CN101037708A - Usage of homocysteine level prediction by polymorphic loci gene type, method and kit - Google Patents
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Abstract
The invention relates to using of polymorphism site genotype of methylenetetrahydrofolate reductase gene (MTHFR) for forecasting cotype cysteine level and/or related diseases of cotype cysteine, polymorphism spoligotyping oligoneucleotide for testing polymorphism site genotype of MTHFR, a method of forecasting cotype cysteine level and/or related diseases of cotype cysteine through testing polymorphism site genotype of MTHFR and reagent case thereof, belongs to medicine field.
Description
Technical field
The pleomorphism site genotype that the present invention relates to Methylene tetrahydrofolate reductase (MTHFR) gene is used to predict the purposes that is associated homocysteine level and/or homocysteine disease takes place.The invention still further relates to a kind of genotypic polymorphism parting oligonucleotide of pleomorphism site that is used to measure mthfr gene, a kind of by measuring the pleomorphism site genotype of mthfr gene, prediction homocysteine level and/or homocysteine method and the test kit that disease takes place that be associated.The invention belongs to field of medicaments.
Background technology
(Hcy homosysteine) as the metabolic intermediate product of methionine(Met), itself does not participate in proteinic synthetic homocysteine 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].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).MTHFR is the key enzyme that the N5-methyl tetrahydrofolate forms, and mthfr gene C677T sudden change can cause enzymic activity and descend, and makes methionine(Met) cycle penalty, and the Hcy level raises in the blood.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) cause that the light moderate of this enzymic activity reduces, 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 diseases, 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 Biol2005,25:115-121] 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.
[Am J Clin Nutr.1999 such as Jacques, 69 (3): 482-489] on the basis of the national for the third time nutrition of the U.S. and health survey research, U.S. population is representational to having, the age detects the 3766 routine male sex more than 12 years old and 4819 routine women's serum Hcy levels.The result shows that in non-Hispanic white man, the geometric mean of serum Hcy concentration is respectively the male sex 9.6 μ mol/L, women 7.9 μ mol/L; Be respectively the male sex 9.8 μ mol/L, women 8.2 μ mol/L non-Hispanic Black people; At the Mexico descendants male sex 9.7 μ mol/L, women 7.4 μ mol/L.Research [the Chinese epidemiology magazine that town and country, Beijing men and women's both sexes in 35～64 years old 1168 routine Plasma Hcy horizontal distribution characteristics and correlative factor are carried out, 2002,23 (1): 32-36] result shows, the Plasma Hcy geometric mean male sex is higher than women (15.4 μ mol/L vs, 12.2 μ mol/L, P＜0.001); Plasma Hcy distributes and exists differentials between town and country, and the rural area male sex (18.0 μ mol/L) is 1.5 times (P＜0.001) of the city male sex (12.0 μ mol/L), and rural area women (12.9 μ mol/L) is 1.3 times (P＜0.001) of Urban Women (9.6 μ mol/L); The morbidity of high Hcy mass formed by blood stasis (Hcy 〉=16 μ mol/L) is 15.3% among 35～64 years old crowd in town and country, Beijing.The correlative factor analysis shows that the distribution of Beijing area crowd's Plasma Hcy level exists difference between age, sex and town and country; The morbidity of Beijing area crowd Hcy level and high Hcy mass formed by blood stasis is apparently higher than western developed country, especially the Rural areas; The difference of Hcy level may reflect Effect of Environmental more between town and country.
In blood plasma, Hcy has three kinds of form: Hcy, two sulphur Hcy-Hcy and two sulphur Hcy-halfcystine.Free type Hcy only be in the blood always about 2% of tHcy in the normal human.About 80%Hcy is with cystine linkage and albumin bound.Two sulphur Hcy-Hcy and Hcy-halfcystine are about 10%～15% of tHcy in the blood.The method of measuring Hcy is more, and commonly used at present has: 1. high performance liquid chromatography (HPLC); 2. enzyme immunoassay (EIA); 3. ion chromatography; 5. fluorescence polarization immunoassay.At present clinical commonly used and mature methods is high performance liquid chromatography and combined gas chromatography mass spectrometry.But all exist operation more complicated, test consuming time long, repeatability is not good enough, cost is higher, be difficult for defective such as penetration and promotion.
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 homocysteine is the particularly Hazard Factor of cardiac and cerebral vascular diseases of a lot of diseases, the level of homocysteine and the generation of disease, development and prognosis etc. are closely related, and there is certain defective in the detection method of clinical mensuration homocysteine commonly used, and shortage foresight, in order earlier to predict homocysteine level, prevent better simultaneously and intervene the homocysteine process that disease takes place that is associated, press for the level of homocysteine is carried out early prediction, the present invention promptly provides a kind of method and test kit and purposes of predicting homocysteine level, and prediction homocysteine method and test kit and the purposes that disease takes place that be associated.
Summary of the invention
The present invention provides a kind of method and test kit for the prediction that disease takes place that is associated of homocysteine level and homocysteine, i.e. homocysteine level prediction by polymorphic loci gene type by measuring mthfr gene and/or the homocysteine possibility that disease takes place that is associated.On the basis of pleomorphism site gene type assay, can predict the possibility that is associated homocysteine level and/or homocysteine disease takes place, therefore can earlier prevent the rising of homocysteine level, prevention or intervene homocysteine the be associated generation and the process of disease better, alleviate the patient suffering, reduce medical treatment cost.
One aspect of the present invention, the pleomorphism site genotype that relates to mthfr gene are used to predict the be associated purposes of disease of homocysteine level and/or homocysteine.
In purposes of the present invention, the pleomorphism site genotype of described MTHFR comprises at least and is selected from the C677T pleomorphism site, 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, there is the pleomorphism site of linkage disequilibrium in the gene polymorphism sites that the pleomorphism site of described MTHFR can also further comprise the disease that is associated with above-mentioned or other prediction homocysteine levels and/or homocysteines, comprises nonsense mutation site, missense mutation site and the pleomorphism site that is positioned at gene intron position, generegulation position.
In the present invention, the described homocysteine disease that is associated comprises atherosclerosis, coronary heart disease, cerebrovascular disease, kidney function damage, peripheral vascular disease, arteriovenous thrombotic diseases, hypertension, hyperlipidemia, diabetes, psychotic disorder.Concrete, when
When (1) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the prediction homocysteine level was higher, and the possibility that homocysteine level raises is bigger;
When (2) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype and/or 677CT heterozygous, the prediction homocysteine level was lower, and the possibility that homocysteine level does not raise is bigger;
When (3) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, it is bigger that the be associated possibility of disease of homocysteine takes place in prediction;
When (4) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype and/or 677CT heterozygous, it is bigger that the be associated possibility of disease of homocysteine does not take place in prediction.
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, there is the pleomorphism site of linkage disequilibrium in the gene polymorphism sites that can also further comprise the disease that is associated with above-mentioned prediction homocysteine level and/or homocysteine, comprises nonsense mutation site, missense mutation site and the pleomorphism site that is positioned at gene intron position, generegulation position.
One side more of the present invention, relate to the be associated method of disease of homocysteine level in a kind of polymorphism site genotype estimation biological sample that utilizes mthfr gene and/or homocysteine, 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) according to the homocysteine level prediction by polymorphic loci gene type of described mthfr gene and/or the homocysteine disease that is associated.
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, there is the pleomorphism site of linkage disequilibrium in the gene polymorphism sites that can also further comprise the disease that is associated with above-mentioned prediction homocysteine level and/or homocysteine, comprises nonsense mutation site, missense mutation site and the pleomorphism site that is positioned at gene intron position, generegulation position.
In the present invention, the described homocysteine disease that is associated comprises atherosclerosis, coronary heart disease, cerebrovascular disease, kidney function damage, peripheral vascular disease, arteriovenous thrombotic diseases, hypertension, hyperlipidemia, diabetes, psychotic disorder.Concrete, when
When (1) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the prediction homocysteine level was higher, and the possibility that homocysteine level raises is bigger;
When (2) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype and/or 677CT heterozygous, the prediction homocysteine level was lower, and the possibility that homocysteine level does not raise is bigger;
When (3) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, it is bigger that the be associated possibility of disease of homocysteine takes place in prediction;
When (4) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype and/or 677CT heterozygous, it is bigger that the be associated possibility of disease of homocysteine does not take place in prediction.
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-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 the be associated method of disease of homocysteine level in the biological sample and/or homocysteine 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 the be associated method of disease of homocysteine level in the biological sample and/or homocysteine.
Another aspect of the present invention, also relate to the be associated test kit of disease of homocysteine level in a kind of polymorphism site genotype estimation biological sample that utilizes mthfr gene and/or homocysteine, the pleomorphism site genotype of wherein said MTHFR comprises at least and is selected from the C677T pleomorphism site, the wherein said homocysteine disease that is associated comprises atherosclerosis, coronary heart disease, cerebrovascular disease, kidney function damage, peripheral vascular disease, the arteriovenous thrombotic diseases, hypertension, hyperlipidemia, diabetes, psychotic disorder, 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 prediction homocysteine level was higher, and the possibility that homocysteine level raises is bigger;
When (2) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype and/or 677CT heterozygous, the prediction homocysteine level was lower, and the possibility that homocysteine level does not raise is bigger;
When (3) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, it is bigger that the be associated possibility of disease of homocysteine takes place in prediction;
When (4) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype and/or 677CT heterozygous, it is bigger that the be associated possibility of disease of homocysteine does not take place in prediction.
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 gene polymorphism sites of above-mentioned prediction homocysteine level 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 predict homocysteine level and/or the homocysteine disease that is associated 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, detect the pleomorphism site genotype and the homocysteine level of mthfr gene respectively analyzes for the individuality that participates in research, 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; therefore usually can have influence on enzyme function and the activity of MTHFR, have influence on the be associated generation of disease of homocysteine level and/or homocysteine indirectly or directly.Promptly by measuring the pleomorphism site genotype of MTHFR, can predict homocysteine level and/or the homocysteine disease that is associated.
Under the polymorphism situation of the C677T of known mthfr gene, the level of homocysteine in the body inner blood of comparison Different Individual, find the C677T pleomorphism site genotype of different mthfr genes, the level difference of homocysteine, genotype is that 677TT homozygous mutation type individuality is that 677CC wild-type individuality and/or 677CT heterozygous are relatively individual with genotype, homocysteine level raises more obvious in the body, when prompting MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the prediction homocysteine level was higher; When described genotype was 677CC wild-type and/or 677CT heterozygous, the prediction homocysteine level was lower.
Be divided into different levels by further concentration according to homocysteine, analyze as dividing value with different concentration levels respectively, found that, the C677T pleomorphism site genotype of different mthfr genes can be predicted the homocysteine concentration of different levels, has the positive predictive value and the negative predictive value of higher prediction respectively.
Further analyze and find, homocysteine for the different concns level, the C677T pleomorphism site genotype of different mthfr genes has different odds ratios, and increasing along with homocysteine level, the 677TT homozygous mutation type of mthfr gene is compared with 677CC wild-type and/or 677CT heterozygous, odds ratio increases thereupon, and when prompting MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the possibility that the prediction homocysteine level raises was bigger; Described MTHFR pleomorphism site genotype is a 677CC homozygous wildtype and or during the 677CT heterozygous, the possibility that the prediction homocysteine level does not raise is bigger.
One side more of the present invention, also provide and utilized functional gene prediction homocysteine level and/or homocysteine the be associated method and the test kit of disease, can be used as the be associated indication mechanism of the possibility that disease takes place of homocysteine level and/or homocysteine, promptly be used to predict homocysteine level and/or the homocysteine disease that is associated by the pleomorphism site genotype of measuring mthfr gene.On the basis of pleomorphism site gene type assay, can predict homocysteine level and/or the homocysteine disease that is associated, can therefore earlier predict homocysteine level, prevent better simultaneously and intervene the be associated process of disease of homocysteine, 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, the dsSNP ID:rs1801133) pleomorphism site of mthfr gene and predict homocysteine level
(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 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#21.5 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 homocysteine level
When MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the prediction homocysteine level was higher, and the possibility that homocysteine level raises is bigger;
When MTHFR pleomorphism site genotype was 677CC homozygous wildtype and/or 677CT heterozygous, the prediction homocysteine level was lower, and the possibility that homocysteine level does not raise is bigger;
When MTHFR pleomorphism site genotype was 677TT homozygous mutation type, it is bigger that the be associated possibility of disease of homocysteine takes place in prediction;
When MTHFR pleomorphism site genotype was 677CC homozygous wildtype and/or 677CT heterozygous, it is bigger that the be associated possibility of disease of homocysteine does not take place in prediction.
Above method is verified by epidemiological study, earlier individuality is divided into three groups according to MTHFR C677T genotype: 677CC homozygous wildtype group, 677CT heterozygous group and 677TT homozygous mutation type group, measure homocysteine level respectively and other are as physiological parameters such as sex, age, height, body weight, analyze different genotype and the relation between the baseline homocysteine level, the result is as follows:
Homocysteine level difference in the body of different MTHFR C677T pleomorphism site genotype individualities, individual relatively individual in 677TT homozygous mutation type with 677CC homozygous wildtype and/or 677CT heterozygous, homocysteine level raises obviously, still reach the significance,statistical level through the average difference after the multiplefactor correction, prompting mthfr gene C677T pleomorphism site genotype is relevant with homocysteine level, wherein, the individual homocysteine level higher (18.65 ± 11.03 μ mol/L) of 677TT homozygous mutation type, there was a significant difference (p＜0.01) to compare (10.74 ± 3.60 μ mol/L) with 677CC homozygous wildtype individuality; And the individual homocysteine level of CT heterozygous slightly high (11.19 ± 4.30 μ mol/L) is compared (10.74 ± 3.60 μ mol/L) difference and is not had significance (p=0.23) (seeing Table 1) with 677CC homozygous wildtype individuality.
Table 1 mthfr gene C677T pleomorphism site genotype with
The relation of homocysteine level
| Genotype * | The example number | Homocysteine level (means standard deviation | β# | SE# | P value # |
| CC CT TT | 337 509 241 | 10.74±3.60 11.19±4.30 18.65±11.03 | --- 1.1 9.8 | --- 0.8 0.9 | --- 0.230 0.000 |
Annotate:
*Genotype: CC represents MTHFR 677CC homozygous wildtype; TT represents MTHFR 677TT homozygous mutation type; CT represents MTHFR 677CT heterozygous;
# has adjusted factors such as sex, age, height, body weight.
We further adopt the research method of case-contrast, respectively according to homocysteine level be 10 μ moll/L, 15 μ mol/L, 20 μ mol/ as 3 cut off value (cut off), analyze the predicting function of MTHFR C677T pleomorphism site genotype and homocysteine level.The result is as shown in table 2, when with homocysteine level=when 10 μ mol/L are cut off value, positive predictive value is 76.8%, and prompting is when the mthfr gene type is TT homozygous mutation genotype, and prediction the efficient of homocysteine level 〉=10 μ mol/L is 76.8%; When so that homocysteine level=when 15 μ mol/L were cut off value, negative predictive value was 89.3%, prompting when the mthfr gene type be CC when isozygotying the wild gene type, predict that the efficient of homocysteine level＜15 μ mol/L is 89.3%; When so that homocysteine level=when 20 μ mol/L were cut off value, negative predictive value was 98.3%, to point out when the mthfr gene type is the CC homozygous wildtype, prediction the efficient of homocysteine level＜20 μ mol/L is 98.3.%.Result of study shows that when described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the prediction homocysteine level was higher; When described MTHFR pleomorphism site genotype was the 677CC homozygous wildtype, the prediction homocysteine level was lower.
Table 2 mthfr gene C677T pleomorphism site genotype with
The predicting function of homocysteine level
| Cut off | TT | CC | Sensitivity (%) | Specific degree (%) | Positive predictive value (%) | Negative predictive value (%) | ||
| High | Low | High | Low | |||||
| ≥10(μmol/L) ≥15(μmol/L) ≥20(μmol/L) | 185 102 68 | 56 139 173 | 181 36 6 | 156 301 331 | 50.5 73.9 91.9 | 73.6 68.4 69.8 | 76.8 42.3 28.2 | 46.3 89.3 98.2 |
Annotate: CC, represent MTHFR 677CC homozygous wildtype; TT represents MTHFR 677TT homozygous mutation type.
We further analyze the genotypic TT homozygous mutation of MTHFR C677T pleomorphism site genotype and compare with the CC homozygous wildtype, the danger that homocysteine level increases takes place, the possibility that the individuality generation homocysteine level of promptly more different mthfr gene types raises.The result is as shown in table 3, when with homocysteine level=when 10 μ mol/L are cut off value, the possibility of the homocysteine level 〉=10 μ mol/L of MTHFR 677TT homozygous mutation type individuality is 2.8 times of possibility of the homocysteine level 〉=10 μ mol/L of 677CC homozygous wildtype individuality, be that MTHFR 677TT homozygous mutation type individuality is compared with 677CC homozygous wildtype individuality, the danger of homocysteine level 〉=10 μ mol/L is 2.8 (OR=2.8,95%CI=2.1-4.8), there was a significant difference; When with homocysteine level=when 15 μ moll/L are cut off value, the possibility of the homocysteine level 〉=15 μ mol/L of MTHFR 677TT homozygous mutation type individuality is 6.1 times of possibility of the homocysteine level 〉=15 μ mol/L of 677CC homozygous wildtype individuality, be that MTHFR 677TT homozygous mutation type individuality is compared with 677CC homozygous wildtype individuality, the danger of homocysteine level 〉=15 μ mol/L is 6.1 (OR=6.1,95%CI=4.2-9.7), there was a significant difference; When with homocysteine level=when 20 μ mol/L are cut off value, the possibility of the homocysteine level 〉=20 μ mol/L of MTHFR 677TT homozygous mutation type individuality is 23.5 times of possibility of the homocysteine level 〉=20 μ mol/L of 677CC homozygous wildtype individuality, be that MTHFR 677TT homozygous mutation type individuality is compared with 677CC homozygous wildtype individuality, the danger of homocysteine level 〉=20 μ mol/L is 23.5 (OR=23.5,95%CI=11.3-52.1), there was a significant difference.Result of study shows that when described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the possibility that the prediction homocysteine level raises was bigger; When described MTHFR pleomorphism site type was the 677CC homozygous wildtype, the possibility that the prediction homocysteine level does not raise was bigger.
Table 3 mthfr gene C677T pleomorphism site genotype with
The Logistic regression analysis of high Hcy mass formed by blood stasis incidence relation
| Genotype * | Sample size | High Hcy mass formed by blood stasis | OR value # | 95％CI | P value # | |
| Number | ％ | |||||
| ≥10(μmol/L) CC TT ≥15(μmol/L) CC TT ≥20(μmol/L) CC TT | 337 241 337 241 337 241 | 181 185 36 112 6 68 | 53.7 76.8 10.7 46.5 1.8 28.2 | 1.00 2.8 1.00 6.1 1.00 23.5 | --- 2.1-4.8 --- 4.2-9.7 --- 11.3-52.1 | --- 0.00 --- 0.00 --- 0.00 |
Annotate:
*Genotype: CC represents MTHFR 677CC homozygous wildtype; TT represents MTHFR 677TT homozygous mutation type; # has adjusted factors such as sex, age, height, body weight.
By result in the table 1 as can be known, the homocysteine level of the homocysteine level of CT heterozygous and CC homozygous wildtype does not have significant significant difference, we merge into one group with CT heterozygous and CC homozygous wildtype individuality, with TT homozygous mutation type as other one group, still according to the research method of as above case-contrast, be 10 μ mol/L according to homocysteine level respectively, 15 μ mol/L, 20 μ mol/L are as 3 cut off value (cut off), analyze the predicting function of MTHFRC677T pleomorphism site genotype and homocysteine level, obtain as above similar result.Concrete outcome is as shown in table 4, when with homocysteine level=when 10 μ mol/L are cut off value, positive predictive value is 76.8%, and prompting is when the mthfr gene type is TT homozygous mutation type, and prediction the efficient of homocysteine level 〉=10 μ mol/L is 76.8%; When so that homocysteine level=when 15 μ mol/L were cut off value, negative predictive value was 84.1%, to point out when the mthfr gene type is CC homozygous wildtype and/or CT heterozygous, prediction the efficient of homocysteine level＜15 μ mol/L is 84.1%; When so that homocysteine level=when 20 μ mol/L were cut off value, negative predictive value was 96.7%, to point out when the mthfr gene type is CC homozygous wildtype and/or CT heterozygous, prediction the efficient of homocysteine level＜20 μ mol/L is 96.7%..Result of study shows that when described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the prediction homocysteine level was higher; When described MTHFR pleomorphism site genotype was 677CC homozygous wildtype and/or 677CT heterozygous, the prediction homocysteine level was lower.
Table 4 mthfr gene C677T pleomorphism site genotype with
The predicting function of homocysteine level
| Cut off | TT | CC+CT | Sensitivity (%) | Specific degree (%) | Positive predictive value (%) | Negative predictive value (%) | ||
| High | Low | High | Low | |||||
| ≥10(μmol/L) ≥15(μmol/L) ≥20(μmol/L) | 185 102 68 | 56 139 173 | 461 110 28 | 385 736 818 | 28.6 48.1 70.8 | 87.3 84.1 82.5 | 76.8 42.3 28.2 | 42.3 84.1 96.7 |
Annotate: CC, represent MTHFR 677CC homozygous wildtype; TT represents MTHFR 677TT homozygous mutation type; CT represents MTHFR 677CT heterozygous.
Equally, we further analyze the genotypic TT homozygous mutation of MTHFR C677T pleomorphism site genotype and compare with CC homozygous wildtype and/or 677CT heterozygous, the danger that homocysteine level increases takes place, the possibility that the individuality generation homocysteine level of promptly more different mthfr gene types raises.The result is as shown in table 5, when with homocysteine level=when 10 μ mol/L are cut off value, the possibility of the homocysteine level 〉=10 μ mol/L of MTHFR 677TT homozygous mutation type individuality is 5.7 times of possibility of the homocysteine level 〉=10 μ mol/L of 677CC homozygous wildtype individuality, be that MTHFR 677TT homozygous mutation type individuality is compared with 677CC homozygous wildtype and/or 677CT heterozygous individuality, the danger of homocysteine level 〉=10 μ mol/L is 5.7 (OR=5.7,95%CI=2.1-8.6), there was a significant difference; When with homocysteine level=when 15 μ mol/L are cut off value, the possibility of the homocysteine level 〉=15 μ mol/L of MTHFR677TT homozygous mutation type individuality is 11.2 times of possibility of the homocysteine level 〉=15 μ mol/L of 677CC homozygous wildtype and/or 677CT heterozygous individuality, be that MTHFR677TT homozygous mutation type individuality is compared with 677CC homozygous wildtype and/or 677CT heterozygous individuality, the danger of homocysteine level 〉=15 μ mol/L is 11.2 (OR=11.2,95%CI=3.7-32.4), there was a significant difference; When with homocysteine level=when 20 μ mol/L are cut off value, the possibility of the homocysteine level 〉=20 μ mol/L of MTHFR 677TT homozygous mutation type individuality is 23.5 times of possibility of the homocysteine level 〉=20 μ mol/L of 677CC homozygous wildtype and/or 677CT heterozygous individuality, be that MTHFR 677TT homozygous mutation type individuality is compared with 677CC homozygous wildtype individuality, the danger of homocysteine level 〉=20 μ mol/L is 27.5 (OR=27.5,95%CI=12.1-87.9), there was a significant difference.Result of study shows that when described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the possibility that the prediction homocysteine level raises was bigger; When described MTHFR pleomorphism site genotype was 677CC homozygous wildtype and/or 677CT heterozygous, the possibility that the prediction homocysteine level does not raise was bigger.
Table 5 mthfr gene C677T pleomorphism site genotype with
The Logistic regression analysis of high Hcy mass formed by blood stasis incidence relation
| Genotype * | Sample size | High Hcy mass formed by blood stasis | OR value # | 95％CI | P value # | |
| Number | ％ | |||||
| ≥10(μmol/L) CC+CT TT ≥15(μmol/L) CC+CT TT ≥20(μmol/L) CC+CT TT | 846 241 846 241 846 241 | 461 185 110 102 28 68 | 54.5 76.8 13.0 42.3 3.3 28.2 | 1.00 5.7 1.00 11.2 1.00 27.5 | --- 2.1-8.6 --- 3.7-32.4 --- 12.1-87.9 | --- 0.00 --- 0.00 --- 0.00 |
Annotate:
*Genotype: CC represents MTHFR 677CC homozygous wildtype; TT represents MTHFR 677TT homozygous mutation type; CT represents MTHFR 677CT heterozygous;
# has adjusted factors such as sex, age, height, body weight.
Embodiment 2: the test kit of measuring MTHFR C677T homocysteine level prediction by polymorphic loci gene type
(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) for the prediction of homocysteine level:
According to above-mentioned steps (threes') genotype result, with reference to the identical Forecasting Methodology of step (two) of embodiment 1, predict for homocysteine level.
When MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the prediction homocysteine level was higher, and the possibility that homocysteine level raises is bigger;
When MTHFR pleomorphism site genotype was 677CC homozygous wildtype and/or 677CT heterozygous, the prediction homocysteine level was lower, and the possibility that homocysteine level does not raise is bigger;
When MTHFR pleomorphism site genotype was 677TT homozygous mutation type, it is bigger that the be associated possibility of disease of homocysteine takes place in prediction;
When MTHFR pleomorphism site genotype was 677CC homozygous wildtype and/or 677CT heterozygous, it is bigger that the be associated possibility of disease of homocysteine does not take place in prediction.
Claims (16)
1.MTHFR the pleomorphism site genotype of gene is used to predict the purposes that is associated homocysteine level and/or homocysteine disease takes place.
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 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 gene polymorphism sites of above-mentioned prediction homocysteine level 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, the wherein said homocysteine disease that is associated comprises atherosclerosis, coronary heart disease, cerebrovascular disease, kidney function damage, peripheral vascular disease, arteriovenous thrombotic diseases, hypertension, hyperlipidemia, diabetes, psychotic disorder.
5. purposes as claimed in claim 1, wherein
When (1) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the prediction homocysteine level was higher, and the possibility that homocysteine level raises is bigger;
When (2) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype and/or 677CT heterozygous, the prediction homocysteine level was lower, and the possibility that homocysteine level does not raise is bigger;
When (3) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, it is bigger that the be associated possibility of disease of homocysteine takes place in prediction;
When (4) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype and/or 677CT heterozygous, it is bigger that the be associated possibility of disease of homocysteine does not take place in prediction.
6. 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.
7. polymorphism parting oligonucleotide as claimed in claim 6, the pleomorphism site genotype of wherein said MTHFR can also comprise the pleomorphism site that is selected from A1298C, G1793A, G215A, G482A and A1317G, there is the pleomorphism site of linkage disequilibrium in the gene polymorphism sites that can also further comprise the disease that is associated with above-mentioned prediction homocysteine level and/or homocysteine, comprises nonsense mutation site, missense mutation site and the pleomorphism site that is positioned at gene intron position, generegulation position.
8. homocysteine level and/or the homocysteine method that disease takes place that is associated in the polymorphism site genotype estimation biological sample that utilizes mthfr gene, 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 claim 6 or 7 described polymorphism parting oligonucleotide to detect pleomorphism site genotype from mthfr gene described in the biological sample; (2) take place according to the homocysteine level prediction by polymorphic loci gene type of described mthfr gene and/or the homocysteine disease that is associated.
9. method as claimed in claim 8, 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 there is linkage disequilibrium in gene polymorphism sites that the disease that is associated with above-mentioned prediction homocysteine level and/or homocysteine takes place 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. method as claimed in claim 8, the wherein said homocysteine disease that is associated comprises atherosclerosis, coronary heart disease, cerebrovascular disease, kidney function damage, peripheral vascular disease, arteriovenous thrombotic diseases, hypertension, hyperlipidemia, diabetes, psychotic disorder.
11. method as claimed in claim 8, wherein
When (1) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the prediction homocysteine level was higher, and the possibility that homocysteine level raises is bigger;
When (2) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype and/or 677CT heterozygous, the prediction homocysteine level was lower, and the possibility that homocysteine level does not raise is bigger;
When (3) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, it is bigger that the be associated possibility of disease of homocysteine takes place in prediction;
When (4) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype and/or 677CT heterozygous, it is bigger that the be associated possibility of disease of homocysteine does not take place in prediction.
12. method as claimed in claim 8 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.
13. homocysteine level and/or the homocysteine test kit that disease takes place that is associated in the polymorphism site genotype estimation biological sample that utilizes mthfr gene, the pleomorphism site genotype of wherein said MTHFR comprises at least and is selected from the C677T pleomorphism site, the wherein said homocysteine disease that is associated comprises atherosclerosis, coronary heart disease, cerebrovascular disease, kidney function damage, peripheral vascular disease, the arteriovenous thrombotic diseases, hypertension, hyperlipidemia, diabetes, psychotic disorder, described test kit comprises at least a as each described polymorphism parting oligonucleotide in claim 6 or 7, and, choose wantonly, be used for the suitable buffer system and the color development system of detection reaction.
14. test kit as claimed in claim 13, 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 gene polymorphism sites of above-mentioned prediction homocysteine level 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.
15. test kit as claimed in claim 13, wherein
When (1) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, the prediction homocysteine level was higher, and the possibility that homocysteine level raises is bigger;
When (2) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype and/or 677CT heterozygous, the prediction homocysteine level was lower, and the possibility that homocysteine level does not raise is bigger;
When (3) described MTHFR pleomorphism site genotype was 677TT homozygous mutation type, it is bigger that the be associated possibility of disease of homocysteine takes place in prediction;
When (4) described MTHFR pleomorphism site genotype was 677CC homozygous wildtype and/or 677CT heterozygous, it is bigger that the be associated possibility of disease of homocysteine does not take place in prediction.
16. the described test kit of method as claimed in claim 8 and/or claim 13, 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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