Genotyping of single nucleotide polymorphism in gamma-glutamyl hydrolase gene by capillary electrophoresis.

摘要

The gamma-glutamyl hydrolase (GGH) gene plays an important role in methotrexate (MTX) metabolism, ensuring that MTX polyglutamates (MTX-(Glu)(n)) could be converted back into MTX. Accumulation of MTX-(Glu)(n) is a problem in MTX therapy. SNP 452 C>T has been reported to associate with lower catalytic activity and higher accumulation of long-chain MTX-(Glu)(n) in patients treated with higher doses of MTX treatment. We propose and establish a simple and effective CE method for detecting SNP in GGH gene. The DNA samples after amplification were analyzed by SSCP-CE method. The CE conditions were generated by using 1x TBE buffer containing 1.5% w/v hydroxypropyl methyl cellulose under reverse polarity at 25 degrees C. This method was applied to detect genotyping of acute lymphoblastic leukemia patients receiving MTX treatment. The results were confirmed by DNA sequencing with good agreement. Concentrations of MTX-(Glu)(n) in whole blood were analyzed by on-line stacking CE method. MTX-(Glu)(n) levels and genotypes in GGH gene of acute lymphoblastic leukemia patients were evaluated. The SSCP-CE method was found to be feasible for SNP screening in the GGH gene.