Cell Modeling and Rescue of a Novel Non-coding Genetic Cause of Glycogen Storage Disease IX

Area of Science:

• Genetics
• Molecular Biology
• Rare Diseases

Background:

• Delayed diagnosis of Mendelian diseases hinders timely treatment.
• Interpreting non-coding variants impacting splicing and gene expression is a diagnostic challenge.
• Two siblings presented with glycogen storage disease (GSD) type IX γ2, having one known pathogenic PHKG2 variant but no second identified variant.

Purpose of the Study:

• To identify the missing pathogenic variant in PHKG2 in siblings with GSD type IX γ2.
• To functionally validate a novel deep intronic splicing variant.
• To demonstrate a therapeutic strategy for reversing aberrant splicing.

Main Methods:

• Whole genome sequencing (WGS) to detect variants.
• RNA sequencing (RNA-seq) using patient blood and a CRISPR-edited HEK293T cell model.
• CRISPR gene editing to install the variant.
• Antisense splice-switching oligonucleotides (ASOs) for therapeutic validation.

Main Results:

• A deep intronic splicing variant in PHKG2 was identified in both siblings.
• Functional studies confirmed the variant's impact on splicing and cellular function consistent with GSD IX γ2.
• Aberrant splicing was successfully reversed using ASOs in the cell model.

Conclusions:

• Deep intronic variants can be a significant cause of rare genetic diseases like GSD IX γ2.
• WGS combined with functional RNA-seq and cell models provides a robust method for identifying and validating non-coding pathogenic variants.
• ASOs offer a promising therapeutic avenue for reversing splicing defects in rare genetic disorders.