title: Detection of the G>C SNP and rare mutations in the 28-bp repeat of TYMS using gel-based capillary electrophoresis.
abstract: OBJECTIVE
  Polymorphisms in the 5' regulatory region of the thymidylate synthase gene (TYMS) have been shown to modulate thymidylate synthase expression and are associated with resistance to fluoropyrimidine-based therapies. These polymorphisms include a two repeat (2R) or three repeat (3R) of a 28-bp sequence and a G>C SNP in the second repeat of the 3R allele (TSER*3 G>C). Genotyping methods for the TYMS 5'-UTR polymorphisms have typically involved visualizing PCR and RFLP products on agarose gels. This article describes the use of a robust capillary electrophoresis assay for TYMS 5'-UTR genotyping.
  
  
  METHODS
  As part of pharmacogenetic studies, we performed TYMS genotyping for the 5'-UTR polymorphisms in 314 colorectal cancer patients. A gel-based capillary electrophoresis method, employing a high-resolution gel cartridge on a QIAxcel(®) system, was developed to detect PCR products and RFLP fragment sizes.
  
  
  RESULTS
  The high resolution of the capillary electrophoresis technique allowed identification of a 6-bp insertion in the second repeat of the 3R allele in three patients. The frequency of the insertion allele was 0.4% in Caucasians and 1.3% in African-Americans. We also found 3.3% of Caucasian patients were heterozygous for a G>C SNP in the first repeat of the 2R allele, but this allele was not observed in the African-American patients.
  
  
  CONCLUSIONS
  We describe a robust RFLP genotyping technique that employs size discrimination by capillary electrophoresis to genotype the TYMS TSER*3 G>C SNP. The technique also allows identification of a 6-bp insertion in the 3R allele, and we report the allelic frequencies for two uncommon TSER alleles.
authors.affiliation: University of North Carolina, Chapel Hill, NC, USA. thomas.fabienne@claudiusregaud.fr
authors.forename: Fabienne
authors.initials: F
authors.lastname: Thomas
authors.forename: Janelle M
authors.initials: JM
authors.lastname: Hoskins
authors.forename: Anne
authors.initials: A
authors.lastname: Dvorak
authors.forename: Benjamin R
authors.initials: BR
authors.lastname: Tan
authors.forename: Howard L
authors.initials: HL
authors.lastname: McLeod
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D015331:Cohort Studies; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics*; D019075:Electrophoresis, Capillary; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D009154:Mutation; D010597:Pharmacogenetics; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D020080:Tandem Repeat Sequences / Q000235:genetics*; D013940:Thymidylate Synthase / Q000235:genetics*
chemical_list: D020121:5' Untranslated Regions; D013940:Thymidylate Synthase