Detection of the G>C SNP and rare mutations in the 28-bp repeat of TYMS using gel-based capillary electrophoresis

Fabienne Thomas, Janelle M. Hoskins, Anne Dvorak, Benjamin R. Tan, Howard L. McLeod

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

Aims: 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. Materials & 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. Conclusion: 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.

Original languageEnglish
Pages (from-to)1751-1756
Number of pages6
JournalPharmacogenomics
Volume11
Issue number12
DOIs
StatePublished - Dec 2010

Keywords

  • 2RC allele frequency
  • 5′-UTR 6-bp insertion
  • QIAxcel
  • TSER*3 G≥C n TSER tandem repeat
  • TYMS
  • TYMS 2RC allele
  • capillary gel electrophoresis

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