Multiplex Genotyping of Allelic Variants of Genes Involved in Metabolizing Antileukemic Drugs
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Abstract
A biochip, primer set, and genotyping protocol were developed to simultaneously address 16 single nucleotide polymorphisms in antileukemic drug metabolism genes, including TPMT, ITPA, MTHFR, SLCO1B1, SLC19A1, NR3C1, GRIA1, ASNS, MTRR, and ABCB1. The genotyping procedure included a one-round multiplex polymerase chain reaction (PCR) with simultaneous incorporation of a fluorescent label into the PCR product and subsequent hybridization on a biochip with immobilized probes. The method was used to test 65 DNA samples of leukemia patients. Fluorescence signal intensity ratios in pairs of wildtype and respective mutant sequence probes were analyzed for all polymorphic markers and demonstrated high accuracy of genotyping. The reliability of genotype determination using the biochip was confirmed by direct Sanger sequencing.
Keywords
genotyping pharmacogenetics biochip biotransformation genes single nucleotide polymorphism single-round PCRAbbreviations
- MTX
methotrexate
- AL
acute leukemia
- ALL
acute lymphoblastic leukemia
- PCR
polymerase chain reaction
- ABCB1
ATP binding cassette subfamily B member 1 gene
- ASNS
asparagine synthetase (glutamine-hydrolyzing) gene
- dNTP
deoxynucleoside triphosphate
- dUTP
deoxyuridine triphosphate
- GRIA1
glutamate ionotropic receptor AMPA type subunit 1 gene
- ITPA
inosine triphosphate pyrophosphatase gene
- SLCO1B1
solute carrier organic anion transporter family member 1B1 gene
- MTHFR
methylenetetrahydrofolate reductase gene
- MTRR
5-methyltetrahydrofolate–homocysteine methyltransferase reductase gene
- NR3C1
nuclear receptor subfamily 3 group C member 1 gene
- SLC19A1
solute carrier family 19 member 1 gene
- TPMT
thiopurine S-methyltransferase gene
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References
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