No abstract

Background and Purpose The cause of initial ischemic stroke in up to 30% of young patients remains unclear. Fabry disease, due to deficient α-galactosidase A (α-Gal A) activity, is a vascular endothelial glycosphingolipid storage disease typically presenting in childhood. With advancing age, patients develop renal, cardiac, and cerebrovascular disease and die prematurely. A European study suggested an increased prevalence of unrecognized Fabry disease in patients with cryptogenic stroke. We hypothesized that α-Gal A deficiency is a rare cause of initial early-onset ischemic stroke in men. Methods The Stroke Prevention in Young Men Study enrolled >550 men (15 to 49 years) with first ischemic stroke in the Baltimore–Washington area in 2004 to 2007. Frozen plasma samples were assayed for α-Gal A activity, and DNA from patients with consistently low plasma α-Gal A activities were sequenced. Results The study sample consisted of 558 men (42% African-American; median age 44 years). Stroke was cryptogenic in 154 men (40% African-American). In 10 patients with low plasma α-Gal A activities, DNA sequencing identified alterations in the α-Gal A gene in 2 patients. The polymorphism, D313Y, which results in low plasma enzyme activity, but near normal levels of cellular activity was seen in one European-American male. The Fabry disease-causing A143T mutation was seen in an African-American male with cryptogenic stroke (0.18% of all strokes: upper 95% CI=0.53%; 0.65% of cryptogenic strokes: upper 95% CI=1.92%). Conclusions In this biracial population, unrecognized Fabry disease is a rare but treatable cause of initial ischemic stroke in young men.

Human alpha-galactosidase A (EC 3.2.1.22; alpha-Gal A) is the homodimeric glycoprotein that hydrolyses the terminal alpha-galactosyl moieties from glycolipids and glycoproteins. The type, site occupancy and function of the N-linked oligosaccharide chains on this lysosomal hydrolase were determined. Endoglycosidase treatment of the purified recombinant enzyme and mutagenesis studies indicated that three (Asn-139, Asn-192 and Asn-215) of the four potential N-glycosylation consensus sequences were occupied by complex, high-mannose and hybrid-type oligosaccharides respectively. When expressed in COS-1 cells, glycoforms with glycosylation site 1 or 2 obliterated had more than 70% of wild-type activity, and both glycoforms were secreted. In contrast, the glycoform with only site 3 eliminated had decreased activity (less than 40%); little, if any, was secreted. Expressed mutant glycoforms in which site 3 and site 1 or 2 were obliterated had little, if any, intracellular or secreted enzymic activity, and immunofluorescence microscopy revealed that the expressed mutant glycoforms were retained in the endoplasmic reticulum, presumably where they were degraded. Thus glycosylation at site 3 was crucial to the formation of soluble, active enzyme, as well as transport to the lysosome. Absence of the site 3 hybrid-type oligosaccharide exposed an adjacent, normally protected, hydrophobic region, resulting in aggregation of the enzyme polypeptide in the endoplasmic reticulum. In support of this concept, endoglycosidase H-treated enzyme or mannose-terminated enzyme expressed in Autographa californica cells also aggregated when concentrated, emphasizing that site 3 occupancy by a hybrid-type oligosaccharide was required for enzyme solubility.

Gaucher disease, the most prevalent lysosomal storage disease, occurs in three subtypes, all resulting from mutations in the acid ␤ -glucosidase gene. Molecular studies in five severely affected type 1 and two type 2 Gaucher disease patients of non-Jewish descent identified six new mutations: K74X, W179X, G195E, S271N, V352L, and a two-base deletion in exon 10 (1450del2). Two additional mutations identified in these patients (R48W and G202R) have been reported previously, but were not expressed or characterized. Heterologous expression in Sf9 cells using the baculovirus system revealed that the missense mutations, R48W and V352L, had 14 and 7%, respectively, of the specific activity based on cross-reacting immunologic material expressed by the normal allele. In contrast, the G195E, G202R, and S271N mutant alleles were more severely compromised with only 1-2% of the normal expressed specific activity based on cross-reacting immunologic material. Structural distortion at the active site was probed by comparing the interaction of the mutant enzymes with active site-directed inhibitors (castanospermine, conduritol B epoxide and deoxynojirimycin). R48W, G202R, and S271N were normally inhibited, whereas the V352L and G195E mutant enzymes had significantly decreased binding affinity. These mutations further expand the genetic heterogeneity in the lesions causing Gaucher disease types 1 and 2, and further delineate genotype/phenotype correlations and functional domains within the acid ␤ -glucosidase gene. ( J. Clin. Invest. 1997. 99:2530-2537.)