A study of the molecular basis of the lysosomal storage disorder fucosidosis

Fucosidosis is a rare, autosomal recessive, lysosomal storage disorder resulting from a deficiency of the enzyme fucosidase. This defect leads to the accumulation in the lysosomes of tissues and excretion in urine of fucose-containing oligosaccharides, glycoasparagines, and glycolipids. The gene encoding lysosomal fucosidase has been mapped to the short arm of chromosome 1 at position 1p34.1-36.1 and consists of eight exons spanning 23kb of DNA. In this thesis the molecular basis of the enzyme defect has been investigated in thirteen fucosidosis patients. The residual fucosidase activity in extracts of fibroblasts, leukocytes and plasma has been characterised and the urinary oligosaccharides investigated by thin layer chromatography. Patient DNA was haplotyped using two previously described restriction fragment length polymorphisms observed with the restriction enzymes, PvuII and BglI. Single strand conformation polymorphism analysis (SSCP) was used to screen the 13 fucosidosis patients for mutations. 8 of the 9 classic fucosidosis patients revealed conformational alterations (89%). These alterations were subsequently sequenced resulting in eight new mutations being identified. One mutation, P5R, was located in the signal peptide of fucosidase. A 10bp deletion, G96fs (TAG), resulting in a frame shift and the production of a stop codon was also identified in exon 1. Analysis of exon 3 revealed three further mutations, W138X, Y211X and a 1bp deletion in the donor splice site, S216fs(TGA). A g→a point mutation was found in the highly conserved splice site sequences of intron 5. Exon 6 contained two mutations, a missense point mutation N329Y and a 1bp insertion, Y330fs(TAA), which results in the production of a stop codon. The latter mutation was found in the same patient as the P5R mutation, both were homozygous. A new polymorphism has also been identified in exon 5, Q281R, which has been shown to be responsible for the Fu1/Fu2 polymorphism of α-fucosidase isoenzymes observed by starch gel electrophoresis. Transient expression of the normal α-fucosidase cDNA in COS cells was established which allowed the further investigation of the mutations identified. W138X, S216fs(TAG), and both alleles of the polymorphism, Q281R, were created by site directed mutagenesis of normal α-fucosidase cDNA and characterisation of the expressed protein was carried out following transient expression