Multiplex Genotyping of Allelic Variants of Genes Involved in Metabolizing Antileukemic Drugs
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
Preview
Unable to display preview. Download preview PDF.
References
- 1.Otterness D., Szumlanski C., Lennard L., et al. 1997. Human thiopurine methyltransferase pharmacogenetics: Gene sequence polymorphisms. Clin. Pharmacol. Ther. 62, 60–73.CrossRefPubMedGoogle Scholar
- 2.Matimba A., Li F., Livshits A., et al. 2014. Thiopurine pharmacogenomics: Association of SNPs with clinical response and functional validation of candidate genes. Pharmacogenomics. 15, 433–447.CrossRefPubMedPubMedCentralGoogle Scholar
- 3.Kang H.J., Oh Y., Chun S.M., et al. 2008. TotalPlex gene amplification using bulging primers for pharmacogenetic analysis of acute lymphoblastic leukemia. Mol. Cell. Probes. 22, 193–200.CrossRefPubMedGoogle Scholar
- 4.Dulucq S., Laverdière C., Sinnett D., Krajinovic M. 2014. Pharmacogenetic considerations for acute lymphoblastic leukemia therapies. Exp. Opin. Drug Metab. Toxicol. 10, 699–719.CrossRefGoogle Scholar
- 5.Adam de Beaumais T., Fakhoury M., et al. 2011. Determinants of mercaptopurine toxicity in paediatric acute lymphoblastic leukemia maintenance therapy. Br. J. Clin. Pharmacol. 71, 575–584.CrossRefGoogle Scholar
- 6.Marinaki A.M., Ansari A., Duley J.A., et al. 2004. Adverse drug reactions to azathioprine therapy are associated with polymorphism in the gene encoding inosine triphosphate pyrophosphatase (ITPase). Pharmacogenetics. 14, 181–187.CrossRefPubMedGoogle Scholar
- 7.Elaine R. 2008. Mardis The impact of next-generation sequencing technology on genetics. Trends Genet. 24, 133–141.CrossRefGoogle Scholar
- 8.Treviño L.R., Shimasaki N., Yang W., et al. 2009. Germline genetic variation in an organic anion transporter polypeptide associated with methotrexate pharmacokinetics and clinical effects. J. Clin. Oncol. 35, 5972–5978.CrossRefGoogle Scholar
- 9.Gervasini G., Vagace J.M. 2012. Impact of genetic polymorphisms on chemotherapy toxicity in childhood acute lymphoblastic leukemia. Front. Genet. 3, 249–260.CrossRefPubMedPubMedCentralGoogle Scholar
- 10.Tanfous M.B., Sharif-Askari B., Ceppi F., et al. 2015. Polymorphisms of asparaginase pathway and asparaginase-related complications in children with acute lymphoblastic leukemia. Clin. Cancer Res. 21, 329–334.CrossRefPubMedGoogle Scholar
- 11.Jewell C.M., Cidlowski J.A. 2007. Molecular evidence for a link between the n363s glucocorticoid receptor polymorphism and altered gene expression. J. Clin. Endocrinol. Metab. 92, 3268–3277.CrossRefPubMedPubMedCentralGoogle Scholar
- 12.Russcher H., Smit P., van den Akker E.L., et al. 2005. Two polymorphisms in the glucocorticoid receptor gene directly affect glucocorticoid-regulated gene expression. J. Clin. Endocrinol. Metab. 90, 5804–5810.CrossRefPubMedGoogle Scholar
- 13.Rubina A.Y., Pan’kov S.V., Dementieva E.I., et al. 2004. Hydrogel drop microchips with immobilized DNA: properties and methods for large-scale production. Anal. Biochem. 325, 92–106.CrossRefPubMedGoogle Scholar
- 14.Fesenko D.O., Zasedatelev A.S., Nasedkina T.V., Kalennik O.V., Barsky V.E. 2012. Biochip development for determining Y-haplogroups that occur in Russian populations. Mol. Biol. (Moscow). 46 (5), 731–734.CrossRefGoogle Scholar
- 15.Fesenko D.O., Mityaeva O.N., Nasedkina T.V., Rubtsov P.M., Lysov Yu.P., Zasedatelev A.S. 2010. HLA-DQA1, AB0, and AMEL genotyping of biological material with biochips. Mol. Biol. (Moscow). 44 (3), 401–406.CrossRefGoogle Scholar
- 16.Fesenko D.O., Kornienko A.E., Chudinov A.V., Nasedkina T.V. 2011. Preparing of single-stranded DNA in single-stage PCR with low-melt excess primer for hybridization on biochips. Mol. Biol. (Moscow). 45 (2), 237–240.CrossRefGoogle Scholar