Functional studies of new GLA gene mutations leading to conformational fabry disease

Abstract

Fabry Disease (FD) is an X-linked multisystemic lysosomal disorder caused by mutations of α-galactosidase (GLA) gene. Only a few of the 450 genetic lesions identified so far have been characterised by in vitro expression studies. Thus the significance of newly identified GLA nucleotide variants sin FD patients which lead to α-galactosidase (GAL-A) amino acid substitutions or intronic changes can be uncertain. We identified three GLA mutations: c155G>A (p.C52Y), c548G>C (p.G183A), c647A>G (p.Y216C) in as many individuals (two male; one female), and performed in vitro expression studies and Western Blot analysis, in order to clarify their functional effects. Reduced GAL-A activity and normal or partially reduced mutant proteins were present in all overexpressed mutant systems in which, three-dimensional structural analysis showed that the active site was not directly involved. We hypothesize that the three new mutations affect the GAL-A protein, leading to conformational FD. When, mutant proteins overexpressed in COS-1 cells and in patients’ lymphocytes were tested in the presence of the 1-deoxygalactonojirimicin (DGJ) chaperone, the p.G183A and p.Y216C systems showed increased GAL-A enzyme activities and protein stabilisation, while p.C52Y was not responsive. We underline that genetic, biochemical and functional studies are helpful in clarifying the consequences of the missense genetic lesions detected in FD. ERT is the elective therapy for Fabry patients but it is not always possible to issue the enzyme’s active form in all involved organs. Our study endorses the hypothesis that an active-site-specific chemical chaperone, which could be administered orally, might be effective in treating GAL-A conformational defects.

Cerebrovascular Involvement in Fabry Disease

“…3 Mutations are not limited to active site residues, but include those predicting changes related to stability, and indirectly, to catalytic activity. 44,45 Most mutations are family specific, but few occur with sufficient frequency to permit genotype-phenotype correlation. 46 Even within families, phenotypic heterogeneity is often present, suggesting the possibility of gene-environment interaction.…”

Molecular Basis of 1-Deoxygalactonojirimycin Arylthiourea Binding to Human α-Galactosidase A: Pharmacological Chaperoning Efficacy on Fabry Disease Mutants

“…Recent investigations on the molecular basis of FD support that α-Gal A deficiency can be attributed to the interplay of defective biosynthesis, loss of kinetic capability, and excess degradation of the mutant enzyme. 33 Aggregation of mutant α-Gal A, for instance, has been shown to negatively affect the correct trafficking of the enzyme, aggravating the FD phenotype. 34 Moreover, aggregation-prone mutants are less susceptible to be responsive to PC treatment probably due to impaired access to the catalytic site in the aggregates, as demonstrated by the failure of response to DGJ.…”

Progressive renal failure despite long-term biweekly enzyme replacement therapy in a patient with Fabry disease secondary to a new α-galactosidase mutation of Leu311Arg (L311R)

“…The rate of eGFR decrease per year was calculated to be 9.9 mL/min/ 1.73 m 2 of the protein from the endoplasmic reticulum (ER) to the lysosome, resulting in an accumulation of a-GAL in the ER. This could result from a loss of binding activity to chaperone proteins, as seen for missense mutations at C172, G183 [7], R211, Q312 [8], and G411. The patient described here has a gene mutation at L311R.…”