Novel mutations of the GLA gene in Japanese patients with Fabry disease and their functional characterization by active site specific chaperone

Abstract

Fabry disease is an X-linked recessive inborn metabolic disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (EC 3.2. throughout the 7 exons of the α-galactosidase A gene (GLA). Mutation hotspots for Fabry disease do not exist. We examined 62 Fabry patients in Japan and found 24 GLA mutations, including 11 novel ones. A potential treatment reported for Fabry disease is active site specific chaperone (ASSC) therapy using 1-deoxygalactonojirimycin (DGJ), an inhibitor of α-galactosidase A, at subinhibitory concentrations. We transfected COS-7 cells with the 24 mutant GLAs and analyzed the α-galactosidase A activities. We then treated the transfected COS-7 cells with DGJ and analyzed its effect on the mutant enzyme activities. The activity of 11 missense mutants increased significantly with DGJ. Although ASSC therapy is useful only for misfolding mutants and therefore not applicable to all cases, it may be useful for treating many Japanese patients with Fabry disease. © 2007 Wiley-Liss, Inc.KEY WORDS: α-galactosidase A; GLA; lysosomal storage disease; 1-deoxygalactonojirimycin; chaperone therapy INTRODUCTIONFabry disease (FD; MIM# 301500) is a pan-ethnic, X-linked, lysosomal storage disorder caused by a deficiency in the lysosomal enzyme α-galactosidase A (EC 3.2.1.22) (Brady et al., 1967). Human α-galactosidase A is a homodimeric glycoprotein with three N-linked oligosaccharide chains on each subunit. Enzyme deficiency results in a systemic lysosomal accumulation of glycolipids, primarily globotriosylceramide (Gb3), in the vascular endothelium and other tissues. In the classical form of the disease, the patient develops angiokeratoma, hypohidrosis, and episodic pain crises in the extremities during childhood or adolescence. With advancing age, the morbidity of renal failure, cardiac disease, and early onset of stroke increases. The severity of the clinical manifestations depends on the amount of residual α-galactosidase A activity. Hemizygous male patients with no or very low α-galactosidase A activity usually have severe clinical symptoms and die as young adults. Heterozygous female Fabry patients exhibit a wide range of severity, from a virtually symptom-free course (Marguery et al., 1993) to one comparable to that of their male counterparts (Whybra et al., 2001), although they usually have no symptoms or very mild manifestations.We examined Fabry patients in Japan and sequenced the patients' α-galactosidase A gene (GLA) (MIM# 300644). To analyze the mutant α-galactosidase A activities, we transfected COS-7 cells with the mutant GLAs. In addition, since 1-deoxygalactonojirimycin (DGJ) stabilizes the α-galactosidase A conformation and improves its stability (Asano et al., 2000;Ishii et al., 2000;Yam et al., 2006), we added DGJ to the incubation medium and examined its effect on the mutant α-galactosidase A activities. MATERIALS AND METHODS PatientsWe examined 62 Fabry patients from 31 unrelated families. Diagnosis was based on reduced or absent α-galactosidase A activity and typical signs an...

High-throughput screening identified disease-causing mutants and functional variants of α-galactosidase A gene in Japanese male hemodialysis patients

“…It should be addressed that previous studies included subjects with the p.E66Q allele as a 'renal variant' of Fabry disease. 14,15,17,26 However, Lee et al 22 reported that the allele A g ea te n r o l l m e n t( y e a r ) 5 5 6 1 4 5 8 3 7 6 7 0 7 6 6 7 7 6 8 0 frequency of p.E66Q in Korean individuals was remarkably higher (1.046% (95% confidence interval, 0.458-1.634%)) than the prevalence of Fabry disease. They also demonstrated that p.E66Q was a functional polymorphism rather than a pathogenic mutation using COS-7 cells overexpressing a-GLA harboring p.E66Q.…”

Screening of Male Dialysis Patients for Fabry Disease by Plasma Globotriaosylsphingosine

“…p.E66Q is a class 2 mutation that retains a high residual enzyme activity (17). Therefore, plasma lyso-Gb3 could be difficult to detect in patients with this mutation.…”

“…The DNA and RNA were extracted from white blood cells. Genetic testing was performed as described elsewhere (17).…”

Identification of a Novel Mutation and Prevalence Study for Fabry Disease in Japanese Dialysis Patients

“…16 To date, a few variant types of Fabry disease have been identified; their manifestations are primarily limited to the heart, kidneys, or brain. 8,[20][21][22][23][24] It is, however, very difficult to identify Fabry disease in dialysis patients because patients with variants of Fabry disease often lack symptoms that are observed in the classic form. Recently, several studies have investigated the prevalence of Fabry disease in dialysis patients.…”

“…The family members (children) of these two patients, who were asymptomatic, were also identified as heterozygotes for Fabry disease. Previous reports included patients with E66Q mutations, indicating the substitution of glutamine for glutamic acid at residue 66; 11,17,21 this mutation is currently under debate as to whether it is a disease-caused mutation or not. Lee et al 25 reported that the allele frequency of E66Q was approximately 1% among 833 newborns in Korea, suggesting that E66Q is a polymorphism because no globotriaosylceramide is found in patients with E66Q.…”