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

Abstract

Total or partial deficiency of the human lysosomal hydrolase a-galactosidase A is responsible for Fabry disease, the X-linked inborn error of glycosphingolipid metabolism. Together with the predominant a-galactosidase A gene mRNA product encoding the lysosomal enzyme, a weakly regulated alternatively spliced a-galactosidase A transcript is expressed in normal tissues, but its overexpression, due to the intronic g.9331G4A mutation, leads to the cardiac variant. We report the molecular characterization of five Fabry patients including two siblings. Sequencing analysis of the a-galactosidase A gene coding region and intron/exon boundaries identified the new c.124A4G (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 a-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 a-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.9273C4T). These findings strongly suggest a correlation between this new intronic mutation and the unbalanced a-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 a-galactosidase A mRNAs might play a crucial role in revealing possible genetic lesions and in confirming the pathogenetic mechanisms underlying Fabry disease.

Identification of Cryptic Novel α-Galactosidase A Gene Mutations: Abnormal mRNA Splicing and Large Deletions

“…In most intronic GLA gene mutations in subjects with FD, mutations are located in only the GT/AG exon/intron boundary or near the boundary; however, this mutation is located deep in the intron. There have been several other reports of deep intronic mutation in the GLA gene that might affect the mRNA splicing process (Ishii et al 2002; Filoni et al 2008;Pisani et al 2012). Especially those reports show that deep intronic point mutation could have a function of alternative splicing enhancer switch, because it recruits an alternative exon 4 and downregulates the normal GLA mRNA expression level (Ishii et al 2002;Filoni et al 2008).…”

Individuals With Normal GLA Gene Sequence May Present Abnormally Spliced Alpha-Galactosidase mRNA Transcripts

“…Bioinformatic analyses of the g.9273C > T variant indicated that this transition does not significantly change the predicted acceptor site score, in comparison to the wildtype sequence, and that the highly predominant expression of the c.639ins + 57 transcript might be explained by the creation of a novel ESE (8).…”

“…A further, peculiar type of splicing defect identified in FD patients (4,8) is the overexpression of the non-canonical c.639ins + 57 transcript, in association with two deep intronic single nucleotide variants in intron 4, allegedly mediated by exonic splicing enhancer (ESE)-related mechanisms.…”

Use of lissamine rhodamine ceramide trihexoside as a functional assay for alpha-galactosidase A in intact cells

“…4 , the heterozygous status of a female patient could be determined even though the activity against 4-MUG in homogenates of her fi broblasts was found to be in the low normal range. In addition, studies have shown that patients can be symptomatic despite the presence of a normal AGA gene ( 11,24 ). In contrast, at least one known AGA variant (D313Y) can produce a defi ciency of AGA activity in plasma in otherwise asymptomatic patients ( 25 ).…”

“…After 18 h incubation, the cells were collected by centrifugation, washed twice with PBS, and resusease ( 10 ), genetic screening of women suspected of having the disorder requires a complete sequencing of the GLA gene unless the familial mutation is known. In addition, Filoni et al recently reported a patient and his sister with symptomatic disease but with no mutation in the GLA gene ( 11 ). Overproduction of a splice variant of the AGA resulted in interference of the function of the normal enzyme with resultant accumulation of CTH.…”