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The Impact of Genetics In The Care of Patients With Rare Pediatric Epilepsies: Past, Present Future

Epigenetic approaches to epilepsy diagnosis
Heather Mefford

Around 50% of patients with DEEs do not have a known genetic cause. To determine the molecular cause(s) of unsolved DEE, we must examine genetic variants that are beyond the capabilities of current sequencing approaches. DNA methylation is an epigenetic modification of DNA that plays a role in X-chromosome inactivation, imprinting, and regulation of gene expression, and aberrant methylation is found in neurodevelopmental disorders such as Angelman, Prader-Willi, and Fragile X syndromes. Often, rare differentially methylated regions (DMRs) are due to underlying DNA-sequence variations (e.g., GC-rich repeat expansions) that are not detected by standard sequencing technologies. Methylation patterns can also serve as biomarkers of disease; the diagnostic test EpiSign evaluates genome-wide methylation signatures for about 120 monogenic neurodevelopmental disorders (i.e. disorders caused by a single-gene variant), including more than 30 epilepsy syndromes and DEE. To investigate the role of methylation in DEE, we are generating genome-wide methylation data for a large cohort of unsolved patients to identify rare DMRs and methylation signatures. We are validating rare DMRs with long-read sequencing approaches and investigating the effect of DMRs on gene expression using engineered and patient-derived cells. Using the approach, we have identified novel candidate etiologies for DEE.

 

 

Mosaic variants detectable in blood extend the clinico-genetic spectrum of GLI3-related Hypothalamic Hamartoma
Michael Hildebrand

Hypothalamic hamartoma (HH) can be syndromic (e.g. Pallister-Hall syndrome (PHS), HH and mesoaxial polydactyly) or non-syndromic. Most PHS cases have germline variants in GLI3, but a minority remain unresolved. Some non-syndromic HH cases have GLI3 mosaic variants in brain. PHS and non-syndromic HH are regarded as two separate GLI3-related disorders, clinically and genetically. Here we searched for mosaic variants in unsolved cases. High depth exome sequencing was performed on leukocyte-derived DNA in one unsolved PHS and 25 non-syndromic HH cases. We searched for mosaic variants in GLI3 and other HH associated genes. Mosaic variants were confirmed by droplet-digital PCR. The PHS case had a GLI3 stop-gain variant at 6.9% variant allele fraction (VAF). Two non-syndromic cases had GLI3 variants – a stop-gain (VAF 3.7%) and a frameshift (VAF 7.8%). One non-syndromic patient with 3.7% VAF in blood had 35.8% VAF in HH tissue. He had a vestigial extra digit removed adjacent to his left 5th finger. GLI3 mosaicism is associated with a phenotypic spectrum from PHS to HH with subtle extra PHS features, to isolated non-syndromic HH. High depth sequencing permits detection of low-level mosaicism, which is an important cause of both syndromic and non-syndromic HH.

The role of non-coding variants in the pediatric genetic epilepsies
Gemma Carvill

For individuals with pediatric epilepsies without a molecular diagnosis, the genetic underpinnings are likely to be diverse, thus a creative and multi-faceted approach is required to identify causative genetic variants. This challenge is one of the major focuses of our group and we use short and long read genome sequencing to identify novel genetic etiologies outside of the coding regions. Here we will demonstrate how deletion of a long non-coding RNA, CHASERR, can lead to altered gene dosage of the known epilepsy gene, CHD2. We will describe the mechanism of action and the downstream effects of both too much/too little CHD2 and associated clinical and molecular phenotypes. Moreover, we reveal how aberrant splicing of a poison exon in SCN1A leads to haploinsufficiency and clinical phenotypes synonymous with classic truncating SCN1A variants. We will also describe our efforts to characterize poison exons across human brain development and detect other epilepsy-associated genes that harbor these exons, and disease-associated variants. Collectively, these new mechanisms of disease involving non-coding regions of the genome are likely to account for a significant proportion of unexplained cases of rare epilepsies. In addition, the discovery of these elements, and our understanding of how they control gene expression also presents a unique opportunity for gene targeting therapies.

 

The genetic architecture of the pediatric epilepsies
Laura Swanson

The widespread implementation of high-throughput DNA sequencing has revolutionized our understanding of the genetic basis of the pediatric epilepsies, especially the Developmental and epileptic encephalopathies (DEEs). We now know that ~50% of patients have a genetic diagnosis, and that over 300 genes have been identified. In most high-income countries, gene panel testing is available for all new onset epilepsy cases in infants, and young children. Meanwhile in low-middle income countries, these tests are more difficult to access, though several groups are developing targeted-strategies to address these inequities and restrictions to access. A genetic diagnosis is also changing the way we treat and care for patients with DEEs, and there is an exciting move towards precision therapies. At present, this primarily involves avoiding specific anti-seizure medications (ASMs), such as sodium channel blockers in SCN1A-related epilepsies, or recommending other ASMs, such as sodium channel blockers in SCN8A-related epilepsies. However, there is now also much enthusiasm from pharmaceutical and biotech companies in rare disease and gene-targeting approaches to curing DEEs. There are now several clinical trials, and these approaches have the ability to transform lives, by treating the cause, rather than the symptoms of seizures. Finally, the remaining 50% of patients do not have a known genetic cause and in this symposium, we discuss the numerous approaches to finding answers for patients.