Improving molecular diagnosis of fabry disease: functional validation of novel splicing variants in GLA

Background Fabry disease (FD) is an X-linked lysosomal storage disorder caused by pathogenic variants in the GLA gene. Although genetic testing is the gold standard for FD diagnosis, the identification of numerous splicing variants of unknown pathogenicity poses a significant diagnostic challenge. This uncertainty limits the clinical utility of sequencing in guiding patient management and therapeutic intervention. Methods A combination of in silico prediction tools and in vitro minigene splicing assays was utilized to explore the pathogenicity and underlying molecular mechanism of novel deep non-coding variants identified via long-read sequencing in clinically suspected but genetically unsolved patients. Results Functional assays revealed that the large deep-intronic insertion c.640-613ins1715 fundamentally disrupts local splicing pattern and introduces new non-canonical splicing sites. It induces a complex array of aberrant events, including partial exon skipping, intron retention, and pseudoexon inclusion, generating five distinct abnormal transcripts alongside residual normal transcript. These aberrant transcripts introduce frameshifts predicted to yield truncated, non-functional proteins. Consequently, this variant was definitively classified as Pathogenic. Conversely, neither the two deep-intronic single nucleotide variants (c.640-363C > T and c.639+761 G > A) nor their cis configuration exhibited aberrant splicing in our minigene system. However, due to the proband’s later-onset FD phenotype, ambiguous population frequencies (gnomAD), and the potential for tissue-specific splicing missed by in vitro models, both variants remain classified as VUS. Conclusions This study expands the mutational spectrum of the GLA gene and underscores the complexity of deep-intronic variants. Our findings highlight the critical role of robust functional splicing assays in interpreting variant pathogenicity, establishing accurate genotype-phenotype correlations, and ultimately facilitating precision medicine for FD.