title: Genotyping of single nucleotide polymorphism in γ-glutamyl hydrolase gene by capillary electrophoresis.
abstract: The γ-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 1× TBE buffer containing 1.5% w/v hydroxypropyl methyl cellulose under reverse polarity at 25°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.
authors.affiliation: School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Hui-Ling
authors.initials: HL
authors.lastname: Cheng
authors.forename: Shyh-Shin
authors.initials: SS
authors.lastname: Chiou
authors.forename: Yu-Mei
authors.initials: YM
authors.lastname: Liao
authors.forename: Yen-Ling
authors.initials: YL
authors.lastname: Chen
authors.forename: Shou-Mei
authors.initials: SM
authors.lastname: Wu
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000627:therapeutic use; D019075:Electrophoresis, Capillary / Q000379:methods*; D005838:Genotype; D006801:Humans; D065347:Hypromellose Derivatives; D008727:Methotrexate / Q000627:therapeutic use; D008747:Methylcellulose / Q000031:analogs & derivatives / Q000737:chemistry; D020641:Polymorphism, Single Nucleotide; D018807:Polymorphism, Single-Stranded Conformational; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D013696:Temperature; D011623:gamma-Glutamyl Hydrolase / Q000235:genetics*
chemical_list: D000964:Antimetabolites, Antineoplastic; D065347:Hypromellose Derivatives; D008747:Methylcellulose; D011623:gamma-Glutamyl Hydrolase; D008727:Methotrexate