Identification of Novel and Targetable Pathogenic Non-coding Variants

Abstract

Delayed diagnosis of Mendelian disease substantially prevents early therapeutic intervention that could improve symptoms and prognosis. Although clinical genetics has historically focused on identifying protein-coding variants that cause disease, growing evidence indicates a substantial role for non-coding variants, especially those that alter splicing and/or gene expression. This likely contributes to the incomplete diagnostic yield of exome sequencing in patients with suspected genetic disease. However, non-coding variants present additional challenges in detection, functional interpretation, and classification as pathogenic or benign. In a cohort of genetically undiagnosed Mendelian disease patients, we identified two siblings with glycogen storage disease (GSD) type IX γ2, both of whom had a classic clinical presentation, enzyme deficiency, and a known pathogenic splice acceptor variant on one allele of PHKG2. Despite the autosomal recessive nature of that disease, no variant on the second allele was identified by gene panel sequencing. To identify a potential missing second pathogenic variant, a combination of genome sequencing, short-read RNA-seq, and long-read RNA-seq detected putative deep intronic splicing variant and a corresponding pseudoexon inclusion in both siblings. We confirmed the functional splicing effects of this variant by generating a HEK293T cell model in which we installed the variant using CRISPR editing. That cellular model has genetic, biochemical, and cellular impacts that are consistent with GSD IX γ2 and allow us to screen candidate antisense therapeutics that target aberrant splicing, optimizing a potential personalized therapeutic prior to evaluation in the patient’s own cells. This demonstrates a robust pathway for detecting, validating, and reversing the impacts of novel non-coding causes of rare disease. Expanding the use of this approach could markedly improve the diagnostic yield of genetic sequencing and advance precision medicine.