Olivia Grischow – Molecular Genetics

Oral presentation (ppt):

Abstract:

Confirmation of somatic variants at ultra-low frequencies in seizure disorders using targeted amplicon sequencing methods

“Background: Somatic mosaicism results from spontaneous mutations arising during embryonic development and yields a genetic variant found only in specific cells in the affected tissue. Next-generation sequencing (NGS) of DNA from disease-affected tissues is revealing somatic mosaicism in increasing numbers of disorders. Epilepsy is a prominent neurological disorder that can be attributed to somatic mutations in brain tissue. We undertook a study of surgically resected brain tissues from pediatric epilepsy patients with treatment-intractable epilepsy and attempted to identify somatic mutations using whole exome sequencing (WES). We found that sensitive detection of low-frequency, mosaic variants was challenging due to the low read depth coverage of WES (~250-fold). If only a few variant-containing reads were present, the actual prevalence of the variant was difficult to ascertain. One solution to this dilemma is the production of high-depth coverage by targeted amplicon sequencing to accurately confirm the variant’s presence and determine its prevalence.

Methods: To date, we have used WES to study 78 brain tissue samples from 25 epilepsy patients. Somatic variants were detected in 11/25 (44%) patients, some of whom had more than one variant detected. In order to validate and better determine the prevalence of somatic candidates discovered through WES, we used a targeted sequencing approach. PCR primers were designed amplify a ~250bp region containing each variant of interest, and the resulting products were used to construct an NGS library. All libraries were pooled and sequenced using either the Illumina MiniSeq or iSeq.

Results: We were able to confirm somatic mosaic variants with tissue prevalence as low as 0.02% and validated all but one candidate variant. All validated variants were present in affected brain tissues but not in comparator blood samples from the same patient. We identified known pathogenic variants in epilepsy genes (e.g., MTOR, SLC35A2) and also identified variants in novel, candidate epilepsy genes (e.g., IGF1R, EEF2).

Conclusions/Discussion: As somatic mosaicism becomes an increasingly prominent contributor to the etiology of neurological disorders, ensuring the validity of a detected variant as well as determining its precise tissue-specific prevalence is increasingly vital. Targeted amplicon sequencing is a rapid and inexpensive technique that permits confident identification of pathogenic mosaic variants at ultra-low frequencies in disease-associated tissue genomes.”