RU2756203C1 - Method for determining human genotype associated with acetylation of xenobiotics - Google Patents

Abstract

FIELD: medical biotechnology.SUBSTANCE: invention relates to medical biotechnology. The invention is a method for determining the human genotype associated with fast and slow acetylation of xenobiotics. Genotyping of arylamine-N-acetyltransferase is carried out by detecting tag SNP rs1495741 using specific primers NAT2Fand NAT2Rand two fluorescently labeled probes containing “closed nucleotide” sites, NAT2andin the amplification mode, namely one 95° cycle 5 min, 40 95° cycles 10 s, 65° cycles 15 s, 72° cycles 15 s. The results are evaluated by recording the fluorescence signal via the FAM and R6G channels by detecting the signal between cycles 18-38.EFFECT: invention makes it possible to reduce the determination time, as well as to increase sensitivity and specificity.1 cl, 4 ex

Description

The alleged invention relates to medicine, namely to pharmacogenetics, molecular diagnostics, and relates to oligonucleotide primers, fluorescent DNA probes and a method for determining the human genotype associated with fast and slow acetylation of xenobitotics.

Arylamine-N-acetyltransferase 2 (NAT2) is an enzyme of the second phase of biotransformation of xenobiotics, which carries out the acetylation of arylamines and hydrazines. The enzyme is synthesized in all cells of the body, but its greatest activity is observed in liver cells, where the detoxification of substances is completed. The enzyme gene is localized on chromosome 8 (8p22) and has a number of polymorphisms, the combination of which led to the existence of two haplotypes associated with slow or fast acetylation of the substrate (Hein, David W. "N-acetyltransferase SNPs: emerging concepts serve as a paradigm for understanding complexities of personalized medicine. "Expert opinion on drug metabolism & toxicology 5.4 (2009): 353-366). The combination of allelic variants of the NAT2 gene leads to a trimodal distribution of the population on fast, slow, and intermediate acetylators.

NAT2 participates in the acetylation of aromatic and heterocyclic amines, which are contained in tobacco smoke, environmental pollutants, combustion products, endogenous compounds (serotonin, histamine, dopamine), some medicinal substances, and are also released into the atmospheric air during the production of paints, rubber, coal gas ...

In medicine, the assessment of the rate of acetylation of drugs is of the greatest practical importance. In studies by a number of scientists on the efficacy and safety of isoniazid use for the treatment of patients with tuberculosis, the dependence of the frequency of the neuro- and hepatotoxic effect of the drug on the genotype was shown: slow acetylators had higher risks of developing toxic effects when using isoniazid (Ho, Hsin-Tien, et al. " The NAT2 tag SNP rs1495741 correlates with the susceptibility of antituberculosis drug-induced hepatotoxicity. "Pharmacogenetics and Genomics 23.4 (2013): 200-207). In addition, slow acetylators have a predisposition to the development of medicinal systemic lupus erythematosus with long-term treatment with antiarrhythmic drugs (procainamide, hydralazine), hematological toxicity with the use of dapsone, toxic epidermal necrolysis in the treatment of infectious diseases with antibiotics of the sulfonamide group. Fast acetylators, in comparison with slow ones, are less susceptible to toxic effects, but the effectiveness of the use of drugs in this group is reduced due to the rapid metabolism and excretion of the active substance from the body, which leads to a decrease in the time of its effect on target cells. Determination of the genotype of the NAT2 gene allows prescribing drugs in accordance with the individual characteristics of the patient, which is relevant for the development of personalized medicine. In addition, a slowdown in the rate of acetylation is associated with an increased risk of cancer due to a decrease in the deactivation of aromatic amines. A number of studies have shown a relationship between the predisposition to cancer, such as breast cancer and bladder cancer, with the NAT2 genotype, especially among smokers (Umberto Gelatti et al. "N-Acetyltransferase-2, glutathione S-transferase M1 and T1 genetic polymorphisms, cigarette smoking and hepatocellular carcinoma: A case-control study. "Int. J. Cancer: 115, (2005): 301-306. Thus, genetically determined characteristics of the biotransformation of xenobiotics determine the individual reactions of the organism to a variety of toxic substances and drugs.

There are several known methods for genotyping the NAT2 gene, based on the determination of seven SNPs: rs1801279 (G191A), rs1041983 (C282T), rs1801280 (T341C), rs1799929 (C481T), rs1799930 (G590A), rs1208 (rs7993AG), rs1208 (rs7803AG), , Selinski S, Dannappel D, Blaszkewicz M, Golka K. Reinvestigation of the concordance of hu-man NAT2 phenotypes and genotypes. Arch Toxicol. 2005; 79: 196-200].

There is a known method of sequencing the second exon of the NAT2 gene and determining the genotype using the NAT2PRED classification algorithm (Kuznetsov, Igor V., Michael McDuffie, and Roxana Moslehi. "A web server for inferring the human N-acetyltransferase-2 (NAT2) enzymatic phenotype from NAT2 genotype . "Bioinformatics 25.9 (2009): 1185-1186). However, the application of this method is limited in medical practice due to the complexity of sample preparation, the duration of the test and the high cost of equipment and kits.

The known method of genotyping, based on the analysis of polymorphism of the length of restriction DNA fragments (polymerase chain reaction in real time) [Miller, MA, Grigg, ME, Kreuder, S., James, ER, Melli, A.S. , Crosbie, PR, Jessup, DA, Boothroyd, JC, Brownstein, D., Conrad, PA, 2004. Anunusual genotype of Toxoplasma gondiiis common in California sea otters (Enhydra lutris nereis) and is a cause of mortality. Int. J. Parasitol. 34, 275-284.]. The disadvantage of this method is its time and labor input, since it involves carrying out two polymerase chain reactions and the use of 7 restriction enzymes, followed by detection in an agarose gel.

A known method for determining the polymorphism of the NAT2 gene (rs1801280) in combination with genes of other biotransformation enzymes using the method of hybridization on a biological chip. A diagnostic system "PF-Biochip" (Reg.Ud. No. FS 012b2006 / 5317-06) has been developed to identify and diagnose a predisposition to the development of oncological diseases and to determine individual sensitivity to certain drugs (5-fluorouracil, methotrexate, omeprazole, etc. ). The disadvantages of this method include the duration of the analysis (Gra, O.A., et al. "Genetic polymorphism of GST, NAT2, and MTRR and susceptibility to childhood acute leukemia." Molecular Biology 42.2 (2008): 187). In addition, the rs1801280 polymorphism is often identified in combination with rs1041983 to more accurately predict the NAT2 genotype.

The closest way is "Kit and method for detecting polymorphism of the NAT2 gene using fluorescent quantitative real-time PCR" (Reg. Beats. No. WO / 2014/036924). To detect a key single nucleotide polymorphism (SNP), a set of oligonucleotide primers is used, followed by real-time fluorescent quantitative polymerase chain reaction.

The above methods have a number of disadvantages - high labor input and cost of the kits, the duration of the test, which makes it difficult to use them in practical medicine.

The technical result of the proposed method is to reduce the time of determination, as well as to increase the sensitivity and specificity with the availability of equipment and reagents.

и NAT2R

и двух флюоресцентно-меченных зондов, содержащих участки «замкнутых нуклеотидов» NAT2

и

в режиме амплификации, а именно 1 цикл 95° - 5 мин, 40 циклов 95° - 10 с, 65° - 15 с, 72° - 15 с.New in the proposed method is that genotyping of arylamine-N-acetyltransferase is carried out based on the detection of tag SNP rs1495741 using specific primers NAT2F

and NAT2R

and two fluorescently labeled probes containing regions of "closed nucleotides" NAT2

and

in the amplification mode, namely 1 cycle 95 ° - 5 min, 40 cycles 95 ° - 10 s, 65 ° - 15 s, 72 ° - 15 s.

It is also new that the evaluation of the results is carried out by recording the fluorescence signal in the FAM and R6G channels by detecting the signal between 18-38 cycles.

To identify the key single nucleotide polymorphism (tag SNP) rs1495741, synthetic oligonucleotide primers and DNA probes designed by the authors were used:

и NAT2R

1) Primers NAT2F

and NAT2R

и

2) Fluorescently labeled probes NAT2

and

The advantage of the proposed method is that the determination of the NAT2 genotype includes the isolation of DNA from blood, saliva and other biological samples, carrying out the polymerase chain reaction with real-time detection (RT-PCR) with the developed oligonucleotide primers and fluorescently labeled probes in the amplification mode (1 cycle 95 ° C - 5 min; 40 cycles 95 ° C - 10 s, 65 ° C - 15 s, 72 ° C - 15 s) and evaluate the result. Evaluation of the result is carried out by recording the fluorescence signal through the FAM (520 nm) and R6G (557 nm) channels by detecting the signal between 18-38 cycles. The analysis of the patent and scientific literature showed that the proposed method differs from other solutions in this area. Thus, the primers and probes required for the detection of NAT2 haplotypes using RT-PCR were not found. Compared to the used PCR-RFLP, biochip hybridization and sequencing methods, the designed primers and DNA probes make it possible to quickly, with high sensitivity and specificity, determine the NAT2 genotype and the type of acetylation based on SNP at position 18415371 of chromosome 8: A; A - slow, A ; G - intermediate, G; G - fast. The proposed method is characterized by a high speed of implementation, the availability of equipment and reagents for its implementation, and high sensitivity and specificity. It has been shown that the results of genotyping using this SNP correlate with the type of acetylation, which was predicted based on seven SNPs in the sequence of the second exon of the NAT2 gene (Selinski, Silvia, et al. "Genotyping NAT2 with only two SNPs (rs1041983 and rs1801280) outperforms the tagging SNP rs1495741 and is equivalent to the conventional 7-SNP NAT2 genotype. "Pharmacogenetics and genomics 21.10 (2011): 673-678.).

The design of primers and probes was carried out in manual mode based on the structure of the description of SNP rs1495741 in the reference genomes (nucleotide sequence to the right and left of the target position) [https://www.ncb.nlm.nih.gov/snp/rs1495741].

NAT2F

NAT2R

Probe 1

Probe 2

The chemical synthesis of primers and probes is carried out by our order at the Sintol Research and Production Company (Moscow), where the concentration of oligonucleotides is determined. For work, the authors use the dilution of oligonucleotide primers and probes from freeze-dried material. Probe 1 is labeled with the R6G fluorophore at the 5 'end and the fluorescence quencher BHQ2 at the 3' end. Signal detection was performed via the "yellow" (R6G / Yellow) channel (wavelength 557 nm). Probe 2 is labeled with the FAM fluorophore at the 5 'end and the fluorescence quencher RTQ1 at the 3' end. The signal was detected via the green (FAM / Green) detection channel in a RotorGene6000 thermal cycler (wavelength 520 nm).

These primers were selected based on the analysis of the gene structure in the range of 200 bp. around the SNP tag rs1495741.

и NAT2R

и двух флюоресцентно-меченных зондов, содержащих участки «замкнутых нуклеотидов»: NAT2

и

в режиме амплификации, а именно 1 цикл 95° - 5 мин, 40 циклов 95° - 10 с, 65° - 15 с, 72° - 15 с, а оценку результатов проводят регистрацией сигнала флуоресценции по каналам FAM и R6G путем детекции сигнала между 18-38 циклами, что соответствует критерию «новизна».Comparative analysis of the proposed method and the prototype shows that the proposed method differs in that genotyping of arylamine-N-acetyltransferase is carried out by detecting tag SNP rs1495741 using specific primers NAT2F

and NAT2R

and two fluorescently labeled probes containing regions of "closed nucleotides": NAT2

and

in the amplification mode, namely 1 cycle 95 ° - 5 min, 40 cycles 95 ° - 10 s, 65 ° - 15 s, 72 ° - 15 s, and the results are assessed by recording the fluorescence signal through the FAM and R6G channels by detecting the signal between 18-38 cycles, which corresponds to the criterion of "novelty".

The proposed method allows you to quickly and with high sensitivity and specificity to determine the NAT2 genotype and the type of acetylation in the test material. The advantage of the proposed method is the reduction in research time, simple and automated interpretation of the results, the availability of the method for clinical laboratories.

The method is carried out as follows.

NAT2R

зонд 1

зонд 2

сульфат магния, смесь четырех 2'-дезоксинуклеозид-5'-трифосфатов, TaqF-полимераза. Конечный объем реакционной смеси - 25 мкл. Проводят полимеразную цепную реакцию с детекцией в реальном времени с разработанными олиглнуклеотидными праймерами и флуоресцентно-меченными зондами в режиме амплификации. Режимы амплификации ДНК на амплификаторе CFX БиоРад: 1 цикл 95°С - 5 мин; 40 циклов 95°С - 10 с, 65°С - 15 с, 72°С - 15 с. Оценку результата проводят путем регистрации сигнала флуоресценции по каналам FAM (520 нм) и R6G (557 нм) путем детекции сигнала между 18-38 циклами. Предложенный способ позволяет быстро, с высокой специфичностью выявить аллель 1495741А, связанный с медленным ацетилированием ксенобиотиков, и аллель 1495741G, связанный с быстрым ацетилированием ксенобиотиков. Присутствие обоих аллелей 1495741А и 1495741G оценивают как генотип, связанный с умеренной скоростью ацетилирования.Isolate DNA from blood, saliva, or other biological samples. The material is placed in a test tube with a screw cap. DNA isolation is performed with a set of DNA-sorb-B according to the manufacturer's protocol (Interlabservice, Moscow). Genotype NAT2 is identified. To 12.5 μl of the sample, add 12.5 μl of the mixture containing the buffer for polymerase chain reaction, primer NAT2F

NAT2R

probe 1

probe 2

magnesium sulfate, a mixture of four 2'-deoxynucleoside-5'-triphosphates, TaqF polymerase. The final volume of the reaction mixture is 25 μl. A polymerase chain reaction with real-time detection is carried out with the developed oliglnucleotide primers and fluorescently labeled probes in the amplification mode. Modes of DNA amplification on the CFX BioRad amplifier: 1 cycle at 95 ° С - 5 min; 40 cycles 95 ° C - 10 s, 65 ° C - 15 s, 72 ° C - 15 s. Evaluation of the result is carried out by recording the fluorescence signal through the FAM (520 nm) and R6G (557 nm) channels by detecting the signal between 18-38 cycles. The proposed method makes it possible to quickly, with high specificity, identify allele 1495741A associated with slow acetylation of xenobiotics, and allele 1495741G associated with rapid acetylation of xenobiotics. The presence of both alleles 1495741A and 1495741G is assessed as a genotype associated with a moderate rate of acetylation.

Examples of specific implementation of the method

NAT2R

зонд 1

зонд 2

сульфат магния; смесь четырех 2'-дезоксинуклеозид-5'-трифосфатов; TaqF-полимераза. Конечный объем реакционной смеси - 25 мкл. При регистрации сигнала наблюдали экспоненциальный рост флюоресценции по FAM между 18-38 циклами, что свидетельствует о наличии двух аллелей 1495741А. Таким образом результат оценивается как генотип, связанный с медленной скоростью ацетилирования субстрата.Example 1. The buccal epithelium was scraped from the inside of the cheek of volunteer A. The material was placed in a test tube with a screw cap. DNA isolation was performed with a DNA-sorb-B kit (Interlabservice, Moscow) according to the manufacturer's protocol. To 12.5 μl of the sample was added 12.5 μl of the mixture containing the PCR buffer; primer NAT2F

NAT2R

probe 1

probe 2

magnesium sulfate; a mixture of four 2'-deoxynucleoside-5'-triphosphates; TaqF polymerase. The final volume of the reaction mixture is 25 μl. When registering the signal, an exponential increase in FAM fluorescence was observed between 18-38 cycles, which indicates the presence of two alleles 1495741A. Thus, the result is assessed as a genotype associated with a slow rate of substrate acetylation.

To confirm the obtained result, the sample was sent to ZAO Evrogen for sequencing from the second exon NAT2. The interpretation of the results was carried out using the NAT2PRED classification algorithm (Kuznetsov I.V., McDuffie M., Moslehi R., "A web server for inferring the human N-acetyltransferase-2 (NAT2) enzymatic phenotype from NAT2 genotype." Bioinformatics 25.9 (2009 ): 1185-1186.). As a result, the genotype with a slow type of acetylation was confirmed.

NAT2R

зонд 1

зонд 2

сульфат магния; смесь четырех 2'-дезоксинуклеозид-5'-трифосфатов; TaqF-полимераза. Конечный объем реакционной смеси - 25 мкл. При регистрации сигнала наблюдали экспоненциальный рост флюоресценции по R6G между 18-38 циклами, что свидетельствует о наличии двух аллелей 1495741G, ассоциированных с быстрой скоростью ацетилирования.Example 2. The buccal epithelium was scraped from the inside of the cheek of volunteer P. The material was placed in a test tube with a screw cap. DNA isolation was performed with a DNA-sorb-B kit (Interlabservice, Moscow) according to the manufacturer's protocol. To 12.5 μl of the sample was added 12.5 μl of the mixture containing the PCR buffer; primer NAT2F

NAT2R

probe 1

probe 2

magnesium sulfate; a mixture of four 2'-deoxynucleoside-5'-triphosphates; TaqF polymerase. The final volume of the reaction mixture is 25 μl. When registering the signal, an exponential increase in R6G fluorescence was observed between 18-38 cycles, which indicates the presence of two 1495741G alleles associated with a rapid rate of acetylation.

To confirm the obtained result, the sample was sent to ZAO Evrogen for sequencing from the second exon NAT2. The results were interpreted using the NAT2PRED classification algorithm (Kuznetsov, Igor V., Michael McDuffie, and Roxana Moslehi. "A web server for inferring the human N-acetyltransferase-2 (NAT2) enzymatic phenotype from NAT2 genotype." Bioinformatics 25.9 (2009) : 1185-1186.). As a result, the genotype with the rapid type of acetylation was confirmed.

NAT2R

зонд 1

зонд 2

сульфат магния; смесь четырех 2'-дезоксинуклеозид-5'-трифосфатов; TaqF-полимераза. Конечный объем реакционной смеси - 25 мкл. При регистрации сигнала наблюдали экспоненциальный рост флюоресценции по R6G и FAM между 18-38 циклами, что свидетельствует о наличии двух аллелей 1495741G и 1495741А. Таким образом, генотип NAT2 ассоциирован с умеренной скоростью ацетилирования субстратов.Example 3. The buccal epithelium was scraped from the inside of the cheek of volunteer K. The material was placed in a test tube with a screw cap. DNA isolation was performed with a DNA-sorb-B kit (Interlabservice, Moscow) according to the manufacturer's protocol. To 12.5 μl of the sample was added 12.5 μl of the mixture containing the PCR buffer; primer NAT2F

NAT2R

probe 1

probe 2

magnesium sulfate; a mixture of four 2'-deoxynucleoside-5'-triphosphates; TaqF polymerase. The final volume of the reaction mixture is 25 μl. When registering the signal, an exponential increase in fluorescence for R6G and FAM was observed between 18-38 cycles, which indicates the presence of two alleles 1495741G and 1495741A. Thus, the NAT2 genotype is associated with a moderate rate of substrate acetylation.

To confirm the obtained result, the sample was sent to ZAO Evrogen for sequencing from the second exon NAT2. The results were interpreted using the NAT2PRED classification algorithm (Kuznetsov, Igor V., Michael McDuffie, and Roxana Moslehi. "A web server for inferring the human N-acetyltransferase-2 (NAT2) enzymatic phenotype from NAT2 genotype." Bioinformatics 25.9 (2009) : 1185-1186). As a result, the genotype with an intermediate (moderate) type of acetylation was confirmed.

Example 4. Approbation of the proposed method was carried out in 50 people who voluntarily took part in the study. Informed consent was obtained from each study participant, and the conduct of this work was approved by the ethics committee of Irkutsk State Medical University. Genetic material (buccal epithelium) was investigated in two ways: sequencing of the second exon NAT2, which was carried out by the company "Evrogen", and according to the proposed method, which was performed on the bases of the FGBNU NTs PZSRCH and GBOU VPO ISMU. When evaluating the results of the proposed method, 29 samples were identified with a slow type of acetylation (A; A), 16 with an intermediate (A; G) and 5 samples with a fast (G; G). Subsequently, the DNA was sent for sequencing of the second exon NAT2 on the basis of ZAO Evrogen. The genotype was determined using the NAT2PRED classification algorithm. ([Kuznetsov, Igor B., Michael McDuffie, and Roxana Moslehi. "A web server for inferring the human N-acetyltransferase-2 (NAT2) enzymatic phenotype from NAT2 genotype." Bioinformatics 25.9 (2009): 1185-1186). As a result, only two samples were misclassified: 1 sample had an intermediate type of acetylation genotype and 1 sample with a slow type of acetylation. Thus, the sensitivity and specificity of the proposed method relative to the reference study were 96.6% and 95.2%, respectively.

Claims (1)

A method for determining the human genotype associated with acetylation of xenobitics by detecting arylamine-N-acetyltransferase gene polymorphism using a fluorescent quantitative polymerase chain reaction in real time, characterized in that genotyping of arylamine-N-acetyltransferase is performed by detecting tag SNP rs1495741 NAT2F 5'-AGCCTCTCTCAGGAAAGGAGCA and NAT2R 5'-GCCACTCATGGTCACTTCGGC and two fluorescently labeled probes containing closed nucleotide regions NAT2 5'-R6G-AAGCTACTGTGAA (T-LNA) GCCCA and B-LNA2 -AAGCTACTGTGAAT (G-LNA) CCCA (T-LNA) ATT-RTQ1 in amplification mode, namely 1 cycle 95 ° - 5 min, 40 cycles 95 ° - 10 s, 65 ° - 15 s, 72 ° - 15 s, and the evaluation of the results is carried out by recording the fluorescence signal in the FAM and R6G channels by detecting the signal between 18-38 cycles.