Simultaneous detection of variable number tandem repeats, single nucleotide polymorphisms, and allelic imbalance in the thymidylate synthase gene enhancer region using denaturing high-performance liquid chromatography

Hany Ezzeldin, Cornelia Hoffmayer, Richie Soong, Martin R. Johnson, Adam Lee, Marty Heslin, Robert B Diasio

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Polymorphisms in the thymidylate synthase enhancer region (TSER) have been reported to be associated with alterations in thymidylate synthase (TS) mRNA protein levels. The TSER is characterized by the presence of variable double (2R) and triple (3R) number tandem repeats (VNTRs). In addition to VNTRs, single nucleotide polymorphisms (SNPs) and allelic imbalance (AI), including loss of heterozygosity (LOH), have recently been associated with response to 5-fluorouracil (5-FU)-based chemotherapy. The aim of the current study was to develop a specific denaturing high-performance liquid chromatography (DHPLC) method for the rapid detection of these variations in the TSER in clinical samples. DHPLC analysis was validated in parallel with agarose gel electrophoresis (AGE), enzyme digestion, and quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR). The optimized DHPLC method resolved 100% of the known TSER variations, differentiated between homozygous and heterozygous genotypes, and allowed the qualitative and quantitative detection of AI, including LOH, in tumor samples. This DHPLC method was developed to permit the rapid, sensitive, and accurate identification of the TSER genotype (VNTRs, SNPs, and AI) in clinical protocols where response to flouropyrimidines may be correlated with TSER polymorphisms.

Original languageEnglish (US)
Pages (from-to)276-283
Number of pages8
JournalAnalytical Biochemistry
Volume334
Issue number2
DOIs
StatePublished - Nov 15 2004
Externally publishedYes

Fingerprint

Allelic Imbalance
Thymidylate Synthase
Minisatellite Repeats
High performance liquid chromatography
Polymorphism
Single Nucleotide Polymorphism
Nucleotides
Genes
High Pressure Liquid Chromatography
Loss of Heterozygosity
Genotype
Tandem Repeat Sequences
Chemotherapy
Agar Gel Electrophoresis
Polymerase chain reaction
Transcription
Clinical Protocols
Electrophoresis
Fluorouracil
Sepharose

Keywords

  • 5-FU
  • Allelic imbalance
  • DHPLC
  • Genetic polymorphism
  • LOH
  • Thymidylate synthase
  • TSER

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Simultaneous detection of variable number tandem repeats, single nucleotide polymorphisms, and allelic imbalance in the thymidylate synthase gene enhancer region using denaturing high-performance liquid chromatography. / Ezzeldin, Hany; Hoffmayer, Cornelia; Soong, Richie; Johnson, Martin R.; Lee, Adam; Heslin, Marty; Diasio, Robert B.

In: Analytical Biochemistry, Vol. 334, No. 2, 15.11.2004, p. 276-283.

Research output: Contribution to journalArticle

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