Davies, Joanna Pauline; (1995) The molecular genetics of Fabry disease. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Anderson-Fabry disease is the lysosomal storage disorder resulting from a deficiency of α-galactosidase A (E.C. 3.2.1.22.), which cleaves α-linked galactose residues from the non-reducing ends of oligosaccharide chains. Loss of activity leads to the progressive accumulation of partially catabolised glycosphingolipids in the lysosomes of all cells but particularly those of the vascular endothelium. The α-galactosidase A gene, which is located at Xq22, spans 12kb and has 7 exons. The disease affects an estimated 1/40 000 and there is no cure. Although hemizygotes can be diagnosed reliably by demonstrating a deficiency of α-galactosidase A, heterozygote detection is unreliable due to mosaicism in the pattern of α-galactosidase A expression, caused by random Inactivation of the X-chromosome. The aims of this study were to develop methods for mutation detection, which would allow conclusive identification of heterozygotes and a study of the relationship between the genotype and the phenotype. Mutation analysis by a combination of single strand conformation polymorphism (SSCP) analysis and sequencing has detected 6 polymorphisms in the non-coding regions of the α-galactosidase A gene and 26 different, putative disease-causing mutations in 28 out of 30 unrelated Fabry patients. The polymorphisms were present in 7/61 unaffected males and in 4/30 Fabry patients. The mutations consisted of 16 missense, 5 nonsense, one splice site, 3 small deletion and 1 insertion mutation, 25 of which had not been identified previously. Two of these mutations were not detected by SSCP analysis but were found by sequencing. The mutations in 2 families were not identified by these methods. The effects of the mutations on the α-galactosidase A activity were investigated by studying the residual activity in cultured cells from the patients and by the transient expression of mutated cDNA in COS-1 cells. Only 1 out of the 9 cell cultures had detectable activity, from a patient with the N215S mutation and a presimied, mild phenotype. Six missense mutations found in this study and a further 3, found by others, were transiently expressed from cDNA tn COS-1 cells. No α-galactosidase A activity was produced by 5 mutant constructs, R49L, R112H, V269A, V316E and Q327K, while 4, G35R, Q279E, R301Q and G361R, produced low activity, compared with the wild-type cDNA. Mutations that produced detectable intracellular α-galactosidase A activity, G35R, Q269E and R301Q, were found in patients with variant phenotypes. The identification of 26 different mutations in 28 families and of 6 polymorphisms within the α-galactosidase A gene has allowed accurate carrier detection and a study of the relationship between genotype and phenotype. This will improve patient management and indicate those most likely to benefit from enzyme therapy in the future.

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