Characterization of arylsulfatase A from different forms of metachromatic leukodystrophy patients
Description
Arylsulfatase A (ASA) is a lysosomal enzyme whose primary physiological function is desulfation of cerebroside sulfate. Deficiency of ASA leads to metachromatic leukodystrophy (MLD) with different ages of onset. Some healthy individuals also show low ASA activity which is sometimes indistinguishable from that in MLD patients. These so-called ASA pseudodeficient (PD) individuals bring complications to the diagnosis of MLD. This dissertation attempts to establish a model to study the relationships between genotype and phenotype for the PD individuals and MLD patients with different ages of onset Mutations were identified in the ASA-deficient variants using the direct solid-phase sequencing method. These base changes lead to amino acid alterations. Three novel mutations were confirmed by restriction analyses of more than 100 normal alleles. ASA was purified from human liver and the rabbit anti-human ASA antiserum was obtained. Attempts were made to produce monoclonal antibodies against ASA and two of the monoclonal antibodies reacted with ASA in Western blots. ASA from fibroblasts of normals and ASA-deficient variants was partially purified. Western blots were performed after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF), and dot blot of fractions from the purification procedure. The Western blotting patterns were different among the controls and ASA-deficient variants The K$\sb{\rm m}$ and heat-inactivation profiles were established from normals and an PD individual. The K$\sb{\rm m}$ of the PD individual was three times that of the controls. The half-lives of ASA from normal subjects were three times that of the PD individual at 60$\sp\circ$C and double that of the PD individual at 65$\sp\circ$C The genotype and phenotype relationships were analyzed and this study provides information for diagnosis, prognosis, genetic counseling and gene therapy for MLD patients