Gene marker, primer, probe and kit for predicting child graft versus host disease and application of gene marker, primer, probe and kit

Abstract

The invention is applicable to the technical field of gene detection, and provides a gene marker, a primer, a probe and a kit for predicting a child graft versus host disease and application of the gene marker, the primer, the probe and the kit. It is found for the first time that a combination of 12 gene markers including TPMT, UGT2B10, BHLHE22, KIAA1715, GSTA1, GSTM1, CTH, MTHFR, CYP2B6, HFE, CPS and HPSE in peripheral blood of a child patient suffering from the graft versus host disease and the hepatic minor vein occlusion disease has significant difference between hematopoietic stem cell transplanted child patients suffering from the GVHD and hematopoietic stem cell transplanted child patients not suffering from the GVHD, the combination can be used as one of laboratory immunity related detection indexes for auxiliary diagnosis of the GVHD, operation is easy and feasible, and the method is suitable for clinical application. The clinical application value is very high. According to the kit for predicting the child graft versus host disease, 17 SNP loci and one genotype homozygous deletion are detected, genetic typing is completed by combining a high-throughput sequencing and capillary electrophoresis combined method, risk assessment can be further given according to the typing result of a patient, and individualized susceptible gene detection is achieved.

Description

Technical Field

[0001] The present application belongs to the field of gene detection technology, and in particular, relates to gene markers, primers and probes, kits and their non-diagnostic applications for predicting graft-versus-host disease in children. Background Art

[0002] Graft-versus-host disease (GVHD) is one of the most common life-threatening complications of allogeneic hematopoietic cell transplantation. It is mainly caused by the T cells in the donor's graft being activated by immune response in the recipient's body after transplantation to acquire cytolytic ability and then attack cells carrying foreign antigens. Its characteristics are similar to the symptoms of autoimmune and other immune diseases, such as scleroderma, S-syndrome, immune cytopenia, etc. At present, the diagnosis of graft-versus-host disease is mainly based on pathological manifestations such as diarrhea and rash after transplantation and related laboratory tests.

[0003] Foreign studies have found that a variety of biomarkers can be used as potential predictors and early detection indicators for GVHD, among which genetic susceptibility is important in the study of the etiology of GVHD, and multiple research evidence supports the immunological basis and genetic tendency of GVHD. However, there have been no reports on the use of gene combination kits for susceptibility genes TPMT, UGT2B10, BHLHE22, KIAA1715, GSTA1, GSTM1, CTH, MTHFR, CYP2B6, HFE, CPS and HPSE in the risk assessment of GVHD in children.

[0004] So far, there is a lack of characteristic pathological diagnostic markers for GVHD in children, and there is even less standard and widely used diagnostic guidelines for GVHD in children. Summary of the invention

[0005] The purpose of the embodiments of the present application is to provide a gene marker, primers and probes, a kit and applications thereof for predicting graft-versus-host disease in children.

[0006] The embodiment of the present application is implemented as follows: the genetic markers for predicting graft-versus-host disease in children are a combination of TPMT gene, UGT2B10 gene, BHLHE22 gene, KIAA1715 gene, GSTA1 gene, GSTM1 gene, CTH gene, MTHFR gene, CYP2B6 gene, HFE gene, CPS1 gene and HPSE gene.

[0007] Preferably, the SNP sites of the TPMT gene are rs1800462, rs1800460 and rs1142345; the SNP site of the UGT2B10 gene is rs17146905; the SNP site of the BHLHE22 gene is rs16931326; the SNP site of the KIAA1715 gene is rs2289971; the SNP sites of the GSTA1 gene are rs3957356, rs11964968 and rs5 8912740; the SNP site of the CTH gene is rs1021737; the SNP sites of the MTHFR gene are rs1801133 and rs1801131; the SNP site of the CYP2B6 gene is rs3745274; the SNP site of the HFE gene is rs1800562; the SNP site of the CPS1 gene is rs1047891; the SNP sites of the HPSE gene are rs4693608 and rs4364254.

[0008] The embodiments of the present application also provide the use of the above-mentioned gene markers for predicting graft-versus-host disease in children and / or their detection reagents in the preparation of a product for detecting graft-versus-host disease in children.

[0009] The present application also provides primers and probes for predicting graft-versus-host disease in children, including SNP site primers, GSTM1 primers, internal reference quality control primers, GSTM1 probes, and internal reference quality control probes; The SNP site primers are designed based on the SNP sites of TPMT gene, UGT2B10 gene, BHLHE22 gene, KIAA1715 gene, GSTA1 gene, CTH gene, MTHFR gene, CYP2B6 gene, HFE gene, CPS1 gene and HPSE gene, and the primer sequences are shown in SEQ ID NOs. 1 to 34; The GSTM1 primers are designed based on the Indel site of the GSTM1 gene, and the primer sequences are shown in SEQ ID NOs. 35 to 36; The internal reference quality control primers are designed based on β-globulin, and the sequences are shown in SEQ ID NOs. 37-38; The GSTM1 probe is designed based on the GSTM1 gene, and the probe sequence is shown in SEQ ID NO.39; The internal reference quality control probe is designed based on the β-globulin, and the probe sequence is shown in SEQ ID NO.40.

[0010] The embodiment of the present application also provides a kit for predicting graft-versus-host disease in children, wherein the kit comprises the above-mentioned detection reagent for the gene marker for predicting graft-versus-host disease in children.

[0011] Preferably, the kit comprises the above-mentioned primers and probes for predicting graft-versus-host disease in children.

[0012] The present application also provides a non-diagnostic application method of a kit for predicting graft-versus-host disease in children, comprising: Using the above-mentioned kit for predicting graft-versus-host disease in children, one round of PCR amplification is performed on the genomic DNA of the peripheral blood sample to be tested to obtain SNP site amplification products and GSTM1 amplification products; After the SNP site amplification product is purified, a second round of PCR library amplification is performed using a library construction adapter primer. After purification, the obtained library product is sequenced on a machine to obtain sequencing data; After quality control of the sequencing data, the data is posted back to the genome, and the coverage information of the genotypes of the positive and negative chains of the candidate sites of the genome are statistically compared, so as to calculate the genotype of the candidate site and whether imbalance occurs based on the coverage information, and determine whether the current genotype is a risk genotype according to a preset genotype risk database; The GSTM1 amplification product is subjected to capillary electrophoresis to determine whether GSTM1 is homozygous deleted.

[0013] This application discovered for the first time that the combination of 12 gene markers, including TPMT, UGT2B10, BHLHE22, KIAA1715, GSTA1, GSTM1, CTH, MTHFR, CYP2B6, HFE, CPS and HPSE, in the peripheral blood of children with graft-versus-host disease and hepatic veno-occlusive disease showed significant differences between children with hematopoietic stem cell transplantation who had GVHD and those who did not develop GVHD. This combination of 12 gene markers can be used as one of the laboratory immune-related detection indicators for auxiliary diagnosis of GVHD. The operation is simple and feasible, and has high clinical application value.

[0014] The kit provided in the present application for predicting graft-versus-host disease in children detects 17 SNP sites and 1 genotype homozygous deletion, and completes genotyping by combining high-throughput sequencing with capillary electrophoresis. Based on the typing results of the patient, a risk assessment can be further given to achieve individualized susceptibility gene testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A diagram showing the results of homozygous deletion (Null) and non-homozygous deletion (Positive) provided in Example 6 of the present application; Figure 2A flowchart of multiple library data analysis provided in Example 7 of the present application; Figure 3 The first-generation sequencing results provided in Example 8 of the present application (some first-generation sequencing results were not detected and are not included in the result comparison); Figure 4 The high-throughput sequencing data analysis results provided in Example 8 of the present application. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0017] The embodiments of the present application provide genetic markers for predicting graft-versus-host disease in children, which are a combination of TPMT gene, UGT2B10 gene, BHLHE22 gene, KIAA1715 gene, GSTA1 gene, GSTM1 gene, CTH gene, MTHFR gene, CYP2B6 gene, HFE gene, CPS1 gene and HPSE gene.

[0018] The present application also provides primers and probes for predicting graft-versus-host disease in children, including SNP site primers, GSTM1 primers, internal reference quality control primers, GSTM1 probes and internal reference quality control probes; as shown in Table 1, the SNP site primers are designed based on the SNP sites of TPMT gene, UGT2B10 gene, BHLHE22 gene, KIAA1715 gene, GSTA1 gene, CTH gene, MTHFR gene, CYP2B6 gene, HFE gene, CPS1 gene and HPSE gene, and the primer sequences are shown in SEQ ID NOs.1 to 34; the GSTM1 primers are designed based on the Indel site of the GSTM1 gene, and the primer sequences are shown in SEQ ID NOs.35 to 36; the internal reference quality control primers are designed based on β-globulin, and the sequences are shown in SEQ ID NOs.37 to 38; the GSTM1 probe is designed based on the GSTM1 gene, and the probe sequence is shown in SEQ ID NOs. As shown in SEQ ID NO.39; the internal reference quality control probe is designed based on the β-globulin, and the probe sequence is shown in SEQ ID NO.40.

[0019] Among them, the above-mentioned primers include upstream primers and downstream primers. The sequence of the upstream primer is designed with the SNP site as the coordinate starting point and the base sequence extending 150bp in the 5' direction as the sequence information; the sequence of the downstream primer is designed with the SNP site as the coordinate starting point and the base sequence extending 150bp in the 3' direction as the sequence information, and the fragment size is about 200bp.

[0020] Among them, the above-mentioned probe includes two genotypes of GSTM1 negative and positive, and the sequence of the probe is designed with the SNP site as the coordinate starting point and the base sequence extending 15bp in the 5' direction as the sequence information; the sequence of the mutated probe is designed with the SNP site as the coordinate starting point and the base sequence extending 15bp in the 3' direction as the sequence information, and the fragment size is about 14~20bp.

[0021] The embodiments of the present application also provide the use of the above-mentioned gene markers for predicting childhood graft-versus-host disease and / or their detection reagents in the preparation of childhood graft-versus-host disease detection products, wherein the detection products can be detection reagents, kits, etc.

[0022] Wherein, the kit comprises the above-mentioned detection reagent for the gene marker for predicting graft-versus-host disease in children. Specifically, the kit includes a combination for amplifying the following DNA sequence polymorphism sites: MTHFR (rs1801133, rs1801131), HPSE (rs4693608, rs4364254), UGT2B10 (rs17146905), BHLHE22 (rs16931326), KIAA1715 (rs2289971), TPMT*2 (rs1800462), TPMT*3A (rs1800460), TPMT*3B (rs1142345), GSTA1*B (rs3957356, rs11964968, rs58912740), CTH (rs1021737), HFE (rs1800562), CPS1 (rs1047891), CYP2B6 (rs3745274) and GSTM1.

[0023] Optionally, the kit comprises Taq DNA Polymerase (Mg 2+) of the PR amplification kit sold by Nanjing Novozyme Biotechnology Co., Ltd., catalog number P101-D1. 2+ plus buffer), and the above-mentioned primers and probes for predicting graft-versus-host disease in children.

[0024] The present application also provides a non-diagnostic application method of a kit for predicting graft-versus-host disease in children, that is, a method for detecting the above-mentioned biomarkers. This detection method only requires two rounds of PCR amplification and magnetic bead purification of the product, and the operation is relatively simple and time-consuming, and includes the following steps: Using the above-mentioned kit for predicting graft-versus-host disease in children, one round of PCR amplification is performed on the genomic DNA of the peripheral blood sample to be tested to obtain SNP site amplification products and GSTM1 amplification products; After the SNP site amplification product is purified, a second round of PCR library amplification is performed using a library construction adapter primer. After purification, the obtained library product is sequenced on a machine to obtain sequencing data; After quality control of the sequencing data, the data is posted back to the genome, and the coverage information of the genotypes of the positive and negative chains of the candidate sites of the genome are statistically compared, so as to calculate the genotype of the candidate site and whether imbalance occurs based on the coverage information, and determine whether the current genotype is a risk genotype according to a preset genotype risk database; The GSTM1 amplification product is subjected to capillary electrophoresis to determine whether GSTM1 is homozygous deleted.

[0025] The amplicon library construction and detection system of the present application is used to simultaneously compare the detection results of clinical samples with their corresponding capillary electrophoresis detection results to perform methodological results and conduct risk confirmation.

[0026] The SNP molecular markers for detecting the marker genes related to the risk of GVHD in children provided in this application include the 17 SNP sites shown in Table 1 below, the primer probe sequences of the internal reference control gene designed based on the GSTM1 gene and β-globulin, and the detection technology combining the second-generation high-throughput sequencing and the first-generation capillary electrophoresis sequencing is selected by collecting peripheral blood of children with hematopoietic stem cell transplantation before transplantation, combining bioinformatics analysis to identify point mutations in related genes, and giving the corresponding genotypes. According to the reported risk of each genotype in the relevant literature guidelines for GVHD detection, the genetic risk assessment of related patients is guided, which can provide a clearer personalized diagnosis and treatment basis for the clinic, and can also be applied to the field of drug research and development. In addition, the probes for the SNP molecular markers provided in the embodiments of this application can detect the SNP sites in both directions with high detection accuracy.

[0027] The unique double tag used in this application, each i5 and i7 is a unique, non-repetitive tag sequence, and the DNA molecule is specifically labeled by the specific molecular tag in the adapter; since each nucleic acid in the starting material has a unique molecular barcode, the bioinformatics analysis software can highly accurately filter out duplicate reads and PCR errors, report unique reads, and thus eliminate identified errors before the final data analysis. The unique double tag is the I5 / I7 sequence (i.e., Index sequence) designed by this application, as shown below:

[0028] The following is a detailed description of gene markers, primers and probes, kits and their applications for predicting graft-versus-host disease in children with specific examples, as shown below. The PCR amplification kits used in the following examples were purchased from Nanjing Novezan Biotechnology Co., Ltd. The primers and probe reagents used in this application are independently developed and are not sold on the market; the experimental methods used in this application are conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0029] Example 1: Screening of GVHD Susceptibility Genes From the relevant guidelines for GVHD detection and published literature, 12 high-frequency genes of pediatric GVHD susceptibility genes were selected, SNP sites related to drug testing and annotated, GSTM1 gene, and internal reference control gene based on β-globulin were selected. A total of 17 target SNP sites and 2 sets of primer-probe combinations were designed, as shown in Table 1.

[0030] Table 1 Target SNP sites & sequences

[0031] Example 2: Probe design and synthesis According to the coordinate position of the SNP site on the genome shown in Table 1, the upstream primer was designed with the SNP site as the starting point of the coordinate and the base sequence extending 150bp bases to the 5' end as the sequence information, and the upstream primer did not include the SNP site sequence; the downstream primer was designed with the SNP site as the starting point of the coordinate and the base sequence extending 150bp bases to the 3' end as the sequence information, and the downstream primer did not include the SNP site sequence; the probe (14bp<probe<20bp) was designed with the GSTM1 insertion & deletion genotype and the β-globin gene as the starting point of the coordinate and the base sequence extending 15bp bases to the 5' end and the 3' end as the sequence information, and the probe can detect two genotypes of the GSTM1 gene negative and positive at this site. According to the peak diagram of the first-generation fragment analysis of the GSTM1 test results, the GSTM1 gene primer probe and the β-globin primer probe were mixed and configured.

[0032] Example 3: Extraction of genomic DNA Genomic DNA was obtained by extracting EDTA-anticoagulated peripheral blood (collected within the hospital and sent to an external laboratory) using the Adlai Genomic DNA Extraction Kit (DN0101).

[0033] The concentration of the extracted DNA was measured by NanoDrop, and 260 / 280 was between 1.7 and 2.1. The total amount of DNA extracted was greater than 100 ng, which met the detection requirements.

[0034] Example 4: One round of PCR multiplex amplification 1. Dilute the DNA sample to 5 ng / μL working solution.

[0035] 2. Dilute the primer master solution to 100uM and prepare the primer working solutions Amplicon-primer Mix1 and GSTM1-Primer Mix2 according to the proportion.

[0036] 3. Use the LAmp DNA Polymerase (Mg2+ plus buffer) kit to complete a round of PCR multiplex amplification with the extracted DNA sample. The specific steps are as follows: (1) Prepare the PCR system reaction mix except DNA, according to the actual PCR quantity * 110%, pipette to mix well, and then centrifuge briefly to collect the reaction mix to the bottom of the tube. After the reaction mix is ​​prepared, place it in an ice box or a 4°C refrigerator for use. The amplification system is shown in Table 2, and the one-round PCR multiple amplification reaction program is shown in Tables 3 and 4. Among them, Amplicon-primer Mix1 is a mixture of primers for each SNP site in Table 1 in equal proportions, and GSTM1-Primer Mix2 is a mixture of GSTM1 primers, internal reference quality control primers, GSTM1 probes, and internal reference quality control probes in equal proportions.

[0037] Table 2: One-round PCR multiplex amplification system

[0038] Table 3: Amplicon-amplification procedures

[0039] Table 4: GSTM1-amplification procedure

[0040] 4. After the PCR reaction is completed, use VAHTS DNA Clean Beads (Cat. No. N411-02, Nanjing Novozyme Biotechnology Co., Ltd.) to purify the amplified product of the primer system Amplicon-primer Mix1 by magnetic beads; the amplified product of the primer system GSTM1-Primer Mix2 is subjected to capillary electrophoresis. The steps of magnetic bead purification are as follows: a. After the beads are equilibrated to room temperature, vortex and mix the VAHTS DNA Clean Beads.

[0041] b. Pipette 40 μL VAHTS DNA Clean Beads into the sample tube containing 20 μL ligation product and pipette gently for more than 20 times to mix thoroughly.

[0042] c. Incubate at room temperature for 5 minutes.

[0043] d. Briefly centrifuge the sample tube and place it on a magnetic rack to separate the magnetic beads and liquid. After the solution is clear (about 5 minutes), carefully remove the supernatant.

[0044] e. Keep the sample tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, incubate at room temperature for at least 30 seconds, rotate the centrifuge tube to wash the magnetic beads, and discard the supernatant; f. Keep the sample tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, incubate at room temperature for at least 30 seconds, rotate the centrifuge tube to wash the magnetic beads, and discard the supernatant; g. Centrifuge the sample tube instantaneously and place it on a magnetic rack to remove the remaining ethanol. After drying at room temperature until there is no bright reflection on the surface of the magnetic beads, remove the sample tube from the magnetic rack, add 24 μL Nuclease-Freewater to resuspend the magnetic beads, and mix well by pipetting.

[0045] h. Let stand at room temperature for 2 minutes, place on the magnetic rack again, and take 20 μL of supernatant into a new centrifuge tube.

[0046] 5. Determination of PCR product concentration The concentration of the purified PCR product was measured using Qubit 3.0, and the measurement steps are as follows.

[0047] a. Add 3 additional portions of Qubit Mix to prepare Qubit dsDNA HS Buffer and Qubit dsDNA HSReagent Concentrate based on the amount of purified product. The configuration system for each product is as follows:

[0048] b. Mix the prepared Qubit Mix by inversion, and prepare the measurement mixture of Qubit Mix and sample in the dedicated Qubit Assay Tubes according to the following ratio:

[0049] Vortex and centrifuge briefly. Store in dark place for 2 min.

[0050] c. Open Qubit 3.0 Fluorometer: Select DNA → Select dsDNA High Sensitivity → Select Standard Tube Measurement, click Run Standards → Insert Standard Tube 1, click "ReadStandard" when you see "Insert Standard 1" → Insert Standard Tube 2, click "Read Standard" when you see "Insert Standard 2". Then the interface will automatically switch to sample measurement, select the sample volume specification of 2μL, and click "Read Tube" to get the measured concentration result in ng / μL.

[0051] Example 5: Second round of PCR amplification and library construction Using the purified first-round multiplex PCR products as samples, the LAmp DNA Polymerase (Mg2+ plusbuffer) kit and library construction adapter primers (M5XX and M7XX, public sequences can be checked) were used to complete the second-round PCR library amplification. The specific steps are as follows: Prepare the PCR system reaction mix except for the first round of purified products. Prepare according to the actual PCR quantity * 110%. Mix by pipetting and centrifuge briefly to collect the reaction mix to the bottom of the tube. After the reaction mix is ​​prepared, place it in an ice box or a 4°C refrigerator for later use. The amplification system is shown in Table 5, and the reaction procedure is shown in Table 6: Table 5: Second round PCR library amplification system

[0052] Table 6: Second round PCR library construction reaction program

[0053] 1. After the library construction reaction is completed, use VAHTS DNA Clean Beads (Cat. No. N411-02, Nanjing Novozyme Biotechnology Co., Ltd.) to purify the amplified product with magnetic beads. The steps of magnetic bead purification are as follows: a. After the beads are equilibrated to room temperature, vortex and mix the VAHTS DNA Clean Beads.

[0054] b. Pipette 30 μL VAHTS DNA Clean Beads into the sample tube containing 20 μL ligation product and pipette gently for more than 20 times to mix thoroughly.

[0055] c. Incubate at room temperature for 5 minutes.

[0056] d. Briefly centrifuge the sample tube and place it on a magnetic rack to separate the magnetic beads and liquid. After the solution is clear (about 5 minutes), carefully remove the supernatant.

[0057] e. Keep the sample tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, incubate at room temperature for at least 30 seconds, rotate the centrifuge tube to wash the magnetic beads, and discard the supernatant; f. Keep the sample tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, incubate at room temperature for at least 30 seconds, rotate the centrifuge tube to wash the magnetic beads, and discard the supernatant; g. Centrifuge the sample tube instantaneously and place it on a magnetic rack to remove the remaining ethanol. After drying at room temperature until there is no bright reflection on the surface of the magnetic beads, remove the sample tube from the magnetic rack, add 24 μL Nuclease-Freewater to resuspend the magnetic beads, and mix well by pipetting.

[0058] h. Let stand at room temperature for 2 minutes, place on the magnetic rack again, and take 20 μL of supernatant into a new centrifuge tube.

[0059] i. The purified library was measured for concentration using Qubit 3.0 and sequenced.

[0060] Example 6: GSTM1-capillary electrophoresis The purpose of GSTM1-capillary electrophoresis is to detect the PCR product of the first round of GSTM1 primer amplification in Example 4, and to determine whether GSMT is homozygous deletion based on whether the product has a peak, and then VOD risk assessment. The specific operation steps are as follows: a. Mix 3 μL of GS500-LIZ internal standard and 7 μL of HIDI, shake and mix thoroughly, and centrifuge briefly.

[0061] b. Take 8 μL of the above mixture and 1 μL of PCR product, mix them, add them to a 96-well plate, cover with sealing film, shake and mix, and centrifuge briefly.

[0062] c. Denature the mixed product and run the PCR at 95°C for 3 minutes, then quickly cool it in a -20°C refrigerator.

[0063] d. Capillary electrophoresis was performed on 3730xl using the fragment analysis protocol.

[0064] Genemapper3.2 was used to analyze the capillary electrophoresis results. The blue and green peak heights were both greater than 500, and the blue-green peak height ratio was between 0.5 and 1.5. There were no obvious product peaks at other positions, and the negative control (NTC) had no obvious peak. Figure 1 shown.

[0065] Example 7: Multiplex library data analysis The sequencing instrument Novasek was used, the sequencing strategy was 150bp (PE150) at both ends, and 1G of sequencing data was obtained for each sample. After sequencing, the qualified sequencing data (quality control standard: Q30>85%, minimum number of reads of the site>500, and heterozygous allele ratio 35%-65%) were subjected to bioinformatics analysis. Figure 2 The specific analysis process is as follows: 1) Perform quality control on sequencing data using fastp software to remove low-quality sequences; 2) Use BWA alignment software to map the quality-controlled reads back to the genome; 3) Use samtools to count the coverage of the genotypes of the positive and negative strands mapped to the candidate genomic sites; 4) Integrate the coverage information of the positive and negative strands to calculate the genotype of the candidate site and whether imbalance occurs; 5) Compare with the known genotype risk database to determine whether the current genotype is a risk genotype; After data analysis, the output is shown in Table 7 below, which gives the gene and the corresponding RS number, and evaluates the genotype risk. The genotypes of the three SNP sites of TPMT and GSTA1 genes jointly determine the VOD risk.

[0066] Table 7: Data output

[0067] Example 8: Detection accuracy verification (1) According to Examples 3, 4, 5, 6, and 7, 20 patients were selected to complete high-throughput sequencing testing of the risk of graft-versus-host disease in children.

[0068] (2) First-generation sequencing technology is still considered the gold standard for genetic testing due to its high accuracy and low false positive rate. The genotype of 17 loci was tested on the selected 20 samples by first-generation sequencing. The GSTM1 gene test result was no longer included in the test because it was a fragment analysis.

[0069] (3) The results showed that the consistency rate of the results of all sites of the 20 patients under the two detection systems reached 100%. The statistical comparison results are shown in Table 8. Figure 3-4 shown.

[0070] Table 8: Comparative analysis of first-generation sequencing and high-throughput sequencing results

[0071] By comparing the results of the first-generation sequencing method with the high-throughput sequencing method, it can be seen that the results given by the bioinformatics analysis process are correct, the clinical test results are credible, the tests are highly consistent, and the accuracy is 100%.

[0072] Example 9 Post-transplant patient testing According to Examples 3, 4, 5, 6, and 7, 78 patients were selected to complete the prediction of the risk of graft-versus-host disease in children. According to the high and low risks corresponding to the specific genotypes of the 17 SNP sites + GSTM1 gene, the number of high-risk genotypes in each patient was calculated, and the threshold of the number of high-risk genotypes for GVHD was analyzed. The specific results are shown in Tables 9 and 10 below.

[0073] Table 9: Analysis of the relationship between GVHD and 12 genes with 18 loci

[0074] Compared with patients who did not develop GVHD after transplantation (as shown in Table 7), the OR values ​​of rs1801133, rs1801131, rs4693608 and rs4364254 in 12 genes and 18 loci in patients who developed GVHD after transplantation were increased (OR>3.00), suggesting that rs1801133, rs1801131, rs4693608 and rs4364254 are more reliable as genetic risk diagnostic indicators for the occurrence of clinical GVHD.

[0075] Table 10: Analysis of the relationship between GVHD and high-risk SNP sites

[0076] In summary, the embodiment of the present application tested 78 clinical samples, and the results showed that 18 loci of 12 genes including TPMT, UGT2B10, BHLHE22, KIAA1715, GSTA1, GSTM1, CTH, MTHFR, CYP2B6, HFE, CPS and HPSE can be used as genetic molecular indicators for diagnosing and predicting graft-versus-host disease in children. It is simple and easy to operate and has high clinical application value.

[0077] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

[0078] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A gene marker for predicting graft-versus-host disease in children, characterized in that: The gene markers are a combination of TPMT gene, UGT2B10 gene, BHLHE22 gene, KIAA1715 gene, GSTA1 gene, GSTM1 gene, CTH gene, MTHFR gene, CYP2B6 gene, HFE gene, CPS1 gene and HPSE gene.

2. The gene marker for predicting graft-versus-host disease in children according to claim 1, characterized in that: The SNP sites of the TPMT gene are rs1800462, rs1800460 and rs1142345; the SNP site of the UGT2B10 gene is rs17146905; the SNP site of the BHLHE22 gene is rs16931326; the SNP site of the KIAA1715 gene is rs2289971; the SNP sites of the GSTA1 gene are rs3957356, rs11964968 and rs589 12740; the SNP site of the CTH gene is rs1021737; the SNP site of the MTHFR gene is rs1801133 and rs1801131; the SNP site of the CYP2B6 gene is rs3745274; the SNP site of the HFE gene is rs1800562; the SNP site of the CPS1 gene is rs1047891; the SNP site of the HPSE gene is rs4693608 and rs4364254.

3. Use of the gene marker for predicting graft-versus-host disease in children and / or its detection reagent according to claim 1 in preparing a product for detecting graft-versus-host disease in children.

4. Primers and probes for predicting graft-versus-host disease in children, characterized in that: Including SNP site primers, GSTM1 primers, internal reference quality control primers, GSTM1 probes and internal reference quality control probes; The SNP site primers are designed based on the SNP sites of TPMT gene, UGT2B10 gene, BHLHE22 gene, KIAA1715 gene, GSTA1 gene, CTH gene, MTHFR gene, CYP2B6 gene, HFE gene, CPS1 gene and HPSE gene, and the primer sequences are shown in SEQ ID NOs. 1 to 34; The GSTM1 primers are designed based on the Indel site of the GSTM1 gene, and the primer sequences are shown in SEQ ID NOs. 35 to 36; The internal reference quality control primers are designed based on β-globulin, and the sequences are shown in SEQ ID NOs. 37-38; The GSTM1 probe is designed based on the GSTM1 gene, and the probe sequence is shown in SEQ ID NO.39; The internal reference quality control probe is designed based on the β-globulin, and the probe sequence is shown in SEQ ID NO.

40.

5. A kit for predicting graft-versus-host disease in children, characterized in that: The kit comprises the detection reagent for the gene marker for predicting graft-versus-host disease in children according to claim 1.

6. The kit for predicting graft-versus-host disease in children according to claim 5, characterized in that: The kit comprises the primers and probes for predicting graft-versus-host disease in children as described in claim 4.

7. A method for non-diagnostic use of a kit for predicting graft-versus-host disease in children, characterized in that: include: Using the kit for predicting graft-versus-host disease in children as described in claim 5, a round of PCR amplification is performed on the genomic DNA of the peripheral blood sample to be tested to obtain a SNP site amplification product and a GSTM1 amplification product; After the SNP site amplification product is purified, a second round of PCR library amplification is performed using a library construction adapter primer. After purification, the obtained library product is sequenced on a machine to obtain sequencing data; After quality control of the sequencing data, the data is posted back to the genome, and the coverage information of the genotypes of the positive and negative chains of the candidate sites of the genome are statistically compared, so as to calculate the genotype of the candidate site and whether imbalance occurs based on the coverage information, and determine whether the current genotype is a risk genotype according to a preset genotype risk database; The GSTM1 amplification product is subjected to capillary electrophoresis to determine whether GSTM1 is homozygous deleted.

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