CN107447024B

CN107447024B - Application of HLA-B allele in preparation of detection reagent for predicting risk of liver injury caused by plant polygonum multiflorum and components thereof

Info

Publication number: CN107447024B
Application number: CN201710811870.0A
Authority: CN
Inventors: 李超鹏; 欧阳冬生; 陈露露; 周露萍; 饶泰
Original assignee: Changsha Metropolitan Medical Inspection Co Ltd
Current assignee: Changsha Metropolitan Medical Inspection Co Ltd
Priority date: 2017-09-11
Filing date: 2017-09-11
Publication date: 2019-12-24
Anticipated expiration: 2037-09-11
Also published as: CN107447024A

Abstract

The invention discloses the use of HLA-B35: 01 alleles. 1. The kit is used as a genetic marker and a treatment target for preparing a detection reagent for predicting the risk of liver injury caused by the polygonum multiflorum and polygonum multiflorum components. 2. Used as a therapeutic target for preparing the medicine for treating the liver injury caused by the polygonum multiflorum. 3. The kit is used as a marker for preparing a detection reagent for screening high-sensitivity people suffering from liver injury caused by fleece-flower root and substances containing fleece-flower root.

Description

Application of HLA-B allele in preparation of detection reagent for predicting risk of liver injury caused by plant polygonum multiflorum and components thereof

Technical Field

The invention relates to an application of HLA-B35: 01 allele, in particular to an application of HLA-B35: 01 allele as a liver injury risk genetic marker of polygonum multiflorum, a pharmaceutical preparation containing polygonum multiflorum and a health care product containing polygonum multiflorum.

Background

In China, the liver injury caused by Chinese herbal medicines accounts for 21 percent of all the medicinal liver injuries, and the liver injury caused by polygonum multiflorum is most remarkable. Polygonum multiflorum (Polygonum multiflorum Thunb.) of Polygonaceae has been used for thousands of years in China and is considered as one of the four immortals. The use of the plant polygonum multiflorum is widespread, and the dried root tuber is called polygonum multiflorum (polygonum multiflorum RADIX) or polygonum multiflorum; the processed product is called "radix Polygoni Multiflori Preparata (POLYGONI MULTIFLORIRADIX PRAEPARATA)"; the dried rattan is called "CAULIS Polygoni Multiflori (POLYGONI MULTIFLORI CAULIS)". The population base for eating the polygonum multiflorum is huge in China, and in recent years, the cases of liver injury caused by oral administration of the polygonum multiflorum and the finished preparations of the polygonum multiflorum increase year by year. However, because the components of the polygonum multiflorum are complex and the use method is various, the toxic components and pathogenic mechanisms of the polygonum multiflorum cannot be determined in the existing research, and no effective prediction, diagnosis and treatment means for polygonum multiflorum liver injury can be used.

The previous epidemiological research shows that the incidence of liver injury caused by polygonum multiflorum is low, the liver injury is frequently caused after l-4 weeks after the medicine is taken, the liver injury is not related to the sex and age of patients, the liver injury can also be caused by low-dose administration, and the re-excitation phenomenon and familial case report also exist. The previous polygonum multiflorum can cause liver damage under the condition of large dose and long-term administration in a normal animal model and is far from clinical hepatotoxicity cases, while the polygonum multiflorum can cause liver damage and histopathological changes of animals at lower dose in a very heterogeneous liver damage animal model based on endotoxin (lipopolysaccharide) immunosensitization, and is similar to clinical cases. Therefore, the liver injury caused by polygonum multiflorum can be presumed to be immunological idiosyncratic liver injury.

Human Leukocyte Antigen (HLA) is a group of closely linked genomes that initiate adaptive immune responses by coding and generating protein molecules associated with immune responses and immune recognition to recognize and present antigenic peptides. An increasing number of human genome-wide association studies have demonstrated that there is also a strong association between HLA genotypes and some drug-specific liver damage. For example: HLA-A02: 01, HLA-DQB1 06:02 alleles have a strong association with amoxicillin clavulanate potassium induced liver injury in European and American white species (OR ═ 2.3(1.8-2.9), p ═ 1.8X 10^-10；OR＝4.2(2.7-6.6)，p＝4.6×10^-10) (ii) a The HLA-DRB1 x 07:01 allele has strong correlation with liver injury caused by tumor targeting drug lapatinib (p is 7.8 x 10)^-11) (ii) a HLA-B57: 01 allele is strongly associated with liver damage caused by flucloxacillin (OR 80.6, p 9.0X 10)^-19) And the like. The existing research proves that the small molecule drug can be combined with the specificity HLA molecule in a non-covalent way to form a compound, and the compound presents the antigen to the surface of the cell membrane for the recognition of a T cell receptor and activates T cellsThe cell has killing effect on liver cells. Therefore, different types of HLA genotypes can be used as genetic markers for predicting different drugs causing immunological idiosyncratic liver damage. The FDA requires that specifications of drugs such as carbamazepine, abacavir, lapatinib, allopurinol, etc., note the association between specific adverse drug reactions and HLA genotypes, and suggests to check the relevant HLA genotypes before taking the drugs for predicting the occurrence of adverse drug reactions.

HLA-B belongs to type I leukocyte antigen, has more complex polymorphism, has 656 different alleles detected, and the coded protein thereof is expressed in almost all nucleated cells, and has the main function of presenting exogenous antigen to the cell surface for recognition by CD8+ T cells. It is proved that different HLA-B alleles have strong association with partial drug-induced liver injury, such as liver injury caused by HLA-B57: 01 and flucloxacillin, liver injury caused by B44: 03 and ticlopidine, liver injury caused by B35: 02 and minocycline, and the like.

Disclosure of Invention

The invention aims to solve the technical problem of providing a new application of HLA-B35: 01 allele as a genetic marker and a treatment target point for predicting liver injury caused by plant fleece-flower root and components thereof, and research results show that HLA-B3501 (the gene sequence is shown in the specification: http:// www.ebi.ac.uk/cgi-bin/ipd/imgt/HLA/get _ allele. cgi? B35: 01) is an important genetic susceptibility factor of fleece-flower root caused liver injury, can be used as a genetic marker for predicting the risk of liver injury of patients taking fleece-flower root and a treatment target point for treating fleece-flower root caused liver injury, and specifically can be used as the following application, 1 is used as the genetic marker and the treatment target point for preparing a detection reagent for predicting the risk of liver injury caused by plant fleece-flower root and fleece-flower root components, 2 is used as the treatment target point for preparing a medicine for treating fleece-flower root caused liver injury, 3 is used as the marker for preparing a detection reagent for screening the sensitive population suffering from liver injury caused by fleece-flower root and substances containing fleece-flower root.

Wherein the polygonum multiflorum comprises: raw fleece-flower root, prepared fleece-flower root and vine of multiflower knotweed; the application form of the polygonum multiflorum can comprise but is not limited to decoction pieces, powder, wall-broken powder, water extract, various organic extracts, polygonum multiflorum extract, polygonum multiflorum granules and the like; the fleece-flower root product refers to a product of a pharmaceutical preparation, a health-care beverage, a health-care food, a traditional Chinese medicine formula, a Chinese patent medicine and the like which contain the fleece-flower root component.

The substance for detecting whether a human has an HLA-B35: 01 allele may be any reagent, kit and/or apparatus known in the art for use in any method for detecting the presence of an allele, including but not limited to the following methods: PCR-based HLA sequencing typing methods, DNA specific hybridization methods, and HLA serotyping methods.

The substance for detecting whether a human has an HLA-B35: 01 allele may further include a reagent, a kit and/or an apparatus which can be used in the method for detecting an equivalent genetic marker of the human HLA-B35: 01 allele. The equivalent genetic marker is a genetic marker associated with the HLA-B35: 01 allele. The isovalent genetic marker can be a single nucleotide polymorphism, a simple repeat sequence and the like, such as rs2596501, rs2844521, rs2308655, rs1140546, rs1051488, rs1131500, rs1130992, rs2308500, rs41541515, rs41551014, rs1050240, rs41546016, rs41545916, rs 1050820823, rs41541216, rs709052, rs61759952, rs2257269, rs2596493, rs1131285, rs1131279, rs 1131271271271271271271275, rs41547422, rs41545114, rs 105060621849, rs1050642, rs12697944 97052, rs4997052, rs 499766150, rs709055, rs 10512736, rs1131112, rs 3131313142, rs 105633414456353148, rs 1072, rs 10705778131779, rs 11330031779, rs 11330056353005635059, rs 1133005635045620, rs 113300316620, rs 11330056375620, rs 3005620, rs 1133005620, rs 30056375637563756375620, rs 300316620, rs 435445, rs 113355620, rs 1133556375620, rs 113355620, rs 3005637563756375620, rs 4354045620, rs 113355620, rs 1133556375620, rs 30056375620, rs 4354045620, rs 43540456375620, rs 4354045620, rs 113355620, rs 1133005620, rs 113no, rs 4354045620, rs 113355620, rs 113no, rs 1133556375620, rs 113no, rs.

Other subtypes of HLA-B35: 01 can also express HLA-B35: 01 polypeptide molecules on cell membranes, and are also susceptible genetic factors of liver damage caused by polygonum multiflorum. Many subtypes of HLA-B35: 01 have been reported, such as: HLA-B35: 01: 01-B35: 01: 45. These subtypes have different distribution frequencies in different populations, and it is believed that other subtypes of HLA-B35: 01 can be used as substitutes for HLA-B35: 01 for risk assessment of liver damage by Polygonum multiflorum.

Furthermore, it should be noted that other genetic markers linked to this gene, in addition to HLA-B35: 01 itself, can also predict liver damage by Polygonum multiflorum. This is due to the fact that genetic markers in the very vicinity of the HLA alleles tend to co-segregate or show linkage disequilibrium with the HLA-B35: 01 gene. The presence of these genetic markers therefore indicates the presence of HLA-B35: 01. For example, there is a strong linkage disequilibrium between MICA 002 and HLA-B35: 01, and the correlation coefficient between both can reach more than 80% in multiple populations.

HLA-B3501 can be used as target in drug development for screening and/or identifying method for treating drug induced hepatic injury induced by Polygoni Multiflori radix preparation. For example, contacting polygonum multiflorum with a cell or humanized animal expressing the allele as a model to cause liver cell damage, and further testing the efficacy of the candidate drug in treating polygonum multiflorum-induced liver damage; or contacting the cell or humanized animal expressing the allele with candidate drug to inhibit the expression and/or function of the allele, and administering Polygoni Multiflori radix product to test the prevention effect of the candidate drug on liver injury of patients susceptible to Polygoni Multiflori radix.

In the examples of the present application, the results of the study on a population consisting of 26 patients with hepatotoxicity induced by administration of polygonum multiflorum and 99 healthy controls showed that the oral administration of polygonum multiflorum-containing fraction to carriers of HLA-B35: 01 allele resulted in 60-fold higher liver damage than non-carriers (OR 61.22 (15.98-290.81)). In practical use, the HLA-B35: 01 test can be used for predicting whether the individual taking the polygonum multiflorum-containing component is likely to suffer from liver injury. The carriers of HLA-B35: 01 gene need to avoid taking or eating the medicine, food and health care product containing the polygonum multiflorum component to avoid liver injury, if the carrier is taken or taken by mistake, the carrier needs to avoid long-term and large-dose use, and the liver function is closely monitored in the taking process. Non-carriers of HLA-B35: 01 gene have a low incidence of liver damage when taking pharmaceuticals, foods and health products containing Polygonum multiflorum components, but also need to avoid long-term, high dose use and monitor liver function.

Drawings

FIG. 1 shows the correlation between HLA region locus and liver injury of Polygonum multiflorum, with the abscissa representing the position of HLA region and the ordinate representing-log₁₀(p) the dotted line indicates the significance limit p of 1 × 10^-5The diamonds are HLA-B35: 01 alleles and the triangles are MICA 002 genes.

Fig. 2 is a graph of the receiver performance characteristics for risk prediction of liver damage by polygonum multiflorum using HLA-B3501 as a predictive marker.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 HLA-B35: 01 alleles as genetic markers for predicting liver damage in the plant Polygonum multiflorum and its constituents

A sample

The samples are human peripheral blood, including case samples, polygonum multiflorum tolerance control samples and healthy control samples.

1. Case sample

20 patients with liver damage from polygonum multiflorum of chinese character were included in the study (11 for the discovery phase of the study, 9 for the validation phase of the study). The time from the beginning of taking fleece-flower root to the occurrence of symptoms of liver injury is 10-240 days, and common symptoms comprise: yellow skin, dark brown urine, nausea, vomiting, loss of appetite, fatigue, fever, etc. Laboratory tests show that glutamate pyruvate transaminase exceeds the upper limit of 3 times normal or total bilirubin exceeds 40. mu. mol/L. The patients are diagnosed as the drug-induced liver injury caused by taking polygonum multiflorum by at least 1 specialist physician in hepatopathy departments, and the RUCAM causal relationship assessment scale score > is 3. Also 6 patients of Han nationality of China (in the validation phase of the study) took a prescription containing Polygoni Multiflori radix (containing 10-15 g/dose of Polygoni Multiflori radix) under the direction of the physician, and monitored liver function once every 20 days, and had more than 2 times of upper limit of normal glutamic-pyruvic transaminase during taking.

2. Polygonum multiflorum tolerance control group sample

In the validation study, the polygonum multiflorum tolerance control group samples comprise 66 patients who were not suffering from liver enzyme abnormality after taking a polygonum multiflorum-containing prescription (containing 10-15 g/dose of polygonum multiflorum) under the guidance of a physician, monitoring liver function once every 20 days, and continuously monitoring for 60 days.

3. Healthy control sample

The discovery phase of the healthy control sample selection literature (Zhou F, Cao H, Zuo X, Zhang T, Zhang X, Liu X, Xu R, Chen G, Zhang Y, ZHEN X, Jin X, Gao J, Mei J, Sheng Y, Li Q, Liang B, Shen J, Shen C, Jiang H, Zhu C, Fan X, Xu F, Yue M, Yi X, Ye C, Zhang C, Liu X, Yu L, Wu J, ChenM, Zhuang X, Tang L, Shao H, Wu L, Li J, Xu Y, Zhang Y, ZHao S, Wang Y, Li G, Xu H, ZengL, Wang J, Bai M, Chen Y, Chen W, Kang T, Wu Y, Xia X, Yang Z, Wang J, Shang X, Zhang X J, Shang X, Shang J, Zhang X, Zhang C, Shang J, Zhang X, Zhang J, zhang J, Yu X, Li Y, Yang S, Yang H, Wang J, Liu J,l, Sun L, Wang J, Zhang X. deep sequencing of the MHC region in the core position constraints to the student sof complex disease. Nat Genet 2016; 48:740-6.) reported HLA genotype frequency data of 20635 Chinese Han population.

Healthy control samples in the validation stage were derived from 99 recruited chinese han volunteers, and entered into the cohort standard: laboratory examination shows no abnormal liver function, no history of other immunological diseases, and no history of infectious diseases.

Second, method

1. Discovery phase

After informed consent of 11 patients, 2ml of whole blood was drawn, whole genomic DNA was extracted, the 6P21.3 region (chr6:28477797-33448354) including the conventional HLA region (about 3.37Mb) and its flanking region (about 1.6Mb) was captured using the NimbleGen SeqCap target region capture kit, sequencing depth 100X, using ATHLATES software and IMGT/HLA database (Release 3.22.0)) And (3) comparing and typing the reference sequences to obtain 122 types of typing, wherein the typing precision reaches 4 bits, and then analyzing the relevance by adopting a Fisher precision test method. The results of the association analysis (fig. 1) showed HLA regions HLA-B35: 01(OR 364.50(51.68-14061.27), p 4.2 × 10^-15) Is highly related to liver damage caused by polygonum multiflorum.

2. Verification phase

In order to further verify the relevance of HLA-B35: 01 allele, the PCR-SBT method is adopted to carry out HLA-B typing verification on other 15 patients with liver injury caused by polygonum multiflorum, 66 polygonum multiflorum tolerant samples and 99 healthy volunteers, and the typing precision reaches 4. And analyzing the relevance of the typing result by adopting a Fisher accurate test method. The results are shown in table 1:

TABLE 1 typing results of HLA-B35: 01 alleles at the validation stage

3. Combined analysis (Table 2)

The data of the two stages of discovery and verification are merged and analyzed to find that: all HLA-B35: 01 carriers were heterozygotes, with no homozygote detection.

TABLE 2 HLA-B35: 01 allelic typing results

4. Sensitivity and specificity

As can be seen from the results in Table 2, the number of true positive samples (HLA-B3501 carriers) and the number of false positive samples (not HLA-B35: 01 carriers) among the 26 patients with liver damage caused by Polygonum multiflorum were 20 in the case group; 66 control groups were treated with polygonum multiflorum component, 61 true negative samples (those not carrying HLA-B35: 01) and 5 false negative samples (HLA-B3501 carriers), according to the formula:

sensitivity is 100% of the number of true positives/(number of true positives + number of false negatives).

The specificity is 100% of the number of true negative persons/(number of true negative persons + number of false positive persons).

Calculating to obtain: sensitivity is 0.7692; specificity is 0.9242.

FIG. 2: receiver operator characteristic curve (ROC): the ROC curve is a graph obtained by plotting a true positive rate and a false positive rate and is used to represent the correlation between sensitivity and specificity. The area under the ROC curve is typically between 1.0 and 0.5. In the case of AUC > 0.5, the closer the AUC is to 1, the better the diagnostic effect. AUC has lower accuracy when being 0.5-0.7, AUC has certain accuracy when being 0.7-0.9, and AUC has higher accuracy when being more than 0.9. When AUC is 0.5, the diagnostic method is completely ineffective and is not valuable. In the present example, AUC under ROC curve is 0.8467(0.7681-0.9423), which indicates that the diagnostic method has a certain accuracy.

Claims

Application of HLA-B allele in preparing a detection reagent for predicting risk of liver injury caused by plant polygonum multiflorum and components thereof; the HLA-B allele is an HLA-B35: 01 allele.
2. The use according to claim 1, wherein said HLA-B allele is used as a genetic marker for the preparation of a test reagent for predicting the risk of liver damage caused by Polygonum multiflorum and its components.
The application of HLA-B allele in preparing medicine for treating liver injury caused by polygonum multiflorum; the HLA-B allele is an HLA-B35: 01 allele.
4, the application of HLA-B allele as a marker in preparing a detection reagent for screening the high-sensitivity population suffering from liver injury caused by fleece-flower root and substances containing fleece-flower root; the HLA-B allele is an HLA-B35: 01 allele.