Detection material for single nucleotide polymorphism of IL23R gene related to risk of non-traumatic femoral head necrosis and its application

Abstract

The present invention discloses a detection material for single nucleotide polymorphism of IL23R gene related to the risk of non-traumatic femoral head necrosis and its application, and the specific single nucleotide polymorphism sites involved are rs4655686, rs6683039, rs11465754, rs1884444, rs1569922, and rs6693831. The present invention firstly describes the association between the above 6SNPs and the risk of non-traumatic femoral head necrosis, which has great value for molecular early warning and molecular level prevention and treatment of non-traumatic femoral head necrosis.

Description

[0001] This application is a Chinese patent Single nucleotide polymorphism of IL23R gene associated with risk of non-traumatic femoral head necrosis Stability detection substances and their applications "The divisional application of the original patent is 2020109056370 , application date is 2020- 09-01 , the applicant is Jilin University , the application publication number is CN111961719A. Technical Field

[0002] The present invention belongs to the field of biotechnology, and specifically relates to a substance for detecting single nucleotide polymorphism of IL23R gene related to the risk of non-traumatic femoral head necrosis, and further using the substance for molecular early warning, drug treatment target and clinical molecular diagnosis and typing of non-traumatic femoral head necrosis. Background Art

[0003] Osteonecrosis of the femoral head (ONFH) is a complex disease caused by the combined effects of genetic and environmental factors. In the past 10 years, the incidence of ONFH has shown an increasing trend year by year. It is estimated that an average of 20,000 to 30,000 patients are newly diagnosed with ONFH in the United States each year, and 150,000 to 200,000 new cases of ONFH are reported in China each year. In addition, there are currently 8.12 million adult ONFH patients in China, and the main disease group is young and middle-aged people between the ages of 40 and 50. The high disability rate leads to serious social and family economic burdens, which is one of the main challenges facing human health. Early detection of ONFH genetic molecular diagnostic markers and subsequent implementation of early molecular warning to advance the prevention and treatment period are the key to reducing the incidence of ONFH.

[0004] In recent years, the important research progress of the molecular pathogenesis of ONFH is that multiple series of gene polymorphisms are related to the risk of ONFH. They either form linkage disequilibrium with other genes to jointly affect the onset of ONFH, or affect the expression function of other susceptible genes, and play a synergistic role in the development of ONFH. These genetic molecular etiology research results clearly indicate that ONFH belongs to a complex disease in which multiple micro-effect genes are jointly involved and induced by environmental factors. However, the main bottleneck problem in the field of gene polymorphism research on ONFH at home and abroad at present is that there are many data on scattered gene loci, and few data on systematic research of multi-gene loci, which is not conducive to the elaboration of the molecular etiology and molecular pathogenesis of multiple micro-effect genes. These bottleneck problems are serious constraints in elaborating the molecular pathogenesis of ONFH, and are also the key obstacles to the docking of the latest achievements of the molecular etiology of ONFH and the implementation of clinical prevention and treatment. The present invention starts with solving this fundamental problem and finds the important application value of the IL23R gene 6SNPs combination in molecular early warning, molecular diagnosis and therapeutic drug targets for non-traumatic femoral head necrosis.

[0005] The IL23R gene is located at chromosome 1p31.3 and contains 13 exons. The gene encodes a subunit of the protein IL23A / IL23 receptor. This protein pairs with the receptor molecule IL12RB1 / IL12Rbeta1, both of which are required for IL23A signal transduction. The protein constitutively binds to Janus kinase 2 (JAK2) and binds to the transcriptional activator STAT3 in a ligand-dependent manner.

[0006] IL23A is a heterodimeric protein composed of two subunits: IL-23R and IL-12Rb1. IL-23R is expressed on activated T cells, dendritic cells, macrophages, and natural killer (NK) cells, and binds specifically to IL-23, which is produced primarily by dendritic cells and activated macrophages from peripheral tissues. IL23R is a proinflammatory cytokine that inhibits osteoclastogenesis through receptor activator of nuclear factor NFκB ligand and affects T cells. In the Korean population, IL23R gene polymorphisms were found to be associated with the expression of this gene in patients with ONFH. Among them, IL23R gene polymorphisms were particularly associated with the risk of idiopathic ONFH, but not with alcohol- and hormone-induced ONFH. Studies on the Chinese population have confirmed that the rs6693831 polymorphism of the IL23R gene is associated with a reduced risk of ONFH caused by drinking in Chinese men. Alcoholism affects fat metabolism and osteocyte stress and is an important risk factor for osteonecrosis.

[0007] ONFH is associated with a variety of factors, such as hormone abuse, alcoholism, infection, bone marrow infiltrating diseases, coagulation defects and some autoimmune diseases, etc. In particular, some autoimmune diseases, including systemic lupus erythematosus (SLE), polymyalgia rheumatica (PMR), rheumatoid arthritis (RA) and inflammatory bowel disease (IBD), are all related to the occurrence of osteonecrosis.

[0008] IL23 is a proinflammatory cytokine composed of the p19 subunit and p40 subunit of IL12. IL23R pairs with IL12R, and both are required for the IL23 signaling pathway. IL23 regulates the activity of immune responses and promotes inflammatory responses through the participation of IL23R. These cytokines are mainly expressed in cells of the immune system, such as T cells, macrophages, and dendritic cells. IL23-deficient mice are resistant to collagen-induced arthritis, and IL23 also inhibits osteoclastogenesis by inhibiting RANK through T cell activation. Many studies in recent years have shown that IL23R is associated with inflammatory diseases. Some variations in the IL23R gene are associated with the risk of skin diseases such as psoriasis. In addition, in a previously published genome-wide association study, IL23R gene polymorphisms were associated with susceptibility to IBD and Crohn's disease (CD). Summary of the invention

[0009] The present invention determines that the single nucleotide polymorphism sites of the IL23R gene or a combination of sites with interactions are associated with the susceptibility to ONFH, and compares the genotypes and allele frequencies of ONFH patients and the control group. The innovative integration of combined application of molecular technology is the first to explain the association between the 6SNPs genotyping, allele frequencies, and combinations of genes of the IL23R gene and the risk of ONFH at home and abroad, and proposes key molecular targets for establishing molecular-level prevention and treatment strategies for ONFH. The present invention is used for screening and early intervention of susceptible populations of ONFH, and promotes the early connection between the research results of molecular etiology and the clinical prevention and treatment of ONFH.

[0010] One object of the present invention is to detect a substance for single nucleotide polymorphism of IL23R gene related to the risk of non-traumatic femoral head necrosis, characterized in that:

[0011] Use of the substance in diagnosing or assisting in diagnosing non-traumatic femoral head necrosis, or in preparing a product for diagnosing or assisting in diagnosing the risk of non-traumatic femoral head necrosis;

[0012] Or the use of the substance in evaluating or assisting in evaluating non-traumatic femoral head necrosis, or in preparing a product for evaluating or assisting in evaluating the risk of non-traumatic femoral head necrosis;

[0013] The combination of sites with interactions related to non-traumatic femoral head necrosis is any one or more of the following (1) to (6):

[0014] (1) The combination of rs6683039 and rs11465754;

[0015] (2) the combination of rs6683039 and rs1884444;

[0016] (3) the combination of rs6683039 and rs1569922;

[0017] (4) Combination of rs11465754 and rs1569922

[0018] (5) the combination of rs1884444 and rs6693831;

[0019] (6) The combination of rs4655686 and rs6693831.

[0020] Further, the substance is a product for preparing a diagnosis or auxiliary diagnosis of the risk of non-traumatic femoral head necrosis, or the substance is a product for preparing an evaluation or auxiliary evaluation of the risk of non-traumatic femoral head necrosis, and the product includes a readable carrier recording at least one of the following conditions A to I:

[0021] A. The risk of non-traumatic femoral head necrosis in the tested person who carries both the minimum homozygous genotype CC of the rs6683039 locus and the minimum homozygous genotype GG of rs11465754 is reduced;

[0022] B. The risk of non-traumatic femoral head necrosis in the tested person who carries both the rs6683039 minimum homozygous genotype CC and the rs1884444 minimum homozygous genotype GG is reduced;

[0023] C. The risk of non-traumatic femoral head necrosis in the tested person who carries both the rs6683039 minimum homozygous genotype CC and the rs1569922 minimum homozygous genotype TT is reduced;

[0024] D. The risk of non-traumatic femoral head necrosis in the tested person who carries both the minimum homozygous genotype GG of the rs11465754 locus and the minimum homozygous genotype TT of rs1569922 is reduced;

[0025] E. The risk of non-traumatic femoral head necrosis in the tested person who carries both the rs1884444 minimum homozygous genotype GG and the rs6693831 minimum homozygous genotype TT is reduced;

[0026] F. The risk of non-traumatic femoral head necrosis in the tested person who carries both the rs4655686 minimum homozygous genotype AA and the rs6693831 minimum homozygous genotype TT is reduced.

[0027] Furthermore, the substance is a reagent for direct sequencing; or a reagent for combining polymerase chain reaction with restriction fragment length polymorphism analysis; or a reagent for combining polymerase chain reaction with direct sequencing; or a reagent for any of the following SNP typing methods: a hybridization-based method, a primer extension-based method, a conformation-based method, or a high-resolution melting curve analysis technique.

[0028] Furthermore, the material used to detect the single nucleotide polymorphism at the rs4655686 site is specifically a primer pair A or a set of single-stranded DNA molecules A; the primer pair A consists of a single-stranded DNA molecule as shown in SEQ ID NO.1 and a single-stranded DNA molecule as shown in SEQ ID NO.2; the set of single-stranded DNA molecules A consists of a single-stranded DNA as shown in SEQ ID NO.1, a single-stranded DNA as shown in SEQ ID NO.2, and a single-stranded DNA as shown in SEQ ID NO.3.

[0029] Furthermore, the substance used to detect the single nucleotide polymorphism at the rs6683039 site is specifically a primer pair B or a set of single-stranded DNA molecules B; the primer pair B consists of a single-stranded DNA molecule as shown in SEQ ID NO.4 and a single-stranded DNA molecule as shown in SEQ ID NO.5; the set of single-stranded DNA molecules B consists of a single-stranded DNA as shown in SEQ ID NO.4, a single-stranded DNA as shown in SEQ ID NO.5, and a single-stranded DNA as shown in SEQ ID NO.6.

[0030] Furthermore, the substance used to detect the single nucleotide gene polymorphism at the rs11465754 site is a primer pair C or a set of single-stranded DNA molecules C; the primer pair C consists of a single-stranded DNA molecule as shown in SEQ ID NO.7 and a single-stranded DNA molecule as shown in SEQ ID NO.8; the set of single-stranded DNA molecules C consists of a single-stranded DNA as shown in SEQ ID NO.7, a single-stranded DNA as shown in SEQ ID NO.8, and a single-stranded DNA as shown in SEQ ID NO.9.

[0031] Furthermore, the substance used to detect the single nucleotide gene polymorphism at the rs1884444 site is a primer pair D or a set of single-stranded DNA molecules D; the primer pair D is composed of a single-stranded DNA molecule as shown in SEQ ID NO.10 and a single-stranded DNA molecule as shown in SEQ ID NO.11; the set of single-stranded DNA molecules D is composed of a single-stranded DNA as shown in SEQ ID NO.10, a single-stranded DNA as shown in SEQ ID NO.11 and a single-stranded DNA as shown in SEQ ID NO.12.

[0032] Furthermore, the substance used to detect the single nucleotide gene polymorphism at the rs1569922 site is a primer pair E or a set of single-stranded DNA molecules E; the primer pair E is composed of a single-stranded DNA molecule as shown in SEQ ID NO.13 and a single-stranded DNA molecule as shown in SEQ ID NO.14; the set of single-stranded DNA molecules D is composed of a single-stranded DNA as shown in SEQ ID NO.13, a single-stranded DNA as shown in SEQ ID NO.14, and a single-stranded DNA as shown in SEQ ID NO.15.

[0033] Furthermore, the substance used to detect the single nucleotide gene polymorphism at the rs6693831 site is a primer pair or a set of single-stranded DNA molecules; the primer pair consists of a single-stranded DNA molecule as shown in SEQ ID NO.13 and a single-stranded DNA molecule as shown in SEQ ID NO.16; the set of single-stranded DNA molecules consists of a single-stranded DNA as shown in SEQ ID NO.17, a single-stranded DNA as shown in SEQ ID NO.14, and a single-stranded DNA as shown in SEQ ID NO.18.

[0034] Further, the rs4655686 site is the 67172321st nucleotide from the 5' end of chromosome 1 of the human genome; the nucleotide at the site is T or A. The rs6683039 site is the 67165650th nucleotide from the 5' end of chromosome 1 of the human genome; the nucleotide at the rs6683039 site is C or T. The rs11465754 site is the 33259408th nucleotide from the 5' end of chromosome 1 of the human genome; the nucleotide at the rs11465754 site is A or G. The rs1884444 site is the 67168129th nucleotide from the 5' end of chromosome 1 of the human genome; the nucleotide at the rs1884444 site is G or T. The rs1569922 site is the 67199280th nucleotide from the 5' end of chromosome 1 of the human genome; the nucleotide at the rs1569922 site is T or C. The rs6693831 site is the 1st nucleotide from the 5' end of chromosome 1 of the human genome; the nucleotide at the rs6693831 site is T or C.

[0035] The present invention also provides a kit comprising the substance for detecting single nucleotide polymorphisms of at least one combination of sites (1) to (6) associated with the risk of non-traumatic femoral head necrosis.

[0036] The present invention also provides a targeted therapeutic drug, which is a drug that uses any one or more of the site combinations (1) to (6) associated with non-traumatic femoral head necrosis as target genes.

[0037] The present invention discloses the application of a marker combination of rs4655686, rs6683039, rs11465754, rs1884444, rs1569922 and rs6693831 of the IL23R gene single nucleotide polymorphism sites in detecting the risk of non-traumatic femoral head necrosis, and further used for ONFH molecular early warning, clinical molecular diagnosis and typing, and drug treatment targets.

[0038] The substance provided by the present invention is a product for detecting rs4655686, rs6683039, rs11465754, rs1884444, rs1569922, rs6693831 loci and their combinations in the risk of ONFH and molecular warning, clinical molecular diagnosis and typing, or the application and product thereof as a target for ONFH drug treatment.

[0039] The inventors of the present invention conducted a clinical case-control study on ONFH in the Han Chinese population and found that the minimum homozygous genotype AA and the minimum allele A frequency at the rs4655686 single nucleotide polymorphism site of the IL23R gene were significantly associated with a reduced risk of ONFH; the genetic recessive model was also significantly associated with a reduced risk of ONFH, and could be used as a molecular protective marker for reducing the risk of ONFH. It was also found that the interaction between rs6683039 and rs11465754 was significantly associated with reducing the risk of ONFH; the interaction between rs6683039 and rs1884444 was significantly associated with reducing the risk of ONFH; the interaction between rs6683039 and rs1884444 was significantly associated with reducing the risk of ONFH; the interaction between rs11455754 and rs1569922 was significantly associated with reducing the risk of ONFH; the interaction between rs18844444 and rs6693831 was significantly associated with reducing the risk of ONFH; and the interaction between rs4655686 and rs6693831 was significantly associated with reducing the risk of ONFH.

[0040] The present invention describes for the first time the association between the above 7SNPs of the IL23R gene and the risk of ONFH, which is of great value for molecular early warning and molecular level prevention and treatment of ONFH. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the flow chart of Massarray SNP typing experiment. DETAILED DESCRIPTION

[0042] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0043] Unless otherwise specified, the experimental materials and reagents used in the following examples can be purchased through commercial channels.

[0044] Example 1: Application of IL23R gene in molecular early warning, molecular diagnosis and therapeutic drug target of non-traumatic femoral head necrosis.

[0045] 1. Establishment of ONFH case-control study system

[0046] Based on informed consent, the ONFH case-control study system included 600 cases, including 300 healthy controls and 300 ONFH patients.

[0047] ONFH cases were selected from inpatients who visited the Department of Orthopedics of the Second and Third Clinical Medical Colleges of Jilin University from June 2012 to October 2018. There were 204 males and 96 females with an age of 54.49±11.8 years. Obvious history of hip trauma, congenital hip diseases, hip infectious diseases, and hip tumors were excluded, and the clinical diagnosis and staging of ONFH were performed according to the Ficat diagnosis and staging criteria.

[0048] The healthy control group was selected from healthy subjects who underwent physical examinations at the Physical Examination Center of the Second Clinical Medical College of Jilin University from November 2013 to March 2014. There were 131 males and 169 females, aged 56.5±8.61 years. Their fasting blood glucose, serum triglycerides and total cholesterol were all within the reference value levels of the normal population. There were no abnormalities in the abdominal organ ultrasound examination and chest X-ray film, and the history of major diseases such as cardiovascular and cerebrovascular diseases was excluded. The above case-control study system was all Chinese Han people, and there was no blood relationship between the individuals.

[0049] 2. Extraction of Genomic DNA from Peripheral Blood Leukocytes

[0050] 2 ml of cubital venous blood was collected in the fasting state, and the whole blood genomic DNA extraction kit was used as described in the instructions. The main steps were as follows: 2 ml of anticoagulated blood was centrifuged at 2000 rpm for 10 min, and plasma was stored at -80 °C; 1 ml of red blood cell lysis solution A was added to the remaining cells and gently inverted to mix; centrifuged at 10000 g for 2 min at room temperature, and the supernatant was discarded; the precipitate was resuspended in 500 μl of red blood cell lysis solution A, and after gently blowing and mixing, centrifuged at 10000 g for min at room temperature, and the supernatant was discarded; 0.6 mL of solution A was added, and gently blown and mixed, and the mixture was allowed to stand at room temperature for 5 min ; Add 0.2mL chloroform and 0.3mL solution B, and immediately invert to mix; centrifuge at 13000g for 3min at room temperature, transfer the supernatant to a new 1.5mL plastic centrifuge tube; add 0.7ml isopropanol, invert and mix 5-8 times, and a flocculent DNA precipitate will appear; add 1mL 75% ethanol, centrifuge at 13000g for min, discard the supernatant and evaporate the ethanol in the air, add 0.2ml Tris-EDTA to dissolve the DNA; take 1μl to extract DNA, and use Nanodrop2000 to detect the DNA content and purity; package the DNA and store it at -80℃.

[0051] 3. Optimization and screening of target genes and their SNPs

[0052] The Hapmap database and related literature were used to query the SNPs of the IL23R gene. The distribution of each SNP site in different countries, ethnic groups, and regional populations was compared through multiple bioinformation databases, especially the distribution data of the Asian population. The distribution of gene polymorphisms conformed to the Hardway equilibrium. Six SNPs were selected as target sites for the study in the gene coding region, promoter region, and intron. The selected target gene SNP sites are shown in Table 1.

[0053] Table 1

[0054]

[0055] The sequence information of 11 SNPs of CREBPA, PPARg and CREBBP genes is shown in Table 2.

[0056] Table 2

[0057]

[0058] 4. Massarray SNP typing experimental process, see Figure 1 .

[0059] (I) Primer design, synthesis and dilution 1. Obtaining gene sequences: ① Register as a user on the Myagena website; ② Enter the name of the SNP site on the NCBI website (http: / / www.ncbi.nlm.nih.gov / projects / SNP / ) and display it in the dbSNPbatchreportor format; ③ Send the sequence of the SNP site to the email address registered on the Myagena website; ④ Select Genotyping in the TOOLS toolbar of the Myagena website; ⑤ Click RSformat and select the file sent to the email address by the NCBI website in the Browse button; ⑥ After the website completes formatting the sequence, select ProxSNP in the Sentto column; ⑦ Start ProxSNP and click BeginStart; ⑧ After completing the above steps, select PreXTEND in the Sentto column; ⑨ Start ProxSNP and click BeginStart; ⑩ In the generated results, select OUTPUT and copy the file content to a new text file .txt. 2. AssayDesigner3.1 software was used to design primers for PCR reaction and single-base extension primers, and the primers were submitted to a biological company for synthesis: ① Select the Browse button in the SNP Group column of the software to find the txt file generated above; ② Select SBEMassExtend in the AssayDesign column, and select iPlex in the SBEstopmix column, and select different reaction multiplicities in MμltiplexLevel according to actual conditions; ③ Select the default parameters for SNPcapture, Extendprimerdesign, and MASSMμltiplexing; ④ After setting the parameters, click Run; ⑤ Find the generated primer sequence file in the corresponding position of the txt file directory. The designed primer sequence is shown in Table 3.

[0060] Table 3

[0061]

[0062] 3. Primer dilution: ①PCRmastermix primer configuration, dilute the single tube PCRmaster to a concentration of 100μM, add deionized water to mix all single tube PCRmasters so that the final reaction PCRmastermix concentration is 0.5μM; ②EXTENDMix primer configuration: dilute the single tube extension primer to a final concentration of 500μM, add primers and mix so that the concentrations of each primer are 8μM, 10μM, and 15μM. Calculate the molecular weight, mass number, and molar number of the primer according to the instructions for use of the DNA synthesis product, and then calculate the amount of deionized water to be added according to the required concentration. ② Take 1 times (less than 6300Da), 1.2 times (6300Da to 7200Da), and 1.5 times (greater than 7200Da) of the mixed single tube extension primer according to the molecular weight and mix them for use. (II) AgenaMassArray system genotyping steps; Typing principle: Amplify the target fragment containing the SNP site to be tested through PCR reaction, then use SAP enzyme to remove the remaining deoxyribonucleoside triphosphate (dNTP) and primers in the PCR system, and then add a single base extension primer, whose 3' terminal base is close to the SNP site and is completely complementary to the base on the target fragment. Four ddNTPs are used to replace dNTPs. In this way, the probe only extends one base at the SNP site, and the connected ddNTP corresponds to the allele of the SNP site. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) is used to detect the molecular weight difference between the extension product and the unextended primer to determine the base at this point. Sequencing primer sequence table related information table 3. 1. PCR amplification reaction: ① Prepare PCR master mix in 1.5 ml EP tube and centrifuge at low speed with oscillation. The reaction components are shown in Table 4 below; ② Use an 8-channel pipette to add 4 μl PCR master mix to each well of the 384-well plate, and finally add 1 μl template DNA (20 ng / μl) to mix well, carefully cover the 384-well sealing film, and press each well firmly to prevent evaporation during the PCR program. Centrifuge at 1000 rpm for 1 minute; ③ Set the following PCR amplification reaction program: 94℃5min; 94℃20sec, 56℃30sec, 72℃1min, 45cycles; 72℃3min; 4℃∞. Place the PCR reaction plate on the PCR instrument and start the program.

[0063] Table 4

[0064]

[0065] 2. Alkaline phosphatase treatment of the product:

[0066] ①After the PCR reaction is completed, the PCR product is treated with SAP (shrimpalkalinephosphatase) to remove free dNTPs in the system; ②Prepare the alkaline phosphatase treatment reaction solution in a new 1.5ml EP tube. The SAP Mix reaction components are shown in Table 5 below; ③Add SAPmix to the 384-well PCR reaction plate. For each alkaline phosphatase treatment reaction well, the total reaction volume is 7μl, including 5μl PCR product and 2μl SAPmix; ④After the transfer is completed, carefully cover the 384-well sealing film and press each well to prevent evaporation during the PCR program. After centrifugation, perform the following reaction program; ⑤Set the SAP reaction program: 37℃20min; 85℃5min; 4℃∞. Place the 384-well reaction plate on the PCR instrument and start the program.

[0067] Table 5

[0068] SAP mix of Reagent Concentration Volume(1rxn) Water(HPLC grade) NA 1.53μl SAPBuffer 10x 0.17μl SAPEnzyme 1U / μl 0.30μl Total volume - 2.00μl

[0069] 3. Single base extension reaction: ① After the alkaline phosphatase treatment, perform a single base extension reaction with a total reaction volume of 9μl; ② Prepare a single base extension reaction solution in a new 1.5ml EP tube, and the EXTENDMix reaction components are shown in Table 6 below; ③ Add EXTENDMix to the 384-well reaction plate accordingly. For each reaction well, the single base extension reaction system EXTENDMix 2μl; SAP+PCRreaction 7μl, TotalVolume 9μ; ④ After the transfer is completed, carefully cover the 384-well sealing film and press each well to prevent evaporation during the PCR program. After centrifugation, perform the following reaction program: 94℃30sec; [94℃5sec, (52℃5sec, 80℃5sec)5cycles]40cycles; 4℃∞.

[0070] Table 6

[0071] EXTENDMixofReagent Conc.in 9μl Volume(1rxm) Water(HPLC grade) NA 0.619μl iPLEXBufferPlus 0.222x 0.200μl iPLEXTerminationmix 1x 0.200μl PrimerMix (7μM:14μM) 0.625uM:1.25uM 0.940μl iPLEXEnzyme 1x 0.041μl

[0072] 4. Resin purification: ① Fill the 384 / 6MG Dimple plate evenly with resin and leave it for 10 minutes to dry; ② Add 16μl of water to each well of the 384 sample plate; ③ Gently turn the 384 sample plate over and place it on the Dimple plate, then tap it to make the resin fall into each well of the sample plate; ④ Place the 384 sample plate in a flip centrifuge and rotate at room temperature for 30 minutes to mix.

[0073] 5. Chip spotting: Start the MassARRAY Nanodispenser RS1000 spotter and transfer the resin-purified extension product to the 384-well SpectroCHIP bioarray.

[0074] 6. Mass spectrometry detection and data output: The spotted SpectroCHIP chip is analyzed using a MALDI-TOF mass spectrometer. The detection results are obtained using TYPER4.0 software to obtain the original data and genotyping diagram. The integrity and correctness of the data file are checked, and the results are saved in the corresponding storage medium and submitted to the bioinformatics room for analysis.

[0075] (iii) statistical analysis;

[0076] Genotype frequencies and allele frequencies were calculated, Hardy-Weinberg equilibrium and MAF were tested, and the genotype and allele distributions between the case and control groups were compared by Person's chi-square test.

[0077] Single SNP association analysis was performed by PLINK chi-square test to analyze the differences in genotype and allele at each locus between the ONFH group and the disease group to find loci associated with the disease.

[0078] The correlation between the pairwise interactions of multiple SNPs and the risk of ONFH was completed through PLINK gene interaction (epistasis) analysis.

[0079] V. Experimental Results Analysis

[0080] (I) Correlation between 6SNPs genotyping of IL23R gene and the risk of ONFH in the ONFH group and the control group. The specific distribution of 6SNPs genotypes in the ONFH group and the control group is shown in Table 7.

[0081] The PLINK chi-square analysis of the sequencing results found that the frequency of the minimum homozygous genotype AA (Minorgenotype) at the rs4655686 (A / T) site of the IL23R gene in the ONFH group was significantly lower than that in the control group, as shown in Table 7, P = 5.249e-005, which means that carriers of the AA type at the rs4655686 (A / T) site of the IL23R gene significantly reduced the risk of ONFH.

[0082] Table 7

[0083]

[0084] (II) Correlation between the allele frequencies of 6 SNPs of IL23R gene and the risk of ONFH in the ONFH group and the control group. The distribution frequencies of SNP allele types in the case group and the control group are shown in Table 8.

[0085] The sequencing results showed that the frequency of the minor allele A (Minor Allele) of the IL23Rrs4655686 (A / T) locus in the ONFH group was significantly lower than that in the control group, P = 0.007019; OR (95% CI) 0.671 (0.502-0.897), that is, the frequency of the A allele of the IL23Rrs4655686 (A / T) locus significantly reduced the risk of ONFH.

[0086] Table 8

[0087]

[0088] *PLINK chi-square test.

[0089] (III) The correlation between different genetic models of IL23R gene 6SNPs and the risk of ONFH is shown in Table 9. The analysis results of different genetic models of IL23R gene 6SNPs sites further found that the recessive model (AAvsAT+TT) of IL23Rrs4655686 (A / T) site in the ONFH group was significantly lower than that in the control group, P=2.94E-056, that is, the frequency of the minimum homozygous genotype AA at the IL23Rrs4655686 (A / T) site significantly reduced the risk of ONFH.

[0090] Table 9

[0091]

[0092]

[0093] (IV) The correlation between gene-gene interaction of 6 SNPs of IL23R gene and the risk of ONFH.

[0094] The PLINK software gene interaction analysis method was used to analyze the pairwise interaction of the six SNPs: rs4655686 (A / T), rs6683039 (C / T), rs11465754 (A / G), rs1884444 (G / T), rs1569922 (T / C), and rs6693831 (T / C). The results are shown in Table 10.

[0095] The rs4655686 site is the 67172321st nucleotide from the 5' end of chromosome 1 of the human genome; the nucleotide of the site is T or A; the rs6683039 site is the 67165650th nucleotide from the 5' end of chromosome 1 of the human genome; the nucleotide of the rs6683039 site is C or T; the rs1569922 site is the 33259408th nucleotide from the 5' end of chromosome 1 of the human genome; the rs11465754 site is the 67165748th nucleotide from the 5' end of chromosome 1 of the human genome; the rs The nucleotide at the 11465754 site is A or G; the rs1884444 site is the 67168129th nucleotide from the 5' end of chromosome 1 of the human genome; the nucleotide at the rs1884444 site is G or T; the rs1569922 site is the 67199280th nucleotide from the 5' end of chromosome 1 of the human genome; the nucleotide at the rs1569922 site is T or C; the rs6693831 site is the 67255184th nucleotide from the 5' end of chromosome 1 of the human genome; the nucleotide at the rs6693831 site is C or T.

[0096] The results of analyzing gene interactions using PLINK software revealed that:

[0097] The interaction between the rs6683039 (C / T) site of the IL23R gene and the rs11465754 (A / G) site of the IL23R gene significantly reduced the risk of ONFH, OR: 0.7132, P = 0.04978. In other words, individuals who simultaneously carry the minimum homozygous genotype CC of the rs6683039 (C / T) site of the IL23R gene and the minimum homozygous genotype GG of the rs11465754 (A / G) site significantly reduce the risk of ONFH, which can serve as a molecular protective marker for reducing the risk of ONFH.

[0098] The interaction between the rs6683039 (C / T) site of the IL23R gene and the rs1884444 (G / T) site of the IL23R gene reduced the risk of ONFH, OR: 0.6878, P = 0.03934, that is, individuals carrying both the minimum homozygous genotype CC of the rs6683039 (C / T) site of the IL23R gene and the minimum homozygous genotype GG of the rs1884444 (G / T) site of the IL23R gene significantly reduced the risk of ONFH, which can be used as a molecular protective marker to reduce the risk of ONFH.

[0099] The interaction between the rs6683039 (C / T) site of the IL23R gene and the rs1569922 (T / C) site of the IL23R gene significantly reduced the risk of ONFH, OR: 0.6871, P = 0.03841, that is, individuals carrying both the minimum homozygous genotype CC of the rs6683039 (C / T) site of the IL23R gene and the minimum homozygous genotype TT of the rs1569922 (T / C) site of the IL23R gene significantly reduced the risk of ONFH, which can be used as a molecular protective marker to reduce the risk of ONFH.

[0100] The interaction between the IL23R gene rs11465754 (A / G) site and the IL23R gene rs1569922 (T / C) site significantly reduced the risk of ONFH, OR: 0.6914, P = 0.03871, that is, individuals carrying both the minimum homozygous genotype GG of the IL23R gene rs11465754 (A / G) site and the minimum homozygous genotype TT of the IL23R gene rs1569922 (T / C) site significantly reduced the risk of ONFH, which can be used as a molecular protective marker to reduce the risk of ONFH.

[0101] The interaction between the rs1884444 (G / T) site of the IL23R gene and the rs6693831 (T / C) site of the IL23R gene significantly reduced the risk of ONFH, OR: 0.6615, P = 0.04742, that is, individuals carrying both the minimum homozygous genotype GG of the rs1884444 (G / T) site of the IL23R gene and the minimum homozygous genotype TT of the rs6693831 (T / C) site of the IL23R gene significantly reduced the risk of ONFH, which can be used as a molecular protective marker to reduce the risk of ONFH.

[0102] The interaction between the rs4655686 (A / T) site of the IL23R gene and the rs6693831 (T / C) site of the IL23R gene was significantly associated with a reduced risk of ONFH. In other words, individuals who simultaneously carried the minimum homozygous genotype AA of the rs4655686 (A / T) site of the IL23R gene and the minimum homozygous genotype TT of the rs6693831 (T / C) site of the IL23R gene significantly reduced the risk of ONFH, and could serve as a molecular protective marker for reducing the risk of ONFH.

[0103] The present invention first discovered that the pairwise interactions of the 6 SNPs of the IL23R gene are significantly associated with the risk of ONFH, and these risk correlations are partially or completely different from the effects of a single SNP on the risk of ONFH. The results of the gene interactions more clearly discovered the protective effects of multiple SNPs on reducing the risk of ONFH, further verifying that ONFH is a complex disease caused by the combination of multiple micro-effect genes, and elaborating the combined effects of these molecular genetic loci is of great value for the establishment of early molecular warning and molecular prevention and control strategies for ONFH. The gene interaction, also known as the epistatic effect, refers to the effect of two pairs of non-allelic genes that affect the same trait, one pair of genes (dominant or recessive) inhibiting (or masking) the effect of another pair of dominant genes.

[0104] Table 10. Correlation between pairwise interactions between IL23R6 SNPs and the risk of ONFH

[0105]

[0106] *PLINK chi-square analysis; # PLINK gene interaction analysis

[0107] Those skilled in the art will be able to perform analysis of nucleotides present on the SNP markers disclosed herein in the nucleic acid of an individual by any method or technique for detecting SNP sites. For example, a person can use the method of the present invention to detect SNP site markers by performing Tagman method, mass spectrometry, DNA microarray method, microsequencing, hybridization, restriction enzyme analysis, oligonucleotide ligation detection, allele-specific PCR-HRM or a combination of the above methods, of course, this list is only exemplary and is in no way intended to limit the present invention. Those skilled in the art can use any suitable method to achieve this detection.

[0108] Those skilled in the art can use any suitable method to achieve such detection.

[0109] The main experimental instruments and equipment involved in the embodiments of the present invention are shown in Table 11 below.

[0110] Table 11

[0111]

[0112]

[0113] The main reagents or software involved in the embodiments of the present invention are shown in Table 12 below.

[0114]

[0115]

[0116] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

[0117] The specific implementation of the present invention described above does not constitute a limitation on the protection scope of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Use of a substance for detecting single nucleotide polymorphisms of site combinations related to non-traumatic femoral head necrosis in the preparation of a product for diagnosing or assisting in diagnosing the risk of a subject suffering from non-traumatic femoral head necrosis, or in the preparation of a product for evaluating or assisting in evaluating the risk of a subject suffering from non-traumatic femoral head necrosis; The site combination related to non-traumatic femoral head necrosis is any one or more of the following (1) to (5): (1) The combination of rs6683039 and rs11465754; (2) The combination of rs6683039 and rs1884444; (3) The combination of rs6683039 and rs1569922; (4) Combination of rs11465754 and rs1569922 (5) The combination of rs1884444 and rs6693831.

2. The use according to claim 1, characterized in that: The product includes a readable carrier recording at least one of the following conditions A to E: A. The risk of non-traumatic femoral head necrosis in the tested person who carries both the minimum homozygous genotype CC of the rs6683039 locus and the minimum homozygous genotype GG of rs11465754 is reduced; B. The risk of non-traumatic femoral head necrosis in the tested person who carries both the rs6683039 minimum homozygous genotype CC and the rs1884444 minimum homozygous genotype GG is reduced; C. The risk of non-traumatic femoral head necrosis in the tested person who carries both the rs6683039 minimum homozygous genotype CC and the rs1569922 minimum homozygous genotype TT is reduced; D. The risk of non-traumatic femoral head necrosis in the tested person who carries both the minimum homozygous genotype GG of the rs11465754 locus and the minimum homozygous genotype TT of rs1569922 is reduced; E. The risk of non-traumatic femoral head necrosis in the tested person who carries both the rs1884444 minimum homozygous genotype GG and the rs6693831 minimum homozygous genotype TT is reduced.

3. The use according to claim 1, characterized in that: The substance used to detect the single nucleotide polymorphism of the rs6683039 site is specifically a primer pair B or a set of single-stranded DNA molecules B; the primer pair B is composed of a single-stranded DNA molecule as shown in SEQ ID NO.4 and a single-stranded DNA molecule as shown in SEQ ID NO.5; the set of single-stranded DNA molecules B is composed of a single-stranded DNA as shown in SEQ ID NO.4, a single-stranded DNA as shown in SEQ ID NO.5, and a single-stranded DNA as shown in SEQ ID NO.6: The substance used to detect the single nucleotide gene polymorphism of the rs11465754 site is a primer pair C or a set of single-stranded DNA molecules C; the primer pair C is composed of a single-stranded DNA molecule as shown in SEQ ID NO.7 and a single-stranded DNA molecule as shown in SEQ ID NO.8; the set of single-stranded DNA molecules C is composed of a single-stranded DNA as shown in SEQ ID NO.7, a single-stranded DNA as shown in SEQ ID NO.8, and a single-stranded DNA as shown in SEQ ID NO.9; The substance used to detect the single nucleotide gene polymorphism of the rs1884444 site is a primer pair D or a set of single-stranded DNA molecules D; the primer pair D is composed of a single-stranded DNA molecule as shown in SEQ ID NO.10 and a single-stranded DNA molecule as shown in SEQ ID NO.11; the set of single-stranded DNA molecules D is composed of a single-stranded DNA as shown in SEQ ID NO.10, a single-stranded DNA as shown in SEQ ID NO.11, and a single-stranded DNA as shown in SEQ ID NO.12; The substance used to detect the single nucleotide gene polymorphism of the rs1569922 site is a primer pair E or a set of single-stranded DNA molecules E; the primer pair E is composed of a single-stranded DNA molecule as shown in SEQ ID NO.13 and a single-stranded DNA molecule as shown in SEQ ID NO.14; the set of single-stranded DNA molecules D is composed of a single-stranded DNA as shown in SEQ ID NO.13, a single-stranded DNA as shown in SEQ ID NO.14, and a single-stranded DNA as shown in SEQ ID NO.15; The material used to detect the single nucleotide gene polymorphism at the rs6693831 site is a primer pair or a set of single-stranded DNA molecules; the primer pair is composed of a single-stranded DNA molecule as shown in SEQ ID NO.16 and a single-stranded DNA molecule as shown in SEQ ID NO.17; the set of single-stranded DNA molecules is composed of a single-stranded DNA as shown in SEQ ID NO.16, a single-stranded DNA as shown in SEQ ID NO.17 and a single-stranded DNA as shown in SEQ ID NO.18.

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