Prediction of response of mutated alpha-galactosidase A to a pharmacological chaperone.

OBJECTIVE: To examine the relationship between types and locations of mutations of the enzyme alpha-galactosidase (Gal) A in Fabry disease and the response to the pharmacological chaperone 1-deoxygalactonojirimycin (DGJ). METHODS: T cells grown from normal individuals or from patients with Fabry disease were tested for response to treatment with DGJ by increased activity of alpha-Gal A. RESULTS: Cells from normal controls responded with a 28% increase in alpha-Gal A activity, whereas response in Fabry individuals was mutation dependent ranging from no increase to fully normal activity. Nine truncation mutations (all nonresponsive) and 31 missense mutations were tested. Three groups of missense mutations were categorized: responders with activity more than 25% of normal, nonresponders, with less than 7% and an intermediate response group. In normal cells and in responders an increase in the mature lysosomal form of alpha-Gal A was observed after DGJ treatment. Nonresponders showed little or no protein with or without DGJ. The intermediate response group showed an increase in band intensity but incomplete processing of the enzyme to the mature form. CONCLUSION: Mapping the missense mutations to the structure of alpha-Gal A identified several factors that may influence response. Mutations in regions that are not in alpha-helix or beta-sheets, neither involved in disulfide bonds nor with an identified functional or structural role were more likely to respond. Predictability is, however, not precise and testing of each mutation for response to pharmacological chaperone therapy is necessary for Fabry disease and related lysosomal storage disorders.

Screening for pharmacological chaperones in Fabry disease.

As a prerequisite for clinical trials of pharmacological chaperone therapy (PCT) for Fabry disease, we developed a rapid screening assay for enhancement of endogenous alpha-galactosidase A (alpha-Gal A) in patient-derived cells. We used a T-cell based system to screen 11 mutations causing Fabry disease for enhanceability using 1-deoxygalactonojirimycin (DGJ). When patient-derived T-cells were grown in the presence of DGJ, alpha-Gal A activity increased to more than 50% of normal in several mutations but was unaffected in others. In addition to the mutation R301Q, reported previously, A97V, R112H, R112C, A143T, and L300P were enhanceable, but R356W, G132R, A143P, R220X, and 30delG were not. The level of alpha-Gal A activity achieved provides a basis for the therapeutic trial of DGJ in patients with similarly enhanceable enzyme. This assay method has general utility in other disorders in assessing the degree of enhancement of activity of mutated proteins by PCT.

Transfer of a mitochondrial DNA fragment to MCOLN1 causes an inherited case of mucolipidosis IV.

A patient with mucolipidosis-IV heterozygous for two mutations in MCOLN1 expressed only her father's cDNA mutation c.1207C>T predicting an R403C change in mucolipin. She inherited a 93bp segment from mitochondrial NADH dehydrogenase 5 (MTND5) from her mother that was inserted in-frame prior to the last nucleotide of exon 2 of MCOLN1 (c.236_237ins93). This alteration abolished proper splicing of MCOLN1. The splice site at the end of the exon was not used due to an inhibitory effect of the inserted segment, resulting in two aberrant splice products containing stop codons in the downstream intron. These products were eliminated via nonsense-mediated decay. This is the first report of an inherited transfer of mitochondrial nuclear DNA causing a genetic disease. The elimination of the splice site by the mitochondrial DNA requires a change in splicing prediction models.