Establishing Personalized Medicine in Fabry Disease Through Functional Analysis of Disease Mutants

Fabry disease is a hereditary metabolic disorder caused by insufficient activity of the enzyme α-galactosidase A, which is required to remove terminal galactose from lysosomal substrates. There are over 1000 gene mutations identified, and single amino acid substitutions form the largest group. Each substitution reduces or eliminates the enzyme activity in patients, and the amount of enzyme activity correlates with the severity of the disease. The monogenic nature of Fabry disease enables genotype-phenotype predictions, but how individual mutations lead to a loss of enzyme activity is still unclear. Functional α-galactosidase A in the lysosome requires many maturation steps, including the folding of the polypeptide, post-translational modifications, trafficking of the enzyme to the lysosome, and substrate binding. We developed a series of assays to check each of the stages in the maturation of the protein, including enzymatic activity, the stability of the protein, post-translational modifications, and trafficking to the lysosome. To identify the molecular basis of disease-causing mutations, we applied these assays to mutant α-galactosidase A proteins representing different classes of Fabry disease variants. Through this process, we identified mutant α-galactosidase A proteins which do not fold correctly, do not traffic correctly, and do not have correct post-translational modifications. We determined X-ray crystallographic structures of mutant α-galactosidase A proteins, showing an atomic basis for the defects leading to Fabry disease. In addition, we tested mutant proteins for response to the pharmacological chaperones for Fabry disease, indicating which classes of mutations are amenable to pharmacological chaperones. This study provides insight at an atomic, molecular, and cellular level into the disease-causing missense mutations and can be used to choose the best therapeutic options for Fabry disease patients