Abstract
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 a 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. This case series reports two unrelated male patients with clinical, enzymatic, and pathological features consistent with FD, who tested negative for pathogenic variants in the alpha-galactosidase A (GLA) via Sanger sequencing and NGS. 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 quantitative real-time PCR showing significantly reduced expression of normal GLA transcripts. 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.