Fabry disease: novel alpha-galactosidase A 3'-terminal mutations result in multiple transcripts due to aberrant 3'-end formation.

Mutations in the gene that encodes the lysosomal exoglycohydrolase, alpha-galactosidase A (alpha-GalA), cause Fabry disease, an X-linked recessive inborn error of glycosphingolipid catabolism. Human alpha-GalA is one of the rare mammalian genes that has its polyadenylation signal in the coding sequence and lacks a 3' untranslated region (UTR). We identified two novel frameshift mutations, 1277delAA (del2) and 1284delACTT (del4), in unrelated men with classical Fabry disease. Both mutations occurred in the 3' terminus of the coding region and obliterated the termination codon, and del2 also altered the polyadenylation signal. To characterize these mutations, 3' rapid amplification of cDNA ends (RACE) and polymerase chain reactions (PCR) were performed, and the amplicons were subcloned and sequenced. Both mutations generated multiple transcripts with various lengths of 3' terminal sequences, some elongating approximately 1 kb. Mutant transcripts were classified as follows: type I transcripts had terminal in-frame thymidines that created termination codons when polyadenylated, type II had downstream termination codons within the elongated alpha-GalA sequence, and type III, the most abundant, lacked termination codons at their 3' ends. To determine if the type III transcripts were degraded by the recently described cytosolic messenger RNA degradation pathway for messages lacking termination codons, northern blot analysis was performed. However, the finding of similar levels of nuclear and cytoplasmic alpha-GalA mRNA in normal and patient lymphoblasts suggested that mRNA degradation did not result from either mutation. Expression of representative transcript types revealed differences in intracellular localization and/or protein stability and catalytic activity, with most mutant proteins being nonfunctional. Characterization of these 3' mutations identified a novel molecular mechanism causing classical Fabry disease.