RU2653492C2 - Method for determining the human genotype by polymorphism in the cytochrome p450 cyp2d6*6 (1707delt) rs5030655 gene - Google Patents

Abstract

FIELD: medicine; biotechnology.

SUBSTANCE: invention relates to medicine, biology and biotechnology and is intended to determine the human genotype by polymorphism in the cytochrome gene P450 CYP2D6*6 (1707delT) rs5030655. Pair of primers is used, fluorescently labeled allele-specific oligonucleotide probes, and a universal oligonucleotide labeled with a fluorescence quencher are used. Assignment of a sample to a homozygote or heterozygote for a given allele is evaluated by the shape of the DNA melting curves, namely, by the maximum of the first derivative of the fluorescence plots.

EFFECT: invention allows to conduct a study in a single tube, which reduces the cost of the study.

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Description

1. The technical field

The proposed method relates to the field of medicine, biology and biotechnology and can be used in compiling an individual “genetic passport”, implementing the concept of individual medicine, to formulate personal recommendations for the appointment and adjustment of chemotherapy using tamoxifen, taking into account the individual genetic characteristics of the patient. Tamoxifen, a selective modulator of estrogen receptors, is the "gold standard" in the treatment of early stage estrogen-positive breast cancer. Long-term studies have shown that its use reduces the risk of relapse and death by 30-50%. Aromatase inhibitors (anastrozole and letrozole) are more effective than tamoxifen, but due to the significant number of side effects, they are suitable for a much smaller number of patients (Lash TL et al., 2009; Goetz MP et al., 2007; Ingelman-Sundberg M. et al., 2005 ) The metabolic activity of tamoxifen in the body is determined by the activity of one of the enzymes of the cytochrome P450 family encoded by the CYP2D6 gene: under the action of the enzyme, tamoxifen passes into a metabolically active form - endoxifen. It was found that various allelic variants of the CYP2D6 gene have different effects on enzyme activity and, ultimately, on the metabolic activity of tamoxifen. The CYP2D6 gene is extremely polymorphic - more than 100 alleles have been described for it. The alleles CYP2D6 * 3, CYP2D6 * 4, CYP2D6 * 6 are inactive (non-functional) alleles associated with a low level of tamoxifen metabolism (poor metabolizer group). Patients with two inactive (non-functional) alleles of CYP2D6 are characterized by a low level of tamoxifen metabolism (poor metabolizer group), for patients with one or two inactive alleles or with one inactive / inactive and one active allele, a reduced level of metabolism (intermediate metabolizer group), for patients with two active alleles - normal metabolic rate (ultrarapid metabolizer group). For the first two groups of patients, the therapeutic effect of tamoxifen is reduced or even absent (Goetz M.R. et al., 2005; Schroth W. et al., 2007; Bradford LD et al., 2002; Ingelman-Sundberg M. et al ., 2007).

2. The level of technology

A widespread method for determining the type of nucleotide located at a specific DNA site is based on the use of allele-specific primers with registration of PCR results at the end of the reaction by electrophoresis. When using PCR with recording the results during the reaction, various methods are known for determining the genotype of the test sample. There are various methods for determining the type of nucleotide located at a specific DNA site, based on the use of allele-specific primers with the registration of PCR results directly during the reaction using fluorescently labeled samples (oligonucleotides) (Andreas R. Tobler at all, "THE SNPlex Genotiping System: A Flexible and Scalable Platform for SNP Genotyping ", Journal of Biomolecular Techniques, V. 16, issue 4, Desember 2005). For example, Applied Biosystems kits use one pair of primers for each allele and two probes with different fluorescence labels, depending on the allele genotype, different labels burn up ("TaqMan SNP Genotyping Assays", Applied Biosistems, Prodakt Bulletin, USA, 06/2006 ) The disadvantage of the method used in these sets is the relatively low reliability of the study results due to the small difference between the obtained fluorescence curves for different alleles. The proposed approach to the detection of genetic polymorphism is the use of two allele-specific and labeled with different fluorescent labels of oligonucleotides, as well as an oligonucleotide carrying the quencher of fluorescence and hybridizing to the matrix next to the allele-specific oligonucleotide. Hybridization of the allele-specific oligonucleotide to the matrix leads to the transfer of energy from the donor fluorophore located on it to the fluorescence quencher of the adjacent quenching oligonucleotide. Amplification results are recorded at the end of PCR by taking the fluorescence spectrum when the temperature of the reaction mixture varies from 25 to 80 degrees Celsius (the so-called "melting curves"). Upon receipt of fluorescence graphs, both heating and cooling of the reaction mixture in the indicated temperature range are possible. The present invention makes the determination of variable positions in DNA more reliable, reduces the cost of such studies through the use of standard equipment.

3. Disclosure of invention

The technical result, the achievement of which the invention is directed, is to increase the availability of such studies, since the method can be implemented on standard known equipment. It also provides the ability to conduct research in one test tube, which reduces research costs. The method of genotyping used by us is a variant of the classical method of “adjoining samples”. When determining substitutions of single nucleotides, PCR was first performed with primers common to both variants of the sequence, then the temperature of the reaction mixture was reduced to hybridize the resulting matrix with oligonucleotide samples. To determine the sequence variant, two types of oligonucleotides were used, hybridizing to the matrix side by side. One of the oligonucleotides was labeled with a fluorophore, the other with a fluorescence quencher. The reaction used one common oligonucleotide with a fluorescence quencher and a pair of allele-specific (sequence-specific) oligonucleotides bearing a fluorophore. Oligonucleotide samples corresponding to one or another variant of the sequence were labeled with various fluorophores, which made it possible to determine both variants in the same tube. The genotype was determined after PCR and hybridization by measuring the level of fluorescence during temperature denaturation of oligonucleotide duplexes and the resulting matrices (or vice versa - hybridization). This measurement was carried out in real time, its result was melting curves. The reaction conditions were chosen so as to maximize the difference in the melting temperatures of perfect and imperfect duplexes. Thus, if the analyzed sample contained only one variant of the sequence, i.e. was homozygous for this polymorphism, the melting point for a sample forming a perfect duplex was significantly higher than for a sample forming an imperfect duplex. If a heterozygous sample was analyzed, the melting points were almost the same. The indicated result is achieved by using fluorescence-labeled allele-specific oligonucleotide probes and primers during PCR.

FAM means fluorescent dye FAM, VIC means fluorescent dye VIC, BHQ1 means a dark fluorescence quencher attached to the 5'-terminal nucleotide.

4. The implementation of the invention

As a material for research, it is recommended to use peripheral blood. Isolation of DNA from biomaterial is performed using a set of reagents for DNA isolation (not the subject of this patent). The polymerase chain reaction and the determination of the melting temperature of oligonucleotide samples were carried out using a DTprime detecting amplifier (NPO DNA-Technology LLC, Russia). The following amplification temperature regime was used: 94 ° C for 10 s, 64 ° C for 30 s for 50 cycles. Upon completion of the amplification reaction, the reaction mixture was cooled to 25 ° C at a rate of 2 ° C / sec. Melting curves were obtained as follows: the temperature of the reaction mixture was increased from 25 ° C to 75 ° C in increments of 1 ° C, measuring the level of fluorescence at each step. Results i.e. the assignment of a sample to a homozygote or heterozygote for a given allele is evaluated by the shape of the DNA melting curves (Fig. 1).

Bibliography

1. Lash TL, Lien EA, Sorensen HT, Hamilton-Dutoit S Genotype-guided tamoxifen therapy: time to pause for reflection? // Lancet Oncol. 2009 Aug; 10 (8): 825-33.

2. Goetz MP, Rae JM, Suman VJ, Safgren SL, Ames MM, Visscher DW, Reynolds C, Couch FJ, Lingle WL, Flockhart DA, Desta Z, Perez EA, Ingle JN. Pharmacogenetics of tamoxifen biotransformation is associated with clinical outcomes of efficacy and hot flashes.// J Clin Oncol. 2005 Dec 20; 23 (36): 9312-8.

3. Goetz MP, Ingle J. Early discontinuation of tamoxifen: a lesson for oncologists.// Cancer. 2007 Dec 1; 110 (11): 2595-6.

4. Schroth W, Antoniadou L, Fritz P, Schwab M, Muerdter T, Zanger UM, Simon W, Eichelbaum M, Brauch H. Breast cancer treatment outcome with adjuvant tamoxifen relative to patient CYP2D6 and CYP2C19 genotypes.// J Clin Oncol. 2007 Nov 20; 25 (33): 5187-93.

5. Bradford L.D. CYP2D6 allele frequency in European Caucasians, Asians, Africans and their descendants. // Pharmacogenomics. 2002 Mar; 3 (2): 229-43.

6. Ingelman-Sundberg M, Sim SC, Gomez A, Rodriguez-Antona C. Influence of cytochrome P450 polymorphisms on drug therapies: pharmacogenetic, pharmacoepigenetic and clinical aspects. // Pharmacol Ther. 2007 Dec; 116 (3): 496-526. Epub 2007 Oct 9.

7. Ingelman-Sundberg M, Rodriguez-Antona C. Pharmacogenetics of drug-metabolizing enzymes: implications for a safer and more effective drug therapy.//. Philos Trans R Soc Lond In Biol Sci. 2005 Aug 29; 360 (1460): 1563-70.

Claims (4)

A method for determining the human genotype by polymorphism in the cytochrome P450 gene CYP2D6 * 6 (1707delT) rs5030655, based on the removal of melting curves with fluorescently-labeled allele-specific oligonucleotide samples, characterized in that they use a pair of primers that are common for all alleles, differing for each -labeled allele-specific oligonucleotide probes and a universal oligonucleotide labeled with a fluorescence quencher, of the following nucleotide composition:

FAM - means fluorescent dye FAM, VIC - means fluorescent dye VIC, BHQ1 - means a dark fluorescence quencher attached to the 5'-terminal nucleotide; the assignment of a sample to a homozygote or heterozygote for a given allele is evaluated by the shape of the DNA melting curves, by the maximum of the first derivative of the fluorescence graphs.

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