CN105567794B - Novel ankylosing spondylitis susceptibility detection method and kit - Google Patents
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Abstract
The invention discloses a new ankylosing spondylitis susceptibility detection method and a new ankylosing spondylitis susceptibility kit, wherein the method for detecting ankylosing spondylitis susceptibility comprises the following steps: detecting MIR146A gene and/or transcript of the individual, comparing with normal MIR146A gene and/or transcript, and indicating that the possibility of the individual suffering from ankylosing spondylitis is higher than that of normal people if difference exists; the kit comprises: the primer specifically amplifies MIR146A gene or transcript, and the primer amplifies an amplification product which is 100-2000bp in length and contains 659 th site in SEQ ID NO. 1. The invention can be used for early auxiliary diagnosis of ankylosing spondylitis, and can ensure that some carriers can take reasonable preventive measures before onset of disease, thereby improving the life cycle and the life quality of the carriers and having great application value and social benefit.
Description
Technical Field
The invention relates to a method and a kit for detecting susceptibility of ankylosing spondylitis, in particular to a novel method and a kit for detecting susceptibility of ankylosing spondylitis by using genotypes.
Background
Ankylosing spondylitis, also known as von bechterev's disease or maritstrumepell's disease, is a chronic, progressive, chronic inflammatory disease in which the medial-axial joints are involved. Mainly affecting the sacroiliac joint, the spinal joints and the paraspinal tissues of the pelvis. The main symptoms are low back pain, spinal stiffness and limited range of motion, and the bilateral sacroiliitis (sacrolitis) is shown by X-ray. Since the disease usually attacks the sacroiliac joint first and then affects the spine repeatedly, and finally causes the bony ankylosis of the spine, it is currently called Ankylosing Spondylitis (AS) at home and abroad.
Ankylosing spondylitis has a significant ethnicity, and the incidence rates vary greatly among different ethnic groups. The incidence rate of Indians is the highest, and the incidence rate of North American Indians is 2.7-6.3%. The second is caucasian, and the incidence rate of the caucasian is 0.1 to 1.4 percent. Yellow people are lower than white people, and China is about 0.3 percent. The incidence rate of black people is the lowest, about 1/4 of that of white people, and is only 0.2% in Africa.
Ankylosing spondylitis is well developed in adults aged 20 to 40 years, especially young males aged 20 to 30 years.
Ankylosing spondylitis is autosomal dominant inheritance, has obvious family inheritance tendency, and the genetic factor is more than 90%. According to investigation, the incidence of relatives of ankylosing spondylitis patients is about twice AS high AS that of common people, AS patients with the sibling recurrence risk ratio of more than 82.90% have HLA-B27 positive, and the positive rate in normal people is 8%; the positivity of HLA-B27 accounts for 50% for children, and the positivity of HLA-B27 accounts for 25% for generating ankylosing spondylitis. It has been essentially concluded that HLA-B27 plays a role in the pathogenesis of AS AS an independent causative gene. Since only 1% to 5% of HLA-B27-positive individuals developed AS, it was suggested that other genes were involved in the onset of AS.
The MIR146A gene codes a product belonging to a microRNA, and can regulate the expression and translation of a downstream target gene at the level of messenger RNA. A great deal of research already proves that MIR146A is involved in the regulation of human inflammation pathways and is a key gene for regulating a plurality of pro-inflammatory reactions. Polymorphic sites in the MIR146A gene have also been found to be associated with a variety of autoimmune diseases, including psoriasis, systemic lupus erythematosus, sclerosis, and the like. The relevance of the rs57095329 site and the incidence of ankylosing spondylitis has not been proposed.
Disclosure of Invention
The invention aims to provide a novel ankylosing spondylitis susceptibility detection method and a kit, and provides a basis for auxiliary diagnosis (especially early diagnosis) of ankylosing spondylitis and treatment of ankylosing spondylitis.
In a first aspect of the present invention, there is provided a method for genotyping susceptibility to ankylosing spondylitis in an individual, comprising the steps of:
and detecting the MIR146A gene and/or transcript of the individual, and comparing the MIR146A gene and/or transcript with the normal MIR146A gene and/or transcript, wherein the difference indicates that the possibility of suffering from ankylosing spondylitis of the individual is higher than that of the normal population.
In another preferred embodiment, the method detects the gene or transcript of MIR146A and compares the difference with the normal MIR146A nucleotide sequence.
In another preferred embodiment, the difference is the following Single Nucleotide Polymorphism (SNP):
659 position A → G; wherein the nucleotide position numbering is based on SEQ ID NO. 1.
In a second aspect of the present invention, there is provided a method for detecting the presence or absence of a single nucleotide polymorphism of MIR146A gene in a sample, comprising the steps of:
1) amplifying MIR146A gene of the sample by using MIR146A gene specific primers to obtain an amplification product;
2) detecting the presence or absence of the following single nucleotide polymorphisms in the amplification product:
659 position A → G; wherein the nucleotide position numbering is based on SEQ ID NO. 1.
In another preferred example, the MIR146A gene specific primers have the sequences of SEQ ID nos. 2 and 3.
In another preferred embodiment, the amplification product has a length of 100-2000bp and contains 659 th position in SEQ ID NO. 1.
In a third aspect of the present invention, there is provided a kit for detecting ankylosing spondylitis (susceptibility to ankylosing spondylitis) using a genotype, comprising: the primer for specifically amplifying MIR146A gene or transcript, preferably, the primer amplifies an amplification product which has the length of 100-2000bp and contains 659 th site in SEQ ID NO. 1.
In another preferred embodiment, the kit further comprises a reagent selected from the group consisting of:
1) a probe that binds to a mutation at position 659 in SEQ ID NO. 1;
2) a mutant restriction enzyme that recognizes position 659 in SEQ ID NO. 1.
In another preferred embodiment, the mutation is selected from the group consisting of a single nucleotide polymorphism:
659 position A → G; wherein the nucleotide position numbering is based on SEQ ID NO. 1.
The sequence of the primer can be shown as SEQ ID NO.2 and 3.
The length of the amplification product is 100-2000bp and contains the 659 th site in SEQ ID NO. 1.
The kit further comprises: taq enzyme, dNTPs, magnesium ions and conventional PCR reaction buffer solution.
The present invention has been made through intensive and extensive studies to determine and analyze SNPs of a large number of candidate genes. The genomic sequence of MIR146A is found and proved to be closely related to ankylosing spondylitis, wherein the correlation research result shows that the distribution of the SNP at the 659 th position (A → G at the 659 th position) (marked as rs57095329) in the SEQ ID NO.1 of the MIR146A gene in a control group and a case group has a significant difference (P < 0.05), so that the SNP can be used as a specific SNP for auxiliary detection of ankylosing spondylitis (or susceptibility thereof). The present invention has been completed based on this finding.
Specifically, the invention discloses a Single Nucleotide Polymorphism (SNP) of the MIR146A gene and the correlation of the polymorphism and ankylosing spondylitis. The SNP of the invention is the G/A polymorphism of 659 th site of the sequence shown in SEQ ID NO.1, and the frequency of genotype GG in the population with ankylosing spondylitis is lower than that in the population with normal control.
The most convenient method for detecting the SNP of the invention is to amplify the MIR146A gene of a sample by using MIR146A gene specific primers to obtain an amplification product; then detecting whether the following single nucleotide polymorphisms exist in the amplification product: a → G at position 659, wherein the numbering of nucleotide positions is based on SEQ ID NO. 1.
It should be understood that, after the present invention discloses the correlation between the SNP of the MIR146A gene and ankylosing spondylitis, one skilled in the art can easily design an amplification product that can specifically amplify the position containing the SNP, and then determine whether the 659 th A → G exists by sequencing or the like. In general, the length of the primer is 15-50bp, preferably 20-30 bp. Although complete complementarity of the primer to the template sequence is preferred, it is known to those skilled in the art that specific amplification (i.e., amplification of only the desired fragment) is also possible in the presence of a primer that is not necessarily complementary to the template, particularly at the 5' end of the primer.
Kits containing these primers and methods of using these primers are within the scope of the invention, provided that the primers amplify an amplification product containing the corresponding position of the SNP of the invention. A preferred primer pair has the sequences of SEQ ID NO.2 and 3.
Although the length of the amplification product is not particularly limited, the length of the amplification product is usually 100 to 2000bp, preferably 150 to 1500bp, more preferably 200 to 1000 bp. These amplification products should contain the 659 th position in SEQ ID NO. 1.
The SNP of the invention has very high relevance with the ankylosing spondylitis, so the SNP can be used for early auxiliary diagnosis of the ankylosing spondylitis, and can ensure that some carriers can take reasonable preventive measures before the onset of disease without being muzzled, thereby improving the life cycle and the life quality of the carriers, and having great application value and social benefit.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.
Example 1
First, research object
Case samples were collected entirely from outpatients, and the patients were diagnosed by clinically experienced rheumatologists in the physician's physician in the Ji Hospital according to revised New York standards set forth in 1984, and confirmed by multiple follow-up visits. Control samples were selected from southern central panel of age, gender matched, individuals without a history of arthritis.
Peripheral blood samples were randomly collected from 96 AS patients and 95 healthy normal control individuals on an informed consent basis.
Second, Experimental methods and results
1. DNA extraction
DNA was extracted from human peripheral blood samples by the conventional phenol chloroform method (DNA was also extracted using a commercial kit) and corrected to 20 ng/. mu.l for conventional PCR amplification.
2. Primer design for use in PCR and sequencing
Based on the genomic sequence of MIR146A in GenBank, the following primers were designed and synthesized:
sense primer P1: 5'-accgcagaaccaaattaacg-3' (shown in SEQ ID NO. 2)
Antisense primer P2: 5'-aatgaatcctcccaccatca-3' (shown in SEQ ID NO. 3).
3. PCR amplification of MIR146A Gene
PCR amplification was performed using Taq enzyme on a GeneAmp 9700PCR instrument using the Touchdown program using the extracted DNA as a template. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min, denaturation at 94 ℃ for 30 sec, annealing at 63 ℃ for 40 sec, and extension at 72 ℃ for 40 sec for 10 cycles, wherein the annealing temperature decreases by 0.5 ℃ per cycle; 30 cycles of subsequent denaturation at 94 ℃ for 30 seconds, annealing at 58 ℃ for 40 seconds, and extension at 72 ℃ for 40 seconds; final extension at 72 ℃ for 7 min.
The PCR amplification product was verified by agarose gel electrophoresis. As a result, an amplification product of MIR146A gene was obtained.
4. SNP discovery and detection
PCR products were purified with Resin, sequenced using ABI-3730DNA sequencer (ABI), and sequence calling, genotyping, and SNP confirmation using Polypred software (HTTP:// drog. mbt. washington. edu/Polyph. html. university of Washington, USA).
As a result, the following SNPs were found to be included:
a → G at position 659 in SEQ ID NO.1 (rs 57095329).
Wherein rs 57095329:
ttgcagttgcacattccaaaggccttgtacgttctttccagtttgggaaaaggagcagctaagggcacccatggaaacacttactaattgtgtgaccttaggcaagtccccctaacctcaccaaagataaaattcccttatgagtaaaaggggatcacaaaatggagataataattcgggctacctgctatggatgattgctgtgggattagggaagatgatgcacgtgatacatttagcagagtgcctggtgcctagtaggtgcccattaaaatttagctatgactgttctctttagctgacacacaagactgccttgaatgttcacatttccagagaaaggtgctcaggaagatttctcagtgttccgcctgccaggtcagtttacagttcaaagaatccctttgttcaaagggtgagcaaatccaggcctcgtgtatctgcagcaatgaaacaagggagctttctgcctgatcttctccccaaaggcagtctctctttttaaatgcctgcacacatgttatttatttatttttggttaacgtttaaattgaggttttggctgaaactcagcctgcgcgcacttgaaaagccaacaggctcattgggcagccgataaagctctcgggatttccccgcggggctgcggagagtacagAcaggaagcctggggacccagcgcctgaccagaacttcctcgggggaggctgcaggggagcaggcgcatcctgcacagaacgctctagagcgcgcaggccaaagcaccaggctgctcctgacacgtgctgcaagagggtccccgacccgggggtccagaccctgcacgcatgatggggaaggtggaggcttccccctcagctccgcggagagaagctgacactgccaggctggaaccttccattccggcccagcctcttcctccctcgctgtgccgaggagggatctagaagggactttccagagagggttagcgtgcagggtgtggaaatggaataaaagcatatgcaaataggccttagctgccttcctctaccccagcaaataagagtctctccagaaagatgctctttctccaagacgcttgaccgctcttcctttcctggatggcaccagcagggccgattggagtggtaaaccctgggccggaaggcatgccaaagggtggacaggatggacaggagacagtagcacaacgaggag (shown in SEQ ID NO. 1).
Wherein, in the sequence "A"is the SNP site.
5. SNP genotyping and association analysis
SNP genotyping was performed by direct one-way sequencing. I.e., typing and correlation analysis in ankylosing spondylitis patients and normal control groups.
And carrying out descriptive statistical analysis on the genotyping result, and carrying out chi-square test on the tabulation. Whether the genotypes were different between the patients and the controls was observed, and the analysis results were as follows:
according to the research method of case-control correlation analysis, chi-square calculation is carried out on the genotyping result, and the result shows that the genotype GG of the polymorphic site rs57095329 has obvious statistical difference. Wherein, in case samples, the distribution of rs57095329 is (genotype AA 57, genotype AG 36, genotype GG 3); in contrast, the control samples (genotype AA 56, genotype AG 27, and genotype GG 12) were significantly different from each other.
Genotype GG was less in case than control and chi-square test P was 0.015. Individuals with genotype GG are at a 4.5-fold lower risk of developing ankylosing spondylitis than individuals with the other two genotypes (AA + AG) (OR ═ 0.223, 95% CI: 0.06-0.82).
The above results show that: the genotype GG reduces the susceptibility of ankylosing spondylitis, and the genotype GG of the polymorphic site rs57095329 of the MIR146A gene is obviously related to the disease protection of ankylosing spondylitis. Namely: the 659 SNP site in SEQ ID NO.1 is related to the occurrence of ankylosing spondylitis.
Example 2 ankylosing spondylitis susceptibility detection kit
As described in example 1, the A → G mutation at position 659 in SEQ ID NO.1 is closely related to ankylosing spondylitis. Therefore, MIR146A gene specific primers can be designed based on the mutation, and then the DNA of the patient is used as a template for amplification and detection.
A kit (100 human trials) was prepared containing:
1) sense primer P1 (shown in SEQ ID NO. 2) at a concentration of 100 pmol;
2) antisense primer P2 (shown in SEQ ID NO. 3) at a concentration of 100 pmol; and
3) PCR reaction solution containing Taq enzyme, dNTP, magnesium ions and PCR reaction buffer solution.
Randomly selecting a test group of 100 individuals, wherein the test group comprises: the method is used for detecting the ankylosing spondylitis of the patient, and comprises the following steps of (1) not knowing whether the patient is suffering from the ankylosing spondylitis, a patient known to suffer from the ankylosing spondylitis and a normal person without the ankylosing spondylitis.
3ml of peripheral blood of the subject to be detected in the test group was extracted, and DNA was extracted from the blood using a conventional method (or using a specific commercial kit). The PCR primer in the ankylosing spondylitis detection kit is diluted to 10 mu mol/l, and the extracted DNA is used as a template to perform PCR reaction with the provided primer. After purification of the PCR product, sequencing was performed using ABI 3730DNA sequencer, and sequence interpretation and SNP confirmation were performed using Polyphred software.
Alternatively, the amplified product and the normal control are chromatographed by Denaturing High Performance Liquid Chromatography (DHPLC), whereby A → G at position 659 in SEQ ID NO.1 can be detected.
And (3) detection results: a659 th A → G subject exists for MIR146A gene, and is further tested by conventional methods to confirm whether or not the subject has ankylosing spondylitis. The detection result shows that the susceptibility proportion of ankylosing spondylitis of a detection object containing 659 th GG is obviously lower than that of GG type normal population (less than 50% of the normal population).
Therefore, it was revealed that an auxiliary detection of ankylosing spondylitis could be performed by detecting A → G at position 659 of MIR146A gene.
Example 3 adjuvant detection of susceptibility to ankylosing spondylitis
The test of example 2 was repeated except that 70 persons (who did not know whether there was any ankylosing spondylitis symptom before the test) were randomly selected for the test.
A kit (100 human trials) was prepared containing:
1) sense primer P1 (shown in SEQ ID NO. 2) at a concentration of 100 pmol;
2) antisense primer P2 (shown in SEQ ID NO. 3) at a concentration of 100 pmol; and
3) PCR reaction solution containing Taq enzyme, dNTP, magnesium ions and PCR reaction buffer solution.
3ml of peripheral blood of the subject to be examined was extracted, and DNA was extracted from the blood using a conventional method (or using a specific commercial kit). The PCR primer in the ankylosing spondylitis detection kit is diluted to 10 mu mol/l, and the extracted DNA is used as a template to perform PCR reaction with the provided primer. After purification of the PCR product, sequencing was performed using ABI 3730DNA sequencer, and sequence interpretation and SNP confirmation were performed using Polyphred software.
The result also proves that the proportion of ankylosing spondylitis of the test object containing the 659 th GG is obviously lower than that of healthy individuals (which is less than 50% of the normal population) with the 659 th GG.
Claims (4)
1. The application of a genotype detection reagent in preparing a ankylosing spondylitis detection kit is characterized in that the genotype detection reagent comprises: the primer specifically amplifies MIR146A gene or transcript, detects the single nucleotide polymorphism of A → G at the 659 th site in SEQ ID NO.1, and the length of the amplified product is 100-2000 bp.
2. Use according to claim 1, characterized in that: the genotype detection reagent further contains a reagent selected from the group consisting of:
1) a probe that binds to a mutation at position 659 in SEQ ID NO. 1;
2) a mutant restriction enzyme that recognizes position 659 in SEQ ID NO. 1.
3. Use according to claim 1, characterized in that: the sequences of the primers are shown as SEQ ID NO.2 and 3.
4. The use of claim 1, wherein the genotype detection reagent further comprises: taq enzyme, dNTPs, magnesium ions and conventional PCR reaction buffer solution.
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| US20150024390A1 (en) * | 2011-10-27 | 2015-01-22 | Jieruo Gu | Method and kit for detecting specific single nucleotide polymorphism associated with ankylosing spondylitis |
| WO2013155980A1 (en) * | 2012-04-19 | 2013-10-24 | 中国科学院上海生命科学研究院 | Auto-immune disease-related microrna and use thereof |
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| CN101525655A (en) * | 2008-03-05 | 2009-09-09 | 上海人类基因组研究中心 | Method for testing susceptibility of ankylosing spondylitis and kit |
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| Xiaobing Luo等.A Functional Variant in microRNA-146a Promoter Modulates Its Expression and Confers Disease Risk for Systemic Lupus Erythematosus.《PLoS Genetics》.2011,第7卷(第6期),第1-12页. * |
| 李友等.miR-146a基因多态性与腔隙性脑梗死的关系.《山东医药》.2014,第54卷(第16期),第1-3页. * |
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