KR101633783B1 - Method of prediction the blood concentration of tacrolimus using SNP in CYP3A5 and CYP3A7 genes - Google Patents

Abstract

The present invention relates to a method for predicting a drug concentration of an entity with respect to tacrolimus. More specifically, the present invention relates to a composition for predicting a drug concentration of an entity with respect to tacrolimus, wherein the composition comprises rs10211, rs12360, and rs2257401 disposed in CYP3A7 and rs15524 single nucleotide polymorphism (SNP) disposed in CYP3A5; a kit for detecting the SNP; and a method for predicting a drug concentration of an entity with respect to tacrolimus by analyzing a haplotype with respect to the SNP. According to the present invention, the composition preliminarily predicts a drug concentration of an entity with respect to tacrolimus according to a genetic difference of an entity, so the composition may be useful in determining a suitable administration amount of tacrolimus. Thus, in the early stage, a drug concentration in blood may be made to rapidly reach a proper treatment region, so early repulsion and occurrence of drug side effects are reduced. Consequently, the composition is very useful in ensuring effectiveness and safety of tacrolimus.

Description

Methods for predicting drug concentration in tacrolimus using nucleotide sequence polymorphisms of CYP3A5 and CYP3A7 gene regions [

The present invention relates to a method for predicting drug concentration in blood of an individual against tacrolimus, and more particularly, to a method for predicting drug concentration of tacrolimus including rs15524 single nucleotide polymorphism (SNP) located in CYP3A7, rs10211, rs12360, rs2257401 and CYP3A5 A kit for detecting the polymorphic site, and a method for predicting blood drug concentration of an individual to tacrolimus by analyzing a haplotype of the polymorphic site.

Tacrolimus is an immunosuppressive agent used to prevent rejection in transplant patients and to reduce immune responses in autoimmune diseases such as glomerulonephritis. Tacrolimus has a wide range of individual pharmacokinetic differences and narrow therapeutic areas. In the case of transplant patients, when the blood concentration of tacrolimus in the body is low at the initial time of administration, the rejection of the transplant organs increases, and when the blood concentration of the transplant is high, toxic adverse reactions and infections due to drugs may occur frequently. Therefore, it is essential to monitor the blood concentration of the drug in accordance with the narrow therapeutic range in order to demonstrate the appropriate therapeutic effect of the drug and ensure safety (Undre NA, Stevenson P, Schafer A. Pharmacokinetics of tacrolimus: clinically relevant aspects. Proc., 1999; 31 (7A): 21S-24S).

Currently, most medical institutions start dosing patients with the initial dose calculated as body weight for transplant patients, and then monitor the tacrolimus blood levels to correct dosage. However, liver dysfunction, time after transplantation, hematocrit, serum albumin, age, race, drug interactions, and especially genetic polymorphisms have been reported as tacrolimus pharmacokinetic affectors.

The activity of cytochrome P450 3A5 (CYP3A5) as a major metabolic enzyme of tacrolimus has a major effect on pharmacokinetics. Typically, when the intact 6986 base (99672916 base of chromosome 7) is changed from A to G, stop codon CYP3A5 * 3 is not expressed and metabolism is lost. Patients with CYP3A5 * 3 had a higher dose-correction dose, so the dose of tacrolimus should be reduced compared to wild-type patients. (Rojas L, Neumann, Herrero MJ, et al. Effect of CYP3A5 * 3 on kidney transplant recipients treated with tacrolimus: a systematic review and meta-analysis of observational studies., Pharmacogenomics Journal, 2015; 15: 38-48). Conversely, in the case of cytochrome P450 3A4 (CYP3A4), the upstream -392 base (997844573 base of chromosome 7) shifts from A to G, and the expression of CYP3A4 * 1B enzyme increases, And the metabolic rate of tacrolimus is accelerated. Therefore, it is very important to confirm various genotypic information when administering tacrolimus to perform drug therapy. (Shi WL, Tang HL, Zhai SD. Effects of the CYP3A4 * 1B Genetic Polymorphism on the Pharmacokinetics of Tacrolimus in Adult Renal Transplant Recipients: A Meta-Analysis. PLoS One. 2015; 10 (6): e0127995)

Although many studies have shown that tacrolimus has been associated with several factors for pharmacokinetic prediction, it has not yet been fully controlled for tacrolimus concentration over a long period of time from the beginning of the administration, and acute rejection and drug toxicity . Thus, there is a need for the development of a number of biomarkers that enable such tacrolimus pharmacokinetic prediction.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Therefore, the inventors of the present invention conducted a study to develop a technique for predicting the tacrolimus pharmacokinetics by analyzing single nucleotide polymorphism. As a result, it has been found that in the organ transplant group taking tacrolimus, whole exome sequencing (WES) Genetic information showing differences in drug plasma concentrations between individuals was searched by searching various genetic information. Through this study, SNPs related to tacrolimus pharmacokinetics were identified, and the SNPs of cytochrome P450 3A (Cytochrome 3A, CYP3A) SNPs and their haplotypes are closely related to the pharmacokinetics of tacrolimus.

Accordingly, an object of the present invention is to provide a probe or primer capable of detecting single nucleotide polymorphism of a CYP3A gene family selected from the group consisting of NCBI refSNP IDs: rs10211, rs12360, rs2257401, and rs15524 as tacrolimus And to provide a composition for predicting the drug concentration of an individual.

It is another object of the present invention to provide a method for predicting the drug concentration of an individual against tacrolimus in a patient selected from the group consisting of NCBI refSNP ID: rs10211, rs12360, rs2257401 and rs15524 And a method for detecting a single base polymorphism of the CYP3A gene family.

It is a further object of the present invention to provide a method for predicting the drug concentration of an individual against tacrolimus which determines haplotypes for the single nucleotide polymorphic site and determines that the haplotype is highly or lowly pharmacokinetic response to tacrolimus, And to provide a method for analyzing single nucleotide polymorphisms.

It is still another object of the present invention to provide a kit for predicting drug concentration of an individual against tacrolimus comprising primers for screening polymorphism selected from NCBI refSNP ID: rs10211, rs12360, rs2257401 and rs15524 of the CYP3A gene family or genotyping primers .

Another object of the present invention is to provide an individual drug concentration prediction marker for tacrolimus comprising a nucleotide sequence polymorphism selected from the NCBI refSNP IDs of the CYP3A gene family: rs10211, rs12360, rs2257401 and rs15524.

In order to achieve the above object, the present invention provides a probe or primer capable of detecting a single nucleotide polymorphism of a CYP3A gene family selected from the group consisting of NCBI refSNP IDs: rs10211, rs12360, rs2257401 and rs15524, A composition for predicting drug concentration of an individual for tacholimus.

In order to achieve the above object, the present invention provides a method for predicting the drug concentration of tacrolimus in a patient, comprising the steps of: analyzing the NCBI refSNP ID: rs10211, rs12360, rs2257401 And rs15524. ≪ Desc / Clms Page number 2 > The present invention also provides methods for detecting a single nucleotide polymorphism of a CYP3A gene family selected from the group consisting of:

In order to accomplish still another object of the present invention, the present invention provides a method for determining the haplotype of the single nucleotide polymorphism site, thereby determining the drug concentration of the haplotype against the tacrolimus which determines whether the haplotype is high or low in the pharmacokinetic response to tacrolimus Provides a method for single base polymorphism analysis of the CYP3A gene family for prediction.

In order to accomplish still another object of the present invention, the present invention provides a screening primer selected from the NCBI refSNP ID: rs10211, rs12360, rs2257401 and rs15524 of the CYP3A gene family, a primer for screening of genotyping Of the drug concentration of the drug.

In order to accomplish still another object of the present invention, the present invention provides a drug concentration prediction marker for tacrolimus of an individual comprising a nucleotide sequence polymorphism selected from the NCBI refSNP IDs of the CYP3A gene family: rs10211, rs12360, rs2257401 and rs15524.

Hereinafter, the present invention will be described in detail.

The term " single nucleotide polymorphism " in the present invention means a genetic change or mutation showing a difference in one nucleotide sequence (A, T, G, C) in the nucleotide sequence of DNA is more than 2% It refers to genetic trait information on which sequences can occur.

The term " next generation sequencing (NGS) " in the present invention refers to a method of dividing a genome into numerous pieces, analyzing the genetic information of each fragment, combining and analyzing the entire nucleotide sequence.

The term 'whole exome sequencing (WES)' in the present invention means sequencing only gene regions involved in protein production as one of the next-generation sequencing methods.

The term " pharmacokinetic indicator " in the present invention refers to the lowest blood concentration (C ₀ ) of tacrolimus, the lowest blood concentration ratio (C ₀ / D) to the administration dose, the dose ratio to the body weight (D / kg) and a blood concentration ratio _{(C 0 / D / kg)} .

In the present invention, the term " haplotype " refers to a series of SNPs located on one chromosome at the time of germ cell division, and may be divided into a plurality of bundles, , And this haplotype is a combination of several SNPs present on the same chromosome and exhibits a constant SNP pattern.

The composition of the present invention is for predicting the drug concentration of an individual against tacrolimus and is a probe capable of detecting single nucleotide polymorphism of the CYP3A gene family selected from the group consisting of NCBI refSNP IDs: rs10211, rs12360, rs2257401 and rs15524 Or a primer as an active ingredient.

The polymorphic site of the present invention is located in the exon, UTR (untranslated region), intron or adjacent region of the CYP3A gene family, and is a region specified by rs10211, rs12360, rs2257401, or rs15524 as the NCBI refSNP ID.

rs10211 is a SNP located in the 3 'UTR region of CYP3A7, located at position 90302994 of chromosome 7, and is a SNP in which T is mutated to C. rs10211 is composed of 8 to 200 consecutive DNA sequences substituted with C at the 501st base T of the nucleotide sequence shown in SEQ ID NO: 1.

rs12360 is a SNP located in the 3 'UTR region of CYP3A7, located at 99303094th of chromosome 7, and A is a SNP mutated into G. rs12360 can be composed of 8 to 200 consecutive DNA sequences substituted with G at the 501st base A of the nucleotide sequence shown in SEQ ID NO: 2.

rs2257401 is a SNP located in the exon 11 region of CYP3A7, located at position 99306685 of chromosome 7, and is a SNP in which G is mutated into C. rs2257401 can be composed of 8 to 200 consecutive DNA sequences substituted with C at the 501st base G of the nucleotide sequence shown in SEQ ID NO: 3.

rs15524 is a SNP located in the 3 'UTR region of CYP3A5, located at position 99245914 of chromosome 7, and A is a SN variant in G. rs15524 is substituted with G at the 501st base A of the nucleotide sequence shown in SEQ ID NO: 4 and can be composed of 8 to 200 consecutive DNA sequences.

The polymorphisms of rs10211, rs12360, rs2257401, or rs15524 were found to affect the metabolism of tacrolimus in individuals and were associated with blood drug concentrations. Could know. These results suggest that rs10211, rs12360, rs2257401, or rs15524 polymorphisms can be used as predictors of drug concentration in individuals for tacrolimus and as a marker for diagnosis based on them.

The term " diagnosis " as used herein includes any type of analysis used to predict or predict the disease or condition of a patient and to determine the risk of developing a pathological particular condition.

The term " drug concentration " as used herein also means an increase or decrease in the concentration of tacrolimus in a subject through a series of processes such as absorption, metabolism and excretion of tacrolimus in response to pharmacokinetics of the subject to tacrolimus . Preferably, the drug concentration of the individual in the present invention may be the concentration of the drug in the blood of the subject, i.e., tacrolimus.

And the term " polymorphism " in the present invention means the occurrence of two or more alternative sequences or alternate alleles within a genetically determined population. Preferred polymorphic markers have two alleles that exhibit an incidence of 1% or more, more preferably 10% or 20% or more in selected populations.

The term " single nucleotide polymorphism " in the present invention means a variation of the DNA nucleotide sequence in which a single nucleotide A, T, C or G in the genome is different in the same species or between chromosome pairs of each individual.

The present invention relates to a base mutant at each SNP position in a DNA base sequence, but also includes a polynucleotide sequence complementary to the aforementioned nucleotide sequence when such SNP base mutation is found in a double-stranded gDNA (genomic DNA) Is interpreted. Thus, the base at the SNP position in the complementary polynucleotide sequence is a complementary base.

In the present invention, the CYP3A gene family represents a subfamily of the cytochrome P450 present in the CYP3A locus. The CYP3A gene family generally includes CYP3A4, CYP3A5, CYP3A7 and CYP3A43 but represents CYP3A4, CYP3A5 and CYP3A7 in which the SNPs of the present invention are located.

Meanwhile, the composition of the present invention may further include other SNPs of the known CYP3A family, i.e., NCBI refSNP ID: rs776746, rs12333983, or rs2242480.

rs776746 is a SNP located in the intron 3 region of CYP3A5, located at 99672961th position of chromosome 7, and is a SNP in which A is mutated to G. It is known that the expression of CYP3A5 is changed according to the variation of this SNP, and as a result, it is a SNP which is known as a major drug metabolizing effect index of tacrolimus. rs776746 is substituted with G at the 501st base A of the nucleotide sequence shown in SEQ ID NO: 5 and can be composed of 8 to 200 consecutive DNA sequences.

rs12333983 is a SNP located in the downstream region of CYP3A4, located at 99756491th position of chromosome 7, and is a SNP in which A is mutated to T. The effect of SNP on tacrolimus drug concentration log (C ₀ / D) has been reported (Birdwell KA, Grady B, Choi L, et al. characterize pharmacogenomic predictors of tacrolimus dose requirement in kidney transplant recipients. Pharmacogenet Genomics. 2012; 22 (1): 32-42). rs12333983 can be composed of 8 to 200 consecutive DNA sequences substituted with T at position 501 of base sequence of SEQ ID NO: 6.

rs2242480 is a SNP located in intron 10 region of CYP3A4, located at 99763843 of chromosome 7, and C is a SN variant of T. The effect of SNP on tacrolimus drug concentration log (C ₀ / D) has been reported (Li CJ, Li L, Lin L, et al. Impact of the CYP3A5, CYP3A4, COMT, IL-10 and POR genetic polymorphisms on tacrolimus metabolism in Chinese renal transplant recipients. PLoS One. 2014 Jan 21; 9 (1): e86206). rs2242480 can be composed of 8 to 200 consecutive DNA sequences substituted with T at the 501st base C of the nucleotide sequence shown in SEQ ID NO: 7.

In the present invention, tacrolimus is a generic name of a commercial agent used in various fields, and is a kind of immunosuppressant used to prevent rejection in transplant patients. Therefore, the blood drug concentration prediction of a subject to tacrolimus of the present invention is associated with rejection reaction in organ transplantation, and the subject in the present invention may be a human or organ transplant recipient as a subject in need of tacrolimus administration.

In the present invention, transplantation may be kidney transplantation, liver transplantation, heart transplantation, lung transplantation, hematopoietic stem cell transplantation, and preferably kidney transplantation.

In order to provide information necessary for predicting the drug concentration of an individual to tacrolimus, the present invention provides a method for detecting a drug concentration of a drug selected from the group consisting of NCBI refSNP ID: rs10211, rs12360, rs2257401, and rs15524 selected from the group consisting of CYP3A Lt; RTI ID = 0.0 > polymorphism < / RTI > In addition, the single nucleotide polymorphism detected in the method of the present invention may further comprise a SNP of NCBI refSNP ID: rs776746, rs12333983, or rs2242480.

In the present invention, analysis of the nucleotide sequence of the SNP, i.e., SNP genotyping, can be performed according to various methods known in the art. Sequence analysis can be performed by sequencing analysis, microarray hybridization, allele specific PCR, dynamic allele-specific hybridization (DASH), PCR extension analysis, But not limited to, Single Strand Conformation Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE), Restriction Fragment Length Polymorphism (PCR-RFLP), Two Dimensional Gene Scanning (TDGS), Taq-Man or TOGATM Do not.

For example, a method using an Affymetrix SNP chip, a Single Strand Conformation Polymorphism (PCR) method, a PCR-SSO (Specification Sequence Oligonucleotide) method, a dot hybridization method, a PCR- (Allele Specific Oligonucleotide) Hybridization Method, TaqMan Method, Pyrosequencing Method, Invader Method, SNaPShot Method (Allele Specific Gene Amplification Method), MassArray Method, Microplate Array Diagonal Gel Electrophoresis Microplate Array Diagonal Gel Electrophoresis (MADG) method, GoldenGate analysis method, SNPlex method and BeadArray method.

In the method of the present invention, primers for amplifying nucleic acid molecules or probes for detecting nucleic acid molecules may be used for SNP genotyping.

As used herein, the term "primer" refers to a single strand of oligonucleotides that is hybridized under suitable conditions (presence of four different nucleoside triphosphates and polymerases such as DNA or RNA polymerases) Quot; means acting as a starting point at which to initiate directed DNA synthesis. The suitable length of the primer is determined by the characteristics of the primer to be used, but is usually 15 to 30 bp in length. The primer need not be exactly complementary to the sequence of the template, but should be complementary enough to form a hybrid-complex with the template.

In the present invention, the term "probe" means a linear oligomer having a natural or modified monomer or linkage comprising a deoxyribonucleotide and a ribonucleotide that can hybridize to a particular nucleotide sequence. Preferably, the probe is single stranded for maximum efficiency in hybridization. The probe is preferably a deoxyribonucleotide. As the probe used in the present invention, a sequence completely complementary to the sequence including the SNP may be used, but a sequence substantially complementary may be used as long as it does not interfere with the specific hybridization. Suitable conditions for hybridization include, but are not limited to, Nucleic Acid Hybridization, A Practical Approach, Hayes, BD, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, IRL Press, Washington, DC (1985). ≪ / RTI >

The stringent condition used for hybridization can be determined by controlling the temperature, the ionic strength (buffer concentration) and the presence of a compound such as an organic solvent, and the like. This stringent condition can be determined differently depending on the sequence to be hybridized.

Meanwhile, the present invention provides a method for analyzing a single nucleotide polymorphism of the CYP3A gene family for predicting the drug concentration of an individual against tacrolimus, said method comprising the steps of:

(a) isolating genomic DNA from an individual;

(b) determining the nucleotide sequence of the NCBI refSNP ID: rs10211, rs12360, rs2257401 and rs15524 regions;

(c) In the determined nucleotide sequence, T is replaced with C, rs12360 is substituted with G, rs2257401 is replaced with C, and rs15524 is replaced with G to detect a nucleotide sequence polymorphism step;

(d) determining a haplotype with the detected base sequence polymorphism; And

(e) In the case of haplotype H1, it is determined that the drug concentration of the individual for tacrolimus is high, and that for H2, H3 and H4, the drug concentration of the individual for tacrolimus is low.

In the single base polymorphism analysis method of the present invention, step (a) is a step of isolating nucleic acid from an individual.

The term "nucleic acid" in the present invention encompasses both DNA (gDNA and cDNA) and RNA molecules. Nucleotides which are basic constituent units in nucleic acid molecules include not only natural nucleotides but also analogues analogue).

In the present invention, the nucleic acid molecule can be obtained from various types of " biological sample "isolated from an individual or individual and can be obtained, for example, from tissues, whole blood, serum, plasma, body fluids, urine, Preferably, but not limited to, tissue or whole blood.

The step of obtaining the nucleic acid from the specimen can be carried out by a conventional DNA separation method or RNA isolation and reverse transcription. In the present invention, when the nucleic acid molecule is a genomic DNA (gDNA), the separation of gDNA can be performed according to a conventional method known in the art (see Rogers & Bendich (1994)), GDNA can be isolated using a commercial kit known in the art (e.g., QIAamp DNA Blood Maxi Kit). When the starting material is mRNA, the total RNA is isolated by a conventional method known in the art (see Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (1987); and Chomczynski, P. et al., Anal. Biochem. 162: 156 (1987)). The isolated total RNA is synthesized by cDNA using reverse transcriptase. Because the total RNA is isolated from animal cells, it has a poly-A tail at the end of mRNA. CDNA can be easily synthesized using oligo dT primers and reverse transcriptase using such a sequence characteristic (see PNAS USA, 85: 8998 (1988); Libert F, et al., Science, 244: 569 (1989); and Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press )).

(b) is a step of determining the nucleotide sequence of NCBI refSNP ID: rs10211, rs12360, rs2257401 and rs15524, and the determination of each SNP and nucleotide sequence is as described above. On the other hand, NCBI refSNP IDs: rs776746, rs12333983 and rs2242480 can be further analyzed.

In step (c), T is substituted for C in rs10211, A is substituted in G for rs12360, G is substituted for C in rs2257401, and G is substituted for G in rs15524. . When rs776746, rs12333983, and rs2242480 were additionally included in step (b), rs776746 was substituted with G, rs12333983 was substituted with T, rs2242480 was replaced with T, .

(d) is a step of determining a haplotype with the detected base sequence polymorphism. The crystals of monoclust are as follows according to Table 1 for rs10211, rs12360, rs2257401 and rs15524,

Haplotype rs15524 rs10211 rs12360 rs2257401 H1 T A T G H2 C G C C H3 T A T C H4 C A T G

rs10211, rs12360, rs2257401, rs15524 and additionally rs776746, rs12333983 and rs2242480 are determined according to Table 2 below.

Haplotype rs15524 rs776746 rs10211 rs12360 rs2257401 rs12333983 rs2242480 Ht1 T G A T G T C Ht2 C A G C C A T Ht3 C A G C C A C Ht4 T G A T C T C Ht5 C A G C C T C Ht6 C G A T G T C

Step (e) is a step of determining that the individual drug concentration of tacrolimus is high when the haplotype is H1, and that the drug concentration of tacrolimus is low when H2, H3 and H4 are present.

In this step, the drug concentration of tacrolimus is determined according to the haplotype, and the drug concentration for tacrolimus depends on the drug metabolism ability such as the change of the function or activity of CYP3A depending on the genotype determined according to the haplotype do.

The term " drug metabolism " in the present invention indicates drug metabolism ability of tacrolimus administered to an individual. High and low drug metabolism ability means that the concentration of drug in blood is low or high according to the metabolism of tacrolimus, respectively do. That is, the high drug metabolism indicates that the metabolism of tacrolimus rapidly progresses after the administration of tacrolimus and the drug concentration is rapidly lowered. The low drug metabolism indicates that the metabolism of tacrolimus is slow and the concentration of drug in the blood is kept high.

In addition, when the haplotype is Ht1, the drug concentration of the individual for tacrolimus is high, and in the case of Ht2, Ht3, Ht4, Ht5, and Ht6, tacromimus is higher in the case of including rs776746, rs12333983, rs2242480 and rs4646437 in step (e) The drug concentration of the individual is determined to be low.

Meanwhile, the present invention provides a kit for predicting individual drug concentrations for tacrolimus comprising primers for screening polymorphism selected from NCBI refSNP ID: rs10211, rs12360, rs2257401 and rs15524 of the CYP3A gene family or genotyping primers . For screening additional nucleotide sequence polymorphisms in the kit of the present invention, primers for screening polynucleotides or genotyping primers selected from the group consisting of NCBI refSNP ID: rs776746, rs12333983 and rs2242480 may be additionally included.

The kit of the present invention may contain not only primers capable of amplifying to detect the SNP site of the present invention but also reagents necessary for polymerization such as dNTPs, various polymerase and coloring agents.

In one embodiment of the present invention, genomic DNA was obtained from organ transplant recipients who received immunosuppressant triple therapy including tacrolimus, and tacrolimus pharmacokinetic SNPs were selected. As a result, mutations were confirmed from a total of 2512 genes, and 264 genetic mutations related to pharmacokinetics including SNP and InDel were identified. The SNPs of the CYP family of exon and UTR regions, namely NCBI refSNP IDs: rs10211, rs12360, rs2257401 and rs15524, were finally derived based on the genes with both pharmacokinetic indices and influencing factors. NCBI refSNP IDs: rs776746, rs12333983 and rs2242480.

In one embodiment of the present invention, whether or not the linkage disequilibrium (LD) relationship of the four SNPs derived through the NGS analysis was confirmed, and evaluation was made as to whether the integrated SNPs were formed. As a result, it was confirmed that the four SNPs confirmed by the present invention had a haplotype block, and the result including the additional four SNPs revealed that seven SNPs were formed in the entire haplotype block.

In one embodiment of the present invention, the pharmacokinetic effect of tacrolimus on haplotype was confirmed. For this, ANOVA test using a linear model in which the haplotype was divided according to a certain criterion and corrected for the final clinical variables was performed. As a result, , And log (C ₀ / D) and log (C ₀ / D / kg), respectively.

Thus, the present invention provides a drug concentration prediction marker for individuals of tacrolimus comprising a nucleotide sequence polymorphism selected from the NCBI refSNP IDs of the CYP3A gene family: rs10211, rs12360, rs2257401, and rs15524. In addition, the marker may additionally comprise a nucleotide sequence polymorphism selected from the group consisting of NCBI refSNP IDs: rs776746, rs12333983, and rs2242480.

The markers of the present invention may be implemented in microarray form for diagnostic purposes. The microarray of the present invention may comprise DNA or RNA polynucleotides. The microarray comprises a conventional microarray except that the polynucleotide of the present invention is contained in the probe polynucleotide.

Methods for producing microarrays by immobilizing probe polynucleotides on a substrate are well known in the art. The term " probe polynucleotide " means a polynucleotide capable of hybridization, and means an oligonucleotide capable of binding to the complementary strand of the nucleic acid in a sequence-specific manner. Such probes include peptide nucleic acids described in the literature (Nielsen et al., Science, 254, 1497-1500 (1991)). The probe of the present invention is an allele-specific probe in which a polymorphic site exists in a nucleic acid fragment derived from two members of the same species and hybridizes to a DNA fragment derived from one member but does not hybridize to a fragment derived from another member . In this case, the hybridization conditions must be sufficiently stringent to hybridize to only one of the alleles, showing significant differences in the hybridization intensity between alleles. This can lead to good hybridization differences between different allelic forms. The probe of the present invention can be used for a diagnostic method or the like for detecting alleles. The diagnostic methods include detection methods based on hybridization of nucleic acids such as Southern blots, and may be provided in a form pre-bonded to a substrate of a DNA chip in a method using a DNA chip. The hybridization can usually be performed under stringent conditions, for example, a salt concentration of 1 M or less and a temperature of 25 ° C or higher. For example, conditions of 5x SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) and 25-30 [deg.] C may be suitable for allele-specific probe hybridization.

Immobilization of a probe polynucleotide associated with a marker of the present invention on a substrate can also be easily prepared using this conventional technique. In addition, hybridization of nucleic acids on a microarray and detection of hybridization results are well known in the art. The detection can be accomplished, for example, by labeling the nucleic acid sample with a labeling substance capable of generating a detectable signal comprising a fluorescent material, such as Cy3 and Cy5, and then hybridizing on the microarray and generating The hybridization result can be detected.

For reference, the above-mentioned nucleotide and protein work can be referred to the following references (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982); Sambrook et al Inc., San Diego, Calif. (1990), < RTI ID = 0.0 > Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press )).

Thus, according to the present invention, it may be useful to determine an appropriate dosage for tacrolimus by predicting the drug concentration of the individual to tacrolimus according to the genetic difference of the individual. Therefore, it is very effective in securing the efficacy and safety of tacrolimus by reducing the incidence of early rejection reaction and side effects of drug because the drug blood concentration can be rapidly reached to the appropriate therapeutic region at an early stage.

Figure 1 is a haplotype of 4 SNPs related to tacrolimus pharmacokinetics invented through NGS.
Figure 2 is a haplotype of 7 SNPs related to tacrolimus pharmacokinetics.
Figure 3 shows changes in the C ₀ / D / kg index for one month according to the variation of the four SNPs of the present invention.
FIG. 4 shows changes in the C ₀ / D index for one year according to the variation of rs 15524 and rs 10211 of the present invention.
Figure 5 shows changes in the C ₀ / D index for one year according to the rs12360 and rs22574014 variations of the present invention.
FIG. 6 shows changes in C ₀ / D / kg for one month and C ₀ / D index for one year according to the degree of variation of the haplotype for the four SNPs of the present invention. (1: H1 / H1, TATG / TATG, 2: H1 / H2 or H1 / H3 or H1 / H4, TATG / CGCC or TATG / TATC or TATG / CATG,
FIG. 7 shows changes in C ₀ / D / kg for one month and C ₀ / D index for one year according to the degree of haplotypes variation for a total of seven SNPs including the four SNPs of the present invention. (1: Ht1 / Ht1, TGATGTC / TGATGTC; 2: Ht1 / Ht2 or Ht1 / Ht3 or Ht1 / Ht4, TGATGTC / CAGCCAT or TGATGTC / CAGCCAC or TGATGTC / TGATCTC; Ht2 / Ht2, CAGCCAT / CAGCCAT)

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Collection of experimental and experimental samples

The subjects of this study were recruited from Seoul National University Hospital, and were limited to patients with renal transplantation who received triple therapy with immunosuppressive agents including tacrolimus. All information provided was obtained from all patient groups. This study has been validated by the Institutional Ethics Committee of Seoul National University Hospital (IRB No. C-1504-009-662). Demographic characteristics of the recruited patients are shown in Table 3.

Characteristics of recruitment patients taking tacrolimus summary patient Number of patients (N) 60 Age (years) 44.9 ± 15.4 Male (N (%)) 40 (66.7%) Organ Transplant Type (N (%)) 20 (33.3%) Living related 20 (33.3%) Living unrelated 17 (28.3%) Deceased donor 23 (38.3%) Causes of chronic kidney disease Diabetes Mellitus 19 (31.7%) Hypertension) 3 (3.3%) Glomerulonephritis 15 (25%) Polycystis kidney disease 6 (11.7%) Obstruction 2 (3.3%) Unknown 9 (15%) Others 6 (10%) Age of donor (age, years) 44.2 ± 13.6 Donor sex, male (N (%)) 31 (51.7%)

Peripheral blood samples for gene analysis were obtained from 60 renal transplant recipients and genomic DNA was extracted using QuickGene DNA whole blood kit (KURABO, Japan). Absorbance was measured at 260 nm, 280 nm and 230 nm using Nanodrop ^™ (Thermo Fisher Scientific, US), and DNA concentration and purity were measured and stored at -80 ° C.

≪ Example 2 >

Selection of tacrolimus pharmacokinetic SNPs

<2-1> NGS analysis

DNA was isolated, purified, isolated and amplified using a SureSelect Human All Exon + UTR v5 kit (Agilent, USA) to identify the whole exome and untranslated region (UTR) The library was created. Sequence analysis was carried out by Ion Proton using the library and the position of the toxin site was also confirmed by annotation of the sequence analyzed using a torrent server analysis pipeline. Ion reporter software was used to verify that the mapping ratio of all samples as data QC was more than 70% and the coverage depth was more than 90% of 20x.

<2-2> Calculation of pharmacokinetic index

Tacrolimus blood levels were measured in blood samples collected from patients who had been taken prior to the next drug administration for determination of the lowest blood level by liquid chromatography mass spectrometry (LC-MS / MS). The lowest blood concentration ratio (C ₀ / D) and the lowest blood concentration ratio (C ₀ / D / kg) of the tacrolimus as a pharmacokinetic index were selected. Dose, and body weight were retrospectively collected using an electronic medical record (EMR).

The period was 3 days, 1 week, 2 weeks, 1 month (4 weeks), 3 months (12 weeks), 6 months (24 weeks), 12 months ). Table 4 shows the calculated values for the pharmacokinetic indexes for each period.

Pharmacokinetic index calculated by period Parameter Day 3 Week 1 Week 2 Month 1 Month 3 Month 6 Month 12 C _{0 / D 2.45 ± 2.44 2.03 ± 1.65 1.61 ± 1.09 1.66 ± 1.14 1.62 ± 1.26 1.93 ± 1.60 1.96 + 1.67 (ng / mL per mg / day) C0 / D / kg, 1.054 + - 12.794 0.037 + 0.034 0.031 0.030 0.030 0.022 - - - (ng / mL per mg / kg / day)}

<2-3> Statistical analysis of analyzed genes

(Age), hematocrit (Hct), cholesterol (Chol), albumin (Alb), and T (logarithmic) for the linearity of the pharmacokinetic indicators C ₀ / D and C ₀ / - The influence of clinical variables of bilirubin (T.bil) was evaluated by multiple regression analysis. Specifically, we used a regression model with log (PK) values as response variables and six important clinical variables (age, weight, HCT, CHOL, ALB, T.BILL) ) Were performed.

For the 0-3 days and 3-7days cases, the stepwise feature selection is performed for all 6 clinical variables in the case of the value of C ₀ / D except for the case where the log PK value has many missing values. . In the case of C ₀ / D / kg, stepwise feature selection was carried out with 5 clinical variables, excluding body weight, as independent variables, since the weight was already calibrated as a response variable. Finally, the remaining variables, ALB and T.BILL, Respectively.

In order to determine the difference of SNPs between two pharmacokinetic indicators, ANOVA test was performed using a linear model in which each phenotype was encoded according to hetero- mutant and homo-mutant, and the final clinical variables were corrected for each SNP.

In order to extract genetic variants with significant differences through ANOVA analysis, p-value according to multiple testing was verified and corrected using Benjamin-Hochberg method to yield a genotype smaller than 0.05.

Mutations were confirmed from a total of 2512 genes. A total of 264 genetic mutations were identified including SNP and InDel. Finally, four SNPs of the CYP family of exon and UTR regions were derived based on the genes that are both influential in both pharmacokinetic parameters and period.

The information of the derived pharmacokinetic-related SNPs is shown in Table 5, and the significance for each period of 1 month and 1 year is shown in Tables 6 and 7 below.

Pharmacokinetic association SNP information rs # Gene Location start num variant MAF HWE test rs15524 CYP3A5 UTR3 7: 99245914 A> G 0.292 0.113 rs10211 CYP3A7 UTR3 7: 99302994 T > C 0.267 0.094 rs12360 CYP3A7 UTR3 7: 99303094 A> G 0.267 0.094 rs2257401 CYP3A7,
CYP3A7-CYP3A51P exonic 7: 99306685 C> G 0.3 0.125

C ₀ / D of the CYP family SNP Gene Genotype status C _{0 / D (ng / mL per mg / day) day 1-3 day 4-7 day 8-14 day 15-28 month 1 month 3 month 6 month 12 CYP3A5, rs15524 A / A 3.35 ± 2.79 2.76 ± 1.39 2.10 ± 0.90 2.13 ± 1.17 2.44 ± 1.09 2.33 ± 1.57 2.37 ± 1.78 2.48 ± 1.61 A / G 1.96 + 1.24 1.44 ± 0.74 1.12 ± 0.73 1.23 + - 0.46 1.33 + - 0.45 1.26 + - 0.55 1.29 + - 0.53 1.56 ± 0.87 G / G 1.06 ± 0.50 *} ^{0.76 + 0.41 * 0.80 + - 0.25 * 0.91 + 0.39 * 0.87 + - 0.33 * 0.84 + - 0.23 * 0.81 0.26 * 0.88 0.27 * CYP3A7, rs10211 T / T 3.30 ± 2.72 2.72 + 1.36 2.06 ± 0.89 2.09 ± 1.14 2.40 ± 1.07 2.32 ± 1.52 2.36 ± 1.74 2.52 + 1.61 T / C 1.84 ± 1.27 1.33 + - 0.74 1.09 ± 0.37 1.18 ± 0.45 1.27 + - 0.45 1.14 + - 0.44 1.16 0.32 1.34 0.48 C / C 1.09 ± 0.53 * 0.76 + - 0.45 * 0.76 + - 0.25 * 0.91 + 0.42 * 0.86 + 0.35 * 0.83 + - 0.24 * 0.81 0.28 * 0.89 0.29 * CYP3A7, rs12360 A / A 3.30 ± 2.72 2.72 + 1.36 2.06 ± 0.89 2.09 ± 1.14 2.40 ± 1.07 2.32 ± 1.52 2.36 ± 1.74 2.52 + 1.61 A / G 1.84 ± 1.27 1.33 + - 0.74 1.09 ± 0.37 1.18 ± 0.45 1.27 + - 0.45 1.14 + - 0.44 1.16 0.32 1.34 0.48 G / G 1.09 ± 0.53 * 0.76 + - 0.45 * 0.76 + - 0.25 * 0.91 + 0.42 * 0.86 + 0.35 * 0.83 + - 0.24 * 0.81 0.28 * 0.89 0.29 * CYP3A7, C / C 3.51 + - 2.76 2.87 ± 1.32 2.15 + 0.88 2.18 ± 1.16 2.52 + 1.04 2.42 ± 1.55 2.41 ± 1.82 2.61 ± 1.64 CYP3A7-CYP3A51P, rs2257401 C / G 1.75 + - 1.23 1.31 + 0.71 1.08 0.37 1.19 0.43 1.25 0.43 1.17 + - 0.43 1.27 + - 0.42 1.39 + - 0.52 G / G 1.14 ± 0.51 * 0.79 + 0.42 * 0.82 + 0.28 * 0.92 + - 0.39 * 0.90 + 0.35 * 0.83 + - 0.22 * 0.85 + - 0.28 * 0.91 + - 0.27 *}

^* p-value < 0.01

^** month 1: Average for one month

C ₀ / D / kg of CYP family SNP Gene Genotype status log (C _{0 / D / kg) (ng / mL per mg / kg / day) day 1-3 day 4-7 day 8-14 day 15-28 month 1 CYP3A5, rs15524 A / A 1.693 ± 9.390 0.050 0.032 0.042 0.034 0.041 0.025 0.396 + 2.010 A / G 0.032 0.018 0.024 0.014 0.019 ± 0.009 0.022 0.008 0.023 ± 0.008 G / G 0.018 0.013 *} ^{0.014 0.011 * 0.014 ± 0.007 * 0.018 0.010 * 0.016 0.010 * CYP3A7, rs10211 T / T 1.598 ± 9.118 0.049 0.032 0.041 0.033 0.041 0.024 0.385 ± 1.952 T / C 0.030 0.019 0.022 0.014 0.019 ± 0.009 0.021 0.007 0.022 0.008 C / C 0.019 0.014 * 0.014 0.011 * 0.013 0.008 * 0.017 0.011 * 0.015 0.011 * CYP3A7, rs12360 A / A 1.598 ± 9.118 0.049 0.032 0.041 0.033 0.041 0.024 0.385 ± 1.952 A / G 0.030 0.019 0.022 0.014 0.019 ± 0.009 0.021 0.007 0.022 0.008 G / G 0.019 0.014 * 0.014 0.011 * 0.013 0.008 * 0.017 0.011 * 0.015 0.011 * CYP3A7, C / C 1.747 + 9.535 0.052 + 0.031 0.043 + 0.034 0.043 0.025 0.409 + 2.041 CYP3A7-CYP3A51P, rs2257401 C / G 0.028 0.018 0.022 0.013 * 0.018 ± 0.009 0.021 ± 0.008 0.021 ± 0.008 G / G 0.020 0.014 * 0.015 0.011 * 0.015 0.009 * 0.018 0.010 * 0.017 0.011 *}

^* p-value < 0.01

^** month 1: Average for one month

On the other hand, tacrolimus is metabolized by cytochrome p450 enzyme, and it has been reported that drug metabolism is influenced by genes related to CYP family in previous studies. Thus, we confirmed that the SNPs derived from this study had an effect on pharmacokinetics in linkage disequilibrium (LD) relationships with previously reported SNPs affecting tacrolimus pharmacokinetics. Through review of literature, we could identify three genes in total.

- CYP3A5 (chr 7: 99672916), rs776746, intron, A> G

- CYP3A4 (chr 7: 99756491), rs12333983, downstream variant 500B, A > T

- CYP3A4 (chr 7: 99763843), rs2242480, intron, C > T

The above three SNPs were genotyped by SNaPshat (Thermo Fisher Scientific, US), and the results are shown in Table 8 below.

Additional pharmacokinetic association SNP information rs # Gene Location start num variant MAF HWE test rs776746 CYP3A5 intron 3 7: 99672916 A> G 0.269 0.206 rs12333983 CYP3A4 downstream variant 500B 7: 99756491 A> T 0.25 0.233 rs2242480 CYP3A4 intron 10 7: 99763843 C> T 0.2 0.889

&Lt; Example 3 >

Association analysis and determination of haplotype

To evaluate the linkage disequilibrium (LD) relationship between the four SNPs derived from the NGS analysis, we used the Haploview (Broad Institute, USA) program to evaluate the formation of haplotypes by solid spine of LD analysis.

As a result, as shown in FIG. 1, it was confirmed that the four SNPs confirmed by the present invention had a haplotype block, and two haplotypes were found, and the frequencies are shown in Table 9 below.

Pill. rs15524 rs10211 rs12360 rs2257401 Freq. H1 T A T G 0.675 H2 C G C C 0.267 H3 T A T C 0.033 H4 C A T G 0.025

<Example 4>

Evaluation of the influence of pharmacokinetics on haplotype

The haplotype was divided into three groups according to the degree of variation based on frequency: H1 / H1 (TATG / TATG); H1 / H2 or H1 / H3 or H1 / H4 (TATG / CGCC or TATG / TATC or TATG / CATG); H2 / H2 (CGCC / CGCC).

Haplotypes were classified as 1, 2, 3 (1: H1 / H1; 2: H1 / H2 or H1 / H3 or H1 / H4; 3: H2 / H2) and ANOVA test using linear model was performed through SPSS Respectively.

The significance of each period according to the derived haplotype is shown in Tables 10 and 11 below. The pharmacokinetic index according to the daily haplotype is shown in Fig. Finally, although the four inventive SNPs alone exert an influence on the pharmacokinetics of tacrolimus, the haplotype variability, that is, all four SNP variants, exerts a greater influence on the pharmacokinetics of tacrolimus.

C ₀ / D by period according to haplotype H Haplotype C _{0 / D (ng / mL per mg / day) day 1-3 day 4-7 day 8-14 day 15-28 month 1 month 3 month 6 month 12 One 3.57 ± 2.83 2.92 ± 1.35 2.20 ± 0.88 2.22 ± 1.18 2.58 ± 1.05 2.44 ± 1.60 2.43 ± 1.86 2.58 ± 1.65 2 1.80 ± 1.18 1.36 ± 0.70 1.12 0.35 1.21 0.43 1.29 + - 0.42 1.24 ± 0.52 1.34 0.55 1.53 + - 0.84 3 1.09 ± 0.53 *} ^{0.76 + - 0.45 * 0.76 + - 0.25 * 0.91 + 0.42 * 0.86 + 0.35 * 0.83 + - 0.24 * 0.81 0.28 * 0.89 0.29 *}

^* p-value < 0.01

^** month 1: Average for one month

C ₀ / D / kg by period according to haplotype H Haplotype log (C _{0 / D / kg) (ng / mL per mg / kg / day) day 1-3 day 4-7 day 8-14 day 15-28 month 1 One 1.861 ± 9.847 0.053 0.032 0.044 0.034 0.043 0.025 0.434 ± 2.107 2 0.029 0.017 0.023 0.013 0.019 ± 0.009 0.021 ± 0.008 0.022 0.008 3 0.019 0.014 *} ^{0.014 0.011 * 0.013 0.008 * 0.017 0.011 * 0.015 0.011 *}

^* p-value < 0.01

^** month 1: Average for one month

&Lt; Example 5 >

Association analysis and determination of haplotype

To assess the LD relationship for the four haploid and four SNPs reported previously associated with tacrolimus pharmacokinetics, use the Haploview (Broad Institute, USA) program to assess whether a haplotype is formed through a solid spine of LD assay Respectively.

As a result, as shown in FIG. 2, it was confirmed that a haplotype block was formed for 7 SNPs, and two haplotypes were found, and the frequencies are shown in Table 12 below.

Haplotype rs15524 rs776746 rs10211 rs12360 rs2257401 rs12333983 rs2242480 frequency Ht1 T G A T G T C 0.649 Ht2 C A G C C A T 0.183 Ht3 C A G C C A C 0.059 Ht4 T G A T C T C 0.033 Ht5 C A G C C T C 0.025 Ht6 C G A T G T C 0.025

&Lt; Example 7 >

Evaluation of relevance to pharmacokinetics by haplotype

To confirm the correlation between haplotype and acute rejection, haplotype was identified for each patient and all haplotypes expressed were divided into three groups according to the degree of mutation. Ht1 / Ht1 (TGATGTC / CAGCCAT or TGATGTC / CAGCCAC or TGATGTC / TGATCTC, etc.) or other mutations of Ht1 / Ht2 or Ht1 / Ht3 or Ht1 / Ht4.

Haplotypes were classified as 1, 2, and 3 (1: Ht1 / Ht1; 2: Ht1 / Ht2 or Ht1 / Ht3 or Ht1 / Ht4; Ht2 / Ht2) and ANOVA test using a linear model was performed using SPSS .

The significance of each period according to the derived haplotype is shown in Tables 13 and 14 below. The pharmacokinetic index according to the haplotype is shown in Fig. Finally, although the four invented SNPs alone exert an influence on the pharmacokinetics of tacrolimus, the haplotype variability, that is, the variability of all seven SNPs, exerts a greater influence on the pharmacokinetics of tacrolimus.

C ₀ / D by period according to haplotype Ht Haplotype C _{0 / D (ng / mL per mg / day) day 1-3 day 4-7 day 8-14 day 15-28 month 1 month 3 month 6 month 12 One 3.60 ± 2.98 2.81 ± 1.36 2.22 ± 0.91 2.08 ± 1.04 2.54 + 1.07 2.35 ± 1.53 2.38 ± 1.89 2.58 ± 1.67 2 1.89 ± 1.20 1.56 ± 1.06 1.17 + 0.49 1.42 0.94 1.41 + - 0.68 1.39 + 1.01 1.41 ± 0.87 1.54 ± 0.96 3 0.78 + - 0.20 *} ^{0.58 ± 0.18 * 0.69 0.20 * 0.69 + - 0.27 * 0.67 ± 0.19 * 0.79 + 0.38 * 0.89 + 0.42 * 0.89 0.41 *}

^* p-value < 0.01

^** month 1: Average for one month

C ₀ / D / kg by period according to haplotype Ht Haplotype log (C _{0 / D / kg) (ng / mL per mg / kg / day) day 1-3 day 4-7 day 8-14 day 15-28 month 1 One 2.062 ± 10.380 0.053 + 0.033 0.046 0.036 0.042 0.024 0.477 ± 2.221 2 0.030 0.019 0.025 0.016 0.020 0.010 0.025 0.016 0.024 0.011 3 0.011 ± 0.004 *} ^{0.010 0.000 * 0.010 0.000 * 0.013 0.006 * 0.010 0.003 *}

^* p-value < 0.01

^** month 1: Average for one month

As described above, according to the present invention, it is useful to predict the drug concentration of an individual against tacrolimus according to the genetic difference of an individual, and to determine an appropriate dosage for tacrolimus. Therefore, it is very effective in securing the efficacy and safety of tacrolimus by reducing the incidence of early rejection reaction and side effects of drug because the drug blood concentration can be rapidly reached to the appropriate therapeutic region at an early stage.

<110> Seoul National University <120> Method of prediction of blood concentration of tacrolimus using          SNP in CYP3A5 and CYP3A7 genes <130> NP16-0024 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 1001 <212> DNA <213> Homo sapiens <400> 1 agaacacggg ttcccatgaa tttgtctcag gtcaaacctc acacagaata ggtgctccat 60 gaatattttg ttaaatgata gatgatgaat gttctcacta tccaatcttc acacatctta 120 tagagtaagt ataacgaatc caagatttat agtgctgaaa gtagttttta tatgtttaca 180 aagcattatt gtcagtaatt tttttttact ttgatgctat actttctact tttgctttat 240 ttaatgcttc tcaatatgct cgtttaactg ttgcagatcc ccctgaaatt acgctttgga 300 ggacttcttc taacagaaaa acccattgtt ctaaaggctg agtcaaggga tgagaccgta 360 agtggagcct gatttcccta aggacttctg gtttgctctt caagaaagct gtgccccaga 420 acaccagaga cctcaaatta ctttacaaat agaaccctga aatgaagacg ggcttcatcc 480 aatgtgctgc ataaatatc agggattctg tacgtgcatt gtgctctctc atggtctgta 540 tagagtgtta tacttggtaa tatagaggag atgaccaaat cagtgctggg gaagtagatt 600 tggcttctct gcttctcata ggactatctc caccaccccc agttagcacc attaactcct 660 cctgagctct gataacataa ttaacatttc tcaataattt caaccacaat cattaataaa 720 aataggaatt attttgatgg ctctaacagt gacatttata tcatgtgtta tatctgtagt 780 attctatagt aagctttata ttaagcaaat caataaaaac ctctttacaa aagtattatt 840 ggatgtttcc tgcacattaa ggagaaatct atagaactga atgactgaga accaacaact 900 aaatattttg atcattgtaa tcactgttgg tgtgggaact ggagtgcagt ggtgcaatct 960 tggctcactg cgagctctgc ctcccaggtt cacgccattc t 1001 <210> 2 <211> 1001 <212> DNA <213> Homo sapiens <400> 2 attaacttca taagatggtt taaaaagttt accgttatac ctaataatct ggtttaaatt 60 tttaaaactc atccattttc gttaaaattt aaatcaaaaa agaacacggg ttcccatgaa 120 tttgtctcag gtcaaacctc acacagaata ggtgctccat gaatattttg ttaaatgata 180 gatgatgaat gttctcacta tccaatcttc acacatctta tagagtaagt ataacgaatc 240 caagatttat agtgctgaaa gtagttttta tatgtttaca aagcattatt gtcagtaatt 300 tttttttact ttgatgctat actttctact tttgctttat ttaatgcttc tcaatatgct 360 cgtttaactg ttgcagatcc ccctgaaatt acgctttgga ggacttcttc taacagaaaa 420 acccattgtt ctaaaggctg agtcaaggga tgagaccgta agtggagcct gatttcccta 480 aggacttctg gtttgctctt caagaaagct gtgccccaga acaccagaga cctcaaatta 540 ctttacaaat agaaccctga aatgaagacg ggcttcatcc aatgtgctgc ataaataatc 600 ggggattctg tacgtgcatt gtgctctctc atggtctgta tagagtgtta tacttggtaa 660 tatagaggag atgaccaaat cagtgctggg gaagtagatt tggcttctct gcttctcata 720 ggactatctc caccaccccc agttagcacc attaactcct cctgagctct gataacataa 780 ttaacatttc tcaataattt caaccacaat cattaataaa aataggaatt attttgatgg 840 ctctaacagt gacatttata tcatgtgtta tatctgtagt attctatagt aagctttata 900 ttaagcaaat caataaaaac ctctttacaa aagtattatt ggatgtttcc tgcacattaa 960 ggagaaatct atagaactga atgactgaga accaacaact a 1001 <210> 3 <211> 1001 <212> DNA <213> Homo sapiens <400> 3 acaggcatat gatgctactg tattgatgga aagcagtgtc atcactgctt tctttgtcta 60 tctgttgcac taacctataa attctttgaa gattagacgc atgtttttta ttcaaatttg 120 attcctgaag atttgagtat attcaggaaa aaatattcat ttgtgggaca taataatagc 180 attgtgatca tgtgttttta aaagagtcct tacattttgg acacttatga cccataagga 240 cataattgag gaccttcatc ttaagttgct gggactgtga ctggctatag ttttctttta 300 tctagtctgt ggtttgtaaa gtgtgacaat gatcaatttc atgatttgaa aaaacctttt 360 aaacataaaa agacaagcaa acgattgtac aagcccagac tgtcctgtag agaggcagaa 420 tatgcttgaa ccaggctggt tcagggaggg ctcccttccc aggggcctcc tacctttcag 480 ggaggaactt ctcaggctct ctccagtact ttgggtcatg atgaagaaca tagcttggaa 540 tcatcaccac cacccctttg ggaataaaca tcccattgat ttcaacatct tttttgcaga 600 ccctctcaag tctcatagca actgggaata atctgagtgt ttcattcacc accatgtcaa 660 gatactccaa ctgtagcaca gtatcatagg tgggtggtgc ctgaaaagaa agaaacagat 720 ttggataaat tgagattttt gaattaactt ttaactcagt ccatgtagta ctgttgaact 780 gttagaagtt ccagagaaca actcatactg gcaaaggatt gtagcattca taaagtattt 840 taataactgt catgcccttt gatgtgctca atggcccact aagatgtgtg gaggagttat 900 gaaagcagga gacattcggg aaggctctaa ccccaatgac tgaaatcctt agcataacca 960 tgtagtggtt tttctttctc atggaagcaa acatgactaa t 1001 <210> 4 <211> 1001 <212> DNA <213> Homo sapiens <400> 4 agttttaacc tataaaaatt taactttata tagcacttcc aaaatagttt gccataatac 60 ctactaatct ggatttaatt tttaaaactc atccttttaa cttaagattt aaataaaaaa 120 aaaaaaacac gagtccacaa gaatttgtct caggcctggc acagagtcag tgctccataa 180 atattttgtt aaacgatgga tggtgagtgc ttttactatc cagtatttac ccagcttata 240 gattaagtat gaagagttca agatacatgg tgttaagagt cgtttttata tgcttgcaaa 300 gcatttttgt catatttttt ctactttgct tccatctttt cttctttcac ttcatttatt 360 aattattccat atgcttgttt aactattgta gatccccttg aaattagaca cgcaaggact 420 tcttcaacca gaaaaaccca ttgttctaaa ggtggattca agagatggaa ccctaagtgg 480 agaatgagtt attctaagga cttctacttt ggtcttcaag aaagctgtgc cccagaacac 540 cagagatttc aacttagtca ataaaacctt gaaataaaga tgggcttaat ctaatgtact 600 gcatgagtag ttggtgattt tgtacattca ttgagctctc ccagagtctg tgtagagtgt 660 tgtgcattat gtagtataaa ggaggtgacc aggtaagtga cagataggta gactcagctt 720 ctctgcttct cataggacta cctctaccca cctctagtta gcattatcaa ctcctcctga 780 gctctcatca gagaataaat atttctcaac aatttgatcc ataactttta agaaaaataa 840 gaattatcat gatgactcta atagtgacat ttatatcacg ttttatttgt aatattctat 900 aagttttata ttaagcgaag tgataaaatc ccctttacaa aaatattatc tgatgccatc 960 ctgcacacta aagagaaatc tatagaactg aatgactgaa a 1001 <210> 5 <211> 1001 <212> DNA <213> Homo sapiens <400> 5 actgtgattt tataaggtgg tctcagccaa ttgcagcagc tgttccctgt cagaggggct 60 agaggtttgg tgagagcagt ggatgaggtg cagtggtgtg tttgttcact agaagcaagt 120 gggagaaagc tttgcctctt tgtacttctt catcttctcc cctcaagtcc tcagaatcca 180 cagcgctgac tgtggagtgc tgtggagctg gcatggccca tacaggcaac atgacttagt 240 agacagatga cacagctcta gatgtccatg ggccccacac caactgccct tgcagcattt 300 agtccttgtg agcacttgat gatttacctg ccttcaattt ttcactgacc taatattctt 360 tttgataatg aagtatttta aacatataaa acattatgga gagtggcata ggagataccc 420 acgtatgtac cacccagctt aacgaatgct ctactgtcat ttctaaccat aatctcttta 480 aagagctctt ttgtctttca atatctcttc cctgtttgga ccacattacc cttcatcata 540 tgaagccttg ggtggctcct gtgtgagact cttgctgtgt gtcacaccct aatgaactag 600 aacctaaggt tgctgtgtgt cgtacaacta ggggtatgga ttacataaca taatgatcaa 660 agtctggctt cctgggtgtg gctccagctg cagaatcggg ctagtgaagt ttaatcagct 720 ccgttgtccc cacacagaac gtatgaaggt caactccctg tgctggccat cacagatccc 780 gacgtgatca gaacagtgct agtgaaagaa tgttattctg tcttcacaaa tcgaagggta 840 agcatccatt ttttgaaatt taaataatga ttgatccact gattaaattt ttattttgaa 900 aaaaacatat attcacagaa ggttacctaa aaaatgtaca ggaaggttcc atgtactctt 960 catcctgtcc cgcccagtgg taacatcttg caatcttgta t 1001 <210> 6 <211> 1001 <212> DNA <213> Homo sapiens <400> 6 aactttactg aatgtgttta tcagttccaa tagttctttg atggagtctt taggtttttc 60 caaatatcat ttggaaatcg tttgcaagtc atttggaaat gtcatttgga agtcattttc 120 aagtatcatt tccaaagatt acttggaaat tatttgaaaa ttttcttgct tcctggcaca 180 aaatgtgtcc taaacttatc ttgaacattc ttaattcttg ccctggcatc agccatttct 240 ctaaagagct ctggttccct tcagtggaaa atggtattca taagccaaga tcggggcact 300 aagtgtgttc attgctattg aaatgtcatt gttacctagc cctatcagtg gagagaagta 360 gggaatatat gttttttgta cacacacaca aatacagaca tacatttaca tctatctatc 420 tcattatctg tcatctatct gttacctatc tatcatctat ctacccatct atctatctac 480 aagggtatga atatacacgg atacatccat tccaatctaa tttcatagga ttcatgctag 540 ttttctccct ttctgaatcc atgactccct tctgcaaagg tgagaagcct ggcttctatc 600 atccttaata catttactta tttgatcaat cccctttatg tagccaaacc tcacccaacc 660 cctcccactc atggatgccc tcctcgcctc atgtgggttc tccataccct atttaggctc 720 tggctgctct tgcaagccac ccctctgcgt cgaccccttc tctgcttgct tggactccca 780 cacccaggcc aggatgtctc tccacatgaa cctccttggt ctctgacttg gagccaccat 840 gactctctct tctctacctt aatgtgtggc caccatctgg ctgccaccca cctaatggct 900 ttaggactaa attattcagg aagggaaaat aaaggaagaa tttggaaaag aaaaggaatg 960 agtgctttta ggcttattgc tcaatcaatt gaccaatcga c 1001 <210> 7 <211> 1001 <212> DNA <213> Homo sapiens <400> 7 aggctatgac cactagcatc aaataacata tgagaaagtg attgttacct ttcaaaaaaa 60 aaagtcacat gtctgtaacc aagaaaataa aaagagataa aaagagcaaa ttcctgtgtc 120 catatgtgga tacagctaag gggacatcac acaccactat ggtgcctgct gcaaacatgt 180 aattcacaac caaaaccaat gtgaaatgag tgtgagcaac aaatgcttat tgttagatgc 240 cactgaggtt tttggaggtt ttttacttag cattattgta gtaatagaaa gcagatgaac 300 cagagccagc acgttttaca aagatcttac gcttctgcca gtagcaacca tttgctatgt 360 ttctttcttt ttcttttcag agccttccta catagagtca gtgaaagaat cagtgattat 420 gctttttata aaaattctcc tgggaagtgg tgaggaggca tttttgctaa ggtttcacct 480 cctccctcct tctccatgta ccatccactc accttattgg gtaaaactgc atcaatttcc 540 tcctgcagtt tctgctggac atcagggtga gtggccagtt catacataat gaaggagaga 600 acactgctcg tggtttcata gccagcaaaa ataaagataa ttgattgggc cacgagctcc 660 agatcggaca gagctgaaag gagaggaaag acattttagg taaatcagat caatgtaggg 720 catcacagtt tagatgaaga gaaatctaag tgaagccctc aaatccctaa gggaaaagaa 780 tagaaaagca attcagaggt acactggggg tggtttcatt ctgatgtgta tcttacagaa 840 ccagaataag gcaaatcatt ttaatccagt tttccccaag gtcttcgaga atgtgggcca 900 tccactcctc ctcatgccac ccccgccacc tgcacagctg tgtaatttcc agagagagca 960 tttccgcata acatactcag tgttatgggt gtgttccttg a 1001

Claims (15)

A composition for predicting the drug concentration of an individual against tacrolimus comprising, as an active ingredient, a probe or a primer capable of detecting single nucleotide polymorphism of a CYP3A gene family consisting of NCBI refSNP ID: rs12360.
2. The composition of claim 1, wherein said single nucleotide polymorphism further comprises selected from the group consisting of NCBI refSNP IDs: rs10211, rs2257401, rs15524, rs776746, rs12333983 and rs2242480.
2. The composition of claim 1, wherein the tacrolimus is administered after organ transplantation.
2. The composition of claim 1, wherein the subject is a human or organ transplant recipient requiring tacrolimus administration.
[4] The composition according to claim 3, wherein the organ transplantation is selected from the group consisting of renal transplantation, liver transplantation, heart transplantation, lung transplantation, and hematopoietic stem cell transplantation.
2. The composition of claim 1, wherein the drug concentration is a pharmacokinetics response to tacrolimus.
The composition of claim 1, wherein the drug concentration of the individual is the drug concentration in the blood of the subject.
A method for detecting a single nucleotide polymorphism of the corticochrome 3A gene family comprising NCBI refSNP ID: rs12360 by sequencing from a patient sample, in order to provide information necessary for predicting drug concentration of an individual to tacrolimus (tacrolimus).
9. The method of claim 8, wherein the single nucleotide polymorphism further comprises selected from the group consisting of NCBI refSNP IDs: rs10211, rs2257401, rs15524, rs776746, rs12333983, and rs2242480.
A method for analyzing single nucleotide polymorphisms of the Cytochrome 3A gene family for predicting individual drug concentrations for tacholimus comprising the steps of:
(a) isolating genomic DNA from an individual;
(b) determining the nucleotide sequence of the NCBI refSNP ID: rs10211, rs12360, rs2257401 and rs15524 regions;
(c) In the determined nucleotide sequence, T is replaced with C, rs12360 is substituted with G, rs2257401 is replaced with C, and rs15524 is replaced with G to detect a nucleotide sequence polymorphism step;
(d) determining a haplotype with the detected base sequence polymorphism; And

(e) Determining that the individual drug concentration for tacrolimus is high in case of haplotype H1, and that the drug concentration of individual for tacrolimus is low in case of H2, H3 and H4.
11. The method of claim 10, wherein the method further comprises determining the nucleotide sequence to include NCBI refSNP IDs: rs776746, rs12333983, and rs2242480, wherein rs776746 is substituted by G, rs12333983 is substituted by T, rs2242480 Ht2, Ht3, Ht4, Ht5, and Ht6 were detected in haplotypes by detecting the nucleotide sequence polymorphism in which T was replaced by T, and when haplotype was Ht1, the drug concentration was higher for tacrolimus. Characterized in that the drug concentration of the individual is determined to be low.

.
Kit for predicting individual drug concentrations for tacrolimus comprising primers for screening polynucleotide polymorphism consisting of NCBI refSNP ID: rs12360 of the Cytochrome 3A gene family or genotyping primers.
13. The kit according to claim 12, wherein the kit further comprises a screening primer or a genotyping primer selected from the group consisting of NCBI refSNP IDs rs10211, rs2257401, rs15524, rs77646, rs12333983 and rs2242480. .
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