CN104962642A - Aspirin resistance-related GPIIIa gene polymorphic site and application - Google Patents
Aspirin resistance-related GPIIIa gene polymorphic site and application Download PDFInfo
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- CN104962642A CN104962642A CN201510423236.0A CN201510423236A CN104962642A CN 104962642 A CN104962642 A CN 104962642A CN 201510423236 A CN201510423236 A CN 201510423236A CN 104962642 A CN104962642 A CN 104962642A
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Abstract
The invention discloses an aspirin resistance-related GPIIIa gene polymorphic site and an application. An antiplatelet drug (particularly aspirin) plays a crucial role in the prevention of ischemic stroke; however, current investigation results indicate that many ischemic stroke patients taking the antiplatelet drug still suffer relapse of stroke even, and the phenomenon is called antiplatelet drug resistance. The applicant defines an aspirin resistance-related GPIIIa gene polymorphic site GPIIIa (A1166C) and designs a primer for detecting the site polymorphism for the site, wherein the primer can be used for preparing a kit for screening aspirin resistant patients so as to select an antiplatelet drug for ischemic stroke patients and reduce the aspirin resistance and stroke relapse.
Description
Technical field
The present invention relates to medical conditions medicine and select field, be specifically related to the relevant GP III a gene polymorphism sites of aspirin resistance and application.
Technical background
Along with China's aging population, Cerebral Vascular Disease rate rises year by year, latest survey result shows that cerebro-vascular diseases became the first killer surmounting tumour harm Chinese health from 2010, annual China new cases 2,000,000, ischemic cerebrovascular disease accounts for 70 ~ 80%, patients with recurrent accounts for 27%, and the patient of 70% leaves neurologic impairment, and the high lethality rate of cerebro-vascular diseases, disability rate and recurrence rate have caused great burden on society and economical load.Antiplatelet drug (especially acetylsalicylic acid) plays vital effect in the control of cerebro-vascular diseases, even if but current the survey showed that a lot of patients with cerebrovascular disease has taken antiplatelet drug, still repeatedly send out palsy again, this phenomenon is called that antiplatelet drug is resisted.
Antiplatelet drug opposing is relevant to factors, as cerebro-vascular diseases Related Risk Factors (sex, diabetes, smoking, metabolism syndrome, acute coronary syndrome, previously cardiovascular event, hepatic and renal function exception, disease in the blood system etc.), patient compliance or drug dose, drug interaction and gene pleiomorphism etc.Wherein gene pleiomorphism is current study hotspot.Previously study discovery, there is gene pleiomorphism in the factor affecting multiple links of acetylsalicylic acid effect in vivo, this polymorphism directly affects its function, thus different to hematoblastic reaction after making Different Individual Aspirin.According to incompletely statistics, the patient of Clinical Aspirin Resistance is up to 40%, but because the target spot affecting acetylsalicylic acid effect is numerous, up to the present, the gene pleiomorphism that aspirin resistance is relevant is also uncertain.But acetylsalicylic acid is as the important drugs of ischemic cerebrovascular secondary prevention, clear and definite aspirin resistance related gene polymorphism is most important as early as possible.
Platelet glycoprotein GPⅢa is one of cell adhesion receptor integrin family member, and thrombocyte, by GP III a and the attachment proteins such as scleroproein, fibronectin specific binding, participates in platelet adhesion reaction and gathering.Based on the research that applicant is nearest, applicant specify that the GP III a gene polymorphism sites relevant to aspirin resistance: GP III a (A1166C), this site can be used for, for Ischemic Stroke selects antiplatelet drug, reducing aspirin resistance and Stroke Recurrence rate.
Summary of the invention
The object of the present invention is to provide the GP III a gene polymorphism sites that aspirin resistance is relevant: A1166C.This site is positioned at the 1166th of people GP III a full length gene, and its base is A or C, carries the allelic patient of GP III a mutant 1166C compared with wild-type A1166 patient, more easily occurs aspirin resistance.
Another object of the present invention there are provided and detects aspirin resistance and to be correlated with the primer of GP III a gene polymorphism sites A1166C.
Last object of the present invention there are provided the application of primer in preparation screening aspirin resistance patient test kit based on GP III a gene polymorphism sites A1166C.This test kit can be used for screening aspirin resistance patient, better for Ischemic Stroke selects antiplatelet drug, to reduce aspirin resistance and Stroke Recurrence rate.
To achieve these goals, the present invention adopts following technical measures:
The GP III a gene polymorphism sites relevant to aspirin resistance, this site is the sudden change that there occurs A → C 1166 of people GP III a gene, and the patient carrying GP III a mutant 1166C after testing more easily aspirin resistance occurs.Based on the A1166C site design primer of GP III a gene, then this site is detected.
Protection content of the present invention also comprises and detects aspirin resistance and to be correlated with the primer of GP III a gene polymorphism sites A1166C; as long as the primer that this primer designs based on the A1166C site of GP III a gene; and energy Successful amplification goes out to comprise the DNA fragmentation of the A1166C of GP III a gene, is protection content of the present invention.
Preferably, the primer designed according to above-mentioned GP III a gene polymorphism sites A1166C is:
Positive-sense strand: 5 '-TGTCAGACACTACACTCA-3 '
Antisense strand: 5 '-GGATAATCCTAGGAAAAT-3 ';
Or
Positive-sense strand: 5 '-TCCTGAACCTCACGGTCG-3 '
Antisense strand: 5 '-AAGGATGTAGAGGTTAAT-3 ';
Or
Positive-sense strand: 5 '-CTACGGTAGTACGTCCGA-3 ';
Antisense strand: 5 '-GTCATCAGCCCCAAAGAG-3 '.
Based on the application of primer in preparation screening aspirin resistance patient test kit of GP III a gene polymorphism sites A1166C, comprise and utilize the DNA of GP III a (A1166C) site primer pair Ischemic Stroke to detect, the patient of GP III a mutant 1166C is carried if detect, then this patient has the trend more easily producing aspirin resistance, and detection method includes but not limited to the existing detection technique for SNP site at present.
Comprise and utilize GP III a (A1166C) site provided by the invention primer, the pleomorphism site primer relevant to other aspirin resistances combines the test kit formed, the DNA of patient is detected, screens the patient more easily producing aspirin resistance.The pleomorphism site that other described aspirin resistances are relevant is preferably: people P2Y1 gene polymorphism sites (A745G), primer: positive-sense strand: 5 '-CACGGGTGGCCATGTCGC-3 '; Antisense strand: 5 '-TACCAGGTAAATCGATTT-3 '.
Compared with prior art, the present invention has following characteristics:
1) GP III a gene polymorphism sites (A1166C) provided by the invention is relevant to aspirin resistance, is intended to Ischemic Stroke and better selects antiplatelet drug, reduces Stroke Recurrence rate.
2) primer for detecting GP III a gene polymorphism sites A1166C provided by the invention, is intended to the test kit of the patient preparing screening aspirin resistance.
Embodiment
Technical scheme of the present invention, if not otherwise specified, is the conventional scheme of this area.
Embodiment 1:
The excavation of GP III a gene polymorphism sites (A1166C) relevant to aspirin resistance:
2010 ~ 2014 years apoplexy patient data are transferred from palsy database, according to Ischemic Stroke Case definition, screening is diagnosed as Ischemic Stroke, and choose the patient of clear and definite Aspirin as secondary prevention (secondary prevention is for having cerebral apoplexy symptom or postapoplectic patient having occurred, and these people need to prevent again cerebral apoplexy to occur) further for research object is as total sample (totally 5033 people).
1. the number of Stroke Recurrence in clear and definite total sample, inquires into the relation (table 2) of GP III a (A1166C) gene polynorphisms and Stroke Recurrence in total sample;
2. in total sample, return institute further consultation patient and (comprise recurred and patient that is that do not recur, totally 3429 people), gather peripheral blood in patients, VerifyNow instrument is adopted to detect acetylsalicylic acid reaction member (ARU), determine the number of ARU >=550, inquire into the relation (table 1) of GP III a (A1166C) gene polynorphisms and ARU >=550.
In the embodiment of the present invention, two kinds of any one generations of situation are all considered as aspirin resistance occurs below:
If 1. during patient's Aspirin, still there is palsy, be then defined as aspirin resistance;
2. adopt VerifyNow instrument to detect acetylsalicylic acid reaction member (ARU), if ARU >=550, be then defined as thrombocyte hyperergy, show that this patient exists aspirin resistance.
Result shows, Aspirin carries out in the Ischemic Stroke of secondary prevention, compared with wild-type patient, GP III a (A1166C) saltant type group patient acetylsalicylic acid reaction member ARU value is obviously high, and ARU >=550 patient numbers is apparently higher than wild-type group (table 1); Stroke Recurrence rate also obviously high (table 2) simultaneously.
Realize by the following method for the detection of GP III a (A1166C) gene polynorphisms in the present embodiment:
Primer is designed as follows for GP III a gene polymorphism sites (A1166C):
Positive-sense strand: 5 '-TGTCAGACACTACACTCA-3 '
Antisense strand: 5 '-GGATAATCCTAGGAAAAT-3 '.
With patient's STb gene for template, utilize Standard PCR to increase, expanding fragment length is 350bp, order-checking, detects the 186th base of amplified fragments.The DNA sequence dna of wild-type patient is for shown in SEQ ID NO.1, and the DNA sequence dna of saltant type patient is for shown in SEQ ID NO.2.The 186th base pair is adopted in this sequence gone out primer amplification to answer the 1166th site in GP III a gene.
Table 1 GP III a (A1166C) wild-type patient and saltant type patient ARU value
Table 2 GP III a (A1166C) wild-type patient and saltant type patient Stroke Recurrence rate
Result shows, the primer designed based on this site can be used for the screening of GP III a (A1166C) saltant type patient, so that better for Ischemic Stroke selects antiplatelet drug, and reduction aspirin resistance and Stroke Recurrence rate.
Embodiment 2:
GP III a gene polymorphism sites (A1166C) primer relevant to aspirin resistance is preparing the application of screening in aspirin resistance patient test kit:
There is Ischemic Stroke first to all, give acetylsalicylic acid and carry out secondary prevention, after 3 months, patient is followed up a case by regular visits to, detect each patient ARU, and Stroke Recurrence situation.Get peripheral blood in patients simultaneously and whether there is GP III a (A1166C) polymorphism by gene sequencing detection patient, analyze saltant type and wild-type patient aspirin resistance incidence.
Begin one's study from August, 2013, it is in August, 2014 that case includes dead line in, according to Ischemic Stroke Case definition, chooses and is diagnosed as Ischemic Stroke as research object, complete following work:
1., according to Ischemic Stroke and TIA secondary prevention guide, give all patient's acetylsalicylic acid and carry out secondary prevention;
2. all patients follow up a case by regular visits to when starting secondary prevention 90 days, detect ARU and the Stroke Recurrence situation of follow-up investigation object;
3. take peripheral blood in patients, utilize primer provided by the invention (positive-sense strand: 5 '-TGTCAGACACTACACTCA-3 '; Antisense strand: 5 '-GGATAATCCTAGGAAAAT-3 '), carry out pcr amplification, order-checking, the fragment total length 350bp amplified, wherein the 1166th site of the 186th bit base corresponding GP III a gene.Therefore, by judging sequence the 1166th bit base of amplification, detecting patient is GP III a (A1166) wild-type or GP III a (1166G) saltant type.
4. analyze saltant type and wild-type patient ARU value and Stroke Recurrence rate.
Result shows, Aspirin carries out in the Ischemic Stroke of secondary prevention, compared with GP III a (A1166) wild-type patient, GP III a (1166C) saltant type group patient ARU value is higher, and ARU >=550 patient numbers is apparently higher than wild-type group (table 3), Stroke Recurrence rate also obviously high (table 4).
Table 3 GP III a (A1166C) wild-type patient and saltant type patient ARU value
Table 4 GP III a (A1166C) wild-type patient and saltant type patient Stroke Recurrence rate
Embodiment 3:
The excavation of the P2Y1 gene polymorphism sites (A745G) relevant to aspirin resistance:
With the palsy database that embodiment 1 is same, blood sample and method, detect the relation (table 6) of P2Y1 (A745G) gene polynorphisms and Stroke Recurrence in total sample (totally 5033 people, sample is identical with embodiment 1); And return the relation (table 5) of P2Y1 (A745G) gene polynorphisms and ARU >=550 in institute further consultation patient (comprising that recurred and patient that is that do not recur, totally 3429 people, identical with embodiment 1).
Result shows, Aspirin carries out in the Ischemic Stroke of secondary prevention, compared with wild-type patient, P2Y1 (A745G) saltant type group patient acetylsalicylic acid reaction member ARU value is obviously high, and ARU >=550 patient numbers is apparently higher than wild-type group (table 5); Stroke Recurrence rate also obviously high (table 6) simultaneously.
Realize by the following method for the detection of P2Y1 (A745G) gene polynorphisms in the present embodiment:
Primer is designed as follows for P2Y1 gene polymorphism sites (A745G):
Positive-sense strand: 5 '-CACGGGTGGCCATGTCGC-3 '
Antisense strand: 5 '-TACCAGGTAAATCGATTT-3 '.
With patient's STb gene for template, utilize Standard PCR to increase, expanding fragment length is 350bp, order-checking, detects the 185th base of amplified fragments.The DNA sequence dna of wild-type patient is for shown in SEQ ID NO.9, and the DNA sequence dna of saltant type patient is for shown in SEQ ID NO.10.The 185th base pair is adopted in this sequence gone out primer amplification to answer the 745th site in P2Y1 gene.
Table 5 P2Y1 (A745G) wild-type patient and saltant type patient ARU value
Table 6 P2Y1 (A745G) wild-type patient and saltant type patient Stroke Recurrence rate
Result shows, the primer designed based on this site can be used for the screening of P2Y1 (A745G) saltant type patient, so that better for Ischemic Stroke selects antiplatelet drug, and reduction aspirin resistance and Stroke Recurrence rate.
Embodiment 4:
GP III a gene polymorphism sites (A1166C) primer relevant to aspirin resistance is preparing the application of screening in aspirin resistance patient test kit:
Described in the present embodiment, the primer of test kit comprises:
GP III a gene polymorphism sites (A1166C) primer: positive-sense strand: 5 '-TGTCAGACACTACACTCA-3 ', antisense strand: 5 '-GGATAATCCTAGGAAAAT-3 ', with P2Y1 gene polymorphism sites (A745G) primer: positive-sense strand: 5 '-CACGGGTGGCCATGTCGC-3 ', antisense strand: 5 '-TACCAGGTAAATCGATTT-3 '.
Specific operation process is as follows:
The palsy database same with embodiment 1 and blood sample and method, detect when GP III a gene (A1166C) and P2Y1 gene (A745G) are mutant simultaneously and the relation (sample is 5033 people in embodiment 1) of Stroke Recurrence; And GP III a gene (A1166C) and P2Y1 gene (A745G) are the relation (sample is 3429 people in embodiment 1) of during mutant and ARU value >=500 simultaneously.Result as shown in Table 7 and 8.
The detection method of GP III a gene (A1166C) with the detection method of embodiment 2, P2Y1 (A745G) gene with embodiment 3.
Table 7 wild-type patient compares with saltant type patient ARU value
Table 8 wild-type patient compares with saltant type patient Stroke Recurrence rate
Known to table 7 and table 8, when GP III a (A1166C) and P2Y1 (A745G) is mutant, more easily there is aspirin resistance in patient, therefore need for this Ischemic Stroke selects suitable antiplatelet drug, reduce aspirin resistance and Stroke Recurrence rate.
SEQUENCE LISTING
Oerlikon peace bio tech ltd, <110> Wuhan
The GP III a gene polymorphism sites that <120> aspirin resistance is relevant and application
The GP III a gene polymorphism sites that <130> aspirin resistance is relevant and application
<160> 12
<170> PatentIn version 3.1
<210> 1
<211> 350
<212> DNA
<213> artificial sequence
<400> 1
acagtctgtg atgtgagttt ggaggacttg gagtgccagc tgtggctggc atagaatttg 60
tctcctctgc ctttgttttt tgttttcttt taacaggaaa agattggctg gaggaatgat 120
gcatcccact tgctggtgtt taccactgat gccaagactc atatagcatt ggacggaagg 180
ctggcaggca ttgtccagcc caatgacggg cagtgtcatg ttggtagtga caatcattac 240
tctgcctcca ctaccatggt gagatctctg gcacactgtg gtttctattc atgattgtga 300
tacatgagac gtcattaacc tctacatcct tcattttcct aggattatcc 350
<210> 2
<211> 350
<212> DNA
<213> artificial sequence
<400> 2
acagtctgtg atgtgagttt ggaggacttg gagtgccagc tgtggctggc atagaatttg 60
tctcctctgc ctttgttttt tgttttcttt taacaggaaa agattggctg gaggaatgat 120
gcatcccact tgctggtgtt taccactgat gccaagactc atatagcatt ggacggaagg 180
ctggccggca ttgtccagcc caatgacggg cagtgtcatg ttggtagtga caatcattac 240
tctgcctcca ctaccatggt gagatctctg gcacactgtg gtttctattc atgattgtga 300
tacatgagac gtcattaacc tctacatcct tcattttcct aggattatcc 350
<210> 3
<211> 18
<212> DNA
<213> artificial sequence
<400> 3
tgtcagacac tacactca 18
<210> 4
<211> 18
<212> DNA
<213> artificial sequence
<400> 4
ggataatcct aggaaaat 18
<210> 5
<211> 18
<212> DNA
<213> artificial sequence
<400> 5
tcctgaacct cacggtcg 18
<210> 6
<211> 18
<212> DNA
<213> artificial sequence
<400> 6
aaggatgtag aggttaat 18
<210> 7
<211> 18
<212> DNA
<213> artificial sequence
<400> 7
ctacggtagt acgtccga 18
<210> 8
<211> 18
<212> DNA
<213> artificial sequence
<400> 8
gtcatcagcc ccaaagag 18
<210> 9
<211> 350
<212> DNA
<213> artificial sequence
<400> 9
gtgcccaccg gtacagcggt gtggtgtacc ccctcaagtc cctgggccgg ctcaaaaaga 60
agaatgcgat ctgtatcagc gtgctggtgt ggctcattgt ggtggtggcg atctccccca 120
tcctcttcta ctcaggtacc ggggtccgca aaaacaaaac catcacctgt tacgacacca 180
cctcagacga gtacctgcga agttatttca tctacagcat gtgcacgacc gtggccatgt 240
tctgtgtccc cttggtgctg attctgggct gttacggatt aattgtgaga gctttgattt 300
acaaagatct ggacaactct cctctgagga gaaaatcgat ttacctggta 350
<210> 10
<211> 350
<212> DNA
<213> artificial sequence
<400> 10
gtgcccaccg gtacagcggt gtggtgtacc ccctcaagtc cctgggccgg ctcaaaaaga 60
agaatgcgat ctgtatcagc gtgctggtgt ggctcattgt ggtggtggcg atctccccca 120
tcctcttcta ctcaggtacc ggggtccgca aaaacaaaac catcacctgt tacgacacca 180
cctcggacga gtacctgcga agttatttca tctacagcat gtgcacgacc gtggccatgt 240
tctgtgtccc cttggtgctg attctgggct gttacggatt aattgtgaga gctttgattt 300
acaaagatct ggacaactct cctctgagga gaaaatcgat ttacctggta 350
<210> 11
<211> 18
<212> DNA
<213> artificial sequence
<400> 11
cacgggtggc catgtcgc 18
<210> 12
<211> 18
<212> DNA
<213> artificial sequence
<400> 12
taccaggtaa atcgattt 18
Claims (6)
1. for people GP III
athe primer of the 1166th design of full length gene.
2. primer according to claim 1, is specially:
Positive-sense strand: 5 '-TGTCAGACACTACACTCA-3 '
Antisense strand: 5 '-GGATAATCCTAGGAAAAT-3 '.
3. primer according to claim 1, is specially:
Positive-sense strand: 5 '-TCCTGAACCTCACGGTCG-3 '
Antisense strand: 5 '-AAGGATGTAGAGGTTAAT-3 '.
4. primer according to claim 1, is specially:
Positive-sense strand: 5 '-CTACGGTAGTACGTCCGA-3 ';
Antisense strand: 5 '-GTCATCAGCCCCAAAGAG-3 '.
5. the application of primer according to claim 1 in preparation screening aspirin resistance patient test kit.
6., for screening an aspirin resistance patient test kit, comprise two pairs of primers: positive-sense strand: 5 '-TGTCAGACACTACACTCA-3 ', antisense strand: 5 '-GGATAATCCTAGGAAAAT-3 '; And positive-sense strand: 5 '-CACGGGTGGCCATGTCGC-3 '; Antisense strand: 5 '-TACCAGGTAAATCGATTT-3 '.
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| CN201510423236.0A CN104962642B (en) | 2015-07-18 | 2015-07-18 | The related a gene polymorphism sites of GP III of aspirin resistance and application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486943A (en) * | 2018-12-26 | 2019-03-19 | 武汉友芝友医疗科技股份有限公司 | For detecting and the primer sets of aspirin resistance related gene polymorphic site and kit and its application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102010900A (en) * | 2010-06-08 | 2011-04-13 | 广州益善生物技术有限公司 | Liquid chip and specific primer for detecting SNP of GPIIIa gene and liquid chip and specific primer for detecting SNP of GPIIIa and COX-1 genes |
-
2015
- 2015-07-18 CN CN201510423236.0A patent/CN104962642B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102010900A (en) * | 2010-06-08 | 2011-04-13 | 广州益善生物技术有限公司 | Liquid chip and specific primer for detecting SNP of GPIIIa gene and liquid chip and specific primer for detecting SNP of GPIIIa and COX-1 genes |
Non-Patent Citations (2)
| Title |
|---|
| YAN ZHANG等: "Genetic Variation of ITGB3 Is Associated with Asthma in Chinese Han Children", 《PLOS ONE》 * |
| 丁建平等: "血小板膜GPⅢa 受体的基因多态性与阿司匹林在冠心病患者中疗效的关系", 《中国现代医学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486943A (en) * | 2018-12-26 | 2019-03-19 | 武汉友芝友医疗科技股份有限公司 | For detecting and the primer sets of aspirin resistance related gene polymorphic site and kit and its application |
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