Fabry disease, a rare genetic disorder affecting multiple organs, has understudied correlations among enzyme activity, genotype, and clinical manifestations in patients of different sexes with classical and late-onset phenotypes. In this study, clinical data, α-Gal A activity, and GLA gene test results of 311 patients, who were categorized by classical and late-onset phenotypes, ⩽5% and > 5% of the normal mean value of enzyme activity, and truncated and nontruncated mutation groups, were collected. The common clinical manifestations of Fabry disease included acroparesthesia, hypohidrosis/anhidrosis, neuropsychiatric system, and renal and cardiovascular involvement. Multiorgan involvement was higher in males and classical phenotype patients. In both sexes, classical patients commonly presented with acroparesthesia and multiorgan involvement, whereas late-onset patients showed renal, neuropsychiatric, and cardiovascular involvement. Male and classical patients had lower enzyme activity than female and late-onset patients, respectively. Classical males with enzyme activity of ⩽5% of the normal mean level showed higher multiorgan involvement frequency than those with enzyme activity of > 5%, whereas no significant difference was observed among females. Ninety-five gene mutation sites were detected, with significant phenotype heterogeneity in patients with the same mutation. No significant difference in enzyme activity or clinical manifestations was observed between truncated and nontruncated mutations. Overall, male patients with Fabry disease, regardless of classical or late-onset phenotype, have a higher frequency of multiple-organ involvement and lower α-Gal A activity than female patients. α-Gal A activity was closely correlated with clinical symptoms in males but weakly correlated with clinical manifestations in females. The clinical manifestations of patients with the same mutation are heterogeneous, and the correlation between gene mutation and enzyme activity or clinical manifestation is weak.
Adolescent ; Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Young Adult ; Age of Onset ; alpha-Galactosidase/metabolism* ; China ; Fabry Disease/enzymology* ; Genotype ; Mutation ; Phenotype ; Sex Factors ; East Asian People/genetics*

Fabry's disease is an X-linked lysosomal storage disorder caused by abnormalities in the alpha-galactosidase A (GLA) gene, which leads to a GLA deficiency and to the intracellular deposition of globotriaosylceramide (Gb3) within vascular endothelium and other tissues. It manifests as progressive multiple organ dysfunctions caused by the deposition of Gb3. On the other hand, congenital agammaglobulinemia is usually caused by mutations in Bruton's tyrosine kinase (Btk) gene with X-linked dominence, suppresses B cell maturation, and causes recurrent pyogenic infections. In former reports, the distance between the loci in the Xq22 region of the human X chromosome was found to be about 69 kilobases. A 23-yr-old man diagnosed with congenital agammaglobulinemia at age 5, showed typical clinical and laboratory and histopathological findings of Fabry's disease. The genetic basis of this combination of the two syndromes was studied in this patient. Here, we report a case of Fabry's disease with congenital agammaglobulinemia.
Adult ; Agammaglobulinemia/congenital/*genetics/pathology ; Chromosomes, Human, X ; Fabry Disease/diagnosis/*genetics ; Humans ; Kidney/pathology ; Male ; Microscopy, Electron ; Sequence Analysis, DNA ; Skin/pathology ; alpha-Galactosidase/genetics/metabolism

This study was aimed to prepare a reconstructed B. Fragilis-derived recombinant α-galactosidase developed for human B to O blood group conversion. Based on the construction of recombinant E. Coli (DE3) which can express α-galactosidase, the inducing time and inducer concentration were optimized for high expression of α-galactosidase. Then, the expression products in supernatant were purified by cation and anion exchange column chromatography. The purified α-galactosidase was used to treat B group red blood cells in phosphate buffer (pH 6.8) for 2 hours to prepare O group red blood cells. The results showed that the optimal inducing conditions for α-galactosidase expression were IPTG 0.1 mmol/L, 37°C and 2 hours. The specific enzyme activity of purified protein increased from 0.42 U/mg to 2.1 U/mg as compared with pre-purification. And, the conditions of B to O blood group conversion were 26°C, pH 6.8 (neutral pH condition) and 2 hours. Moreover, 225 µg of the enzyme could converse 1 ml B red blood cells to O completely. It is concluded that the technology of expression and purification of recombinant α-galactosidase has been established, and the purified protein can converse B red blood cells to O completely, which means that an effective enzyme conversing B red blood cells to O has been obtained.
ABO Blood-Group System ; immunology ; Bacteroides fragilis ; enzymology ; Cloning, Molecular ; Escherichia coli ; metabolism ; Humans ; Recombinant Proteins ; biosynthesis ; alpha-Galactosidase ; biosynthesis

OBJECTIVETo investigate the interaction of deficiency in thrombosis-related gene in a mouse model.

METHODSTo generate mice carrying mutations in alpha-galactosidase A (Gla) and factor V Leiden (Fvl) and analyze the phenotypes, namely, tissue fibrin deposition and thrombus formation in organs.

RESULTSFibrin deposition in organs of mice carrying both mutations in Gla and Fvl was significantly increased compared with that in mice with single mutaton: [Gla(-/0) Fv(Q/Q)+Gla(-/-)Fv(Q/Q)] vs.[Gla(-/0)Fv(+/+)]=(0.28+/-0.03)% vs.(0.07+/-0.007)%, P<0.01; [Gla(-/0)Fv(Q/Q)+Gla(-/-)Fv(Q/Q)] vs.[Gla(+/0)Fv(Q/Q)+Gla(+/+)Fv(Q/Q)]=(0.28+/-0.03)% vs.(0.11+/-0.02)%, P< 0.01. Meanwhile, the number of thrombi on organ sections of mice carrying both mutations in Gla and Fvl was significantly increased compared with the single mutation carrier: [Gla(-/0)Fv(Q/Q)+Gla(-/-)Fv(Q/Q)] vs.[Gla(-/0)Fv(+/+)]=1.9+/-0.7 vs. 0.0+/-0.0, P<0.05; [Gla(-/0)Fv(Q/Q)+Gla(-/-)Fv(Q/Q)] vs. [Gla(+/0)Fv(Q/Q)+Gla(+/+)Fv(Q/Q)]=1.9+/-0.7 vs. 0.3+/-0.1, P<0.05.

CONCLUSIONThese observations demonstrated that there was synergistic effect in Gla and Fvl deficiency in mice. It suggested that there could be a combination of GLA deficiency and FVL or other thrombosis-related gene defect in patients with genetic severe early-onset thrombosis.

Animals ; Factor V ; genetics ; Fibrin ; metabolism ; Immunohistochemistry ; Mice ; Mutation ; Thrombosis ; genetics ; metabolism ; alpha-Galactosidase ; genetics

BACKGROUND/AIMS: Fabry disease is an X-linked recessive and progressive disease caused by alpha-galactosidase A (alpha-GaL A) deficiency. We sought to assess the prevalence of unrecognized Fabry disease in dialysis-dependent patients and the efficacy of serum globotriaosylceramide (GL3) screening. METHODS: A total of 480 patients of 1,230 patients among 17 clinics were enrolled. Serum GL3 levels were measured by tandem mass spectrometry. Additionally, we studied the association between increased GL3 levels and cardiovascular disease, cerebrovascular disease, or left ventricular hypertrophy. RESULTS: Twenty-nine patients had elevated serum GL3 levels. The alpha-GaL A activity was determined for the 26 patients with high GL3 levels. The mean alpha-GaL A activity was 64.6 nmol/hr/mg (reference range, 45 to 85), and no patient was identified with decreased alpha-GaL A activity. Among the group with high GL3 levels, 15 women had a alpha-GaL A genetics analysis. No point mutations were discovered among the women with high GL3 levels. No correlation was observed between serum GL3 levels and alpha-GaL A activity; the Pearson correlation coefficient was 0.01352 (p = 0.9478). No significant correlation was observed between increased GL3 levels and the frequency of cardiovascular disease or cerebrovascular disease. CONCLUSIONS: Fabry disease is very rare disease in patients with end-stage renal disease. Serum GL3 measurements as a screening method for Fabry disease showed a high false-positive rate. Thus, serum GL3 levels determined by tandem mass spectrometry may not be useful as a screening method for Fabry disease in patients with end stage renal disease.
Adult ; Aged ; Fabry Disease/blood/*diagnosis ; Female ; Humans ; Kidney Failure, Chronic/blood/*therapy ; Male ; Middle Aged ; *Renal Dialysis ; Trihexosylceramides/*blood ; alpha-Galactosidase/genetics/metabolism

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism that results from mutations in the gene encoding the alpha-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.
1-Deoxynojirimycin/*analogs & derivatives/metabolism ; Adolescent ; Adult ; Animals ; Asian Continental Ancestry Group/*genetics ; COS Cells ; Cercopithecus aethiops ; Fabry Disease/*enzymology/genetics ; Gene Expression ; Humans ; Male ; Middle Aged ; Mutation ; Young Adult ; alpha-Galactosidase/*genetics/*metabolism

BACKGROUNDHuman group O red blood cells have great benefit in specialized transfusion areas such as armed conflict and natural calamity. The group B antigen differs structurally from group O antigen only by the addition of one terminal alpha-linked galactose residue. In this study we aimed to remove the terminal galactose from group B red blood cell to get group O red blood cell.

METHODSalpha-galactosidase cDNA was cloned by RT-PCR from Catimor coffee beans grown on Hainan Island of China. The vector for alpha-galactosidase cDNA expression was constructed and transferred into Pichia pastoris cells by electroporation. The transgenic cells were cloned by fermentation and the recombinant alpha-galactosidase was purified by ion exchange chromatography. After studying the biochemical characters of alpha-galactosidase, we have used it in converting human erythrocytes from group B to group O.

RESULTSThe purity of recombinant alpha-galactosidase was higher than 96%, which was thought to be suitable for the use of blood conversion. Enzymatically converted human group O red blood cells (ECHORBC) exhibited membrane integrity, metabolic integrity, normal cell deformation and morphology. There were no coagulation between ECHORBC and any group of human blood. The ECHORBC will keep normal structure and function for a period of 21 days at 4 degrees C in monoammoniumphosphate nutrient solution. Experiments with Rhesus monkeys and gibbons showed that transfusion of enzymatically converted erythrocytes was safe.

CONCLUSIONECHORBC can be easily obtained from group B red blood cell by alpha-galactosidase digestion. This study suggests that ECHORBC could be transfused to patients safely and efficiently.

ABO Blood-Group System ; classification ; metabolism ; Animals ; Blood Transfusion ; Cloning, Molecular ; Coffee ; enzymology ; Erythrocytes ; metabolism ; Humans ; Macaca mulatta ; Quality Control ; Recombinant Proteins ; isolation & purification ; pharmacology ; alpha-Galactosidase ; immunology ; isolation & purification ; pharmacology ; toxicity

In order to meet the demand for safe transfusion in special conditions and to utilize the donated blood supply efficiently, technology has been developed to convert erythrocytes from type A, B, or AB to "universal donor" blood. Conversion of blood type B to O was performed by means of recombinant alpha-galactosidase digestion. The results showed that blood type B to O was converted successfully, 1 transfusion unit of red cells of group B (100 ml totally) could converted to universal blood cells in the optimal conditions including pH 5.6, 26 degrees C, 2 hours, obturation and sterilization. It is concluded that the universal red blood cells converted from group B to group O are conformed to demand of identification rules of biological products, no harmful effects of alpha-galactosidase on cell structure and function are observed. The converted red cells can stored in 4 degrees C for 21 days.
ABO Blood-Group System ; classification ; immunology ; Blood Group Incompatibility ; prevention & control ; Blood Transfusion ; methods ; Coffee ; enzymology ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Erythrocytes ; immunology ; metabolism ; Humans ; Isoantigens ; drug effects ; metabolism ; Recombinant Proteins ; metabolism ; pharmacology ; alpha-Galactosidase ; genetics ; metabolism ; pharmacology

This study was aimed to explore impact of removal of cell membrane G alalpha1-3Gal beta1-4Glc NAc epitopes (called alpha-Gal) and chemical modification of other xenoantigen on bovine red blood cell (bRBC) and porcine red blood cell (pRBC) antigenicity and to compare their modified erythrocytes, in order to provide basis for development of human blood substitute with rich source, high safety and efficacy. bRBC and pRBC were subjected to both enzymatic removal of membrane alpha-Gal with recombinant coffee bean alpha-galactosidase (rC alpha-GalE) and covalent attachment of benzotriazole carbonate-linked methoxypolyethylene glycol (mPEG-BTC, MW = 20 kD). The effects of treatment were measured by hemagglutination, flow cytometric assay of IgG binding and clinical cross-match testing to human sera. The results showed that although alpha-galactosidase treatment reduced hemagglutination titers to levels similar to negative control, the combination of the treatments was most effective. Clinically used cross-match tests between bRBC, pRBC and human sera demonstrated increased compatibility. Bovine RBC were more robust than pRBC, and had less xenoantigens, and had longer half life than pRBC in vivo. These characteristics suggested that bRBCs were more suitable to investigation as an alternatives to hRBC in clinical transfusion than pRBC. These data suggested that strategies to remove or mask xenoantigens on bRBC reduce antigenicity sufficiently to allow in vitro cross-match compatibility to human sera, and therefore bRBC following modification may be considered as human blood substitute.
Animals ; Antigens, Heterophile ; immunology ; Blood Substitutes ; Cattle ; Disaccharides ; immunology ; Epitopes ; immunology ; Erythrocyte Membrane ; immunology ; Erythrocyte Transfusion ; methods ; Erythrocytes ; immunology ; metabolism ; Humans ; Swine ; alpha-Galactosidase ; immunology

OBJECTIVETo explore the strategies which reduce the amount of xenoantigen Galalpha1,3Gal.

METHODSHuman alpha-galactosidase gene and alpha1,2-fucosyltransferase gene were transferred into cultured porcine vascular endothelial cells PEDSV.15 and human alpha-galactosidase transgenic mice were produced. The Galalpha1,3Gal on the cell surface and susceptibility of cells to human antibody-mediated lysis were analyzed.

RESULTSHuman alpha-galactosidase gene alone reduced 78% of Galalpha1,3Gal on PEDSV.15 cell surface while human alpha-galactosidase combined with alpha1,2-fucosyltransferase genes removed Galalpha1,3Gal completely. Decrease of Galalpha1,3Gal could reduce susceptibility of cells to human antibody-mediated lysis, especially during co-expression of alpha-galactosidase gene and alpha1,2-fucosyltransferase gene. RT-PCR indicated positive human alpha-galactosidase gene expression in all organs of positive human alpha-galactosidase transgenic F1 mice including heart, liver, kidney, lung, and spleen, the amount of Galalpha1,3Gal antigens on which was reduced largely. 58% of spleen cells from F1 mice were destroyed by complement-mediated lysis compared with 24% of those from normal mice.

CONCLUSIONSHuman alpha-galactosidase gene and alpha1,2-fucosyltransferase gene effectively reduce the expression of Galalpha1,3Gal antigens on endothelial cell surface and confers resistance to human serum-mediated cytolysis. The expression of human alpha-galactosidase in mice can also eliminate the Galalpha1,3Gal antigens in most tissues and decrease the susceptibility of spleen cells to human serum-mediated cytolysis.

Animals ; Antigens, Heterophile ; metabolism ; Cell Death ; Cells, Cultured ; Disaccharides ; metabolism ; Endothelial Cells ; metabolism ; Fucosyltransferases ; genetics ; metabolism ; Graft Rejection ; genetics ; Humans ; Mice ; Mice, Transgenic ; Spleen ; cytology ; Swine ; Transfection ; alpha-Galactosidase ; genetics ; metabolism