Bunge S, Clements P R, Byers S, Kleijer W J, Brooks D A, Hopwood J J
Institut für Humangenetik, Universitäts-Krankenhaus Eppendorf, Hamburg, Germany.
Biochim Biophys Acta. 1998 Sep 30;1407(3):249-56. doi: 10.1016/s0925-4439(98)00046-5.
Fibroblasts from 16 patients with known alpha-L-iduronidase gene mutations and different clinical phenotypes of mucopolysaccharidosis type I (MPS I) were investigated in order to establish genotype/phenotype correlations. Enzyme kinetic studies were performed using the specific alpha-L-iduronidase substrate iduronosyl anhydro[1-3H]mannitol-6-sulfate. Specific residual enzyme activities were estimated using the kinetic parameters and an immunoquantification assay which determines levels of alpha-L-iduronidase protein. Cells were cultured in the presence of [35S]sulfate and the in vivo degradation of accumulated labelled glycosaminoglycans measured after different chase times. Residual enzyme activity and different amounts of residual enzyme protein were present in extracts from 9 of 16 cell lines covering a wide spectrum of clinical severity. Catalytic capacity, calculated as the product of kcat/Km and ng iduronidase protein per mg cell protein, was shown in most cases to be directly related to the severity of clinical phenotype, with up to 7% of normal values for patients with the attenuated form of MPS I (Scheie) and less than 0.13% for severely affected patients (Hurler) In vitro turnover studies allowed further refinement of correlations between genotype and phenotype. Scheie disease compared to Hurler disease patients were shown to accumulate smaller amounts of glycosaminoglycans that were also turned over faster. A combination of turnover and residual enzyme data established a correlation between the genotype, the biochemical phenotype and the clinical course of this lysosomal storage disorder.
为了建立基因型/表型相关性，对16例已知α-L-艾杜糖醛酸酶基因突变且患有不同临床表型的I型粘多糖贮积症（MPS I）患者的成纤维细胞进行了研究。使用特异性α-L-艾杜糖醛酸酶底物艾杜糖醛酸酐[1-3H]甘露糖醇-6-硫酸盐进行酶动力学研究。使用动力学参数和测定α-L-艾杜糖醛酸酶蛋白水平的免疫定量分析来估计特异性残余酶活性。细胞在[35S]硫酸盐存在下培养，并在不同的追踪时间后测量积累的标记糖胺聚糖的体内降解情况。16个细胞系中有9个的提取物中存在残余酶活性和不同量的残余酶蛋白，涵盖了广泛的临床严重程度范围。在大多数情况下，以kcat/Km与每毫克细胞蛋白中α-L-艾杜糖醛酸酶蛋白纳克数的乘积计算的催化能力与临床表型的严重程度直接相关，I型MPS（Scheie）轻症患者的催化能力高达正常值的7%，而重症患者（Hurler）则低于0.13%。体外周转研究进一步完善了基因型与表型之间的相关性。与Hurler病患者相比，Scheie病患者积累的糖胺聚糖量较少，周转速度也更快。周转和残余酶数据的结合建立了这种溶酶体贮积症的基因型、生化表型和临床病程之间的相关性。
