TY - JOUR
T1 - 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
AU - Ezzeldin, Hany
AU - Hoffmayer, Cornelia
AU - Soong, Richie
AU - Johnson, Martin R.
AU - Lee, Adam
AU - Heslin, Marty
AU - Diasio, Robert
PY - 2004/11/15
Y1 - 2004/11/15
N2 - 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.
AB - 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.
KW - 5-FU
KW - Allelic imbalance
KW - DHPLC
KW - Genetic polymorphism
KW - LOH
KW - TSER
KW - Thymidylate synthase
UR - http://www.scopus.com/inward/record.url?scp=5644228858&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=5644228858&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2004.07.025
DO - 10.1016/j.ab.2004.07.025
M3 - Article
C2 - 15494134
AN - SCOPUS:5644228858
SN - 0003-2697
VL - 334
SP - 276
EP - 283
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -