Cell-based assays of GLA genetic variants of unknown significance

The enzyme α-galactosidase A (GLA) breaks down the fat called Gb3 in lysosomes (the degrading center in our cells). When GLA is missing or does not work properly, due to mutations in the GLA gene, Gb3 accumulates in lysosomes with consequent damage to some organs and tissues. This condition is referred to as Fabry disease (FD). The severity of the disease depends on several factors including the type of mutation. Over 1,000 different types of GLA mutations have been identified. Most of them are already associated with the severity of the symptoms, but for some of them, called Variants of Uncertain Significance (VUS), this association is still controversial or unknown and further studies are needed. One of the reasons that contribute to these controversies can be found in the assay currently used to measure the enzymatic activity of GLA that adopts very artificial conditions and may not reflect the actual activity of GLA in the lysosome. We propose to develop cell-based assays that may overcome these limits and that may become part of the diagnostic toolkit for FD. We will set up assays that assess the distribution of mutant GLA (does it reach the lysosomes?) and the ability to break down Gb3 in lysosomes (can it prevent or rescue Gb3 storage in lysosomes?). With our expertise in cell biology and equipment of the screening facility at TIGEM, we propose that our lab may become a reference center for the in-cell studies of GLA VUS for Italian families.