CN111662975A - Application of product for detecting gene locus mutation in preparation of product for predicting or evaluating metabolic condition of patient taking tacrolimus - Google Patents
Application of product for detecting gene locus mutation in preparation of product for predicting or evaluating metabolic condition of patient taking tacrolimus Download PDFInfo
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Abstract
The invention discloses application of a product for detecting CYP3A4rs2242480 and CYP3A4rs4646437 gene locus mutation in preparation of a product for predicting or evaluating metabolism of patients after taking tacrolimus. According to the invention, single nucleic acid polymorphism (SNP) at CYP3A4rs2242480 and rs 46437 sites of 221 cases of kidney transplant patients is determined, clinical combined medication conditions are discussed, genomics and statistical analysis discover that the combined use of the pentaester capsule (WZC) and CYP3A4rs 2242480-rs 46437 multiple forms are main factors influencing in-vivo metabolism of tacrolimus, and the individual administration of TAC is considered in the aspect of pharmacogenomics and a dose prediction scheme of the TAC is made, so that safe, effective, economical and appropriate individual administration of the TAC is realized, and a theoretical basis is provided for clinical individual administration and adjustment of the administration scheme.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to application of a product for detecting gene locus mutation in preparation of a product for predicting or evaluating metabolic condition of a patient taking tacrolimus.
Background
Organ transplantation is the final means of treatment of end-stage renal disease, and has rapidly progressed at home and abroad in recent years, and with the release of the "chinese guideline for organ donation" in 2015, the kidney transplantation operation performed in china by 2017 has been jumping to the second place in the world. However, immune rejection after organ transplantation is the leading cause of failure of organ transplantation. Therefore, the rational selection of immunosuppressive agents is critical to prevent the occurrence of postoperative organ transplant rejection.
Tacrolimus (TAC) is a family of calcineurin inhibitors and has become a first-line therapeutic drug for preventing immunosuppression after organ transplantation, but the drug has a narrow therapeutic window and obvious inter-and intra-individual differences of Pharmacokinetics (PK) while exerting curative effects. Therefore, clinical use must carefully monitor the safety margin of TAC through Therapeutic Drug Monitoring (TDM) to ensure that graft rejection can be reduced while exerting the best therapeutic effect, avoiding side effects of TAC such as infection, PTDM (post-transplant diabetes). However, due to the hysteresis of TDM, patients are at particular risk for patients in the early post-transplant period because the blood concentration is too low or too high before detection. Meanwhile, tacrolimus is expensive, and transplant patients should take immunosuppressants for a lifetime to avoid transplant failure, which causes great economic burden to the patients. Therefore, if the individualized administration of TAC can be considered from the pharmacogenomics aspect and a dose prediction scheme of TAC can be made, it will be possible to realize safe, effective, economical and appropriate individualized administration of TAC.
Pharmacological and pharmacogenomic studies have shown that statistically, 20-95% of drug disposition and effect differences are related to genetic factors. CYP3A5 and CYP3A4 are enzymes playing an important role in TAC metabolism in vivo, and researches show that the gene mutation of CYP3A4/5 is one of the reasons for obvious individual difference of clinical curative effect of TAC. Among them, the significant effect of the CYP3A5 gene polymorphism on tacrolimus pharmacokinetics is well-defined. Studies have shown that patients with CYP3a5 x 3/' 3 type (CYP3a5 non-expressors) possess higher plasma concentrations than patients with CYP3a5 x 1/' 3 and CYP3a5 x 1/' 1(CYP3a5 expressors). CYP3A4 gene polymorphism also affects metabolism of TAC, CYP3A4 x 1G (rs2242480) is a site with the highest mutation frequency in all CYP3A4 single nucleic acid polymorphisms at present, the mutation frequency in Chinese people is about 30.8%, and the influence of 8.1-17.6% of gene polymorphism on TAC is explained. CYP3A4rs2242480 has influence on TAC pharmacokinetics, and the current research is diverged. CYP3A4rs4646437 is another SNP site which is discovered recently and has a high mutation rate in east Asian population, and rs 46437 is found to be related to metabolism of some drugs, but has little influence on TAC pharmacokinetics. Due to the influence of factors such as heredity and the like, the pharmacokinetic characteristics of the TAC have larger individual difference, so that the clinical significance of finding safe and effective auxiliary medicines for stably improving the blood concentration of the TAC is larger for ensuring that the blood concentration is stably in a treatment window in the clinical use process aiming at various factors influencing the blood concentration. Pentaester capsules, the earliest approved Traditional Chinese Medicine (TCM) for use as a hepatoprotectant, were found to affect the activity of enzymes involved in TAC metabolism, further affecting TAC metabolism. At present, the penta-ester capsule is widely used as a synergist of TAC of a transplanted patient, so that the dosage of the TAC of the transplanted patient can be greatly reduced, and the effect of reducing the medical cost of the transplanted patient is achieved.
In the prior art, the target point for monitoring and evaluating the tacrolimus metabolism condition and the accuracy of the product are low, and the condition that a combined tacrolimus synergist such as a penta-ester capsule cannot be evaluated exists. Therefore, there is a need to find a new target and product for predicting or evaluating tacrolimus metabolism to solve the above problems.
Disclosure of Invention
The invention aims to overcome the defects that the target point for monitoring and evaluating the tacrolimus metabolism condition and the product have low accuracy and cannot evaluate combined tacrolimus synergists in the prior art, and provides the application of the product for detecting the site mutation of CYP3A4rs2242480 and rs4646437 genes in preparing the product for predicting or evaluating the metabolism condition of patients taking tacrolimus.
The technical scheme provided by the invention is as follows:
the application of the product for detecting the site mutation of CYP3A4rs2242480 and rs4646437 gene in preparing the product for predicting or evaluating the metabolism of tacrolimus after a patient takes the tacrolimus.
The PCR primer and the probe are designed for two gene polymorphisms, namely CYP3A4rs2242480 and CYP3A4rs4646437 which significantly influence tacrolimus metabolism through genomics and statistical analysis, and the gene polymorphisms are analyzed in a multi-fold manner; meanwhile, the influence of the patient combined medicine penta-ester capsule on tacrolimus metabolism is comprehensively considered, and the accuracy of the tacrolimus metabolic prediction of the patient is remarkably improved by comprehensive polymorphism analysis results.
The invention subdivides the metabolic types of the tacrolimus of the testee into 2 types, namely a tacrolimus slow metabolic type and a standard metabolic type. The accuracy of the traditional method for detecting single genotyping only on the drug metabolism prediction of a patient is limited, the accuracy can be improved by detecting multiple types of CYP3A4rs2242480 and CYP3A4rs4646437, the metabolic type of the patient can be comprehensively judged by combining the hematocrit, the time after transplantation and the drug administration condition of the patient through an algorithm, the detection result is more accurate, the specificity is better, and more reliable reference data can be provided for safe and effective clinical medication.
The product for detecting the site mutation of the CYP3A4rs2242480 and CYP3A4rs4646437 genes can be any suitable product as long as the product can detect the SNP in the genes. Preferably, in one embodiment of the present invention, the product for detecting mutations at the CYP3a4rs2242480 and CYP3a4rs4646437 gene loci is a primer pair and/or a probe pair for detecting mutations at the gene loci.
Preferably, in the embodiment of the invention, the primer pair for detecting the mutation of the CYP3A4rs2242480 gene locus is shown as SEQ ID No.1-2, and the probe pair for detecting the mutation of the CYP3A4rs2242480 gene locus is shown as SEQ ID No. 3-4; the primer pair for detecting the site mutation of the CYP3A4rs 464646437 gene is shown as SEQ ID No.5-6, and the probe pair for detecting the site mutation of the CYP3A4rs4646437 gene is shown as SEQ ID No. 7-8.
Preferably, in one embodiment of the present invention, the patient is administered tacrolimus simultaneously with a tacrolimus potentiator.
The synergist can be any suitable synergist, such as pentaester capsules, indirubin derivative II7, and the like. Preferably, in one embodiment of the present invention, the tacrolimus synergist is a penta-ester capsule.
Preferably, in one embodiment of the invention, the evaluation is calculated according to the following formula:
P＝M1×M2
m1 ═ combined medication penta-ester capsules, M1 ═ 0.854; a Wuzhi capsule for non-combined medicines, M1 ═ 1
M2-CYP 3A4rs2242480-CYP3A4rs 46437 multifold genotype, CYP3A4rs2242480-CYP3A4rs 46437 slow metabolism (CG/CG) M2-1, and the rest standard metabolism M2-1.24.
Before the evaluation by using the above formula, the site mutation typing of CYP3A4rs2242480 and CYP3A4rs4646437 gene of the sample to be tested is determined.
The primer pair and the probe pair provided by the invention comprise primers and probe pairs for detecting CYP3A4rs2242480 and CYP3A4rs 4646437. The 5 'end of each group of probe pairs for detecting gene mutation sites is respectively marked with different fluorescent groups, and the 3' end is marked with MGB. The invention has no special limitation on the type of the fluorescent group, and the fluorescent group is commonly used in the field.
Preferably, in one embodiment of the present invention, the 5' ends of the probe pairs are labeled with FAM fluorophore and VIC fluorophore, respectively.
In another aspect of the invention, a tacrolimus metabolism gene detection kit is provided, which comprises a product for detecting the site mutation of CYP3A4rs2242480 and CYP3A4rs4646437 genes.
The product for detecting the site mutation of the CYP3A4rs2242480 and CYP3A4rs4646437 genes in the kit can be any suitable product as long as the product can detect the SNP in the genes. Preferably, in one embodiment of the present invention, the product for detecting mutations at the CYP3a4rs2242480 and CYP3a4rs4646437 gene loci is a primer pair and/or a probe pair for detecting mutations at the gene loci.
Preferably, in the embodiment of the invention, the primer pair for detecting the site mutation of the CYP3A4rs2242480 gene in the kit of the invention is shown as SEQ ID No.1-2, and the probe pair for detecting the site mutation of the CYP3A4rs2242480 gene is shown as SEQ ID No. 3-4; the primer pair for detecting the site mutation of the CYP3A4rs 464646437 gene is shown as SEQ ID No.5-6, and the probe pair for detecting the site mutation of the CYP3A4rs4646437 gene is shown as SEQ ID No. 7-8.
In a third aspect, the invention provides the use of the kit as described above in the manufacture of a product for assessing the metabolic profile of a patient after administration of tacrolimus. Preferably, the patient is a patient after organ transplantation.
The kit of the invention also comprises necessary reagents such as a buffer system, enzymes and the like and a kit instruction besides the product for detecting the site mutation of the CYP3A4rs2242480 and CYP3A4rs4646437 genes. The formula required for the evaluation is contained in the kit instructions:
P＝M1×M2
m1 ═ combined medication penta-ester capsules, M1 ═ 0.854; a Wuzhi capsule for non-combined medicines, M1 ═ 1
M2-CYP 3A4rs2242480-CYP3A4rs 46437 multifold genotype, CYP3A4rs2242480-CYP3A4rs 46437 slow metabolism (CG/CG) M2-1, and the rest standard metabolism M2-1.24.
The invention has the beneficial effects that:
the invention subdivides the metabolic types of the tacrolimus of the testee into 2 types, namely a tacrolimus slow metabolic type and a standard metabolic type. The invention discovers that the pentaester capsule and CYP3A4rs2242480-CYP3A4rs4646437 multiple form combined by genomics and statistical analysis are main factors influencing tacrolimus in vivo metabolism, the accuracy of the traditional method for detecting single genotyping only has limit on the drug metabolism prediction of a patient, the accuracy can be improved by detecting the CYP3A4rs2242480-CYP3A4rs4646437 multiple form, the metabolic type of the patient can be comprehensively judged by combining erythrocyte volume, the time after transplantation and the drug combination condition of the patient through an algorithm, the detection result is more accurate, the specificity is better, and more reliable reference data can be provided for safe and effective clinical medication.
Drawings
FIG. 1 is a graph showing the effect of different genotypes of CYP3A4 multiple rs2242480-rs 46437 on the Tacrolimus Clearance (CL), wherein the relationship between CL and different genotypes of CYP3A4 multiple rs2242480-rs 46437 is shown; the upper edge of the box body represents 75% quantiles of data, the lower edge represents 25% quantiles of data, a solid line in the middle of the box body is a median, and the tail end of the shaft whisker represents 1.5 times of a quarter-bit interval;
FIG. 2 is the concentration dose ratio of CYP3A4 multiple rs2242480-rs4646437 genotype tacrolimus (C)0/D) result graph.
DESCRIPTION OF THE SEQUENCES
SEQ ID No.1 is a forward primer for detecting CYP3A4rs2242480 gene site mutation;
SEQ ID No.2 is a reverse primer for detecting the site mutation of the CYP3A4 rsrs224248 gene;
SEQ ID No.3 is a probe 1 for detecting CYP3A4 rsrs224248 gene site mutation;
SEQ ID No.4 is a probe 2 for detecting the site mutation of the CYP3A4 rsrs224248 gene;
SEQ ID No.5 is a forward primer for detecting site mutation of CYP3A4rs4646437 gene;
SEQ ID No.6 is a reverse primer for detecting the site mutation of the CYP3A4rs4646437 gene;
SEQ ID No.7 is a probe 1 for detecting site mutation of CYP3A4rs4646437 gene;
SEQ ID No.8 is a probe 2 for detecting the site mutation of the CYP3A4rs4646437 gene.
Detailed Description
The invention discloses application of a product for detecting gene locus mutation in preparation of a product for predicting or evaluating the metabolic condition of a patient taking tacrolimus. It is expressly intended that all such alterations and modifications which are obvious to those skilled in the art are deemed to be incorporated herein by reference, and that the techniques of the invention may be practiced and applied by those skilled in the art without departing from the spirit, scope and range of equivalents of the invention.
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.
In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.
Example 1:
1. recipient information:
this is a retrospective multicenter study, and 211 cases of patients who underwent kidney transplantation operations in the first subsidiary hospital of Nanchang university, people hospital of Jiangxi province from 11 months to 2019 months in 2005 were collected as study subjects. Inclusion criteria were as follows: (1) patients who first undergo renal transplantation; (2) the age is 18-70 years old; exclusion criteria were as follows: (1) patients who develop graft failure or death after surgery; (2) patients who replace TAC with cyclosporine or other immunosuppressive agents within 1 month post-surgery. All enrolled patients were clear of the study content and signed an informed consent. The time of admission and relevant basic information (demographic and clinical data) of the patient are tracked using a hospital electronic medical record system. The method is divided into a TAC + penta-ester capsule (WZC) group and a single TAC group according to whether the penta-ester capsule is combined or not. The patient characteristic information is shown in table 1.
TABLE 1 demographic and clinical characteristics of 211 total renal transplant patients
2. Immunosuppressant
Renal transplant patients all used the same triple immunization protocol, i.e., TAC, an antimetabolite (mycophenolate mofetil) and a corticosteroid (methylprednisolone). The initial dose of tacrolimus is 0.1-0.15 mg/kg administered to a patient twice a day, the administration amount of TAC is adjusted according to the physiological condition of the patient according to the concentration data of the treatment drug during the hospitalization period (within 40 days). Generally, the steady-state concentration of tacrolimus is maintained at 5-10 ng/ml
3. Analytical method
During hospitalization of a patient after kidney transplantation, blood sample collection is carried out at intervals of 2, 3 days or longer according to clinical treatment requirements, after the patient takes tacrolimus for 12 hours, 1-2 mL of peripheral venous blood is extracted and placed in an EDTA-K2 anticoagulation tube in a 7:50 fasting state in the morning, and the blood sample is stored in a refrigerator at the temperature of-20 ℃ for later use. The concentration of the whole blood thus obtained was measured by a Microparticle Enzyme Immunoassay (MEIA) and the whole blood was subjected to the protocol required by the manufacturer. Linear range of the instrument is 0-30 ng & ml-1The total precision (CV%) of the assay was within 10%. Will be less than 2ng ml in the study-1The drug concentration point of (a) is excluded.
4. Analysis of genetic polymorphisms
By Hardy-Weinberg balance, the result shows that P is more than 0.05 (except CY3A 4rs 4646437), the gene frequency reaches genetic balance, and the research data are representative. Among 211 patients tested, the CYP3A4 genotyping and haplotype frequencies are shown in Table 3 and Table 4, wherein rs2242480 CC CT TT genotype in CYP3A4 is 98, 96 and 15, and the genotype frequencies are 46.89%, 45.93% and 7.18%, respectively, which meet Hardy-Weinberg equilibrium (P > 0.05); the rs4646437 GG AG AA genotypes are 113, 67 and 25 cases respectively, the genotype frequencies are 54.59%, 32.37% and 13.04% respectively, and Hardy-Weinberg balance is not met (P < 0.05).
CYP3A4 haplotype rs2242480C/-rs4646437/G wild type is most common (60%), followed by the variant (TA) haplotype (17.6%) and the variant (CA) haplotype (15%) (Table 4), which in the following study were divided into two groups: rs2242480-rs4646437(CCGG) are defined as slow metabolism, others as standard metabolism.
In order to easily observe the influence of different genotypes of CYP3A4 multiple rs2242480-rs4646437 on the tacrolimus Clearance (CL), a CYP3A4rs 2242480-rs4646437 diagnostic graph is drawn by using an R language, and the influence of the slow metabolism type of rs2242480-rs 46437(CCGG) on the tacrolimus Clearance (CL) is judged to be lower than that of the standard metabolism type according to the diagnostic graph. Through statistical analysis, whether the penta-ester capsule is combined or not, the C0/D value of the slow metabolism group haplotype is higher than that of other haplotypes within 21D after operation, the statistical significance (p is less than 0.05) is found, and the statistical analysis result is shown in figure 2.
TABLE 3 CYP3A4 gene distribution frequency in 211 patients in total
TABLE 4 haplotype frequency for 211 renal transplant recipients based on CYP3A4rs 2242480-rs4646437 SNPs
A kidney transplantation adult receptor tacrolimus metabolism genotype detection method comprises the following steps:
1) determining the CYP3A4rs2242480 and rs4646437 gene locus mutation typing of a sample to be detected;
2) analyzing the result, wherein the result is calculated according to the following formula:
P＝M1×M2
m1 ═ combined medication penta-ester capsules, M1 ═ 0.854; a Wuzhi capsule for non-combined medicines, M1 ═ 1
M2-CYP 3A4rs 2242480-rs 46437 multiple genotype, CYP3A4rs 2242480-rs 46437 slow metabolism (CG/CG) M2-1, and the rest standard metabolism M2-1.24.
The gene polymorphism mutation detection system comprises:
| composition (I) | Volume of |
| qPCR MasterMix | 10uL |
| Upstream primer | 0.5uL |
| Downstream primer | 0.5uL |
| Probe 1 | 0.5uL |
| Probe 2 | 0.5uL |
| Ultrapure water | 8uL |
| In total | 20uL |
Designing and detecting CYP3A4rs 2242480-rs4646437 multi-fold type probe:
CYP3A4 rs2242480
| Type | Sequence | Start bp | Stop bp | Tm |
| Forward Primer | CCACGAGCAGTGTTCTCTCCTTC | 331 | 354 | 72 |
| Reverse Primer | AATAGAAAGCAGATGAACCAGAGCC | 287 | 312 | 72 |
| Probe1 | CAGTGTTCTCTCCTTCGTGCTCC | 338 | 361 | 66 |
| Probe2 | GGCTCTGGTTCATCTGAGTGTAGC | 301 | 325 | 66 |
CYP3A4 rs4646437
| Type | Sequence | Start bp | Stop bp | Tm |
| Forward Primer | GGCAAAGAGATTAGGGCATTGA | 420 | 441 | 59 |
| Reverse Primer | AGGCCAGTGGCTGAGCAA | 545 | 528 | 59 |
| Probe1 | CTGATCTCACTGCTGTAGTGGTGCTCCT | 483 | 510 | 66 |
| Probe2 | CTGATCTCACTGCTGTAGCGGTGCTC | 483 | 508 | 66 |
the foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> first subsidiary Hospital of Nanchang university
<120> use of product for detecting gene locus mutation for preparing product for predicting or evaluating metabolic condition of patient taking tacrolimus
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Claims (10)
1. Use of a product for detecting mutations at the CYP3A4rs2242480 and CYP3A4rs4646437 gene loci in the preparation of a product for predicting or evaluating the metabolic status of patients taking tacrolimus.
2. The use according to claim 1, wherein the product for detecting the site mutation of CYP3A4rs2242480 and CYP3A4rs4646437 gene is a primer pair and/or a probe pair for detecting the site mutation of the gene.
3. The use according to claim 2, wherein the primer pair for detecting the mutation of the CYP3A4rs2242480 gene locus is shown as SEQ ID No.1-2, and the probe pair for detecting the mutation of the CYP3A4rs2242480 gene locus is shown as SEQ ID No. 3-4; the primer pair for detecting the site mutation of the CYP3A4rs 464646437 gene is shown as SEQ ID No.5-6, and the probe pair for detecting the site mutation of the CYP3A4rs4646437 gene is shown as SEQ ID No. 7-8.
4. The use according to any one of claims 1 to 3, wherein the patient is administered tacrolimus concurrently with a tacrolimus potentiator.
5. Use according to claim 4, characterized in that the tacrolimus potentiator is a pentaester capsule.
6. Use according to any one of claims 1 to 3, characterized in that said assessment is calculated according to the following formula:
P＝M1×M2
m1 ═ combined medication penta-ester capsules, M1 ═ 0.854; a Wuzhi capsule for non-combined medicines, M1 ═ 1
M2-CYP 3A4rs2242480-CYP3A4rs4646437 multiple genotype, CYP3A4rs2242480-CYP3A4rs4646437 slow metabolism (CG/CG) M2-1, and the rest standard metabolism M2-1.24.
7. The use according to claim 2 or 3, wherein the probe pairs are labeled with different fluorophores at their 5 'ends and with MGBs at their 3' ends, respectively.
8. The use according to claim 7, wherein the 5' ends of the probe pairs are labeled with FAM fluorophore and VIC fluorophore, respectively.
9. A kit is characterized by comprising a product for detecting site mutation of CYP3A4rs2242480 and CYP3A4rs4646437 gene.
10. Use of a kit according to claim 9 for the manufacture of a product for predicting or assessing the metabolic profile of a patient taking tacrolimus, preferably a patient after organ transplantation.
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| CN111471758A (en) * | 2020-04-09 | 2020-07-31 | 南昌大学第一附属医院 | Application of product for detecting gene locus mutation in preparation of product for evaluating metabolic condition of patient taking tacrolimus |
| CN113889221A (en) * | 2021-12-08 | 2022-01-04 | 首都医科大学宣武医院 | System for accurately selecting treatment dosage of tacrolimus of myasthenia gravis patient and application of system |
| CN115547513A (en) * | 2022-11-22 | 2022-12-30 | 中日友好医院(中日友好临床医学研究所) | Method for predicting using dosage of intrathecal opioid analgesic |
| CN117431312A (en) * | 2023-12-19 | 2024-01-23 | 中日友好医院(中日友好临床医学研究所) | Polymorphic site combination for predicting tacrolimus dosage, prediction model and kit |
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| CN111471758A (en) * | 2020-04-09 | 2020-07-31 | 南昌大学第一附属医院 | Application of product for detecting gene locus mutation in preparation of product for evaluating metabolic condition of patient taking tacrolimus |
| CN111471758B (en) * | 2020-04-09 | 2024-05-14 | 南昌大学第一附属医院 | Use of a product for detecting mutations in the gene locus for the preparation of a product for assessing the metabolic condition of a patient following tacrolimus administration |
| CN113889221A (en) * | 2021-12-08 | 2022-01-04 | 首都医科大学宣武医院 | System for accurately selecting treatment dosage of tacrolimus of myasthenia gravis patient and application of system |
| WO2023104163A1 (en) * | 2021-12-08 | 2023-06-15 | 首都医科大学宣武医院 | System for accurate selection of therapeutic dose of tacrolimus for myasthenia gravis patient, and use |
| CN117296102A (en) * | 2021-12-08 | 2023-12-26 | 首都医科大学宣武医院 | System for accurately selecting tacrolimus treatment dosage of myasthenia gravis patient and application |
| CN115547513A (en) * | 2022-11-22 | 2022-12-30 | 中日友好医院(中日友好临床医学研究所) | Method for predicting using dosage of intrathecal opioid analgesic |
| CN117431312A (en) * | 2023-12-19 | 2024-01-23 | 中日友好医院(中日友好临床医学研究所) | Polymorphic site combination for predicting tacrolimus dosage, prediction model and kit |
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