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.
Filoni
Camilla
C
Metabolic and Muscular Unit, Clinic of Pediatric Neurology, AOU Meyer, University of Florence, Florence, Italy.
Caciotti
Anna
A
Carraresi
Laura
L
Donati
Maria Alice
MA
Mignani
Renzo
R
Parini
Rossella
R
Filocamo
Mirella
M
Soliani
Fausto
F
Simi
Lisa
L
Guerrini
Renzo
R
Zammarchi
Enrico
E
Morrone
Amelia
A
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