Effects of a chemical chaperone on genetic mutations in α-galactosidase A in Korean patients with Fabry disease

Abstract

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism that results from mutations in the gene encoding the α-galactosidase A (GLA) enzyme. We have identified 15 distinct mutations in the GLA gene in 13 unrelated patients with classic Fabry disease and 2 unrelated patients with atypical Fabry disease. Two of the identified mutations were novel (i.e., the D231G missense mutation and the L268delfsX1 deletion mutation). This study evaluated the effects of the chemical chaperones 1-deoxygalactonojirimycin (DGJ) on the function of GLA in vitro, in cells containing missense mutations in the GLA gene. Nine missense and a nonsense mutations, including one novel mutation were cloned into mammalian expression vectors. After transient expression in COS-7 cells, GLA enzyme activity and protein expression were analyzed using fluorescence spectrophotometry and Western blot analysis, respectively. DGJ enhanced GLA enzyme activity in the M42V, I91T, R112C and F113L mutants. Interestingly, the I91T and F113L mutations are associated with the atypical form of Fabry disease. However, DGJ treatment did not have any significant effect on the GLA enzyme activity and protein expression of other mutants, including C142W, D231G, D266N, and S297F. Of note, GLA enzyme activity was not detected in the novel mutant (i.e., D231G), although protein expression was similar to the wild type. In the absence of DGJ, the E66Q mutant had wild-type levels of GLA protein expression and approximately 40% GLA activity, indicating that E66Q is either a mild mutation or a functional single nucleotide polymorphism (SNP). Thus, the results of this study suggest that the chemical chaperone DGJ enhances GLA enzyme activity and protein expression in milder mutations associated with the atypical form of Fabry disease.

Mutations of the GLA gene in Korean patients with Fabry disease and frequency of the E66Q allele as a functional variant in Korean newborns

“…19 In this report, we found remarkably high residual enzyme activity of a protein with the p.E66Q mutation, previously known as a pathogenic mutation in patients with atypical Fabry disease. 9,11,17,20 p.E66Q synthesizes 40% of wild-type enzyme activity when overexpressed in COS-7 cells, indicating that p.E66Q might be a mild mutation or a functional single nucleotide polymorphism (SNP).…”

“…Moreover, the enzyme activity of p.E66Q variant was 43.8±3.03% of normal activity in transiently overexpressed COS-7 cells (Table 2). Confocal microscopy showed that the p.E66Q variant GLA was expressed in lysosomes, a pattern similar to that of wild-type GLA, whereas the enzyme with the p.R112C mutation, a pathogenic mutation in atypical Fabry disease and that retains 4.7±0.13% of normal activity, 19 was not expressed in lysosomes, but was rather retained in the endoplasmic reticulum (ER) (Figure 1). Allele frequencies of the p.E66Q variant in the Korean population Among 1176 alleles tested, 12 were found to harbor the p.E66Q variant.…”

“…From October 1998 to November 2009, 28 unrelated Korean families, comprised of 33 males and 16 females, had been diagnosed at the Asan Medical Center and enrolled in the Korean Fabry Registry. 19,21,22 Clinical manifestations were reviewed in five patients from four families (family 25-28) with the p.E66Q variant. GL3 was measured both in plasma and urine of the patients.…”

“…In vitro assay of GLA activity was conducted as described earlier. 19 DNA purification and TaqMan SNP genotyping assay A total of 833 Korean subjects born in 2008, 490 males and 343 females, were recruited. The genomic DNA of their neonatal dried blood spot was extracted from one or two dried blood spot disks, 3 mm in diameter, using the Generation Capture Card Kit (Qiagen, Hilden, Germany).…”

“…9,11,17,20 p.E66Q synthesizes 40% of wild-type enzyme activity when overexpressed in COS-7 cells, indicating that p.E66Q might be a mild mutation or a functional single nucleotide polymorphism (SNP). 19 Herein, we report 25 distinct mutations in GLA, including six novel mutations, identified from 28 unrelated Korean families with Fabry disease. In addition, we review the clinical and biochemical findings of five patients with the p.E66Q variant and investigate the allele frequency of p.E66Q in the normal Korean population.…”