Unbalanced GLA mRNAs ratio quantified by real-time PCR in Fabry patients' fibroblasts results in Fabry disease.
Total or partial deficiency of the human lysosomal hydrolase alpha-galactosidase A is responsible for Fabry disease, the X-linked inborn error of glycosphingolipid metabolism. Together with the predominant alpha-galactosidase A gene mRNA product encoding the lysosomal enzyme, a weakly regulated alternatively spliced alpha-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 alpha-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 alpha-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 alpha-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 alpha-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 alpha-galactosidase A mRNAs might play a crucial role in revealing possible genetic lesions and in confirming the pathogenetic mechanisms underlying Fabry disease.
Metabolic and Muscular Unit, Clinic of Pediatric Neurology, AOU Meyer, University of Florence, Florence, Italy.
Camilla
C
Filoni
Anna
A
Caciotti
Laura
L
Carraresi
Maria Alice
MA
Donati
Renzo
R
Mignani
Rossella
R
Parini
Mirella
M
Filocamo
Fausto
F
Soliani
Lisa
L
Simi
Renzo
R
Guerrini
Enrico
E
Zammarchi
Amelia
A
Morrone
D017398:Alternative Splicing / Q000235:genetics; D000795:Fabry Disease / Q000201:enzymology* / Q000235:genetics*; D005347:Fibroblasts / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D006801:Humans; D008297:Male; D016366:Open Reading Frames / Q000235:genetics; D017354:Point Mutation; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000235:genetics; D000519:alpha-Galactosidase / Q000096:biosynthesis* / Q000235:genetics*
D012333:RNA, Messenger; D000519:alpha-Galactosidase