Treatment of a methylmalonyl-CoA mutase stopcodon mutation.

There are limited treatment options for the metabolic disorder methylmalonic aciduria. The disorder can be caused by nonsense mutations within the methylmalonyl-CoA mutase gene, resulting in the production of a truncated protein with little or no catalytic activity. We used a genomic reporter assay and mouse primary cell lines which carry a stop-codon mutation in the human methylmalonyl-CoA mutase gene to test the effects of gentamicin and PTC124 for stop-codon read-through potential. Fibroblast cell lines were established from methylmalonic aciduria knockout-stop codon mice. Addition of gentamicin to the culture medium caused a 1.5- to 2-fold increase in mRNA expression of the human methylmalonyl-CoA mutase gene. Without treatment the cells contained 19% of the normal levels of methylmalonyl-CoA mutase enzyme activity which increased to 32% with treatment, suggesting a functional improvement. Treatment with PTC124 increased the amount of human methylmalonyl-CoA mutase gene mRNA by 1.6±0.3-fold and a trend suggesting increased enzyme activity. The genomic reporter assay, BAC_MMA(∗)EGFP, expresses enhanced green fluorescent protein when read-through of the stop codon occurs. Using flow cytometry, RT-real-time PCR and enzyme assay, read-through was measured. Treatment with PTC124 at 20µmol/L resulted in a significant increase in enhanced green fluorescent protein, a 2-fold increase in mRNA expression and a trend to a slight increase in enzyme activity. The clinical relevance of these effects may be tested in mouse models of MMA carrying nonsense mutations in the methylmalonyl-CoA mutase gene. Pharmacological approaches have the advantage of providing a broader effect on multiple tissues, which will benefit many different disorders with similar nonsense mutations.