Article

Unbalanced GLA mRNAs ratio quantified by real-time PCR in Fabry patients' fibroblasts results in Fabry disease.

Metabolic and Muscular Unit, Clinic of Pediatric Neurology, AOU Meyer, University of Florence, Florence, Italy.
European Journal of HumanGenetics (impact factor: 4.4). 06/2008; 16(11):1311-7. DOI:10.1038/ejhg.2008.109
Source: PubMed

ABSTRACT 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.

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Keywords

absolute real-time PCR
 
Fabry disease
 
Fabry patients
 
human lysosomal hydrolase alpha-galactosidase
 
intron/exon boundaries
 
intronic g.9331G>A mutation
 
last patients
 
new intronic lesion
 
new intronic mutation
 
normal tissues
 
pathogenetic mechanisms
 
possible alpha-galactosidase
 
predominant alpha-galactosidase
 
reduced enzyme activity
 
spliced alpha-galactosidase
 
spliced mRNAs
 
total RNA preparations
 
two alpha-galactosidase
 
unbalanced alpha-galactosidase
 
X-linked inborn error