Fabry Disease (FD) is an X-linked multisystemic lysosomal disorder caused by mutations of α-galactosidase (GLA) gene. Only a few of the 450 genetic lesions identified so far have been characterised by in vitro expression studies. Thus the significance of newly identified GLA nucleotide variants sin FD patients which lead to α-galactosidase (GAL-A) amino acid substitutions or intronic changes can be uncertain. We identified three GLA mutations: c155G>A (p.C52Y), c548G>C (p.G183A), c647A>G (p.Y216C) in as many individuals (two male; one female), and performed in vitro expression studies and Western Blot analysis, in order to clarify their functional effects. Reduced GAL-A activity and normal or partially reduced mutant proteins were present in all overexpressed mutant systems in which, three-dimensional structural analysis showed that the active site was not directly involved. We hypothesize that the three new mutations affect the GAL-A protein, leading to conformational FD. When, mutant proteins overexpressed in COS-1 cells and in patients’ lymphocytes were tested in the presence of the 1-deoxygalactonojirimicin (DGJ) chaperone, the p.G183A and p.Y216C systems showed increased GAL-A enzyme activities and protein stabilisation, while p.C52Y was not responsive. We underline that genetic, biochemical and functional studies are helpful in clarifying the consequences of the missense genetic lesions detected in FD. ERT is the elective therapy for Fabry patients but it is not always possible to issue the enzyme’s active form in all involved organs. Our study endorses the hypothesis that an active-site-specific chemical chaperone, which could be administered orally, might be effective in treating GAL-A conformational defects.

Total or partial deficiency of the human lysosomal hydrolase a-galactosidase A is responsible for Fabry disease, the X-linked inborn error of glycosphingolipid metabolism. Together with the predominant a-galactosidase A gene mRNA product encoding the lysosomal enzyme, a weakly regulated alternatively spliced a-galactosidase A transcript is expressed in normal tissues, but its overexpression, due to the intronic g.9331G4A mutation, leads to the cardiac variant. We report the molecular characterization of five Fabry patients including two siblings. Sequencing analysis of the a-galactosidase A gene coding region and intron/exon boundaries identified the new c.124A4G (p.M42V) genetic lesion as well as a known deletion in three patients, whereas in the two remaining patients, no mutations were identified. To evaluate possible a-galactosidase A gene transcription alterations, both predominant and alternatively spliced mRNAs were quantified by absolute real-time PCR on total RNA preparations from the patients' fibroblasts. An impressive reduction in the predominant a-galactosidase A transcript was detected in the last patients (Pt 4 and Pt 5). However, the alternatively spliced mRNA was dramatically overexpressed in one of them, carrying a new intronic lesion (g.9273C4T). These findings strongly suggest a correlation between this new intronic mutation and the unbalanced a-galactosidase A mRNAs ratio, which could therefore be responsible for the reduced enzyme activity causing Fabry disease. The real-time assay developed here to investigate the two a-galactosidase A mRNAs might play a crucial role in revealing possible genetic lesions and in confirming the pathogenetic mechanisms underlying Fabry disease.

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