CN101096705A - Usage, method and reagent case for prediction of cardio-cerebrovascular disease occurrence by polymorphism site genetype - Google Patents
Usage, method and reagent case for prediction of cardio-cerebrovascular disease occurrence by polymorphism site genetype Download PDFInfo
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Abstract
The invention discloses a polymorphism site genetype of MTHFR gene to predict individual cardiovascular disease and prognostic application in the medical domain, which detects polymorphism parting oligonucleotide of MTHFR gene and polymorphism site genetype through MTHFR gene to predict the individual cardiovascular disease and prognostic method and agent box.
Description
Technical field
The pleomorphism site genotype that the present invention relates to Methylene tetrahydrofolate reductase (MTHFR) gene is used to predict that cardiovascular and cerebrovascular diseases takes place and the purposes of prognosis.The invention still further relates to a kind of by measuring the pleomorphism site genotype of mthfr gene, the method and the test kit of generation of prediction cardiovascular and cerebrovascular diseases and prognosis.The invention belongs to field of medicaments.
Background technology
Cerebral apoplexy has sickness rate height, recurrence rate height, disability rate height and lethality rate high 4 big characteristics, and methods of treatment is limited, is one of serious disease of harm humans health, so more and more important to the prevention of various Hazard Factor.The Hazard Factor of cerebral apoplexy are divided into controllability and uncontrollability two portions.The controllability Hazard Factor are cerebral apoplexy primary preventions, mainly comprise having hypertension, hyperlipidemia and hyperglycemic patients to carry out dietary adjustments and drug intervention, give up smoking, give up alcohol, to atrial fibrillation and serious carotid artery stenosis patient's treatment etc.Yet a large amount of clinical practice confirms, the patients with cerebral apoplexy due to quite a few atherosclerosis is arranged, and both do not had bad habits such as smoking, excessive drinking before ill, also do not have hypertension, diabetes, HDH.
Most meta analytical resultss show that (Hcy is to bring out important, independent risk factor of cardiovascular and cerebrovascular diseases homosysteine) to hyperhomocysteinemiainjury.The atheromatous lesions of coronary artery and other arteries may more early appear in high Hcy mass formed by blood stasis patient, is the independent hazard factor of ischemic cardio cerebrovascular diseases.Boushey etc. [JAMA, 1995,274 (8): 10492-10571] carry out Information integration to 27 parts of results of study and analyze discovery, and it is relevant with the dangerous increase of fatal and nonfatal atherosclerotic vascular disease that tHcy raises.The importance classes of this danger is similar to other risk factor such as hypercholesterolemia, hypertension, smoking.They also find to be between Hcy level and the blood vessel danger a kind of linear being correlated with step by step, i.e. dose-dependence from analyze.The importance classes of this relation is similar to plasma cholesterol increases 0.15mmol/L.In addition, 19 " high Hcy and the cardiovascular disorder " cooperation plan [JAMA that carry out at the center in Europe, 1997,277 (14): 17752-17811] investigation also confirms, Hcy is a kind of independent risk factor of ischemic cardio cerebrovascular diseases, and thinks that high Hcy mass formed by blood stasis is more valuable than any other risk factor indication atherosclerosis.
Homocysteine itself does not participate in proteinic synthetic as the metabolic intermediate product of methionine(Met) in the human body.Homocysteine mainly carries out metabolism by two approach: the approach that methylates again and commentaries on classics sulphur approach.Approach again methylates: 50% homocysteine being arranged approximately under the effect of methionine synthase, is cofactor with the vitamin B12, is methyl donor with the N5-methyl tetrahydrofolate, takes place to methylate again, and synthetic methionine is participated in the metabolism of body internal protein again.Methyl donor in this reaction (N-5 methyl tetrahydrofolate) is by N-5, and 10-Methylene tetrahydrofolate reductase (MTHFR, methylene tetrahydrofolate reductase) acts on N-5, and the 10-methylene tetrahydrofolate forms.In addition, about 50% homocysteine also can be by changeing the sulphur approach at CBS (CBS, under the catalysis of cystathionine β-synthase), be condensed into Guang sulphur enzyme with Serine, the latter further generates halfcystine and α-ketone butyric acid, this process need pyridoxal phosphate (active vitamin B6) is as cofactor [TIPS, 1990,11:411-416].
The formation of high Hcy mass formed by blood stasis relates to multiple factor, comprise [Cattaneo M.Hyperhomocysteinemia such as heredity, nutrition, medicine and other factors, atherosclerosis and thrombosis[J] .Thromb Haemost, 1999,81:165-176].Influence the interior Plasma Homocysteine of body mainly from inherited genetic factors and trophic factor two aspects.Heredity Hcy mass formed by blood stasis mainly is because due to the active reduction of cystathionine beta synthetic enzyme, methionine synthetase (MS) and Methylene tetrahydrofolate reductase (MTHFR).In the human mthfr gene sudden change that has been found that, majority is the sudden change that can cause serious decline of enzymic activity even enzymic activity disappearance, rare in the crowd, and the transgenation of the 677th Nucleotide (C → T) causes that the thermolability of this enzyme increases, the light moderate of enzymic activity reduces, and makes methionine(Met) cycle penalty, and homocysteine concentration increases [Circulation, 1996,94:3074-3078].[Circulation such as Brattstrom, 1998,98 (23): 2520-2526] 13 Plasma Hcy levels of being carried out in 3 years and research and 23 case control studies of mthfr gene type have been summed up, relate to 5869 routine cardiovascular patients and 6644 routine normal healthy controls persons altogether, find that TT type Plasma Hcy level is significantly higher than the CC type.
Hyperhomocysteinemiainjury is relevant with the complication of multiple disease and disease.Hyperhomocysteinemiainjury can cause blood vessel endothelium injury and dysfunction, and the stimulated vascular smooth muscle cell hyperplasia causes the disorder of vasomotion factor balance, causes pregnancy induced hypertension syndrome (PIH).Therefore, most researchs think that mthfr gene is the main candidate [Chinese public health, 2004,20,6:762-764] of PIH.Think extensively that at present except that the conventional risk factors of cardiovascular and cerebrovascular disease, homocysteine is another strong predictor of atherosclerotic vascular disease.(total homocysteine, tHcy) level raises and can cause coronary heart disease, cerebro-vascular diseases, peripheral vascular disease, arteriovenous thrombotic disease etc. homocysteine total in the blood.The basis shows all that with clinical study tHcy is independently Hazard Factor of cardiovascular and cerebrovascular diseases in recent years.High tHcy mass formed by blood stasis can inspire all important cardiovascular and cerebrovascular diseases and take place, and comprises coronary heart disease, cerebral apoplexy, kidney function damage, peripheral vascular disease etc. [angiocardiology progress, 2000,21:29].A large amount of facts shows that light to moderate tHcy mass formed by blood stasis (tHcy concentration 〉=12 μ mol/L, but≤100 μ mol/L) is the most extensive, the strongest, the Hazard Factor independently of cardiovascular disorder due to the atherosclerosis.Sutton etc. [Circulation, 1997,96:1745] think that blood Hcy level and isolated systolic hypertension are independent relevant.Have report to think, being related of the gene pleiomorphism of Hcy metabolism key enzyme and isolated systolic hypertension is close.MTHFR may be the tumor susceptibility gene of isolated systolic hypertension, and tHcy is the important intermediate phenotype [Chinese cardiovascular diseases magazine, 2003,31:269-273] of isolated systolic hypertension.Lim etc. [Am J Epidemiol, 2002,156:1105-1113] think that high-caliber Hcy can increase the hypertensive danger of generation.Hyperhomocysteinemiainjury (HHcy) is the dead and low independent prediction factor [Arterioscler Thromb Vasc Biol 2005,25:115-121] of left ventricular ejection fraction (LVEF) of hypertensive patient.The tHcy level is higher in the patients with hypertension, may be the important factor that target organ damages such as the heart, brain, kidney more easily take place.
One pregnant before gene warning diagnostic kit and detection method thereof apply for a patent April 2 calendar year 2001, number of patent application is 01107305.5, Granted publication number is CN1155722C.This invention is gene warning diagnostic kit and detection method thereof before pregnant.Said warning gene comprises Methylene tetrahydrofolate reductase gene and methionine synthases reductase gene, its mutational site is respectively C677T and A66G, and corresponding primer sequence is provided respectively, can before pregnant, has diagnosed filial generation whether neural tube defects takes place.
Because cardiovascular and cerebrovascular diseases is serious health risk disease, and cerebral apoplexy has sickness rate height, recurrence rate height, disability rate height and lethality rate high 4 big characteristics especially, and methods of treatment is limited, be one of serious disease of harm humans health, so more and more important to the prevention of various Hazard Factor.For the prevention of cardiovascular and cerebrovascular diseases certain effect has been arranged for the intervention of some known generally acknowledged Hazard Factor such as hypertension, hyperlipidemia and hyperglycemia and treatment, but some patients have still been arranged because other unknown Hazard Factor of existence cause adverse consequences.In order earlier more fully to prevent the especially generation of cerebral apoplexy of cardiovascular and cerebrovascular diseases, improve its prognosis, the clinical Hazard Factor that need to intervene early and treat cardiovascular and cerebrovascular diseases urgently, promptly press for for cardiovascular and cerebrovascular diseases especially cerebral apoplexy and carry out early prediction, the present invention promptly provides a kind of new prediction cardiovascular and cerebrovascular diseases generation and method and the test kit and the purposes of prognosis.
Summary of the invention
The pleomorphism site genotype that the present invention relates to mthfr gene is used to predict the purposes of the generation and the prognosis of individual cardiovascular and cerebrovascular diseases.
In addition, the present invention provides a kind of method and test kit for the generation of individual cardiovascular and cerebrovascular diseases and the prediction of prognosis, i.e. the generation of the individual cardiovascular and cerebrovascular diseases of polymorphism site genotype estimation by measuring mthfr gene and the possibility of prognosis.On the basis of pleomorphism site gene type assay, can predict the possibility of the generation and the prognosis of individual cardiovascular and cerebrovascular diseases, therefore can earlier prevent the generation of cardiovascular and cerebrovascular diseases, improve the prognosis of cardiovascular and cerebrovascular diseases, reduce case fatality rate, alleviate the patient suffering, reduce medical treatment cost.
In purposes of the present invention, method and test kit, the pleomorphism site genotype of described MTHFR comprises the C677T pleomorphism site at least, and the pleomorphism site genotype of described MTHFR can also comprise the pleomorphism site that is selected among A1298C, G1793A, G215A, G482A, the A1317G.In the present invention, the pleomorphism site of described MTHFR can also further comprise the pleomorphism site that has linkage disequilibrium with the said gene pleomorphism site, comprises nonsense mutation site, missense mutation site and the pleomorphism site that is positioned at gene intron position, generegulation position.
In the present invention, described cardiovascular and cerebrovascular diseases comprises acute cardiovascular and cerebrovascular incident, acute cerebrovascular disease, cerebral infarction, hematencephalon, transient cerebral ischemic attack, cerebral apoplexy, cerebral apoplexy, acute cardiovascular disease, coronary heart disease, stenocardia, acute myocardial infarction, cardiogenic shock, sudden cardiac death, be preferably cerebral apoplexy, prognosis refers in particular to death.Individuality is preferably the hypertension individuality.Concrete, when
When (1) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the possibility of the individual generation of prediction cardiovascular and cerebrovascular diseases was bigger, and prognosis is relatively poor;
When (2) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype or 677CT heterozygous, the possibility of the individual generation of prediction cardiovascular and cerebrovascular diseases was less, and prognosis is better;
When (3) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place was bigger, and prognosis is relatively poor;
When (4) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype or 677CT heterozygous, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place was less, and prognosis is better.
Be particularly related to MTHFR pleomorphism site genotype and blood homocysteine synergy in the purposes of the present invention for individual cardiovascular and cerebrovascular diseases generation and prognosis prediction, when being particularly related to the blood homocysteine level that increases in appropriateness, MTHFR pleomorphism site genotype is for the predicting function of cardiovascular and cerebrovascular diseases generation and prognosis, and the synergy of MTHFR pleomorphism site genotype and hypertension prediction cardiovascular and cerebrovascular diseases generation and prognosis, promptly for the hypertension individuality, MTHFR pleomorphism site genotype is more remarkable for the predicting function of cardiovascular and cerebrovascular diseases generation and prognosis, concrete, when
(1) described MTHFR pleomorphism site genotype is a 677TT homozygous mutation type, when occurring together hyperhomocysteinemiainjury, predicts that the possibility of individual cardiovascular and cerebrovascular diseases generation is bigger, and prognosis is relatively poor;
(2) described MTHFR pleomorphism site genotype is 677CC homozygous wildtype or 677CT heterozygous, when occurring together hyperhomocysteinemiainjury, predicts that the possibility of individual cardiovascular and cerebrovascular diseases generation is less, and prognosis is better;
(3) described MTHFR pleomorphism site genotype is a 677TT homozygous mutation type, and when occurring together hyperhomocysteinemiainjury, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place is bigger, and prognosis is relatively poor;
(4) described MTHFR pleomorphism site genotype is 677CC homozygous wildtype or 677CT heterozygous, and when occurring together hyperhomocysteinemiainjury, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place is less, and prognosis is better.
Hyperhomocysteinemiainjury among the present invention refers in particular to blood homocysteine level appropriateness and increases, be blood homocysteine level≤20umol/L in the present invention, perhaps the blood homocysteine level is lower than 90% of the high value of blood homocysteine level among the crowd.
Another aspect of the present invention relates to a kind of genotypic polymorphism parting oligonucleotide of pleomorphism site that is used to measure mthfr gene.Preferably, described polymorphism parting oligonucleotide is: (1) allele-specific nucleic acid primer, it can detect the pleomorphism site genotype of mthfr gene, perhaps (2) are used to detect the genotypic oligonucleotide probe of pleomorphism site of mthfr gene, its can be specifically with mthfr gene on the nucleic acid hybridization of pleomorphism site, the pleomorphism site genotype of wherein said MTHFR comprises at least and is selected from the C677T pleomorphism site, preferably, the length of oligonucleotide probe is 15-50 Nucleotide.
Among the present invention, the pleomorphism site genotype of described MTHFR can also comprise the pleomorphism site that is selected from A1298C, G1793A, G215A, G482A and A1317G, the pleomorphism site that has linkage disequilibrium with the said gene pleomorphism site be can also further comprise, nonsense mutation site, missense mutation site and the pleomorphism site that is positioned at gene intron position, generegulation position comprised.
One side more of the present invention, relate to a kind of individual cardiovascular and cerebrovascular diseases generation of polymorphism site genotype estimation of mthfr gene and method of prognosis utilized, the pleomorphism site genotype of wherein said MTHFR comprises at least and is selected from the C677T pleomorphism site, and described method comprises step: (1) utilizes above-mentioned polymorphism parting oligonucleotide to detect pleomorphism site genotype from mthfr gene described in the biological sample; (2) take place and prognosis according to the individual cardiovascular and cerebrovascular diseases of the polymorphism site genotype estimation of described mthfr gene.
At least comprise in the pleomorphism site genotype of MTHFR described in the method for the present invention and to be selected from the C677T pleomorphism site, can also comprise the pleomorphism site that is selected from A1298C, G1793A, G215A, G482A and A1317G, the pleomorphism site that has linkage disequilibrium with the said gene pleomorphism site be can also further comprise, nonsense mutation site, missense mutation site and the pleomorphism site that is positioned at gene intron position, generegulation position comprised.
In the present invention, described cardiovascular and cerebrovascular diseases comprises acute cardiovascular and cerebrovascular incident, acute cerebrovascular disease, cerebral infarction, hematencephalon, transient cerebral ischemic attack, cerebral apoplexy, cerebral apoplexy, acute cardiovascular disease, coronary heart disease, stenocardia, acute myocardial infarction, cardiogenic shock, sudden cardiac death, be preferably cerebral apoplexy, prognosis refers in particular to death.Concrete, when
When (1) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the possibility of the individual generation of prediction cardiovascular and cerebrovascular diseases was bigger, and prognosis is relatively poor;
When (2) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype or 677CT heterozygous, the possibility of the individual generation of prediction cardiovascular and cerebrovascular diseases was less, and prognosis is better;
When (3) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place was bigger, and prognosis is relatively poor;
When (4) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype or 677CT heterozygous, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place was less, and prognosis is better.
Be particularly related to MTHFR pleomorphism site genotype in the method for the present invention, blood homocysteine and blood folate level are for the synergy of individual cardiovascular and cerebrovascular diseases generation and prognosis prediction, when being particularly related to the blood homocysteine level that increases in appropriateness, MTHFR pleomorphism site genotype is for the predicting function of cardiovascular and cerebrovascular diseases generation and prognosis, and the synergy of MTHFR pleomorphism site genotype and hypertension prediction cardiovascular and cerebrovascular diseases generation and prognosis, promptly for the hypertension individuality, MTHFR pleomorphism site genotype is more remarkable for the predicting function of cardiovascular and cerebrovascular diseases generation and prognosis, concrete, when
(1) described MTHFR pleomorphism site genotype is a 677TT homozygous mutation type, when occurring together hyperhomocysteinemiainjury, predicts that the possibility of individual cardiovascular and cerebrovascular diseases generation is bigger, and prognosis is relatively poor;
(2) described MTHFR pleomorphism site genotype is 677CC homozygous wildtype or 677CT heterozygous, when occurring together hyperhomocysteinemiainjury, predicts that the possibility of individual cardiovascular and cerebrovascular diseases generation is less, and prognosis is better;
(3) described MTHFR pleomorphism site genotype is a 677TT homozygous mutation type, and when occurring together hyperhomocysteinemiainjury, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place is bigger, and prognosis is relatively poor;
(4) described MTHFR pleomorphism site genotype is 677CC homozygous wildtype or 677CT heterozygous, and when occurring together hyperhomocysteinemiainjury, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place is less, and prognosis is better.
Hyperhomocysteinemiainjury among the present invention refers in particular to blood homocysteine level appropriateness and increases, blood homocysteine level appropriateness increases finger blood homocysteine level≤20umol/L in the present invention, perhaps the blood homocysteine level is lower than 90% of the high value of blood homocysteine level among the crowd, promptly, purposes provided by the invention and method are under crowd that blood homocysteine level appropriateness increases or individual situation, prediction effect is best, at the blood homocysteine level (blood homocysteine level＞20umol/L that significantly raises, perhaps the blood homocysteine level be higher than the high value of blood homocysteine level among the crowd 90%) situation under, the effect of prediction reduces (positive predictive value, negative predictive value, susceptibility or specificity increase the crowd and the individuality of scope less than blood homocysteine level appropriateness).
In method of the present invention, can use the following difference foranalysis of nucleic acids technology that is selected from that comprises: polymerase chain reaction (PCR), polymerase chain reaction one restriction fragment length polymorphism, PCR-alleles-specific oligonucleotide probe method, PCR-sequence specific oligonucleoside acid system, sequencing, PCR-sequence specific primers method, the PCR-fluorescent method, the PCR finger-printing method, oligonucleotide connects to be analyzed, the detection method of fluorescent energy resonance transfer, biochip, nucleic acid chip, mass-spectrometric technique, genescan, single strand conformation polymorphism, denaturing gradient gel electrophoresis, enzyme or chemical mispairing patterning method, the Taqman biological detecting method.
PCR, PCR-RFLP, biochip, sequencing, genescan genotype detection method are the conventional methods of using of those skilled in the art.The Taqman technology is a kind of method of using fluorescence technique to carry out real-time quantitative PCR.Biochip is meant methods such as adopting the synthetic or micro-sampling of Hiroshima original position, with the large number of biological macromole such as nucleic acid fragment, peptide molecule even tissue slice, biological samples such as cell solidify in upholder in an orderly manner (as slide, silicon chip, polyacrylamide gel, carriers such as nylon membrane) surface, form intensive two-dimentional molecular arrangement, then with the molecular hybridization that hits of the biological sample to be measured of mark, by specific instrument such as laser confocal scanning instrument or electric charge coupling photographic camera (CCD) intensity of hybridization signal is carried out fast, parallel, check and analysis efficiently, thereby the quantity of target molecule or quality in the judgement sample.Wherein preferred detection method is PCR, PCR-RFLP, Taqman technology, biochip, nucleic acid chip or test kit. the present invention are not to be restriction for detection method for the explanation of the genotypic detection method of pleomorphism site; Any those skilled in the art adopt conventional biological technique method to predict that by detecting pleomorphism site genotype of the present invention individual cardiovascular and cerebrovascular disease takes place and the method for prognosis all belongs to content of the present invention, can further include adopt the difference of conventional biological technique method by detecting the genotypic transcription product of pleomorphism site functional form of the present invention and/or expression product indirectly the pleomorphism site that is associated of reflection predict that individual cardiovascular and cerebrovascular disease in the biological sample takes place and the method for prognosis.
Another aspect of the present invention, also relate to a kind of individual cardiovascular and cerebrovascular diseases generation of polymorphism site genotype estimation of mthfr gene and test kit of prognosis of utilizing, the pleomorphism site genotype of wherein said MTHFR comprises at least and is selected from the C677T pleomorphism site, wherein said cardiovascular and cerebrovascular diseases comprises acute cardiovascular and cerebrovascular incident, acute cerebrovascular disease, cerebral infarction, hematencephalon, transient cerebral ischemic attack, cerebral apoplexy, cerebral apoplexy, acute cardiovascular disease, coronary heart disease, stenocardia, acute myocardial infarction, cardiogenic shock, sudden cardiac death, be preferably cerebral apoplexy, prognosis refers in particular to death, described test kit comprises at least a polymorphism parting oligonucleotide of the present invention, and, choose wantonly, be used for the suitable buffer system and the color development system of detection reaction.Concrete, when
When (1) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the possibility that the prediction cardiovascular and cerebrovascular diseases takes place was bigger, and prognosis is relatively poor;
When (2) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype or 677CT heterozygous, the possibility that the prediction cardiovascular and cerebrovascular diseases takes place was less, and prognosis is better;
When (3) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place was bigger, and prognosis is relatively poor;
When (4) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype or 677CT heterozygous, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place was less, and prognosis is better.
In addition, when
(1) described MTHFR pleomorphism site genotype is a 677TT homozygous mutation type, when occurring together hyperhomocysteinemiainjury, predicts that the possibility of individual cardiovascular and cerebrovascular diseases generation is bigger, and prognosis is relatively poor;
(2) described MTHFR pleomorphism site genotype is 677CC homozygous wildtype or 677CT heterozygous, when occurring together hyperhomocysteinemiainjury, predicts that the possibility of individual cardiovascular and cerebrovascular diseases generation is less, and prognosis is better;
(3) described MTHFR pleomorphism site genotype is a 677TT homozygous mutation type, and when occurring together hyperhomocysteinemiainjury, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place is bigger, and prognosis is relatively poor;
(4) described MTHFR pleomorphism site genotype is 677CC homozygous wildtype or 677CT heterozygous, the hyperhomocysteinemiainjury that occurs together, and the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place is less, and prognosis is better.
Hyperhomocysteinemiainjury among the present invention refers in particular to blood homocysteine level appropriateness and increases, be blood homocysteine level≤20umol/L in the present invention, perhaps the blood homocysteine level is lower than 90% of the high value of blood homocysteine level among the crowd, promptly, test kit provided by the invention is under crowd that blood homocysteine level appropriateness increases or individual situation, prediction effect is best, at the blood homocysteine level (blood homocysteine level＞20umol/L that significantly raises, perhaps the blood homocysteine level be higher than the high value of blood homocysteine level among the crowd 90%) situation under, the effect of prediction reduces (positive predictive value, negative predictive value, susceptibility or specificity increase the crowd and the individuality of scope less than blood homocysteine level appropriateness).
The pleomorphism site genotype of MTHFR can also further comprise the pleomorphism site that is selected among A1298C, G1793A, G215A, G482A and the A1317G in the test kit provided by the invention, the pleomorphism site that has linkage disequilibrium with the said gene pleomorphism site be can also further comprise, nonsense mutation site, missense mutation site and the pleomorphism site that is positioned at gene intron position, generegulation position comprised.The test kit that the present invention mentions comprises the C677T pleomorphism site of measuring MTHFR at least, can further include one or an above site measuring in above-mentioned other pleomorphism sites of MTHFR, also comprise the different permutation and combination of above-mentioned pleomorphism site.
Be selected from the biological sample described in method of the present invention and the test kit: blood sample, humoral sample, tissue sample and culturing cell, preferred, described sample is a blood sample.Wherein blood sample comprises peripheral blood cells, white corpuscle, serum etc., humoral sample comprises urine, saliva, tissue juice, cerebrospinal fluid, body cavity transudate etc., and tissue sample comprises oral mucosa examination son, hair, skin, biopsy, organizes sample secretory product, fecal matter sample etc.
Described test kit is measured the needed specific primer of above-mentioned pleomorphism site except comprising, the conventional assembly of the test kit that also comprises the utilization pcr amplification and detect, reagent, damping fluid etc., perhaps comprise the conventional assembly, reagent, damping fluid of the test kit that methods such as utilization chip, little detection system detect etc., those skilled in the art are familiar with these conventional assembly and detection methods.
Based on mthfr gene,, can design and obtain various diagnostic reagents and test kit to be used to predicting that individual cardiovascular and cerebrovascular diseases takes place and prognosis at its different pleomorphism site.Various diagnostic reagents and test kit based on Forecasting Methodology of the present invention and purposes acquisition also belong to the scope of the invention.
" test kit " among the present invention is not limited to the natural formation of test kit, can show as microchip, little detection system or depend on the detection system of various carriers, and the unitizing form that comprises aforementioned inspection systems, as microwell plate system, paper carrier, glass carrier, nylon membrane carrier, plastic carrier, silica-gel carrier, gel carrier, membranous carrier etc.
The present invention is based on epidemiological study for many years, adopt perspective nido case-control study to detect the pleomorphism site genotype of mthfr gene respectively for the individuality that participates in research, the baseline blood pressure value, blood homocysteine level and the physical signs that some are basic, for the mthfr gene pleomorphism site, hypertension, blood homocysteine level etc. is analyzed the effect of cardiovascular and cerebrovascular incident and prognosis thereof, developed the reagent corresponding box based on researching and analysing the result, and, established purposes and the method among the present invention by the detection validation of test kit.
Human mthfr gene is positioned on the karyomit(e) 1p36.3, gene comprises 11 exons and 10 introns, the length of exon is respectively 99-252bp, intron length is respectively 192-981bp, cDNA total length 2.2kb, a kind of flavoprotein of encoding, its biochemical function is that catalysis is from 5, the 10-methylene tetrahydrofolate is to reduction reaction [the Zhou J of 5-methyl tetrahydrofolate, Kang SS, Wong P WK, et al.Purification and characterization ofmethylenetetrahydrofolate reductase from human cadaver liver.Biochem Med Metab Bio, 1990,43:234-242.].The 5-methyl tetrahydrofolate is a kind of methyl donor, participates in multiple important bioprocess (as synthesizing of purine, pyrimidine).It also with methionine metabolism in homocysteine answer methylate relevant.The pleomorphism site genotype of MTHFR will change the functionally active of MTHFR.The MTHFR enzymic activity reduces, and can cause Hcy to accumulate in vivo, and Hcy is not only relevant with cardiovascular disorder, and has embryotoxicity, may be a kind of teratogenecity material.The MTHFR defective is relevant with the generation of cardiovascular disorder and congenital malformation.
Hyperhomocysteinemiainjury can promote all important cardiovascular and cerebrovascular diseases to take place, and comprises coronary heart disease, cerebral apoplexy, kidney function damage, peripheral arterial disease etc.The tHCY level is higher in the patients with hypertension, may be the important factor that target organ damages such as the heart, brain, kidney more easily take place.
Exon position, intron position and non-coding region position that common single nucleotide polymorphism (SNP) site can be positioned at gene comprise regulator site, are preferably the exon position, especially can change the pleomorphism site of amino acid sequence coded.
The inventor discovers:
The common pleomorphism site of MTHFR shows as but is not limited to following form: C677T, A1298C, G1793A, G215A, G482A, A1317G, is preferably C677T.The pleomorphism site genotype of MTHFR; especially above-mentionedly be positioned at the pleomorphism site that the exon position can have influence on the corresponding encoded aminoacid sequence; usually can have influence on enzyme function and the activity of MTHFR; therefore have influence on the be associated generation of disease of homocysteine level and/or homocysteine indirectly or directly, therefore can further predict the generation and the prognosis of cardiovascular and cerebrovascular diseases.Promptly, can predict the generation and the prognosis of individual cardiovascular and cerebrovascular diseases by measuring the pleomorphism site genotype of MTHFR.Especially, our research has had beyond thought result, MTHFR pleomorphism site genotype, hypertension, blood homocysteine level have synergy for the predicting function of individual cardiovascular and cerebrovascular diseases generation and prognosis, and promptly the factor more than the said two devices is stronger for the predicting function of individual cardiovascular and cerebrovascular diseases generation and prognosis.Be that MTHFR pleomorphism site genotype takes place for individual cardiovascular and cerebrovascular diseases and prognosis has predicting function; When associating hypertension, predicting function strengthens, and is promptly stronger for the effect of its cardiovascular and cerebrovascular diseases generation of the individual prediction of hypertension and prognosis; When uniting hypertension and blood homocysteine level simultaneously, predicting function is stronger, and the three is taken place for the individual cardiovascular and cerebrovascular diseases of prediction jointly and prognosis has synergy.
Meet the qualified research object 2～10 years of studying selected condition by following up a case by regular visits to 30,000 many cases, collect 204 routine object generation cardiovascular and cerebrovascular diseases (comprising death), wherein 148 examples had complete MTHFR C677T loci gene type data, according to 1: 1 coupling sex, age and the regional similar control group of life.After proofreading and correct a plurality of cardiovascular risk factors, carry the risk high 6.4 times (2.3-17.6) of total cardiovascular and cerebrovascular incident generation of the more non-hypertension of the genotypic hyperpietic of MTHFR 677TT, no 677TT genotype patient; The risk high 8.2 times (2.7-24.7) that stroke event takes place; The risk high 5.3 times (1.7-16.5) that the cardiovascular and cerebrovascular mortality ratio takes place.After proofreading and correct Plasma Homocysteine, forecasting risk slightly reduces, but still remarkable.Because Plasma Homocysteine and MTHFR 677TT genotype height correlation, this research has been carried out layer analysis according to the baseline homocysteine level, 1) in having removed each control group after the research object of 90% fen position of Plasma Homocysteine, carry the more non-hypertension of the genotypic hyperpietic of 677TT, do not have the risk high 14.5 times (2.8-75.3) that 677TT genotype patient's total cardiovascular and cerebrovascular incident takes place, the risk high 15.5 times (2.8-86.0) that stroke event takes place, mortality ratio risk high 26.8 times (3.0-236.3).2) removed the object of Plasma Homocysteine＞20 μ mol/L after, carry the more non-hypertension of the genotypic hyperpietic of 677TT, do not have the risk high 15.0 times (2.9-77.7) that 677TT genotype patient's total cardiovascular and cerebrovascular incident takes place, the risk high 16.0 times (2.9-88.6) that stroke event takes place, mortality ratio risk high 20.2 times (2.2-187.0).
The present invention also provides and has utilized functional gene to predict that individual cardiovascular and cerebrovascular diseases takes place and the method and the test kit of prognosis, can be used as the indication mechanism of the possibility of cardiovascular and cerebrovascular diseases generation and prognosis, promptly be used to predict the generation and the prognosis of cardiovascular and cerebrovascular diseases by the pleomorphism site genotype of measuring mthfr gene.On the basis of pleomorphism site gene type assay, can further predict cardiovascular and cerebrovascular diseases better in conjunction with blood pressure level, blood homocysteine level etc., can therefore earlier predict the generation and the prognosis of cardiovascular and cerebrovascular diseases, prevent and intervene the process of cardiovascular and cerebrovascular diseases better, reduce medical treatment cost.
In the content of the present invention, we have designed the primer sequence that is used to measure pleomorphism site genotype site especially, and according to this primer sequence and target sequence characteristics determined the amplification efficiency height, specificity is good and the timesaving detection method, make things convenient for those of ordinary skill in the art to grasp and use, good practical value is arranged.
Particularly the C677T pleomorphism site genotype at MTHFR has designed the used pcr amplification primer of following detection, compares with the amplimer of routine, and the amplification efficiency height, specificity is good and save time, and better use value is arranged.The genotypic specific PCR amplimer of the C677T pleomorphism site of MTHFR is as follows, and the fragment length that amplification obtains is 274bp:
Forward primer: 5 '-CTT TGA GGC TGA CCT GAA GC-3 '
Reverse primer: 5 '-CTG GGA AGA ACT CAG CGA AC-3 '
Embodiment
Embodiment 1: measure C677T (Ala222Val, dsSNP ID:rs1801133) the pleomorphism site genotype of mthfr gene and predict that cardiovascular and cerebrovascular diseases takes place and prognosis
(1) the C677T pleomorphism site genotype of mensuration mthfr gene
(1) genomic dna of extraction host cell
(a) add the 30ml erythrocyte cracked liquid in whole blood, slowly shake up, room temperature left standstill 10 minutes, during, shake for several times, thoroughly splitting erythrocyte;
(b) in 4 ℃, 2000 rev/mins centrifugal 10 minutes, remove supernatant, the white corpuscle that will precipitate is broken up on the oscillator in rotation, adds proteolytic enzyme 40 μ l, RNA enzyme 50 μ l, shakes up, and adds write cell lysis buffer and puts 15ml, 37 ℃ of water-baths of mixing were taken out after 20 minutes, put in the cold water;
(c) add cold albumen precipitation liquid 4ml, be placed on-20 ℃ of refrigerators 5 minutes behind the mixing, take out in 4 ℃, 3000 rev/mins centrifugal 10 minutes, supernatant liquor poured into slowly shakes in the 50ml centrifuge tube that has added the 15ml Virahol for several times, separate out to the DNA floss;
(d) the DNA floss of separating out is moved in another 1.5ml centrifuge tube, after 75% ethanol 1ml washes the DNA floss, drying at room temperature;
(e) add DNA hydrating fluid 1.0ml, put shaking table, shaken over night, standby;
(f) mensuration of DNA concentration adopts ultraviolet spectrophotometry, measures the OD value under two wavelength of 260nm and 280nm respectively, is DNA concentration with OD260nm * 50 income values.And with OD260nm/OD280nm ratio estimation DNA purity.
(2) use PCR and restriction fragment length polymorphism analysis method (PCR-RFLP) to detect the MTHFRC677T pleomorphism site
According to MTHFR C677T gene order design PCR Auele Specific Primer, comprise PCR forward primer and PCR reverse primer, carry out conventional pcr amplification by following condition.
Primer sequence:
Forward primer: 5 '-CTT TGA GGC TGA CCT GAA GC-3 '
Reverse primer: 5 '-CTG GGA AGA ACT CAG CGA AC-3 '
The PCR reaction system:
Genomic dna 45ng, upstream and downstream primer 10pmol (20 μ mol/L), dNTPs 2.0mmol/l, 10 * buffer1.0 μ l, Gold Taq archaeal dna polymerase 3U, ddH2O supplies cumulative volume to 10 μ l.
The PCR reaction conditions:
Behind 95 ℃ of pre-sex change 10min: 94 ℃ of sex change 30sec, 59 ℃ of annealing 45sec, 68 ℃ are extended 45sec, 35 loop cycles: last 68 ℃ are extended 7min; Obtain the amplified fragments of 274 bp.
Enzyme tangent condition and system (15 μ l):
MTHFR C677T site PCR product purpose fragment length is 245bp, and total enzyme system of cutting is 15 μ l, PCR product 10 μ l wherein, and 10 * NEBuffer#2,1.5 μ l, HinfI restriction endonuclease 4U (0.4 μ l) and 3.1 μ l ddH2O, 37 ℃ are spent the night.
HinfI restriction endonuclease recognition site is:
Genotype result judges:
Product point sample after the DNA enzyme cut is on 2.5% agarose gel, and electrophoresis read glue figure and carries out gene type assay after 1 hour under the 200V voltage under ultraviolet lamp.Idiotype is identified as follows:
Endonuclease bamhi is 274bp, and the mthfr gene type is 677CC;
Endonuclease bamhi is 274+228+46bp, and the mthfr gene type is 677CT;
Endonuclease bamhi is 228+46bp, and the mthfr gene type is 677TT.
(2) prediction of cardiovascular and cerebrovascular diseases generation and prognosis
When MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the possibility that the prediction cardiovascular and cerebrovascular diseases takes place was bigger, and prognosis is relatively poor;
When MTHFR pleomorphism site genotype was 677CC homozygous wildtype or 677CT heterozygous, the possibility that the prediction cardiovascular and cerebrovascular diseases takes place was less, and prognosis is better;
When MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place was bigger, and prognosis is relatively poor;
When MTHFR pleomorphism site genotype was 677CC homozygous wildtype or 677CT heterozygous, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place was less, and prognosis is better;
MTHFR pleomorphism site genotype is a 677TT homozygous mutation type, and the hyperhomocysteinemiainjury that occurs together predicts that the possibility of individual cardiovascular and cerebrovascular diseases generation is bigger, and prognosis is relatively poor;
MTHFR pleomorphism site genotype is 677CC homozygous wildtype or 677CT heterozygous, and the hyperhomocysteinemiainjury that occurs together predicts that the possibility of individual cardiovascular and cerebrovascular diseases generation is less, and prognosis is better;
MTHFR pleomorphism site genotype is a 677TT homozygous mutation type, the hyperhomocysteinemiainjury that occurs together, and the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place is bigger, and prognosis is relatively poor;
MTHFR pleomorphism site genotype is 677CC homozygous wildtype or 677CT heterozygous, the hyperhomocysteinemiainjury that occurs together, and the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place is less, and prognosis is better.
Above method is verified by epidemiological study, we adopt perspective nido case one contrast research, by following up a case by regular visits to selected qualified research object 39,165 examples (on average following up a case by regular visits to 6.2 years), collect 204 routine object generation cardiovascular and cerebrovascular diseases (comprising death), wherein 148 examples had complete MTHFR C677T loci gene type data, according to 1: 1 coupling sex, age and the regional similar control group of life.
Case can be divided into total cardiovascular and cerebrovascular diseases, cerebral apoplexy and dead three groups in the perspective nested case-control study, has carried out sex, age, life area coupling in the control group.Totally 148 pairs of total cardiovascular and cerebrovascular diseases/control groups, totally 133 pairs of cerebral apoplexy/control groups, death/control group totally 102 pairs have intermediate phenotype data such as complete genotype, Plasma Homocysteine, blood pressure.Measure blood pressure, homocysteine level, blood sugar, blood fat respectively and other are as physiological parameters such as sex, age, height, body weight, obtain individual MTHFR C677T pleomorphism site genotype data simultaneously, analyze the relation between different genotype, blood pressure, homocysteine level and cardiovascular and cerebrovascular diseases generation and the prognosis.Three case/control group crowds' baseline characteristic comprises age, sex, height, body weight, weight index (BMI), smoking, drink etc. in different groups, distribute similar substantially, no significant difference.
As shown in table 1, MTHFR 677TT genotype distributes to have in total cardiovascular and cerebrovascular diseases, cerebral apoplexy, dead group and increases trend gradually, and difference distribution is more obvious under recessive model instructs especially, and credit is analysed the result there was a significant difference by statistics.Analytical results shows, MTHFR C677T pleomorphism site genotype has predicting function for total cardiovascular and cerebrovascular diseases, cerebral apoplexy and death thereof, be that MTHFR pleomorphism site genotype is when being 677TT homozygous mutation type, the possibility that prediction cardiovascular and cerebrovascular diseases, cerebral apoplexy take place is bigger, dead possibility is bigger, and prognosis is relatively poor; When MTHFR pleomorphism site genotype was 677CC homozygous wildtype or 677CT heterozygous, the possibility that prediction cardiovascular and cerebrovascular diseases, cerebral apoplexy take place was less, and dead possibility is less, and prognosis is better.
The relation of table 1.MTHFR C677T pleomorphism site genotype and cardiovascular and cerebrovascular diseases and prognosis
| Variable name | Total cardiovascular and cerebrovascular incident case-control group | Cerebral apoplexy case-control group | Cardiovascular and cerebrovascular incident death control group | |||
| Control group | The case group | Control group | The case group | Control group | The case group | |
| The recessive MODEL C C+CT of example number MTHFR C677T CC CT TT TT | 148 N(％) 55(37.2) 76(51.4) 17(11.5) 131(88.5) 17(11.5) | 148 N(％) 50(33.8) 72(48.6) 26(17.6) 122(82.4) 26(17.6) | 133 N(％) 49(36.8) 68(51.1) 16(12.0) 117(88.0) 16(12.0) | 133 N(％) 45(33.8) 65(48.9) 23(17.3) 110(82.7) 23(17.3) | 102 N(％) 34(37.8) 46(51.1) 10(11.1) 80(88.9) 10(11.1) | 102 N(％) 29(32.2) 43(47.8) 18(20.0) 72(80.0) 18(20.0) |
Annotate: CC, represent MTHFR 677CC homozygous wildtype; TT represents MTHFR 677TT homozygous mutation type; CT represents MTHFR 677CT heterozygous
We analyze the relation of homocysteine level and cardiovascular and cerebrovascular diseases, and are as shown in table 2, and in total cardiovascular and cerebrovascular diseases group, the baseline Plasma Homocysteine is 18.1 ± 13.0 μ mol/L, and significance is higher than control group (14.9 ± 6.8 μ mol/L); Homocysteine level after denary logarithm transforms distributes in two groups and highly significant difference is still arranged (2.8 ± 0.4 vs, 2.6 ± 0.3 μ mol/L).The Plasma Homocysteine of same significant difference is also observed (cerebral apoplexy/control group: 18.2 ± 13.6 vs 15.2 ± 7.0 in other two groups; Die of illness/control group: 19.1 ± 14.2 vs 15.4 ± 7.9).The results of univariate logistic analysis prompting, homocysteine are total cardiovascular and cerebrovascular incident, the risk of stroke event generation and the significant risk factor of mortality ratio thereof.Be that independent blood homocysteine level can predict that cardiovascular and cerebrovascular diseases takes place and prognosis, the possibility that cardiovascular and cerebrovascular diseases takes place increases along with the rising of blood homocysteine level, and its prognosis is along with the increase of blood homocysteine level is poorer, and dead risk increases and increases along with the blood homocysteine level.
Simultaneously, our result of study shows, the ill ratio of hypertension distributes in case/contrast separately also marked difference, and the result is as shown in table 2, hyperpietic's proportion in total cardiovascular and cerebrovascular diseases group and control group is respectively 79.1% and 50.0%, and credit analyses that there was a significant difference by statistics; Proportion is 82.0% and 48.9% in cerebral apoplexy group and control group, and credit analyses that there was a significant difference by statistics; Proportion is 74.4% and 47.8% in dead group of cardiovascular and cerebrovascular diseases and control group, credit analyses that there was a significant difference by statistics, The results of univariate logistic analysis prompting hypertension is the risk that takes place of total cardiovascular and cerebrovascular diseases, cerebral apoplexy and because of the significant risk factor of cardiovascular and cerebrovascular diseases, cerebral apoplexy death, and promptly independent blood pressure situation and blood pressure level can be predicted the generation and the prognosis of cardiovascular and cerebrovascular diseases.
Crowd's essential characteristic in the total cardiovascular event of table 2., cerebral apoplexy, dead group and the control group thereof
| Variable name | Total cardiovascular and cerebrovascular diseases | Cerebral apoplexy | Dead | |||
| Control group | The case group | Control group | The case group | Control group | The case group | |
| Example number blood plasma tHcy μ mol/L logarithm transforms tHcy, and mol/L sex man hypertension is | 148 Mean±SD 14.9±6.8 2.6±0.3 N(％) 107(72.3) 74(50.0) | 148 Mean±SD 18.1±13.0 ** 2.8±0.5 ** N(％) 107(72.3) 117(79.1) ** | 133 Mean±SD 15.2±7.0 2.7±0.3 N(％) 96(72.2) 65(48.9) | 133 Mean±SD 18.2±13.6 * 2.8±0.5 * N(％) 96(72.2) 109(82.0) ** | 102 Mean±SD 15.4±7.9 2.7±0.4 N(％) 67(74.4) 43(47.8) | 102 Mean±SD 19.1±14.2 * 2.8±0.5 ** N(％) 67(74.4) 67(74.4) ** |
Table is annotated:
*P＜0.01,
*P＜0.05
Analyze homocysteine level for cardiovascular and cerebrovascular diseases and stroke onset influence and dead relation, analytical results shows that homocysteine level can predict cardiovascular and cerebrovascular diseases and cerebral apoplexy and dead generation.The result is as shown in table 3, at whole cardiovascular and cerebrovascular diseases case group/control groups, whole cerebral apoplexy case group/control groups, in cardiovascular and cerebrovascular diseases death group/control group and four sample population of cerebral apoplexy death group/control group, the morbidity ratio of 10≤Hcy in each sample population＜20 μ mol/L group and Hcy 〉=20 μ mol/L group all is significantly higher than Hcy＜10 μ mol/L group and (has proofreaied and correct the age, sex, BMI, smoking, drink, hyperlipemia, factor such as impaired fasting glucose and hypertension), credit analyses that there was a significant difference by statistics, and, cardiovascular and cerebrovascular diseases, cerebral apoplexy and dead onset risk increase along with increasing of Hcy level.Trend Test analytical results further confirms, the possibility of the generation of the high more cardiovascular and cerebrovascular diseases of Hcy level, cerebral apoplexy and death is big more, promptly along with the increase of homocysteine level, the possibility that cardiovascular and cerebrovascular diseases, cerebral apoplexy take place increases, because of the possibility increase of cardiovascular and cerebrovascular diseases death or cerebral apoplexy death.
Table 3. homocysteine level is to the influence (adopting the Logistic regression analysis) of stroke onset
| Hcy level (μ mol/L) | Total routine number (N) | Case group N (%) | After the correction * | ||
| OR | 95％CI | P | |||
| Whole cardiovascular and cerebrovascular diseases case groups/control group sample | |||||
| Hcy＜10 10≤Hcy＜20 Hcy≥20 Trend Test | 42 282 54 --- | 13(31.0) 141(50.0) 35(64.8) --- | 1.0 2.2 3.7 1.9 | --- 1.0-4.6 1.4-9.4 1.2-3.0 | --- 0.039 0.006 0.007 |
| Whole cerebral apoplexy case groups/control group sample | |||||
| Hcy＜10 10≤Hcy＜20 Hcy≥20 Trend Test | 35 248 49 --- | 12(34.3) 122(49.2) 32(65.3) --- | 1.0 1.9 4.1 1.8 | --- 0.9-4.1 1.5-10.7 1.1-2.9 | --- 0.094 0.005 0.030 |
| Cardiovascular and cerebrovascular diseases death group/control group sample | |||||
| Hcy＜10 10≤Hcy＜20 Hcy≥20 Trend Test | 33 188 47 --- | 7(21.2) 97(51.6) 30(63.8) --- | 1.0 3.9 5.7 2.2 | --- 1.5-9.8 1.9-17.5 1.3-3.8 | --- 0.004 0.002 0.004 |
| Cerebral apoplexy death group/control group sample | |||||
| Hcy＜10 10≤Hcy＜20 Hcy≥20 Trend Test | 26 159 43 --- | 6(23.1) 80(50.3) 28(65.1) --- | 1.0 3.3 5.1 2.1 | --- 1.2-9.2 1.5-17.2 1.2-3.7 | --- 0.021 0.010 0.014 |
*Correction factor comprises, age, sex, BMI, smoking, drink, blood fat, blood sugar, blood pressure.
Further analysis revealed, hypertension and homocysteine level have combined action to cardiovascular and cerebrovascular incident and dead prediction thereof.Analytical results sees Table 4, analysis to above-mentioned four samples, the cardiovascular and cerebrovascular diseases patient ratio that all shows the high Hcy of hypertensive patients group is significantly higher than the normal Hcy group of normal arterial pressure (proofreaied and correct age, sex, BMI, smoking, drink, factors such as blood fat and fasting plasma glucose), the OR value is organized both OR value sums greater than high Hcy group of normal arterial pressure and the normal Hcy of hypertension, and prompting hypertension and homocysteine level are to the combined action that has of cardiovascular and cerebrovascular incident.
Table 4. hypertension and homocysteine level are to the combined action of cardiovascular and cerebrovascular incident
| Blood pressure | The Hcy level | Total routine number (N) | Case group N (%) | After the correction * | ||
| OR | 95％CI | P | ||||
| Whole cardiovascular and cerebrovascular diseases case groups/control group sample | ||||||
| Normal hypertension hypertension | Normal high normal high | 21 110 21 226 | 3(14.3) 38(34.6) 10(47.6) 138(61.1) | 1.0 3.3 4.9 9.5 | --- 0.9-11.9 1.1-22.0 2.7-33.7 | --- 0.071 0.040 ＜0.001 |
| Whole cerebral apoplexy case groups/control group sample | ||||||
| Normal hypertension hypertension | Normal high normal high | 17 98 18 199 | 2(11.8) 31(31.6) 10(55.6) 123(61.8) | 1.0 3.6 8.2 12.1 | --- 0.8-16.8 1.4-48.3 2.6-55.6 | --- 0.106 0.020 0.001 |
| Cardiovascular and cerebrovascular diseases death group/control group sample | ||||||
| Normal hypertension hypertension | Normal high normal high | 19 80 14 155 | 3(15.8) 29(36.3) 4(28.6) 98(63.2) | 1.0 3.2 1.9 9.4 | --- 0.9-12.1 0.3-10.6 2.6-34.4 | --- 0.085 0.456 ＜0.001 |
| Cerebral apoplexy death group/control group sample | ||||||
| Normal hypertension hypertension | Normal high normal high | 15 71 11 131 | 2(13.3) 24(33.8) 4(36.4) 84(64.1) | 1.0 3.5 3.3 11.8 | --- 0.7，17.0 0.5，23.1 2.5，55.4 | --- 0.119 0.232 0.002 |
Table is annotated:
*Correction factor comprises, age, sex, BMI, smoking, drink, hyperlipemia and impaired fasting glucose;
Blood pressure: SBP 〉=140mmHg or DBP 〉=90mmHg are defined as hypertension, otherwise are defined as normal arterial pressure;
The horizontal Hcy of Hcy＜0 μ mol/L is defined as normal Hcy level, and Hcy 〉=10 μ mol/L are defined as high Hcy level.
A plurality of risk factors take place the cardiovascular and cerebrovascular incident and the risk of mortality ratio in order further to understand, and the different polynary Logistic regression models of table 5 employing have been assessed the combined action between hypertension and MTHFR C677T pleomorphism site genotype.A plurality of risk factor conditions such as model I further proofreaies and correct age, sex, BMI, smoking, drinks, blood fat, blood sugar, the result shows, the risk high 6.4 times (2.3-17.6) that carry the more non-hypertension of the genotypic hyperpietic of MTHFR 677TT, total cardiovascular and cerebrovascular diseases of (promptly carry MTHFR 677CC genotype or the carry MTHFR 677CT genotype) patient that do not have the 677TT genotype takes place, there was a significant difference for statistical analysis; After model II further proofreaies and correct Plasma Homocysteine, still high (the OR=4.6 of risk that carry the more non-hypertension of the genotypic hyperpietic of MTHFR 677TT, total cardiovascular and cerebrovascular incident of (promptly carry MTHFR 677CC genotype or the carry MTHFR 677CT genotype) patient that do not have the 677TT genotype takes place, 1.5-13.8), there was a significant difference for statistical analysis.In cerebral apoplexy risk assessment, find: adopt model I to proofread and correct under a plurality of risk factor conditions, carry the more non-hypertension of the genotypic hyperpietic of 677TT, do not have the risk high 8.2 times (2.7-24.7) that 677TT genotype patient's stroke event takes place; After model II further proofreaies and correct Plasma Homocysteine, carry the more non-hypertension of the genotypic hyperpietic of 677TT, do not have 677TT genotype patient and compare, (OR=7.0,2.1-23.5), there was a significant difference for statistical analysis still to have higher cerebral apoplexy risk.In the risk assessment of cardiovascular and cerebrovascular diseases death, find equally: adopt model I to proofread and correct under a plurality of risk factor conditions, carry the more non-hypertension of the genotypic hyperpietic of 677TT, do not have the risk high 5.3 times (1.7-16.5) that 677TT genotype mortality in said patients takes place; After model II further proofreaies and correct Plasma Homocysteine, carry the genotypic hyperpietic of MTHFR 677TT still have higher mortality risk (OR=3.4,1.0-11.6).The result shows, MTHFR C677T pleomorphism site genotype and hypertension have independent effect and synergy for the influence of the generation of cardiovascular and cerebrovascular diseases, cerebral apoplexy and cerebral apoplexy death, the acting in conjunction of the two is greater than independent effect, be that MTHFR pleomorphism site genotype is when being 677TT homozygous mutation type, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension, cerebral apoplexy take place is bigger, dead possibility is bigger, and prognosis is relatively poor; When MTHFR pleomorphism site genotype was 677CC homozygous wildtype or 677CT heterozygous, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension, cerebral apoplexy take place was less, and dead possibility is less, and prognosis is better.
Table 5.Logistic regression model assessment MTHFR C677T pleomorphism site
With the combined action of hypertension to total cardiovascular event, cerebral apoplexy and mortality risk thereof
| Blood pressure 1 | MTHFR C677T 4 | Total routine number N | Case load N | Case per-cent % | Proofread and correct 2 | Proofread and correct 3 | ||||
| OR | 95％CI | P | OR | 95％CI | P | |||||
| Total cardiovascular and cerebrovascular incident case-control sample | ||||||||||
| Normal hypertension hypertension | 677CC or 677CT 677TT 677CC or 677CT 677TT | 92 13 161 30 | 27 4 95 22 | 29.3 30.8 59.0 73.3 | 1.0 0.9 3.3 6.4 | 0.3-3.5 1.8-6.2 2.3-17.6 | 0.938 ＜0.001 ＜0.001 | 1.0 0.7 3.1 4.6 | 0.2-2.9 1.7-5.9 1.5-13.8 | 0.622 ＜0.001 0.007 |
| Cerebral apoplexy case-control sample | ||||||||||
| Normal hypertension hypertension | 677CC or 677CT 677TT 677CC or 677CT 677TT | 80 12 147 27 | 21 3 89 20 | 26.3 25.0 60.5 74.1 | 1.0 0.8 4.3 8.2 | 0.2-3.6 2.2-8.6 2.7-24.7 | 0.820 ＜0.001 ＜0.001 | 1.0 0.7 4.2 7.0 | 0.2-3.4 2.1-8.5 2.1-23.5 | 0.702 ＜0.001 0.002 |
| Cardiovascular and cerebrovascular incident death check sample | ||||||||||
| Normal hypertension hypertension | 677CC or 677CT 677TT 677CC or 677CT 677TT | 69 8 103 24 | 22 3 60 17 | 31.9 37.5 58.3 70.8 | 1.0 1.1 2.8 5.3 | 0.2-5.6 1.4-5.8 1.7-16.5 | 0.889 0.005 0.004 | 1.0 0.7 2.6 3.4 | 0.1-4.2 1.2-5.3 1.0-11.6 | 0.742 0.012 0.055 |
Table is annotated: 1 blood pressure: SBP 〉=140mmHg or DBP 〉=90mmHg are defined as hypertension, otherwise are defined as normal arterial pressure:
2 correction factors comprise: age, sex, BMI, smoking, drink, blood fat, blood sugar;
3 correction factors comprise: age, sex, BMI, smoking, drink, blood fat, blood sugar and logarithm transform back tHcy;
4 CC represent MTHFR 677CC homozygous wildtype; TT represents MTHFR 677TT homozygous mutation type; CT represents the MTHFR677CT heterozygous
Further layer analysis, in having removed each control group after the research object of 90% fen position of Plasma Homocysteine, proofread and correct age, sex, BMI, smoking, drink, under the blood fat, a plurality of risk factor conditions of blood sugar, adopt Logistic regression model assessment MTHFR C677T pleomorphism site and hypertension interaction total cardiovascular disorder, cerebral apoplexy and mortality risk thereof.The result is as shown in table 6, the risk high 14.5 times (2.8-75.3) that carry the more non-hypertension of the genotypic hyperpietic of 677TT, total cardiovascular and cerebrovascular diseases of (promptly carry MTHFR 677CC genotype or the carry MTHFR 677CT genotype) patient that do not have the 677TT genotype takes place, the risk high 15.5 times (2.8-86.0) that cerebral apoplexy takes place, mortality risk high 26.8 times (3.0-236.3), significance has been analysed in credit by statistics.After further proofreading and correct Plasma Homocysteine, carry the more non-hypertension of the genotypic hyperpietic of 677TT, (promptly carry MTHFR 677CC genotype or the carry MTHFR 677CT genotype) patient that do not have the 677TT genotype still has the risk (OR=16.3 that higher total cardiovascular and cerebrovascular incident takes place, 3.1-86.7), risk (the OR=18.5 that higher stroke event takes place, 3.2-105.3), risk (the OR=25.6 that higher mortality ratio takes place, 2.8-234.2), credit analyses that there was a significant difference by statistics.
The result is as shown in table 6, after having removed the object of Plasma Homocysteine＞20 μ mol/L, proofread and correct under a plurality of risk factor conditions, the risk high 15.0 times (2.9-77.7) that carry the more non-hypertension of the genotypic hyperpietic of 677TT, total cardiovascular and cerebrovascular diseases of (promptly carry MTHFR 677CC genotype or the carry MTHFR 677CT genotype) patient that do not have the 677TT genotype takes place, the risk high 16.0 times (2.9-88.6) that cerebral apoplexy takes place, dead risk high 20.2 times (2.2-187.0), credit analyses that there was a significant difference by statistics.After further proofreading and correct Plasma Homocysteine, carry the more non-hypertension of the genotypic hyperpietic of 677TT, (promptly carry MTHFR 677CC genotype or the carry MTHFR 677CT genotype) patient that do not have the 677TT genotype still has the risk (OR=16.7 that higher total cardiovascular and cerebrovascular incident takes place, 3.2-88.3), risk (the OR=19.1 that higher stroke event takes place, 3.4-108.9), higher mortality risk (OR=20.7,2.2-196.3), credit analyses that there was a significant difference by statistics.
Hyperhomocysteinemiainjury among the present invention refers in particular to blood homocysteine level appropriateness and increases, be blood homocysteine level≤20umol/L in the present embodiment, perhaps the blood homocysteine level is lower than 90% of the high value of blood homocysteine level among the crowd.The result shows under appropriate homocysteine level, MTHFR C677T pleomorphism site and hypertension have independent action and interaction to total cardiovascular disorder, cerebral apoplexy and mortality risk thereof, be that MTHFR pleomorphism site genotype is a 677TT homozygous mutation type, hyperhomocysteinemiainjury occurs together, the possibility of predicting individual cardiovascular and cerebrovascular diseases, cerebral apoplexy generation is bigger, dead possibility is bigger, and prognosis is relatively poor; MTHFR pleomorphism site genotype is 677CC homozygous wildtype or 677CT heterozygous, and the hyperhomocysteinemiainjury that occurs together predicts that the possibility of individual cardiovascular and cerebrovascular diseases, cerebral apoplexy generation is less, and dead possibility is less, and prognosis is better; MTHFR pleomorphism site genotype is a 677TT homozygous mutation type, the hyperhomocysteinemiainjury that occurs together, and the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place is bigger, and dead possibility is bigger, and prognosis is relatively poor; MTHFR pleomorphism site genotype is 677CC homozygous wildtype or 677CT heterozygous, the hyperhomocysteinemiainjury that occurs together, and the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place is less, and dead possibility is less, and prognosis is better.
Table 6.MTHFR C677T pleomorphism site and hypertension
Interaction to total cardiovascular disorder, cerebral apoplexy and mortality risk thereof
| Blood pressure 1 | MTHFR C677T 4 | Total routine number N | Case load N | Case per-cent % | Proofread and correct 2 | Proofread and correct 3 | ||||
| OR | 95％CI | P | OR | 95％CI | P | |||||
| Appropriateness homocysteine level (being lower than each control group tHcy 90%) | ||||||||||
| Total cardiovascular and cerebrovascular incident case-control sample | ||||||||||
| Normally | 677CC or 677CT | 86 | 23 | 26.7 | 1.0 | 1.0 | ||||
| Normally | 677TT | 9 | 2 | 22.2 | 0.8 | 0.1-4.3 | 0.795 | 0.9 | 0.2-5.2 | 0.935 |
| Hypertension | 677CC or 677CT | 142 | 80 | 56.3 | 3.3 | 1.7-6.5 | ＜0.001 | 3.5 | 1.8-7.1 | ＜0.001 |
| Hypertension | 677TT | 13 | 11 | 84.6 | 14.5 | 2.8-75.3 | 0.001 | 16.3 | 3.1-86.7 | 0.001 |
| Cerebral apoplexy case-control sample | ||||||||||
| Normally | 677CC or 677CT | 75 | 18 | 24.0 | 1.0 | 1.0 | ||||
| Normally | 677TT | 9 | 2 | 22.2 | 0.9 | 0.2-5.1 | 0.928 | 1.2 | 0.2-6.9 | 0.839 |
| Hypertension | 677CC or 677CT | 130 | 76 | 58.5 | 4.6 | 2.1-9.7 | ＜0.001 | 5.2 | 2.4-11.4 | ＜0.001 |
| Hypertension | 677TT | 11 | 9 | 81.8 | 15.5 | 2.8-86.0 | 0.002 | 18.5 | 3.2-105.3 | 0.001 |
| Cardiovascular and cerebrovascular incident death check sample | ||||||||||
| Normally | 677CC or 677CT | 65 | 19 | 29.2 | 1.0 | 1.0 | ||||
| Normally | 677TT | 6 | 2 | 33.3 | 1.2 | 0.2-8.0 | 0.839 | 1.2 | 0.2-7.9 | 0.863 |
| Hypertension | 677CC or 677CT | 89 | 49 | 55.1 | 2.8 | 1.3-6.1 | 0.010 | 2.7 | 1.2-6.1 | 0.014 |
| Hypertension | 677TT | 11 | 10 | 90.9 | 26.8 | 3.0-236.3 | 0.003 | 25.6 | 2.8-234.2 | 0.004 |
| The appropriateness homocysteine level (≤20umol/L) | ||||||||||
| Total cardiovascular and cerebrovascular incident case-control sample | ||||||||||
| Normally | 677CC or 677CT | 87 | 23 | 26.4 | 1.0 | 1.0 | ||||
| Normally | 677TT | 9 | 2 | 22.2 | 0.8 | 0.2-4.4 | 0.809 | 0.9 | 0.2-5.2 | 0.939 |
| Hypertension | 677CC or 677CT | 143 | 81 | 56.6 | 3.5 | 1.8-6.8 | ＜0.001 | 3.7 | 1.9-7.4 | ＜0.001 |
| Hypertension | 677TT | 13 | 11 | 84.6 | 15.0 | 2.9-77.7 | 0.001 | 16.7 | 3.2-88.3 | ＜0.001 |
| Cerebral apoplexy case-control sample | ||||||||||
| Normally | 677CC or 677CT | 76 | 18 | 23.7 | 1.0 | 1.0 | ||||
| Normally | 677TT | 9 | 2 | 22.2 | 0.9 | 0.2-5.2 | 0.943 | 1.2 | 0.2-7.1 | 0.817 |
| Hypertension | 677CC or 677CT | 130 | 76 | 58.5 | 4.7 | 2.2-10.0 | ＜0.001 | 5.4 | 2.5-11.7 | ＜0.001 |
| Hypertension | 677TT | 11 | 9 | 81.8 | 16.0 | 2.9-88.6 | 0.002 | 19.1 | 3.4-108.9 | ＜0.001 |
| Cardiovascular and cerebrovascular incident death check sample | ||||||||||
| Normally | 677CC or 677CT | 65 | 19 | 29.2 | 1.0 | 1.0 | ||||
| Normally | 677TT | 6 | 2 | 33.3 | 1.3 | 0.2-8.4 | 0.797 | 1.3 | 0.2-8.8 | 0.785 |
| Hypertension | 677CC or 677CT | 87 | 47 | 54.0 | 2.6 | 1.2-5.6 | 0.017 | 2.6 | 1.2-5.8 | 0.019 |
| Hypertension | 677TT | 9 | 8 | 88.9 | 20.2 | 2.2-187.0 | 0.008 | 20.7 | 2.2-196.3 | 0.008 |
Table is annotated: 1 blood pressure: SBP 〉=140mmHg or DBP 〉=90mmHg are defined as hypertension, otherwise are defined as normal arterial pressure;
2 correction factors comprise: age, sex, BMI, smoking, drink, blood fat, blood sugar;
3 correction factors comprise: age, sex, BMI, smoking, drink, blood fat, blood sugar and logarithm transform back tHcy;
4 CC represent MTHFR 677CC homozygous wildtype; TT represents MTHFR 677TT homozygous mutation type; CT represents the MTHFR677CT heterozygous.
Our perspective study concluding prompting, the risk and the death thereof of total cardiovascular disorder, cerebral apoplexy can be significantly predicted in combined action between MTHFR C677T site and the hypertension, under appropriate homocysteine level condition, this combined action is remarkable unusually to cardiovascular and cerebrovascular incident and predict mortality thereof especially.
Embodiment 2: the test kit of measuring cerebrovascular disease generation of MTHFR C677T polymorphism site genotype estimation and prognosis
(1) moiety of test kit: nucleic acid extracting reagent, the PCR reaction reagent, MTHFR C677T pleomorphism site genotype Auele Specific Primer, nucleic acid polymerase, restriction enzyme, endonuclease reaction mixed solution and positive control template, negative control template, the test kit that is assembled into after the packing respectively.Wherein the positive control template comprises MTHFR C677T homozygous wildtype, heterozygous, homozygous mutation type positive control template; Auele Specific Primer is for can specific amplification containing the primer of MTHFR C677T pleomorphism site at least.
(2) step of Jian Ceing:
(1) genomic dna of extraction host cell:
(a) get 400 μ l erythrocyte cracked liquids and add in the 1.5ml centrifuge tube, add 100 μ l left and right sides fresh whole blood or anticoagulated whole bloods.37 ℃ of water-baths 5 minutes, centrifugal 1 minute of 15000g;
(b) remove supernatant, add 100 μ l write cell lysis buffers, at a high speed vibrate 30 seconds, 37 ℃ of water-baths 5 minutes to the liquid homogeneous.Add 35 μ l albumen precipitation liquid, 15000g is centrifugal 90 seconds after vibrating 20 seconds at a high speed, visible brown precipitation at the bottom of the centrifuge tube;
(c) the whole immigration of supernatant liquor is equipped with in the 1.5ml centrifuge tube of 100 μ l Virahols, soft back and forth several to the adularescent floss that shakes up occurs;
(d) abandon supernatant liquor, note keeping white precipitate, add 100 μ l, 75% ethanol (with the dehydrated alcohol preparation), centrifugal 90 seconds of 15000g abandons supernatant, the drying at room temperature precipitation;
(e) add nucleic acid storage liquid 100 μ l, gained solution is Whole Blood Genomic DNA;
(f) mensuration of DNA concentration adopts ultraviolet spectrophotometry, measures the OD value under two wavelength of 260nm and 280nm respectively, is DNA concentration with OD260nm * 50 income values.And with OD260nm/OD280nm ratio estimation DNA purity.
(2) use PCR and restriction fragment length polymorphism analysis method (PCR-RFLP) to detect the MTHFR677CT pleomorphism site:
According to MTHFR 677CT gene order design PCR Auele Specific Primer, comprise PCR forward primer and PCR reverse primer, carry out conventional pcr amplification by following condition.
Primer sequence:
Forward primer: 5 '-CTT TGA GGC TGA CCT GAA GC-3 '
Reverse primer: 5 '-CTG GGA AGA ACT CAG CGA AC-3 '
The PCR reaction system:
Genomic dna 45ng, upstream and downstream primer 10pmol (20 μ mol/L), dNTPs 2.0mmol/l,, 10 * buffer1.0 μ l, Gold Taq DNA polymerase 3U, ddH2O supplies cumulative volume to 10 μ l.
The PCR reaction conditions:
Behind 95 ℃ of pre-sex change 10min: 94 ℃ of sex change 30sec, 59 ℃ of annealing 45sec, 68 ℃ are extended 45sec, 35 loop cycles; Last 68 ℃ are extended 7min.Obtain the fragment of 274bp.
Enzyme tangent condition and system (15 μ l):
MTHFR C677T site PCR product purpose fragment length is 274bp, and total enzyme system of cutting is 15 μ l, PCR product 10 μ l wherein, and 10 * NEBuffer#2,1.5 μ l, HinfI restriction endonuclease 4U (0.4 μ l) and 3.1 μ l ddH2O, 37 ℃ are spent the night.
HinfI restriction endonuclease recognition site is:
(3) result of genotype detection judges:
Product point sample after the DNA enzyme cut is on 2.5% agarose gel, and electrophoresis read glue figure and carries out gene type assay after 1 hour under the 200V voltage under ultraviolet lamp.Idiotype is identified as follows:
Endonuclease bamhi is 274bp, and the mthfr gene type is the 677CC homozygous wildtype;
Endonuclease bamhi is 274+228+46bp, and the mthfr gene type is the 677CT heterozygous:
Endonuclease bamhi is 228+46bp, and the mthfr gene type is a 677TT homozygous mutation type.
(4) take place for cerebrovascular disease and the prediction of prognosis:
According to above-mentioned steps (threes') genotype result, with reference to the identical Forecasting Methodology of step (two) of embodiment 1, predict for cerebrovascular disease and prognosis.
When MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the possibility that the prediction cardiovascular and cerebrovascular diseases takes place was bigger, and prognosis is relatively poor;
When MTHFR pleomorphism site genotype was 677CC homozygous wildtype or 677CT heterozygous, the possibility that the prediction cardiovascular and cerebrovascular diseases takes place was less, and prognosis is better;
When MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place was bigger, and prognosis is relatively poor;
When MTHFR pleomorphism site genotype was 677CC homozygous wildtype or 677CT heterozygous, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place was less, and prognosis is better;
MTHFR pleomorphism site genotype is a 677TT homozygous mutation type, when occurring together hyperhomocysteinemiainjury, predicts that the possibility of individual cardiovascular and cerebrovascular diseases generation is bigger, and prognosis is relatively poor;
MTHFR pleomorphism site genotype is 677CC homozygous wildtype or 677CT heterozygous, when occurring together hyperhomocysteinemiainjury, predicts that the possibility of individual cardiovascular and cerebrovascular diseases generation is less, and prognosis is better;
MTHFR pleomorphism site genotype is 677TT homozygous mutation type or hyperhomocysteinemiainjury, and the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place is bigger, and prognosis is relatively poor;
MTHFR pleomorphism site genotype is 677CC homozygous wildtype or 677CT heterozygous, and when occurring together hyperhomocysteinemiainjury, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place is less, and prognosis is better.
Claims (21)
1.MTHFR the pleomorphism site genotype of gene is used to predict that individual cardiovascular and cerebrovascular diseases takes place and the purposes of prognosis.
2. purposes as claimed in claim 1, the pleomorphism site genotype of wherein said mthfr gene comprises the C677T pleomorphism site at least.
3. purposes as claimed in claim 1, the pleomorphism site genotype of wherein said mthfr gene can also comprise the pleomorphism site that is selected from A1298C, G1793A, G215A, G482A and A1317G, the pleomorphism site that has linkage disequilibrium with the said gene pleomorphism site be can also further comprise, nonsense mutation site, missense mutation site and the pleomorphism site that is positioned at gene intron position, generegulation position comprised.
4. purposes as claimed in claim 1, wherein said cardiovascular and cerebrovascular diseases comprises acute cardiovascular and cerebrovascular incident, acute cerebrovascular disease, cerebral infarction, hematencephalon, transient cerebral ischemic attack, cerebral apoplexy, cerebral apoplexy, acute cardiovascular disease, coronary heart disease, stenocardia, acute myocardial infarction, cardiogenic shock, sudden cardiac death, is preferably cerebral apoplexy.
5. purposes as claimed in claim 1, the wherein individual hypertension individuality that refers to especially.
6. purposes as claimed in claim 1, wherein
When (1) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the possibility of the individual generation of prediction cardiovascular and cerebrovascular diseases was bigger, and prognosis is relatively poor;
When (2) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype or 677CT heterozygous, the possibility of the individual generation of prediction cardiovascular and cerebrovascular diseases was less, and prognosis is better;
When (3) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place was bigger, and prognosis is relatively poor;
When (4) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype or 677CT heterozygous, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place was less, and prognosis is better.
7. purposes as claimed in claim 6, wherein
(1) described MTHFR pleomorphism site genotype is a 677TT homozygous mutation type, when occurring together hyperhomocysteinemiainjury, predicts that the possibility of individual cardiovascular and cerebrovascular diseases generation is bigger, and prognosis is relatively poor;
(2) described MTHFR pleomorphism site genotype is 677CC homozygous wildtype or 677CT heterozygous, when occurring together hyperhomocysteinemiainjury, predicts that the possibility of individual cardiovascular and cerebrovascular diseases generation is less, and prognosis is better;
(3) described MTHFR pleomorphism site genotype is a 677TT homozygous mutation type, and when occurring together hyperhomocysteinemiainjury, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place is bigger, and prognosis is relatively poor;
(4) described MTHFR pleomorphism site genotype is 677CC homozygous wildtype or 677CT heterozygous, and when occurring together hyperhomocysteinemiainjury, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place is less, and prognosis is better.
8. genotypic polymorphism parting oligonucleotide of pleomorphism site that is used to measure mthfr gene, preferably, described polymorphism parting oligonucleotide is: (1) allele-specific nucleic acid primer, it can detect the pleomorphism site genotype of mthfr gene, perhaps (2) are used to detect the genotypic oligonucleotide probe of pleomorphism site of mthfr gene, its can be specifically with mthfr gene on the nucleic acid hybridization of pleomorphism site, the pleomorphism site genotype of wherein said MTHFR comprises at least and is selected from the C677T pleomorphism site, preferably, the length of oligonucleotide probe is 15-50 Nucleotide.
9. polymorphism parting oligonucleotide as claimed in claim 8, the pleomorphism site genotype of wherein said MTHFR can also comprise the pleomorphism site that is selected from A1298C, G1793A, G215A, G482A and A1317G, the pleomorphism site that has linkage disequilibrium with the said gene pleomorphism site be can also further comprise, nonsense mutation site, missense mutation site and the pleomorphism site that is positioned at gene intron position, generegulation position comprised.
10. one kind is utilized the individual cardiovascular and cerebrovascular diseases generation of polymorphism site genotype estimation of mthfr gene and the method for prognosis, the pleomorphism site genotype of wherein said MTHFR comprises the C677T pleomorphism site at least, and described method comprises step: (1) utilizes claim 8 or 9 described polymorphism parting oligonucleotide to detect pleomorphism site genotype from mthfr gene described in the biological sample; (2) according to the generation and the prognosis of the individual cardiovascular and cerebrovascular diseases of the polymorphism site genotype estimation of described mthfr gene.
11. method as claimed in claim 10, the pleomorphism site genotype of wherein said MTHFR comprises the C677T pleomorphism site at least, can also comprise the pleomorphism site that is selected from A1298C, G1793A, G215A, G482A and A1317G, the pleomorphism site that has linkage disequilibrium with the said gene pleomorphism site be can also further comprise, nonsense mutation site, missense mutation site and the pleomorphism site that is positioned at gene intron position, generegulation position comprised.
12. method as claimed in claim 10, wherein said cardiovascular and cerebrovascular diseases comprises acute cardiovascular and cerebrovascular incident, acute cerebrovascular disease, cerebral infarction, hematencephalon, transient cerebral ischemic attack, cerebral apoplexy, cerebral apoplexy, acute cardiovascular disease, coronary heart disease, stenocardia, acute myocardial infarction, cardiogenic shock, sudden cardiac death, is preferably cerebral apoplexy.
13. method as claimed in claim 10, the wherein individual hypertension individuality that refers to especially.
14. method as claimed in claim 10, wherein
When (1) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the possibility that the prediction cardiovascular and cerebrovascular diseases takes place was bigger, and prognosis is relatively poor;
When (2) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype or 677CT heterozygous, the possibility that the prediction cardiovascular and cerebrovascular diseases takes place was less, and prognosis is better;
When (3) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place was bigger, and prognosis is relatively poor;
When (4) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype or 677CT heterozygous, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place was less, and prognosis is better.
15. method as claimed in claim 14, wherein
(1) described MTHFR pleomorphism site genotype is a 677TT homozygous mutation type, when occurring together hyperhomocysteinemiainjury, predicts that the possibility of individual cardiovascular and cerebrovascular diseases generation is bigger, and prognosis is relatively poor;
(2) described MTHFR pleomorphism site genotype is 677CC homozygous wildtype or 677CT heterozygous, when occurring together hyperhomocysteinemiainjury, predicts that the possibility of individual cardiovascular and cerebrovascular diseases generation is less, and prognosis is better;
(3) described MTHFR pleomorphism site genotype is a 677TT homozygous mutation type, and when occurring together hyperhomocysteinemiainjury, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place is bigger, and prognosis is relatively poor;
(4) described MTHFR pleomorphism site genotype is 677CC homozygous wildtype or 677CT heterozygous, and when occurring together hyperhomocysteinemiainjury, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place is less, and prognosis is better.
16. method as claimed in claim 11 is wherein used the following difference foranalysis of nucleic acids technology that is selected from that comprises: polymerase chain reaction, polymerase chain reaction-restriction fragment length polymorphism analysis, PCR-alleles-specific oligonucleotide probe method, PCR-sequence specific oligonucleoside acid system, sequencing, PCR-sequence specific primers method, the PCR-fluorescent method, the PCR finger-printing method, oligonucleotide connects to be analyzed, the detection method of fluorescent energy resonance transfer, biochip, nucleic acid chip, mass-spectrometric technique, genescan, single strand conformation polymorphism, denaturing gradient gel electrophoresis, enzyme or chemical mispairing patterning method, with the Taqman biological detecting method.
17. one kind is utilized the individual cardiovascular and cerebrovascular diseases generation of polymorphism site genotype estimation of mthfr gene and the test kit of prognosis, the pleomorphism site genotype of wherein said MTHFR comprises at least and is selected from the C677T pleomorphism site, wherein said cardiovascular and cerebrovascular diseases comprises acute cardiovascular and cerebrovascular incident, acute cerebrovascular disease, cerebral infarction, hematencephalon, transient cerebral ischemic attack, cerebral apoplexy, cerebral apoplexy, acute cardiovascular disease, coronary heart disease, stenocardia, myocardial infarction, cardiogenic shock, sudden cardiac death, be preferably cerebral apoplexy, described test kit comprises at least a as each described polymorphism parting oligonucleotide in claim 8 or 9, and, choose wantonly, be used for the suitable buffer system and the color development system of detection reaction.
18. test kit as claimed in claim 17, the pleomorphism site genotype of wherein said MTHFR can also comprise the pleomorphism site that is selected from A1298C, G1793A, G215A, G482A and A1317G, the pleomorphism site that has linkage disequilibrium with the said gene pleomorphism site be can also further comprise, nonsense mutation site, missense mutation site and the pleomorphism site that is positioned at gene intron position, generegulation position comprised.
19. test kit as claimed in claim 17, wherein
When (1) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the possibility that the prediction cardiovascular and cerebrovascular diseases takes place was bigger, and prognosis is relatively poor;
When (2) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype or 677CT heterozygous, the possibility that the prediction cardiovascular and cerebrovascular diseases takes place was less, and prognosis is better;
When (3) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place was bigger, and prognosis is relatively poor;
When (4) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype or 677CT heterozygous, the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place was less, and prognosis is better.
20. test kit as claimed in claim 17, wherein
(1) described MTHFR pleomorphism site genotype is a 677TT homozygous mutation type, and the hyperhomocysteinemiainjury that occurs together predicts that the possibility of individual cardiovascular and cerebrovascular diseases generation is bigger, and prognosis is relatively poor;
(2) described MTHFR pleomorphism site genotype is 677CC homozygous wildtype or 677CT heterozygous, and the hyperhomocysteinemiainjury that occurs together predicts that the possibility of individual cardiovascular and cerebrovascular diseases generation is less, and prognosis is better;
(3) described MTHFR pleomorphism site genotype is a 677TT homozygous mutation type, the hyperhomocysteinemiainjury that occurs together, and the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place is bigger, and prognosis is relatively poor;
(4) described MTHFR pleomorphism site genotype is 677CC homozygous wildtype or 677CT heterozygous, the hyperhomocysteinemiainjury that occurs together, and the possibility that the individual cardiovascular and cerebrovascular diseases of prediction of hypertension takes place is less, and prognosis is better.
21. the described test kit of method as claimed in claim 10 or claim 17, wherein said biological sample is selected from blood sample, humoral sample, tissue sample and culturing cell, and preferred, described biological sample is a blood sample.
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| CNA2006100900937A CN101096705A (en) | 2006-06-27 | 2006-06-27 | Usage, method and reagent case for prediction of cardio-cerebrovascular disease occurrence by polymorphism site genetype |
| US11/638,634 US20070134709A1 (en) | 2005-12-14 | 2006-12-13 | Usages of MTHFR gene polymorphisms in predicting homocysteine level, disease risk, and treatment effects and related methods and kit |
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