Enhancing Fabry disease screening and diagnostic efficiency: Integration of enzyme, biomarker, and next-generation sequencing testing.

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by a deficiency of alpha-galactosidase A (α-Gal A) due to pathogenic variants of the GLA gene. This study reports findings from data collected through The Lantern Project, including results from α-Gal A enzyme activity and globotriaosylsphingosine (lyso-Gb3) biomarker assays from dried blood spots, as well as next-generation sequencing (NGS). A total of 513 enzyme tests, 284 lyso-Gb3 tests, and 994 NGS tests were conducted on 1380 individuals (708 female, 661 male, 11 sex unknown) with clinical suspicion, or family history or abnormal newborn screening (NBS) results who participated in the project from December 2018-April 2023. Among these individuals, 21 % (103 male and 2 sex unknown) had abnormal α-Gal A levels (range 0.054-1.069 μM/h, normal level ≥ 1.10 μM/h), and 70 % (79 female, 115 male and 2 sex unknown) had elevated lyso-Gb3 levels (range 1.12-130.56 ng/mL, normal level ≤ 1.11 ng/mL). A total of 137 different reportable variants have been identified in this cohort including novel variants c.[351T>G;361G>C] (p.I117M;A121P), c.370-558_370-1del, c.548del, and c.1165C>T (p.P389S). All female patients with loss-of-function (LOF) variants and biomarker results were found to have elevated lyso-Gb3 levels. In contrast to those with LOF variants, females with missense GLA variants had various lyso-Gb3 results. Pathogenic (P)/likely pathogenic (LP) missense variants [including c.1087C>T (p.R363C), c.1088G>A (p.R363H), c.593T>C (p.I198T), c.644A>G (p.N215S), c.335G>A (p.R112H), c.337T>C (p.F113L), and c.835C>G (p.Q279E)] were identified in 16 female patients with normal lyso-Gb3 levels. These findings suggest that the use of lyso-Gb3 testing without GLA sequencing may result in missed diagnosis in some female patients with FD missense variants. Different biochemical and DNA variant profiles have been observed between NBS and non-NBS male patients. Among the non-NBS males, both enzyme and biomarker results correlate with age at the time of testing, which appear to be an approximated indicator for age of onset and disease severity. Among all the NBS males with both enzyme and lyso-Gb3 results, abnormal α-Gal A levels were found in all having P/LP variants except for the 11 who had c.427G>A (p.A143T) or c.870G>C (p.M290I), while normal lyso-Gb3 results have been found in many males with various P/LP variants - though it should of course be noted that lyso-Gb3 levels may increase over time. Our observation in NBS male patients may suggest that the enzyme test has a higher sensitivity, while lyso-Gb3 test has a higher specificity, and when combined with DNA test results, can provide a more comprehensive and reliable result. This manuscript presents the largest-to-date, comprehensive, multi-testing Fabry cohort with demographic information and biochemical phenotypes from a single clinical laboratory. The results from this dataset demonstrate that the integration of enzyme, biomarker, and NGS testing can enhance screening/diagnostic efficiency for FD, particularly for female patients.