Heterozygous mutations at the mut locus in fibroblasts with mut0 methylmalonic acidemia identified by polymerase-chain-reaction cDNA cloning.

Genetic defects in the enzyme methylmalonyl CoA mutase cause a disorder of organic acid metabolism termed "mut methylmalonic acidemia." Various phenotypes of mut methylmalonic acidemia are distinguished by the presence (mut-) or absence (mut0) of residual enzyme activity. The recent cloning and sequencing of a cDNA for human methylmalonyl CoA mutase enables molecular characterization of mutations underlying mut phenotypes. We identified compound heterozygous mutations in a mut0 fibroblast cell (MAS) line by cloning the methylmalonyl CoA mutase cDNA by using the polymerase chain reaction (PCR), sequencing with internal primers, and confirming the pathogenicity of observed mutations by DNA-mediated gene transfer. Both mutations alter amino acids common to the normal human, mouse, and Propionibacterium shermanii enzymes. This analysis points to evolutionarily preserved determinants critical for enzyme structure or function. The application and limitation of cDNA cloning by PCR for the identification of mutations are discussed.