WO2013060004A1 - Method for detecting specific single nucleotide polymorphism related to ankylosing spondylitis and kit therefor - Google Patents

Method for detecting specific single nucleotide polymorphism related to ankylosing spondylitis and kit therefor Download PDF

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WO2013060004A1
WO2013060004A1 PCT/CN2011/081359 CN2011081359W WO2013060004A1 WO 2013060004 A1 WO2013060004 A1 WO 2013060004A1 CN 2011081359 W CN2011081359 W CN 2011081359W WO 2013060004 A1 WO2013060004 A1 WO 2013060004A1
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ankylosing spondylitis
base
pcr
dna
reaction
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PCT/CN2011/081359
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French (fr)
Chinese (zh)
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古洁若
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Gu Jieruo
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Priority to CN201180067457.9A priority Critical patent/CN103502451B/en
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Publication of WO2013060004A1 publication Critical patent/WO2013060004A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a specific single nucleotide polymorphism associated with detecting ankylosing spondylitis
  • Ankylosing spondylitis is a subtype of spondyloarthropathies (SPA). Its main clinical manifestations are central axis manifestations such as inflammatory low back pain, morning stiffness, and restricted spinal activity, or / and concurrent peripheral arthritis, tendon end, ligament attachment point inflammation, to the advanced stage of the disease, there will be spinal rigidity or deformity.
  • Previous studies have suggested that the role of genetic factors plays a more important role in the pathogenesis of AS, and previous twin-based studies have shown that high consistent incidence in monozygotic and fraternal twins suggests genetic factors. The role in the pathogenesis of AS exceeds 90%.
  • HLA-B27 is an important gene that is also thought to be related to the genetic aggregation of AS family.
  • MHC also known as the major histocompatibility complex
  • HLA-B27 is a tightly linked group of genes encoding major histocompatibility antigens on a chromosome of the vertebrate, associated with immune responses, immune regulation, and transplant rejection. Studies have shown that in monozygotic twins, the consistent probability of HLA-B27 is 63%, and the consistent probability of HLA-B27 in twins twins is 23%.
  • HLA-B27-positive first-degree relatives in AS patients are 6-16 times more likely to have a family history of HLA-B27-positive individuals, suggesting that non-HLA-B27 familial factors are present in the disease. Development also plays an important role. This opens up a whole new way of thinking about the mechanism of the occurrence and development of ankylosing spondylitis. Through the whole-genome SNP chip scanning and analysis, we can find the susceptible SNP associated with ankylosing spondylitis and explore the ankylosing spondylitis. Pathogenic or susceptible genes.
  • Single nucleotide polymorphism mainly refers to DNA sequence polymorphism caused by single nucleotide variation at the genomic level, which is the most common one in human heritable variation. Species, accounting for more than 90% of all known polymorphisms. SNPs are widespread in the human genome, with an average of one in every 500 to 1000 base pairs, with an estimated total of 3 million or more. The polymorphisms exhibited by SNPs involve only a single base variation, which can be caused by a single base transition or transversion, or by the insertion or deletion of a base. In genomic DNA, any base may be mutated, so SNPs may be both within the gene sequence and possibly non-coding sequences outside the gene.
  • cSNP SNP located in the coding region
  • the mutation rate is only 1/5 of the surrounding sequence, but it is of great significance in the study of hereditary diseases. Therefore, the research of cSNP is more concerned.
  • cSNP can be further divided into two types: one is synonymous cSNP (synonymous cSNP), that is, the change of the coding sequence caused by SNP does not affect the amino acid sequence of the translated protein.
  • the mutated base has the same meaning as the unmutated base; the other is a non-synonymous cSNP, which means that the change in the base sequence can change the protein sequence translated by the blueprint, thereby affecting the protein.
  • the function, this change is often the direct cause of changes in biological traits, about half of cSNPs are non-synonymous cSNPs; in addition, SNPs can also be used as genetic markers to help find and identify disease susceptibility genes.
  • Brown et al. performed a full scan and verification of a large sample of AS patients and healthy volunteers based on a self-made intensive SNP chip. It also pointed out that the two genes IL23R and ARTS1 may be new to AS. Sense gene, followed by the research of ARTS1 gene in Korean and Chinese research institutions, the results suggest that ARTS1 is associated with AS; Chinese research institutions have also verified IL-23R, the results No correlation was found between the two, suggesting that there may be differences between different populations. Moreover, although the above-mentioned Brown's research in 2007 used a dense SNP chip, the chip did not perform a full scan of the dense SNP for this genome, but only customized the intensive SNP chip for the location of interest, thus causing the chip.
  • Illumina Human lM-duo chip With the rapid development of chip technology, Illumina Human lM-duo chip, Illumina Human 610-Quad chip and Illumina OmniExpress chip have emerged to provide more dense SNP mark, covering a wider range of areas.
  • Illumina Human lM-duo chip With the rapid development of chip technology, Illumina Human 610-Quad chip and Illumina OmniExpress chip have emerged to provide more dense SNP mark, covering a wider range of areas.
  • GWAS Genome-wide association analysis
  • ankylosing spondylitis is a relatively common disease, the course of the disease is lingering, and it is easy to cause disability, it should be an early diagnosis and treatment, especially for young people aged 16-25.
  • SNPs that are closely related to ankylosing spondylitis, and to design methods and kits for detecting the specificity of ankylosing spondylitis.
  • the present invention is directed to the deficiencies of the currently lacking specificity of detection methods and related kits in the early diagnosis of AS, providing a highly specific examination method and providing a dedicated kit.
  • SNP rs4552569 can be used as a specific SNP for detecting ankylosing spondylitis. It is an important auxiliary diagnostic indicator for ankylosing spondylitis in the early stage, and the present invention has been completed.
  • One of the objects of the present application is to provide a nucleotide fragment of a pathogenicity-related single nucleotide polymorphism (SNP) of ankylosing spondylitis at an rs4552569 site, ankylosing spondylitis (AS)
  • SNP pathogenicity-related single nucleotide polymorphism
  • AS ankylosing spondylitis
  • One of the objects of the present application is to provide a method for early auxiliary diagnosis of ankylosing spondylitis, which is achieved by the following scheme: obtaining a nucleotide sequence from a sample to be tested, and detecting the position at rs4552569 Whether the base on the base is C, and if the base of the site is C, the incidence of ankylosing spondylitis is significantly increased.
  • the sample includes blood, body fluid or tissue.
  • the method includes the following steps:
  • the method of obtaining the nucleotide sequence from the sample to be tested further includes fluorescent quantitative PCR, denaturing high performance liquid chromatography, restriction fragment length polymorphism, ligase detection or DNA fragment sequencing.
  • One of the objects of the present application is to provide a method for detecting an ankylosing spondylitis-specific single nucleotide polymorphism, the method comprising the steps of:
  • PCR amplification primers and single base extension primers of rs4552569 site were designed using Sequenom Genotyping Tools and MassARRAY Assay Design software;
  • DNA from tissues, cells or blood samples was extracted using the TIANamp Blood DNA kit or NucleoSpin Tissue (MN), quantified by spectrophotometry, agarose gel electrophoresis, and the quality of the qualified DNA was adjusted to 5 ( ig / L, -20 ° C storage standby;
  • reaction conditions are 94 °C for 4 minutes; 94 °C20 seconds, 56 °C for 30 seconds, 72 °C for 1 minute, 45 cycles; 72 °C for 3 minutes; 4 °C ⁇ ;
  • the PCR reaction product was treated with 0.5 U SAP (shrimp alkaline phosphatase) to remove free dNTPs from the system.
  • the reaction system was 7 ⁇ , of which the PCR product was 5 ⁇ , and the SAP mixture was 2 ⁇ (SAP 0.5).
  • U, buffer 0.17 ⁇ ) reaction procedure 37 ° C for 20 minutes; 85 ° C for 5 minutes; 40 °C ⁇ ;
  • the total volume of 9 ⁇ reaction system contains 7 ⁇ of the PCR product after SAP treatment, wherein each extension reaction primer mixture is 0.804 ⁇ , iPLEX enzyme is 0.041 ⁇ 1, the extension mixture is 0.2 ⁇ , and the reaction procedure is 94 ° C for 30 seconds; 94 ° C for 5 seconds; °C 5 seconds, 80 °C 5 seconds 5 cycles; return 94 °C 5 seconds, a total of 40 cycles; 72 °C 3 minutes, 4 °C oo;
  • Chip spotting and qualifier detection
  • the purified product was transferred to a 384-well SpectroCHIP (Sequenom) chip for measurement.
  • the SpectroCHIP chip was analyzed by MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) matrix-assisted laser desorption ionization time-of-flight mass spectrometry. 4.0 software (sequenom) typing and outputting results;
  • kits for detecting a specific single nucleotide polymorphism of ankylosing spondylitis comprising: PCR amplification primer, extension primer, DNA designed for a region near the base of rs4552569 Polymerase, deionized water, dNTP, MgC12, PCR buffer, SAP, clean resin tree.
  • One of the objects of the present application is to provide the above-mentioned ankylosing spondylitis-specific single nucleotide polymorphism (SNP) in the treatment of ankylosing spondylitis, that is, when the base at the rs4552569 site is C, it is risky. , is the application of T in the treatment of ankylosing spondylitis.
  • SNP single nucleotide polymorphism
  • ankylosing spondylitis-specific single nucleotide polymorphism-associated susceptibility or pathogenicity Because the base at the rs4552569 locus is cytosine C.
  • the above applications include the use of susceptibility or pathogenic genes associated with ankylosing spondylitis-specific single nucleotide polymorphism (SNP) in the preparation of a medicament for the treatment of ankylosing spondylitis.
  • SNP single nucleotide polymorphism
  • the application includes the function of susceptibility or pathogenic genes involved in the intervention of rs4552569, including translation, transcription, and protein synthesis processes, preparation of therapeutic drugs, improvement of bone metabolism abnormalities in patients, and in vivo The immune inflammatory response to achieve therapeutic goals.
  • the control population of 4231 healthy volunteers mainly included healthy volunteers without AS symptoms or medical history in three places in China, Guangdong, Anhui and Singapore. These healthy volunteers were also informed. Later agreed to draw blood for the study and signed informed consent.
  • the basic conditions of the case and healthy volunteers were recorded, including: gender, age and place of origin. 4 ml of whole blood was taken, placed in an EDTA tube, and brought back to the laboratory for DNA extraction.
  • rheumatologists also carefully inquired and recorded the situation of each AS patient and healthy volunteers, especially the AS patients also recorded basic conditions (including: gender, age, age of onset, duration) And clinical phenotype (including: waxy fingers, hip involvement, peripheral arthritis, inflammatory low back pain).
  • the composition of the kit is: Cell Lysis Night, Buffer GS, Buffer GB, Buffer Liquid GD, rinse PW, elution buffer TB, proteinase K, adsorption column CB3, collection tube (2 ml), 1.5 mL sterile collection tube.
  • the study used the Illumina HuamHap 610-Quad SNP chip, the Illumina Human lM-duo chip and the Illumina OmniExpress chip to perform a full-genome SNP scan of the sample.
  • the following are three types of Illumina chips;
  • the chip cartridge can detect four samples in parallel on one chip, which significantly increases the amount of sample output information and reduces errors in experimental operations.
  • the chip is widely used in the content of the HumanHap550 chip, with an additional 100,000 genetic markers.
  • Human 610-quad The genome-wide information covered is authoritative for both known and recently reported CNV regions.
  • the SNPs of the Illumina HuamHap 610-Quad SNP chip are evenly distributed on the genome at a density of about 5 kb.
  • the Illumina HuamHap 610-Quad SNP chip was designed for specific studies targeting highly polymorphic CNV regions in the genome (fragment replication regions and genomic regions without SNPs).
  • the chip contains more than one million probe information.
  • the chip contains probes for gene SNPs, tagged SNPs, copy number changes (CNV), and other high-value genomic regions, as well as new sites such as: increased disease-associated SNP sites Flexible selection of high-density SNP sites in some genomic coding regions.
  • the chip provides high sample throughput and comprehensive genomic content.
  • the OmniExpress can hold up to 12 samples.
  • the chip interrogates more than 700,000 variations per sample, with a total of more than 8 million data points on a single chip.
  • the marker on OmniExpress is part of Illumina's HumanOmnil-Quad content, which combines optimized SNP tag combinations from all three phases of the international HapMap program, with the highest data quality and best content, including copy number variation (CNV) ) Comprehensive support for the application.
  • CNV copy number variation
  • the heterozygosity range is statistically analyzed from the mean of the heterozygosity mean (Mean) -3 ⁇ standard deviation (SD) to the mean heterozygosity (Mean) +3 ⁇ standard deviation (SD). Samples not in this interval Will be removed. Not included in further statistics.
  • the estimated SNP for analysis in the three chips is done by the IMPUTE2 software (version number: 2.1.0), which maximizes the chip's coverage of the chromosome.
  • C 6.91 x 10- 5, i.e., the presence of significant correlation ankylosing spondylitis, C is the SNP nucleotide risk type, when T is a protected type.
  • the 3702 healthy volunteers were mainly from the healthy volunteers collected by the Third affiliated Hospital of Sun Yat-sen University and the Cancer Center of Sun Yat-sen University. The population were healthy volunteers without AS symptoms or medical history. These healthy volunteers Also, after being informed, agreed to draw blood for the study and signed an informed consent form.
  • the composition of the kit is: Cell Lysis Night, Buffer GS, Buffer GB, Buffer GD, rinse solution PW, elution buffer TB, proteinase K, adsorption column CB3, collection tube (2 ml), 1.5 mL sterile collection tube.
  • Primer design (including amplification primers and extension primers) was performed using the iPLEX® Gold snp genotyping technique of the Sequenom Massarray® DNA Mass Spectrometry System. Primer information for SNP rs4552569 is as follows:
  • the template DNAlul was added to each well in a 384-well plate, and 4 ul of the above reaction system was added.
  • the 384-well plate was centrifuged at 1000 rpm for 1 minute, and after mixing, amplification was carried out according to the following procedure: 94. C 4 minutes pre-denaturation, 94. C 20 seconds, 56 ° C 30 seconds, 72 ° C 1 minute for a total of 45 cycles, 72 ° C 3 minutes, 4 ° C cooling.
  • the main feature of matrix-assisted laser desorption ionization time-of-flight mass spectrometry is that The target sequence was amplified by PCR, and then the snp sequence-specific extension primer was added, and one base was extended at the SNP site.
  • the prepared sample analyte is co-crystallized with the chip substrate, and the crystal is placed in a vacuum tube of the mass spectrometer, and then excited by a transient nanosecond (1 (T 9 s) strong laser, resulting in energy due to the energy absorbed by the matrix molecules through the radiation.
  • the matrix crystals sublimate the nucleic acid molecules are desorbed and converted into metastable ions, and the generated ions are mostly singly charged ions, which obtain the same kinetic energy in the accelerating electric field, and then A non-electric field drift region is separated according to its mass-to-charge ratio, and flies in a vacuum tube to reach the detector.
  • the ions generated by MALDI are commonly detected by a Time-of-Flight (TOF) detector, and the smaller the ion mass, the smaller the ion mass is. The faster the arrival, the genotype of SNP in the above PCR reaction product can be quickly and conveniently detected by this method.
  • TOF Time-of-Flight
  • the subjects in the study were all AS and healthy controls in Examples 1 and 2, and these AS cases were diagnosed by experienced rheumatologists based on clinical findings and imaging findings. New York revised standard AS patients. For each AS patient, blood was drawn for use in the study and the informed consent was signed after being informed of the purpose of the study. Finally, a total of 3937 AS patients and 7727 healthy volunteers were included in the study.
  • the TIANamp Blood DNA kit is used to extract DNA from human peripheral blood samples.
  • the composition of the kit is: Cell Lysis Night, Buffer GS, Buffer GB, Buffer GD , rinse solution PW, elution buffer TB, proteinase K, adsorption column CB3, collection tube (2 ml) and 1.5 mL sterile collection tube.
  • the TIANamp Blood DNA kit is used to extract DNA from human peripheral blood samples.
  • the composition of the kit is: Cell Lysis Night, Buffer GS, Buffer GB, Buffer GD , rinse solution PW, elution buffer TB, proteinase K, adsorption column CB3, collection tube (2 ml), 1.5 ml sterile collection tube.
  • the template DNAlul was added to each well of the plate to be tested, and 4 ul of the above reaction system was added, and centrifuged at 1000 rpm for 1 minute. After mixing, the amplification was carried out according to the following procedure: 94. C 4 minutes pre-denaturation, 94. C 20 seconds, 56 ° C 30 seconds, 72 ° C 1 minute for a total of 45 cycles, 72 ° C 3 minutes, 4 ° C cooling.
  • the prepared sample analyte is co-crystallized with the chip substrate, and the crystal is placed in a vacuum tube of the grammar instrument, and then excited by a transient nanosecond (10-9 s) strong laser, which can be quickly and conveniently detected by the method.
  • a transient nanosecond (10-9 s) strong laser which can be quickly and conveniently detected by the method.
  • test result When the test result is C, it is a risk type, and when the test result is T, it is a protection type.
  • the kit includes: PCR amplification primers, extension primers, DNA polymerase, deionized water, dNTP, MgC12, PCR buffer, SAP, clean resin designed for the region near rs4552569 base.
  • the amplification primers and extension primers are shown in the following table:
  • the steps of the detection method are as follows:
  • DNA from tissues, cells or blood samples was extracted using the TIANamp Blood DNA kit or NucleoSpin Tissue (MN), quantified by spectrophotometry, agarose gel electrophoresis, and the quality of the qualified DNA was adjusted to 5 ( ig / L, -20 ° C storage standby;
  • each reaction is 5 ⁇ 1, including template DNA 10 g, Hotstar Taq 0.5 U, amplification primers 0.5pmol each, 25mM dNTP ⁇ . ⁇ , reaction conditions 94 °C 4 minutes; 94 ° C20 seconds, 56 °C for 30 seconds, 72 °C for 1 minute, 45 cycles; 72 °C for 3 minutes; 4 °C ⁇ ;
  • the PCR reaction product was treated with 0.5 U SAP (shrimp alkaline phosphatase) to remove the body.
  • Free dNTP in the system 7 ⁇ reaction system, 5 ⁇ PCR product, 2 ⁇ SAP solution (SAP 0.5 U, buffer 0.17 ⁇ ), reaction procedure at 37 °C for 20 minutes; 85 °C for 5 minutes; 40 °C ⁇ ;
  • the total volume of 9 ⁇ reaction system contains 7 ⁇ of the PCR product after SAP treatment, wherein each extension reaction primer mixture is 0.804 ⁇ , iPLEX enzyme is 0.041 ⁇ 1, the extension mixture is 0.2 ⁇ , and the reaction procedure is 94 ° C for 30 seconds; 94 ° C for 5 seconds; °C 5 seconds, 80 °C 5 seconds 5 cycles; return 94 °C 5 seconds, a total of 40 cycles; 72 °C 3 minutes, 4 °C oo;
  • Chip spotting and qualifier detection
  • the purified product was transferred to a 384-well SpectroCHIP (Sequenom) chip for measurement.
  • the SpectroCHIP chip was analyzed by MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) matrix-assisted laser desorption ionization time-of-flight mass spectrometry. 4.0 software (sequenom) typing and outputting results;

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Abstract

Provided is a nucleotide fragment with single nucleotide polymorphism related to ankylosing spondylitis. Among ankylosing spondylitis patients, the frequency of C at the site of rs4552569 of the nucleotide fragment is significantly higher than that in healthy controls, whereas the frequency of T at the same site is significantly lower than that in healthy controls; that is, when the base at the site of rs4552569 is C, the diagnosis of AS disease can effectively be assisted. Also provided are a method for detecting corresponding single nucleotide polymorphism related to ankylosing spondylitis and a kit therefor, and the method and kit can be used to perform specific auxiliary diagnosis of ankylosing spondylitis at an early stage.

Description

强直性脊柱炎相关特异性单核苷酸多态性的检测方法及其试剂盒 技术领域  Method for detecting specific single nucleotide polymorphism related to ankylosing spondylitis and kit thereof
本发明申请涉及一种检测强直性脊柱炎相关的特异性单核苷酸多态性 The present invention relates to a specific single nucleotide polymorphism associated with detecting ankylosing spondylitis
( SNP ) 的方法及专用试剂盒， 属于生物医学检测技术领域。 (SNP) method and special kit belong to the field of biomedical detection technology.
背景技术 Background technique
强 直性脊柱炎 ( ankylosing spondylitis , AS ) 是脊柱关节 病 ( spondyloarthropathies, SpA ) 的一种亚型， 它主要的临床表现为炎性腰背痛、 晨僵、 脊柱活动受限等中轴表现， 或 /和并发外周关节炎、 肌腱末端、 韧带附着 点炎， 至疾病晚期， 会出现脊柱强直或畸形。 以往的研究中认为遗传因素的作 用在 AS的发病机制中占据了更为重要的作用， 既往的基于双胞胎的研究表明， 在单卵双生和异卵双生的双胞胎中高的一致的发病率提示遗传因素在 AS 的发 病机制中的作用超过了 90%。  Ankylosing spondylitis (AS) is a subtype of spondyloarthropathies (SPA). Its main clinical manifestations are central axis manifestations such as inflammatory low back pain, morning stiffness, and restricted spinal activity, or / and concurrent peripheral arthritis, tendon end, ligament attachment point inflammation, to the advanced stage of the disease, there will be spinal rigidity or deformity. Previous studies have suggested that the role of genetic factors plays a more important role in the pathogenesis of AS, and previous twin-based studies have shown that high consistent incidence in monozygotic and fraternal twins suggests genetic factors. The role in the pathogenesis of AS exceeds 90%.
在有关 AS致病基因的研究过程中， HLA-B27是最早也是被认为与 AS的家 族聚集性的遗传相关的重要的基因。 但在基因风险方面， 研究认为 HLA-B27在 疾病的总的遗传风险大约只占了 20-30%, 而整个 MHC占的比例约是 40-50%。 MHC又叫主要组织相容性复合体， 是存在于脊推动物某一染色体上编码主要组 织相容性抗原的一组紧密连锁的基因群， 与免疫应答、 免疫调节和移植排斥等 有关。 研究表明， 在单卵双生的双胞胎中， HLA-B27的一致的机率是 63% , 在 双卵双生的双胞胎中 HLA-B27的一致的机率是 23%。 研究还显示： AS患者的 HLA-B27 阳性的一级亲属患病机率是没有家族史的 HLA-B27 阳性的个体的 6-16倍，这些都提示非 HLA-B27的家族性的因素在疾病的发展中也起到重要的 作用。 这为研究强直性脊柱炎的发生和发展的机制开辟了全新的思路， 通过对全基因 组的 SNP芯片全扫并分析， 寻找与强直性脊柱炎相关的易感 SNP, 从而探讨强直 性脊柱炎的致病或易感基因。 In the course of research on AS pathogenic genes, HLA-B27 is an important gene that is also thought to be related to the genetic aggregation of AS family. However, in terms of genetic risk, the study concluded that the total genetic risk of HLA-B27 in disease is only about 20-30%, and the proportion of the whole MHC is about 40-50%. MHC, also known as the major histocompatibility complex, is a tightly linked group of genes encoding major histocompatibility antigens on a chromosome of the vertebrate, associated with immune responses, immune regulation, and transplant rejection. Studies have shown that in monozygotic twins, the consistent probability of HLA-B27 is 63%, and the consistent probability of HLA-B27 in twins twins is 23%. Studies have also shown that: HLA-B27-positive first-degree relatives in AS patients are 6-16 times more likely to have a family history of HLA-B27-positive individuals, suggesting that non-HLA-B27 familial factors are present in the disease. Development also plays an important role. This opens up a whole new way of thinking about the mechanism of the occurrence and development of ankylosing spondylitis. Through the whole-genome SNP chip scanning and analysis, we can find the susceptible SNP associated with ankylosing spondylitis and explore the ankylosing spondylitis. Pathogenic or susceptible genes.
单核苷酸多态性 (single nucleotide polymorphism, SNP),主要是指在基因组水 平上由单个核苷酸的变异所引起的 DNA序列多态性， 它是人类可遗传的变异中 最常见的一种， 占所有已知多态性的 90%以上。 SNP在人类基因组中广泛存在， 平均每 500 ~ 1000个碱基对中就有 1个， 估计其总数可达 300万个甚至更多。 SNP所表现的多态性只涉及到单个碱基的变异， 这种变异可由单个碱基的转换 (transition)或颠换 (transversion)所引起， 也可由碱基的插入或缺失所致。 在基因 组 DNA中， 任何碱基均有可能发生变异， 因此 SNP既有可能在基因序列内， 也 有可能在基因以外的非编码序列上。 总的来说， 位于编码区内的 SNP(coding SNP, cSNP)比较少， 因为在外显子内， 其变异率仅及周围序列的 1/5, 但它在遗 传性疾病研究中却具有重要意义， 因此 cSNP的研究更受关注。 从对生物的遗传 性状的影响上来看， cSNP又可分为 2种： 一种是同义 cSNP(synonymous cSNP), 即 SNP所致的编码序列的改变并不影响其所翻译的蛋白质的氨基酸序列，突变碱 基与未突变碱基的含义相同； 另一种是非同义 cSNP(non-synonymous cSNP), 指 碱基序列的改变可使以其为蓝本翻译的蛋白质序列发生改变， 从而影响了蛋白 质的功能， 这种改变常是导致生物性状改变的直接原因， cSNP中约有一半为非 同义 cSNP; 此外， SNP也可作为遗传标记， 帮助寻找和确定疾病的易感基因。  Single nucleotide polymorphism (SNP), mainly refers to DNA sequence polymorphism caused by single nucleotide variation at the genomic level, which is the most common one in human heritable variation. Species, accounting for more than 90% of all known polymorphisms. SNPs are widespread in the human genome, with an average of one in every 500 to 1000 base pairs, with an estimated total of 3 million or more. The polymorphisms exhibited by SNPs involve only a single base variation, which can be caused by a single base transition or transversion, or by the insertion or deletion of a base. In genomic DNA, any base may be mutated, so SNPs may be both within the gene sequence and possibly non-coding sequences outside the gene. In general, the SNP (coding SNP, cSNP) located in the coding region is relatively small, because in the exon, the mutation rate is only 1/5 of the surrounding sequence, but it is of great significance in the study of hereditary diseases. Therefore, the research of cSNP is more concerned. From the perspective of the influence on the genetic traits of organisms, cSNP can be further divided into two types: one is synonymous cSNP (synonymous cSNP), that is, the change of the coding sequence caused by SNP does not affect the amino acid sequence of the translated protein. The mutated base has the same meaning as the unmutated base; the other is a non-synonymous cSNP, which means that the change in the base sequence can change the protein sequence translated by the blueprint, thereby affecting the protein. The function, this change is often the direct cause of changes in biological traits, about half of cSNPs are non-synonymous cSNPs; in addition, SNPs can also be used as genetic markers to help find and identify disease susceptibility genes.
在 2007年， Brown等就根据自制的密集型 SNP芯片对大样本的 AS患者和健康 志愿者对照人群间进行了全扫并验证， 同时指出了 IL23R和 ARTS1 这两个基因 可能是 AS的新易感基因，随后韩国和中国的研究机构对 ARTS1基因之进行验证， 结果认为 ARTS1与 AS存在关联； 中国的研究机构还对 IL-23R进行了验证， 结果 未发现两者之间存在关联，这提示了不同种群间可能存在着差异。而且上述 2007 年的 Brown的研究中虽然采用密集型 SNP芯片， 但是该芯片并未对这个基因组都 做密集型 SNP的全扫， 只是针对感兴趣的位置来定制密集型 SNP芯片， 所以会导 致芯片在其它未覆盖到的或 SNP标记不够密集全扫的区域出现遗漏。于是在 2010 年 Brown等采用了的 Illumina HumHap 370 芯片对欧洲人种后代的澳大利亚、 英国和北美 AS患者和健康志愿者对照人群进行全扫并验证， 结果除了发现之前 研究提出的 ARTS1和 IL-23R^因可能是 AS的易感基因之外，还提示 ANTXR也可 能与 AS相关联， 同时还提示了 2pl5和21q22存在与AS相关联的SNP。 In 2007, Brown et al. performed a full scan and verification of a large sample of AS patients and healthy volunteers based on a self-made intensive SNP chip. It also pointed out that the two genes IL23R and ARTS1 may be new to AS. Sense gene, followed by the research of ARTS1 gene in Korean and Chinese research institutions, the results suggest that ARTS1 is associated with AS; Chinese research institutions have also verified IL-23R, the results No correlation was found between the two, suggesting that there may be differences between different populations. Moreover, although the above-mentioned Brown's research in 2007 used a dense SNP chip, the chip did not perform a full scan of the dense SNP for this genome, but only customized the intensive SNP chip for the location of interest, thus causing the chip. Omissions occur in other areas that are not covered or where the SNP mark is not sufficiently dense. So in 2010, Brown and other Illumina HumHap 370 chips were used to fully scan and validate the European, American and North American AS patients and healthy volunteers in the European race. The results were found in addition to the ARTS1 and IL-23R proposed in the previous study. ^Because it may be the susceptibility gene of AS, it also suggests that ANTXR may also be associated with AS, and also suggests that there are SNPs associated with AS in 2pl5 and 21q22.
随着芯片技术的迅速发展， Illumina HumHap 370 芯片之后又出现了 Illumina Human lM-duo芯片 、 Illumina Human610-Quad 芯片 和 Illumina OmniExpress芯片提供了更为密集的 SNP标记， 覆盖的区域更加广泛。 考虑上文 提到的研究背景， 我们设计了以 Illumina Human lM-duo芯片、 Illumina Human610-Quad 芯片和 Illumina OmniExpress芯片在中国汉族 AS患者和健康志 愿者对照人群中进行全扫并进行关联分析， 即全基因组关联分析研究（ GWAS ), 以探索是否能发现 AS的相关的易感基因。  With the rapid development of chip technology, Illumina Human lM-duo chip, Illumina Human 610-Quad chip and Illumina OmniExpress chip have emerged to provide more dense SNP mark, covering a wider range of areas. Considering the research background mentioned above, we designed a full-sweep and correlation analysis in the Chinese Han AS patients and healthy volunteers with Illumina Human lM-duo chip, Illumina Human 610-Quad chip and Illumina OmniExpress chip. Genome-wide association analysis (GWAS) to explore whether AS-related susceptibility genes can be found.
由于强直性脊柱炎是较为常见的疾病， 病程缠绵， 且易造成残疾， 故应争 取早期诊断、 治疗， 尤其对于 16-25岁的青年更是如此。 为了加强早期诊断强直 性脊柱炎， 本领域迫切需要寻找出与强直性脊柱炎密切相关的 SNP, 并设计出 检测强直性脊柱炎特异性的方法和试剂盒。  Because ankylosing spondylitis is a relatively common disease, the course of the disease is lingering, and it is easy to cause disability, it should be an early diagnosis and treatment, especially for young people aged 16-25. In order to strengthen the early diagnosis of ankylosing spondylitis, there is an urgent need to find SNPs that are closely related to ankylosing spondylitis, and to design methods and kits for detecting the specificity of ankylosing spondylitis.
目前为止， 对于强直性脊柱炎的诊断， 除了从临床症状以及影像学资料来 进行辅助判断外， 还未有特异性的实验室诊断指标和相应的方法以及试剂盒对 该疾病进行特异性诊断的报道。 发明内容 So far, for the diagnosis of ankylosing spondylitis, in addition to the clinical judgment and imaging data to assist the judgment, there is no specific laboratory diagnostic indicators and corresponding methods and kits for the specific diagnosis of the disease. Report. Summary of the invention
本发明申请即是针对目前在 AS 的早期诊断中尚缺乏特异性的检测方法及 相关试剂盒的不足之处， 提供一种特异性强的检查方法， 并提供专用的试剂盒。  The present invention is directed to the deficiencies of the currently lacking specificity of detection methods and related kits in the early diagnosis of AS, providing a highly specific examination method and providing a dedicated kit.
本发明申请经过深入而广泛的研究， 通过密集型的全基因组 SNP芯片对大 样本的强直性脊柱炎患者和健康对照人群进行全基因组关联分析， 在国际上首 次发现并证明了 SNP rs4552569与强直性脊柱炎存在明显相关性（该 SNP碱基 是 C (胞嘧啶） 时为风险型， 是 T (胸腺嘧啶） 时为保护型， Ρ=8.77χ 10—1(), 该 项研究已经被国际权威杂志 Nature Genetic刊用， 将于近期发表） ， 因此， SNP rs4552569可作为检测强直性脊柱炎的特异性 SNP, 是强直性脊柱炎早期重要的 辅助诊断指标， 并在此基础上完成了本发明。 After extensive and extensive research, the intensive genome-wide SNP chip is used for genome-wide association analysis of large samples of ankylosing spondylitis patients and healthy control populations, and SNP rs4552569 and tonicity are first discovered and proved internationally. There is a significant correlation between spondylitis (the SNP base is C (cytosine) when it is risky, T (thymine) is protective, Ρ = 8.77 χ 10-1 () , the study has been internationally authoritative The journal Nature Genetic is published in the near future. Therefore, SNP rs4552569 can be used as a specific SNP for detecting ankylosing spondylitis. It is an important auxiliary diagnostic indicator for ankylosing spondylitis in the early stage, and the present invention has been completed.
本发明申请的目的之一是提供一种强直性脊柱炎致病相关性单核苷酸多态 性（SNP )的核苷酸片段，该核苷酸片段在 rs4552569位点上，强直性脊柱炎（AS ) 患者碱基为 C的频率显著高于健康对照， 相反， 该位点上碱基为 T的频率则显 著低于健康对照， 即在 rs4552569位点上碱基为 C时， 能有效帮助 AS疾病的诊 断。  One of the objects of the present application is to provide a nucleotide fragment of a pathogenicity-related single nucleotide polymorphism (SNP) of ankylosing spondylitis at an rs4552569 site, ankylosing spondylitis (AS) The frequency of patients with base C was significantly higher than that of healthy controls. Conversely, the frequency of base T at this site was significantly lower than that of healthy controls, that is, when the base was C at rs4552569, it could effectively help. Diagnosis of AS disease.
本发明申请的目的之一是提供一种对强直性脊柱炎进行早期辅助性诊断的 方法， 该方法是通过下述方案实现的： 从待测样品中得到核苷酸序列， 检测位 于 rs4552569位点上的碱基是否为 C, 如果该位点的碱基为 C, 则强直性脊柱炎 的患病几率显著增大。  One of the objects of the present application is to provide a method for early auxiliary diagnosis of ankylosing spondylitis, which is achieved by the following scheme: obtaining a nucleotide sequence from a sample to be tested, and detecting the position at rs4552569 Whether the base on the base is C, and if the base of the site is C, the incidence of ankylosing spondylitis is significantly increased.
进一步的， 所述的样品包括血液、 体液或组织。  Further, the sample includes blood, body fluid or tissue.
进一步的， 所述的方法包括下述的步骤：  Further, the method includes the following steps:
1、 提取待测样本中的 DNA;  1. Extract DNA from the sample to be tested;
2、 以该 DNA为模板， 以针对 rs4552569碱基附近的编码区设计 PCR引 物进行 PCR反应， 得到 PCR反应产物； 2. Using the DNA as a template, design a PCR primer for the coding region near the rs4552569 base. Carrying out a PCR reaction to obtain a PCR reaction product;
3、 测出该 PCR反应产物的碱基序列组成；  3. Detecting the base sequence composition of the PCR reaction product;
4、 将得到的碱基序列与正常基因序列进行比较， 确定 rs4552569位点上 的碱基是否为 C。  4. Compare the obtained base sequence with the normal gene sequence to determine whether the base at the rs4552569 site is C.
在上述方法中， 从待测样品中得到的核苷酸序列的方法还包括荧光定量 PCR、 变性高效液相色谱、 限制性片段长度多态性、 连接酶检测或 DNA片段测 序。  In the above method, the method of obtaining the nucleotide sequence from the sample to be tested further includes fluorescent quantitative PCR, denaturing high performance liquid chromatography, restriction fragment length polymorphism, ligase detection or DNA fragment sequencing.
本发明申请的目的之一是提供对上述强直性脊柱炎特异性单核苷酸多态性 进行检测的方法， 所述的方法包括如下步骤：  One of the objects of the present application is to provide a method for detecting an ankylosing spondylitis-specific single nucleotide polymorphism, the method comprising the steps of:
1. 引物设计：  1. Primer design:
使用 Sequenom公司 Genotyping Tools及 MassARRAY Assay Design软件设 计 rs4552569位点的 PCR扩增引物及单碱基延伸引物；  PCR amplification primers and single base extension primers of rs4552569 site were designed using Sequenom Genotyping Tools and MassARRAY Assay Design software;
2. 组织、 细胞及血样 DNA提取：  2. Tissue, cells and blood samples DNA extraction:
使用 TIANamp Blood DNA kit血液基因组 DNA提取试剂盒或 NucleoSpin Tissue(MN)提取组织、 细胞或血样中的 DNA, 用分光光度计定量， 琼脂糖凝胶 电泳质检， 质检合格的 DNA将浓度调整到 5( ig/L, -20 °C储存备用；  DNA from tissues, cells or blood samples was extracted using the TIANamp Blood DNA kit or NucleoSpin Tissue (MN), quantified by spectrophotometry, agarose gel electrophoresis, and the quality of the qualified DNA was adjusted to 5 ( ig / L, -20 ° C storage standby;
3. PCR扩增：  3. PCR amplification:
采用多重 PCR扩增技术， 每个反应总体积 5μ1, 包含模板 DNA 10 η^ , Hotstar Taq 0.5 U, 扩增引物每条 0.5pmol, 25mM dNTP Ο. ΐμΐ,反应条件为 94 °C 4 分钟； 94 °C20 秒， 56 °C30 秒， 72 °C 1 分钟， 45个循环； 72 °C 3分钟； 4°C∞ ； Using multiplex PCR amplification technology, the total volume of each reaction is 5μ1, including template DNA 10 η^ , Hotstar Taq 0.5 U, amplification primers 0.5pmol each, 25mM dNTP Ο. ΐμΐ, reaction conditions are 94 °C for 4 minutes; 94 °C20 seconds, 56 °C for 30 seconds, 72 °C for 1 minute, 45 cycles; 72 °C for 3 minutes; 4 °C∞;
4. PCR产物纯化： 4. Purification of PCR products:
PCR反应产物使用 0.5 U SAP ( shrimp alkaline phosphatase )处理， 去除体 系中游离的 dNTP,反应体系 7 μΐ,其中 PCR产物 5 μΐ, SAP混合液 2 μΐ (SAP 0.5 U, buffer 0.17 μΐ), 反应程序 37°C 20分钟； 85 °C 5分钟； 40 °C ∞; The PCR reaction product was treated with 0.5 U SAP (shrimp alkaline phosphatase) to remove free dNTPs from the system. The reaction system was 7 μΐ, of which the PCR product was 5 μΐ, and the SAP mixture was 2 μΐ (SAP 0.5). U, buffer 0.17 μΐ), reaction procedure 37 ° C for 20 minutes; 85 ° C for 5 minutes; 40 °C ∞;
5. 单碱基延伸：  5. Single base extension:
总体积 9 μΐ反应体系包含 SAP处理后 PCR产物 7 μΐ, 其中各延伸反应引物 混合物 0.804 μΐ, iPLEX酶 0.041μ1, 延伸混合物 0.2 μΐ,反应程序为 94°C 30秒； 94 °C 5秒； 52 °C 5秒， 80 °C 5秒 5个循环； 返回 94 °C 5秒， 共 40个循环； 72 °C 3分钟， 4°C oo;  The total volume of 9 μΐ reaction system contains 7 μΐ of the PCR product after SAP treatment, wherein each extension reaction primer mixture is 0.804 μΐ, iPLEX enzyme is 0.041 μ1, the extension mixture is 0.2 μΐ, and the reaction procedure is 94 ° C for 30 seconds; 94 ° C for 5 seconds; °C 5 seconds, 80 °C 5 seconds 5 cycles; return 94 °C 5 seconds, a total of 40 cycles; 72 °C 3 minutes, 4 °C oo;
6. 树脂纯化：  6. Resin purification:
每个延伸反应产物用 6mg Clean Resin树脂纯化；  Each extended reaction product was purified with 6 mg of Clean Resin resin;
7. 芯片点样及质语检测：  7. Chip spotting and qualifier detection:
将纯化产物移至 384 孔 SpectroCHIP (Sequenom)芯片上进行测定， SpectroCHIP 芯 片 使 用 MALDI-TOF ( matrix-assisted laser desorption/ionization-time of flight )基质辅助激光解吸附电离飞行时间质谱分析， 检测结果使用 TYPER 4.0软件 ( sequenom )分型并输出结果；  The purified product was transferred to a 384-well SpectroCHIP (Sequenom) chip for measurement. The SpectroCHIP chip was analyzed by MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) matrix-assisted laser desorption ionization time-of-flight mass spectrometry. 4.0 software (sequenom) typing and outputting results;
8. 将 rs4552569位点检出的单碱基进行分析， 如果该位点的碱基为胞嘧啶 ( C ) , 则强直性脊柱炎的患病几率显著增大。  8. Analysis of the single base detected at the rs4552569 locus. If the base of the locus is cytosine (C), the risk of ankylosing spondylitis is significantly increased.
本发明申请的目的之一是提供检测强直性脊柱炎特异性单核苷酸多态性的 试剂盒， 该试剂盒包括： 针对 rs4552569碱基附近的区域设计的 PCR扩增引物、 延伸引物、 DNA聚合酶、 去离子水、 dNTP、 MgC12、 PCR緩沖液、 SAP、 clean resin树月旨。  One of the objects of the present application is to provide a kit for detecting a specific single nucleotide polymorphism of ankylosing spondylitis, the kit comprising: PCR amplification primer, extension primer, DNA designed for a region near the base of rs4552569 Polymerase, deionized water, dNTP, MgC12, PCR buffer, SAP, clean resin tree.
本发明申请的目的之一是提供上述强直性脊柱炎特异性单核苷酸多态性 ( SNP )在强直性脊柱炎治疗中的应用， 即 rs4552569位点上的碱基是 C时为风 险型， 是 T时为保护型在强直性脊柱炎治疗中的应用。  One of the objects of the present application is to provide the above-mentioned ankylosing spondylitis-specific single nucleotide polymorphism (SNP) in the treatment of ankylosing spondylitis, that is, when the base at the rs4552569 site is C, it is risky. , is the application of T in the treatment of ankylosing spondylitis.
进一步的， 所述强直性脊柱炎特异性单核苷酸多态性相关的易感或致病基 因是在 rs4552569位点上的碱基为胞嘧啶 C。 Further, the ankylosing spondylitis-specific single nucleotide polymorphism-associated susceptibility or pathogenicity Because the base at the rs4552569 locus is cytosine C.
上述的应用包括强直性脊柱炎特异性单核苷酸多态性（ SNP )相关的易感或 致病基因在制备治疗强直性脊柱炎的药物中的应用。  The above applications include the use of susceptibility or pathogenic genes associated with ankylosing spondylitis-specific single nucleotide polymorphism (SNP) in the preparation of a medicament for the treatment of ankylosing spondylitis.
具体来讲， 该应用包括通过干预 rs4552569 的相关的强直性脊柱炎的易感 或致病基因的功能， 包括翻译、 转录及蛋白质合成的过程， 制备成治疗药物， 改善患者的骨代谢异常以及体内的免疫炎症反应， 从而达到治疗目的。  Specifically, the application includes the function of susceptibility or pathogenic genes involved in the intervention of rs4552569, including translation, transcription, and protein synthesis processes, preparation of therapeutic drugs, improvement of bone metabolism abnormalities in patients, and in vivo The immune inflammatory response to achieve therapeutic goals.
具体实施方式 detailed description
以下结合具体的实施方式， 对本发明申请所述的强直性脊柱炎易感性检测 方法及其试剂盒进行描述， 目的是为了公众更好的理解本发明申请所述的技术 内容， 而不是对所述技术内容的限制， 事实上， 在以相同或近似的原理， 对所 述方法步骤、 试剂、 反应条件已经对所述试剂盒做出的任何改进， 以实现基本 相同的效果为目的， 则都在本发明申请所要求保护的技术方案之内。 实施例一  Hereinafter, the method for detecting susceptibility to ankylosing spondylitis and the kit thereof according to the present application are described in conjunction with specific embodiments, in order to better understand the technical content of the present application, but not for the public. Limitations of the technical content, in fact, on the same or similar principle, for the purpose that the method steps, reagents, and reaction conditions have been modified for the kit to achieve substantially the same effect, Within the technical solution claimed by the present application. Embodiment 1
SNP rs4552569与强直性脊柱炎的发病相关性研究一  Study on the relationship between SNP rs4552569 and the pathogenesis of ankylosing spondylitis
1.1 研究对象 1.1 Research object
研究中的 1837例散发 AS病例， 主要是 2005-2009年于中山大学附属第三医院 门诊就诊或住院的 AS患者， 这些 AS患者均是由经验丰富的风湿病学专家们根据 临床表现和影像学检查结果诊断的符合 1984年纽约修订标准的 AS患者。 对于每 个 AS患者， 在被告知之该研究目的之后同意抽血用于该研究， 并签署知情同意 书。  Among the 1837 cases of sporadic AS in the study, mainly AS patients who were admitted to the outpatient clinic of the Third Affiliated Hospital of Sun Yat-sen University from 2005 to 2009, these AS patients were based on clinical manifestations and imaging studies by experienced rheumatologists. The results of the diagnosis were diagnosed in patients with AS who met the revised 1984 New York standard. For each AS patient, blood was drawn for the study and the informed consent was signed after being informed of the purpose of the study.
4231例健康志愿者对照人群则主要包括了广东、安徽和新加坡的华人三个地 方的无 AS症状或病史的健康志愿者对照人群， 这些健康志愿者同样在被告知之 后同意抽血用于该研究， 并签署知情同意书。 The control population of 4231 healthy volunteers mainly included healthy volunteers without AS symptoms or medical history in three places in China, Guangdong, Anhui and Singapore. These healthy volunteers were also informed. Later agreed to draw blood for the study and signed informed consent.
收集病例时， 同时记录好病例和健康志愿者对照人群的基本情况， 包括： 性别、 年龄和籍贯。 抽取全血 4ml, 放于 EDTA管， 带回实验室提取 DNA。 另 外， 除了抽血， 风湿病专家还仔细询问并记录下每个 AS患者和健康志愿者对照 人群的情况， 尤其是 AS患者时还记录下基本情况（包括： 性别、 年龄、 发病年 龄、 病程）和临床表型 （包括： 腊样指、 髋关节受累、 外周关节炎、 炎性腰背 痛）。 而对于健康志愿者对照人群， 则仔细询问既往病史， 包括呼吸系统、 心血 管系统、 内分泌系统、 血液系统、 消化系统、 骨 几肉系统、 神经系统等各个 专科的情况， 另外重点排除风湿免疫科的病史， 询问是否有腰背痛等既往病史。 1.2 实验方法和结果  When collecting cases, the basic conditions of the case and healthy volunteers were recorded, including: gender, age and place of origin. 4 ml of whole blood was taken, placed in an EDTA tube, and brought back to the laboratory for DNA extraction. In addition, in addition to blood sampling, rheumatologists also carefully inquired and recorded the situation of each AS patient and healthy volunteers, especially the AS patients also recorded basic conditions (including: gender, age, age of onset, duration) And clinical phenotype (including: waxy fingers, hip involvement, peripheral arthritis, inflammatory low back pain). For healthy volunteers, the patient's medical history, including the respiratory system, cardiovascular system, endocrine system, blood system, digestive system, bone system, nervous system and other specialties, and the Department of Rheumatology History, ask if there is a history of low back pain and other past medical history. 1.2 Experimental methods and results
1.2.1 DNA的提取 1.2.1 Extraction of DNA
在该研究中，我们使用的是 TIANamp Blood DNA kit血液基因组 DNA提取试 剂盒， 从人的外周血样本中提取 DNA, 该试剂盒的组成是： 细胞裂解夜、 緩沖 液 GS、 緩沖液 GB、 緩沖液 GD、 漂洗液 PW, 洗脱緩沖液 TB、 蛋白酶 K、 吸附柱 CB3、 收集管 （2ml )、 1.5mL无菌收集管。  In this study, we used the TIANamp Blood DNA Kit Blood Genomic DNA Extraction Kit to extract DNA from human peripheral blood samples. The composition of the kit is: Cell Lysis Night, Buffer GS, Buffer GB, Buffer Liquid GD, rinse PW, elution buffer TB, proteinase K, adsorption column CB3, collection tube (2 ml), 1.5 mL sterile collection tube.
1.2.2.研究采用的密集型的全基因组的 SNP芯片 1.2.2. Intensive whole-genome SNP chip used in the study
研究采用了 Illumina HuamHap 610-Quad SNP 芯片、 Illumina Human lM-duo 芯片和 Illumina OmniExpress芯片对样本进行全基因组 SNP全扫， 下面是三种 Illumina芯片的筒介；  The study used the Illumina HuamHap 610-Quad SNP chip, the Illumina Human lM-duo chip and the Illumina OmniExpress chip to perform a full-genome SNP scan of the sample. The following are three types of Illumina chips;
1、 Illumina HuamHap 610-Quad SNP 芯片  1, Illumina HuamHap 610-Quad SNP chip
该芯片筒介： 该款芯片在一张芯片上可平行进行 4个样品的检测， 显著的 增加了样本输出信息量， 减少了实验操作中的误差。 该芯片广泛地采用了 HumanHap550芯片的内容，以及额外增加了 100,000个遗传标记。 Human 610-quad 涵盖的全基因组信息， 对于已知的和新近报道的 CNV区域来说都具有权威性。 Illumina HuamHap 610-Quad SNP 芯片的 SNPs以约 5kb的密度均匀分布在基因组 上。针对于基因组中高多态性的 CNV区域（片段复制区和无 SNP的基因组区域）， Illumina HuamHap 610-Quad SNP 芯片设计了一些特异的靶标来进行研究。 The chip cartridge: The chip can detect four samples in parallel on one chip, which significantly increases the amount of sample output information and reduces errors in experimental operations. The chip is widely used in the content of the HumanHap550 chip, with an additional 100,000 genetic markers. Human 610-quad The genome-wide information covered is authoritative for both known and recently reported CNV regions. The SNPs of the Illumina HuamHap 610-Quad SNP chip are evenly distributed on the genome at a density of about 5 kb. The Illumina HuamHap 610-Quad SNP chip was designed for specific studies targeting highly polymorphic CNV regions in the genome (fragment replication regions and genomic regions without SNPs).
Illumina Human lM-duo芯片。 Illumina Human lM-duo chip.
该芯片中包含了超过一百万个探针信息。 该款芯片门包含针对基因 SNPs, 标签 SNPs, 拷贝数变化（CNV ), 及其他高价值的基因组区域设计探针， 同时还 增加了一些新的位点， 如： 增加了疾病相关的 SNP位点， 灵活的选择一些基因组 编码区高密度的 SNP位点。  The chip contains more than one million probe information. The chip contains probes for gene SNPs, tagged SNPs, copy number changes (CNV), and other high-value genomic regions, as well as new sites such as: increased disease-associated SNP sites Flexible selection of high-density SNP sites in some genomic coding regions.
2、 Illumina OmniExpress芯片  2, Illumina OmniExpress chip
该芯片提供了高样品通量和全面的基因组内容， OmniExpress上可容纳 12 个样品， 芯片拷问每个样品的 70多万个变异， 单张芯片上总共有超过 800万个数 据点。 OmniExpress上的标志物是 Illumina的 HumanOmnil-Quad内容的一部分, 它从国际 HapMap计划所有三个阶段中选择的优化 SNP标签组合， 有了最高的数 据质量和最佳内容， 包括对拷贝数变异（CNV )应用的全面支持。  The chip provides high sample throughput and comprehensive genomic content. The OmniExpress can hold up to 12 samples. The chip interrogates more than 700,000 variations per sample, with a total of more than 8 million data points on a single chip. The marker on OmniExpress is part of Illumina's HumanOmnil-Quad content, which combines optimized SNP tag combinations from all three phases of the international HapMap program, with the highest data quality and best content, including copy number variation (CNV) ) Comprehensive support for the application.
1.2.3研究步骤 1.2.3 Research steps
我们通过上述三种芯片对所有的散发 AS病例以及健康志愿者对照进行全基 因组 SNP分型。 然后进行关联分析， 分析中对性别、 年龄和主成份分析结果的数 据分别进行校正， 寻找具有统计意义的 SNP(s), 即可能与疾病相关的 SNP(s)。 1.2.3.1 样本准备和基因分型  We performed a full genomic SNP typing on all sporadic AS cases and healthy volunteer controls using the above three chips. Correlation analysis is then performed, and the data for gender, age, and principal component analysis results are corrected separately to find statistically significant SNP(s), the SNP(s) that may be associated with the disease. 1.2.3.1 Sample preparation and genotyping
在从全血样本中提取 DNA后， 所有的 DNA均标准化为 50ng/ml的浓缩液。 对于高密度 SNP芯片分型， 我们根据 Illumina推荐的操作程序， 通过芯片， 我们 使用大约 200ng的 DNA进行样本的基因分型，并利用 Illumina Beadstudio进行数据 读取和基因型转换。 数据结果中有 272386个 SNP是 3个芯片都均有覆盖， 另外有 通过统计学分析后推算出 1083964个 SNP也用于分析。 After DNA was extracted from whole blood samples, all DNA was normalized to a 50 ng/ml concentrate. For high-density SNP chip typing, we used the chip, we used approximately 200 ng of DNA for genotyping the sample and used Illumina Beadstudio for data processing according to the Illumina recommended procedure. Read and genotype conversion. Among the data results, 272,386 SNPs were covered by all three chips. In addition, 1083964 SNPs were also calculated by statistical analysis.
1.2.3.2数据分析的质量监控 ( quality control, QC ) 1.2.3.2 Quality control (QC) of data analysis
研究对 AS病例、健康志愿者对照人群分别进行 SNP的质量控制和监控，其中 包括可读率（ call rate ) <95%, 次等位基因（ MAF , minor allele frequency ) <3%, 以及不符合哈迪 -温伯格平衡 (Hardy- Weinberg Equilibrium, HEW, P < 10-6) , 以 尽可能去除分型错误的 SNP; 在得到全扫的样本的基因型数据之后，我们分析并 计算杂合度， 取杂合度范围从杂合度平均值（Mean ) -3χ标准差 （ SD )到杂合 度平均值（Mean ) +3χ标准差 （ SD ) 区间内的样本资料进行统计学分析， 不在 这区间的样本将移除。 不纳入进一步的统计中。 然后， 我们利用 PLINK软件和 基于 IBS ( identical by state ) 的方法， 分别对这些数据进行遗传关系检验； 对于 那些基于遗传分析的重复或具有亲缘关系的一对标本， 我们去除其中一个可读 率较低的。 我们利用主成份分析 ( principle component analysis, PCA )方法， 以 及 HapMap数据库 z中人群数据， 检测样本中的人群分层现象， 并去除显著离群 的样本。 最后， 再取这几个独立数据的交集。  The study conducted quality control and monitoring of SNPs in AS cases and healthy volunteers, including call rate <95%, MAF (minor allele frequency) <3%, and non-conformity. Hardy-Weinberg Equilibrium (HEW, P < 10-6) to remove as much as possible SNPs; after obtaining the genotype data of the full-sweep sample, we analyze and calculate the heterozygosity The heterozygosity range is statistically analyzed from the mean of the heterozygosity mean (Mean) -3χ standard deviation (SD) to the mean heterozygosity (Mean) +3χ standard deviation (SD). Samples not in this interval Will be removed. Not included in further statistics. Then, we use PLINK software and IBS (isolated by state)-based methods to perform genetic relationship tests on these data. For those pairs of specimens based on repeated or related genetic analysis, we remove one of the read rates. low. We used the principle component analysis (PCA) method and the population data in the HapMap database to detect population stratification in the sample and remove significant outliers. Finally, take the intersection of these several independent data.
1.2.3.3统计学分析 1.2.3.3 Statistical analysis
对于全扫数据进行的分析， 由于存在一定程度的人群离散、 分层现象， 所有 成分分析（PCA ) 并得到 3个主成份， 分析这 3个组分之间是否存在样本聚类现 象， 以明确是否需要将这些样本的群体离散度作为校正的参数； 然后， 我们在 根据这些样本的数据绘制百分位图 （Quantile-Quantile plot ), 在绘制之前， 需要 对所有需要校正的因素进行校正， 这些因素包括性别、 年龄（如果散发病例和 健康志愿者对照人群间存在统计学上差异） 以及上述 PCA分析后如果存在聚类 现象的主成份， 结果会得到 GC值， 在分析数据结果时， 我们用软件将每个散发 病例的等位基因的频率与健康志愿者对照人群的基因频率进行卡方检验， 会得 到卡方值 A , 而
Figure imgf000012_0001
, 如果 QC越接近 1 , 即表示样本的效力越高， 如 果出现偏移， 则提示可能有两种情况： 一种是样本存在分层现象即人群离散较 为明显； 第二种是存在与疾病风险相关的 SNP。 For the analysis of full-scan data, due to the existence of a certain degree of population dispersion and stratification, all component analysis (PCA) and three principal components are obtained, and whether there is sample clustering between the three components is analyzed. Is it necessary to use the population dispersion of these samples as a parameter for correction; then, we draw a Quantile-Quantile plot based on the data from these samples, and all the factors that need to be corrected need to be corrected before drawing. Factors include gender, age (if there is a statistical difference between sporadic cases and healthy volunteers) and if there is clustering after PCA analysis above The main component of the phenomenon, the result will get the GC value. When analyzing the data results, we use the software to perform the chi-square test on the frequency of the alleles of each sporadic case and the gene frequency of the healthy volunteer control population, and the chi-square value will be obtained. A , and
Figure imgf000012_0001
If the QC is closer to 1, it means that the sample is more effective. If there is an offset, there are two possible situations: one is that the sample is stratified, that is, the population is more discrete; the second is the existence and disease risk. Related SNPs.
经过上述分析后，如果 PCA分析结果提示主成份之间存在分层现象， 即存在 人群离散度的差异， 我们将该存在差异的离散度也作为协变量进行校正， 此外 我们将年龄和性别 （如果散发病例和健康志愿者对照人群间存在差异）作为协 变量， 利用 PLINK软件进行 logistic回归分析， 寻找与疾病关联的 SNP位点。 我们 用 Haploview (v4.1)软件进行分析结果的展示， 即 SNP染色体位置及其 -loglOP表 示的 Manhattan图。  After the above analysis, if the PCA analysis results suggest that there is stratification between the main components, that is, there is a difference in the dispersion of the population, we also correct the dispersion of the difference as a covariate, in addition we will age and gender (if Differences between sporadic cases and healthy volunteers were used as covariates. Logistic regression analysis was performed using PLINK software to find SNP loci associated with disease. We use Haploview (v4.1) software to display the results of the analysis, namely the SNP chromosome location and the Manhattan map represented by -loglOP.
1.2.3.4 SNP的推算及连锁不平衡模式分析  1.2.3.4 SNP estimation and linkage disequilibrium model analysis
对 3个芯片中的用于分析的推算 SNP是通过 IMPUTE2软件（版本号： 2.1.0 ) 来完成的， 使芯片对染色体的覆盖范围达到最大化。  The estimated SNP for analysis in the three chips is done by the IMPUTE2 software (version number: 2.1.0), which maximizes the chip's coverage of the chromosome.
1.2.4结果 1.2.4 Results
SNP rs4552569在 1837例 AS和 4231例健康志愿者对照中存在明显差异， P值 SNP rs4552569 showed significant differences in 1837 AS and 4231 healthy volunteer controls, P value
=6.91 x 10— 5, 即与强直性脊柱炎存在明显相关性， 该 SNP碱基是 C时为风险型， T 时为保护型。 = 6.91 x 10- 5, i.e., the presence of significant correlation ankylosing spondylitis, C is the SNP nucleotide risk type, when T is a protected type.
上述结果表明， 36.3Genome Buildde人类组基因图谱中的 5号染色体的 83209349位的 SNP rs4552569中， 碱基 T→C改变， 增加了 AS的易感性， 等位基因 型 C与 AS的发生显著相关。 SNP rs4552569与强直性脊柱炎的发病相关性研究二 The above results indicated that in the SNP rs4552569 of the 83209349 locus on chromosome 5 in the 36.3 Genome Buildde human genome map, the base T→C changed, increasing the susceptibility of AS, and the allele type C was significantly correlated with the occurrence of AS. SNP rs4552569 and the incidence of ankylosing spondylitis
2.1 研究对象 2.1 Research object
研究中的 2205例散发 AS病例， 主要是 2005-2009年于中山大学附属第三医院 门诊就诊或住院的 AS患者及部分其它医院取得的样本， 这些 AS患者均是由经验 丰富的风湿病学专家们根据临床表现和影像学检查结果诊断的符合 1984年纽约 修订标准的 AS患者。 对于每个 AS患者， 在被告知之该研究目的之后同意抽血用 于该研究， 并签署知情同意书。  2205 cases of sporadic AS in the study were mainly obtained from AS patients and some other hospitals in the outpatient clinic of the Third Affiliated Hospital of Sun Yat-sen University from 2005 to 2009. These AS patients were all experienced rheumatologists. AS patients who were diagnosed according to clinical manifestations and imaging findings in accordance with the 1984 New York revision criteria. For each AS patient, consent was drawn to the study and an informed consent was signed after being informed of the purpose of the study.
3702例健康志愿者对照人群则主要来自中山大学附属第三医院及中山大学 肿瘤防治中心收集的健康志愿者人群， 该人群均为无 AS症状或病史的的健康志 愿者对照人群， 这些健康志愿者同样在被告知之后同意抽血用于该研究， 并签 署知情同意书。  The 3702 healthy volunteers were mainly from the healthy volunteers collected by the Third Affiliated Hospital of Sun Yat-sen University and the Cancer Center of Sun Yat-sen University. The population were healthy volunteers without AS symptoms or medical history. These healthy volunteers Also, after being informed, agreed to draw blood for the study and signed an informed consent form.
2.2 实验方法和结果 2.2 Experimental methods and results
2.2.1 DNA的提取 2.2.1 Extraction of DNA
在该研究中，我们使用的是 TIANamp Blood DNA kit血液基因组 DNA提取试 剂盒从人的外周血样本中提取 DNA, 该试剂盒的组成是： 细胞裂解夜、 緩沖液 GS、緩沖液 GB、緩沖液 GD、漂洗液 PW,洗脱緩沖液 TB、蛋白酶 K、吸附柱 CB3、 收集管 （2ml )、 1.5mL无菌收集管。  In this study, we used the TIANamp Blood DNA Kit Blood Genomic DNA Extraction Kit to extract DNA from human peripheral blood samples. The composition of the kit is: Cell Lysis Night, Buffer GS, Buffer GB, Buffer GD, rinse solution PW, elution buffer TB, proteinase K, adsorption column CB3, collection tube (2 ml), 1.5 mL sterile collection tube.
2.2.2研究步骤 2.2.2 Research steps
2.2.2.1引物设计 2.2.2.1 Primer design
用 Sequenom公司 Massarray® DNA质谱阵列基因分析系统的 iPLEX® Gold snp基因型分析技术进行引物设计（包括扩增引物和延伸引物）。 SNP rs4552569 的引物信息如下：  Primer design (including amplification primers and extension primers) was performed using the iPLEX® Gold snp genotyping technique of the Sequenom Massarray® DNA Mass Spectrometry System. Primer information for SNP rs4552569 is as follows:
为区别扩增引物和延伸引物， 提高 PCR效率和质语质量， 在扩增引物的 5， 端加上了一个十个碱基的标记， 即 5，-ACGTTGGATG-3，。 并在下列实验条件下 进行扩增延伸及分析。 In order to distinguish between amplification primers and extension primers, to improve PCR efficiency and quality of the proverb, in the amplification of primers 5, A ten base mark is added to the end, namely 5,-ACGTTGGATG-3. Amplification extension and analysis were carried out under the following experimental conditions.
Figure imgf000014_0001
Figure imgf000014_0001
2.2.2.2基因组 DNA扩增 （聚合酶链式反应 PCR ):  2.2.2.2 Genomic DNA amplification (polymerase chain reaction PCR):
2.2.2.2.1 反应体系如下：  2.2.2.2.1 The reaction system is as follows:
Figure imgf000014_0002
Figure imgf000014_0002
2.2.2.2.2 PCR步骤： 2.2.2.2.2 PCR steps:
在 384孔板中每孔加入模板 DNAlul, 再加入 4ul上述反应体系， 将 384孔板 1000转离心 1分钟，混匀后按照下列程序进行扩增： 94。 C 4 minutes预变性， 94。 C 20 秒， 56° C 30秒， 72° C 1 分钟共 45个循环， 72° C 3 minutes, 4° C 冷却。  The template DNAlul was added to each well in a 384-well plate, and 4 ul of the above reaction system was added. The 384-well plate was centrifuged at 1000 rpm for 1 minute, and after mixing, amplification was carried out according to the following procedure: 94. C 4 minutes pre-denaturation, 94. C 20 seconds, 56 ° C 30 seconds, 72 ° C 1 minute for a total of 45 cycles, 72 ° C 3 minutes, 4 ° C cooling.
2.2.2.3 质谱分析： 2.2.2.3 Mass spectrometry:
基质辅助激光解吸电离飞行时间质谱 （MALDI-TOF MS) 的主要特点是，先 通过 PCR扩增目标序列， 然后加入 snp序列特异延伸引物， 在 SNP位点上， 延 伸 1个碱基。 将制备的样品分析物与芯片基质共结晶， 将该晶体放入质谱仪的 真空管 , 而后用瞬时纳秒 （l(T9s) 强激光激发， 由于基质分子经辐射所吸收的 能量， 导致能量蓄积并迅速产热， 从而使基质晶体升华， 核酸分子就会解吸附 并转变为亚稳态离子， 产生的离子多为单电荷离子， 这些单电荷离子在加速电 场中获得相同的动能， 进而在一非电场漂移区内按照其质荷比率加以分离， 在 真空小管中飞行到达检测器。 MALDI产生的 离子常用 飞行时间 ( Time-of-Flight,TOF )检测器来检测， 离子质量越小， 就越快到达。 用该方法 可快捷方便的检测出上述 PCR反应产物中 SNP的基因型。 The main feature of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is that The target sequence was amplified by PCR, and then the snp sequence-specific extension primer was added, and one base was extended at the SNP site. The prepared sample analyte is co-crystallized with the chip substrate, and the crystal is placed in a vacuum tube of the mass spectrometer, and then excited by a transient nanosecond (1 (T 9 s) strong laser, resulting in energy due to the energy absorbed by the matrix molecules through the radiation. Accumulating and rapidly producing heat, so that the matrix crystals sublimate, the nucleic acid molecules are desorbed and converted into metastable ions, and the generated ions are mostly singly charged ions, which obtain the same kinetic energy in the accelerating electric field, and then A non-electric field drift region is separated according to its mass-to-charge ratio, and flies in a vacuum tube to reach the detector. The ions generated by MALDI are commonly detected by a Time-of-Flight (TOF) detector, and the smaller the ion mass, the smaller the ion mass is. The faster the arrival, the genotype of SNP in the above PCR reaction product can be quickly and conveniently detected by this method.
2.2.2.4 统计分析 2.2.2.4 Statistical analysis
研究对 AS病例、 健康志愿者对照人群分别进行 SNP的质量控制和监控， 其 中包括可读率（ call rate ) <95%, 次等位基因 （ MAF, minor allele frequency ) <3%, 以及不符合哈迪 -温伯格平衡 (Hardy- Weinberg Equilibrium, HEW, P < 10-6) 的样本将不用于进一步分析。 通过质量控制后， 2,100例患者和 3,496 例健康对 照通过了质量控制， 用于进一步的统计分析。 利用 PLINK软件及 R方法对验证的 样本及所有的样本进行 logistic回归分析， 寻找与疾病关联的 SNP位点。 并用 Haploview (v4.1)软件绘制 SNP位点所在区域的连锁图。  The study conducted quality control and monitoring of SNPs in AS cases and healthy volunteers, including call rate <95%, minor allele frequency (MAF) <3%, and non-conformity. Samples of Hardy-Weinberg Equilibrium, HEW, P < 10-6 will not be used for further analysis. After quality control, 2,100 patients and 3,496 healthy controls passed quality control for further statistical analysis. Logistic regression analysis was performed on the validated samples and all samples using PLINK software and R method to find SNP loci associated with the disease. The linkage map of the region where the SNP locus is located is drawn by Haploview (v4.1) software.
2.2.3 结果 2.2.3 Results
SNP rs4552569在 2205例 AS和 3702例健康志愿者对照中存在明显差异， P值 =2.92χ10"06, 即与强直性脊柱炎存在明显相关性， 该 SNP碱基是 C时为风险型， Τ 时为保护型。 上述结果表明， 36.3Genome Buildde人类组基因图谱中的 5 号染色体的 83209349位的 SNP rs4552569中， 碱基 T→C改变， 增加了 AS的易感性， 等位 基因型 C与 AS的发生显著相关。 实施例三 There were significant differences between SNP rs4552569 in 2205 AS and 3702 healthy volunteer controls. P value=2.92χ10" 06 , which was significantly associated with ankylosing spondylitis. The SNP base was risky when C, Τ For protection type. The above results indicated that in the SNP rs4552569 of 83209349 located on chromosome 5 of the 36.3 Genome Buildde human genome, the base T→C changed, increasing the susceptibility of AS, and the allele C was significantly correlated with the occurrence of AS. Embodiment 3
3.1.1研究对象 3.1.1 Research object
研究中的对象是所有实施例 1和实施例 2中的 AS人群和健康对照人群， 这些 AS病例， 均是由经验丰富的风湿病学专家们根据临床表现和影像学检查结果诊 断的符合 1984年纽约修订标准的 AS患者。 对于每个 AS患者， 在被告知之该研究 目的之后同意抽血用于该研究， 并签署知情同意书。 最终， 共计有 3937例 AS患 者和 7727例健康志愿者对照纳入该研究。  The subjects in the study were all AS and healthy controls in Examples 1 and 2, and these AS cases were diagnosed by experienced rheumatologists based on clinical findings and imaging findings. New York revised standard AS patients. For each AS patient, blood was drawn for use in the study and the informed consent was signed after being informed of the purpose of the study. Finally, a total of 3937 AS patients and 7727 healthy volunteers were included in the study.
3.2.2 DNA的提取 3.2.2 Extraction of DNA
在对待检测的标本， 使用 TIANamp Blood DNA kit血液基因组 DNA提取试 剂盒， 从人的外周血样本中提取 DNA, 该试剂盒的组成是： 细胞裂解夜、 緩沖 液 GS、 緩沖液 GB、 緩沖液 GD、 漂洗液 PW, 洗脱緩沖液 TB、 蛋白酶 K、 吸附柱 CB3、 收集管 ( 2ml )和 1.5mL无菌收集管。  The TIANamp Blood DNA kit is used to extract DNA from human peripheral blood samples. The composition of the kit is: Cell Lysis Night, Buffer GS, Buffer GB, Buffer GD , rinse solution PW, elution buffer TB, proteinase K, adsorption column CB3, collection tube (2 ml) and 1.5 mL sterile collection tube.
3.2.3 实验步骤 3.2.3 Experimental steps
我们将经过数据质量控制后的实施例一中的所有 AS患者和健康对照的有关 SNP rs4552569 的数据提取出来，同时提出经过数据质量控制后的实施例二中的 所有 AS患者和健康对照的有关 SNP rs4552569 的数据； 经过整理放入同一数据 库进行分析。统计学分析则是利用 PLINK软件所有的样本的 SNP rs4552569 的数 据进行 logistic回归分析， 并且校正了年龄、 性别等干扰因素， 从而寻找 SNP rs4552569 的数据与疾病间是否存在关联。 3.3 实验结果： We extracted data on SNP rs4552569 from all AS patients and healthy controls in Example 1 after data quality control, and presented relevant SNPs for all AS patients and healthy controls in Example 2 after data quality control. Rs4552569 data; after sorting into the same database for analysis. Statistical analysis was performed using logistic regression analysis of SNP rs4552569 data from all samples of PLINK software, and the interference factors such as age and gender were corrected to find out whether there was any correlation between SNP rs4552569 data and disease. 3.3 Experimental results:
结果 SNP rs4552569在 AS患者和健康志愿者对照中存在明显差异， P值 =8.77χ 10"10, 即 SNP rs4552569位点上的碱基与强直性脊柱炎存在明显相关性， 该 SNP碱基是 C时为风险型， T时为保护型。 结果与实施例一和实施例二的研究 结果均一致。 实施例四 RESULTS: SNP rs4552569 was significantly different between AS patients and healthy volunteers. P value=8.77χ 10" 10 , ie, the base of SNP rs4552569 was significantly associated with ankylosing spondylitis. The SNP base is C. The time is risk type, and T is protection type. The results are consistent with the results of the first embodiment and the second embodiment.
4.1.1 研究对象 4.1.1 Research object
所有欲检测是否携带有强直性脊柱炎易感基因的患者及家属。  All patients and their families who want to test for the presence of ankylosing spondylosis susceptibility genes.
4.2.2 DNA的提取 4.2.2 DNA extraction
在对待检测的标本， 使用 TIANamp Blood DNA kit血液基因组 DNA提取试 剂盒， 从人的外周血样本中提取 DNA, 该试剂盒的组成是： 细胞裂解夜、 緩沖 液 GS、 緩沖液 GB、 緩沖液 GD、 漂洗液 PW, 洗脱緩沖液 TB、 蛋白酶 K、 吸附柱 CB3、 收集管 （2ml )、 1.5ml无菌收集管。  The TIANamp Blood DNA kit is used to extract DNA from human peripheral blood samples. The composition of the kit is: Cell Lysis Night, Buffer GS, Buffer GB, Buffer GD , rinse solution PW, elution buffer TB, proteinase K, adsorption column CB3, collection tube (2 ml), 1.5 ml sterile collection tube.
4.2.3 实验步骤 4.2.3 Experimental steps
4.2.3.1 PCR步骤： 4.2.3.1 PCR steps:
在待检测板中每孔加入模板 DNAlul, 再加入 4ul上述反应体系， 1000转离 心 1分钟， 混匀后按照下列程序进行扩增： 94。 C 4 minutes预变性， 94。 C 20 秒， 56° C 30秒， 72° C 1 分钟共 45个循环， 72° C 3 minutes, 4° C 冷却。  The template DNAlul was added to each well of the plate to be tested, and 4 ul of the above reaction system was added, and centrifuged at 1000 rpm for 1 minute. After mixing, the amplification was carried out according to the following procedure: 94. C 4 minutes pre-denaturation, 94. C 20 seconds, 56 ° C 30 seconds, 72 ° C 1 minute for a total of 45 cycles, 72 ° C 3 minutes, 4 ° C cooling.
3.2.3.2质谱分析： 3.2.3.2 Mass spectrometry:
经树脂纯化后， 将制备的样品分析物与芯片基质共结晶， 将该晶体放入质 语仪的真空管 , 而后用瞬时纳秒 （10-9s) 强激光激发， 用该方法可快捷方便的 检测出上述 PCR^应产物中 SNP的基因型。 4.3 实验结果： After purification by the resin, the prepared sample analyte is co-crystallized with the chip substrate, and the crystal is placed in a vacuum tube of the grammar instrument, and then excited by a transient nanosecond (10-9 s) strong laser, which can be quickly and conveniently detected by the method. The genotype of the SNP in the above PCR product was determined. 4.3 Experimental results:
检测结果为 C时为风险型， 检测结果为 T时为保护型。  When the test result is C, it is a risk type, and when the test result is T, it is a protection type.
该试剂盒包括： 针对 rs4552569碱基附近的区域设计的 PCR扩增引物、 延伸 引物、 DNA聚合酶、 去离子水、 dNTP、 MgC12、 PCR緩沖液、 SAP、 clean resin 树脂。 其中扩增引物、 延伸引物如下表所示：  The kit includes: PCR amplification primers, extension primers, DNA polymerase, deionized water, dNTP, MgC12, PCR buffer, SAP, clean resin designed for the region near rs4552569 base. The amplification primers and extension primers are shown in the following table:
Figure imgf000018_0001
检测方法步骤如下：
Figure imgf000018_0001
The steps of the detection method are as follows:
1. 引物设计：  1. Primer design:
使用 Sequenom公司 Genotyping Tools及 MassARRAY Assay Design软件设 计待测 SNP位点的 PCR扩增引物及单碱基延伸引物；  PCR amplification primers and single base extension primers for SNP sites to be tested using Sequenom Genotyping Tools and MassARRAY Assay Design software;
2. 组织、 细胞及血样 DNA提取：  2. Tissue, cells and blood samples DNA extraction:
使用 TIANamp Blood DNA kit血液基因组 DNA提取试剂盒或 NucleoSpin Tissue(MN)提取组织、 细胞或血样中的 DNA, 用分光光度计定量， 琼脂糖凝胶 电泳质检， 质检合格的 DNA将浓度调整到 5( ig/L, -20 °C储存备用；  DNA from tissues, cells or blood samples was extracted using the TIANamp Blood DNA kit or NucleoSpin Tissue (MN), quantified by spectrophotometry, agarose gel electrophoresis, and the quality of the qualified DNA was adjusted to 5 ( ig / L, -20 ° C storage standby;
3. PCR扩增：  3. PCR amplification:
采用多重 PCR扩增技术， 每个反应总体积 5μ1, 包含模板 DNA 10 g, Hotstar Taq 0.5 U, 扩增引物每条 0.5pmol, 25mM dNTP Ο.ΐμΐ,反应条件为 94 °C 4 分钟； 94 °C20 秒， 56 °C30 秒， 72 °C 1 分钟， 45个循环； 72 °C 3分钟； 4°C∞ ； Using multiplex PCR amplification technology, the total volume of each reaction is 5μ1, including template DNA 10 g, Hotstar Taq 0.5 U, amplification primers 0.5pmol each, 25mM dNTP Ο.ΐμΐ, reaction conditions 94 °C 4 minutes; 94 ° C20 seconds, 56 °C for 30 seconds, 72 °C for 1 minute, 45 cycles; 72 °C for 3 minutes; 4 °C∞;
4. PCR产物纯化： 4. Purification of PCR products:
PCR反应产物使用 0.5 U SAP ( shrimp alkaline phosphatase )处理， 去除体 系中游离的 dNTP,反应体系 7 μΐ,其中 PCR产物 5 μΐ, SAP混合液 2 μΐ (SAP 0.5 U, buffer 0.17 μΐ), 反应程序 37 °C 20分钟； 85 °C 5分钟； 40 °C ∞; The PCR reaction product was treated with 0.5 U SAP (shrimp alkaline phosphatase) to remove the body. Free dNTP in the system, 7 μΐ reaction system, 5 μΐ PCR product, 2 μΐ SAP solution (SAP 0.5 U, buffer 0.17 μΐ), reaction procedure at 37 °C for 20 minutes; 85 °C for 5 minutes; 40 °C ∞ ;
5. 单碱基延伸：  5. Single base extension:
总体积 9 μΐ反应体系包含 SAP处理后 PCR产物 7 μΐ, 其中各延伸反应引物 混合物 0.804 μΐ, iPLEX酶 0.041μ1, 延伸混合物 0.2 μΐ, 反应程序为 94°C 30秒； 94 °C 5秒； 52°C 5秒， 80°C 5秒 5个循环； 返回 94 °C 5秒， 共 40个循环； 72°C 3分钟， 4°C oo;  The total volume of 9 μΐ reaction system contains 7 μΐ of the PCR product after SAP treatment, wherein each extension reaction primer mixture is 0.804 μΐ, iPLEX enzyme is 0.041 μ1, the extension mixture is 0.2 μΐ, and the reaction procedure is 94 ° C for 30 seconds; 94 ° C for 5 seconds; °C 5 seconds, 80 °C 5 seconds 5 cycles; return 94 °C 5 seconds, a total of 40 cycles; 72 °C 3 minutes, 4 °C oo;
6. 树脂纯化：  6. Resin purification:
每个延伸反应产物用 6mg Clean Resin树脂纯化；  Each extended reaction product was purified with 6 mg of Clean Resin resin;
7. 芯片点样及质语检测：  7. Chip spotting and qualifier detection:
将纯化产物移至 384孔 SpectroCHIP (Sequenom)芯片上进行测定， SpectroCHIP芯 片使用 MALDI-TOF ( matrix-assisted laser desorption/ionization— time of flight )基 质辅助激光解吸附电离飞行时间质谱分析， 检测结果使用 TYPER 4.0软件 ( sequenom )分型并输出结果； The purified product was transferred to a 384-well SpectroCHIP (Sequenom) chip for measurement. The SpectroCHIP chip was analyzed by MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) matrix-assisted laser desorption ionization time-of-flight mass spectrometry. 4.0 software (sequenom) typing and outputting results;
8. 将 rs4552569位点检出的单碱基进行分析， 如果该位点的碱基为胞嘧啶， 则强直性脊柱炎的患病几率显著增大。  8. Analysis of the single base detected at the rs4552569 locus. If the base of the locus is cytosine, the risk of ankylosing spondylitis is significantly increased.

Claims

1、 一种强直性脊柱炎致病相关性单核苷酸多态性的核苷酸片段, 其特征在 于-.该核苷酸片段在 rs4552569位点上，强直性脊柱炎患者碱基为 C的频率显著 高于健康对照， 相反， 该位点上碱基为 T的频率则显著低于健康对照。 A nucleotide fragment of a disease-associated single nucleotide polymorphism of ankylosing spondylitis, characterized in that - the nucleotide fragment is at the rs4552569 site, and the base of the ankylosing spondylitis patient is C The frequency was significantly higher than that of healthy controls. Conversely, the frequency of base T at this locus was significantly lower than that of healthy controls.
2、 一种对强直性脊柱炎进行早期辅助性诊断的方法， 其特征在于， 所述的 方法是通过下述方案实现的：从待测样品中得到核苷酸序列，检测位于 rs4552569 位点上的碱基是否为 C,如果该位点的碱基为 C， 则强直性脊柱炎的患病几率增 大。  2. A method for early auxiliary diagnosis of ankylosing spondylitis, characterized in that the method is achieved by: obtaining a nucleotide sequence from a sample to be tested, and detecting at a position of rs4552569 Whether the base is C or not, if the base of the site is C, the incidence of ankylosing spondylitis increases.
3、 根据权利要求 2所述的方法， 其特征在于： 所述的样品包括血液、 体液 或组织。  3. A method according to claim 2 wherein: said sample comprises blood, body fluids or tissue.
4、根据权利要求 2所述的方法， 其特征在于： 所述的方法包括下述的步骤: 4. The method of claim 2 wherein: said method comprises the steps of:
1) 提取待测样本中的 DNA; 1) extracting DNA from the sample to be tested;
2) 以该 DNA为模板， 以针对 rs4552569碱基附近的编码区设计 PCR引物 进行 PCR反应， 得到 PCR反应产物；  2) using the DNA as a template, designing PCR primers for the coding region near the base of rs4552569 for PCR reaction, and obtaining a PCR reaction product;
3) 测出该 PCR反应产物的碱基序列组成；  3) measuring the base sequence composition of the PCR reaction product;
4) 将得到的碱基序列与正常基因序列进行比较， 确定 rs4552569位点上的 碱基是否为（。  4) Compare the obtained base sequence with the normal gene sequence to determine whether the base at the rs4552569 site is (.
5、 根据权利要求 2所述的方法， 其特征在于： 所述的从待测样品中得到的 核苷酸序列的方法还包括荧光定量 PCR、 变性高效液相色谱、 限制性片段长度 多态性、 连接酶检测或 DNA片段测序。 '  5. The method according to claim 2, wherein: the method for obtaining a nucleotide sequence obtained from the sample to be tested further comprises real-time PCR, denaturing high performance liquid chromatography, restriction fragment length polymorphism , ligase detection or sequencing of DNA fragments. '
6、 一种强直性脊柱炎特异性单核苷酸多态性的检测方法， 其特征在于： 所 述的检测方法包括如下步骤：  6. A method for detecting a specific single nucleotide polymorphism of ankylosing spondylitis, characterized in that: the detection method comprises the following steps:
1 ) 引物设计：  1) Primer design:
使用 Sequenom公司 Genotyping Tools及 MassAR AY Assay Design软件设 更正页 （细则第 91条） 计 rs4552569位点的 PCR扩增引物及单碱基延伸引物； Correction page using Sequenom's Genothing Tools and MassAR AY Assay Design software (Article 91) a PCR amplification primer and a single base extension primer of rs4552569;
2 ) 组织、 细胞及血样 DNA提取：  2) Tissue, cell and blood samples DNA extraction:
使用 TIANamp Blood DNA kit血液基因组 DNA提取试剂盒或 NucleoSpin Tissue(M )提取组织、 细胞或血样中的 DNA， 用分光光度计定量， 琼脂糖凝胶 电泳质检， 质检合格的 DNA将浓度调整到 5( ig/L, - 20°C储存备用；  DNA from tissues, cells or blood samples was extracted using the TIANamp Blood DNA kit or NucleoSpin Tissue (M), quantified by spectrophotometry, agarose gel electrophoresis, and the quality of the qualified DNA was adjusted to 5 ( ig / L, - 20 ° C storage standby;
3 ) PCR扩增：  3) PCR amplification:
采； 多重 PCR扩增技术,每个反应总体积 5μ1, 包含模板 DNA 10 η^, Hotstar Taq 0.5 U, 扩增引物每条 0.5pmol, 25mM dNTP 0.1 μΐ ,反应条件为 94 °C 4分钟； 94 °C20秒， 56 "C30秒， 72 °C 1 分钟， 45个循环； 72°C 3分钟； 4°C∞ ； Multiplex PCR amplification technique, total reaction volume 5μ1, containing template DNA 10 η^, Hotstar Taq 0.5 U, amplification primers 0.5pmol each, 25mM dNTP 0.1 μΐ, reaction conditions 94 °C 4 minutes; 94 °C20 seconds, 56 "C30 seconds, 72 °C 1 minute, 45 cycles; 72 °C 3 minutes; 4 °C∞;
4 ) PCR产物纯化： 4) Purification of PCR products:
PCR反应产物使用 0.5 U SAP ( shrimp alkaline phosphatase )处理， 去除体 系中游离的 dNTP,反应体系 7 μΐ，其中 PCR产物 5 μΐ, SAP混合液 2 μΐ (SAP 0.5 U， buffer 0.17 μ1)， 反应程序 37°C 20分钟； 85°C 5分钟； 40 °G ∞;  The PCR reaction product was treated with 0.5 U SAP (shrimp alkaline phosphatase) to remove free dNTPs from the system. The reaction system was 7 μΐ, of which the PCR product was 5 μΐ, the SAP mixture was 2 μΐ (SAP 0.5 U, buffer 0.17 μ1), and the reaction procedure was 37. °C 20 minutes; 85 °C 5 minutes; 40 °G ∞;
5 ) 单碱基延伸 ··  5) Single base extension ··
总体积 9 μΐ反应体系包含 SAP处理后 PCR产物 7 μΐ, 其中各延伸反应引物 混合物 0.804 μΐ, iPLEX酶 0.041μ1,延伸混合物 0.2 μΐ, 反应程序为 94°C 30秒； 94V 5秒； 52°C 5秒， 80°C 5秒 5个循环； 返回 94°C 5秒， 共 40个循环； 72°C 3分钟， 4°C oo;  The total volume of 9 μΐ reaction system contains 7 μΐ of the PCR product after SAP treatment, wherein each extension reaction primer mixture is 0.804 μΐ, iPLEX enzyme is 0.041 μ1, the extended mixture is 0.2 μΐ, and the reaction procedure is 94 ° C for 30 seconds; 94 V 5 seconds; 52 ° C 5 seconds, 80 ° C 5 seconds 5 cycles; return 94 ° C 5 seconds, a total of 40 cycles; 72 ° C 3 minutes, 4 ° C oo;
6 ) 树脂纯化： '  6) Resin purification: '
每个延伸反应产物用 6mg Clean Resin树脂纯化；  Each extended reaction product was purified with 6 mg of Clean Resin resin;
7 ) 芯片点样及质谱检测：  7) Chip spotting and mass spectrometry detection:
将纯化产物移至 384 孔 SpectroCfflP (Sequenom)芯片上进行测定， SpectroCfflP 芯 片 使 用 MALDI-TOF ( matrix-assisted laser 更正页 （细则第 91条） desorption/ionization-time of flight )基质辅助激光解吸附电离飞行时间质普分析， 检测结果使用 TYPER 4.0软件 ( sequenom )分型并输出结果； The purified product was transferred to a 384-well SpectroCfflP (Sequenom) chip for measurement, and the SpectroCfflP chip was subjected to MALDI-TOF (matrix-assisted laser correction page (Rule 91) Desorption/ionization-time of flight) Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis, using TYPER 4.0 software (sequenom) for typing and outputting results;
8 )将 rs4552569位点检出的单碱基进行分析， 如果该位点的碱基为胞嘧啶， 则强直性脊柱炎的患病几率显著增大。  8) Analysis of the single base detected at the rs4552569 locus. If the base of the locus is cytosine, the risk of ankylosing spondylitis is significantly increased.
7、 一种强直性脊柱炎特异性单核苷酸多态性检测的试剂盒， 其特征在于： 所述的试剂盒包括： 针对 rs4552569碱基附近的区域设计的 PCR扩增引物、 延 伸引物、 DNA聚合酶、去离子水、 dNTP、 MgC12、 PCR緩冲液、 SAP和 clean resin 树脂。  7. A kit for detecting specific single nucleotide polymorphism of ankylosing spondylitis, characterized in that: the kit comprises: a PCR amplification primer designed to target a region near the base of rs4552569, an extension primer, DNA polymerase, deionized water, dNTP, MgC12, PCR buffer, SAP and clean resin.
8、 权利要求 1所述的强直性脊柱炎致病相关性单核苷酸多态性在强直性脊 柱炎治疗中的应用。  8. The use of a disease-associated single nucleotide polymorphism of ankylosing spondylitis according to claim 1 in the treatment of ankylosing spondylitis.
9、 根据权利要求 8所述的应用， 其特征在于： 所述的应用包括强直性脊柱 炎特异性单核苷酸多态性相关的易感或致病基因在制备治疗强直性脊柱炎的药 物中的应用。  9. The use according to claim 8, wherein: the application comprises a susceptibility or pathogenic gene associated with ankylosing spondylitis-specific single nucleotide polymorphism in the preparation of a medicament for treating ankylosing spondylitis Application in .
10、 根据权利要求 9所述的应用， 其特征在于： 所述强直性脊柱炎特异性 单核苷酸多态性相关的易感或致病基因为 rs4552569位点上的碱基为胞嘧啶。  10. The use according to claim 9, wherein the susceptibility or pathogenic gene associated with the ankylosing spondylitis-specific single nucleotide polymorphism is a cytosine at the rs4552569 site.
11、 根据权利要求 8 所述的应用， 其特征在于： 所述的应用包括通过干预 rs455256.9的相关的强直性脊柱炎的易感或致病基因的功能， 包括翻译、 转录及 蛋白质合成的过程， 制备成治疗药物， 改善患者的骨代谢异常以及体内的免疫 炎症反应， 从而达到治疗 PJ的。  11. Use according to claim 8, characterized in that said application comprises susceptibility or pathogenic gene function, including translation, transcription and protein synthesis, by interfering with rs455256.9 related to ankylosing spondylitis The process, which is prepared into a therapeutic drug, improves the abnormality of bone metabolism in the patient and the immune inflammatory reaction in the body, thereby achieving treatment of PJ.
更正页 （细则第 91条) Correction page (Article 91)
PCT/CN2011/081359 2011-10-27 2011-10-27 Method for detecting specific single nucleotide polymorphism related to ankylosing spondylitis and kit therefor WO2013060004A1 (en)

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* Cited by examiner, † Cited by third party
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEN CHAO: "Association of ERAPl, ANTXR2, IL1R2, IL23R, JAK2 and STAT3 Polymorphisms with Ankylosing Spondylitis", CHINESE DOCTORAL DISSERTATIONS FULL-TEXT.-28 DATABASE (MEDICINE AND HEALTH SCIENCES), 15 September 2010 (2010-09-15), pages 24 - 28 *
DATABASE GENBANK 18 August 2010 (2010-08-18), accession no. s253586435 *
DATABASE GENBANK 26 February 2008 (2008-02-26), accession no. s93156442 *

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