenic variants, 26/235 had variants supporting precision therapy. Multivariate regression modelling highlighted neonatal or infantile-onset seizures and movement abnormalities as predictive of a positive genetic finding. We used this, coupled with an emphasis on precision medicine outcomes, to propose the pragmatic Think-Genetics strategy for early recognition of a possible genetic aetiology. Conclusion: Our findings emphasise the importance of an early genetic diagnosis in DEE. We designed the Think-Genetics strategy for early recognition, appropriate interim management and genetic testing for DEE in resource-constrained settings.