| ObjectiveThe mucopolysaccharidoses(MPS) are lysosomal storage disorders characterized by deficient degradation of glycosaminoglycans(GAG) caused by the underlying enzymatic defect. There are at least seven types of MPS, including typeⅠ,Ⅱ,Ⅲ,Ⅳ,Ⅵ,Ⅶ,Ⅸ,involving 11 kinds of hydrolytic enzymes. With the development of hematopoietic stem cell transplantation and enzyme replacement therapy, MPS have become treatable genetic metabolic diseases. In order to improve prognosis, it is crucial to diagnose and treat MPS in early phrases. Enzymatic analysis is an important basis for disease diagnosis. Purposes of this research are as follows:1. To establish enzymatic assays of MPS.2. To have an enzymatic diagnosis for the patients who were clinically highly suspected to have MPS.3. To evaluate the validity of urinary GAG and serum chitotriosidase in MPS screening.4. To summarize the clinical features of various types of MPS.Subjects and Assays1.Subjects: (1) Case group: According to the clinical manifestations and the results of X-ray examination, we identified thirty-six patients with highly suspected MPS at the Department of Endocrinology and Metabolism in Guangzhou Women and Children's Medical Center from June 2008 to December 2010. Among them 25 were males and 11 were females. Their ages spanned from 0.2 to 10.8 years, with the average age (4.2±2.3) years. We carried out quantitative and electrophoretic analysis of urinary GAG for all patients. Out of the 36 cases, 32 cases were collected peripheral blood, which we used to conduct enzymatic analysis for MPS in leukocytes in our hospital. In all MPS patients confirmed by emzymatic assays, serum chitotriosidase were measured by fluorescent substrates. (2) Control group: Sixty-three healthy children, aged from 1.3 to 13 years old, enrolled for physical examination in Department of Health Care of Guangzhou Women and Children's Medical Center.2. Enzymatic assays: Six common MPS enzymes were included: alpha-L-iduronidase(MPSⅠ), iduronate-2-sulfatase(MPSⅡ), N-acetylgalactosamine-6- sulfatase (MPSⅣA),β-galactosidase (MPS IVB), arylsulfatase B (MPSⅥ),β-glucuronidase (MPS VII). Leukocytes were isolated with dextran.Cell homogenates were prepared by ultrasonic cracker. Protein content of cell homogenates was determinated by Bicinchoninic Acid protein assay kit (BCA). Enzymatic activity assays for common MPS were measured by specific artificial fluorescent substrates. Urinary GAG were detected quantitatively by dimethylmethylene-tris spectrophotometry and qualitatively analyzed by agarose gel electrophoresis. Serum chitotriosidase were measured by specific artificial fluorescent substrates. Activities of arylsulfatase B by spectrophotometry in leukocytes were also measured in patients with highly suspected MPS VI.Results1. We established the fluorescent substrates enzymatic assays for six common MPS in leukocytes and the spectrophotometry for arylsulfatase B. The intra variation coefficients of various assays were less than 6% and inter coefficient variation less than 10%.2. We established normal reference values of various enzymatic assays, such as alpha-L-iduronidase (MPSⅠ) 69.7±23.8nmol / (mg. h), iduronate-2-sulfatase (MPSⅡ) 77.1±33.9nmol / (mg.4h), N-acetylgalactosamine -6- sulfatase (MPSⅣA) 153.7±37.9nmol/(mg.17h),β-galactosidase (MPS IVB) 94.2±32.1nmol/(mg. h), arylsulfatase B (MPSⅥ) 116.6±37.7nmol / (mg. h),β-glucuronidase (MPS VII) 151.6±45.8nmol / (mg. h). Arylsulfatase B activity by spectrophotometry 1.7±0.5 U/g.3. The enzymatic types of the patients highly suspected with MPS: All 32 cases who were collected peripheral blood in our hospital were confirmed MPS by enzymatic assay. Among them, 4 cases(12.5%) were typeⅠ, 11 cases (34.3%) typeⅡ, 12 cases (37.5%) type IVA , 4 cases (12.5%) typeⅥ,1 case (3.2%) typeⅦ. No typeⅣB was found in this study. Both results of spectrophotometry and fluorescence substrate assays showed that arylsulfatase B activity was deficient in leukocytes in patients with MPSⅥ.4. Among the 32 cases with MPS, the urinary GAG concentration and urinary ratio GAG / Cr in 31 cases were significantly elevated, the other one case with typeⅣA had normal urinary GAG. The positive rate of urinary GAG in MPS patients was 96.9%. At the same time, urinary GAG electrophoretic analysis showed that 31 cases were abnormal, typeⅠ,Ⅱ,Ⅵ,Ⅶpatients with dermatin sulfate(DS)positive and typeⅣA patients with chondroitin sulfate (CS) positive except one IVA case. The coincidence rate of quantitative and electrophoretic analysis of urinary GAG was 100%. Serum chitotriosidase activtities were elevated slightly in 6 (18.8%) out of 32 MPS patients.5. The clinical features of various types of MPS: MPS exhibited significant genetic heterogeneity and various degrees of multi-system injury. Patients with typeⅠ,Ⅱ,Ⅵdisplayed coarse facial features, stiff joints, growth retardation and significantly increased GAG in urine. The main clinical manifestations of typeⅣA patients were growth retardation, skeletal deformities, laxity joints, normal intelligence and slightly increased or normal GAG in urine. MPSⅦwas rare and showed hydrops fetalis, thrombocytopenia,Alder-Reilly granules in the leucocytes. Conclusions:1. We established the fluorescence substrate assays for measuring the six common MPS enzymatic activity in leukocytes, with good stability, specificity and suitable for clinical application.2. We established the normal reference values of six common MPS enzyme activities in leukocytes.3. We firstly found that the most common type is MPS IVA, followed by the MPSⅡ, and reported the first case of MPS VII in China.4. Except for a few MPSⅣA patients, the quantitative and electrophoresis of urinary GAG are feasible and reliable screening method for MPS.5. Serum chitotriosidase assay can not yet be used as a routine biochemical marker for MPS screening and diagnosis because it detected elevation only in a few MPS patients.6. MPS have significant heterogeneity and involve multiple organs. Multi-disciplinary cooperation can improve the diagnosis and prognosis. |
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