A New Class of Pathogenic Non-Coding Variants in GLA

Background: Fabry disease (FD) exhibits a spectrum of clinical manifestations ranging from mild to severe, posing a diagnostic challenge, particularly in non-classic subtypes. Genetic testing remains a gold standard for precise diagnosis of FD and is pivotal in genetic counseling. Although conventional approaches such as Sanger sequencing and short-read next-generation sequencing (NGS) have been successfully used to diagnose FD, they often fail to detect deep intronic variants, complex rearrangements, or large deletions or duplications. In contrast, long-read sequencing (LRS) enables comprehensive coverage of intronic and repetitive regions, facilitating precise identification of atypical variants missed by conventional methods. Objective: This case series report two unrelated male patients with clinical, enzymatic, and pathological features consistent with FD, who tested negative for alpha-galactosidase A (GLA) mutations via Sanger sequencing and NGS. LRS, RNA sequencing, quantitative real-time PCR (qRT-PCR), and bioinformatic analysis were employed to investigate potential atypical GLA variants. Results: LRS identified novel non-coding variants in both patients. Patient 1 carried a ~1.7-kb insertion within intron 4, corresponding to part of a long interspersed nuclear element-1, while RNA sequencing revealed two new GLA transcripts. Patient 2 harbored a ~2.5-kb insertion within a SINE-VNTR-Alu retroposon element located in the 5′-untranslated region, with qRT-PCR showing significantly reduced expression of normal GLA transcripts. Conclusions: These findings reveal non-coding variants that contribute to the missing heritability in FD, highlight this genomic region as a priority for future investigation, and demonstrate the potential utility of LRS in diagnostic workflows for unresolved FD cases.