A generic assay for the identification of splicing variants that induce nonsense-mediated decay in Pompe disease

Area of Science:

• Genetics
• Molecular Biology
• Biochemistry

Background:

• Genetic variants impacting mRNA expression and processing are often overlooked in genetic diseases.
• Pompe disease, a monogenic disorder, results from acid α-glucosidase (GAA) deficiency.
• Previous assays could miss splicing variants due to mRNA degradation.

Purpose of the Study:

• To develop an extended generic splicing assay capable of detecting aberrant splicing even with mRNA degradation.
• To identify novel splicing variants in Pompe disease patients.
• To enable functional analysis of unknown splicing variants and guide targeted therapeutic development.

Main Methods:

• Inhibition of mRNA degradation using cycloheximide.
• Unbiased splicing analysis of all GAA exons using exon flanking RT-PCR and exon internal RT-qPCR.
• Identification and characterization of novel splicing events and variants.

Main Results:

• Ten novel splicing events were detected in four Pompe disease patients with suspected splicing variants after cycloheximide treatment.
• Natural inclusion of GAA introns 6 and 12 sequences in transcripts from both patients and controls indicated inefficient canonical splicing.
• Aberrant splicing caused by the c.546G>T variant was identified, leading to the development of an antisense oligonucleotide that improved GAA splicing and enzymatic activity.

Conclusions:

• The extended generic splicing assay effectively detects aberrant splicing in the presence of mRNA degradation.
• This assay facilitates the functional analysis of previously undetected splicing variants.
• The approach aids in developing targeted treatment options for genetic diseases like Pompe disease.