CN102747075A - Coronary heart disease associated sixth chromosome susceptible region tagging single nucleotide polymorphism sites, haplotypes thereof, and applications of sites and haplotypes - Google Patents
Coronary heart disease associated sixth chromosome susceptible region tagging single nucleotide polymorphism sites, haplotypes thereof, and applications of sites and haplotypes Download PDFInfo
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Abstract
The invention relates to coronary heart disease associated sixth chromosome susceptible region tagging single nucleotide polymorphism sites, haplotypes thereof, and applications of the sites and the haplotypes. The tagging single nucleotide polymorphism sites comprise sites of rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and rs2076529 in a segment of a closely linked region in C6orf10-BTNL2 genes on a chromosome 6p21.32. The single nucleotides of the haplotype at the sites of rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and rs2076529 are G, A, A, C, C and T respectively, A, G, G, T, T and C respectively, or A, A, A, C, C and T respectively. The tagging single nucleotide polymorphism sites and the haplotypes have important application prospects in the prevention and/or the diagnosis of the coronary heart disease.
Description
Technical field
The present invention relates to the haplotype and the application of regional tagged single-nucleotide polymorphic loci of No. 6 karyomit(e) susceptibles relevant and composition thereof, be specifically related to karyomit(e) 6p21.32 and go up haplotype and detection method thereof that a series of regional tagged single-nucleotide polymorphic locis, said mononucleotide polymorphism site are formed in the intergenic one section close linkage zone of C6orf10-BTNL2 and the application that is used for detecting the ill risk of crowd's coronary heart disease with coronary heart disease.
Background technology
The atherosclerotic cardiovascular and cerebrovascular disease have become the main health problem that world wide is paid close attention to.The World Health Organization's report in 2004 shows that the annual cardiovascular disorder in the whole world is taken the death toll Da Gaoda 1,720 ten thousand that causes as the leading factor with coronary heart disease and apoplexy, accounts for 1/3rd of all death tolls.Estimate that this numeral of the year two thousand twenty will further increase by 50%, up to 2,500 ten thousand, cardiovascular disorder is global human " No.1 killer ".An extensive perspective study of carrying out in China shows that also heart disease has become the major causes of death of Chinese population, apportion man, women's cause of death the 2nd and the 1st.China annual New Development myocardial infarction 500,000 people, the Hazard Factor of being correlated with along with the change and the atherosclerosis of mode of life continue to increase, and coronary heart disease and myocardial infarction morbidity yet will be lasting ascendant trend.
A large amount of research datas show that coronary heart disease is a kind of complex disease, are by due to a plurality of minor genes and the long-term interaction of environmental factors.Therefore identify tumor susceptibility gene relevant with coronary heart disease or Disease-causing gene, further the tumor susceptibility gene of screening increase disease risks is confirmed susceptible individual in the crowd, will help cause of coronary heart disease risk profile, new drug development, diagnosis and individualized treatment.From the basis to clinical; People have carried out a large amount of research to this; And accumulating a large amount of knowledge aspect the Hazard Factor of coronary heart disease and the pathogenetic physiopathology of coronary disease; But the definite genetic molecule mechanism about coronary heart disease and myocardial infarction generation is but known little about it, and for how to identify inheritance susceptible gene and the coronary heart disease genetic predisposition of identifying the experimenter, lacks comprehensive system effective recognition method always.
Summary of the invention
To the problems referred to above, an object of the present invention is to provide a kind of tagged single-nucleotide polymorphic loci of karyomit(e) 6p21.32 regional gene; Another object of the present invention provides a kind of karyomit(e) 6p21.32 regional gene haplotype of being made up of the tagged single-nucleotide polymorphic loci of karyomit(e) 6p21.32 regional gene, and a kind of dangerous haplotype of being made up of the tagged single-nucleotide polymorphic loci of karyomit(e) 6p21.32 regional gene of karyomit(e) 6p21.32 regional gene particularly is provided; Another object of the present invention provides a kind of method that detects the tagged single-nucleotide polymorphic loci of karyomit(e) 6p21.32 regional gene of the present invention; Another object of the present invention provides a kind of reagent that detects the tagged single-nucleotide polymorphic loci of karyomit(e) 6p21.32 regional gene and detects the preparation of karyomit(e) 6p21.32 regional gene haplotype or the application in the test kit in preparation; Another object of the present invention provides a kind of method that detects karyomit(e) 6p21.32 regional gene haplotype; Another object of the present invention provides a kind of method of vitro detection coronary heart disease dependent genes; Another object of the present invention provides a kind of reagent that detects the tagged single-nucleotide polymorphic loci of karyomit(e) 6p21.32 regional gene is used for the preparation or the test kit of vitro detection coronary heart disease dependent genes in preparation application; The reagent that another object of the present invention provides a kind of tagged single-nucleotide polymorphic loci that detects karyomit(e) 6p21.32 regional gene is used for the vitro detection testing sample in preparation and whether contains the preparation of the dangerous haplotype of karyomit(e) 6p21.32 regional gene or the application in the test kit; Another object of the present invention provides the method that whether contains the dangerous haplotype of karyomit(e) 6p21.32 regional gene in a kind of vitro detection testing sample; Another object of the present invention provide a kind of vitro detection from the reagent of the tagged single-nucleotide polymorphic loci of karyomit(e) 6p21.32 regional gene in the sample of individuality to be measured in the preparation of the preparation prediction individual danger of suffering from coronary heart disease to be measured or the application in the test kit; Another object of the present invention provides a kind of external prediction individual method of suffering from the danger of coronary heart disease to be measured; Another object of the present invention provides a kind of test kit of polymorphum of the tagged single-nucleotide polymorphic loci that detects karyomit(e) 6p21.32 regional gene.
At first; The invention provides tagged single-nucleotide polymorphic loci (the tagging SNPs of karyomit(e) 6p21.32 regional gene; TSNPs), this site is respectively mononucleotide site rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and the rs2076529 of karyomit(e) 6p21.32 regional gene.Above-mentioned pleomorphism site is on the basis of a large amount of experiments, to draw.In embodiment of the present invention, at first use full genome chip (Affymetrix Axiom
TMGenome-Wide CHB 1Array Plate) chip carries out gene type to 1515 routine patients with coronary heart disease and 5019 routine normal peoples to SNP in the full genome range; The data of utilizing chip to obtain; Estimate the incidence relation of genotype and phenotype, thus obtain maybe with coronary heart disease between have potential related chromosomal region; Next selects the representational site of corresponding chromosomal region to use FludigmEP1
TMGENETIC ANALYSIS system, Taqman MGB probe method is carried out the gene type experiment, further in 15460 routine patients with coronary heart disease and 11472 routine check samples, verifies.(Linkage disequilibrium, LD) situation is used r to linkage disequilibrium in data analysis between use Haploview computed in software chromosomal region site
2Expression (0-1) makes up relatively its haplotype difference between patient and normal control of haplotype (haplotype), the Susceptible population of checking and definite coronary heart disease simultaneously.Use logistic regression analysis assessment each SNP site and Coronary Heart Disease, (Odds ratio is OR) with 95% credibility interval to calculate dangerous allelic relative risk.Among the present invention, draw first through the lot of experiments analysis, (it is interregional that more particularly this zone is positioned at the last 32368kb～32520kb of karyomit(e) 6p21.32 upward in the intergenic one section close linkage zone of C6orf10-BTNL2 to be positioned at karyomit(e) 6p21.32; Data Source: human genome DB (Human, build36) series of SN-striking P site (rs9268402, rs442406; Rs9268473, rs3817973, rs2076533; Rs2076529, particularly rs9268402) and the coronary disease susceptibility significant correlation, on a large amount of experimental results of the present invention basis; These SNPs can be used as the label SNPs of karyomit(e) 6p21.32 regional gene, for research karyomit(e) 6p21.32 regional gene function provides new method, under the prerequisite that guarantees the research power of a test; Can effectively reduce the research link, reduce research cost.
On a large amount of experimental results of the present invention basis, of the present inventionly further discover this a series of SNPs site and coronary disease susceptibility significant correlation; See table 1, the dangerous allelotrope of these SNPs is respectively: the rs9268402 pleomorphism site is G, and the rs442406 pleomorphism site is A; The rs9268473 pleomorphism site is A; The rs3817973 pleomorphism site is C, and the rs2076533 pleomorphism site is C, and the rs2076529 pleomorphism site is T.Carry these dangerous allelic individual relative risks that coronary heart disease take place and be do not have the carrier 1.14-1.17 doubly.Wherein karyomit(e) rs9268402 pleomorphism site G allelotrope carrier's incidence of coronary heart disease risk is that (OR=1.17,95%CI:1.07-1.27), risk is the highest for 1.17 times of the allelic carrier of A.Rs9268402 polymorphic site and rs442406, rs9268473, rs3817973, rs2076533, rs2076529 pleomorphism site have strong linkage disequilibrium (r
2＞0.7).Result of study of the present invention has confirmed that further Chr6p21.32 zone polymorphic site and coronary heart disease are closely related.
On the basis of above-mentioned label SNP, the present invention furthers investigate the haplotype of being made up of rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and rs2076529 again.The result finds: made up 3 kinds of haplotype: GAACCT, AGGTTC and AAACCT by Chr6p21.32 zone rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and rs2076529; Wherein haplotype GAACCT and AGGTTC are the most common in the crowd; Frequency is respectively 0.597 and 0.331; These two haplotypes are associated with the susceptibility of coronary heart disease, and there is significant difference in its frequency between patients with coronary heart disease and normal controls.Coronary heart disease risk haplotype (dangerous haplotype) is GAACCT, and the frequency in the coronary heart disease case is 0.626, is higher than the frequency 0.589 of control group significantly.
Thereby; On the one hand; The invention provides the karyomit(e) 6p21.32 regional gene haplotype of being made up of tagged single-nucleotide polymorphic loci of the present invention, this haplotype is respectively G, A, A, C, C, T at the mononucleotide in rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and rs2076529 site; A, G, G, T, T, C; Or A, A, A, C, C, T.Wherein, The haplotype that mononucleotide in rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and rs2076529 site is respectively G, A, A, C, C, T is the dangerous haplotype of karyomit(e) 6p21.32 regional gene; The present invention has explained this problem through a large amount of definite experimental results; After proofreading and correct age, BMI, hypertension, smoking, drinking, carry the individuality of this haplotype and suffer from of dangerous significantly the raising of the danger of coronary heart disease than the individuality trouble coronary heart disease of carrying other haplotypes.
On the other hand, the present invention provides a kind of method that detects the tagged single-nucleotide polymorphic loci of karyomit(e) 6p21.32 regional gene of the present invention.Available multiple technologies known in the art detect the tagged single-nucleotide polymorphic loci of karyomit(e) 6p21.32 regional gene of the present invention at dna level, rna level.For example:
Can adopt the method for direct order-checking; Can directly disclose crt gene and carry the sequence difference between the mutator gene through the dna direct order-checking; Specifically can be that traditional commercial sequencing kit of use or automatic DNA sequencer DNA checks order to dna direct, or the tetra-sodium of development in recent years order-checking (Pyrosequencing), micrometering preface (SNaPshot) etc.The tetra-sodium sequencing technologies is suitable for the sequencing analysis to known short sequence; After its principle is primer and template DNA annealing; Under the synergy of archaeal dna polymerase (DNA polymerase), ATP sulfurylase (ATP sulfurytase), luciferase (1uciferase) and 4 kinds of enzymes of apyrase (Apyrase); The polymerization of each dNTP on the primer and the release coupling of first order fluorescence signal are got up; Through detecting the release and the intensity of fluorescence, reach the purpose of The real time measure dna sequence dna.The SnaPshot technology platform is Applied Biosystems; ABI company has released to aim at detects SNP analysis of design software and test kit; Can carry out gene type simultaneously to a plurality of SNP site, also be called as minisequencing, after the primer SNaPshot reaction of this method to different mutational site design different lengthss; Product is analyzed through electrophoretic separation, multicolored fluoroscopic examination, Gene mapper, can in a running gel, detect a plurality of SNP site.
Also can adopt method, specifically comprise the Taqman probe method, DNA chip method etc. based on hybridization.TaqMan SNP gene type principle: utilize exonuclease that the excision of 5 ' the special allelotrope dye marker is produced the check signal that continues, reaction system comprises: with genomic dna or PCR product is template; One couple of PCR primers, and two types of 2 MGB probe in detecting SNP of using FAM and VIC mark respectively; Read the somatotype data at the PCR reaction end.The DNA chip technology: testing gene is after extracting; Be cut into fragment different in size; Behind fluorescence chemical material mark, be expelled on the slide glass that is embedded with chip, because the degree of DNA and probe hybridization is relevant with fluorescence intensity; Therefore through laser scanning, can measure the variation of sequence to be detected according to the fluorescence power.
Can also adopt method, resolve ion flight time mass spectrum (MALDI-Tof-MS) like ground substance assistant laser based on primer extension.Ground substance assistant laser is resolved the ion flight time mass spectrum and is detected principle: one section probe of adjacent SNP site design; In reaction system, substitute dNTP, make probe only extend a base and promptly stop, according to the difference in SNP site in the SNP site with ddNTP; Probe will combine different ddNTP; Thereby have different molecular weight, mass spectrograph can detect this molecular weight difference, thereby realizes the purpose of SNP somatotype.
Can also adopt method based on conformation; Concrete example such as restriction fragment length polymorphism (RFLP) are analyzed, single strand conformation polymorphism (single-strand conformational polymorphism; SSCP) analysis, denaturing gradient gel electrophoresis (denaturing gradient gel electrophoresis; DGGE) (denaturing high performance liquid chromatography dHPLC) waits the technology of analysis for analysis, sex change high-efficient liquid phase chromatogram technology.Wherein, The principle of RFLP: some SNP polymorphic site just in time is in the recognition site place of restriction enzyme; Wherein a kind of pcr amplification segment of polymorphic correspondence can be cut by corresponding restriction endonuclease; And another kind can not, therefore can know through the fragment length analysis behind the PCR product electrophoresis after enzyme is cut and detect the genotype of sample in this site; There is not proper restriction site if detect the SNP site; Often can introduce restriction enzyme digestion sites, can realize that through this primer method of modifying the somatotype in most SNP site can both realize with rflp analysis through changing indivedual bases at PCR primer 3 ' end.SSCP: under the condition of non-sex change, single stranded DNA has certain pleated sheet structure, and this pleated sheet structure is by its nucleotide sequence decision; The susceptibility of SSCP depends on SNP to folding influence and the folding electrophoretic mobility that how to influence aim sequence; Its strategy is, the fragment to be measured of pcr amplification is mixed with deionized formamide, and then 95 ℃ of sex change are unwind; Be quenched to again in the ice, separate through native polyacrylamide gel electrophoresis then; Under the stable ideal conditions, the isolating result of gel electrophoresis is that heterozygote comprises two very near bands of separation in a certain position range, and normal dna fragmentation occupy a wherein band, and the dna fragmentation of homozygous mutation SNP occupy another band.DGGE: utilize double chain DNA molecule in the gel of certain gradient sex change agent concentration, during electrophoresis, can issue first portion in certain denaturing agent concentration and unwind, cause electrophoretic mobility to descend; Even have the difference of having only a base pair between a kind of two kinds of dna moleculars of SNP allelotype respectively, also can part take place at different time and unwind, thereby be separated into two bands.DHPLC: this technology is the mutating technology of a detection SNP who on SSCP and DGGE basis, grows up, and this technology is mixed with the dna fragmentation and the wild-type DNA of the unknown, after heat denatured, makes its cooling reannealing again; If the DNA of sudden change is arranged, this moment, formed duplex had two kinds, was homologous duplex and allos duplex; Different based on homologous duplex and allos duplex melting temperature(Tm); Through the temperature of control DHPLC, it is maintained near the operation down of dna molecular Tm value, carry out wash-out then; The more little dna molecular and the avidity of post are more little, just elute more easily; After DNA expands through PCR because the existence of base mismatch forms heteroduplex because single stranded DNA with charge ratio double-stranded few, eluted earlier, and separated with normal pairing two strands, last peak type or number according to chromatographic peak confirmed having of SNP or do not had.
Can also adopt high resolving power solubility curve analytical technology (HRM).This analytical technology is according in certain TR, the product of pcr amplification being carried out sex change, during fluorescent signal in the detection architecture in real time.Fluorescent value can be drawn solubility curve along with temperature variation.Each segment DNA all has its unique sequence, thereby unique melting curve shape has also just been arranged, and is the same as dna fingerprinting, has very high specificity, stability and repeated.Accurately distinguish wild-type homozygote, heterozygote and mutagenicity homozygote according to curve.
In the specific implementation, those skilled in the art can select the tagged single-nucleotide polymorphic loci of above-mentioned any technological vitro detection karyomit(e) 6p21.32 of the present invention regional gene according to practical situation.Also can adopt the combination of multiple technologies to come the mutational site of vitro detection karyomit(e) 6p21.32 regional gene sequence.For example, when the restriction fragment length polymorphism analytical procedure with after PCR combines, detection sensitivity and specificity are higher.When direct sequencing was used in combination with PCR, detection sensitivity improved greatly.In an embodiment of the present invention, application be Taqman MGB method, use be FludigmEP1
TMGENETIC ANALYSIS system platform; Because this platform is the gene type platform that belongs to higher flux; When sample number is 96 sites of 96 pattern detection (using 96.96 dynamic chips) or 48 sites of 48 pattern detection (using 48.48 dynamic chips), has the advantage of economical and efficient.And when being applied to the analyzing and testing of a small amount of testing sample, adopt FludigmEP1 specific to scheme of the present invention
TMGENETIC ANALYSISsystem platform does not just possess the advantage on economy and the efficient; Can adopt the real-time fluorescence quantitative PCR system this moment; Like 7900HT (Fast) the real time PCR system of AppliedBiosystems, 7500real time PCR system etc., use the Taqman-MGB probe method gene type is carried out in single site; Also can PCR RFLP, other methods of genotyping such as HRM.
On the basis of the method for the tagged single-nucleotide polymorphic loci of above-mentioned detection karyomit(e) 6p21.32 of the present invention regional gene, the present invention also provides the reagent of the mononucleotide polymorphism site rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and/or the rs2076529 that detect karyomit(e) 6p21.32 regional gene to detect the preparation of karyomit(e) 6p21.32 regional gene haplotype or the application in the test kit in preparation.More specifically, the reagent of the mononucleotide polymorphism site of said detection karyomit(e) 6p21.32 regional gene can be the reagent that is used for direct sequencing; Or be used for combine with the restriction fragment length polymorphism analysis reagent of (PCR-RFLP) of polymerase chain reaction; Or be used for the reagent that the polymerase chain reaction combines with direct sequencing; Or be used for the reagent of following any SNP classifying method: based on the method for hybridization, based on the method for primer extension, based on the method or the high resolving power solubility curve analytical technology of conformation.
On the other hand, the present invention also provides a kind of method that detects the dangerous haplotype of karyomit(e) 6p21.32 regional gene, and this method comprises the tagged single-nucleotide polymorphic loci that detects karyomit(e) 6p21.32 regional gene.
Detect the method for any detection SNP that the method for the tagged single-nucleotide polymorphic loci of karyomit(e) 6p21.32 regional gene can be known in the art, for example can be for the above-mentioned direct sequencing of this specification sheets, based on the method for hybridization, based on the method for primer extension, based on detection methods such as the method for conformation or high resolving power solubility curve analytical technologies.In addition, the present invention's method of detecting the dangerous haplotype of karyomit(e) 6p21.32 regional gene also can comprise the detected result of said tagged single-nucleotide polymorphic loci is carried out statistical study.For example; In an embodiment of the invention; This method is utilized the Haploview program, after proofreading and correct age, BMI, hypertension, smoking, drinking, the result by tagged single-nucleotide polymorphic loci of the present invention is carried out statistical study; Especially the haplotype of being made up of tagged single-nucleotide polymorphic loci of the present invention is carried out statistical study, thereby drawn the dangerous haplotype of karyomit(e) 6p21.32 regional gene.
On the other hand; The present invention also provides a kind of method of vitro detection coronary heart disease dependent genes, and this method comprises the tagged single-nucleotide polymorphic loci that detects karyomit(e) 6p21.32 regional gene: rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and/or rs2076529.The present invention is on the basis of a large amount of experiments and statistical study; Chosen the normal artificial research object of 16975 routine patients with coronary heart disease and 16491 examples; Proved karyomit(e) 6p21.32 regional gene and Coronary Heart Disease with definite experimental data, thereby coronary heart disease dependent genes is provided.
Thereby the present invention also provides the tagged single-nucleotide polymorphic loci that detects karyomit(e) 6p21.32 regional gene: the reagent of rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and/or rs2076529 is used for the application of the preparation or the test kit of vitro detection coronary heart disease dependent genes in preparation.
The present invention also provides the tagged single-nucleotide polymorphic loci that detects karyomit(e) 6p21.32 regional gene: the reagent of rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and/or rs2076529 is used for the vitro detection testing sample in preparation and whether contains the preparation of the dangerous haplotype of karyomit(e) 6p21.32 regional gene or the application in the test kit; Wherein, the mononucleotide of the dangerous haplotype of karyomit(e) 6p21.32 regional gene in the rs9268402 site is G.Further, the mononucleotide of the dangerous haplotype of karyomit(e) 6p21.32 regional gene in rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and rs2076529 site is respectively G, A, A, C, C, T.
On the other hand, the present invention also provides the method that whether contains the dangerous haplotype of karyomit(e) 6p21.32 regional gene in a kind of vitro detection testing sample, and this method comprises:
1) DNA of extraction testing sample carries out pcr amplification to the tagged single-nucleotide polymorphic loci of karyomit(e) 6p21.32 regional gene: rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and/or rs2076529 design primer;
2) whole PCR products are analyzed;
3) identify the mononucleotide polymorphic in rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and/or rs2076529 site, whether the haplotype of its formation is: G-A-A-C-C-T, A-G-G-T-T-C or A-A-A-C-C-T.
On the other hand, the present invention also provides the tagged single-nucleotide polymorphic loci of a kind of vitro detection from karyomit(e) 6p21.32 regional gene in the sample of individuality to be measured: the reagent of rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and/or rs2076529 is in the preparation of the danger of preparation prediction individual trouble coronary heart disease to be measured or the application in the test kit.From another angle; The present invention also provides a kind of external prediction the individual method of suffering from the danger of coronary heart disease to be measured, and this method comprises the tagged single-nucleotide polymorphic loci of vitro detection from karyomit(e) 6p21.32 regional gene in the sample of individuality to be measured: rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and/or rs2076529.Wherein, the mononucleotide that the carries the rs9268402 site remarkable rising of danger that to be the individual of G or the individuality that carries the haplotype G-A-A-C-C-T that is made up of the mononucleotide of site rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and the rs2076529 danger of suffering from coronary heart disease suffer from coronary heart disease than the individuality that carries the haplotype A-G-G-T-T-C that is made up of the mononucleotide of site rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and rs2076529 or A-A-A-C-C-T.The testing sample that contains gene of the present invention can obtain Tathagata autoblood, urine, saliva, gastric juice, hair, the cell of examination of living tissue and necrotomy material from experimenter's cell.Preferably come autoblood.Can obtain to contain the testing sample of gene of the present invention earlier from experimenter's cell, extract the DNA of gene then according to ordinary method.Said individuality to be measured is preferably Chinese han population.The present invention is through a large amount of experiments, after proofreading and correct age, BMI, hypertension, smoking, drinking, compares with haplotype A-G-G-T-T-C or A-A-A-C-C-T, and the individuality that carries haplotype G-A-A-C-C-T is suffered from the dangerous of coronary heart disease and significantly raise.On the basis of the statistical study of large sample of the present invention; Can use method of the present invention separately; The haplotype that vitro detection constitutes from the tagged single-nucleotide polymorphic loci of karyomit(e) 6p21.32 regional gene in the sample of individuality to be measured: rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and rs2076529; With the external prediction individual danger of suffering from coronary heart disease to be measured; Also can consider other environmental risk factors simultaneously, whether whether for example whether smoking, hypertension, mellitus, blood fat disorder and whether have the inclination to obstruct family history etc. whether reach the external prediction individual dangerous purpose of coronary heart disease of suffering to be measured.
On the other hand, the present invention also provides a kind of test kit of polymorphum of the tagged single-nucleotide polymorphic loci that detects karyomit(e) 6p21.32 regional gene, and test kit contains respectively:
1) primer in amplification rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and/or rs2076529 site;
2) pcr amplification enzyme and corresponding damping fluid;
3) measure rs9268402, rs442406, rs9268473, rs3817973, the polymorphic reagent of rs2076533 and/or rs2076529 site.
In the said test kit one or more containers can be housed, one or more components that contain the karyomit(e) 6p21.32 regional gene of said pleomorphism site in order to detection are housed in the container.According to the difference of concrete detection method and detection pleomorphism site, test kit can contain different components.What provide simultaneously with it can be through medication management mechanism of government audit, relevant medicine or biological products manufacturing, the information using and sell.Those of ordinary skills are known; The increase primer of said pleomorphism site can be generally 15-30 base according to the known nucleotide sequence design of the present invention; GC content is about 45%-50%; Under suitable temperature, combine with template specificity, it can utilize special computer programming, for example (Oligo 6.53 softwares).The enzyme of pcr amplification can be TaqDNA polysaccharase, Tth archaeal dna polymerase, and VENT archaeal dna polymerase etc. can be used in the enzyme of pcr amplification.The reagent that detects said polymorphum in the test kit is according to the difference of detection method and difference.For example, adopt the Taqman detecting probe method, can contain the taqman-MGB probe that is useful on the special SNP of detection site in the test kit; Adopt the MALDI-TOF-MS method, can contain the single-basic extension primer that is used to detect special SNP site in the test kit; When adopting the polymerase chain reaction to detect said pleomorphism site, can contain restriction enzyme and corresponding restriction enzyme mapping in the test kit with the restriction fragment length polymorphism analysis method of combining.The test kit of the polymorphum of the tagged single-nucleotide polymorphic loci of said detection karyomit(e) 6p21.32 regional gene can be used for the vitro detection coronary heart disease dependent genes, and the vitro detection individual danger of suffering from coronary heart disease to be measured.
In sum, the present invention has found karyomit(e) 6p21.32 regional gene 6 important variant sites: rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and the rs2076529s relevant with coronary heart disease.Wherein the particular combination in particularly said 6 sites, rs9268402 site has increased the individual risk of suffering from coronary heart disease greatly.This discovery has the potential using value clinically: genotype identification is carried out in these 6 sites to karyomit(e) 6p21.32 regional gene; Carry the G-A-A-C-C-T haplotype if find certain individuality; Then should compare with the individuality that carries A-G-G-T-T-C or A-A-A-C-C-T haplotype by individuality, the risk of suffering from coronary heart disease significantly raises.In view of the above, the doctor can instruct and should change bad life habits by individuality, reduces environmental factors bringing out disease.It is thus clear that the haplotype that karyomit(e) 6p21.32 regional gene label SNP forms has important application prospects in the prediction of coronary heart disease and prevention.
Description of drawings
Fig. 1 is for showing the association diagram of chr6p21.32 region S NP site and coronary heart disease.Wherein, X-coordinate is a chromosome position, and ordinate zou is the SNP site and the related significance P value (log of coronary heart disease
10P), represent site rs9268402 with ◆ the expression.
Fig. 2 uses FludigmEP1 among the embodiment
TMGENETIC ANALYSIS system when Taqman MGB probe method is carried out the gene type experiment, carries out the application of sample process synoptic diagram of Assay and Sample.
Fig. 3 shows that 96 samples (94 samples to be tested and 2 negative controls) represent site rs9268402 gene type result (dendrogram).Wherein, the X axle is represented the VIC fluorescence intensity, and the Y axle is represented FAM fluorescence; Genotype result: 1, redness is the A/A homozygote, 2, green is the G/G homozygote, 3, blueness is the A/G heterozygote, 4, the negative contrast of black (NTC).
The snapshot in rs9268402 site when Fig. 4 carries out data analysis for using SNP Genotyping Analysis software version 3.0 softwares.
Fig. 5 is that 96 samples (wherein comprising two negative control NTC) rs9268402 gene type data derive result's (part) sectional drawing.
Embodiment
In order more to be expressly understood the present invention, further describe the present invention with reference to the following example and accompanying drawing at present.Embodiment only is used for explaining and does not limit the present invention in any way.The experimental technique of unreceipted actual conditions is ordinary method and the normal condition that affiliated field is known among the embodiment, or the condition of advising according to manufacturers.
Selection and the detection of embodiment one chr6p21.32 gene label SNPs
One, case-control sample inclusion criteria
The coronary heart disease case comprises myocardial infarction patient and patient with angina pectoris.The selected Case definition of myocardial infarction case is Diagnosis of Acute Myocardial Infarction standard (according to WHO a Case definition in 1979): promptly the typical chest pain symptom duration is more than 30 minutes; Continuous 2 the ST sections of leading of electrocardiogram(ECG are raised (limb leads 0.1mv, chest leads 0.2mv) and serial dynamic change are arranged; The serum marker substrate concentration of myocardial necrosis raises, and raises like troponin (TNT/TNI), and myocardium isozyme (CK-MB) raises greater than high 2 times of limitting of normal value.The selected Case definition of stenocardia case surpasses 70% for finding that through coronary angiography at least one main branch of coronary artery is narrow.Valvular heart disease, congenital heart disease, heart failure, serious kidney and hepatic diseases, secondary hypertension, myocardosis, familial hypercholesterolemia and suspicious patients with coronary heart disease except each item inspection.The case that meets above-mentioned diagnosis can be gone into anthology research.The control group inclusion criteria: previously there are not coronary heart disease or other Atheromatosis histories, no pectoralgia, cardiac symptom such as uncomfortable in chest, electrocardiogram(ECG does not have obvious ischemic change.Case group and control group are Chinese han population, and consanguinity-less relation.
Study population's essential characteristic: the study population comprises the chip detection sample (comprising 1515 routine patients with coronary heart disease and 5019 example contrasts) in examination stage, essential characteristic such as following table:
Two, research experiment scheme
In Chinese han population, choose the normal artificial research object of 1515 routine patients with coronary heart disease and 5019 examples.Use full genome chip (Affymetrix Axiom
TMGenome-Wide CHB 1Array Plate) SNP in the full genome range is carried out gene type.The data of utilizing chip to obtain are estimated the incidence relation of genotype and phenotype, thus obtain maybe with coronary heart disease between have potential related chromosomal region.(Linkage disequilibrium, LD) situation is used r to linkage disequilibrium in data analysis between use Haploview computed in software chromosomal region site
2Expression (0-1) makes up relatively its haplotype difference between patient and normal control of haplotype (haplotype), the Susceptible population of checking and definite coronary heart disease simultaneously.Use logistic regression analysis assessment each SNP site and Coronary Heart Disease, (Odds ratio is OR) with 95% credibility interval to calculate dangerous allelic relative risk.
Chr6p21.32 region S NP site sequence
Three, experimental result
Each site strength of association (OR value) and significance level (P value) through related (GWAS) analytical calculation SNP of full genome chip; Discovery is positioned at karyomit(e) 6p21.32 and goes up that (32368kb～32520kb is interregional) has a correlation signal (P value is less than 0.001) (see Fig. 1, provided the rs9268402 upstream and downstream regional all sites of the 300K significance related with coronary heart disease altogether) between the C6orf10-BTNL2 gene.Series of SN-striking P site in this section close linkage zone (rs9268402, rs442406, rs9268473, rs3817973, rs2076533 rs2076529) with the coronary disease susceptibility significant correlation, sees table 1.This a series of SNP site and both wings correlated series thereof are following:
rs9268402：AAATCTTGAGAGGGATATGAACATCC[A/G]GATTGAAGACGATCAAAGCATCCCA(SEQIDNo.1)
rs4424066：ATGAAAGTGGACACACCATGACTGGA[A/G]ATACTATAGGACTTAATTTTAAGAA(SEQ?IDNo.2)
rs9268473：CAAGAAAACTCCAAACAAGTTCATGG[A/G]TTTCTCATCTGTTTTCATGCTGAGT(SEQIDNo.3)
rs3817973：GTGGACTCTGACTAAAGAAGACTTGT[T/C]TTGTTCGCACACTAGGTACCTGTAA(SEQ?IDNo.4)
rs2076533：CATAGAATGATGTATAAAATGTTTTC[T/C]GAGTAACAAAGTGTCACACAATAAT(SEQIDNo.5)
rs2076529：TACCGTCTCATGTCTCCCTCCTGACA[T/C]GACCACTCACTGCGGTAACTGCTCC(SEQ?IDNo.6)
The dangerous allelotrope in above-mentioned SNP site is respectively: the rs9268402 pleomorphism site is G; The rs442406 pleomorphism site is A, and the rs9268473 pleomorphism site is A, and the rs3817973 pleomorphism site is C; The rs2076533 pleomorphism site is C, and the rs2076529 pleomorphism site is T.Carry these dangerous allelic individual relative risks that coronary heart disease take place and be do not have the carrier 1.14-1.17 doubly, see table 1.Wherein karyomit(e) rs9268402 pleomorphism site G allelotrope carrier's incidence of coronary heart disease risk is that (OR=1.17,95%CI:1.07-1.27), risk is the highest for 1.17 times of the allelic carrier of A.This zone rs9268402 polymorphic site and rs442406, rs9268473, rs3817973, rs2076533, rs2076529 pleomorphism site have strong linkage disequilibrium (r
2＞0.7, see table 2).
By Chr6p21.32 zone rs9268402, rs442406, rs9268473, rs3817973, rs2076533, rs2076529 have made up 3 kinds of haplotypes, and wherein haplotype GAACCT and AGGTTC are the most common in the crowd, and frequency is respectively 0.597 and 0.331.These two haplotypes are associated with the susceptibility of coronary heart disease, and there is significant difference (seeing table 3) in its frequency between patients with coronary heart disease and normal controls.The coronary heart disease risk haplotype is GAACCT, and the frequency in the coronary heart disease case is 0.626, is higher than the frequency 0.589 of control group significantly.
The association results of table 1.Chr6p21.32 region S NP site and coronary heart disease
Zone: the chromosomal region band of representing this pleomorphism site place; Risk allele: risk allelotrope; Other allele: with reference to allelotrope; Gene frequency is the risk gene frequency.OR (95%CI) representes to compare with carrying the allelic individuality of reference, carries the allelic individual relative risk that coronary heart disease takes place of risk.
Table 2.Chr6p21.32 zone rs9268402 and other SNP linkage disequilibriums relation (r
2)
| The SNP polymorphic site | The zone | Chromosome position | r 2 |
| rs9268402 | 6p21.32 | 32449331 | 1.00 |
| rs4424066 | 6p21.32 | 32462406 | 0.74 |
| rs9268473 | 6p21.32 | 32463661 | 0.74 |
| rs3817973 | 6p21.32 | 32469089 | 0.74 |
| rs2076533 | 6p21.32 | 32471505 | 0.74 |
| rs2076529 | 6p21.32 | 32471933 | 0.74 |
The association results of table 3.Chr6p21.32 region S NP haplotype and coronary heart disease
* haplotype is respectively by rs9268402, rs442406, and rs9268473, rs3817973, rs2076533, rs2076529 forms.
The detection of embodiment two chr6p21.32 gene label SNPs
In the present embodiment, further in 15460 routine patients with coronary heart disease and 11472 routine check samples, detect to the representational site of embodiment one selected corresponding chromosomal region, conduct is to implementing the checking of a selected label SNPs simultaneously.
One, case-control sample inclusion criteria
Case-control sample inclusion criteria is with embodiment one.The study population attaches most importance to and rechecks test card sample (comprising 15460 routine patients with coronary heart disease and 11472 example contrasts), essential characteristic such as following table:
Two, detection method
The duplicate detection checking of present embodiment is to use FludigmEPl
TMGENETIC ANALYSIS system; This system is the high-throughput gene type system of U.S. chip Fludigm company exploitation, is made up of integrated fluid pipeline (IFC) chip, integrated fluid conduit controller (IFC controller), heat circulating system and fluorescent signal acquisition system (EP1reader); Adopt the conventional reagent of Taqman gene type, can carry out gene type to 96 SNP sites of 96 samples simultaneously; Be a kind of high-throughout open genetic analysis platform (Wang, J., Lin, M., Crenshaw, A.; Hutchinson, A., Hicks, B., Yeager, M.; Berndt, S., Huang, W., Hayes, R.B.; Chanock, S.J., Jones, R.C., and Ramakrishnan, R.2009Nov 28.High-throughput single nucleotide polymorphism genotyping using nanofluidic Dynamic Arrays.BMC Genomics).
Taqman MGB probe method is carried out the gene type experiment, and this gene type experiment is specific as follows: instrument, material
Instrument: FludigmEP1
TMGENETIC ANALYSIS system reagent, sample:
Consumptive material:
| 96.96 gene typing chips | ?96.96Dynamic?Array(138X) |
| 96 hole PCR plates and disposable shrouding film | ?96well?plates?and?seal(one-off) |
Experimental technique
(1) operation steps:
1.Assay preparation: on 96 hole PCR plates, prepare Assay mix with reference to following table
| ?Component | Each well volume (μ l) |
| ?40×Taqman?Genotyping?Probe | ?1.25 |
| ?2×Assay?Loading?Reagent | 2.5 |
| ?ROX(50×) | 0.25 |
| ?DNase/RNase-free?water | 1.0 |
| TV | 5.0 |
After adding, cover disposable shrouding film, abundant mixing on the microwell plate vibrator, centrifugal 30 seconds of 2500～3000rpm on the microwell plate whizzer (about 500g cf-) places subsequent use on ice.
2.sample preparation: in 96 orifice plate PCR, prepare Sample mix with reference to following form:
| Component | Each well volume (μ l) |
| Master?Mix(2×) | 3 |
| 20×GT?Sample?Loading?Reagent | 0.3 |
| AmpliTaq Gold archaeal dna polymerase | 0.06 |
| Genomic?DNA | 2.52 |
| DNase/RNase-free?water | 0.12 |
| TV | 6.0 |
After adding, cover disposable shrouding film, abundant mixing on the microwell plate vibrator, centrifugal about 30 seconds of 2500～3000rpm on the microwell plate whizzer (about 500g cf-) places subsequent use on ice.
3. application of sample and amplification procedure
Chip is in integrated fluid conduit controller HX after the initialize, with reference to Fig. 2 application of sample.(the application of sample amount of Assay is 4.2 μ l/ holes, and the application of sample amount of Sample is 5.2 μ l/ holes).To add excellent chip and reappose in integrated fluid conduit controller HX, start " Load Mix (138X) " program, carry out separatory and mixing.
After said process is accomplished; Take out chip (in one hour) from integrated fluid conduit controller HX at once; Remove the blue pad pasting in chip back, chip is placed heat circulating system (Stand alone Thermo Cycler), start Thermo Cycler PCR appearance; The PROGTM96 program is carried out the PCR process.After PCR finishes, turn off PCR appearance below vacuum pump (Vacuum Pump), open PCR appearance upper cover, take off chip.
(2) fluorescent signal data gathering: start fluorescent signal acquisition system (EPl reader);
DataCollection Software version 3 softwares are gathered the fluorescent signal data.
(3) gene data analysis mode and result output: data using SNP Genotyping Analysis software version 3.0 softwares with collecting, carry out data analysis.Represent the FAM fluorescence signal intensity with the X axle, the Y axle is represented the VIC fluorescence signal intensity, according to mononucleotide polymorphic site not two kinds of fluorescent signals of isoallele highly carry out cluster relatively by force, the gene type result exports with the excel form.Software parameter can design voluntarily, as: represent the VIC fluorescence signal intensity with the X axle, the Y axle is represented the FAM fluorescence signal intensity; Represent XX, YY homozygote respectively with red and green, on behalf of heterozygote, black, blueness represent negative control (NTC).The gene type result exports with the excel form.
Three, experimental result
The rs9268402 locus gene somatotype dendrogram that detects is seen Fig. 3.The screenshotss in rs9268402 site were referring to Fig. 4 when using SNP Genotyping Analysis software version 3.0 softwares carried out data analysis.96 samples (wherein comprising two negative control NTC) rs9268402 gene type data derive result's (part) sectional drawing referring to Fig. 5.
In the present embodiment, 6p21.32 Regional Representative site rs9268402 further verifies in 15460 routine patients with coronary heart disease and 11472 routine check samples, remarkable related (P=1.04 * 10 of rs9268402 and coronary heart disease
-12) see table 4, further merge all 16975 routine cases and related more significantly (P=2.77 * 10 of 16491 check sample analyses demonstration rs9268402 with coronary heart disease
-15) (seeing table 5), this result has confirmed that further Chr6p21.32 zone polymorphic site and coronary heart disease are closely related.
The association results of table 4.Chr6p21.32 Regional Representative site rs9268402 in the repeated authentication sample
HWE_P is the P value of HWE check.
The association results of table 5.Chr6p21.32 Regional Representative site rs9268402 in 16975 routine cases and 16491 routine check samples
Zone: the chromosomal region band of representing this pleomorphism site place; Risk allele (Freq): expression risk allelotrope and gene frequency thereof.OR (95%CI) representes to compare with carrying the allelic individuality of reference, carries the allelic individual relative risk that coronary heart disease takes place of risk.
Through above-mentioned experimental study of the present invention; The analysis of haplotype frequency difference between patient and normal people of forming to said SNP site rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and/or rs2076529 and by these SNP sites can draw the variation of each concrete base in site and by its haplotype difference formed and the relation of coronary disease susceptibility.As the crowd who belongs to one of following characteristic is the Susceptible population of coronary heart disease: the intergenic rs9268402 pleomorphism site of C6orf10-BTNL2 is G in karyomit(e) 6p21.32 zone, and the rs442406 pleomorphism site is A, and the rs9268473 pleomorphism site is A; The rs3817973 pleomorphism site is C, and the rs2076533 pleomorphism site is C, and the rs2076529 pleomorphism site is T; And by the SNP site rs9268402 that is positioned at this gene region; Rs442406, rs9268473, rs3817973; Rs2076533, the individuality of the haplotype GAACCT that rs2076529 forms.
The variation property of the haplotype of forming by the SNP site in one section C6orf10-BTNL2 gene region of karyomit(e) 6p21.32 and by series of SN-striking P site according to the present invention is judged the method for coronary heart disease Susceptible population; Can be to the crowd of the early stage clinical symptom of coronary heart disease not occurring; Especially the coronary heart disease high risk population who has conventional risk factors does not have the fast and convenient examination of wound, early discovery coronary heart disease susceptible object; And take corresponding hygienic measures, reduce the sickness rate of coronary heart disease and myocardial infarction.This is an important use of the present invention.
Also can scientific basis be provided according to the present invention for researching and developing coronary heart disease gene therapy targetedly.The present invention further detects gene expression regulation, protein function; The mechanism of causing a disease of further investigation coronary heart disease; Development of new medicine and gene therapy realize having established important foundation based on the individuation diagnosis and treatment of genetic background, and bring important medical health-benefiting and economic benefit.
Claims (10)
1. karyomit(e) 6p21.32 regional gene haplotype, this haplotype is respectively G, A, A, C, C, T at the mononucleotide in rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and rs2076529 site; A, G, G, T, T, C; Or A, A, A, C, C, T.
2. haplotype according to claim 1, wherein, said karyomit(e) 6p21.32 regional gene is meant that being positioned at karyomit(e) 6p21.32 goes up the interregional gene of 32368kb～32520kb.
3. a reagent that detects mononucleotide polymorphism site rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and/or the rs2076529 of karyomit(e) 6p21.32 regional gene detects the preparation of karyomit(e) 6p21.32 regional gene haplotype or the application in the test kit in preparation.
4. application according to claim 3, the reagent of the mononucleotide polymorphism site of wherein said detection karyomit(e) 6p21.32 regional gene is: the reagent that is used for direct sequencing; Or be used for the reagent that the polymerase chain reaction combines with the restriction fragment length polymorphism analysis; Or be used for the reagent that the polymerase chain reaction combines with direct sequencing; Or be used for the reagent of following any SNP classifying method: based on the method for hybridization, based on the method for primer extension, based on the method or the high resolving power solubility curve analytical technology of conformation.
5. detect the tagged single-nucleotide polymorphic loci of karyomit(e) 6p21.32 regional gene: the reagent of rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and/or rs2076529 is used for the application of the preparation or the test kit of vitro detection coronary heart disease dependent genes in preparation.
6. detect the tagged single-nucleotide polymorphic loci of karyomit(e) 6p21.32 regional gene: the reagent of rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and/or rs2076529 is used for the vitro detection testing sample in preparation and whether contains the preparation of the dangerous haplotype of karyomit(e) 6p21.32 regional gene or the application in the test kit; Wherein
The mononucleotide of the dangerous haplotype of karyomit(e) 6p21.32 regional gene in the rs9268402 site is G;
Further, the mononucleotide of the dangerous haplotype of karyomit(e) 6p21.32 regional gene in rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and rs2076529 site is respectively G, A, A, C, C, T.
7. vitro detection is from the tagged single-nucleotide polymorphic loci of karyomit(e) 6p21.32 regional gene in the sample of individuality to be measured: the reagent of rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and/or rs2076529 is in the preparation of the preparation prediction individual danger of suffering from coronary heart disease to be measured or the application in the test kit; Wherein, the mononucleotide that the carries the rs9268402 site remarkable rising of danger that to be the individual of G or the individuality that carries the haplotype G-A-A-C-C-T that is made up of the mononucleotide of site rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and the rs2076529 danger of suffering from coronary heart disease suffer from coronary heart disease than the individuality that carries the haplotype A-G-G-T-T-C that is made up of the mononucleotide of site rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and rs2076529 or A-A-A-C-C-T.
8. according to claim 6 or 7 described application, wherein said testing sample comes autoblood, urine, saliva, gastric juice, hair, examination of living tissue or necrotomy material.
9. according to claim 6 or 7 described application, wherein said testing sample is from Chinese han population.
10. the test kit of the polymorphum of a tagged single-nucleotide polymorphic loci that detects karyomit(e) 6p21.32 regional gene, this test kit contains respectively:
1) primer in amplification rs9268402, rs442406, rs9268473, rs3817973, rs2076533 and/or rs2076529 site;
2) pcr amplification enzyme and corresponding damping fluid;
3) measure rs9268402, rs442406, rs9268473, rs3817973, the polymorphic reagent of rs2076533 and/or rs2076529 site.
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| CN112820349A (en) * | 2020-12-31 | 2021-05-18 | 融智生物科技（青岛）有限公司 | Single nucleotide polymorphism genotyping method |
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| WO1998040517A1 (en) * | 1997-03-10 | 1998-09-17 | Medical Science Systems, Inc. | Prediction of coronary artery disease |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112820349A (en) * | 2020-12-31 | 2021-05-18 | 融智生物科技（青岛）有限公司 | Single nucleotide polymorphism genotyping method |
| CN112820349B (en) * | 2020-12-31 | 2023-05-23 | 融智生物科技（青岛）有限公司 | Single nucleotide polymorphism genotyping method |
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