CN101041855A - Method for determining susceptibility of bleeding-out brain dead in Chinese people by using angiogenin-2 mononucleotide polymorphism sites - Google Patents

Method for determining susceptibility of bleeding-out brain dead in Chinese people by using angiogenin-2 mononucleotide polymorphism sites Download PDF

Info

Publication number
CN101041855A
CN101041855A CN 200610135866 CN200610135866A CN101041855A CN 101041855 A CN101041855 A CN 101041855A CN 200610135866 CN200610135866 CN 200610135866 CN 200610135866 A CN200610135866 A CN 200610135866A CN 101041855 A CN101041855 A CN 101041855A
Authority
CN
China
Prior art keywords
snp
angiogenesis hormone
pleomorphism site
polymorphism
patient
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN 200610135866
Other languages
Chinese (zh)
Inventor
惠汝太
蔡明清
王继征
于晖
张伟丽
孙凯
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fuwai Hospital of CAMS and PUMC
Original Assignee
Fuwai Hospital of CAMS and PUMC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fuwai Hospital of CAMS and PUMC filed Critical Fuwai Hospital of CAMS and PUMC
Priority to CN 200610135866 priority Critical patent/CN101041855A/en
Publication of CN101041855A publication Critical patent/CN101041855A/en
Pending legal-status Critical Current

Links

Images

Landscapes

  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a method to assure susceptibility in bleeding cerebral apoplexy of patient (especially including cerebral hemorrhage or subarachnoid cavity hemorrhage), which is characterized by the following: assuring polymorphism site 1084A/A on stimulating angiogenin -2 specifically; involving substance, composition, agent box and usage of the method.

Description

Utilize angiogenesis hormone-2 mononucleotide polymorphism site to determine the method for hemorrhagic apoplexy susceptibility in the Chinese population
Invention field
The present invention relates to utilize angiogenesis hormone-2 (Angiopoietin-2 is hereinafter to be referred as Ang2) mononucleotide polymorphism site to determine the method for patient, be used for material, composition, test kit of this method and uses thereof the genetic predisposition of hemorrhagic apoplexy.
Background of invention
It is the disease of main clinical manifestation with brain ischemic and hemorrhagic damage symptom that cerebral apoplexy is one group, is divided into hemorrhagic apoplexy (hematencephalon or subarachnoid hemorrhage) and cerebral infarction (cerebral infarction and cerebral thrombosis) two big classes.
In China, cerebral apoplexy is the second largest cause of the death that is only second to cancer, and average per 100,000 philtrums just have 137 people to die from this disease, and the patient who dies from cerebral apoplexy every year has 1,500,000 approximately.Even more serious is that cerebral apoplexy is the high disease of disability rate.In existing about 5,000,000 the patients with cerebral apoplexy of China, 75% has deformity in various degree.Therefore, the cause of disease of active research cerebral apoplexy and treatment plan reduce the incidence and mortality of cerebral apoplexy significantly, for prolonging patient's life-span, improve the quality of living, and reduce health care expenditures, and be significant.
Cerebral apoplexy is a kind of multi-factor disease of complexity, is the coefficient result of inherited genetic factors and environmental factors [59]Hemorrhagic apoplexy is divided into hematencephalon and subarachnoid hemorrhage according to the bleeding part.The caused hematencephalon of arteria cerebri media branch, intracranial aneurysm and cerebrovascular malformation are common causes of bleeding.Showing as one group in pathological change is the syndrome that feature, vessel wall dysfunction cause with the atherosclerosis, comprises angiorrbagia, oozes out and tissue edema etc.
The hemorrhagic apoplexy pathogeny is not bright.But discover at present, have substantial connection with following Hazard Factor: 1) hypertension [60]: being the most important Hazard Factor of cerebral apoplexy, also is its pathologic variation-atherosclerotic basis, and hypertension can cause cerebrovascular generation lipid hyaline degeneration and cause cerebral apoplexy, and high salt diet also inspires cerebral apoplexy by hypertension; 2) smoking: the second largest Hazard Factor that are stroke onset.Smoking can improve plasma fibrinogen content, can increase the incidence of the subarachnoid hemorrhage due to the aneurysm rupture [60]As independent hazard factor, smoking cessation can prevention of brain palsy, particularly patients such as complicated hypertension below 60 years old, myocardosis, diabetes early; 3) cardiovascular disorder: heart trouble (as rheumatic heart disease, coronary heart disease, heart failure, atrial fibrillation etc.) particularly with irregular pulse or myocardial infarction person, is the Hazard Factor of hemorrhagic apoplexy.4) diabetes [61]: diabetic subject's stroke onset rate is 1,549.05/10 ten thousand, and the ND is 333.00/10 ten thousand, and both have significant difference, and (P＜0.01 RR=4.64), illustrates that diabetes are Hazard Factor of cerebral apoplexy [61]Regular Insulin lowering blood glucose level can obviously reduce the cerebral apoplexy extent of disease; 5) hyperlipemia: high lipoprotein a (Lp (a)) is the independent hazard factor of hemorrhagic apoplexy [62], present widespread use is as the index of the Hazard Factor of measuring atherosclerosis, hemorrhagic apoplexy; 6) other: comprise and drinking, infect, medicine etc.
In the Chinese population, hemorrhagic apoplexy accounts for 21%～48% of total class, is 2～3 times of Caucasians.The difference of this race's morbidity, there is different pathogeny in prompting.Although can search out various cerebral apoplexy common susceptibility locis [63], but this site follows the relation of hemorrhagic apoplexy to prove as yet.The inventor finds, in the above-mentioned various Hazard Factor, have only that Lp (a) level can the reasonable dismissal hemorrhagic apoplexy in interethnic morbidity difference.The effect of Hazard Factor, key link-vessel wall pathology that disease is taken place plays and brings out and promoter action the most at last.Therefore, the growth mechanism of research blood vessel, and, will help finally to illustrate this sick genesis mechanism in the variation of hemorrhagic apoplexy.
That the characteristic pathological change of hemorrhagic apoplexy comprises is hemorrhage, ooze out and tissue edema etc., and this is the syndrome that artery is atherosis, the vessel wall dysfunction causes.Known angiogenesis hormone-the 2nd, living back blood vessel is reinvented necessary, can cause new vessel and form, and also integrates new vessel with Ang1 and VEGF coordinative role simultaneously, finally forms functional blood vessel.When the Ang2 gene knockout, embryo's blood vessel in period takes place unaffected, but after coming to the ripening period, owing to lose the integration stabilization of Ang2, the vessel wall integrity is destroyed, and permeability increases, and shows as oedema, and is hemorrhage and dead [1]Therefore, the blood vessel that the Ang2 functional defect causes is integrated incomplete, may be one of atherosclerotic mechanism.
The spontaneously hypertensive cerebral apoplexy easily trouble type rat (stroke-prone spontaneouslyhypertensive rat is that the research inherited genetic factors is to the good model of cerebral apoplexy effect SHRSP).SHRSP can spontaneous formation cerebral apoplexy in experimentation, and damage is aggravation constantly.This damaged portion is owing to cerebral vessels heteroplasia causes.Discover that in various types of cerebral apoplexy animal models, Ang2 expresses all and raises, and increases the most obvious the SHRSP rat [65]In contrast to this, Ang1 and Tie2 express and descend, but do not have notable difference between SHR and WKY rat.At arteria cerebri media infraction (middle cerebral artery occlusion, MCAO) in the model, Ang2 obviously raise in morbidity in back 6 hours, mainly be distributed in the endotheliocyte bitter end portion of infraction and peripheral region, and Ang1 mainly expresses at spongiocyte and neurocyte zone, and does not have considerable change.The expression of Ang2 is carried out synchronously with VEGF, can promote the propagation of endotheliocyte and the generation of new vessel [66](intrauterine growthrestricted fetuses IUGR) can cause comprising coronary heart disease, type ii diabetes to intrauterine growth retardation, hypertension, cerebral apoplexies etc. are in interior adult syndromes, and the appearance of this syndromes takes place closely related unusually with IUGR blood vessel in period.The uniqueness of Ang2 in IUGR expressed and to this pathogenetic influence, may be the major reason of IUGR syndromes [67]
In sum, the Ang2 functional defect, it is incomplete to cause vessel wall to integrate, and shows as and oozes out, and hemorrhage, the surrounding tissue oedema may be to be the important pathogenic factor of the hemorrhagic apoplexy of feature with the vessel wall functional defect.Inquire into status and the effect of Ang2 in hemorrhagic apoplexy dysfunction blood vessel, will help to illustrate early the pathogeny of this complicacy disease, be its prevention, diagnosis and treatment provide useful guidance.
In recent years, on genomic level, inquire into inherited genetic factors and become one of research focus with the association of people's quasi-complexity disease.Case control study is the most frequently used association study [68]Single nucleotide polymorphism (single nucleotide polymorphism SNP) is modal heritable variation because content is abundant, stability is high, can high throughput analysis etc. characteristics, extensively be used as the selection markers thing of human colony's genetic research [68]With the disease linkage analysis [69]Haplotype is meant on the same karyomit(e) in a certain specific gene, the pattern of a plurality of variant sites coexistences.With SNP by comparison, haplotype has the heterozygosity of height, multiple allelotrope coexistence, and therefore higher stability can provide how valuable information in the linkage analysis of complicacy disease relatively [70]An outstanding illustration is from the research of asthma patient to the individual reaction otherness of beta receptor agonist [71]The result shows that the beta 2-adrenergic receptor gene haplotype is closely related with this reactivity, even also can be verified among the crowd on a small scale at one, and independent snp analysis is not suitable for little cohort study.In addition, therefore haplotype can reflect the situation of a certain specific crowd gene genetic evolutional structure owing to be the direct description of genome mutation structure.This evolution information is extremely responsive to the research inherited genetic factors with the relation between the complex disease [72-74]
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, the potential important tool that becomes the gene of location participation human diseases situation of SNP, see as Wang etc., Science 280:1077-1082 (1998), it discloses an exploratory study, wherein 2,227 SNP are positioned in the DNA zone of one 2.3 megabasse by mapping.
Relation between single nucleotide polymorphism and the particular phenotype is not represented or needed SNP is the reason of this phenotype.On the contrary, this relation may only represent that SNP is positioned at the phenotype factor of determination near the location on the genome, more may be found related with these factor of determinations and related with interested phenotype thus thus.Therefore, may there be linkage disequilibrium (LD) in SNP with the function variation of " really ".When the allelic dependency on two different positionss of genome is higher than the dependency of expecting, there is LD, is called allelotrope relevant (allelicassociation) again.Therefore, SNP can cause that owing to it is approaching the sudden change of particular phenotype has value with marking.
The present invention is in the case-control study of angiogenesis hormone-2 as hemorrhagic apoplexy (particularly comprising hematencephalon or subarachnoid hemorrhage) susceptible candidate gene, by the relation of these gene SNP 1084 pleomorphism sites and hemorrhagic apoplexy is carried out association study, study the dependency of this site and hemorrhagic apoplexy, thereby determine the tumor susceptibility gene of Chinese's hemorrhagic apoplexy disease.Simultaneously, the hemorrhagic apoplexy related SNP of being differentiated according to the present invention is determined the target site of medicine, and guiding policy can be provided for follow-up drug screening.
Summary of the invention
One aspect of the present invention relates to the instrument that is used to detect the pleomorphism site 1084A/A on the angiogenesis hormone-2.In one embodiment of the invention, described instrument can be the pleomorphism site 1084A/A polymorphism parting oligonucleotide on the angiogenesis hormone-2, preferably, described polymorphism parting oligonucleotide is: (1) allele-specific nucleic acid primer, it can detect the polymorphism of the pleomorphism site 1084A/A of angiogenesis hormone-2, perhaps (2) are used for detecting the oligonucleotide probe of angiogenesis hormone-2 polymorphism, it can be specifically and the nucleic acid hybridization with the pleomorphism site 1084A/A on the angiogenesis hormone-2, preferably, the length of oligonucleotide probe is 17-50 Nucleotide.
In one embodiment of the invention, described instrument is a restriction enzyme.
Another aspect of the present invention relates to the test kit that is used for above-mentioned detection, wherein at least a above-mentioned instrument, and, choose wantonly, be used for the suitable buffer system and the color development system of detection reaction.
Another aspect of the present invention, the method that relates to a kind of diagnosis patient's bleeding cerebral apoplexy (comprising hematencephalon or subarachnoid hemorrhage), described method comprise the existence of detection from the pleomorphism site 1084A/A on the angiogenesis hormone-2 in described patient's the sample.
Another aspect of the present invention, the method that relates to the susceptibility of a kind of definite patient's bleeding cerebral apoplexy (particularly comprising hematencephalon or subarachnoid hemorrhage), described method comprise the existence of detection from the pleomorphism site 1084A/A on the angiogenesis hormone-2 in described patient's the sample.
One side more of the present invention also relates to the method for a kind of analysis from the isolating stripped biological sample of patient, comprising: the Nucleotide of determining the pleomorphism site 1084A/A on the angiogenesis hormone in the described sample-2.In a preferred embodiment of the invention, use comprises, but be not limited to, be selected from the test of following difference foranalysis of nucleic acids technology: to the direct mass analysis of PCR product, to invasive cleaved products direct analysis, direct sequence analysis, allele specific amplification, allele-specific hybridization, oligonucleotide connection test, effractor's test, DNA chip analysis and restriction fragment length polymorphism, detect the existence of the Nucleotide of the pleomorphism site 1084A/A on the angiogenesis hormone in the described sample-2 with mass spectrum.
Of the present inventionly also relate to a kind of microarray more on the one hand, wherein comprise the parting oligonucleotide of the pleomorphism site 1084A/A on the angiogenesis hormone-2 of above-mentioned definition.In one embodiment of the invention, described microarray is the form of DNA chip.
One side more of the present invention, also relate to detection of biological and imitate the material of the existence of the pleomorphism site 1084A/A on the angiogenesis hormone-2 in the product in the purposes of preparation diagnostic reagent, described diagnostic reagent is used to diagnose the patient's bleeding cerebral apoplexy genetic predisposition of (particularly comprising hematencephalon or subarachnoid hemorrhage).In the present invention, described material is selected from:
Be used to detect the instrument of the pleomorphism site 1084A/A on the angiogenesis hormone-2, for example, pleomorphism site 1084A/A polymorphism parting oligonucleotide on the angiogenesis hormone-2, preferably, described polymorphism parting oligonucleotide is: (1) allele-specific nucleic acid primer, it can detect the polymorphism of the pleomorphism site 1084A/A of angiogenesis hormone-2, perhaps (2) are used for detecting the oligonucleotide probe of angiogenesis hormone-2 polymorphism, it can be specifically and the nucleic acid hybridization with the pleomorphism site 1084A/A on the angiogenesis hormone-2, preferably, the length of oligonucleotide probe is 17-50 Nucleotide;
The antibody of the sudden change of the pleomorphism site 1084A/A on can specific recognition angiogenesis hormone-2; With
Primer or probe or the restriction enzyme of pleomorphism site 1084A/A on can specific recognition angiogenesis hormone-2.
Detailed Description Of The Invention
About the existing report of the research of the SNP of Ang2 [75]This research is to be based upon on 10 normal people's gene sequencing bases of random choose.Though obtained at exon 2, three SNP on 4,5 because sample size is too little, are not enough to reflect the detailed information of Ang2 genome mutation comprehensively, can't carry out haplotype analysis, more can't be based upon the distributed model in the disease.
For illustrating the regularity of distribution of Ang2 haplotype in Chinese hemorrhagic apoplexy crowd, whether relevant with hemorrhagic apoplexy on genomic level so as to research Ang2, we have selected for use the geographic cerebral apoplexy crowd in Xi'an to carry out case-check analysis research.Our PRELIMINARY RESULTS shows, SNP 1084Other genotype in site are susceptible factors of hemorrhagic apoplexy different ages morbidity to the conversion of A/A.SNP 1084Site A/A genotype can be regarded as a risk factor of different ages morbidity.Ang2 may be a susceptible mark of hemorrhagic apoplexy.
Angiogenesis hormone-2 gene order is corresponding to NCBI Acc No:004327.
In this article, angiogenesis hormone-2 gene order and encoded protein matter sequence are numbered according to ordinary method.The base of transcription initiation site is numbered 2.
The numbering of Nucleotide and amino-acid residue is meant and Ang2 cDNA sequence (corresponding Nucleotide and the amino-acid residue of the sequence of NCBI Acc No:004327 or its amino acid sequence coded same position (or numbering) in nucleotide sequence that the present invention is mentioned and the aminoacid sequence.
" correspondence " be meant nucleic acid molecule of the present invention or proteinic sequence with position relative, the best comparison of sequence (alignment) back shown in the cDNA sequence of reference sequences with this reference sequences position.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.
Angiogenesis hormone-2 gene mononucleotide polymorphism site " 1084A/A " is meant this gene the 2914th polymorphism A and A.
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), ALEX (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 sample available from the patient that carries described polymorphism nucleic acid can obtain from individuality easily, includes but not limited to serum, urine sample, saliva, body fluid, (biopsy) tissue samples etc.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.
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 of the diagnostic primers of polymorphism in detection angiogenesis hormone-2 gene, this diagnostic primers can with the nucleic acid hybridization that in sequence, comprises polymorphism (for example angiogenesis hormone-2 gene locus 1084A/A or its reverse complementary sequence) in angiogenesis hormone-2 gene locus 1084 places.
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 ARMS 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 angiogenesis hormone-2 gene, this probe can be specifically and the nucleic acid hybridization that comprises the polymorphism (for example angiogenesis hormone-2 gene locus 1084A/A or its reverse complementary sequence) that is positioned at 1084 of angiogenesis hormone-2 gene locuss in sequence.
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 angiogenesis hormone-2 gene are particularly expressed the human angiogenesis hormone-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 invention still further relates to a kind of test kit that is used to detect the pleomorphism site 1084A/A on the angiogenesis hormone-2, it comprises the pleomorphism site 1084A/A polymorphism parting oligonucleotide at least a angiogenesis hormone-2, or restriction enzyme.The parting oligonucleotide of polymorphism described in the present invention is: (1) allele-specific nucleic acid primer, it can detect the polymorphism of the pleomorphism site 1084A/A of angiogenesis hormone-2, perhaps (2) are used for detecting the oligonucleotide probe of angiogenesis hormone-2 polymorphism, it can be specifically and the nucleic acid hybridization with the pleomorphism site 1084A/A on the angiogenesis hormone-2, preferably, the length of oligonucleotide probe is 17-50 Nucleotide.In one embodiment of the invention, optional buffer system and the color development system that is suitable for described detection reaction that contain in the described test kit.
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.
Angiogenesis hormone of the present invention-2 gene type oligonucleotide 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 angiogenesis hormone-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.
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.
The invention still further relates to the in vitro method of a kind of diagnosis patient's bleeding cerebral apoplexy (comprising hematencephalon or subarachnoid hemorrhage), described method comprises the existence of detection from the pleomorphism site 1084A/A on the angiogenesis hormone-2 in described patient's the sample.
The invention still further relates to the in vitro method of the susceptibility of a kind of definite patient's bleeding cerebral apoplexy (comprising hematencephalon or subarachnoid hemorrhage), described method comprises the existence of detection from the pleomorphism site 1084A/A on the angiogenesis hormone-2 in described patient's the sample.
The invention still further relates to the method for a kind of analysis, comprising: the Nucleotide of determining the pleomorphism site 1084A/A on the angiogenesis hormone in the described sample-2 from the isolating stripped biological sample of patient.In the method for the invention, 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.
In the method for the invention, wherein said sample from the patient is selected from: serum, urine sample, saliva, body fluid and/or (biopsy) tissue samples, optional, described sample can carry out purifying in advance, for example separates total DNA.
The invention still further relates to a kind of microarray that is used to detect the pleomorphism site 1084A/A on the angiogenesis hormone-2, wherein comprise the parting oligonucleotide of the pleomorphism site 1084A/A on the angiogenesis hormone-2 of aforementioned definitions.In one embodiment of the invention, described microarray is the form of DNA chip.
The invention still further relates to detection of biological and imitate the material of the existence of the pleomorphism site 1084A/A on the angiogenesis hormone-2 in the product in the purposes of preparation diagnostic reagent, described diagnostic reagent is used to diagnose the susceptibility of patient's bleeding cerebral apoplexy (hematencephalon or subarachnoid hemorrhage).In the present invention, described material is selected from:
Be used to detect the instrument of the pleomorphism site 1084A/A on the angiogenesis hormone-2, for example, pleomorphism site 1084A/A polymorphism parting oligonucleotide on the angiogenesis hormone-2, preferably, described polymorphism parting oligonucleotide is: (1) allele-specific nucleic acid primer, it can detect the polymorphism of the pleomorphism site 1084A/A of angiogenesis hormone-2, perhaps (2) are used for detecting the oligonucleotide probe of angiogenesis hormone-2 polymorphism, it can be specifically and the nucleic acid hybridization with the pleomorphism site 1084A/A on the angiogenesis hormone-2, preferably, the length of oligonucleotide probe is 17-50 Nucleotide;
The antibody of the sudden change of the pleomorphism site 1084A/A on can specific recognition angiogenesis hormone-2; With
Primer or probe or the restriction enzyme of pleomorphism site 1084A/A on can specific recognition angiogenesis hormone-2.
Description of drawings
Fig. 1: the single nucleotide polymorphism measurement result that shows the Ang-2 sequence: SNP 1456: 176, C/T.
Fig. 2: the single nucleotide polymorphism measurement result that shows the Ang-2 sequence: SNP 757: 151, T/T.
Fig. 3: the single nucleotide polymorphism measurement result that shows the Ang-2 sequence: SNP 757: 152, G/C.
Fig. 4: the single nucleotide polymorphism measurement result that shows the Ang-2 sequence: SNP 757: 151, C/C.
Fig. 5: the single nucleotide polymorphism measurement result that shows the Ang-2 sequence: SNP 757: 153, T/C.
Fig. 6: the single nucleotide polymorphism measurement result that shows the Ang-2 sequence: SNP 757: 168, T/G.
Fig. 7: the single nucleotide polymorphism measurement result that shows the Ang-2 sequence: SNP 1063: 195, G/G; SNP 1084: 216, A/A.
Fig. 8: the single nucleotide polymorphism measurement result that shows the Ang-2 sequence: SNP 1063: 189, G/T; SNP 1084: 210, A/G.
Fig. 9: the single nucleotide polymorphism measurement result that shows the Ang-2 sequence: SNP 1063: 196, G/G; SNP 1084: 217, A/G.
Figure 10: the single nucleotide polymorphism measurement result that shows the Ang-2 sequence: SNP 1063: 145, G/T; SNP 1084: 166, G/G.
Figure 11: the single nucleotide polymorphism measurement result that shows the Ang-2 sequence: SNP 1063: 145, G/G; SNP 1084: 166, G/G.
Figure 12: the single nucleotide polymorphism measurement result that shows the Ang-2 sequence: SNP 1231: 154, G/G.
Figure 13: the single nucleotide polymorphism measurement result that shows the Ang-2 sequence: SNP 1231: 145, A/G.
Figure 14: the single nucleotide polymorphism measurement result that shows the Ang-2 sequence: SNP 1456: 169, T/T.
Figure 15: show each haplotype evolutionary relationship figure of Ang2.
Be used for the present invention is further illustrated below in conjunction with drawings and Examples, and the scope that does not limit the present invention in any way.
Embodiment
Embodiment 1 MethodsThe cases enrolled and research method
MethodsThe cases enrolled and contrast
This research is subsidized by China national 973 problems (investigation of Chinese cerebral apoplexy Hazard Factor and prevention, Hui Rutai professor), by Sino-German laboratory united and coordinating, collects sample.Geographic hemorrhagic apoplexy sample 77 examples in Xi'an are chosen in this research, contrast 77 examples.All participants all sign Informed Consent Form.The diagnosis of hemorrhagic apoplexy is from CT and mr, and standard meets " International Classification of Diseases (the 9th edition) ", and patient details slightly.
Genotype detection
1. the extraction of sample DNA [76]: according to conventional molecular biology method.
9 exons 2.PCR increase: the design of primers document that sees reference [50], but make improvement at exon1 and exon3.Sequence is as follows:
Exon1-1#：5’-CGG GAC TCT GGA CGT GTG TTT-3’
Exon1-2#：5’-GCT CTA ATC CTC TTC ATC CTC CTT CT-3’
Exon3-1#：5’-GTT ATG TTT TCC TGT AGC TTG GGT A-3’
Exon3-2#：5’-CTC TCC CTT CTC CTC CCT CA-3’
3.PCR amplification:
20 μ l system: 10 * buffer, 2 μ l, the upstream primer 1 μ l of 5pmol/ μ l, the downstream primer 1 μ l of 5pmol/ μ l, 10mM dNTP 0.3 μ l, genomic dna 0.2 μ l, 5U/ μ l Taq polysaccharase 0.3ul mends H 2O to 20 μ l.
95℃，5’；40cycle(95℃，30”；54℃，30”；72℃，1’)；72℃，10’(for exon2，4，5，6).
95℃，5’；40cycle(95℃，30”；58℃，30”；72℃，1’)；72℃，10’(for exon1，3，8，9).
95℃，5’；40cycle(95℃，30”；50℃，30”；72℃，2’)；72℃，10’(for exon7).
4.PCR product purification:
1) the NaAc/ dehydrated alcohol of PCR product+5 times volume (5M NaAc: dehydrated alcohol=1: 5)
2)-80 ℃, 2 hours to spending the night.
3) centrifugal: 13,000g, 4 ℃, 20 minutes.
4) 75% washing with alcohol.
5) room temperature is dried, and adds distilled water, and 2% agarose electrophoresis is identified, determines concentration, send order-checking.ABI377 sequenator (PE Applied Biosystems) is adopted in order-checking.
Snp analysis:
The Ang2 cDNA sequence (NCBI AccNo:004327) of sequencing result and G.D.Yancopoulos report is compared [8], seek variant sites.Use SPSS10.0 software to carry out statistical study.
Haplotype analysis
1. haplotype makes up [77]:
We have used according to the Haplotyper software that improves the Bayesian algorithm development.In Unix running environment, parameter is as follows: htyperv2 input output snp peopleround, wherein htperv2 is a dbase, input is the starting material file name of input, output is the result name of output, and snp is the number of SNP, and people is a number, round is an operation number of turns amount, generally selects 20.Illustrate, follow procedure represents to analyze 5 SNP, the result of 154 samples.Input_all is the raw data file title of input, and output_all is the result name of output, moves 20 circle: htyperv2 input_alloutput_all 5 154 20 altogether.
2. haplotype evolutionary analysis [78]:
The software that is adopted: PPH (perfect phylogeny haplotyper) with the haplotype of original not somatotype, is split as two monomer haplotypes (somatotype) in theory, and provides its mutual evolutionary relationship.
Experimental result
One. the physical data of selected patient and contrast
Show hemorrhagic apoplexy and do not have significant difference to impinging upon aspects such as age, sex, weight index, glucose, smoking history by table 1.On systolic pressure, diastolic pressure, total cholesterol, have significant differences (P＜0.01), on high-density lipoprotein (HDL), have significant difference (P＜0.05).It seems that totally two groups are mated; The difference that exists itself is exactly the Hazard Factor of cerebral apoplexy.
Table 1: the general clinical data of MethodsThe cases enrolled and contrast
Hemorrhagic apoplexy Contrast
Example number sex (man/woman) age (year) the total triglycerides of body mass index SBP (mmHg) * DBP (mmHg) * T-CHOL (mmol/L) * (mmol/L) HDL (mmol/L) LDL (mmol/L) glucose (mmol/L) smoking history (with/without) 77 51/26 58.42±8.31 24.07±3.69 147±19 90±10 4.41±0.89 1.96±1.33 35.49±8.79 2.73±1.13 6.57±2.82 35/42 77 50/27 56.92±7.97 24.64±3.56 128±12 81±8 5.03±1.04 2.00±1.11 40.61±9.57 2.77±1.16 5.81±1.61 25/52
Annotate: SBP: systolic pressure; DBP: diastolic pressure; HDL: high-density lipoprotein (HDL); LDL: low-density lipoprotein.*: have significant differences (P＜0.01) between case-control; : have significant difference (P＜0.05) between case-control.
Two .SNP interpretations of result
1.SNP loci frequency
The Ang2 gene comprises 9 exons and 8 introns [75]9 pairs of primer amplifications of this experimental design increase out with whole coding regions of Ang2 gene.Amplified production PCR is through behind the NaAc/ ethanol purification, the ABI377 evaluation of checking order.We carry out the BLAST comparison with the total length Ang2 cDNA sequence (NCBI No.AF004327) of reported first as standard, therefrom seek SNP.In detected 5 SNP, there are three to report: SNP 757, SNP 1084And SNP 1231Two is latest report: SNP 1063And SNP 1456All SNP are nonsense mutation.Each allelotrope sequencing result is seen Fig. 1 to Figure 14, and its distribution frequency sees Table 2.
The distribution of table 2:Ang2 gene coding region SNP and the relative content of each loci
The exon position Nucleotide position Standard sequence SNP Frequency (%)
All Case Contrast Age of onset (year) Hypertension
＜50 ＞50 Have Do not have
2 4 5 7 757 1063 1084 1231 1456 C TGA C GGT A AAA AAAT TGTG C/T C/C T/T C/G G/T G/T G/G A/G A/A G/G A/G G/G C/T T/T 31.8 38.8 14.3 8.1 6.5 9.7 87.7 42.2 26.6 30.5 14.3 85.1 20.8 79.2 33.8 40.3 14.3 7.8 5.2 9.1 89.6 44.2 23.4 31.2 13 87 23.4 76.6 29.9 36.4 14.3 10.4 7.8 10.4 85.7 40.3 29.9 29.9 15.6 83.1 18.2 81.8 29.0 41.9 9.7 14.2 6.5 16.1 83.9 48.4 16.1 35.5 9.7 90.3 19.4 80.6 40.9 31.8 18.2 1.3 9.1 4.5 95.5 31.8 40.9 22.7 22.7 77.3 22.7 77.3 33.7 39.8 12.0 7.3 8.4 9.6 90.4 37.3 28.9 32.5 15.7 84.3 22.9 77.1 28.3 35.0 18.3 11.3 5.0 10 83.3 48.3 25.0 26.7 13.3 85 15.0 85.0
2. whether the SNP frequency ratio is and meaningful between different groups
We are divided into case/contrast, age group (be lower than morbidity in 50 years old and be higher than 50 years old morbidity group) with the study population, and hypertension group (have or do not have history of hypertension) compares.x 2Check finds that in case-control group and the hypertension group, each allele distributions does not have difference.In age group, SNP 1084A/A allelotrope be lower than 50 years old cerebral apoplexy patient and the distribution frequency that is higher than in 50 years old is respectively 16.1% and 40.9%, distributing has significant difference (P＜0.05).Other distributional differences relatively see table 3 for details.
Table 3: the comparison of each allele distributions between different groups
Exon SNP The P value
Case/contrast ＜50 years old/＞50 years old SH/SNH or SNH/NSNH
SNP 757 SNP 1063 SNP 1084 SNP 1231 SNP 1456 C/T C/C T/T C/G G/T G/T G/G A/G A/A G/G A/G G/G C/T T/T 0.604 0.619 1.000 0.548 0.513 0.786 0.462 0.625 0.362 0.861 0.645 0.498 0.427 0.427 0.368 0.454 0.368 N 0.720 0.190 0.190 0.228 0.043*0.319 0.191 0.191 0.765 0.765 0.869/0.667 0.896/0.494 0.215/0.505 N/N N/N N/N 0.763/0.317 0.349/0.438 0.479/0.381 0.817/1.000 0.540/0.951 0.540/0.951 0.747/0.278 0.747/0.278
Note: SH: the concurrent hyperpietic of cerebral apoplexy (n=53); SNH: cerebral apoplexy does not have hyperpietic (n=12); NSNH: no cerebral apoplexy does not have hyperpietic (n=48).*：P＜0.05。
3. haplotype analysis
Haplotype makes up
According to the SNP genotype of being measured, this experiment has adopted improved Bayesian algorithm and relevant Haplotyper software to carry out the reckoning of haplotype.This method adopts Gibbssampler, with theoretical haplotype dividing processing, gathers at last again.The small sample data that is particularly useful for random choose also is suitable for the crowd who does not reach the Hardy-Weinberg distribution equilibrium.Table 4 has provided in the different groups, and each infers genotypic distribution proportion.We have adopted 5% as accepting or rejecting boundary.Being lower than 5% haplotype will be rejected, because this requires related software to have very strong reckoning ability, be that regular software is inaccessible.
Table 4:Ang2 gene haplotype structure and distribution frequency table
Haplotype Distribution frequency (%)
Totally Case Contrast ＜50 ＞50 SH SNH NSH NSNH
00100 00000 10000 10100 10001 00010 00001 01100 37.66 12.34 16.88 10.06 N N 5.52 N 38.96 15.58 14.29 10.39 5.84 N N N 31.17 10.39 22.08 11.04 N 5.19 6.49 N 46.77 9.68 14.52 9.68 6.45 N N N 15.91 31.82 13.64 18.18 N N N N 36.79 16.98 15.09 10.38 N N N N 46.15 11.54 15.38 7.69 N N N N 36.67 11.67 18.33 10.00 N N 13.33 N 25.53 13.83 20.21 14.89 N 7.45 N 6.38
Haplotype is with the allelic numeral of common expression: 0 expression heterozygous, 1 expression homozygous wildtype.At SNP 757, SNP 1063, SNP 1084, SNP 1231And SNP 1456In, 0 and 1 represents C/T and C/C, G/T and G/G, A/G and A/A, A/G and G/G, C/T and T/T respectively.In the table, N represents that distribution frequency is lower than 5% or distribution-free.SH: the concurrent hypertension of cerebral apoplexy (n=53); SNH: cerebral apoplexy does not have hypertension (n=12); NSH: no cerebral apoplexy has hypertension (n=30); NSNH: no cerebral apoplexy does not have hypertension (n=48).
The distribution of haplotype relatively
The comparison of haplotype between different groups, easier reflection gene is with the relation of disease.x 2Check shows, in age group, haplotype 00100 (Hap1) is 46.77% and 15.91% at the distribution proportion that was lower than 50 years old and be higher than among the patient of the courageous and upright cerebral apoplexy of 50 annual expenditures, the distribution frequency of 00000 (Hap2) in above-mentioned two groups is respectively 9.68% and 31.82%, and the two distributes and all has significant difference (the P value is respectively 0.023 and 0.042).Haplotype distribution indifference (table 5) between other groups.
4, haplotype evolutionary analysis
The evolutionary analysis of haplotype can be pointed out in a specific crowd, genotypic variation direction.In case-control study, according to the analysis of correlation results, can point out the variation direction of disease related gene, thereby provide a kind of new research approach for the disease genesis mechanism.This paper adopts the PPH method that 8 kinds of haplotypes of inferring are carried out evolutionary analysis, and purpose is in order to find out the haplotype trend of evolution, thus the evolution site of potential disease-related relatively [53]Haplotype is split as the detailed Sites Combination on two karyomit(e)s, is defined as epna and epnb respectively, and wherein n is the code of haplotype, is 1～8 (Figure 15 and table 6).
Table 5: each haplotype distributional difference in different groups compares
Hap The P value
Sick/right ＜50/＞50 SH/SNH SH/NSH SH/NSNH SNH/NSH SNH/NSNH NSH/NSNH
00100 10000 00000 10100 0.311 0.338 0.210 0.797 0.023 0.0420.857 0.368 0.434 0.453 0.892 0.763 0.923 0.660 0.556 0.853 0.169 0.742 0.452 0.625 0.426 0.651 0.804 0.868 0.091 0.569 0.747 0.797 0.272 0.877 0.926 0.556
Note: disease: case group; Right: control group; Surplus with table 2-4.
To 8＞5% haplotype analysis, find to co-exist in 5 kinds of single times of hypotypes by PPH.The result is as follows:
5 kinds of common haplotype sequences that exist among the table 6:Ang2
Hap No Sequence Other are the person roughly the same
1a 1b 2a 2b 4a TGAGT CTAGT CTGGT CGAAT CGAGC 8a 4b 3a，5a，6a，7a 3b，5b，6b，8b 7b
The present invention has carried out the analysis of Ang2 coding region haplotype to case-contrast data of the hemorrhagic apoplexy crowd that distribute in area, Chinese Xi'an.By 154 routine sample analyses, except 3 SNP site (SNP that reported 757, SNP 1084And SNP 1231) outside, our new discovery two SNP site (SNP 1063And SNP 1456).The allelotrope statistical result showed of each SNP, SNP 1084The A/A genotype, be lower than 50 years old and be higher than 50 years old and have significant difference (P＜0.05) in the patients with cerebral apoplexy.Haplotype analysis is found, all possible 2 5In (=32) haplotype, only obtain 8 kinds of haplotypes.Wherein Hap1 and Hap2 are in being lower than and being higher than 50 years old two groups, and distributing has significant difference (P＜0.05).
The detection of coding region SNP, except scanning potential pathogenic mutation site, comprehensively nonsense mutation is as genetic marker, for haplotype analysis provides the basis.
Detected 5 the SNP sites of the present invention all do not have amino acid and change.Be positioned at the SNP on the exon 2 757Belong to multidigit point SNP, have T, C, the conversion between the G three, all the other all are two classical site conversion.Population genetics discovers that the allelotrope that occurrence frequency is maximum among the crowd is the my late grandfather site often [79]Infer SNP in view of the above 1063, SNP 1231, SNP 1456Only contain two kinds of wild homozygous and heterozygous, SNP 1084It is homozygous also to contain sudden change.Explanation is aspect evolution, and this is conservative relatively, and is especially obvious with former three.The crowd evolves to the crowd that illustrates in special rare site and learns, and particularly the location to diseases predisposing gene among many my late grandfathers crowd has vital role [80]
The inventor does not find the SNP site of the above-mentioned type, but as previously mentioned, and each site is except the situation of SNP2 presents a kind of multidigit point variation, and other 4 SNP sites are two traditional site mutation models.This conservative evolution results is, in case sudden change has taken place in some site, just prompting has the intensive dynamics of evolution to act on this fixed group probably, the change on the perhaps this stability can bring certain special phenotype, as disease etc.SNP 1084May belong to this type of.
The Ang2 coding region has detected 5 SNP sites, is distributed on 4 exons.Be the restriction of troubleshoot system errors and general algorithm reckoning ability, we have adopted 5% as the choice boundary of calculating haplotype [85], the haplotype number of the Ang2 that is extrapolated by Gibbs sampler method is 8, far below theoretical maximum (2 5=32).This be because, the first, on exon 4, have two SNP sites, present the height linksystem, weakened the ability that participates in haplotype analysis as independent factor; The second, the present invention has only analyzed the SNP site on the coding region, and from the evolution angle, the coding region keeps relative conservative property, can help the stability of body.Therefore among the present invention except SNP 757Outside the conversion, other all are classical wild/mutational site conversion, have shown its conservative property between three bases.。
In theory, if a basic haplotype is arranged, and sudden change is the unique dynamics of evolution that produces novel site, and the theoretical value of the haplotype of n SNP generation is n+1 so.When the numerical value of actual or reckoning surpassed n+1, there were other dynamicses of evolution in prompting, as reorganization, repeatedly suddenlyd change etc.The detected SNP number of the present invention is 5, and the haplotype number of reckoning is 8, and may there be the combination of above-mentioned several dynamicses of evolution in prompting.In measured 8 haplotypes of this paper, Hap1～Hap4 is that each group crowd is common, accounts for more than 90% of all total amounts, belongs to total haplotype.Hap5～Hap8 only occurs in the part group, and distribution frequency is between 5%～10%, and every group only have 1～2 kind basically, and it is special to illustrate that this type of haplotype belongs to group.x 2Assay prompting, Hap1 and Hap2 have significant difference (P＜0.05) being lower than 50 years old and being higher than 50 years old to distribute among the cerebral apoplexy crowd.
As previously shown, total haplotype is a conservative relatively class on evolving, and when difference occurring between disease-ordinary person, the variation in rare relatively site has more reference value.Therefore, analyze the reason that causes Hap1 and Hap2 distributional difference between two groups, will help to illustrate two groups of isolating trend on evolving, can seek the potential Hazard Factor thus.Evolutionary analysis shows that on genotype, only there is SNP in the ep1b with ep2a belongs to the source of evolving together 1084Difference.This (SNP that conforms to the SNP statistics 1084The A/A genotype be lower than 50 years old and be higher than in two groups of 50 years old cerebral apoplexy patient also have significant difference).This explanation is just because of SNP 1084Other genotype in site have caused a susceptible factor of hemorrhagic apoplexy different ages morbidity to the conversion of A/A.SNP 1084Site A/A genotype can be regarded as a risk factor of different ages morbidity.
In sum, in detected 5 SNP, SNP 1084The A/A variation in site may be the difference basis at hemorrhagic apoplexy different onset age, and haplotype Hap1 and Hap2 distributional difference provide further evidence for it.

Claims (13)

1. be used to differentiate the instrument of the pleomorphism site 1084A/A on the angiogenesis hormone-2.
2. the instrument of claim 1, it is the pleomorphism site 1084A/A polymorphism parting oligonucleotide on the angiogenesis hormone-2, preferably, described polymorphism parting oligonucleotide is: (1) allele-specific nucleic acid primer, it can detect the polymorphism of the pleomorphism site 1084A/A of angiogenesis hormone-2, perhaps (2) are used for detecting the oligonucleotide probe of angiogenesis hormone-2 polymorphism, it can be specifically and the nucleic acid hybridization with the pleomorphism site 1084A/A on the angiogenesis hormone-2, preferably, the length of oligonucleotide probe is 17-50 Nucleotide.
3. according to the instrument of claim 2, it is a restriction enzyme.
4. test kit, it comprises among at least a claim 1-3 each instrument, and, optional, be used for the suitable buffer system and the color development system of detection reaction.
5. diagnose the method for patient's bleeding cerebral apoplexy (comprising hematencephalon or subarachnoid hemorrhage), described method comprises the existence of detection from the pleomorphism site 1084A/A on the angiogenesis hormone-2 in described patient's the sample.
6. determine the method for the susceptibility of patient's bleeding cerebral apoplexy (comprising hematencephalon or subarachnoid hemorrhage), described method comprises the existence of detection from the pleomorphism site 1084A/A on the angiogenesis hormone-2 in described patient's the sample.
7. analyze method, comprising: the Nucleotide of determining the pleomorphism site 1084A/A on the angiogenesis hormone in the described sample-2 from the isolating stripped biological sample of patient.
8. each method among the claim 5-7, 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.
9. each method among the claim 5-7, wherein said sample from the patient is selected from: peripheral blood cells, white corpuscle, serum, urine sample, saliva, body fluid and/or (biopsy) tissue samples, choose wantonly, described sample can carry out purifying in advance, for example separates total DNA.
10. microarray wherein comprises the parting oligonucleotide of the pleomorphism site 1084A/A on the claim 2 defined angiogenesis hormone-2.
11. the microarray of claim 10, it is the form of DNA chip.
The material of the existence of the pleomorphism site 1084A/A on the angiogenesis hormone-2 is in the purposes of preparation diagnostic reagent in the product 12. detection of biological imitates, and described diagnostic reagent is used to diagnose the patient's bleeding cerebral apoplexy genetic predisposition of (comprising hematencephalon or subarachnoid hemorrhage).
13. the purposes of claim 12, wherein said material is selected from:
The instrument of claim 1-3;
The antibody of the sudden change of the pleomorphism site 1084A/A on can specific recognition angiogenesis hormone-2; With
Primer or probe or the restriction enzyme of pleomorphism site 1084A/A on can specific recognition angiogenesis hormone-2.
CN 200610135866 2005-10-17 2006-10-17 Method for determining susceptibility of bleeding-out brain dead in Chinese people by using angiogenin-2 mononucleotide polymorphism sites Pending CN101041855A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 200610135866 CN101041855A (en) 2005-10-17 2006-10-17 Method for determining susceptibility of bleeding-out brain dead in Chinese people by using angiogenin-2 mononucleotide polymorphism sites

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CN200510109373 2005-10-17
CN200510109373.3 2005-10-17
CN 200610135866 CN101041855A (en) 2005-10-17 2006-10-17 Method for determining susceptibility of bleeding-out brain dead in Chinese people by using angiogenin-2 mononucleotide polymorphism sites

Publications (1)

Publication Number Publication Date
CN101041855A true CN101041855A (en) 2007-09-26

Family

ID=38807649

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 200610135866 Pending CN101041855A (en) 2005-10-17 2006-10-17 Method for determining susceptibility of bleeding-out brain dead in Chinese people by using angiogenin-2 mononucleotide polymorphism sites

Country Status (1)

Country Link
CN (1) CN101041855A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111480081A (en) * 2017-12-13 2020-07-31 豪夫迈·罗氏有限公司 Circulating angiopoietin-2 (Ang-2) and insulin-like growth factor binding protein 7(IGFBP7) for prediction of stroke

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111480081A (en) * 2017-12-13 2020-07-31 豪夫迈·罗氏有限公司 Circulating angiopoietin-2 (Ang-2) and insulin-like growth factor binding protein 7(IGFBP7) for prediction of stroke
US11946938B2 (en) 2017-12-13 2024-04-02 Roche Diagnostics Operations, Inc. Circulating Angiopoietin-2 (Ang-2) and insulin-like growth factor-binding protein 7 (IGFBP7) for the prediction of stroke

Similar Documents

Publication Publication Date Title
JP2005130764A (en) Method for examining disease caused by lipid dysbolism using polymorphism on narc-1 gene, and application of the polymorphism for drug design
US10793910B2 (en) Cystic fibrosis gene mutations
AU2005318278A1 (en) Compositions and methods for treating mental disorders
US7732139B2 (en) Multiple SNP for diagnosing cardiovascular disease, microarray and kit comprising the same, and method of diagnosing cardiovascular disease using the same
CN1863928A (en) Diagnostic and therapeutics for osteoporosis
US20080254451A1 (en) Compositions and Methods for Treating Schizophrenia and Related Disorders
US20050233321A1 (en) Identification of novel polymorphic sites in the human mglur8 gene and uses thereof
CN101037708A (en) Usage of homocysteine level prediction by polymorphic loci gene type, method and kit
CN101041855A (en) Method for determining susceptibility of bleeding-out brain dead in Chinese people by using angiogenin-2 mononucleotide polymorphism sites
JP5904501B2 (en) Method for detecting type 2 diabetes
US9165108B2 (en) Probes and methods for determining the presence or absence of genetic segments
CN100338228C (en) Method and reagent for predicting diabetes II susceptibility
WO2012079008A2 (en) Single nucleotide polymorphism biomarkers for diagnosing autism
JP5002746B2 (en) Gene polymorphisms useful for predicting responsiveness to antidepressants
KR101929383B1 (en) Novel gene marker for discriminating the composition of omega fatty acid of pigs and use thereof
CN1847257A (en) Mononucleotide polymorphism related to hypertrophic degree of central muscle of hypertrophic cardiomyopathy and its use
CN1847407A (en) Method of determining ischemic cerebral apoplexy susceptibility with angiogenesis hormone-1 polymorphism
KR20150092937A (en) SNP Markers for hypertension in Korean
US20070243528A1 (en) Methods for detecting polymorphisms using arms or rflp
CN1738907A (en) Process for detecting predisposition to a cardiovascular disease
JPWO2006068111A1 (en) Method for determining phenotype associated with polymorphism of PPARγ gene
KR20040032838A (en) Genetic marker for decreased activity of cytochrome P450 3A7 protein
CN1952173A (en) Method for determining susceptibility of blood vessel disease by using polymorphism of vitamin K epoxide reductase gene mononucleotide
CN1519370A (en) Polymorphism of first exon in TRPC7 gene
CN101063165A (en) Method for predicting action effect of angiotensin conversion enzyme inhibitor medicament

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C02 Deemed withdrawal of patent application after publication (patent law 2001)
WD01 Invention patent application deemed withdrawn after publication

Open date: 20070926