Abstracts

Rationale: Next-generation sequencing (NGS) is a transformative technology for the diagnostic evaluation of developmental and epileptic encephalopathy (DEE). Evaluation of NGS panel, exome (ES) and whole genome sequencing (WGS) is required to guide clinical utilization.  Using a trio ES approach, we undertook a first study with a cohort of 30 children with DEE at the Sydney Children’s Hospital (SCH) Genetic Epilepsy Clinic (GEC) who had negative comprehensive first-tier testing. The diagnostic yield was 50% (15/30), was cost-effective and led to the identification of 3 novel causes of DEE (KCNT2, ARV1 and PUM1) (Cell, 2018;172;924-932 and Mol Genet Genomic Med,2018;6;186-199). With the availability of WGS we then sought to evaluate its role in the diagnosis of DEE in a second study. Methods: We defined a cohort of 30 children of SCH GEC with DEE: 15 children undiagnosed after the ES study and 15 additional children undiagnosed after standard diagnostic testing, now including NGS panel.  Trio WGS was performed using the Illumina HiSeqX platform and the in-house platforms Seave, Mity, ClinSV and ROHmer for variant annotation and filtration and identification of mitochondrial and structural variants and regions of homozygosity.  Regular collaborative research meetings discussed findings and collaborations were initiated to clarify pathogenicity of novel variants. Variants were confirmed by Sanger sequencing and results reported back to families. Results: Seven additional diagnoses were made in the 15 ES negative patients: the cumulative diagnostic rate in this cohort was 73%. These diagnoses consisted of novel genetic causes of DEE/ variants in known DEE genes with new supportive functional data (n=4), as well as genetic mechanisms undetectable by ES such as complex structural variants (n=3).A diagnosis was confirmed or suspected in 9 of the 15 panel negative patients (60%) with several additional novel findings being further evaluated. The majority (n=5) were in genes excluded from the panel, 1 variant was missed by the panel for technical reasons, and 3 were novel genetic causes. Conclusions: This study of NGS has provided molecular diagnoses to some of our most complex children with DEE.Our study shows:(i)             the power of WGS to detect variants undetectable by ES and chromosomal microarray,(ii)            the intrinsic limitations of a panel approach,(iii)           the value of reanalysis over time,(iv)          the importance of collaborative multidisciplinary evaluation of novel findings, and(v)           some commonalities in the phenotype of children remaining undiagnosed after NGS; they were more likely to have infantile spasms/ seizures quiescent by age 5, and/or autistic features.Approaches to further optimise diagnostic yield, including exploration of somatic mosaicism and non-coding variants, will be discussed. Funding: NHMRC, NSW Health, Kinghorn Foundation.