Abstract
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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Acknowledgements
The cell lines of the two brothers and two additional patients, one male and one female, with the classic phenotype were obtained from the ‘Cell Line and DNA Bank from Patients Affected by Genetic Diseases’ collection (http://www.gaslini.org/labdppm.htm). This paper was partially financed by grants: Genzyme grant n.104 19/2007, Fondi Ateneo (MURST ex 60%). We gratefully thank Professor Claudio Orlando from the Department of Clinical Physiopathology University of Florence, Florence, Italy, for the technical support for real-time PCR setting-up and a critical discussion of the manuscript.
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Filoni, C., Caciotti, A., Carraresi, L. et al. Unbalanced GLA mRNAs ratio quantified by real-time PCR in Fabry patients' fibroblasts results in Fabry disease. Eur J Hum Genet 16, 1311–1317 (2008). https://doi.org/10.1038/ejhg.2008.109
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DOI: https://doi.org/10.1038/ejhg.2008.109
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