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 alpha-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 in FD patients which lead to alpha-galactosidase (GAL-A) amino acid substitutions or intronic changes can be uncertain. We identified three GLA mutations, c.155G>A (p.C52Y), c.548G>C (p.G183A), c.647A>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.

Figure 1

GAL-A three-dimensional structural map of the three novel amino acid substitutions.

Figure 2. Western blot analyses of lysates from COS-1 cells transfected with wild type and mutant constructs

10 µg of total proteins were used in the blots. Western blot were carried out with the anti GAL-A antibody kindly provided by Genzyme Corporation. Mock. Negative controls. B-Actin is reported as a control protein. Where not indicated, DGJ concentration is 20µM.

FIGURE 3. GAL-A activity resulting from DGJ and galactose administration in transfected COS-1 cells and from DGJ administration in lymphocytes

Three independent experiments performed in triplicate both for DGJ (20µM) and galactose (200mM) administration in COS-1 transfections and for DGJ (20µM) administration in lymphocytes, were performed. In the expression systems the value of GAL-A activity of non-transfected COS-1 cells was used to set up the GAL-A intrinsic activity in each experiment. Standard deviation was obtained using Microsoft Excel 97 SR-2. Values are expressed as the percentage of wild type lymphocytes for the DGJ administration in patients’ lymphocytes and as the percentage of COS-1 cells transfected with wild type GAL-A for the expression systems. T- lymphocytes arise from primary cultures and they were not immortalized.