Analysis of splice‐site mutations of the α‐galactosidase A gene in Fabry disease

Abstract

Fabry disease is an X-linked disease caused by a defective lysosomal enzyme, alpha-galactosidase A, and characterized by skin lesions and multiorgan involvement, including kidney, heart, and the central nervous system. Currently more than 200 genotypes have been identified, including several aberrant splicing. However, most of the mutation analyses were performed using genomic sequencing only, and therefore some of the splicing mutations were misclassified as missense mutations. In order to predict the splicing event caused by each mutation, we conducted a literature search for all published mutations located near the splice sites, including exonic point mutations, and performed a splice-site score (SSS) analysis. The literature search identified 13 donor-site mutations, including four exonic mutations (S65T, D183S, K213N, and M267I), located at the end of exons 1, 3, 4, and 5, respectively, six acceptor-site mutations, and one new exon creation. All mutated splice sites, except for the one associated with the new exon creation, had a lower SSS than their respective natural sites. Cryptic or newly created sites were identified with SSS from 0.09 to 1.0. The predictions, based on SSS analysis, are in agreement with all six mutations with known cDNA sequence from the literature, including five mutations with exon skipping and one mutation with creation of a new acceptor site. For the S65T genotype, we performed reverse transcription-polymerase chain reaction (RT-PCR) analysis using RNA isolated from the whole-blood sample. We verified that a weak cryptic site (SSS = 0.09) 14 nucleotides downstream was activated and resulted in an insertion of 14 bp and a frameshift stop at codon 106. This change is more consistent with the clinical presentation of the patient, the classical Fabry disease, than the amino acid substitution (S65T), which does not affect the enzyme function. In conclusion, the SSS analysis is very useful for predicting splicing events and genotype/phenotype correlation in Fabry disease. As different mechanisms may be involved in pre-mRNA splicing, it is important to obtain cDNA sequencing for molecular diagnosis.

Angiokeratoma Corporis Diffusum in a Patient With No Recognizable Enzyme Abnormalities

“…17,19 Perhaps gene mutational analysis could be helpful in this regard, as over 200 mutations have been described in Fabry disease alone. 27 Alternatively, our patient's lipid droplets may represent a very early manifestation of a glomerular disorder, such as minimal change disease. 28 Regardless, his renal function will need to be monitored.…”

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

“…The overexpression of a cryptic exon in intron 4, leading to extremely unbalanced ratios of the c.639ins + 57 over the wildtype GLA transcript, has already been identified as the underlying cause of deficient αGal activity in ( The hypothesis that the non-canonical c.639ins + 57 GLA transcript is overexpressed in patients carrying the g.9331G > A SNP due to increased recognition of the alternative splicing by an A/C-rich enhancer-type ESE (4), generated by the G > A transition, has been disputed (22) since that SNP occurs at a non-consensus site of the cryptic donor splice site and there are no functional ESE motifs (23) within the surrounding intronic region. 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).…”

Hemophilia A Patients with Undetectable Mutations: Current Knowledge and Future Directions*

“…Detailed analysis of cDNA by reverse transcriptase PCR (RT-PCR) represents a powerful tool for searching causative splicing mutations or gene rearrangements. This strategy is applied with disease-related genes in patients who do not show mutations in the coding regions at the DNA level [9,10]. The same strategy was previously applied as the first-line mutation screening method for the F8 and F9 genes [11][12][13].…”