c.640-814T>C mutation in deep intronic region of alpha-galactosidase A gene is associated with Fabry disease via dominant-negative effect.

Fabry disease (FD) is a lysosomal storage disorder resulting from mutations in the alpha-galactosidase A (GLA) gene, characterized by pain, skin lesions, renal failure, and cardiac disease. A 60-year-old proband was hospitalized for recurrent atrial fibrillation (AF) that was unresponsive to medication, with cardiac magnetic resonance imaging (CMRI) revealing left ventricular wall hypertrophy and fat infiltration. Whole-exome sequencing (WES) did not reveal any suspicious pathogenic variants. To further assess the diagnosis, endomyocardial biopsy (EMB) and electron microscopy were performed, revealing abundant zebra bodies in cardiomyocytes, consistent with FD. The diagnosis was ultimately confirmed by GLA enzyme activity analysis (<1.00). Further genetic investigations identified a deep intronic variant (c.640-814T>C) within the GLA gene. Minigene experiments demonstrated that this variant affected the splicing of GLA, resulting in the production of a truncated protein (p.Pro214SerfsTer10). Western blotting (WB) showed that the truncated protein was retained, while immunofluorescence (IF) analysis indicated partial lysosomal localization. In vitro assays confirmed that the retained protein was non-functional and exerted a dominant-negative effect on the normal GLA protein. Molecular docking analysis further revealed that the truncated protein could bind to the wild GLA monomer, significantly reducing cellular GLA enzyme activity. These findings indicate that, beyond being non-functional, the c.640-814T>C mutation may also exerts a dominant-negative effect that impairs the function of the wild GLA protein. These results highlight the importance of recognizing deep intronic mutations in the diagnosis and treatment of FD, contributing to a deeper understanding of the molecular mechanisms, enriching mutation databases, and providing insights into genotype-phenotype correlations.