Fabry disease: Characterization of ?-galactosidase A double mutations and the D313Y plasma enzyme pseudodeficiency allele

Abstract

Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the gene encoding the lysosomal exoglycohydrolase, alpha-galactosidase A (alpha-Gal A; GLA). In two unrelated classically affected males, two alpha-Gal A missense mutations were identified: R112C + D313Y (c.334C>T + c.937G>T) and C172G + D313Y (c.514T>G + c.937G>T). The D313Y lesion was previously identified in classically affected males as the single mutation [Eng et al., 1993] or in cis with another missense mutation, D313Y + G411D (c.937G>T + c.1232G>A) [Guffon et al., 1998]. To determine whether the D313Y mutation was a deleterious mutation or a coding region sequence variant, the frequency of D313Y in normal X-chromosomes, as well as its enzymatic activity and subcellular localization in COS-7 cells was determined. D313Y occurred in 0.45% of 883 normal X-chromosomes, while the R112C, C172G, and G411D missense mutations were not detected in over 500 normal X-chromosomes. Expression of D313Y in COS-7 cells resulted in approximately 60% of wild-type enzymatic activity and showed lysosomal localization, while R112C, C172G, G411D, and the double-mutated constructs had markedly reduced or no detectable activity and were all retained in the endoplasmic reticulum. The expressed D313Y enzyme was stable at lysosomal pH (pH 4.6), while at neutral pH (pH 7.4), it had decreased activity. A molecular homology model of human alpha-Gal A, based on the X-ray crystal structure of chicken alpha-galactosidase B (alpha-Gal B; alpha-N-acetylgalactosaminidase) was generated [Garman et al., 2002], which provided evidence that D313Y did not markedly disrupt the alpha-Gal A enzyme structure. Thus, D313Y is a rare exonic variant with about 60% of wild-type activity in vitro and reduced activity at neutral pH, resulting in low plasma alpha-Gal A activity.

The Modulatory Effects of the Polymorphisms in GLA 5′-Untranslated Region Upon Gene Expression Are Cell-Type Specific

“…Second: in females coinheriting a negatively acting GLA 5 0 UTR SNP in trans with a pathogenic GLA mutation, the compound heterozygosity may significantly decrease the REA and aggravate the clinical phenotype since the aGal activity would be subnormal in all cells, irrespective of which of the two X chromosomes is inactivated. Third: in individuals carrying GLA variants associated with small decreases in REA, which otherwise would not be the cause of FD clinical phenotypes -like the p.Arg118Cys (Ferreira et al 2015) and the p.Asp313Tyr (Yasuda et al 2003;Niemann et al 2013) -the additive effect of a negatively acting GLA 5 0 UTR SNP in cis could decrease REA into the typical range of mutations associated with later-onset phenotypic variants of FD (Ferreira et al 2015), thereby modifying the expected phenotype. This might also affect the probability of identifying individuals carrying such mutations in large case-finding studies of FD among high-risk patients, when the primary screening method is based on aGal activity analysis.…”

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

“…Although the p.D313Y variant was unstable at neutral pH, the enzyme showed up to 60% of mean normal activity in transiently overexpressed cells, and was localized in lysosomes. 33 Considering the genotypic and phenotypic heterogeneities found in Fabry disease, environmental factors or modifying genes may affect phenotypic severity among patients with p.E66Q. 11,34 In addition, because underlying diseases of the five patients in the current report are still undetermined, isolated proteinuria or hypertrophic cardiomyopathy can be the only manifestation in atypical Fabry disease, the age of patient 1 at the latest evaluation was only eight, and patient 3 and 5 are heterozygous carriers of p.E66Q, atypical Fabry disease might not be ruled out confidently in these patients.…”

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

“…COS-7 cells (Originator, Gluzman Y; Riken Cell Bank, RCB 0539) were grown at 37°C in Dulbecco's modified Eagle's medium (DMEM; Gibco, Rockville, MD, U.S.A.) containing 10% fetal bovine serum (FBS; Gibco) (Yasuda et al, 2003). The cells were harvested by trypsin treatment, washed in 10 ml of DMEM containing 10% FBS, and resuspended at 5×10 6 cells/ml in DMEM containing 10% FBS.…”