CN101886121B - Method for detecting locus rs873457 of hypertension susceptibility genes and detection kit - Google Patents
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Abstract
The invention belongs to the fields of molecular biology and medicine and relates to a method for detecting hypertension susceptibility genes and a detection kit thereof. The method for detecting the essential hypertension susceptibility genes comprises the step of detecting the gene type of the locus rs873457 of mitochondria fusion genes 2 (Mfn2)/hyperplasia suppressor genes (HSG) People having the locus rs873457 of the C gene type are more susceptible to the hypertension than the common crowd. The invention also discloses a relevant detection kit containing primers for amplifying the locus rs873457 and primers for amplifying a zone containing the locus rs873457 in the intron No.2 of the Mfn2 genes. The method is easy to operate, can be used for quickly and effectively detecting the gene type of the locus rs873457 and has low cost, thereby being a simple new method for diagnosing and treating the hypertension..
Description
Technical field
The present invention relates to molecular biology and medical field.More specifically, relate to human mitochondrion fusion gene 2 (Mfn2 gene) SNP (single nucleotide polymorphism, SNP) site rs873457 and with the detection of essential hypertension dependency.The invention still further relates to the method and the test kit that detect this SNP site.
Background technology
(essential hypertension EH) is a kind of multifactor, multigenic disease to essential hypertension, by the common morbific common and multiple cardiovascular disorder of environment and heredity factor, human health has been caused great influence.Along with the development of molecular medicine, kind surplus the hypertension relative gene that has been found that has at present had 150, but the pathogenesis of EH still imperfectly understands, and hypertensive early diagnosis and proactive problem still fail to solve fully.EH is the coefficient result of gene and environment, and the variation 30%-60% of blood pressure is owing to heredity.Because environmental factors can be controlled and confirm, is changeless and hypertension genetic is learned factor.Therefore; Control to variable factor; Like prevention, can delay and prevent hypertensive morbidity to a certain extent, but the insufficient recognition of changeless inherited genetic factors is but being had a strong impact on hypertension incidence, diagnosis and treatment to a certain extent the hypertension Hazard Factor.Research ten minutes necessity of therefore hypertension genetic being learned (Wang Zuoguang, Wen Shaojun, Wu Zhaosu. hypertension, tumor susceptibility gene and SNP [J]. the hypertension magazine, 2001,9:259-264).
Nearly more than two decades comes, and about the research of hypertension therapeutic concentrates on to the control of blood pressure with to the protection of target organ more, and has obtained the development of advancing by leaps and bounds.But these means are controlling blood pressure fundamentally, at present to the research of human endogenous hypertension gene also seldom; Therefore; Research endogenous hypertension mechanism is one of main direction of hypertension prevention and control research from now on, has important researching value and application prospect, if through regulating endogenous hypertension mechanism treatment essential hypertension; Will promote the development of China's biologic medical technology, and be the huge medical expense of the annual saving of China.
The hypertension relative gene of being found is at present summed up and is got up may be summarized to be short shr gene and hypertension gene, and Mfn2 belongs to the latter.The predecessor of Mfn2 is intelligent [the Chen GH such as grade of Chinese scholar Chen Guang; Zhang CH; Zhu YQ; Et al.Expression of a novel gene related to hypertension [J] .Natl Med J China, 1997,77:823-828.] in 1997 the spontaneous hypertensive rat (SHR) of cultivation and the VSMC of normal Wistar Kyoto (WKY) rat are carried out the difference demonstration; Clone a new gene---and hyperplasia suppressor gene (hyperplasia suppressor gene, HSG).It includes the base pair of 4160bp, 661 amino acid of encoding altogether (NM_014874, GeneBank Access U41803).After a series of bodies are interior, experiment in vitro is found [Chen KH, Guo XM, Dalong Ma; Et al.Dysregulation of HSG triggers vascular proliferativedisorders [J] .Nature Cell Biol; 2004,6,872-883.]; HSG can suppress Ras-Raf albumen-Si significantly and split plain activated protein kinase genetic expression; And can activate the expression of antioncogene, check cell cycle and the propagation that suppresses various kinds of cell effectively, and this restraining effect realize through apoptotic mode.Therefore the HSG gene is one of candidate gene of essential hypertension; After gene functional research shows; The HSG gene is participated in plastosome and is merged; Therefore definite designation is that (mitofusin 2, and is Mfn2) still at present less to the research of Mfn2 and EH, do not confirm the report of rs873457 and EH dependency for plastosome fusion gene 2.
In sum, for the final treatment hypertension that realizes, this area presses for seeks the essential hypertension tumor susceptibility gene, and method, the test kit of exploitation detection essential hypertension tumor susceptibility gene, and relevant medicine.
Summary of the invention
The method and the detection kit that the purpose of this invention is to provide a kind of detection (comprising early diagnosis) hypertension susceptible gene.
The invention provides a kind of detection method of hypertension susceptibility gene, promptly detect the genotype in Mfn2 gene rs873457 site, rs873457 has the hypertensive onset risk of the genotypic individuality of T and is significantly higher than the general population.
Described rs873457, the intron 2 (fragment contig contig:NT_021937.18 position 6583701 G/C) that is arranged in Mfn2 wherein, thymus nucleic acid (DNA) sequence numbering: the rs873457 position is based on SEQ ID NO:1/6; Primer 1 is based on SEQ ID NO:2/6; Primer 2 is based on SEQ ID NO:3/6; Probe 1 is based on SEQ ID NO:4/6; Probe 2 is based on SEQ ID NO:5/6; Amplified production is based on SEQ ID NO:6/6.
Particularly, the method comprising the steps of: (a) genomic dna of extracting sample, and amplification obtains Mfn2 gene intron 2; (b) genotype of mononucleotide polymorphism site rs873457 in detection step (a) product.
The primer sequence of described amplification Mfn2 gene intron 2 is shown in SEQ ID NO 2 and SEQ ID NO 3.
The genotypic probe sequence of described detection rs873457 is shown in SEQ ID NO 4 and SEQ ID NO 5.
Technology such as the order-checking that relates in the aforesaid method, amplification, extracting genomic dna all can adopt the routine operation method of this area.
The invention provides a kind of test kit that detects hypertension susceptible gene, it contains and site rs873457 bonded probe.
In one embodiment of the invention, with the sequence of site rs873457 bonded probe shown in SEQ ID NO:4 and SEQ ID NO:5.
The mentioned reagent box can also comprise the primer that comprises the zone in rs873457 site in the specific amplification Mfn2 gene intron 2.
In one embodiment of the invention, with specific amplification Mfn2 gene intron 2 in comprise the rs873457 site the sequence of primer in zone shown in SEQ ID NO:2 and SEQ ID NO:3.
The present invention is through research for many years; Proved that first Mfn2 gene mononucleotide polymorphism site rs873457 (fragment contig contig:NT_021937.18 position 6583701G/C) and hypertension are closely related; And found its new function: Mfn2 gene mononucleotide polymorphism site rs873457 is positioned at Mfn2; (fragment contig contig:NT_021937.18 position 6583701 G/C) genotypic change will cause hypertensive onset risk to raise in the intron 2; Wherein the association study result shows; Have significant difference (p＜0.05) in the distribution of Mfn2 gene rs873457G → C in case and control group, this SNP polymorphum can change the transcription factor binding site point.Accomplished the present invention on this basis.
The invention provides a kind of hypertension susceptibility to individuality and carry out method of diagnosing, it comprises step: detect the genotype in this individual Mfn2 gene rs873457 site, judge with this whether should individuality suffer from hypertensive onset risk is higher than the general population.
In a preference, described difference is the SNP that is selected from rs873457.Rs873457 is positioned at Mfn2, in the intron 2 (fragment contig contig:NT_021937.18 position 6583701 G/C).Wherein, thymus nucleic acid (DNA) sequence numbering: the rs873457 position is based on SEQ ID NO:1/6.
The invention provides the method whether a kind of test sample exists the SNP of Mfn2 gene, comprise step:
(a) with the genomic dna of Mfn2 gene intron 2 primer amplified samples, obtain amplified production; With
(b) genotype of mononucleotide polymorphism site rs873457 in the detection amplified production.
The detailed sequence of Mfn2 gene can be the nucleotide sequence (can referring to network address http://www.ncbi.nlm.nih.gov/) of rs873457 referring to accession number.
The inventor checks order to (fragment contig contig:NT_021937.18 position 6583701) zone in the intron in the Mfn2 gene 2.
The polymorphum in Mfn2 gene rs873457 site can directly be used for hypertensive personalized treatment.When using the polymorphum in Mfn2 gene rs873457 of the present invention site, also can use the hypertensive medicament of other treatment simultaneously.
The present invention also provides a kind of pharmaceutical composition, and it contains Mfn2 albumen of the present invention safely and effectively and pharmaceutically acceptable carrier or vehicle.This type carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical prepn should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example prepares through ordinary method with the saline water or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can prepare through ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule are made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day 0.1 microgram/kg body weight-Yue 10 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
When making pharmaceutical composition; Be that the Mfn2 albumen of safe and effective amount or its antagonist, agonist are applied to general Mammals; Wherein this safe and effective dosage is usually at least about 0.1 microgram/kg body weight; And in most of the cases be no more than about 10 mg/kg body weight, preferably this dosage is about 0.1 microgram/kg body weight-Yue 100 mg/kg body weight.Certainly, concrete dosage is factor such as considered route of administration, patient health situation also, and these all are within the skilled practitioners skill.
The genotype that detects the rs873457 of Mfn2 gene also can be used for diagnosing hypertension.Detection can be directed against genomic dna, also can be to the amplified fragments of Mfn2 gene.The genotype of the rs873457 of Mfn2 gene can use existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization to detect sudden change.
The invention provides a kind of method that detects the essential hypertension susceptible gene; It comprises the genotype in the rs873457 site of detection line plastochondria fusion gene 2 (Mfn2); Rs873457 has the genotypic individuality of C, and hypertensive susceptibility is significantly higher than the general population.The invention also discloses the relevant detection test kit, this test kit contains the primer in amplification rs873457 site, can also comprise the primer in No. 2 intron zone of amplification Mfn2 gene.Utilize the present invention to detect the genotype in rs873457 site, method is simple, and is rapidly and efficiently with low cost, for hypertensive diagnosis and treatment provide a simple and direct new way.
Description of drawings
Fig. 1 is the detected result of MFn2 gene rs873457 loci gene type.
Fig. 2 is the sequencing result sectional drawing.The genotypic sequencing result of a kind of SNP that has shown rs873457.Rs873457 is positioned at Mfn2, in the intron 2 (fragment contig contig:NT_021937.18 position 6583701 G/C).
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning; Condition described in the laboratory manual (New York:Cold Spring Harbor LaboratoryPress, 1989), or the condition of advising according to manufacturer.
Embodiment 1 fluorescent PCR detects
One, experiment material
The 7900HT quantitative real time PCR Instrument is available from American AB I company, and pcr reaction solution (TaqMan EXPress MasterMix) is synthetic by u.s.a. applied biosystem company (ABI) customization.
Two, primer and probe design and synthetic:
Partial sequence with MFn2 gene intron 1 is a template, uses Primer ExpressTM 2.0 software analysis TaqMan primer and probe site, and synthetic by u.s.a. applied biosystem company (ABI) customization.
Detect and use primer:
MFn2 gene rs873457 upstream primer sequence: 5 '-CAGGACCTTGTGCAACATAC-3 ' (SEQ ID NO 2)
MFn2 gene rs873457 downstream primer sequence: 5 '-GCCACCCTAATCTTACTGCTCA-3 ' (SEQ ID NO3)
Fluorescent probe:
MFn2 gene rs873457 fluorescent probe 1:5 '-VIC-AGCACACGTCAAAAACT-TAMRA-3 ' (SEQ ID NO4)
MFn2 gene rs873457 fluorescent probe 2:5 '-FAM-AGCACACGTGAAAAACT-TAMRA-3 ' (SEQ ID NO5)
Three, pattern detection:
Experiment detects 907 routine hypertension cases and 471 routine normal control crowds altogether, and every example is collected the about 2ml of blood sample sample, and with phenol/chloroform drawer genomic dna, the extracting result detects with micro-ultraviolet spectrophotometer (INFINIGEN company).
Carry out the fluorescent PCR amplification by following system, at last with SDS 2.3 scanning and cluster analyses.
| 384 orifice plates (ul) | |
| TaqMan?EXPress?Master?Mix(2X) | ?2.5 |
| 20X working stock of SNP Genotyping Assay | ?0.25 |
| GDNA (about 3ng/ul) | ?2.25 |
| The reaction TV | ?5 |
Four, type detected result:
The detected result of genome DNA extraction:
The genomic dna coincidence detection of all samples requires (260/280＞1.8, concentration＞10ng/ul, the detected result that No. 661, sample as shown in the table)
The detected result of MFn2 gene rs873457 loci gene type
Embodiment 2
Detect the rs873457 site of essential hypertension tumor susceptibility gene MFn2 gene with PCR sequencing PCR.Selecting each 10 example of above-mentioned hypertension case-control group sample checks order and judges the genotype of rs873457.
One, experimental technique
The PCR sequencing primer still adopts above-mentioned fluorescent PCR to use primer, the directly order-checking of the purified back of the product of amplification.The instrument of order-checking is the 3130xl genetic analyzer of ABI company, analyzes with sequence analysis 5.2 analysis software, and the result also can check with chromas.
Two, experimental result
Finally, the gene type assay result of the sequencing result of 20 examples and 7900 fluorescent PCRs is in full accord.
Three, the association analysis of MFn2 gene rs873457 genotype and hypertension susceptible
The relatively employing RxC χ that MFn2 gene rs873457 distributes in hypertensive patient and contrast
2Check. carry out statistical study with SPSS software, detected result and SPSS software analysis result are as shown in the figure:
All documents of mentioning in the present invention are all quoted in this application and are done reference, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can make various changes or modification to the present invention, these forms fall within the application's appended claims institute restricted portion equally.
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Claims (6)
1. the detection method of a hypertension susceptibility gene; It is characterized in that; Detect the genotype in Mfn2 gene rs873457 site; Specifically comprise step: (a) genomic dna of extracting sample, amplification obtains to comprise in the Mfn2 gene intron 2 zone in rs873457 site, and amplified production is based on SEQ ID NO:6; (b) genotype of mononucleotide polymorphism site rs873457 in detection step (a) product.
2. detection method as claimed in claim 1 is characterized in that, described rs873457 site is positioned at Mfn2 gene intron 2.
3. detection method as claimed in claim 2 is characterized in that, the primer sequence in zone that comprises the rs873457 site in the amplification Mfn2 gene intron 2 like SEQ ID NO 2 with shown in the SEQ ID NO 3.
4. detection method as claimed in claim 2 is characterized in that, the genotypic probe sequence that detects rs873457 is shown in SEQ ID NO 4 and SEQ ID NO 5.
5. a test kit that detects hypertension susceptible gene is characterized in that, it contains and site rs873457 bonded probe, and the sequence of this probe is shown in SEQ ID NO:4 and SEQ ID NO:5.
6. test kit as claimed in claim 5 is characterized in that, it also comprises the primer that comprises the zone in rs873457 site in the specific amplification Mfn2 gene intron 2, and the sequence of this primer is shown in SEQ ID NO:2 and SEQ ID NO:3.
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| CN102586408B (en) * | 2011-01-12 | 2015-08-19 | 首都医科大学附属北京安贞医院 | The detection method of hypertension susceptible gene Mfn2 mononucleotide polymorphism site rs3820189 and detection kit |
| CN102732602A (en) * | 2011-04-14 | 2012-10-17 | 首都医科大学附属北京安贞医院 | Method for detecting gene rs2255390 site of hypertension susceptible gene WNK1 and detection kit thereof |
| CN105349632A (en) * | 2015-10-23 | 2016-02-24 | 首都医科大学附属北京安贞医院 | Gene variant of primary hypertension susceptibility gene Mfn2 and detection method thereof |
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| CN101103124A (en) * | 2004-11-19 | 2008-01-09 | Oy朱里拉布有限公司 | Method and kit for detecting a risk of essential arterial hypertension |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101103124A (en) * | 2004-11-19 | 2008-01-09 | Oy朱里拉布有限公司 | Method and kit for detecting a risk of essential arterial hypertension |
| CN101608219A (en) * | 2008-06-20 | 2009-12-23 | 上海主健生物工程有限公司 | Hypertension genetic test kit |
| CN101671723A (en) * | 2008-09-11 | 2010-03-17 | 上海市高血压研究所 | Haplotype consisting of single nucleotide polymorphism of kynureninase gene |
Non-Patent Citations (1)
| Title |
|---|
| Yurii S Aulchenko等人.Genetic variation in the KIF1B locus influences susceptibility to multiple sclerosis.《Nature Genetics》.2008,第40卷(第12期),1402-1403. * |
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