Abstract
We describe a diagnostic workflow integrating deep intronic and RNA sequencing to resolve genetically unsolved Pompe cases. A five-year-old girl with hypertrophic cardiomyopathy, muscle weakness, recurrent respiratory tract infections, elevated CK levels, and low alpha-glucosidase enzyme activity was referred to the metabolism department. Genetic tests for Pompe disease, conducted by two different genetic laboratories, failed to detect any mutation. Two years later, the patient was referred for further genetic testing. As the third-step genetic test in our diagnostic algorithm, following exonic sequencing and MLPA, the GAA whole gene sequencing test was performed and revealed the homozygous NM_000152.5: c.1327-419 A > G variant. RNA sequencing confirmed exon extension, showing that this novel variant created a new donor splice site. Ultimately, the patient was genetically diagnosed and received appropriate treatment. This study highlights the importance of incorporating deep intronic and RNA sequencing is an essential subsequent step in the molecular diagnosis of unsolved Pompe cases, and it further reveals a novel pathogenic variant in the GAA gene.