Enzyme Replacement Therapy in the Treatment of Fabry Disease / 罕见病研究

Fabry disease is an X-linked inherited lysosomal storage disease caused by the mutation of GLA gene that encodes α-galactosidase A (α-Gal A). GLA gene mutation causes the decline or deficiency in the activity of α-Gal A, leading to the accumulation of its substrates in the lysosomes of multiple organs and tissue that causes systemic damage and threatens the life of patients. Enzyme replacement therapy (ERT) is the standard of care for Fabry disease. ERT slows or prevents the progression of organ damage, effectively improving heart, brain, kidney functions and significantly improves quality of life. At present, there are two main ERT drugs, agalsidase alpha and agalsidase beta, which have the same amino acid sequence and different N-terminal sugars, and are used for the treatment of Fabry disease. In this paper, the efficacy and safety of ERT in the treatment of Fabry disease were summarized by reviewing the relevant literature at home and abroad and combining the results of some cases treated in Department of Nephrology, Provincial Hospital affiliated to Shandong First Medical University. Previous literature has shown that enzyme replacement therapy is the most important specific treatment for Fabry disease, which has a protective effect on important organs such as kidney, heart, and nervous system.

A novel GLA mutation in a Korean boy with an early cardiac manifestation of Fabry disease

Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by the deficiency of α-galactosidase A. Patients with classical FD present acroparesthesia, hypohidrosis, cornea verticillata, disseminated angiokeratoma, and microalbuminuria in childhood, and develop life-threatening renal, cardiac, and cerebrovascular complications typically after the fourth decade of life. To date, more than 700 mutations responsible for FD have been identified in the human GLA gene. Herein, we report a novel GLA mutation, c.1117_1141del25 (p.Gly373Profs*10), identified in an 11-year-old Korean boy with FD presenting early cardiac and neurologic manifestation and in other affected family members. The boy had acroparesthesia, hypohidrosis, cornea verticillata, and left ventricular hypertrophy. His mother and sister also had acroparesthesia. Two males on the mother's side had similar pain and died of unknown causes. The plasma α-galactosidase A activity (4.1 nmol/hr/mg protein) of the patient was markedly lower than the mean value of the controls. The plasma level of globotriaosylsphingosine was elevated in the patient and all the carriers. We concluded the novel GLA mutation c.1117_1141del25 is a pathogenic mutation for FD, probably related to the early cardiac manifestation of FD.

Production of α-galactosidase from Aspergillus foetidus MTCC 6322 by solid state fermentation and its application in soymilk hydrolysis.

The production of α-galactosidase from the wild fungal strain Aspergillus foetidus MTCC 6322 using solid state fermentation (SSF), its characterization, and its efficacy in the hydrolysis of soymilk using response surface methodology were studied. The optimum conditions for production of α-galactosidase by SSF were: wheat bran (10 g), moisture content (64%), inoculum volume (1.0 mL; 6 × 107 spores/mL) with a yield of 4.1 × 103 units per gram dry substrate (U/gds) at 96 h. The enzyme showed optimum activity at pH 6.0, temperature 40°C, pH stability between 5.0-8.0, and temperature stability between 30-40°C. The enzyme was stable in the presence of trypsin, lipase, and collagenase and it showed susceptibility of the substrates such as raffinose, melibiose, guar gum and soymilk to hydrolysis in varying degrees. The optimized conditions for soymilk hydrolysis were: soymilk (10 mL) from defatted soybean meal (1.5%), α-galactosidase (0.15 UmL-1) at 30°C, pH 6.0 and duration of 1 h.

Blood type conversion in human liver-an experimental study on α-galactosidase / 中华肝胆外科杂志

Objective To study blood type B antigen elimination with α-galactosidase in human liver tissue,and discuss the feasibility of blood type conversion in human liver.Methods The liver specimens from patients with blood type B in liver transplantation were collected,and an in vitro liver perfusion model was established.The in vitro livers were perfused with UW solution +/-α-galactosidase.The effect of enzyme in B antigen of human liver were analyzed by immunofluorescence.Results With UW solution containing α-galactosidase to perfuse the in vitro livers,immunohistochemistry showed the level of blood type B antigen in liver was significantly reduced after hypothermic perfusion and preservation.The B antigen level in 1 h perfusion was reduced to approximate 58％ of this figure prior to perfusion,in 2 h was 10％,and in 4 h was 4％.Among the different intervals,the blood group antigen levels showed significant differences (P ＜ 0.05).In the control group,the blood group antigen levels showed no obvious change on statistical analysis.Conclusions α-galactosidase was effective to clear blood type B antigen in isolated liver tissue.In the experimental group,Although the B antigen did not fall to a undetectable level,liver blood type conversion from B→O remains a promising potential which has been meaningful for related researches on blood type conversion of human organs.

Enzymatic removal of α-Gal antigen in porcine skin / 军事医学

Objective To reduce immunogenicity of porcine skin by removingα-Gal epitopes expressed in cell surface and extracellular matrix using recombinant α-galactosidase produced by Bacteroides fragilis.Methods The porcine skin was harvested from healthy 2-month-old pigs without any skin disorders before being sterilized by iodine and 75%alcohol, respectively.Enzymatic removal of α-Gal antigen was followed by washing with PBS.The α-Gal antigen in the prepared porcine skin was measured with immunofluorostaining of cryosections and the residual enzyme was measured with a double-antibody sandwich ELISA method.Enzymatic removal procedures were optimized by detecting residual enzyme and the effi-cacy ofα-Gal removal under different enzymatic and washing conditions.Results Efficient enzymatic and washing methods were established to removeα-Gal antigen.Theα-Gal removal efficacy was above 90% and residual enzyme was undetect-able (αprescribed minimum ofα-galactosidase detection with indirect ELISA was 1 ng/ml) .Conclusion It is feasible to efficiently removeα-Gal antigen under these enzymatic and washing conditions, and a method of producing low-immunoge-nicity pig skin dressing for burn is established.

Preparation of a novel monoclonal antibody againstα-galactosidase from Bacteroides fragilis for detection of minimal residual enzyme in universal red blood cells / 军事医学

Objective To establish a method of quantiying trace α-galactosidase from Bacteroides fragilis in enzymatic conversion of blood group B to O red blood cells ( B-ECO RBCs) .Methods BALB/c mice were immunized with purified recombinant B.fragilisα-galactosidase ( the purity>90%) to prepare monoclonal antibodies.The ascites were prepared using hybridoma cell lines stably secreting antibody and purified by HiTrap rProtein A column.The antibody titer and spe-cificity were detected by ELISA and Western blotting, respectively.Purified monoclonal antibody and rabbit polyclonal an-tibody were applied to detect residual enzyme in B-ECO RBCs and the washing solution was analyzed by indirect ELISA. Results A high titer and purity antibody was obtained.Western blotting showed that the antibody specifically reacted with B.fragilisα-galactosidase.Moreover, indirect ELISA was sensitive enough to detect the minimal amount of residualα-gal-actosidase at the concentration of 1 ng/ml.After four repeat washing cycles with 1∶4 ( v/v) phosphate-buffered saline, the amount of residual enzyme in B-ECO RBCs was less than 10 ng/ml.Conclusion An effective method of detecting the min-imal amount of residual α-galactosidase in blood conversion is established for safety evaluation of universal RBCs prepara-tion by enzymatic treatment.

Clinical significance of α-galactosidase A activity assay in the screening and diagnosis of Febry disease / 中华内分泌代谢杂志

α-Galactosidase A (α-Gal A ) activities in plasma and peripheral blood granulocytes of 100healthy subjects and one patient with Fabry disease was determined by means of fluorogenic substrate.The results showed that the enzymatic activities of peripheral blood granulocytes and plasma in 100 subjects were (51.97 ± 15.24)and(148.08±26.30) nmol · h-1 · ml-1 respectively.The α-Gal A activities in plasma and granulocytes were positively correlated( r=0.533,P＜0.01 ).The enzymatic activities in peripheral blood granulocytes and plasma of the patients with Fabry disease were 1.05 and 10.06 nmol · h-1 · ml-1 respectively,both much lower than those of 100healthy subjects.These results suggest that α-Gal A activity in plasma and peripheral blood granulocytes can be used for diagnosis and screening of Fabry disease.

Gene mutation and enzymatic activity analysis of α-galactosidase A in a patient with Fabry disease / 中华内分泌代谢杂志

Objective To analyse the clinical characteristics,gene mutation and enzymatic activity of αgalactosidase A(α-GalA)in a 15-year-old male patient with typical Fabry disease,whose mother was without any clinical manifestations.Methods Clinical features and laboratory data were collected from the patient and his mother.Genomie DNA was extracted from peripheral blood of the patient.his mother,and a healthy control subject.Seven exons of the GLA gene were amplified by PCR.PCR products were purified.cloned into T vector,and then sequenced.The enzymatic activity of α-GalA Was measured by fluorimetrie substrate assay. Results DNA sequencing results showed that a missense mutation of 10036-10038delAAG in exon 7 WaS identified in the patient,resulting in the replacement of 374 lysine and 375 glyeine by arginine,which Was not previously reported.The patient Was a hemizygote with gene mutation,his mother WaS a heterozygote carrying gene mutation,and the healthy control without mutation.α-GalA enzymatic activity assay showed that the enzymatic activity of the patient with GLA gene mutation was only 50%of the healthy control subject,while the enzymatic activity of the patient's mother Was about 70%of the heahhy control SObject.Conclusiolls Detecting GLA gene mutation and α-GalA enzymatic activity in patients with Fabry disease who have been clinically diagnosed seelns to be helpful in finding other patients in the family and in further understanding the molecular pathogenesis of that disease.

Cardiac manifestations of Fabry's disease / 中华内科杂志

Objective Fabry' s disease is a rare X-linked recessive disease. Its cardiac manifestations are not well recognized. Methods The data of 3 patients from different Chinese kindreds with Fabry's disease and cardiac manifestations who seeked medical advice in our department in 2007 were analyzed. The age, sex, family history, main symptoms, ECG and echocardiographic findings were recorded for all the patients. The diagnostic criteria of Fabry's disease was based on α-galactosidase (α-GAL) quantity in white blood cells. Results All of the patients were female. Their age was from 41 to 57. Two of them had the typical symptoms of Fabry's disease in their young age. All of them had family history of the disease and cardiac symptoms. ECG showed ST-T change and echocardiography showed hypertrophy of left ventricule of different degrees. Their α-galactosidase level in white blood cells was lower than normal. The α-galactosidase level in patient 1 was the lowest. Her cardiac symptoms were most serious in the three patients and she had involvement of other organs. Conclusion Patients with Fabry's disease may have cardiac manifestations. Family history, typical symptoms in young age and the characteristics of multi-systemic disorder are helpful clues to the diagnosis.

A galactomannan-hydrolysing α-galactosidase from jack fruit (Artocarpus integrifolia) seed: Affinity chromatographic purification and properties.

An acid α-galactosidase from the seeds of the jack fruit seed (Artocarpus integrifolia) has been purified to homogeneity by affinity chromatography on a matrix formed by cross-linking the soluble α-galactose-bearing guar seed galactomannan. The 35kDa enzyme was a homotetramer of 9·5kDa subunits. Its carbohydrate part (5·5%) was composed of galactose and arabinose. The Km with p-nitrophenyl α-D-galactoside as substrate was 0·35 mM. The Ki values indicated inhibition by galactose, 1-O-methyl α-galactose and melibiose in the decreasing order. Among α-galactosides, the enzyme liberated galactose from melibiose, but not from raffinose or stachyose at its pH optimum (5·2). The guar seed galactomannan was however efficiently degalactosidated; limited enzyme treatment abolished the precipitability of the polysaccharide by the α-galactose-specific jack fruit seed lectin, and complete hydrolysis yielded insoluble polysaccharide. Though similar in sugar specificity and subunit assembly, α-galactosidase and the lectin coexisting in the jack fruit seed gave no indication of immunological identity.

α-Galactosidase from germinating guar (Cyamopsis tetragonolobus) seeds.

The changes in α-galactosidase activity in guar (Cyamopsis tetragonolobus) seeds was followed during seven days of germination. The enzyme activity was maximal on the first day of germination and gradually decreased during subsequent days. On the second day of germination the partially purified enzyme upon ion-exchange chromatography on CMSephadex C-50 was resolved into α-galactosidase-A (anionic), α-galactosidase-C1 (cationic) and α-galactosidase-C2 (cationic) and their relative proportions were 28,12 and 60%, respectively. The combined α-galactosidase C1 and C2 activities increased in the first two days of germination followed by significant decrease after the 3rd day onwards, whereas α- galactosidase-A remained fairly constant throughout the germination period, α- Galactosidase-A and C2 had different Km and Vmax values with p-nitrophenyl α-Dgalactopyranoside, raffinose and melibiose as substrates and also differed in their thermal stabilities.