Cloning and expression of mutations demonstrating intragenic complementation in mut0 methylmalonic aciduria

Area of Science:

• Biochemistry
• Genetics
• Metabolic Disorders

Background:

• Methylmalonic aciduria is a metabolic disorder caused by methylmalonyl CoA mutase (MCM) deficiency.
• The mut0 mutation leads to undetectable MCM enzyme activity and is unresponsive to hydroxycobalamin.
• Fibroblast cell lines are crucial for studying complementation in inherited metabolic diseases.

Purpose of the Study:

• To identify the genetic basis of mut0 methylmalonic aciduria in a patient with compound heterozygous mutations.
• To characterize the functional impact of novel MCM mutations (G623R and G703R) on enzyme activity.
• To investigate the complementation potential of these novel mutations with a known MCM mutation (R93H).

Main Methods:

• Genetic sequencing (cDNA cloning and sequencing) to identify mutations in the MCM gene.
• Cell culture and complementation assays using patient-derived fibroblast cell lines.
• Propionate incorporation assays to measure MCM enzyme function.
• Transfection studies to assess the functional impact of specific mutations.

Main Results:

• Compound heterozygosity for two novel mutations, G623R and G703R, were identified in the MCM gene of the patient.
• These novel mutations were found to be in cis with known polymorphisms (H532R and V671I).
• Transfection of G623R and G703R individually into a mut0 cell line did not restore propionate uptake, but they independently complemented the R93H mutation.

Conclusions:

• The identified G623R and G703R mutations are responsible for MCM deficiency and methylmalonic aciduria in this patient.
• These novel mutations are functionally distinct and capable of complementing other MCM mutations, providing insights into MCM structure-function relationships.
• The study highlights the utility of complementation assays in elucidating the pathogenicity of novel mutations in inherited metabolic disorders.