Identification of two novel mutations in the methylmalonyl-CoA mutase gene with decreased levels of mutant mRNA in methylmalonic acidemia.

Genetic defects in the methylmalonyl-CoA mutase (MCM) gene result in methylmalonic acidemia which is inherited as an autosomal recessive disease. We investigated fibroblast cultures obtained from two Japanese patients with MCM deficiency. MCM mRNA was not detected by Northern blot analysis, suggesting that MCM mRNA was markedly decreased. Reverse transcription/polymerase chain reaction (RT-PCR) of MCM mRNA followed by analysis on a fluorescent fragment analyzer indicated that the level of MCM mRNA in these fibroblasts was less than 1% of normal controls. This minute amount of MCM mRNA was successfully amplified by nested RT-PCR and subjected to primary structure analysis. Sequence analysis revealed two novel mutations: a G-to-T substitution at nucleotide position 425 and a 2 bp deletion at nucleotide positions 769 and 770. The first mutation (G425T) resulted in the substitution of a termination codon for glutamic acid at amino acid position 117. The second mutation (769 delta CA) resulted in a frame shift which created a premature termination codon 508 amino acid upstream of the C-terminus of the protein. Patient 1 was homozygous for G425T and patient 2 was a compound heterozygote for G425T and 769 delta CA. Our report is the first to identify MCM mutations that affect the stability of MCM mRNA. An analysis of 16 Japanese patients revealed the presence of G425T in six patients, suggesting a relatively high incidence of the mutation among Japanese patients. This is in sharp contrast to a previous report describing diverse heterogeneity of MCM mutations among Caucasians.