Total or partial deficiency of the human lysosomal hydrolase α-galactosidase A is responsible for Fabry disease, the X-linked inborn error of glycosphingolipid metabolism. Together with the predominant α-galactosidase A gene mRNA product encoding the lysosomal enzyme, a weakly regulated alternatively spliced α-galactosidase A transcript is expressed in normal tissues, but its overexpression, due to the intronic g.9331G > A mutation, leads to the cardiac variant. We report the molecular characterization of five Fabry patients including two siblings. Sequencing analysis of the α-galactosidase A gene coding region and intron/exon boundaries identified the new c.124A > G (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 α-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 α-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.9273C>T). These findings strongly suggest a correlation between this new intronic mutation and the unbalanced α-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 α-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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