Method for detecting single nucleotide polymorphism susceptibility that is target of drug resistance in ankylosing spondylitis

Abstract

This invention provides a method for detecting susceptibility single nucleotide polymorphisms (SNPs) for ankylosing spondylitis and its drug resistance markers. This invention uses genome-wide association analysis to identify differences in ankylosing spondylitis susceptibility SNPs among different drug treatments and explores the association between human leukocyte antigen-B27 and ankylosing spondylitis susceptibility SNPs in the Taiwanese population. The results show that different ethnic groups, sexes, and drug treatment groups exhibit different ankylosing spondylitis susceptibility SNPs.

Description

Technical Field

[0001] The present invention relates to a method for detecting susceptible single-nucleotide polymorphism (SNP) of ankylosing spondylitis (AS) and its drug resistance marker. Prior Art

[0002] AS is a chronic immune-induced arthritis that mainly affects the patient's axial spinal joints. In severe cases, it will extend to peripheral joints, tendons and tendon sheaths, and even inflammation of parts outside the joints (such as the eyes and gastrointestinal tract). According to statistics, about one in every 200 people in the world is affected by AS. In addition, the age group of AS patients is mostly young and middle-aged. Young and middle-aged people are not only an important economic pillar of a family, but also one of the keys to a country's prosperity. However, after suffering from AS, the body's functions will gradually be limited, which will affect the patient's work ability and quality of life. Therefore, how to prevent AS or control the progression of AS has always been a very important public health issue.

[0003] In general, there are many risk factors for AS, and it is still unclear how the interactions between genes, microorganisms, gender, environment and lifestyle factors cause patients to suffer from AS. However, past studies have also clearly shown that genetic and environmental factors play an important role in the development of AS.

[0004] At present, the treatment goal of AS is to improve and maintain spinal flexibility and normal posture, relieve symptomatic pain and reduce the occurrence of complications. The main treatment method for AS is drug therapy. Although daily physical therapy and regular exercise cannot effectively improve the patient's disease, they can relieve the patient's pain and relax the joints to prevent the disease from affecting daily activities. The drugs for AS are generally divided into four types: non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, disease-modifying antirheumatic drugs (DMARDs) and biological agents (such as tumor necrosis factor (TNF) inhibitors and interleukin-17 (IL-17) inhibitors). If the patient still complains of pain after using the drug, analgesics will be used as auxiliary treatment.

[0005] Currently, the first-line drug for the clinical treatment of AS is NSAIDs. Through the anti-inflammatory effect of the drug, the pain of joint inflammation can be relieved and controlled first. However, the effect of the drug varies from person to person. Some AS patients do not respond well to NSAIDs, or have drug-related side effects. Such patients need other types of drugs for treatment. When NSAIDs fail to control the patient's condition well, DMARDs will be given, and if pain still occurs, local steroid injections will be given to relieve local inflammation and achieve the effect of alleviating pain. If the above drugs are ineffective, they will progress to the use of biological agents for treatment.

[0006] The current clinical drugs for AS still have shortcomings such as poor effects and side effects, and people with risk factors for AS also hope to use more accurate methods to detect and predict the incidence, severity and prognosis of AS. However, there are currently no relevant accurate methods and kits that can meet the needs of people with such needs.

[0007] In order to solve the above problems, technicians in this field are in urgent need of developing novel methods for detecting susceptible SNPs of AS and its drug resistance targets to benefit the vast population in need. Summary of the invention

[0008] In view of this, the object of the present invention is to provide a method for detecting susceptible SNPs of ankylosing spondylitis (AS) and its drug resistance target, comprising: when the target population is Taiwanese, the target SNP is rs142577772, rs7756294, rs2073716, rs76977405, rs7766452, rs9368671, rs28862571, rs9266267, rs6936035, rs2251396, rs3094228, rs9688839, rs5006724, rs5006725, rs9266514, rs2844555, rs2507985, rs5025317, rs5025315 or rs4463302.

[0009] Another object of the present invention is to provide a method for detecting susceptible SNPs of AS and its drug resistance targets, comprising: when the gender is male and the medication is traditional medicine, the susceptible SNP includes at least one target SNP, and the at least one target SNP is selected from the following group: rs141230152, rs111322268, rs11757571, rs13198903, rs13202464, rs192624123, rs 2524069, rs2844623, rs3134792, rs4463302, rs4711287, rs4959053, rs41549014, rs76465289, rs77372868, rs77449597, rs7756294, rs78564856, rs79424709, rs9266267, rs9348863 and rs9688839.

[0010] In one embodiment of the present invention, when the target SNP is rs2524069 with an A / T allele, rs4463302 with a T / C allele, rs77449597 with a C / A allele, rs78564856 with a G / A allele, or rs79424709 with a G / A allele, the risk level of ankylosing spondylitis in a person who is male and takes traditional medicine is a high risk level.

[0011] Another object of the present invention is to provide a method for detecting susceptible SNPs of AS and its drug resistance targets, comprising: when the gender is male and the medication is a biological agent, the at least one target SNP is selected from the following group: rs11753208, rs141230152, rs1625792, rs2523586, rs2524069, rs74751264, rs76465289, rs78564856 and rs78725346.

[0012] In one embodiment of the present invention, when the target SNP is rs11753208 allele C / T, rs141230152 allele A / G, rs2523586 allele T / G, rs2524069 allele A / T, rs76465289 allele C / T, rs78564856 allele G / A or rs78725346 allele G / A, the risk level of ankylosing spondylitis in the patient who is male and taking biological drugs is a high risk level.

[0013] Another object of the present invention is to provide a method for detecting susceptible SNPs of AS and its drug resistance targets, comprising: when the gender is male and the medication is a biological agent, and the control group is a traditional medication user, the at least one target SNP is selected from the following group: rs12035845, rs6016758, rs34362099, rs2060596, rs75942205, rs296670, rs521882, rs1646650, rs17057519, rs58532073 and rs61770398.

[0014] In one embodiment of the present invention, when the target SNP is rs6016758 with an allele of G / A, rs58532073 with an allele of C / T, rs296670 with an allele of G / T, or rs75942205 with an allele of T / C, the risk level of ankylosing spondylitis in a male patient taking a biological drug is a high risk level.

[0015] Another object of the present invention is to provide a method for detecting susceptible SNPs of AS and its drug resistance targets, comprising: when the gender is female and the medication is traditional medication, the at least one target SNP is selected from the following group: rs1625792, rs2524069, rs76492679, rs79040988, rs77449597, rs7766452, rs78564856 and rs9266259.

[0016] In one embodiment of the present invention, when the target SNP is rs2524069 with an A / T allele, rs77449597 with a C / A allele, or rs7766452 with a G / A allele, the risk level of ankylosing spondylitis in a female patient who takes traditional medication is a high risk level.

[0017] Another object of the present invention is to provide a method for detecting susceptible SNPs of AS and its drug resistance targets, comprising: when the gender is female and the medication is a biological agent, the at least one target SNP is selected from the following group: rs11753208, rs151361, rs75252698, rs77449597 and rs59356242.

[0018] In one embodiment of the present invention, when the target SNP is rs75252698, the allele is G / A, the allele is C / A, or the allele is C / A of rs77449597, or the allele is C / A of rs59356242, the risk level of ankylosing spondylitis in the patient who is female and taking biological drugs is a high risk level.

[0019] Another object of the present invention is to provide a method for detecting susceptible SNPs of AS and its drug resistance targets, comprising: when the gender is female and the medication is biological preparations, and the control group is traditional medication users, the at least one target SNP is rs10205553.

[0020] In one embodiment of the present invention, when the target SNP is rs10205553 and the allele is G / A, the risk level of ankylosing spondylitis in the patient who is female and taking biological drugs is a high risk level.

[0021] In summary, the efficacy of the present invention is to analyze the differences in AS susceptibility SNPs between different drug treatments using GWAS, and to explore the association between HLA-B27 and AS susceptibility SNPs in the Taiwanese population. The results showed that different ethnic groups, genders, and drug groups had different AS susceptibility SNPs. The present invention can find out the risk-related gene polymorphism sites between different drug treatment effects in AS patients through GWAS analysis, and can provide accurate treatment options and improve medical quality and medical effectiveness in the future clinical drug use of AS.

[0022] The following will further illustrate the implementation mode of the present invention. The following examples are used to illustrate the present invention, not to limit the scope of the present invention. Anyone skilled in the art can make some changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the scope of the attached patent application. Simple diagram description

[0023] FIG. 1 shows a schematic diagram of the method of the present invention. FIG2 shows a flow chart of the method of the present invention, wherein GWAS stands for genome-wide association study, SNP stands for single-nucleotide polymorphism, and HLA-B27 stands for human leukocyte antigen-B27. FIG3 shows the Manhattan plot of male patients using traditional medicine (TPMI). FIG. 4 shows a linkage disequilibrium relationship diagram (TPMI) of 136 significant loci. FIG5 shows a heat map of significant SNPs. FIG. 6 shows the Manhattan plot (TWB) of male patients using traditional medicine. FIG. 7 shows the linkage disequilibrium relationship diagram (TWB) of 165 significant loci. FIG8 shows the Manhattan plot of male patients using biologics (TPMI). FIG. 9 shows the linkage disequilibrium relationship diagram (TWB) of 136 significant loci. FIG. 10 shows the Manhattan plot (TWB) of male patients using biologics. FIG. 11 shows the linkage disequilibrium relationship diagram (TWB) of 7 significant loci. FIG. 12 shows the Manhattan plot of female patients using traditional medicine (TPMI). FIG. 13 shows a linkage disequilibrium relationship diagram (TPMI) of 52 significant loci. FIG14 shows a heat map of significant SNPs. FIG. 15 shows the Manhattan plot (TWB) of female patients using traditional medicine. FIG. 16 shows a linkage disequilibrium relationship diagram (TWB) of 36 significant loci. FIG17 shows a heat map of significant SNPs. FIG. 18 shows the Manhattan plot of female patients using biologics (TPMI). FIG. 19 shows a linkage disequilibrium relationship diagram (TPMI) of 12 significant loci. FIG20 shows a heat map of significant SNPs. FIG. 21 shows the Manhattan plot (TWB) of female patients using biologics. FIG. 22 shows the linkage disequilibrium relationship diagram (TWB) of 8 significant loci. FIG23 shows a heat map of significant SNPs. FIG. 24 shows the Manhattan plot for male patients. FIG25 shows a heat map of significant SNPs. FIG. 26 shows the Manhattan plot for female patients. FIG27 shows a heat map of significant SNPs. Implementation

[0024] [definition]

[0025] According to the present invention, the term "ankylosing spondylitis (AS)" is an immune-induced chronic arthritis that mainly affects the patient's axial spinal joints and, in severe cases, extends to the peripheral joints. In the process of drug treatment of AS, patients need to try many different drugs, which causes many patients who are ineffective with traditional AS drugs to delay the course of the disease and worsen the AS condition.

[0026] Drug treatments for controlling AS are divided into non-steroidal anti-inflammatory drugs, steroids, DMARDs and biologics. Among them, patients first try traditional AS drugs (i.e. non-biologics) for a period of time before switching to biologics when they are ineffective (drug resistance). As a result, many AS patients delay the course of their disease, causing their condition to worsen and become irreversible.

[0027] Therefore, the present invention has the following objectives: 1. To explore the risk-related gene polymorphic loci of AS in the Taiwanese population by genome-wide association study (GWAS); 2. To explore the differences in risk-related gene polymorphic loci of AS susceptibility between different genders by GWAS; 3. To explore the differences in AS susceptibility gene polymorphic loci between patients in different medication groups by GWAS.

[0028] According to the present invention, the genotyping data of subjects enrolled in a medical center in the northern region of Taiwan Precision Medicine Initiative (TPMI) were used to identify the susceptible SNPs of AS using GWAS using the case-control study method.

[0029] According to the present invention, the term "non-steroidal anti-inflammatory drugs (NSAIDs)" is one of the most commonly used drugs for relieving pain and inflammation. NSAIDs include, but are not limited to: Aspirin, Acemetacin, Celecoxib, Diclofenac, Etodolac, Etoricoxib, ibuprofen, Meloxicam, Naproxen and Salsalate.

[0030] According to the present invention, the term "glucocorticoid" refers to a type of adrenocortical hormone secreted by the adrenal cortex, which is a common steroid. Its main effects are anti-inflammatory and immunomodulatory. It can be used to treat many diseases, such as rheumatoid arthritis, lupus erythematosus, allergic diseases, etc. It can be taken orally or injected locally in the treatment of AS. However, the use of steroids has many side effects. Past literature also clearly pointed out that the use of steroids to treat AS is related to the complication of osteoporosis. Therefore, special attention should be paid to the related side effects when using steroids for treatment.

[0031] According to the present invention, the term "disease-modifying antirheumatic drugs (DMARDs)" refers to drugs used to treat inflammatory arthritis, including rheumatoid arthritis, psoriatic arthritis and AS. Common DMARDs are two drugs, Salfasalazine and Methotrexate. Among them, the drug that is more effective in treating AS is Salfasalazine, which mainly treats ulcerative colitis and rheumatoid arthritis, but when used to treat AS, it only treats peripheral spondyloarthritis and has no special effect on axial spondyloarthritis; the same is true for Methotrexate, which is suitable for a variety of cancers, such as blood cancer, bone cancer, etc., and can also be used to treat rheumatoid arthritis and psoriasis, but has no significant effect in treating AS. Despite this, studies have shown that the combined use of Salfasalazine and Methotrexate can significantly control the course of AS, so many patients also take a combined medication course.

[0032] According to the present invention, the term "biologic therapy" refers to drugs developed through biotechnology that can suppress the immune system and reduce inflammation. However, because the use of biologics suppresses the immune system, people who take biologics may be more susceptible to infection. Currently, biologics suitable for AS can be divided into two types: interleukin-17 (IL-17) inhibitors and tumor necrosis factor-α (TNF-α) inhibitors. A total of seven drugs have been approved by the U.S. Food and Drug Administration (CFDA) for use in AS, and the biologics that Taiwan National Health Insurance can currently apply for are Adalimumab (trade name Fumai), Etanercept (trade name Enbo), Golimumab (trade name Simponi) and Secukinumab (trade name Cosentin).

[0033] According to the present invention, the present invention provides a method for detecting susceptible SNPs of ankylosing spondylitis (AS) and its drug resistance target, comprising: when the target population is Taiwanese, the target SNP is rs142577772, rs7756294, rs2073716, rs76977405, rs7766452, rs9368671, rs28862571, rs9266267, rs6936035, rs2251396, rs3094228, rs9688839, rs5006724, rs5006725, rs9266514, rs2844555, rs2507985, rs5025317, rs5025315 or rs4463302.

[0034] The present invention is further illustrated by the following examples. These examples are provided for illustration only and are not intended to limit the scope of protection of the present invention. The scope of protection of the present invention is as shown in the attached patent application scope. [Example] [1.] [Process and structure of the method of the present invention]

[0035] The present invention aims to use GWAS to find out the differences in AS susceptibility SNPs between groups of AS patients using different clinical drugs, see Figure 1.

[0036] The selection of research subjects and data collection are described as follows. The present invention mainly comes from AS patients in a medical center in Taipei City, and they have participated in the TPMI jointly implemented by the hospital and the Academia Sinica. After applying for the genotype sequence data of the patients from the precision medicine and genomics center of the hospital, they also applied to connect the genomic data with the patient's medication, test and medical history diagnosis data. Then, the genotype sequence data of the Taiwan Biobank (TWB) was used as the control group for analysis.

[0037] The study variables and operational definitions are shown in Table 1 below. Table 1 Variable Name Operational Definition type gender The first digit of the ID card is the boundary category age Calculated by birth year continuous Medication history 1. Use traditional medications, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and disease-modifying antirheumatic drugs (DMARDs) 2. Use of first-line biologics 3. Use of second-line biologics category Comorbidities Other diseases recorded in the medical record category

[0038] A flowchart of the method of the present invention is shown in FIG2. FIG2 shows a flowchart of the method of the present invention, wherein GWAS stands for genome-wide association study, SNP stands for single-nucleotide polymorphism, and HLA-B27 stands for human leukocyte antigen-B27. As shown in FIG2, the method of the present invention comprises the following steps: (a) collecting genotyping data of ankylosing spondylitis patients from multiple databases; (b) performing data processing on the genotyping data to obtain a processed genotyping data; (c) grouping the processed genotyping data to obtain a grouped genotyping data; (d) performing GWAS on the grouped genotyping data; (e) screening susceptible SNPs of AS to find sites that have significant differences in whether or not the disease occurs; and (f) determining the expression level of genes related to AS and the correlation between the genes related to AS and the susceptible SNP, thereby detecting and predicting the severity and prognosis of AS.

[0039] Preferably, in FIG. 2 , the databases are databases from TPMI, Taiwan Biobank, or a combination thereof. [Example] [2.] [Data processing and analysis]

[0040] The present invention then performed a logistic regression analysis to compare the susceptibility of gene polymorphism to AS after controlling for variables such as gender, ethnic stratification, age, and relevant medical history, and presented the results as odds ratios (OR) and 95% confidence intervals.

[0041] The Manhattan plot is a plot of the p-values of all SNP sites after GWAS analysis from left to right on the entire genome, and in order to express the results more intuitively, the p-value is usually converted to -log10 (p-value). Therefore, the height of the gene site on the Y axis corresponds to the degree of association with the phenotypic trait or disease. The general threshold is set at 5×10-8. The higher the height, the stronger the association with the phenotypic trait or disease. [Example] [3.] [Analysis results of male patients using traditional medicine] 1. TPMI analysis results

[0042] The control group of this embodiment uses male patients who are not rheumatologists in TPMI. According to the GWAS results, 451 SNPs are significantly associated with male AS patients who use traditional medicines, reaching p < 1-e8. The significant sites of the results of three different analyses (Logis regression and Fisher's exact probability test) were used to find the intersection of 136 SNPs. After using the Haploview program to draw a linkage disequilibrium plot (LD plot), 14 haplotype blocks with high linkage disequilibrium were found. One of the most representative sites (the site with the smallest p-value in the haplotype block) was selected from each haplotype block as a tag SNP. The relevant information of the tag SNP is listed in Tables 2 to 4. Tables 2 to 4 show the analysis results of male patients using traditional medicines in TPMI. Table 2 SNP CHR Location Allele a MAF b in case group MAF b in the control group -log 10(P) OR rs141230152 6 30573616 A / G 0.24 0.04 14.72 8.31 (4.93-14.02) 16.45 8.87 (5.34-14.73) 28.88 7.47 (5.14-10.86) rs2524069 6 31277012 A / T 0.36 0.04 26.57 19.08 (11.19-35.54) 29.28 20.91 (12.38-35.29) 59.65 14.42 (9.99-20.81) rs3134792 6 31344549 T / G 0.43 0.15 12.88 4.65 (3.09-6.98) 13.51 4.54 (3.07-6.71) 30.60 4.31 (3.37-5.52) rs4463302 6 31375890 T / C 0.43 0.07 23.53 13.63 (8.24-22.56) 24.92 13.95 (8.52-22.85) 58.05 9.59 (7.15-12.86) rs4711287 6 32108059 G / A 0.17 0.03 9.91 6.38 (3.63-11.22) 10.41 6.24 (3.63-10.75) 19.02 6.54 (4.25-10.07) a. Allele = Major allele / Minor allele b. MAF = Minor Allele Frequency Table 3 SNP CHR Location Allele a MAF b in case group MAF b in the control group -log 10(P) OR rs4959053 6 31131800 G / A 0.42 0.13 12.98 4.87 (3.21-7.38) 12.78 4.52 (3.03-6.75) 33.90 4.83 (3.74-6.24) rs77449597 6 31072761 C / A 0.34 0.06 20.23 10.99 (6.67-18.13) 22.14 11.46 (7.05-18.63) 44.88 8.81 (6.38-12.18) rs7756294 6 31044579 G / A 0.37 0.09 15.79 6.77 (4.30-10.67) 16.45 6.69 (4.30-10.40) 37.71 6.18 (4.65-8.22) rs78564856 6 31246231 G / A 0.37 0.06 20.82 11.37 (6.90-18.75) 22.93 12.12 (7.45-19.74) 47.17 8.67 (6.33-11.84) rs79424709 6 31303829 G / A 0.40 0.07 20.01 12.24 (7.42-20.18) 23.66 12.81 (7.85-20.93) 50.17 8.56 (6.35-11.53) a. Allele = Major allele / Minor allele b. MAF = Minor Allele Frequency Table 4 SNP CHR Location Allele a MAF b in case group MAF b in the control group -log 10(P) OR rs9266267 6 31358250 A / C 0.41 0.07 25.92 19.77 (11.44-34.19) 27.72 20.37 (11.94-34.74) 53.47 9.20 (6.82-12.41) rs9348863 6 31294684 T / C 0.50 0.25 8.60 2.86 (2.02-4.03) 8.78 2.77 (1.99-3.86) 20.69 3.03 (2.41-3.80) rs9688839 6 31471882 A / G 0.39 0.14 9.53 3.35 (2.30-4.88) 10.47 3.49 (2.41-5.04) 26.11 4.00 (3.10-5.16) a. Allele = Major allele / Minor allele b. MAF = Minor Allele Frequency 2. Analysis results of Taiwan Human Biodatabase

[0043] The control group of this embodiment uses male subjects from the Taiwan Human Biodatabase. According to the GWAS results, 575 SNPs are significantly associated with male AS patients using traditional medicine (p < 1-e8). The significant sites of two different analysis results of Logistic regression and Fisher's exact probability test were used to find the intersection of 165 SNPs. After using the Haploview program to draw a linkage disequilibrium plot (LD plot), 20 haplotype blocks with high linkage disequilibrium were found. One of the most representative sites (the site with the smallest p-value in the haplotype block) was selected from each haplotype block as the tag SNP. The relevant information of the tag SNP is listed in Tables 5 and 6. Tables 5 and 6 show the analysis results of male patients using traditional medicine in the Taiwan Human Biodatabase. If arthritis is excluded, the analysis results are similar, but the OR values all show signs of improvement. Table 5 SNP CHR Location Allele a MAF b in case group MAF b in the control group -log 10(P) OR rs111322268 6 31299767 A / G 0.40 0.08 23.14 8.01 (5.39-12.16) 57.70 7.71 (6.2-9.88) rs11757571 6 31465010 C / T 0.47 0.22 11.88 3.01 (2.22-4.09) 26.16 3.22 (2.61-3.98) rs13198903 6 31376380 C / T 0.43 0.08 27.20 9.90 (6.57-14.91) 68.80 8.98 (7.03-11.47) rs13202464 6 31376806 A / G 0.43 0.08 26.51 9.83 (6.49-14.87) 68.06 8.99 (7.03-11.50) rs141230152 6 30573616 A / G 0.24 0.04 18.57 8.83 (5.49-14.21) 33.83 6.83 (5.04-9.26) rs192624123 6 28884376 T / C 0.16 0.03 10.36 5.46 (3.30-9.05) 21.78 5.92 (4.17-8.41) rs2524069 6 31277012 A / T 0.36 0.04 35.15 21.08 (13.07-33.99) 75.61 14.84 (10.97-20.08) rs2844623 6 31264766 C / T 0.43 0.13 15.41 4.34 (3.05-6.17) 43.81 5.10 (4.08-6.39) rs3134792 6 31344549 T / G 0.43 0.16 15.85 3.89 (2.82-5.36) 32.72 3.91 (3.14-4.86) rs41549014 6 29942810 G / A 0.21 0.04 15.37 7.12 (4.44-11.44) 25.74 5.61 (4.12-7.65) a. Allele = Major allele / Minor allele b. MAF = Minor Allele Frequency Table 6 SNP CHR Location Allele a MAF b in case group MAF b in the control group -log 10(P) OR rs4711287 6 32108059 G / A 0.17 0.03 13.45 7.02 (4.24-11.62) 25.25 7.22 (5.00-10.41) rs76465289 6 31167056 C / T 0.36 0.07 21.65 7.25 (4.87-10.81) 52.29 7.49 (5.80-9.67) rs77372868 6 31334231 C / T 0.43 0.11 19.72 5.80 (4.00-8.41) 52.60 6.30 (4.99-7.94) rs77449597 6 31072761 C / A 0.34 0.06 22.74 8.88 (5.78-13.63) 52.54 8.02 (6.15-10.45) rs7756294 6 31044579 G / A 0.37 0.08 20.68 7.16 (4.77-10.75) 47.91 6.48 (5.06-8.28) rs78564856 6 31246231 G / A 0.37 0.07 21.29 7.16 (4.77-10.75) 52.74 7.43 (5.76-9.57) rs79424709 6 31303829 G / A 0.40 0.08 23.41 8.29 (5.01-12.47) 57.93 7.76 (6.06-9.94) rs9266267 6 31358250 A / C 0.41 0.07 29.47 14.35 (9.08-22.67) 63.25 8.76 (6.80-11.27) rs9688839 6 31471882 A / G 0.39 0.13 14.43 4.01 (2.84-5.67) 35.72 4.48 (3.57-5.63) a. Allele = Major allele / Minor allele b. MAF = Minor Allele Frequency

[0044] This example found 136 and 165 SNPs significantly associated with male AS patients using traditional medicine in the Taiwan Precision Medicine Project and the Taiwan Human Biodatabase, respectively. Then, this example found a tag SNP with the lowest significance in each haploid genotype block with high linkage disequilibrium, which were 14 and 20 respectively. Overall, 126 SNPs were repeated in the SNPs found in the two databases, and there were up to 11 repeated SNPs in the tag SNPs. After further investigation, it was found that the 14 tag SNPs found in the analysis results of TPMI all appeared in the analysis results of TWB, but rs192624123 located in Block 1 in the analysis results of TWB did not appear in the analysis results of TPMI, but was replaced by rs140333527 also located in Block 1 in the analysis results of TWB, with an OR value of 4.33 (95CI = 3.01-6.22, p-value = 1e-14).

[0045] Except for rs192624123, rs4463302 and rs9348863 in the TPMI analysis results were in the same highly linkage-disequilibrium haploid genotype block with rs13198903 (Block 15) and rs111322268 (Block 10) in the TWB analysis results, respectively, while rs77372868 in the TWB analysis results was in the same highly linkage-disequilibrium haploid genotype block with rs79424709 (Block 8) in the TPMI analysis results. From the above results, it can be seen that when the tag SNP is rs2524069 with an A / T allele, rs4463302 with a T / C allele, rs77449597 with a C / A allele, rs78564856 with a G / A allele, or rs79424709 with a G / A allele, the risk level of ankylosing spondylitis in the male patient who takes traditional medicine is a high risk level. [Example] [4.] [Analysis results of male patients using biologics] 1. TPMI analysis results

[0046] The control group of this embodiment uses male patients who are not rheumatologists in TPMI. According to the GWAS results, 152 SNPs are significantly associated with male AS patients using biological agents, reaching p < 1-e8. The significant sites of three different analysis results were used to find the intersection of 64 SNPs. After using the Haploview program to draw a linkage disequilibrium plot (LD plot), 9 haplotype blocks with high linkage disequilibrium were found. One of the most representative sites (the site with the smallest p-value in the haplotype block) was selected from each haplotype block as the tag SNP. The relevant information of the tag SNP is listed in Tables 7 and 8. Tables 7 and 8 show the analysis results of male patients using biological agents in TPMI. Table 7 SNP CHR Location Allele a MAF b in case group MAF b in the control group -log 10(P) OR rs11753208 6 31037655 C / T 0.40 0.09 10.72 11.65 (5.69-23.86) 16.41 8.96 (5.38-14.93) 10.71 7.01 (4.15-11.84) rs141230152 6 30573616 A / G 0.29 0.04 11.20 13.82 (6.53-29.23) 16.41 8.96 (5.38-14.93) 10.16 9.58 (5.24-17.52) rs1625792 6 31338643 G / A 0.46 0.15 8.31 6.91 (3.62-13.19) 13.49 4.78 (3.19-7.16) 8.41 4.84 (2.94-7.98) rs2523586 6 31359658 T / G 0.53 0.08 13.19 15.88 (7.71-32.71) 23.65 10.73 (6.80-16.94) 18.89 12.44 (7.41-20.89) rs2524069 6 31277012 A / T 0.41 0.04 16.01 54.18 (21.13-139.00) 28.97 21.61 (12.69-36.80) 18.97 17.82 (10.07-31.55) a. Allele = Major allele / Minor allele b. MAF = Minor Allele Frequency Table 8 SNP CHR Location Allele a MAF b in case group MAF b in the control group -log 10(P) OR rs74751264 6 31303735 C / T 0.47 0.08 12.28 9.24 (5.05-16.90) 18.78 7.68 (4.93-11.95) 15.57 10.24 (6.09-17.23) rs76465289 6 31167056 C / T 0.41 0.07 11.98 17.09 (7.83-37.32) 20.71 9.81 (6.13-15.71) 13.78 9.73 (5.71-16.59) rs78564856 6 31246231 G / A 0.37 0.06 12.09 19.16 (8.54-42.99) 22.93 12.12 (7.44-19.74) 11.84 8.74 (5.07-15.07) rs78725346 6 31059792 G / A 0.40 0.06 13.07 20.89 (9.40-46.42) 21.12 10.56 (6.53-17.08) 14.43 10.98 (6.37-18.92) a. Allele = Major allele / Minor allele b. MAF = Minor Allele Frequency 2. Analysis results of Taiwan Human Biodatabase

[0047] The control group of this embodiment uses male subjects from the Taiwan Human Biodatabase. According to the GWAS results, 71 SNPs are significantly associated with male AS patients using biological agents (p < 1-e8). The significant sites of two different analysis results of Logistic regression and Fisher's correct probability test were used to find the intersection of 7 SNPs. After using the Haploview program to draw a linkage disequilibrium plot (LD plot), it was found that the 7 SNPs were in the same haplotype block with high linkage disequilibrium. The most representative site in the haplotype block (the site with the smallest p-value in the haplotype block) was taken as the tag SNP. If arthritis is excluded, the analysis results are similar, but the OR values all show signs of improvement.

[0048] This example found 64 and 7 SNPs significantly associated with male AS patients using biological agents in the Taiwan Precision Medicine Project and the Taiwan Human Biodatabase. Then, this example found a tag SNP with the lowest significance in each haploid genotype block with high linkage disequilibrium, which were 9 and 1 respectively. In summary, the 7 SNPs found in the Taiwan Human Biodatabase all appeared in the analysis results of the Taiwan Precision Medicine Project, and the tag SNP in the Taiwan Human Biodatabase analysis results was rs2524069, whose OR value was 112.00 (95CI = 23.59-531.30, p-value = 1e-8) and also appeared in the analysis results of the Taiwan Precision Medicine Project, with an OR value of 54.18 (95CI = 21.13-139.00, p-value = 1e-16).

[0049] In the analysis results of the Taiwan Precision Medicine Project, in addition to rs2524069, rs78725346 is also in the Taiwan Human Biodatabase. In the analysis results of the Taiwan Precision Medicine Project, rs78725346 (Block 3) and rs2524069 (Block 6) exist in different highly linkage-disequilibrium haploid genotype blocks, respectively. However, in the Taiwan Human Biodatabase, the two SNPs exist in the same highly linkage-disequilibrium haploid genotype block. From the above results, it can be seen that when the tag SNP is rs11753208 allele is C / T, rs141230152 allele is A / G, rs2523586 allele is T / G, rs2524069 allele is A / T, rs76465289 allele is C / T, rs78564856 allele is G / A or rs78725346 allele is G / A, the risk level of ankylosing spondylitis in the male and the drug used is a high risk level. [Example] [5.] [Analysis results of female patients using traditional medicine] 1. TPMI analysis results

[0050] The control group of this embodiment uses female patients who are not rheumatologists in TPMI. According to the GWAS results, 144 SNPs are significantly associated with female AS patients who use biological agents, reaching p < 1-e8. The significant sites of the results of three different analyses (Logis regression and Fisher's exact probability test) were used to find the intersection of 52 SNPs. After using the Haploview program to draw a linkage disequilibrium plot (LD plot), 6 haplotype blocks with high linkage disequilibrium were found. One of the most representative sites (the site with the smallest p-value in the haplotype block) was selected from each haplotype block as the tag SNP. The relevant information of the tag SNP is listed in Table 9, which shows the analysis results of female patients using traditional medicines in TPMI. Table 9 SNP CHR Location Allele MAF in case group MAF in the control group -log10(P) OR rs1625792 6 31338643 G / A 0.34 0.13 9.35 4.66 ( 2.87 - 7.56 ) 9.39 4.67 ( 2.88 - 7.57 ) 15.92 3.52 ( 2.64 - 4.68 ) rs76492679 6 31304014 T / C 0.30 0.06 11.12 7.59 ( 4.25 - 13.56 ) 11.42 7.89 ( 4.41 - 14.14 ) 26.10 6.34 ( 4.56 - 8.81 ) rs77449597 6 31072761 C / A 0.27 0.05 13.30 10.80 ( 5.81 - 20.06 ) 13.45 10.90 ( 5.88 - 20.22 ) 26.09 7.30 ( 5.11 - 10.43 ) rs7766452 6 31282015 G / A 0.26 0.04 13.97 12.04 ( 6.41 - 22.62 ) 14.42 12.68 ( 6.73 - 23.88 ) 28.15 8.89 ( 6.06 - 13.05 ) rs78564856 6 31246231 G / A 0.29 0.06 10.98 8.25 ( 4.49 - 15.15 ) 11.30 8.57 ( 4.66 - 15.77 ) 25.88 6.52 ( 4.66 - 9.12 ) rs9266259 6 31358139 C / T 0.30 0.08 9.99 5.88 ( 3.44 - 10.07 ) 9.98 5.87 ( 3.43 - 10.04 ) 21.01 4.90 ( 3.58 - 6.71 ) 2. Analysis results of Taiwan Human Biobank

[0051] The control group of this embodiment uses female subjects in the Taiwan Human Biodatabase. According to the GWAS results, 129 SNPs are significantly associated with female AS patients who use traditional medicines (p < 1-e8). The significant sites of two different analysis results of Logistic regression and Fisher's exact probability test were used to find 36 SNPs that intersect. After using the Haploview program to draw a linkage disequilibrium plot (LD plot), 5 haplotype blocks with high linkage disequilibrium were found. One of the most representative sites (the site with the smallest p-value in the haplotype block) was selected from each haplotype block as the tag SNP. The relevant information of the tag SNP is listed in Table 10, which shows the analysis results of female patients who use traditional medicines in the Taiwan Human Biodatabase. If arthritis is excluded, the analysis results are similar, but the OR values all show signs of improvement. Table 10 SNP CHR Location Allele MAF in case group MAF in the control group -log10(P) OR rs1625792 6 31338643 G / A 0.34 0.15 8.77 4.29 ( 2.67 - 6.89 ) 12.77 3.04 ( 2.28 - 4.04 ) rs2524069 6 31277012 A / T 0.25 0.04 12.09 11.04 ( 5.72 - 21.31 ) 26.69 9.04 ( 6.05 - 13.52 ) rs76492679 6 31304014 T / C 0.30 0.08 8.76 5.27 ( 3.07 - 9.05 ) 21.13 5.06 ( 3.66 - 6.98 ) rs79040988 6 31216641 C / T 0.29 0.08 9.23 5.78 ( 3.32 - 10.07 ) 20.23 4.97 ( 3.59 - 6.89 ) rs9266259 6 31358139 C / T 0.30 0.08 9.27 5.23 ( 3.10 - 8.81 ) 21.79 5.24 ( 3.79 - 7.24 )

[0052] The analysis results of the Taiwan human biodatabase of this embodiment are shown in Figures 15 to 17.

[0053] This example found 52 and 36 SNPs significantly associated with female AS patients using traditional medicines in the Taiwan Precision Medicine Project and the Taiwan Human Biodatabase, and then this study found a tag SNP with the lowest significance in each haploid genotype block with high linkage disequilibrium, which were 6 and 5, respectively. In summary, 35 SNPs were repeated in the analysis results of the two databases, and 3 SNPs in the tag SNP results were repeated in the analysis results of the two databases. However, rs7766452 (OR = 12.04 (6.41-22.62), p-value = 1e-13) in Block 3 of TPMI analysis results also appeared in Block 2 of TWB analysis results, and was replaced by rs2524069 (OR = 11.04 (5.72-21.31), p-value = 1e-12); rs78564856 in Block 2 (OR = 8.25 (4.49-15.15), p-value = 1e-10) and rs77449597 in Block 1 (OR = 10.80 (5.81-20.06), p-value = 1e-13) also appeared in the analysis results of TWB, but they were all located in Block 1 and replaced by rs79040988 (OR = 5.78 (95CI = 3.32 - 10.07, p-value = 1e-9). From the above results, it can be seen that when the tag SNP is rs2524069 with an A / T allele, rs77449597 with a C / A allele, or rs7766452 with a G / A allele, the risk level of ankylosing spondylitis in females and those who use traditional medicines is a high risk level. [Example] [6.] [Female patients using biologics] 1. TPMI analysis results

[0054] The control group of this embodiment uses female patients from non-rheumatology department in TPMI. According to the GWAS results, although the significance did not reach p < 1-e8, there are still 66 SNPs that are significantly associated with female AS patients using biological agents (reaching p < 1-e4). The significant sites of the results of three different analyses (Logis regression and Fisher's exact probability test) were used to find the 12 SNPs that intersect. After drawing the linkage disequilibrium map (LD plot) using the Haploview program, 5 haplotype blocks (Haplotype Block) with high linkage disequilibrium were found, and one of the most representative sites (the site with the smallest p-value in the haplotype block) was selected from each haplotype block as the tag SNP.

[0055] The analysis results of the Taiwan Precision Medicine Project of this embodiment are shown in FIGS. 18 to 20 . 2. Analysis results of Taiwan Human Biobank

[0056] The control group of this embodiment uses female subjects from the Taiwan Human Biodatabase. According to the GWAS results, although the significance did not reach p < 1-e8, there are still 27 SNPs that are significantly associated with female AS patients using biological agents (reaching p < 1-e4). The significant sites of two different analysis results of Logistic regression and Fisher's exact probability test were used to find the intersection of 8 SNPs. After using the Haploview program to draw a linkage disequilibrium plot (LD plot), 5 haplotype blocks with high linkage disequilibrium were found. One of the most representative sites (the site with the smallest p-value in the haplotype block) was selected from each haplotype block as a tag SNP. The relevant information of the tag SNP is listed in Table 11, which shows the analysis results of female patients using biological agents. If arthritis is excluded, the analysis results are similar, but the OR values all show signs of improvement. Table 11 SNP CHR Location Allele MAF in case group MAF in the control group -log10(P) OR rs11753208 6 31037655 C / T 0.41 0.09 4.62 7.85 ( 3.02 - 20.42 ) 4.71 7.28 ( 2.93 - 18.11 ) 4.26 7.43 ( 3.11 - 17.77 ) rs151361 20 59038947 A / G 0.45 0.11 4.38 7.80 ( 2.92 - 20.82 ) 4.56 7.20 ( 2.86 - 18.12 ) 4.18 6.67 ( 2.83 - 15.70 ) rs75252698 14 97525603 G / A 0.36 0.06 5.32 11.14 ( 3.96 - 31.29 ) 4.79 7.18 ( 2.93 - 17.57 ) 4.29 8.41 ( 3.43 - 20.60 ) rs77449597 6 31072761 C / A 0.32 0.05 4.97 17.43 ( 4.88 - 62.19 ) 4.87 16.23 ( 4.63 - 56.94 ) 4.10 9.12 ( 3.60 - 23.15 ) rs59356242 6 31033902 C / A 0.41 0.06 5.10 29.05 ( 6.63 - 127.30 ) 5.18 10.09 ( 3.69 - 27.58 )

[0057] This example found 12 and 8 SNPs significantly associated with female AS patients using biologics in the Taiwan Precision Medicine Project and the Taiwan Human Biobank, and then this study found a tag SNP with the lowest significance in each haploid genotype block with high linkage disequilibrium, which were 4 and 1 respectively. Overall, there were 4 repeated SNPs in the analysis results of the two databases, but no repeated SNPs in the tag SNP results.

[0058] The OR value of rs59356242, which is a tag SNP in the Taiwan Human Biodatabase analysis results, is 29.05 (95CI = 6.63-27.30, p-value = 1e-5). This SNP also exists in the Taiwan Precision Medicine Project analysis results, with an OR value of 8.13 (95CI = 3.04-21.74, p-value = 1e-4), and is located in Block 1 (Ch 6), replaced by rs11753208, with an OR value of 7.85 (95CI = 3.02-20.42, p-value = 1e-4), while rs11753208 has an OR value of 25.26 (95CI = 5.87-108.60, p-value = 1e-4) in the Taiwan Human Biodatabase analysis results. From the above results, it can be seen that when the tag SNP is rs75252698 allele is G / A, the allele of rs77449597 is C / A or the allele of rs59356242 is C / A, the risk level of ankylosing spondylitis in the patient who is female and taking biological drugs is a high risk level. [Example] [7.] [Use of biologics and traditional medicines] [SNP] [difference] 1. SNP differences between male patients using biologics and traditional drugs

[0059] This example is a male patient in the rheumatology department of TPMI, and the patients were grouped according to the medication. According to the GWAS results, although the significance did not reach p < 1-e8, there were still 21 SNPs that were significantly associated with male AS patients using traditional drugs, reaching p < 1-e4. The significant sites of three different analysis results (Logis regression and Fisher's exact probability test) were used to find the intersection of 15 SNPs. Tables 12 to 14 show the analysis results of male patients. Table 12 SNP CHR Location Allele a MAF b in case group MAF b in the control group -log 10(P) OR rs12035845 1 246193448 T / C 0.61 0.38 4.28 2.76 (1.69-4.50) 4.16 2.72 (1.66-4.46) 4.26 2.53 (1.61-3.99) rs13135284 4 153943598 C / T 0.17 0.40 4.30 0.29 (0.16-0.53) 4.31 0.29 (0.16-0.53) 4.72 0.31 (0.18-0.55) rs1646650 7 134335944 C / A 0.39 0.18 4.53 2.89 (1.76-4.28) 4.64 2.96 (1.79-4.90) 4.52 2.86 (1.78-4.60) rs17057519 5 160210808 A / G 0.16 0.40 4.75 0.26 (0.14-0.48) 4.63 0.27 (0.14-0.49) 5.21 0.29 (0.16-0.52) rs2060596 14 80720782 C / A 0.47 0.23 4.77 2.71 (1.72-4.28) 4.52 2.71 (1.70-4.34) 5.24 2.96 (1.87-4.69) a. Allele = Major allele / Minor allele b. MAF = Minor Allele Frequency Table 13 SNP CHR Location Allele a MAF b in case group MAF b in the control group -log 10(P) OR rs296670 9 90938166 G / T 0.24 0.09 4.03 3.15 (1.77-5.59) 4.19 3.26 (1.82-5.81) 4.20 3.35 (1.90-5.90) rs34362099 17 57686728 A / G 0.69 0.43 4.57 2.78 (1.73-4.49) 4.49 2.76 (1.71-4.46) 5.29 2.92 (1.82-4.70) rs521882 8 16786917 A / G 0.48 0.26 4.19 2.57 (1.62-4.09) 4.07 2.54 (1.60-4.04) 4.43 2.64 (1.67-4.15) rs58532073 2 151637439 C / T 0.17 0.04 5.08 5.60 (2.63-7.66) 5.10 5.67 (2.65-12.12) 4.38 4.66 (2.31-9.38) rs6016758 20 42353228 G / A 0.20 0.06 4.38 3.96 (2.05-7.66) 4.41 4.00 (2.07-7.73) 4.44 4.04 (2.15-7.59) a. Allele = Major allele / Minor allele b. MAF = Minor Allele Frequency Table 14 SNP CHR Location Allele a MAF b in case group MAF b in the control group -log 10(P) OR rs61156287 1 3940897 C / A 0.43 0.22 4.10 2.63 (1.63-4.25) 4.15 2.65 (1.64-4.29) 4.11 2.64 (1.66-4.19) rs61770398 1 3935847 T / C 0.41 0.21 4.23 2.81 (1.70-4.65) 4.32 2.87 (1.73-4.76) 4.16 2.63 (1.65-4.19) rs7124658 11 73190400 C / T 0.20 0.07 4.43 4.18 (2.12-8.24) 4.29 4.09 (2.07-8.10) 4.01 3.64 (1.95-6.77) rs75942205 11 116662878 T / C 0.22 0.07 4.70 3.94 (2.10-7.40) 4.63 3.91 (2.08-7.35) 4.89 4.11 (2.24-7.54) a. Allele = Major allele / Minor allele b. MAF = Minor Allele Frequency 2. SNP differences between female patients using biologics and traditional drugs

[0060] This example is a female patient in the rheumatology department of TPMI, and the patients were grouped according to the medication. According to the GWAS results, although the significance did not reach p < 1-e8, there were still 10 SNPs that were significantly associated with female AS patients using traditional drugs (reaching p < 1-e4). The significant sites of three different analysis results (Logis regression and Fisher's exact probability test) were used to find the intersection of 4 SNPs. Table 15 shows the analysis results of female patients. Table 15 SNP CHR Location Allele a MAF b in case group MAF b in the control group -log 10(P) OR rs10205553 2 211695223 G / A 0.47 0.11 4.50 9.07 (3.21-25.59) 4.52 9.23 (3.25-26.22) 5.58 7.19 (3.29-15.73) rs61629263 17 43782280 A / G 0.38 0.09 4.01 6.97 (2.63-18.52) 4.01 6.98 (2.63-18.54) 4.42 6.37 (2.80-14.47) rs7693524 4 189579129 T / A 0.72 0.35 4.18 8.42 (2.95-23.98) 4.17 8.53 (2.97-24.49) 4.13 4.82 (2.15-10.79) rs9927798 16 83945947 C / A 0.28 0.04 4.30 11.02 (3.45-35.18) 4.36 11.56 (3.57-37.41) 4.538 9.46 (3.61-24.77) a. Allele = Major allele / Minor allele b. MAF = Minor Allele Frequency

[0061] In this embodiment, patients in the rheumatology department of the Taiwan Precision Medicine Project were grouped according to the use of biological agents and traditional drugs, and stratified by gender. In the analysis results of males, 15 significant SNPs were found. In addition to the fact that these 15 SNPs were not associated with each other, they were not repeated with the analysis results of females, and the analysis results of females were also not associated with each other. In addition, it can be seen from the above results that for the analysis of females, when the tag SNP is rs10205553, when the allele is G / A, the risk level of ankylosing spondylitis in females and the medication is biological agents is a high risk level.

[0062] The above description is for illustrative purposes only and is not intended to be limiting. Any equivalent modifications or changes made to the present invention without departing from the spirit and scope of the present invention shall be included in the scope of the attached patent application.

[0063] none

Claims

1. A method for in vitro detection of susceptible SNPs for ankylosing spondylitis (AS) and its drug resistance targets, comprising: when the genotype sequence data of an in vitro sample originates from an individual who is female and a conventional drug user, at least one target SNP is selected from the group consisting of: rs1625792, rs2524069, rs76492679, rs79040988, rs77449597, rs7766452, rs78564856, and rs9266259.

2. The method as described in claim 1, wherein when the allele of the target SNP rs2524069 is A / T, the allele of rs77449597 is C / A, or the allele of rs7766452 is G / A, the risk level of the individual from whom the genotype sequence data of the in vitro sample is derived is high if the individual is female and is using conventional medication.

Images