CN1940086A - Method for determining patient's brain ictus genetic susceptibility - Google Patents

Abstract

A method for determining cerebrovascular disease genetic infectivity, its substance, composition and reagent kit are disclosed. The method includes determining VEGFR-2 gene polymorphic site-604T/G and/or +1192C/T and/or +1719A/T genetic polymorphic style. It can be used for cerebral thrombosis, spatial cerebral infarction and cerebral hemorrhage.

Description

Determine the method for patient to the cerebral apoplexy genetic predisposition

Technical field

The present invention relates to determine the method for patient, be used for material, composition, test kit of this method and uses thereof the genetic predisposition of cerebrovascular disease (cerebral apoplexy that particularly comprises cerebral thrombosis, lacunar infarction, hematencephalon).

Background technology

The sternness of cerebro-vascular diseases fashion trend, harm are seriously

Cerebral apoplexy (Stoke) claims apoplexy or cerebrovascular accident (Cerebrovascularaccident) again, is one group of unexpected onset, is the acute cerebrovascular disease of common trait with focal neurological deficit.Cerebral apoplexy has high case fatality rate and disability rate, and in China, cerebral apoplexy then is the second largest deadly disease that is only second to cancer, dies from 1,000,000 people that surpass of cerebral apoplexy every year, is 2-3 times of coronary heart disease death number.The control of cerebral apoplexy has become an important topic in the sanitary work, more and more causes the particularly attention of educational circles of neurology department of domestic and international medical circle.

Cerebral apoplexy is the multi-factor disease of clinical symptom complexity

The clinical symptom complexity of cerebral apoplexy, but mainly can be divided into two big classes: Ischemic Stroke and hemorrhagic apoplexy.The former accounts for 60-70%, and the latter accounts for 30-40%.Ischemic Stroke is further divided into again: the great vessels occlusive disease is due to the final obstructing arterial, to be called cerebral thrombosis owing to forming thrombus gradually in stricture of artery, the tube chamber, and its modal cause of disease is an atherosclerosis; Little vascular obstruction disease is, both not had thrombus in the tube chamber and also do not had embolus due to the final obturation because the brain arteriole is narrow gradually, is called lacunar infarction, and hypertensive arteriosclerosis is modal reason; Heart source property cerebral apoplexy is because thrombus comes off or other embolus enters in the blood flow and to block cerebral arteries and cause, can cause cerebral embolism just come off as the embolus in the atrial fibrillation or the patients with coronary heart disease chambers of the heart.Hemorrhagic apoplexy is divided into hematencephalon and subarachnoid hemorrhage again according to the difference of bleeding part.Hematencephalon is commonly called as " Intracerebral hemorrhage ", is that blood spills in the cerebral tissue owing to arteriorrhexis in the brain.Subarachnoid hemorrhage then is the angiorrhexis of brain surface or brain bottom, and blood directly enters in the subarachnoid space and the ventricles of the brain that contains cerebrospinal fluid.Secular essential hypertension and atherosclerosis are the major causes of hemorrhagic apoplexy.

The genetic risk factor that the research cerebral apoplexy is new is significant

A large amount of clinical practices prove, to the cerebral apoplexy that has taken place, in any case perfectly treatment is all not really desirable to the influence of its case fatality rate and natural history.Therefore, the policy of more answering lay special stress on " to put prevention first " to the control of cerebral apoplexy, and seek, determine the Hazard Factor (risk factor) of cerebral apoplexy, the infringement of managing to reduce or remove these Hazard Factor then then is the most basic means of prevention of brain palsy.The people of dangerous factor can be referred to as " palsy is easily suffered from individuality " (stroke-prone individual).Present fixed Hazard Factor comprise: race, sex, advanced age and the unmodifiable factor of this class of cerebro-vascular diseases family history; The bad life habits that smoking, excessive drinking, obesity, salt Excessive Intake etc. can change, and the disease [5] that can control to a certain extent such as hypertension, diabetes, cardiovascular diseases, hyperlipidaemia.In addition, homocysteine mass formed by blood stasis, plasma fibrinogen level unusual [7], the high aggregation of thrombocyte [8] etc. also are the Hazard Factor of cerebral apoplexy.

Cerebral apoplexy is a kind of multi-factor disease, its morbidity be subjected between the gene and gene and environmental factors between interactional influence jointly [9], interactions such as specific heritable variation and Hazard Factor that some is specific such as salt Excessive Intake, obesity, smoking, excessive drinking, hypertension, diabetes, hyperlipidaemia, may increase some individual danger of suffering from cerebral apoplexy, otherwise, even some individualities have these traditional Hazard Factor, the cardiovascular and cerebrovascular disease incident does not but take place.Therefore, the relevant protecting group of research cerebral apoplexy because of or deleterious gene, and further study relation between these genes and the environmental risk factor on this basis, be the important channel that the cerebral apoplexy individuation is prevented and treated.

Because racial difference, Chinese population has very big difference with the west crowd at everyways such as genetic background, living habit, dietary structure and social-economic structures, thereby the fashion trend of cardiovascular and cerebrovascular disease is also inequality.According to the foreign statistic data, in developed country's cerebro-vascular diseases with ischemia for seeing more, cerebral infarction accounts for 59.2%～85%, hematencephalon is except that Japan, generally below 20%.See that though China's cerebral infarction sickness rate is more the hematencephalon proportion is 30-40%, obviously high than external.Its reason it be unclear that, and also we carry out the point of interest of cerebral apoplexy inheritance susceptible Journal of Sex Research just for this.

Blood vessel takes place and the vital role of vascular endothelial growth factor in stroke onset

In angiopoietic process, the various kinds of cell factor is being brought into play important regulation, comprise vascular endothelial growth factor (Vascular endothelial growth factor, VEGF) and acceptor (VEGFR), angiogenin (Angiopoietin) and acceptor Tie thereof, and cytokine such as EphrinB2/EphB4.Wherein, VEGF and acceptor VEGFR thereof not only start vascularization, and are all bringing into play important effect in mature blood vessel stabilization process thereafter.Vascularization is unusual relevant with the pathophysiological process of multiple disease, as tumour, psoriasis, diabetic oculopathy and obesity, asthma, atherosclerosis etc.

VEGF is the somatomedin of specific action in vascular endothelial cell, plays important regulation in vascular development, maturation, restructuring procedure.Its effect is mainly: (1) promotes the mitotic division of endotheliocyte, induction of vascular endothelial hyperplasia and migration; (2) increase vascular permeability, (3) are the chemoattractants of monocyte and vascular smooth muscle cell (SMC).More and more evidences shows that the unstable of VEGF and atherosclerotic progress and patch has substantial connection recently.(ATVB such as Chen, 1999,19:131-139) using the original position immunohistochemical method confirms in atherosclerotic plaque, smooth muscle cell and scavenger cell all have the strongly expressed of VEGF, VEGF positive cell number and inner membrance vascularization degree are proportionate, and distribution is consistent with the distribution height of new vessel.Change the VEGF of people's reorganization over to the atherosclerosis susceptible mouse model, find that VEGF can promote the progress of atherosclerotic lesions by the mechanism such as formation of inflammatory infiltration and new vessel, scavenger cell in the patch and vascular endothelial cell quantity also significantly increase (NatureMedicine.2001; 7:425-429.).Suppress angiopoietic antagonist endostatin act on Apo E-/-mouse model, can significantly suppress the development of atheromatous plaque damage, the patch volume also dwindles (Circulation.1999 thereupon; 99:1726-1732.).The atherosclerosis mechanism that research such as Lemstrom KB cardiac muscle is transplanted, other cytokine actings in conjunction in results suggest VEGF and the graft microenvironment of living in, the formation of promotion atherosclerotic plaque (Circulation.2002,105:2524-2530).The VEGF level raises in Slevin etc. report acute ischemic cerebral apoplexy patient's the serum, and observes dependency (Stroke, 2000,31 (8): 1863-1870) are arranged between infarct size and the serum VEGF.

Up to the present, vegf receptor finds there are 4 kinds: VEGFR-1 (fms-relatedtyrosine kinase 1; FLT1), VEGFR-2 (kinase insert domainreceptor; KDR), VEGFR-3 and a kind of endothelial cell surface sphaeroprotein neuropilin-1.VEGFR-1 and VEGFR-2's is the high-affinity receptor of VEGF, but the tyrosine kinase activity of VEGFR-1 is lower, and its exact function and signal transduction path it be unclear that.VEGFR-2 is mainly at the vascular endothelial cell specifically expressing, and this external precursor cell such as newborn hemopoietic stem cell are also expressed, and is low expression level at the vascular endothelial cell of Adulthood quiescent condition.VEGF mainly promotes the interaction between endotheliocyte and the formation of new vessel tube chamber by VEGFR-2 in vascularization.The experiment of knock out mice shows that the mouse of isozygotying that VEGFR-2 lacks died from the acute vascular defective in 9.5 days at E, and pathological manifestations is that endotheliocyte and hemopoietic forebody cell lack, and VEGFR-2 is indispensable in the angiopoietic starting stage in prompting.

Single nucleotide polymorphism

Sequence variations in the human genome mainly is made up of single nucleotide polymorphism (" SNP "), and all the other sequence variations are that short series connection repeats (comprising little satellite), long series connection repeats (moonlet) and other insertion and disappearance.But SNP is the position that occurs two replaceable bases in the colony with measured frequency (promptly＞1%).SNP is known as " homotopic ", because because the existence of this polymorphism, some members of species may have not mutant nucleotide sequence (that is, original " allelotrope "), and other member may have mutant nucleotide sequence (being variant or mutation allele).Under the simplest situation, may only there be a mutant nucleotide sequence, this polymorphism is called as two pairs of polymorphic alleles.The generation of replaceable sudden change can produce three pairs of polymorphic alleles etc.SNP extensively is present in the genome, and the SNP that changes gene function may directly help phenotypic variation.Because their popular and general essence, SNP has become the important tool that the location participates in the gene of human diseases situation.

Research purpose:

As candidate gene, adopt the case-control method research gene pleiomorphism of many clinical center and the dependency of stroke onset risk with the VEGFR-2 gene.

Summary of the invention

The present invention relates to be used for to determine VEGFR-2 gene polymorphic site-604T/C and/or+1192C/T and/or+instrument of the genetic polymorphism pattern of 1719A/T.It can be VEGFR-2 gene polymorphic site-604T/C and/or+1192C/T and/or+1719A/T polymorphism parting oligonucleotide, preferably, described polymorphism parting oligonucleotide is: (1) allele-specific nucleic acid primer, it can detect VEGFR-2 gene polymorphic site-604T/C and/or+1192C/T and/or+polymorphism of 1719A/T, perhaps (2) are used for detecting the oligonucleotide probe of the polymorphism of VEGFR-2 gene, its can be specifically with have VEGFR-2 gene polymorphic site-604T/C and/or+1192C/T and/or+nucleic acid hybridization of 1719A/T polymorphism, preferably, the length of oligonucleotide probe is 17-50 Nucleotide.Instrument of the present invention can be a restriction enzyme.

The invention still further relates to the diagnostic kit that is used for cerebrovascular disease (cerebral apoplexy that particularly comprises cerebral thrombosis, lacunar infarction, hematencephalon), comprise instrument of the present invention.

The invention still further relates to diagnosis patient cerebrovascular disease and (particularly comprise cerebral thrombosis; lacunar infarction; hematencephalon is at interior cerebral apoplexy) method; described method comprise detection from VEGFR-2 gene-604T/C in described patient's the sample and/or+1192C/T and/or+existence of 1719A/T; wherein; VEGFR-2 gene promoter area-604C allelotrope is relevant with the ill risk of cerebral thrombosis palsy of reduction; carry+the allelic individuality of 1192T and carrying+the ill risk of the allelic individual hematencephalon of 1719T increases; haplotype (604/+1192/+1719) C/C/T has provide protection to cerebral thrombosis, and haplotype (604/+1192/+1719) the ill risk increase of T/T/T haplotype carrier hematencephalon.

The invention still further relates to and determine that the patient (particularly comprises cerebral thrombosis to cerebrovascular disease; lacunar infarction; hematencephalon is at interior cerebral apoplexy) the method for genetic predisposition; described method comprise detection from VEGFR-2 gene-604T/C in described patient's the sample and/or+1192C/T and/or+existence of 1719A/T; wherein; VEGFR-2 gene promoter area-604C allelotrope is relevant with the ill risk of cerebral thrombosis palsy of reduction; carry+the allelic individuality of 1192T and carrying+the ill risk of the allelic individual hematencephalon of 1719T increases; haplotype (604/+1192/+1719) C/C/T has provide protection to cerebral thrombosis, and haplotype (604/+1192/+1719) the ill risk increase of T/T/T haplotype carrier hematencephalon.

The invention still further relates to the method for analysis, comprising from the isolating stripped biological sample of patient: determine VEGFR-2 gene polymorphic site-604T/C in the described sample and/or+1192C/T and/or+Nucleotide of 1719A/T.Preferably; this method also comprises determines that described patient's cerebrovascular disease (particularly comprises cerebral thrombosis; lacunar infarction; hematencephalon is at interior cerebral apoplexy) genetic predisposition or diagnosis patient cerebrovascular disease (particularly comprise cerebral thrombosis; lacunar infarction; hematencephalon is at interior cerebral apoplexy); wherein; VEGFR-2 gene promoter area-604C allelotrope is relevant with the ill risk of cerebral thrombosis palsy of reduction; carry+the allelic individuality of 1192T and carrying+the ill risk of the allelic individual hematencephalon of 1719T increases; haplotype (604/+1192/+1719) C/C/T has provide protection to cerebral thrombosis, and haplotype (604/+1192/+1719) the ill risk increase of T/T/T haplotype carrier hematencephalon.

In aforesaid method, can use following any difference foranalysis of nucleic acids technology to determine gene pleiomorphism: with mass spectrum to the direct mass analysis of PCR product, invasive cleaved products direct analysis, direct sequence analysis, allele specific amplification, allele-specific hybridization, oligonucleotide are connected test, effractor's test, DNA chip analysis and restriction fragment length polymorphism.

The invention still further relates to a kind of microarray, VEGFR-2 gene polymorphic site-604T/C wherein defined herein and/or+1192C/T and/or+1719A/T polymorphism parting oligonucleotide.Microarray can be the form of DNA chip.

The invention still further relates to detection of biological imitate VEGFR-2 gene polymorphic site-604T/C in the product and/or+1192C/T and/or+material of the existence of 1719A/T is used to prepare the purposes of diagnostic reagent, described diagnostic reagent is used to diagnose patient's cerebrovascular disease (cerebral apoplexy that particularly comprises cerebral thrombosis, lacunar infarction, hematencephalon) or is used for determining the genetic predisposition of patient to cerebrovascular disease (cerebral apoplexy that particularly comprises cerebral thrombosis, lacunar infarction, hematencephalon).Wherein said material can be selected from: instrument of the present invention; The antibody of Val297Ile sudden change and/or Gln472His sudden change in can specific recognition VEGFR-2; With can specific recognition VEGFR-2 gene VEGFR-2 gene polymorphic site-604T/C and/or+1192C/T and/or+primer or probe or the restriction enzyme etc. of 1719A/T.

The accompanying drawing summary

Gene type figure+the 32AA of Fig. 1 a.VEGFR-2 gene rs7667298 order-checking is polymorphic.

Gene type figure+the 32AG of Fig. 1 b.VEGFR-2 gene rs7667298 order-checking is polymorphic.

Gene type figure+the 32GG of Fig. 1 c.VEGFR-2 gene rs7667298 order-checking is polymorphic.

Gene type figure-the 65TT of Fig. 2 a.VEGFR-2 gene rs9994560 order-checking is polymorphic.

Gene type figure-the 65CC of Fig. 2 b.VEGFR-2 gene rs9994560 order-checking is polymorphic.

Gene type figure-the 604TC of Fig. 3 a.VEGFR-2 gene rs2071559 order-checking is polymorphic.

Gene type figure-the 604CC of Fig. 3 b.VEGFR-2 gene rs2071559 order-checking is polymorphic.

Gene type figure-the 604TT of Fig. 3 c.VEGFR-2 gene rs2071559 order-checking is polymorphic.

Gene type figure+the 1192CC of Fig. 4 a.VEGFR-2 gene rs2305948 order-checking is polymorphic.

Gene type figure+the 1192TT of Fig. 4 b.VEGFR-2 gene rs2305948 order-checking is polymorphic.

Gene type figure+the 1192CT of Fig. 4 c.VEGFR-2 gene rs2305948 order-checking is polymorphic.

Gene type figure+the 1719TT of Fig. 5 a.VEGFR-2 gene rs1870377 order-checking is polymorphic.

Gene type figure+the 1719AA of Fig. 5 b.VEGFR-2 gene rs1870377 order-checking is polymorphic.

Gene type figure+the 1719AT of Fig. 5 c.VEGFR-2 gene rs1870377 order-checking is polymorphic.

Fig. 6: VEGFR-2 gene rs2305948 (+1192C/T) with the RFLP somatotype result of restriction endonuclease BstZ171.

Fig. 7: VEGFR-2 gene rs2071559 (604T/C) the RFLP somatotype result of usefulness restriction endonuclease Bsm I.

Fig. 8: VEGFR-2 gene rs1870377 (+1719A/T) with the RFLP somatotype result of restriction endonuclease Alu I.

Fig. 9: the uciferase activity analysis of VEGFR-2 gene promoter-604 wild-type and mutant allele report carrier.

*: P＜0.001, pGL3/-604C and pGL3/-604T are relatively;

The plain unit of enzyme activity of relative fluorescence (ratio firefly/renilla luciferase) represents with mean ± standard deviation.

Embodiment

The present invention relates to method that the single nucleotide polymorphism in the VEGFR-2 gene and patient disease (as cerebrovascular disease dilated cardiomyopathy particularly) are associated.The invention still further relates to and determine the patient to the genetic predisposition of cerebrovascular disease (cerebral apoplexy that particularly comprises cerebral thrombosis, lacunar infarction, hematencephalon) or the method for these diseases of diagnosis patient, this method comprises at least one single nucleotide polymorphism that detects in the VEGFR-2 gene.Particularly, this method comprise detect at least VEGFR-2 gene locus-604 in the described sample and/or+1192 and/or+297 in 1719 Nucleotide or VEGFR-2 protein or 472 s' amino acid, and determine individual state with reference to the polymorphism in the VEGFR-2 gene.The invention still further relates to the isolating nucleic acid of the locational polymorphism that limits above being included in sequence, relating to can be with the nucleic acid primer of this nucleic acid hybridization and oligonucleotide probe and relate to and comprise the diagnostic kit that one or more such primers and probe are used for detecting the polymorphism of VEGFR-2 gene.

The VEGFR-2 gene is positioned at karyomit(e) 4q11-q12, the about 5.8kb of cDNA total length, and NCBIRefSeq mRNA accession No. is NM_002253, is made up of 30 exons and 29 introns.The encoded polypeptides chain is made up of 1356 amino acid.In this article, VEGFR-2 gene order and encoded protein matter sequence are numbered according to ordinary method.

The numbering of Nucleotide and amino-acid residue is meant with NCBI RefSeq mRNA accession No. to be sequence or corresponding Nucleotide and the amino-acid residue of its amino acid sequence coded same position (or numbering) of NM_002253 in nucleotide sequence that the present invention is mentioned and the aminoacid sequence.

" correspondence " is meant nucleic acid molecule of the present invention or the proteinic sequence position relative with this reference sequences position after comparing (alignment) with reference sequences sequence shown in the cDNA sequence of NM_002253 is best.Therefore, nucleic acid molecule of the present invention or protein " correspondence position " or " corresponding Nucleotide " or " corresponding amino acid " are not necessarily numbered identical position or Nucleotide or amino acid with reference sequences.

VEGFR-2 gene " 604T/C " is meant the-604 polymorphism T of this gene and C, and "+1192C/T " is meant the+1192 polymorphism C and T, and "+1719A/T " is meant the+1719 polymorphism A and T.

Single nucleotide polymorphism and detection thereof

Term " patient " is meant animal, preferred mammal, and more preferably primate is most preferably human.

Term " polymorphism " broadly is defined as and comprises known all variations that occur in the nucleotide sequence, comprises insertion, disappearance, displacement and tumor-necrosis factor glycoproteins (comprising that two-fold repeats).

Can be used by any appropriate method that use is used to detect single nucleotide variations according to aforesaid method of the present invention, such as with the direct mass analysis of mass spectrum to the PCR product, direct analysis to invasive cleaved products (invasive cleavage product), directly sequential analysis, allele specific amplification (promptly, the ARMSTM-allele specific amplification, ARMS refers to the sudden change system (amplification refractory mutationsystem) of being obstructed that increases), ALEXTM (amplification refractory mutation system linear extension (amplificationrefractory mutation system linear extension) and COPS (competitive oligonucleotide initiation system), allele-specific hybridization (ASH), oligonucleotide connects test (OLA), effractor's test, (summary is referring to genome research (Genome Research) for DNA chip analysis and restriction fragment length polymorphism (RFLP), 1998,8 volumes, the 769-776 page or leaf; Pharmacogenomics, 2000,1 volume, 95-100 page or leaf; Human mutant (HumanMutation), calendar year 2001,17 volumes, 475-492 page or leaf).

The nucleic acid sample that carries described polymorphism is blood, bronchoalveolar lavage fluid, phlegm, urine or other body fluid or the tissue samples that obtains from individuality easily.These samples can carry out purifying in advance, for example separate total DNA.Can understand, sample can be the nucleotide sequence corresponding to sequence in the sample equally, that is to say that before allelic variation was analyzed, all or part of zone in the nucleic acid samples can be earlier with any technology easily such as polymerase chain reaction (PCR) or ligase chain reaction (LCR) amplification.

Polymorphism in the VEGFR-2 gene can check order by the nucleic acid samples to the patient and this sequence and contrast are relatively come to determine, or determines by the fragment (coding region and the regulatory region of containing the VEGFR-2 gene) of 400-600 base pair in the irrelevant DNA of individual of the different ethnic derivations of pcr amplification.Fragment available forward in these samples and reverse primer order-checking, polymorphism can the gene frequency of variant can be determined (human mutant (Human Mutation) with (through the University of Washington's permission) detection of PolyPhred software, calendar year 2001,17 volumes, 243-254 page or leaf).

Obviously, whether exist for there being a large amount of analytical procedures can be used to detect for a person skilled in the art at one or more polymorphic position variation Nucleotide of the present invention.In general, the detection of the allelic variation recognition techniques of need suddenling change, randomly amplified reaction and randomly signal generating system.International Patent Application WO 00/06768 has been listed many amplification techniques and sudden change detection technique, and some of them are based on round pcr.These technology can be united use with many signal generating systems, and optionally signal generating system is also listed in WO00/06768.

The many current methods that are used to detect allelic variation are summarized in Nollau etc., clinical chemistry (Clin.Chem.),, 43 phases, 1114-1120 page or leaf in 1997; Standard textbook for example " sudden change test experience guide " (Laboratory Protocols for MutationDetection) U.Landegren edit, the Oxford University Press, 1996 with " PCR " second edition Newton ﹠amp; Graham edits, BIOS Scientific Publishers Limited, 1997.

The present invention relates to be used as the allele-specific nucleic acid primer that detects the diagnostic primers of polymorphism in the VEGFR-2 gene, this diagnostic primers can with in sequence in VEGFR-2 gene locus-604 and/or+1192 and/or+1719 comprise polymorphism (for example VEGFR-2 gene-604T/C and/or+1192C/T and/or+1719A/T or its reverse complementary sequence) nucleic acid hybridization.

Allele-specific primers is used from such as in the amplified reactions such as PCR reaction with constant primer one usually, made a distinction by an allelic selective amplification a particular sequence position is made between the allelotrope, for example be used in the primer in the ARMSTM test.The preferred 17-50 of the length of an allele-specific primers Nucleotide, more preferably about 17-35 Nucleotide, most preferably about 17-30 Nucleotide.

Preferably, allele-specific primers is fully corresponding with allelotrope to be detected, but also can consider its derivative, wherein nearly 6 to 8 Nucleotide of 3 ' end are corresponding with allelotrope to be detected and be no more than 10, such as being no more than 8,6,4,2 or 1 residual nucleus thuja acids can change under the situation of not remarkably influenced primer characteristic.Usually at the Nucleotide of the 2nd and/or the 3rd (with respect to 3 ' end) by mispairing to optimize the combination of difference primer and preferentially only to distinguish primer extension from correct allelotrope.

The invention still further relates to the oligonucleotide probe of the polymorphism that is used for detecting the VEGFR-2 gene, this probe can be specifically with in sequence, comprise be positioned at VEGFR-2 gene the-604 and/or+1192 and/or+1719 polymorphism (for example VEGFR-2 gene-604T/C and/or+1192C/T and/or+1719A/T or its reverse complementary sequence) nucleic acid hybridization.

Term " oligonucleotide probe " refers to that length has a kind of nucleotide sequence of 17 Nucleotide at least, and this sequence and part or all of human VEGFR-2 gene are particularly expressed the human VEGFR-2 Gene Partial correspondence of polymorphism.17 to 50 Nucleotide of preferred length.In general such probe comprise with gene in the complete complementary base sequence of corresponding wild type or variant seat.

Yet, if desired, can introduce one or more mispairing, prerequisite is that the resolving ability of oligonucleotide probe is not by excessive influence.Probe of the present invention can carry one or more marks so that detect, such as in Moleaular Beacons.

The present invention also comprises a kind of diagnostic kit, this test kit contains one or more nucleic acid primers and/or one or more oligonucleotide probes with VEGFR-2 gene mononucleotide polymorphism, described polymorphism is selected from: VEGFR-2 gene the-604 and/or+1192 and/or+1719 polymorphism (for example VEGFR-2 gene-604T/C and/or+1192C/T and/or+1719A/T or its reverse complementary sequence), and their reverse complementary sequence.

In some embodiments, a kind of composition contains two or more gene type oligonucleotide of isolabeling not that are useful on the oligonucleotide identity of surveying two or more pleomorphism sites simultaneously.Also can consider to contain two covers or more cover allele-specific primerses to target contains two or more regional primer sets compounds of pleomorphism site with increasing to allow simultaneously.

VEGFR-2 gene type oligonucleotide of the present invention also can be fixed on solid surface or synthetic at solid surface, as chip, pearl or slide (as seeing WO98/20020 and WO98/20019).This fixed gene type oligonucleotide can be used for various polymorphisms and detect test, includes but not limited to the test of probe hybridization and polymerase extension.Fixed VEGFR-2 gene type oligonucleotide of the present invention can be included as rapid screening DNA sample simultaneously in a plurality of genes polymorphism and the orderly oligonucleotide arrays that designs.

Allele specific oligonucleotide primer of the present invention preferably has the only a kind of Nucleotide complementary 3 ' terminal nucleotide with specific SNP, or 3 ' penult Nucleotide preferably, have only thus that it just can be as the primer of polymerase-mediated extension when having the allelotrope that contains this Nucleotide.Comprise in the present invention with the allele specific oligonucleotide primer of coding strand or noncoding strand hybridization.Use technology well known by persons skilled in the art can develop the ASO primer that detects the VEGFR-2 gene pleiomorphism.

Other gene type oligonucleotide of the present invention and the target region hybridization that is positioned at one in one of pleomorphism site described here downstream to several Nucleotide place.This oligonucleotide can be used in the polymerase-mediated primer extension method to detect one of polymorphism as described herein, and therefore this gene type oligonucleotide is called " primer extension oligonucleotide " here.In preferred embodiments, 3 ' end of primer extension oligonucleotide is the Nucleotide complementary deoxynucleotide that is close to pleomorphism site.

In another embodiment, the invention provides and comprise the test kit that is packaged at least two gene type oligonucleotide in the container separately.This test kit also can contain other composition such as the hybridization buffer (this moment, oligonucleotide was treated as probe) that is packaged in the container separately.Alternatively, when oligonucleotide is ready to use in amplified target when zone, this test kit can contain be packaged in the polysaccharase in the container separately and be used for polymerase-mediated primer extension such as PCR through optimizing reaction buffer.

Above-mentioned oligonucleotide composition and test kit are useful in the gene type of individual VEGFR-2 gene and/or haplotype classifying method.

The invention still further relates to the method for diagnosis patient's cerebrovascular disease (cerebral apoplexy that particularly comprises cerebral thrombosis, lacunar infarction, hematencephalon), described method comprise detection from VEGFR-2 gene-604T/C in described patient's the sample and/or+1192C/T and/or+existence of 1719A/T.

The invention still further relates to determine the in vitro method of patient to the genetic predisposition of cerebrovascular disease (cerebral apoplexy that particularly comprises cerebral thrombosis, lacunar infarction, hematencephalon), described method comprise detection from VEGFR-2 gene-604T/C in described patient's the sample and/or+1192C/T and/or+existence of 1719A/T.

The invention still further relates to the in vitro method of analysis, comprising from the isolating stripped biological sample of patient: determine VEGFR-2 gene polymorphic site-604T/C in the described sample and/or+1192C/T and/or+Nucleotide of 1719A/T.Preferably, this method also comprise described analytical results determine described patient's cerebrovascular disease (cerebral apoplexy that particularly comprises cerebral thrombosis, lacunar infarction, hematencephalon) genetic predisposition or the diagnosis patient's cerebrovascular disease (cerebral apoplexy that particularly comprises cerebral thrombosis, lacunar infarction, hematencephalon).

In above method; the allelic existence of VEGFR-2 gene promoter area-604C is relevant with the ill risk of cerebral thrombosis palsy of reduction; carry+the allelic individuality of 1192T and carrying+the ill risk of the allelic individual hematencephalon of 1719T increases; haplotype (604/+1192/+1719) C/C/T has provide protection to cerebral thrombosis, and haplotype (604/+1192/+1719) the ill risk increase of T/T/T haplotype carrier hematencephalon.

In aforesaid method, can use following any difference foranalysis of nucleic acids technology to determine gene pleiomorphism: with mass spectrum to the direct mass analysis of PCR product, invasive cleaved products direct analysis, direct sequence analysis, allele specific amplification, allele-specific hybridization, oligonucleotide are connected test, effractor's test, DNA chip analysis and restriction fragment length polymorphism.

The invention still further relates to detection of biological imitate VEGFR-2 gene polymorphic site-604T/C in the product and/or+1192C/T and/or+material of the existence of 1719A/T is in the purposes of preparation diagnostic reagent, described diagnostic reagent is used to diagnose patient's cerebrovascular disease (cerebral apoplexy that particularly comprises cerebral thrombosis, lacunar infarction, hematencephalon) or is used for determining the genetic predisposition of patient to cerebrovascular disease (cerebral apoplexy that particularly comprises cerebral thrombosis, lacunar infarction, hematencephalon).Wherein said material can be selected from:

VEGFR-2 gene polymorphic site-604T/C and/or+1192C/T and/or+1719A/T polymorphism parting oligonucleotide, preferably, described polymorphism parting oligonucleotide is: (1) allele-specific nucleic acid primer, it can detect VEGFR-2 gene polymorphic site-604T/C and/or+1192C/T and/or+polymorphism of 1719A/T, perhaps (2) are used for detecting the oligonucleotide probe of the polymorphism of VEGFR-2 gene, its can be specifically with have VEGFR-2 gene polymorphic site-604T/C and/or+1192C/T and/or+nucleic acid hybridization of 1719A/T polymorphism, preferably, the length of oligonucleotide probe is 17-50 Nucleotide;

The antibody of Val297Ile sudden change and/or Gln472His sudden change in can specific recognition VEGFR-2; With

Can specific recognition VEGFR-2 gene VEGFR-2 gene polymorphic site-604T/C and/or+1192C/T and/or+primer or probe or the restriction enzyme etc. of 1719A/T.

The invention still further relates to the diagnostic kit that is used for cerebrovascular disease (cerebral apoplexy that particularly comprises cerebral thrombosis, lacunar infarction, hematencephalon), comprise detection of biological that any (one or more) just defined imitate VEGFR-2 gene polymorphic site-604T/C in the product and/or+1192C/T and/or+material of the existence of 1719A/T.

If applicable, in all respects, preferably, described VEGFR-2 gene the-604 and/or+1192 and/or+1719 polymorphism particularly VEGFR-2 gene-604T/C and/or+1192C/T and/or+1719A/T or its reverse complementary sequence.

Following examples are used for the present invention is further illustrated, and the scope that does not limit the present invention in any way.

Embodiment

One, patients with cerebral apoplexy and contrast crowd's is selected:

Research project " hypertension is easily suffered from the molecular mechanism of cerebral apoplexy " is state key basic research project (973 projects that the Ministry of Science and Technology subsidizes, and obtain Ethics Committee of the Ministry of Health, the agreement of each clinical center Ethics Committee and all participators' Informed Consent Form problem G200056901).

During November in November, 2000 to calendar year 2001, China has participated in large-scale case control study in 7 clinical center Beijing, Tianjin, Wuhan Tongji University, Wuhan consonance, Xi'an, Chongqing, Yanzhous, be selected in 2000 routine patients with cerebral apoplexy, and correspondingly be selected in 2000 example contrasts according to the principle of sex, age-matched at areal.The standard that case is selected: according to disease International Classification standard the 9th edition (the International Classification of Disease, ninth revision), collect the patients with cerebral apoplexy of three class hypotypes, comprised cerebral thrombosis (cerebral thrombosis), lacunar infarction (Lacunar infarction), hematencephalon (intracerebral hemorrhage).Diseases such as the outbreak of transience cerebral infarction, subarachnoid hemorrhage, cerebral embolism, cerebral tumor and cerebrovascular arteriovenous malformotion are excluded.Neurologic examination, the CT of the diagnosis basis strictness of cerebral apoplexy, and/or MRI checks.Contrast crowd's cerebral apoplexy exclusion standard is identical with the case inclusion criteria, no cerebral apoplexy symptom or cerebral apoplexy medical history.When carrying out data analysis, have 1849 patients with cerebral apoplexy (60.4 ± 9.23 years old mean age, the male sex accounts for 63.4%) and 1798 contrasts (in mean age 59.6+8.5 year, the male sex accounts for 59.2%).Wherein, patients with cerebral apoplexy comprises 812 examples (43.9%) cerebral thrombosis, 530 routine patient (28.7%) lacunar infarctions, 507 examples (27.4%) hematencephalon.

Patients with cerebral apoplexy and contrast crowd all carry out the standard survey of cardiovascular and cerebrovascular disease, living habit such as investigation content comprises sacred disease history, history of hypertension, diabetic history, systolic pressure, diastolic pressure, height, body weight, smoking, drink.Extract peripheric venous blood simultaneously, carry out every blood biochemistry index and measure, comprise blood sugar, total cholesterol (TC), total triglyceride level (TG), high density lipoprotein cholesterol (HDL-C), NHDL cholesterol (non-HDL-C) etc.Hypertensive Case definition is: 3 blood pressure measurement systolic pressure 〉=140mmHg, or diastolic pressure 〉=90mmHg, the person that perhaps takes depressor.The diagnosis of diabetes standard is: fasting blood glucose level 〉=7.8mmol/L, or 2 hours after the meal blood sugar 〉=11.1mmol/L.

" cardiovascular diseases epidemiology survey handbook prescriptive procedure is finished, and blood biochemistry index detects and all finished by centralab by pressing through the professional who trains for survey, blood pressure, height, measured body weight and blood drawing.

Two, the extraction of genomic dna

Phenol-chloroform extraction method according to standard carries out.

Three, the selection of VEGFR-2 gene SNP s

The VEGFR-2 gene is positioned at karyomit(e) 4q11-q12, the about 5.8kb of cDNA total length, and NCBIRefSeq mRNA accession No. is NM_002253, is made up of 30 exons and 29 introns.The encoded polypeptides chain is made up of 1356 amino acid, is tyrosine kinase receptor.The proteic structure of VEGFR-2 is divided into 3 parts: extracellular region comprises 7 Ig-like structural domains; Stride the film district; Tyrosine kinase domain in the born of the same parents.1-19 amino acid coded signal peptide, 20-764 the proteic ectodomain of amino acid coding VEGFR-2,765-789 amino acid coding striden membrane portions, 790-1356 amino acid coding endochylema intracellular domain.The characteristics of this gene promoter area are not have the TATA box and enrichment GC, and have binding site such as AP-2, GATA, the NF-κ B etc. of 5 Sp1 controlling elements and other a plurality of transcription factors.

We choose 24 patients with cerebral apoplexy at first respectively and contrast as research object, nucleotide sequence to the VEGFR-2 genetic transcription initiation site about 2.0Kb in upstream checks order, to detect mononucleotide polymorphism site and the frequency (method one) thereof in the Chinese population.We find 3 mononucleotide polymorphism sites at VEGFR-2 gene 5 '-control region,-604 T → C and-65T → C is positioned at the transcription initiation site upstream, + 32 A → G is positioned at 5 '-UTR, they are difference called after rs2071559 in dbSNP (http://www.ncbi.nlm.nih.gov/SNP/) database, rs9994560 and rs7667298 (Fig. 1-3).Wherein the frequency of the rare allele C of rs9994560 is 0.04, and the frequency of the rare allelotrope G of rs7667298 is 0.02, rs2071559 the frequency of rare allele C be 0.28.Bioinformatic analysis is carried out in the rs2071559 site, utilize ClustalW software comparison people and mouse to comprise the nucleotide sequence of rs2071559, find that this region height is conservative; Analyze binding site (gTtgg) disappearance that discovery rs2071559 polymorphism-change of 604 T → C causes transcription factor CP-2 with TranscriptionElement Search System (TESS).Therefore in further extensive case control study, we have carried out gene type to rs2071559.

We have carried out order-checking and screening (method two) according to dbSNP database (http://www.ncbi.nlm.nih.gov/SNP/) to the mononucleotide polymorphism site in all 30 exons of VEGFR-2 gene.In addition, Walter studies the sudden change of VEGFR-2 gene in 15 routine teenager's vascular tumor patients, find to exist in 1 routine patient's the vascular tumor tissue missense mutation P1147S of VEGFR-2 gene, this sudden change is positioned at the kinase domain of VEGFR-2 gene, cause proline(Pro) to change Serine into, and in the healthy tissues around the vascular tumor, do not detect this sudden change (Genes Chromosomes Cancer 33:295-303,2002).We have also carried out examination to this sudden change.We find only to have 2 pleomorphism sites after 12 SNPs are carried out examination by direct order-checking, are in respectively among the Exon 7 Rs2305948,, cause nonsynonymous mutation Val297Ile (Fig. 4) at VEGFR-2 genetic transcription initiation site downstream+1192 C/T; Rs1870377 among the Exon 11, in the transcription initiation site downstream+1719 A/T, cause nonsynonymous mutation Gln472His (Fig. 5).Therefore, we choose the association study that rs2305948 and rs1870377 carry out next step case-control.

Method one:

1. with following primer VEGFR-2 gene 5 '-control region is checked order:

Primer	The PCR product	Annealing temperature
			1 F5’-actgtaaataaggctgatag-3’ R5’-cactatgttgcctaagctgg-3’ 2 F5’-gatggaaggatggagctttg-3’ R5’-ccaggctggtctccaatgc-3’ 3 F5’-gaggcaggaggatggactga-3’ R5’-cttttccaagttgacaggt-3’ 4 F5’-tgcctctgccaaaagaaaag-3’ R5’-acacctggggagtgacatca-3’ 5 F5’-aggcatctggaagattcata-3’ R5’-gaaggcttttgtccatcgtg-3’ 6 F5’-AGCCACAAGGGAGAAGCGGATA-3’ R5’-CAAACTTTCACTAGGGCTCTTCGT-3’ 7 F5’-caaaactactggcaggagtc-3’ R5’-catttccccacacaactg-3’ 8 F5’-cccagcgcagtccagttgtgt-3’ R5’-ggtgccggtaggagaggata-3’ 9 F5’-gggagagcggtcaatgtg-3’ R5’-cacccgacctgtctgccttcct-3’	291bp 255bp 330bp 365bp 353bp 290bp 347bp 315bp 394bp	60 62 62 63 63 63 60 63 63

2.PCR (polymerase chain amplified reaction) system sees the following form:

The PCR primer, (5pmol/ μ l) 10 * ExTaq polymerase buffer 10mM dNTP mixed solution template DNA ExTaq archaeal dna polymerase, (5U/ μ l) ddH2O

Each 0.5 μ l, 2.5 μ l, 0.5 μ l, 1.0 μ l, 0.25 μ l, 19.75 μ l

The PCR parameter:

3.PCR product purification

(1) dehydrated alcohol of 25 μ l PCR reaction product with its 6 times of volumes mixed with 5M Ammoniom-Acetate (5: 1) mixing solutions, place-80 ℃ more than 30 minutes;

(2) 14,000rpm, 4 ℃ centrifugal 15 minutes, supernatant discarded;

(3) add 75% alcohol of 200 μ l, 14,000rpm, 4 ℃ centrifugal 5 minutes, suct clearly and discard;

(4) after room temperature or the lyophilize, add 20 μ l TE dissolving;

(5) inhale 2 μ l point samples, 2% sepharose (preparation of 0.5 * tbe buffer liquid) electrophoresis is quantitative.

4. terminal dideoxy method order-checking

Instrument: ABI PRISM 377 sequenators (U.S. Perkin Elmer company)

Method two:

The mononucleotide polymorphism site of VEGFR-2 gene coding region is (according to dbSNP database (http://www.ncbi.nlm.nih.gov/SNP/)

rs ID	Allelotrope	Amino-acid residue	The position of amino acid in polypeptide chain	Exon
					rs1139777	A→T	Thr[T]→Ser[S]	1347	Exon 30
rs11540507	G→C	Ala[A]→Pro[P]	1210	Exon 27
					rs13129474	A→G	Ile[I]→Val[V]	952	Exon 21
rs1139776	A→T	Glu[E]Val[V]	848	Exon 18
					rs1139775	C→G	Asn[N](Lys [K]	835	Exon 17
rs1139774	G(C	Gly[G](Arg[R]	787	Exon 16
					rs1062832	A(G	Thr[T]Ala[A]	772	Exon 16
rs1870377	T(A	His[H]→Gln[Q]	472	Exon 11
					rs2034964	A→G	Asn[N]→Asp[D]	392	Exon 9
rs1824302	A→G	Lys[K](Arg[R]	349	Exon 8
					rs2305948	A(G	Ile[I](Val[V]	297	Exon 7

The pcr amplification system, reaction parameter, the purifying of PCR product and order-checking etc. are all with method one.

Four, 3 mononucleotide polymorphism site rs2071559 in the VEGFR-2 gene, rs2305948 and rs1870377 are genotypic to be determined

The used primer 1.PCR increase

Polymorphic site	Primer	Purpose fragment (bp)	Restriction enzyme	Allelotrope	Endonuclease bamhi (bp)
						rs2071559 (-604T/C) rs2305948 (+1192C/T) rs1870377 (+1719A/T)	F5’-AGCCACAAGGGAGAAGCGGATA-3’ R5’-CAAACTTTCACTAGGGCTCTTCGT-3’ F5’-AAATGTACAATCCTTGGTCACTCCGGGGTA-3’ R5’-TGAGGTTAAAAGTTCTGGTGTCCCTGTT-3’ F5’-GCCTCACATATTATTGTACCATCC-3’ R5’-CCTCCTGTATCCTGAATGAATCT-3’	290 262 404	BsmI BstZ171 AluI	C T T C T A	174,116 290 232,30 262 191,213 404

2.PCR (polymerase chain amplified reaction) system sees the following form:

The PCR primer, (5pmol/ μ l) 10 * Takara Taq polymerase buffer 10mM dNTP mixed solution template DNA Takara Taq polysaccharase, (5U/ μ l) ddH2O

Each 0.2 μ l, 1.0 μ l, 0.2 μ l, 1.0 μ l, 0.1 μ l, 7.3 μ l

The PCR parameter:

3.rs2071559 the detection (Fig. 6) that (-604T/C) is polymorphic

Annotate: be the recognition sequence of restriction endonuclease in the bracket, arrow and boldface type are the allelotrope of SNP.There is not Bsm I restriction enzyme site in the genotypic amplified fragments of T/T, a 290bp fragment will occur; There is Bsm I restriction enzyme site in the C/C genotype, two fragments of 232bp and 30bp will occur; The T/C genotype then produces 290bp, 232bp and three fragments of 30bp.

(2) PCR product enzyme is cut system following (20 μ l):

PCR product 8.0 μ l

10×NEBuffer 2 2.0μl

Bsm I(10U/μl) 0.3μl

ddH ₂O 9.7μl

Behind (3) 65 ℃ of incubation 3h, add 10 * sample-loading buffer, 1 μ l termination reaction;

(4) get the above-mentioned product of 8 μ l, 2% agarose gel electrophoresis is identified.

4.rs2305948 the detection (Fig. 7) that (+1192C/T) is polymorphic

Annotate: be the recognition sequence of restriction endonuclease in the bracket, arrow and boldface type are the allelotrope of SNP.There is not BstZ17 I restriction enzyme site in the genotypic amplified fragments of C/C, a 262bp fragment will occur; There is BstZ17 I restriction enzyme site in the T/T genotype, two fragments of 232bp and 30bp will occur; The C/T genotype then produces 262bp, 232bp and three fragments of 30bp.

(2) PCR product enzyme is cut system following (20 μ l):

PCR product 8.0 μ l

10×NEBuffer 3 2.0μl

BstZ171(5U/μl) 0.5μl

ddH ₂O 9.5μl

Behind (3) 37 ℃ of incubation 3h, add 10 * sample-loading buffer, 1 μ l termination reaction;

(4) get the above-mentioned product of 8 μ l, 4% agarose gel electrophoresis is identified.

5.rs1870377 the detection (Fig. 8) that (+1719A/T) is polymorphic

Annotate: be the recognition sequence of restriction endonuclease in the bracket, arrow and boldface type are the allelotrope of SNP.There is not Alu I restriction enzyme site in the genotypic amplified fragments of A/A, a 404bp fragment will occur; There is Alu I restriction enzyme site in the T/T genotype, two fragments of 191bp and 213bp will occur; The T/A genotype then produces 404bp, 191bp and three fragments of 213bp.

(2) PCR product enzyme is cut system following (20 μ l):

PCR product 8.0 μ l

10×NEBuffer 2 2.0μl

Alu I(10U/μl) 0.3μl

ddH ₂O 9.7μl

Behind (3) 37 ℃ of incubation 3h, add 10 * sample-loading buffer, 1 μ l termination reaction;

(4) get the above-mentioned product of 8 μ l, 2% agarose gel electrophoresis is identified.

Five, statistical analysis: adopt SSPS 10.0 statistical packages, the significance level P value of all statistics is decided to be 0.05 (bilateral).

1. the Clinical symptoms of cerebral apoplexy case group and control group is relatively:

(1) Mann-Whitney method: the total triglyceride level of serum (TG) carries out the natural logarithm conversion, relatively each group difference for skewness distributes before the analysis.

(2) non-matching student ' s t check: mean compares (mean+SD) between the group of blood biochemistry index such as age, blood plasma HDL-C, blood plasma non-HDL-C, total plasma cholesterol, blood pressure, weight index, blood sugar.

(3) Chi square check: compare between the group of classification such as sex, smoking, history of hypertension and diabetic history data.

2. frequency is added up: the gene frequency of mononucleotide polymorphic site SNPs and genotype frequency are in the distribution of case and control group

3.Chi square check:

(1) judges that each polymorphic site gene frequency and distribution of genotypic frequency have or not area, gender difference.

(2) judge whether meet Hardy-Weinberg's balance by each polymorphic site (Hardy-Weinbergequilibrium, HWE), samples met HWE is thought in P＞0.05.

(3) the unit point association analysis in each site.

4. many first Logistic return:

(1) estimate traditional cardiovascular and cerebrovascular disease Hazard Factor age, sex, smoking, drink, the relation of blood plasma HDL, total plasma cholesterol, blood pressure, weight index, blood sugar etc. and cerebral apoplexy,

(2) after the above-mentioned Hazard Factor of correction, detect the SNPs of VEGFR-2 gene and the dependency of cerebral apoplexy.

(3) (odds ratio, OR) (confidenceinterval CI) represents dependency with 95% credibility interval with odds ratio.

5. the linkage disequilibrium analysis between genetic marker and the association analysis of haplotype: metric D, D ', the γ of the linkage disequilibrium degree between the genetic marker ²Value, (http://request.mdacc.tmc.edu/～qhuang/Software/pub.htm) software calculates by 2LD (http://www.iop.kcl.ac.uk/iop/departments/psychmed/gepibst/softw are/21d.stm) and EMLD.Adopt PHASE 2.0 softwares [48] to estimate the frequency (http://www.stat.washington.edu/stephens/phase.html) of every kind of haplotype, and calculating the whole difference of haplotype frequency in case group and the control group, P＜0.05 is as statistical significant difference.Adopt each haplotype frequency of chi square test comparison whether significant difference to be arranged then in the distribution of case group and control group.

Six, VEGFR-2 gene promoter area SNP-604T/C is to the influence of promoter activity

The heritable variation of promoter region can influence the gene transcription activity.-604T/C is positioned at the promoter region of VEGFR-2 genetic transcription initiation site upstream, and we find this region, site high conservative with the promoter region sequence of Clustal W software comparison people and mouse.Utilize TranscriptionElement Search System (TESS) software package to analyze this polymorphic site place sequence possibility bonded transcription factor, find that this section sequence may combine with transcription factor CP2, NF-1 (nuclearfactor-1) and YY-1 (Yin and Yang 1).The change of VEGFR-2 gene-604 base T → C, may influence the binding ability of transcription factor CP-2, NF-1 or YY-1 and this section sequence, perhaps, change the promoter activity and the expression level of gene, thereby influence the blood vessel biological effect of VEGF by the interaction between transcription factor.Therefore we make up VEGFR-2 gene promoter/luciferase reporting study on the carrier SNP-604T/C to the active influence of genetic transcription (square method three).

Method three,

1. make up luciferase reporter gene plasmid pGL3/-604TT:

1.1 PCR method amplification VEGFR-2 gene promoter region sequence:

Select genotype to be-the individual DNA of 604TT, contain the dna fragmentation of the whole promoter regions of VEGFR-2 gene, comprise transcription initiation site and exons 1 (Genbank accession no.AC021220.7) with PCR method and Pyrobest Taq polymeric enzymatic amplification.Primer sequence is: upstream primer 5 '-tag cga gct ctg cca caa gaa gtc cac aca-3 ' (containing restriction enzyme SacI recognition sequence GAGCTC); Downstream primer 5 '-cac ccg acc tgt ctg cct tcc-3 '.Amplification condition: 95 ℃ of sex change 10 minutes; Carry out 30 circulations subsequently, each circulation comprises 94 ℃ of sex change 30 seconds, anneals 30 seconds for 65 ℃, and 72 ℃ were extended 90 seconds; At last, 72 ℃ were extended 10 minutes.Expanding fragment length 1.3Kb has the SacI/XhoI double enzyme site.

1.2 PCR product purification:

(1) add the Ammonium Acetate/ethanol (1: 5) of 6 times of volumes ,-70 ℃ 30 minutes.

(2) 4,000rpm (rev/min) * 10 minutes, 12,000rpm * 10 minute.

(3) 75% washing with alcohol.

(4) treat that ethanol is evaporated completely, add the dissolving of 50 μ l aqua sterilisas.

1.3 PCR purified product sequence verification contains wild gene type-604TT

1.4 make up luciferase reporter gene plasmid pGL3/-604T:

The PCR product of the VEGFR-2 gene promoter area after the above-mentioned sequence verification is cut glue with 1% agarose to be reclaimed, behind restriction enzyme Sac I and Xho I double digestion 4 hours (37 ℃), recovery/purifying purpose fragment (1182bp,-887～+ 295), simultaneously pGL3-Basic luciferase reporting carrier is carried out Sac I and Xho I double digestion and the big fragment of recovery/purifying, the fragment that above-mentioned digestion is reclaimed connects with the T4 dna ligase in 16 ℃ of water-baths at last, transforms, the picking clone extracts plasmid.Enzyme is cut and is identified, the positive colony order-checking determines to insert the segmental exactness of purpose.The order-checking universal primer is the T7 primer: 5 ' TAA TAC GAC TCA CTA TAG GG 3 '; SP6 primer: 5 ' ATT TAGGTG ACA CTA TAG AA 3 '.

2. the sudden change legal system is equipped with luciferase reporter gene plasmid pGL3/-604CC

2.1 make up pGEM-T Easy/-604TT plasmid:

(contain-604TT) add the A tail with Takara Taq enzyme 3 ', and connect into pGEM-T Easy carrier, transform, the picking clone extracts plasmid, enzyme is cut and identified with the pcr amplified fragment of the VEGFR-2 gene promoter area after the sequence verification.The segmental direction of insertion of purpose is determined in the positive colony order-checking.

3 ' adds the A end reaction: 70 ℃ 30 minutes

10×Taq polymerase buffers 1.0μl

dATP(2mM) 1.0μl

Takara Taq(5U/μl) 1.0μl

PCR product 7.0 μ l behind the purifying

Ligation: 4 ℃ 14-16 hour

2×Rapid ligation buffer 5.0μl

pGEM-T Easy Vector 1.0μl

T4 dna ligase 1.0 μ l

The PCR product 3.0 μ l that add the A tail

2.2 the sudden change legal system be equipped with pGEM-T Easy/-604C subclone plasmid (GeneEditor-in vitroSite-Directed Mutagenesis System, Promega)

2.2.1 5 ' end phosphorylation of mutant primer:

The mutant primer sequence: 5 '-cgg gaa tgC tgg cga ac-3 ', wherein C is-604 mutational sites.

Phosphorylation reaction:

Mutant primer (200pmol) 2.5 μ l

T4 kinase 10×buffer 0.5μl

T4 kinase(5U/μl) 1.0μl

ATP(10mM) 2.5μl

Sterile purified water 18.5 μ l

Above-mentioned reaction system was hatched 30 minutes for 37 ℃, 70 ℃ of 10 minutes deactivation T4 kinase ,-20 ℃ of preservations.

2.2.2 the alkaline denaturation of plasmid pGEM-T Easy/-604T

Plasmid DNA (2ng) 12.0 μ l

2M NaOH，2mM EDTA 2.0μl

Sterile purified water 6.0 μ l

Above-mentioned reaction system room temperature was placed 5 minutes, added 2M Ammonium Acetate 2 μ l and dehydrated alcohol (4 ℃) 75 μ l deposit D NA ,-70 ℃ 30 minutes, 4 ℃ are centrifugal 13, supernatant is carefully removed in 000rpm * 15 minute.Use 70% ethanol (4 ℃) washing then, centrifugal 13, supernatant is removed in 000rpm * 15 minute, dries, and adds 50 μ l aseptic distillation water dissolution.0.8% agarose gel electrophoresis is identified.

2.2.3 the hybridization of mutant primer and plasmid DNA

The plasmid DNA 10.0 μ l of sex change

The mutant primer 3.0 μ l of 5 ' phosphorylation

The Selection primer 1.0 μ l of 5 ' phosphorylation

Annealing 10×buffer 2.0μl

Sterile purified water 4.0 μ l

Reduction gradually with pcr amplification instrument control hybridization temperature: 75 ℃ of sex change made temperature slowly drop to 37 ℃ after 5 minutes, and average per minute descends 1.0 ℃ about 40 minutes of whole process.

Chain synthesizes and is connected 2.2.4 suddenly change

Assorted hybridization reaction solution 20.0 μ l

Synthesis 10×buffer 3.0μl

T4 DNA Polymerase 1.0μl

T4 DNA Ligase 1.0μl

Sterile purified water 5.0 μ l

In 37 ℃ of water-baths 90 minutes.

2.2.5 transform BMH 71-18 mutS competent cell

BMH 71-18 mutS is the intestinal bacteria E.Coli bacterial strain of mispairing repairing effect defective, can prevent the reparation of the non-methylate DNA chain of new synthetic, thereby improves the efficient that sudden change produces.The BMH 71-18 mutS competent cell that sudden change liquid is transformed shakes bacterium, cultivation 16-18 hour in 37 ℃ in the LB substratum that contains GeneEditorAntibiotic Selection Mix.

2.2.6 transform the screening of JM109 competent cell and muton

The alkaline lysis method of extracting plasmid transforms the JM109 competent cell, screens muton on the LB flat board that contains Amp resistance and Antibiotic Selection Mix.Hatched 14-16 hour at 37 ℃, picking mono-clonal bacterium was cultivated 12-14 hour in containing the LB nutrient solution of Amp resistance, the results intestinal bacteria, extract plasmid in a small amount with alkaline lysis, the existence of mutational site-604C is determined in order-checking, and the insertion fragment of insertion segmental other sequences of purpose and wild plasmid pGL3/-604T is in full accord.

2.3 make up luciferase reporter gene plasmid pGL3/-604CC

After pGEM-T Easy/-604C subclone plasmid after the above-mentioned sequence verification and pGL3-Basic luciferase reporting carrier used restriction enzyme Sac I and Xho I double digestion 4 hours (37 ℃) respectively, 0.8% agarose gel electrophoresis also reclaims the purpose fragment, in 16 ℃ of water-baths, connect the purpose fragment that above-mentioned digestion is reclaimed with the T4 dna ligase, transform, alkaline lysis extracts plasmid DNA in a small amount.Order-checking is determined to insert the segmental exactness of purpose, obtains the luciferase reporter gene plasmid pGL3/-604C of mutator gene type.

3. cell cultures

3.1 it is foster that Human umbilical vein endothelial cells (HUVEC) former is commissioned to train.

3.1.1 experimental procedure:

(1) opens aseptic pallet, pour stroke-physiological saline solution into.Neonatal umbilical cord in 24 hours is put into pallet rinsing number time, wash down remaining clot in the blood vessel.

Whether (2) cut the umbilical cord two ends, a termination is gone into straight mouthful of threeway syringe, and the other end inserts suction pipe-sebific duct-syringe needle triplet, constantly twitches piston, check to leak gas, and remaining blood in the umbilical cord is driven out of.

(3) sebific duct-syringe needle diad is connected into threeway syringe side mouth, then syringe needle is inserted in the aseptic PBS bottle of preheating, 1 * PBS is gone in siphon, flushing umbilical cord 3～5 times.

(4) syringe needle is extracted, inserted in the 0.25% trypsinase bottle of room temperature preheating, the umbilical cord the other end steps up with vascular clamp, constantly twitches piston, is full of trypsinase fully and does not have plugged vents in umbilical vein.Clamp side chewing-gum pipe.The whole immigration of umbilical cord contained in the Porcelain Jar of 37 ℃ of 1 * PBS water-bath digestion 15 minutes.

(5) take out umbilical cord, the Digestive system in the umbilical vein is collected in the 50ml centrifuge tube, splash into several inactivated serums, Digestive system centrifugal (＜1000rpm, 5～10 minutes, room temperature).

(6) draw cell mass and be precipitated to 25cm ²In the culturing bottle, add an amount of M199 nutrient solution (20cm umbilical cord cell adds 3ml～5ml nutrient solution, contains 15%FBS, 150 μ g/ml ECGS, 75 μ g/ml heparin sodiums), carefully dispel, in 37 ℃, 95%O ₂, 5%CO ₂Cultivate in the incubator.

(7) cell density is to go down to posterity in 70%～90% o'clock, adds the cell dissociation buffer of 0.125% trypsinase, 0.02%EDTA, and observation of cell digestion situation treats that cell and cell separate under the inverted microscope, and cell space becomes circle and brightens, and Digestive system is removed in suction.

(8) add the M199 nutrient solution that contains 10%FBS, carefully dispel cell, inoculate new culturing bottle.

3.2 the cultivation of 293S cell:

3.2.1 the recovery of 293S cell:

(1) freeze-stored cell is taken out from liquid nitrogen container, put into 40 ℃ of water-baths rapidly, rock, make it to melt fully as early as possible (1～1.5 minute).

(2) centrifugal, get supernatant;

(3) add the fresh DMEM substratum (containing 10%FBS) of 5ml, be seeded to 25cm ²In the culturing bottle.

3.2.2 the cultivation of going down to posterity of 293S cell:

(1) cell density is to go down to posterity in 70%～90% o'clock, adds the cell dissociation buffer of 0.125% trypsinase, 0.02%EDTA.

(2) observation of cell digestion situation (about 5 minutes) under the inverted microscope treats that cell and cell separate, and cell space becomes circle and brightens, and inhales and removes Digestive system, removes most of cell.

(3) add the DMEM substratum that 5ml contains 10%FBS, in 37 ℃, 95%O ₂, 5%CO ₂Cultivate in the incubator.

4. transfection and luciferase analysis:

4.1 experiment grouping:

Experimental group: pGL3/-604T wild plasmid

PGL3/-604C mutant plasmid

Empty carrier group: pGL3-Basic luciferase reporting carrier

Positive controls: pGL3-Control luciferase reporting carrier.

Above-mentioned each group cotransfection pRL-CMV report carrier is simultaneously proofreaied and correct transfection efficiency as confidential reference items.

4.2 cell transfecting and luciferase analysis

(1) with Lipofectamine 2000,, goes into HUVEC cell or 293S cell with 0.8 μ g confidential reference items pRL-CMV cotransfection respectively with 20 μ g pGL3/-604T, pGL3/-604C report carrier or pGL3-Basic empty carrier, pGL3-Control positive control carrier.For every kind of report carrier, repeat independently transfection experiment three times.

(2) transfection was abandoned substratum after 24 hours, washed once with PBS, removed PBS.

(3) detect uciferase activity with two luciferase reporting analytical systems.Add 300 μ l, 1 * PLB, incubated at room is 15 minutes on shaking table, makes the complete cracking of cell.

(4) lysate is moved in the 1.5ml centrifuge tube centrifugal, remove precipitation.

(5) get 100 μ l LAR II and 20 μ l lysates move in the fluorescent tube mixing; Measure the activity of Photinus pyralis LUC.

(6) add 100 μ l stop ﹠amp again; Gol Reagent, the activity of measurement sea urchin luciferase.

5. data processing

Carry out 3 independently cell transfecting and uciferase activity analyses, (Relative luciferase unit RLU) represents, i.e. the ratio of fireflyluciferase and renilla luciferase promoter activity with the plain unit of enzyme activity of relative fluorescence.RLU represents with mean ± standard deviation, the difference between non-matching Student ' s t check comparative group, and P＜0.05 is as significance level.

Seven, result:

One) the general clinical data of cerebral apoplexy case group and control group

The Hazard Factor of traditional cardiovascular and cerebrovascular disease comprise that diabetic history, history of hypertension and smoker are more common in the case group.Case group systolic pressure, diastolic pressure, fasting blood glucose level, triglyceride level (TG) all are significantly higher than control group, and high density lipoprotein cholesterol (HDL-C) significantly is lower than control group (table 1).

Two) the VEGFR-2 gene polymorphic is relevant with cerebral apoplexy

VEGFR-2 gene polymorphic site-604T/C ,+1192C/T and+1719A/T distributes in the genotype of case group and control group and all meets the Hardy-Weinberg balance, frequency distribution does not have the significant difference of area, sex.The genotype of each polymorphic site and allelotrope are as shown in table 2 in case group and control group frequency distribution.

In cerebral thrombosis case group, the frequency of polymorphic site-604 allele C is 0.27, and (frequency is 0.30, P=0.001) significantly to be lower than control group, it (is respectively 45.1% and 52.1%, P=0.004) that genotype TC/CC carrier's frequency also significantly is lower than control group.In hematencephalon case group, the frequency of polymorphic site+1192 allelotrope T is 0.18, is significantly higher than control group (frequency is 0.11, P＜0.0001), genotype CT/TT carrier's frequency is significantly higher than control group (being respectively 33.8% and 20.5%, P＜0.0001).In the hematencephalon group, the frequency of polymorphic site+1719 allelotrope T is 0.43, and (frequency is 0.38, P=0.004) to be significantly higher than control group; AA, AT, TT genotype frequency are respectively in the hematencephalon group: 38.9%, 45.4%, 15.7%, in control group, be respectively: and 31.8%, 49.7%, 18.5%, be distributed with remarkable significant difference (P=0.01) between group.

For adjusting the influence of cerebral apoplexy environmental factors and other traditional vascular risk factors, so that whether cerebral apoplexy is a dependent variable, be independent variable(s) with VEGFR-2 gene polymorphic and conventional risk factors, carry out polynary non-conditional Logistic Regression.After having proofreaied and correct conventional risk factors such as age, sex, smoking, blood plasma HDL, total plasma cholesterol, blood pressure, weight index, blood sugar, the ill risk of cerebral thrombosis palsy relevant (OR=0.78,95% CI:0.65-0.95 of VEGFR-2 gene promoter area-604C allelotrope and reduction; P=0.025.).Carry+1192T and+the ill risk of the allelic individual hematencephalon of 1719T increases (OR=2.25,95% CI:1.70-2.96, P=0.0001; OR=1.33,95% CI:1.03-1.73, P=0.01).

Three) the VEGFR-2 gene monomer type is relevant with cerebral apoplexy

We have calculated the linkage disequilibrium degree (table 3) between 3 SNP sites of VEGFR-2 gene, discovery-604T/C and+1192C/T between, and+1192C/T and+exist faint linkage disequilibrium between the 1719A/T, their D ' is respectively 0.1636 and 0.2663.Utilize this 3 SNP sites to make up haplotypes, the association analysis result shows between cerebral infarction group and control group, and the distribution of haplotype frequency all has whole difference (the cerebral infarction group: P=0.01 of significance between hematencephalon group and the control group; Hematencephalon group: P=0.01; Table 17).

(604/+1192/+1719) as reference, further more every kind of haplotype is in the not difference (table 4a, table 4b) of the frequency between on the same group with wild haplotype TCA.We find that haplotype C-C-T significantly is lower than frequency (10.8%) (OR:0.67 in the control group in the frequency (7.6%) of cerebral infarction group; 95% CI:0.54～0.84; P=0.0004), Bonferroni ' s multiple check still has significant difference (P=0.0021) after proofreading and correct.In the hematencephalon group, contain+the allelic haplotype T-T-A of 1192T, the frequency of T-T-T, C-T-A and C-T-T all is significantly higher than the frequency (P＜0.01) in the control group.Wherein haplotype T-T-T is respectively 4.5% and 1.6% in the frequency of hematencephalon and control group, with the onset risk of hematencephalon strong dependency is arranged, and the OR value is 3.43 (95%CI:2.28～5.14; Bonferroni ' s proofreaies and correct P=2.7 * 10 ^-9); And the relative risk of haplotype C-T-T and hematencephalon morbidity is reduced to 2.30 (95%CI:1.39～3.79; Bonferroni ' s proofreaies and correct P=0.007), the frequency in hematencephalon and control group is respectively 2.5% and 1.3%.

Four)-604T/C reduces the transcriptional activity of VEGFR-2 gene promoter area

Because VEGFR-2 mainly expresses at vascular endothelial cell, we with the wild-type that makes up and mutant reporter gene plasmid respectively transient transfection go into Human umbilical vein endothelial cells (HUVEC), in serum free medium, cultivate after 24 hours, whether by measuring the activity of luciferase, having detected mononucleotide polymorphic-604T/C influences transcribing of VEGFR-2 gene promoter area.Simultaneously, with the 293-S cell in contrast, observe the startup activity of this section promoter sequence in non-endotheliocyte of VEGFR-2 gene.The result as shown in Figure 9, VEGFR-2 gene promoter sequence (887～+ 296) almost is not activated the expression activity of luciferase in the 293-S cell; In the HUVEC cell, compare with wild-type-604T, the polymorphic expression activity of luciferase that makes of-604C reduces by 2.93 times of (pGL3/-604C:5.81 ± 2.42; PGL3/-604T:17.03 ± 4.89; P＜0.001).

Eight, conclusion:

VEGFR-2 is the high-affinity receptor of VEGF, is a kind of tyrosine kinase receptor.The multiple physiology of VEGF, pathology effect comprise that the division of promotion vascular endothelial cell, hyperplasia, migration and increase vascular permeability etc. mainly are to finish by the signal transduction pathway of VEGFR-2.

Polymorphic-604C the allelotrope of VEGFR-2 gene promoter area relevant with the ill risk of cerebral thrombosis palsy of reduction (OR=0.78,95% CI:0.65-0.95).Cytologic experiment is the result show, the promoter activity of mutant allele-604C significantly is lower than wild-type-604T allelotrope, therefore, VEGFR-2 gene-604C allelotrope may be by weakening the promoter transcription activity, reduce the expression level of VEGF, weaken it and promote the effect that capillary blood vessel forms, thereby have influence on the progress and the stability of plaque lesions in the atherosclerotic plaque zone.

Association analysis is the result show, carry+1192T and+the ill risk of the allelic individual hematencephalon of 1719T increases (OR=2.25,95% CI:1.70-2.96; OR=1.33,95%CI:1.03-1.73).We have carried out bioinformatic analysis to these two sites.Polymorphic+1192C/T is positioned at VEGFR-2 gene extron 7, causes the amino-acid residue of the 297th of VEGFR-2 mRNA to become Isoleucine (isoleucine) by Xie Ansuan (valine).Though the hydrophobicity of this amino-acid residue does not change, because it is positioned at the 2nd Ig-like structural domain of VEGFR-2 gene extracellular region, and this structural domain is to most important (the J BiolChem.1998 with combining of its part VEGF of VEGFR-2; 273:11197-11204).Adopting the PROSITE database analysis of ExPASy analysis of protein system, show that this site is in the proteinic framework ingredient, be imbedded in protein conformation inside, is N-Semen Myristicae acidylate site, and this site still exists after the sudden change.Polymorphic+1719A/T is positioned at VEGFR-2 gene extron 11, causes the amino-acid residue of the 472nd of VEGFR-2 mRNA to become Histidine (histidine) by glutamine (glutamine), and amino acid whose character is changed into alkalescence by neutrality.This site is in the 5th Ig-like structural domain of VEGFR-2 gene extracellular region.Adopt the PROSITE database analysis of ExPASy analysis of protein system, show the surface of this site at protein steric structure, after glutamine sported histidine, the change of electric charge may cause the change of electrostatic interaction between the amino acid, thereby influenced the binding characteristic of acceptor and part.Therefore, we have reason to infer that the amino acid change of VEGFR-2 gene coding region may influence the space conformation of VEGFR-2, thereby change the binding characteristic of part and acceptor, then influence the downstream signal path, finally change VEGF and promote physiopathology effects such as blood vessel hyperplasia, migration and increase vascular permeability.

It is less that single genetic marker is carried out the effectiveness of genetic analysis, and the haplotype of being made up of several chain sites then can provide more more reliable information, and it is usually more reliable than unit point to detect linkage disequilibrium with haplotype.The conclusion of above-mentioned unit point association analysis has further been supported in the association analysis of haplotype; be that haplotype C/C/T (604/+1192/+1719) is low-riskhaplotype; cerebral thrombosis there is provide protection (OR=0.59; 95%CI:0.43-0.82); and the ill risk increase of T/T/T haplotype carrier hematencephalon (OR=3.18,95%CI:1.79-5.67).

Table 1: the general clinical data of cerebral apoplexy case group and control group

Characteristics	Controls (n＝1798)	Stroke
						Total (n＝1849)	Thrombosis (n＝812)	Lacunar (n＝530)	Hemorrhage (n＝507)
		Age，y Male，％ BMI，kg/m 2 SBP，mmHg DBP，mmHg TC，mmol/L TG，mmol/L Glucose，mmol/L HDL-C，mmol/L Non-HDL-C，mmol/L Cigarrette smoking，％ History of hypertension，％ History of DM，％	59.6(8.5) 59.2 24.1(3.3) 129(17) 79(10) 4.95(1.00) 1.47(15.1) 5.86(1.75) 1.06(0.30) 3.08(0.94) 38.3 26.2 4.8	60.4(9.2)  63.4  24.3(3.4) 147(23)  88(13)  4.75(1.02)  1.64(13.8)  6.60(2.63)  0.90(0.29)  2.97(0.96)  48.5  63.1  12.4 	61.5(9.2)  63.7  24.4(3.5) 147(23)  87(13)  4.86(1.04)  1.70(8.4)  6.76(2.81)  0.90(0.27)  3.06(0.96)  50.9  63.8  16.6 					61.1(8.5)  62.8  24.4(3.2)  143(20)  85(12)  4.79(0.99)  1.71(12.9)  6.39(2.58)  0.93(0.27)  2.92(0.95)  44.9  59.8  12.3 	58.1(9.7) * 63.5  24.0(3.5) 152(23)  92(13)  4.54(0.99)  1.45(13.7) 6.57(2.36)  0.89(0.32)  2.87(0.96)  48.5  65.3  5.9

^*P＜0.05，P＜0.01 versus control.

The genotype in table 2:VEGFR-2 gene polymorphic site and allelotrope are in the frequency distribution of case group and control group

Polymorphism and grouping	Gene frequency		*P	Genotype frequency, n (%)			P	 proofreaies and correct OR (95%CI)
									VEGFR-2-604T → C control group (n=1798) case group (n=1849) cerebral thrombus group (n=812) chamber stalk group (n=530) cerebral hemorrhage group (n=507) VEGFR-2+1192C → T control group (n=1798) case group (n=1849) cerebral thrombus group (n=812) chamber stalk group (n=530) cerebral hemorrhage group (n=507) VEGFR-2+1719A → T control group (n=1798) case group (n=1849) cerebral thrombus group (n=812) chamber stalk group (n=530) cerebral hemorrhage group (n=507)	T 0.69 0.70 0.73 0.68 0.67 C 0.89 0.86 0.88 0.88 0.82 A 0.62 0.61 0.64 0.61 0.57	C 0.31 030 0.27 0.32 0.33 T 0.11 0.14 0.12 0.12 0.18 T 0.38 0.39 0.36 0.39 0.43	0.3 0.001 * 0.6 0.3 0.000 * 0.4 0.3 0.000 * 0.5 0.2 0.5 0.004 *	TT 862(47.9) 920(49.8) 446(54.9) 247(46.6) 227(44.8) CC 1429(79.5) 1367(73.9) 620(76.4) 411(77.5) 336(66.3) AA 699(38.9) 709(38.3) 343(42.2) 205(38.7) 161(31.8)	TC 751(41.8) 750(40.6) 298(36.7) 227(42.8) 225(44.4) CT 351(19.5) 449(24.3) 182(22.4) 112(21.1) 155(30.6) AT 817(45.4) 830(44.9) 347(42.7) 231(43.6) 252(49.7)	CC 185(10.3) 179(9.7) 68(8.4) 56(10.6) 55(10.8) TT 18(1.0) 33(1.8) 10(1.2) 7(1.3) 16(3.2) TT 282(15.7) 310(16.8) 122(15.0) 94(17.7) 94(18.5)	0.5 0.004  0.9 0.5 0.000  0.07 0.6 0.000  0.7 0.3 0.5 0.01 	0.97(0.83-1.13) 0.78(0.65-0.95)  1.08(0.88-1.34) 1.26(0.99-1.62) 1.46(1.22-1.75)  1.21(0.94-1.45) 1.07(0.82-1.39) 2.25(1.70-2.96)  0.99(0.85-1.16) 0.82(0.68-1.03) 1.00(0.80-1.24) 1.33(1.03-1.73) 

^*P and ^P: the chi square test between case group and control group

* many first Logistic return: proofread and correct age, sex, smoking, drink, behind the blood plasma HDL, total plasma cholesterol, blood pressure, weight index, blood sugar, detect VEGFR-2 gene and the dependency of cerebral apoplexy and the dependency of cerebral apoplexy.

The linkage disequilibrium in table 3 VEGFR-2 gene polymorphic site

	+1192C/T				+1719A/T
									D	D’	r 2	P	D	D’	r 2	P
	-604T/C +1192C/T	0.0141	0.1636	0.0105	＜0.0001	-0.0006 -0.0012	0.0046 0.2663	0.0077 0.0004									0.83 ＜0.0001

Annotate: D, D ' and r ²For weighing the index of linkage disequilibrium degree; The P value is to calculate according to the D value to get.

Each haplotype of table 4a VEGFR-2 gene is (604/+1192/+1719) in the distribution of cerebral infarction and hematencephalon group

Haplotype	Control group (n=1798)	Cerebral infarction (n=812)	Cerebral infarction vs contrast
						*OR (95％CI)	P	P corr
			TCA TCT TTA TTT CCA CCT CTA CTT	0.381 0.247 0.044 0.016 0.156 0.108 0.035 0.013	0.399 0.255 0.058 0.021 0.143 0.076 0.037 0.011				1.00 0.99 (0.85-1.15) 0.99 (0.74-1.33) 1.24 (0.80-1.91) 0.81 (0.68-0.98) 0.67 (0.54-0.84) 1.01 (0.73-1.39) 0.81 (0.47-1.41)	NS NS NS 0.025 0.0004 NS NS	NS NS NS 0.174 0.0021 NS NS
Global P		0.01

Each haplotype of table 4b VEGFR-2 gene is (604/+1192/+1719) in the distribution of hematencephalon group

Haplotype	Control group (n=1798)	Hematencephalon (n=507)	Hematencephalon vs contrast
						*OR(95％CI)	P	P corr
			TCA TCT TTA TTT CCA CCT CTA CTT	0.381 0.247 0.044 0.016 0.156 0.108 0.035 0.013	0.313 0.249 0.064 0.045 0.138 0.116 0.052 0.025				1.00 1.23 (1.02-1.48) 1.78 (1.30-2.43) 3.43 (2.28-5.14) 1.08 (0.86-1.35) 1.31 (1.03-1.67) 1.82 (1.29-2.56) 2.30 (1.39-3.79)	0.031 0.00027 3.8×10 -10 NS 0.025 0.001 0.001	0.217 0.0019 2.7×10 -9 NS 0.175 0.007 0.007
Global P		0.01

^*Global P:, and compare the whole difference of haplotype frequency distribution in case group and the control group by PHASE 2.0 software estimate sheet build frequencies

^*OR (95%CI) and ^P: chi square test is the distribution (with wild haplotype TCA is reference) of each certain monomers type between case group and control group relatively;

^P _Corr: after Bonferroni ' s multiple check is proofreaied and correct

Claims (13)

1. be used for determining VEGFR-2 gene polymorphic site-604T/C and/or+1192C/T and/or+instrument of the genetic polymorphism pattern of 1719A/T.

2. the instrument of claim 1, it be VEGFR-2 gene polymorphic site-604T/C and/or+1192C/T and/or+1719A/T polymorphism parting oligonucleotide, preferably, described polymorphism parting oligonucleotide is: (1) allele-specific nucleic acid primer, it can detect VEGFR-2 gene polymorphic site-604T/C and/or+1192C/T and/or+polymorphism of 1719A/T, perhaps (2) are used for detecting the oligonucleotide probe of the polymorphism of VEGFR-2 gene, its can be specifically with have VEGFR-2 gene polymorphic site-604T/C and/or+1192C/T and/or+nucleic acid hybridization of 1719A/T polymorphism, preferably, the length of oligonucleotide probe is 17-50 Nucleotide.

3. according to the instrument of claim 2, it is a restriction enzyme.

4. diagnostic kit comprises each instrument of claim 1-3.

5. diagnosis patient cerebrovascular disease (particularly comprises cerebral thrombosis; lacunar infarction; hematencephalon is at interior cerebral apoplexy) method; described method comprise detection from VEGFR-2 gene-604T/C in described patient's the sample and/or+1192C/T and/or+existence of 1719A/T; wherein; VEGFR-2 gene promoter area-604C allelotrope is relevant with the ill risk of cerebral thrombosis palsy of reduction; carry+the allelic individuality of 1192T and carrying+the ill risk of the allelic individual hematencephalon of 1719T increases; haplotype (604/+1192/+1719) C/C/T has provide protection to cerebral thrombosis, and haplotype (604/+1192/+1719) the ill risk increase of T/T/T haplotype carrier hematencephalon.

6. determine that the patient (particularly comprises cerebral thrombosis to cerebrovascular disease; lacunar infarction; hematencephalon is at interior cerebral apoplexy) the method for genetic predisposition; described method comprise detection from VEGFR-2 gene-604T/C in described patient's the sample and/or+1192C/T and/or+existence of 1719A/T; wherein; VEGFR-2 gene promoter area-604C allelotrope is relevant with the ill risk of cerebral thrombosis palsy of reduction; carry+the allelic individuality of 1192T and carrying+the ill risk of the allelic individual hematencephalon of 1719T increases; haplotype (604/+1192/+1719) C/C/T has provide protection to cerebral thrombosis, and haplotype (604/+1192/+1719) the ill risk increase of T/T/T haplotype carrier hematencephalon.

7. analyze method, comprising from the isolating stripped biological sample of patient: determine VEGFR-2 gene polymorphic site-604T/C in the described sample and/or+1192C/T and/or+Nucleotide of 1719A/T.

8. the method for claim 7; also comprise and determine that described patient's cerebrovascular disease (particularly comprises cerebral thrombosis; lacunar infarction; hematencephalon is at interior cerebral apoplexy) genetic predisposition or diagnosis patient cerebrovascular disease (particularly comprise cerebral thrombosis; lacunar infarction; hematencephalon is at interior cerebral apoplexy); wherein; VEGFR-2 gene promoter area-604C allelotrope is relevant with the ill risk of cerebral thrombosis palsy of reduction; carry+the allelic individuality of 1192T and carrying+the ill risk of the allelic individual hematencephalon of 1719T increases; haplotype (604/+1192/+1719) C/C/T has provide protection to cerebral thrombosis, and haplotype (604/+1192/+1719) the ill risk increase of T/T/T haplotype carrier hematencephalon.

9. each method of claim 5 to 8, wherein use to comprise the test that is selected from following difference foranalysis of nucleic acids technology: with mass spectrum to the direct mass analysis of PCR product, invasive cleaved products direct analysis, direct sequence analysis, allele specific amplification, allele-specific hybridization, oligonucleotide are connected test, effractor's test, DNA chip analysis and restriction fragment length polymorphism.

10. microarray, wherein comprise in the claim 2 defined VEGFR-2 gene polymorphic site-604T/C and/or+1192C/T and/or+1719A/T polymorphism parting oligonucleotide.

11. the microarray of claim 10, it is the form of DNA chip.

12. detection of biological imitates in the product VEGFR-2 gene polymorphic site-604T/C and/or+1192C/T and/or+material of the existence of 1719A/T is used to prepare the purposes of diagnostic reagent, described diagnostic reagent is used to diagnose patient's cerebrovascular disease (cerebral apoplexy that particularly comprises cerebral thrombosis, lacunar infarction, hematencephalon) or is used for determining the genetic predisposition of patient to cerebrovascular disease (cerebral apoplexy that particularly comprises cerebral thrombosis, lacunar infarction, hematencephalon).

13. the purposes of claim 12, wherein said material is selected from:

The instrument of claim 1-4;

The antibody of Val297Ile sudden change and/or Gln472His sudden change in can specific recognition VEGFR-2; With

Can specific recognition VEGFR-2 gene VEGFR-2 gene polymorphic site-604T/C and/or+1192C/T and/or+primer or probe or the restriction enzyme of 1719A/T.

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