CLONING AND EXPRESSION OF MUTATIONS DEMONSTRATING INTRAGENIC COMPLEMENTATION IN MUT(0) METHYLMALONIC ACIDURIA

The mut(0) mutation resulting in methylmalonyl CoA mutase (MCM) apoenz yme deficiency and methylmalonic aciduria is characterized by undetect able enzyme activity in cell extracts and low incorporation of propion ate into cultured cells which is not stimulated by hydroxycobalamin. A mut(0) fibroblast cell line (WG1681) from an African-American male in fant complemented another mut(0) cell line (WG 1130). Cloning and sequ encing of cDNA from WG 1681 demonstrated compound heterozygosity for t wo novel changes at highly conserved sites: G623R and G703R. In additi on, two previously described homozygous polymorphisms, H532R and V671I , were found. Hybridization of allele-specific oligonucleotides to PCR amplified MCM esons from the proband and family members identified a clinically normal mother, half-sister, and half-brother as carriers of the G703R change in cis with both polymorphisms. Transfection of each change into a mut(0) cell line with very low MCM mRNA (GM1673) demons trated a lack of stimulation of propionate uptake in the absence and p resence of hydroxycobalamin. Cotransfection of each mutation with the previously identified R93H mutation of WG 1130 stimulated propionate u ptake, indicating that G623R and G703R are independently capable of co mplementing the R93H mutation.