title: Treatment of a methylmalonyl-CoA mutase stopcodon mutation.
abstract: 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.
authors.affiliation: Metabolic Research, Murdoch Childrens Research Institute, The University of Melbourne Department of Paediatrics, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia. nicole.buck@mcri.edu.au
authors.forename: Nicole E
authors.initials: NE
authors.lastname: Buck
authors.forename: Leonie R
authors.initials: LR
authors.lastname: Wood
authors.forename: Natasha J
authors.initials: NJ
authors.lastname: Hamilton
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Bennett
authors.forename: Heidi L
authors.initials: HL
authors.lastname: Peters
mesh_terms: D000592:Amino Acid Metabolism, Inborn Errors / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D018389:Codon, Nonsense / Q000187:drug effects / Q000235:genetics; D018388:Codon, Terminator / Q000187:drug effects* / Q000235:genetics; D004195:Disease Models, Animal; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D005839:Gentamicins / Q000494:pharmacology*; D006801:Humans; D008765:Methylmalonyl-CoA Mutase / Q000172:deficiency / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D010069:Oxadiazoles / Q000494:pharmacology*
chemical_list: D018389:Codon, Nonsense; D018388:Codon, Terminator; D005839:Gentamicins; D010069:Oxadiazoles; D008765:Methylmalonyl-CoA Mutase; C515878:ataluren