CN101892305B - Method for detecting rs2295281 locus of hypertension susceptibility gene and detection kit - Google Patents
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Abstract
The invention belongs to the field of molecular biology and medicine, relating to a method for detecting a hypertension susceptibility gene and a detection kit thereof. The method for detecting a primary hypertension susceptibility gene comprises the following step of: detecting the genotype of the rs2295281 locus of a mitochondrion fusion gene 2(Mfn2)/a hyperplasia suppressor gene (HSG). The hypertension susceptibility of an rs2295281 individual with a genotype C is remarkably higher than that of the general public. The invention also discloses the corresponding detection kit. The detection kit contains a primer for amplifying the rs2295281 locus and can also contain the primer for amplifying a region comprising the rs2295281 locus in an eighth intron of the Mfn2 gene. The genotype of the rs2295281 locus is detected by adopting the method. The invention has simple and easy method, quick speed and high efficiency and low cost and provides a novel shortcut approach for diagnosing and treating 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) single nucleotide polymorphism (single nucleotide polymorphism, SNP) site rs2295281 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 (essential hypertension, EH) is a kind of multifactor, multigenic disease, and the common and multiple cardiovascular disorder by the environment and heredity factor is caused a disease has jointly caused great impact to human health.Along with the development of molecular medicine, the hypertension relative gene that has been found that has at present had more than 150 to plant, 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, such as the prevention to Risk Factors of Hypertension, can delay to a certain extent and prevent hypertensive morbidity, but the understanding deficiency of changeless inherited genetic factors is but being had a strong impact on to a certain extent to hypertension incidence, diagnosis and treatment.Therefore the research of hypertension genetic being learned very necessary (Wang Zuoguang, Wen Shaojun, Wu Zhaosu. hypertension, tumor susceptibility gene and single nucleotide polymorphism [J]. the hypertension magazine, 2001,9:259-264).
Nearly more than two decades comes, and about the research of hypertension therapeutic concentrates on more to the control of blood pressure with to the protection of target organ, and has obtained the development of advancing by leaps and bounds.But, these means can not fundamentally be controlled blood pressure, at present to the research of human endogenous hypertension gene also seldom, therefore, Study on Endogenous hypertension mechanism is one of main direction of from now on hypertension prevention and control research, has important researching value and application prospect, if by regulating endogenous hypertension mechanism Hypertension, will promote the development of China's bio-medical technology, and be the huge medical expense of the annual saving of China.
The hypertension relative gene of finding 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 vascular smooth muscle cell of normal Wistar Kyoto (WKY) rat are carried out the difference demonstration, clone a new gene---hyperplasia suppressor gene (hyperplasia suppressorgene, HSG).It includes the base pair of 4160bp, 661 amino acid of encoding altogether (NM_014874, GeneBank AccessU41803).Find [Chen KH by a series of In vitroandin vivotrials, Guo XM, Dalong Ma, et al.Dysregulationof HSG triggers vascular proliferative disorders[J] .Nature Cell Biol, 2004,6,872-883.], HSG can suppress significantly Ras-Raf albumen-Si and split plain activated protein kinase genetic expression, and can activate the expression of antioncogene, effectively check the propagation of cell cycle and inhibition various kinds of cell, and this restraining effect realizes by apoptotic mode.Therefore the HSG gene is one of candidate gene of essential hypertension, show by gene functional research, the HSG gene participates in mitochondrial fusion, therefore definite designation is that (mitofusin 2 for mitochondrial fusion gene 2, Mfn2) but at present less to the research of Mfn2 and EH, do not confirm the report of rs2295281 and EH dependency.
In sum, for the final treatment hypertension that realizes, this area is in the urgent need to seeking the essential hypertension tumor susceptibility gene, and exploitation detects method, the test kit of essential hypertension tumor susceptibility gene, and relevant medicine.
Summary of the invention
The purpose of this invention is to provide a kind of method and detection kit that detects hypertension susceptible gene.
The invention provides a kind of detection method of hypertension susceptibility gene, namely detect the genotype in Mfn2 gene rs2295281 site, rs2295281 is significantly higher than the general population with the hypertensive onset risk of the genotypic individuality of C.
Described rs2295281 is arranged in the intron 8 (fragment contig contig:NT_021937.19 position 8064144C/T) of Mfn2.Thymus nucleic acid (DNA) sequence numbering: the rs2295281 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 8; (b) genotype of mononucleotide polymorphism site rs2295281 in detecting step (a) product.
The primer sequence of described amplification Mfn2 gene intron 8 is shown in SEQID NO 2 and SEQID NO 3.
The genotypic probe sequence of described detection rs2295281 is shown in SEQ ID NO 4 and SEQID NO 5.
The 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 the probe of being combined with site rs2295281.
The sequence of the probe of being combined with site rs2295281 in one embodiment of the invention, is shown in SEQ ID NO:4 and SEQID NO:5.
The mentioned reagent box can also comprise the primer that comprises the zone in rs2295281 site in the specific amplification Mfn2 gene intron 8.
Comprise the sequence of primer in zone in rs2295281 site shown in SEQ ID NO:2 and SEQ ID NO:3 in one embodiment of the invention, with in the specific amplification Mfn2 gene intron 8.
The present invention is through for many years research, proved that first Mfn2 gene mononucleotide polymorphism site rs2295281 (is arranged in Mfn2 intron 8, fragment contig contig:NT_021937.19 position 8064144C/T) closely related with hypertension, and found its new function: Mfn2 gene mononucleotide polymorphism site rs2295281 is positioned at Mfn2, (fragment contig contig:NT_021937.19 position 8064144C/T) genotypic change will cause hypertensive onset risk to raise in the intron 8, wherein the association study result shows, have significant difference (p＜0.05) in the distribution of Mfn2 gene rs2295281C → T in case and control group, this SNP polymorphism can change the transcription factor binding site point.Finished on this basis the present invention.
The inventive method can be used for the hypertension susceptibility of individuality is carried out early diagnosis, and it comprises step: detect the genotype in this individual Mfn2 gene rs2295281 site, judge that with this this individuality suffers from hypertensive onset risk and whether be higher than the general population.
In a preference, described difference is the single nucleotide polymorphism that is selected from rs2295281.Rs2295281 is positioned at Mfn2, in the intron 8 (fragment contig contig:NT_021937.19 position 8064144C/T).Wherein, thymus nucleic acid (DNA) sequence numbering: the rs2295281 position is based on SEQ ID NO:1/6.
The invention provides the method whether a kind of test sample exists the single nucleotide polymorphism of Mfn2 gene, comprise step:
(a) with the genomic dna of Mfn2 gene intron 8 primer amplified samples, obtain amplified production; With
(b) genotype of mononucleotide polymorphism site rs2295281 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 rs2295281 referring to accession number.
The inventor is to (NT_021937.19 position 8064144C/T) in the intron 8 in the Mfn2 gene) zone checks order.
The polymorphism in Mfn2 gene rs2295281 site can be directly used in hypertensive personalized treatment.When using the polymorphism in Mfn2 gene rs2295281 of the present invention site, also can use simultaneously the hypertensive medicament of other treatment.
The present invention also provides a kind of pharmaceutical composition, and it contains safely and effectively Mfn2 albumen of the present invention and pharmaceutically acceptable carrier or vehicle.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as Tablet and Capsula can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, Tablet and Capsula are made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day the 10 mg/kg body weight of 0.1 microgram/kg body weight-Yue.In addition, polypeptide of the present invention also can use with the other treatment agent.
When making pharmaceutical composition, 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 approximately 10 mg/kg body weight, preferably this dosage is about 100 mg/kg body weight of 0.1 microgram/kg body weight-Yue.Certainly, concrete dosage also should be considered the factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
The genotype that detects the rs2295281 of Mfn2 gene also can be used for office hypertension.Detection can be for genomic dna, also can be for the amplified fragments of Mfn2 gene.The genotype of the rs2295281 of Mfn2 gene can detect sudden change with existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization.
The invention provides a kind of method that detects the essential hypertension susceptible gene, it comprises the genotype in the rs2295281 site of detection line plastochondria fusion gene 2 (Mfn2), rs2295281 is with the genotypic individuality of C, and hypertensive susceptibility is significantly higher than the general population.The invention also discloses corresponding detection kit, this test kit contains the primer in amplification rs2295281 site, can also comprise the primer in No. 8 intron zone of amplification Mfn2 gene.Utilize the present invention to detect the genotype in rs2295281 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 rs2295281 loci gene type.
Fig. 2 is the sequencing result sectional drawing.Wherein, Mfn2rs2295281 is positioned at Mfn2, the genotypic sequencing result of a kind of SNP of (fragment contig contig:NT_021937.19 position 8064144C/T) in the intron 8.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the 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 Laboratory Press, 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 Master Mix) is synthetic by Applied biosystems (ABI) customization.
Two, primer and probe design and synthetic:
Take the partial sequence of MFn2 gene intron 8 as template, use Primer ExpressTM 2.0 software analysis TaqMan primer and probe site, and synthetic by Applied biosystems (ABI) customization.
Detect and use primer:
MFn2 gene rs2295281 upstream primer sequence: 5 '-CCACTTTGCTGGATGCAC-3 ' (SEQ ID NO 2)
MFn2 gene rs2295281 downstream primer sequence: 5 '-AAGTCACCAGTAAGAACCAGT-3 ' (SEQ ID NO 3)
Fluorescent probe:
MFn2 gene rs2295281 fluorescent probe 1:5 '-VIC-CTTCAGTGCCGGTGCT-TAMRA-3 ' (SEQ ID NO 4)
MFn2 gene rs2295281 fluorescent probe 2:5 '-FAM-CTTCAGTGCTGGTGCT-TAMRA-3 ' (SEQ ID NO 5)
Three, pattern detection:
Experiment detects 917 routine hypertension cases and 494 routine normal control crowds altogether, and every example is collected approximately 2ml of blood sample, and with phenol/chloroform drawer genomic dna, the extracting result detects with micro-ultraviolet/visible light spectrophotometer (INFI NIGEN 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 cumulative volume | 5 |
Four, type detected result:
The detected result of genome DNA extraction:
The genomic dna of all samples meets testing requirement (260/280＞1.8, concentration＞10ng/ul, the as shown in drawings detected result of No. 1178, sample).
The detected result of MFn2 gene rs2295281 loci gene type
Embodiment 2
Detect the rs2295281 site of essential hypertension tumor susceptibility gene MFn2 gene with sequencing.Selecting each 10 example of above-mentioned hypertension case-control group sample checks order and judges the genotype of rs2295281.
One, experimental technique
The PCR sequencing primer still adopts above-mentioned fluorescent PCR primer, the purified rear direct Sequencing 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 rs2295281 genotype and hypertension susceptible
The relatively employing RxC x that MFn2 gene rs2295281 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 table:
Sequence table
SEQUENCE LISTING
＜110〉loyal hospital is pacified in the attached Beijing of the Capital University of Medical Sciences
Fudan University
＜120〉detection method and the detection kit in the rs2295281 site of hypertension susceptible gene
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Claims (2)
1. detect the application of primer in the genotypic test kit of preparation detection mononucleotide polymorphism site rs2295281 in the genotypic probe of rs2295281 and the zone that amplification comprises the rs2295281 site, it is characterized in that, detect the genotype in Mfn2 gene rs2295281 site, the method for this detection comprises step:
(a) genomic dna of extracting sample, amplification obtains to comprise in the Mfn2 gene intron 8 zone in rs2295281 site;
(b) genotype of mononucleotide polymorphism site rs2295281 in detecting step (a) product;
Comprise the primer sequence in zone in rs2295281 site in the described amplification Mfn2 gene intron 8 shown in SEQ ID NO 2 and SEQ ID NO 3;
The genotypic probe sequence of described detection rs2295281 is shown in SEQ ID NO 4 and SEQ ID NO 5.
2. a test kit that detects hypertension susceptible gene is characterized in that, it contains the probe of being combined with site rs2295281,
The sequence of the probe of being combined with site rs2295281 is shown in SEQ ID NO:4 and SEQ ID NO:5;
It also comprises the primer that comprises the zone in rs2295281 site in the specific amplification Mfn2 gene intron 8,
Comprise the sequence of primer in zone in rs2295281 site in the specific amplification Mfn2 gene intron 8 shown in SEQ ID NO:2 and SEQ ID NO:3.
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| CN104450690A (en) * | 2014-10-28 | 2015-03-25 | 首都医科大学附属北京安贞医院 | Primary hypertension susceptibility gene variation locus and detecting method thereof |
| CN104450688A (en) * | 2014-10-28 | 2015-03-25 | 首都医科大学附属北京安贞医院 | Hypertension susceptibility relevant gene variation locus and detecting method thereof |
| CN104450689A (en) * | 2014-10-28 | 2015-03-25 | 首都医科大学附属北京安贞医院 | Hypertension susceptibility relevant gene locus and detecting method thereof |
| CN104450884A (en) * | 2014-10-28 | 2015-03-25 | 首都医科大学附属北京安贞医院 | Hypertension susceptibility relevant gene variation locus and detecting method thereof |
| CN105349630A (en) * | 2015-10-23 | 2016-02-24 | 首都医科大学附属北京安贞医院 | Hypertension susceptibility gene locus rs11553681 and detection method thereof |
| CN105349634A (en) * | 2015-10-23 | 2016-02-24 | 首都医科大学附属北京安贞医院 | Gene variation locus related to hypertension susceptibility and detection method of gene variation locus |
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| CN105349636A (en) * | 2015-10-23 | 2016-02-24 | 首都医科大学附属北京安贞医院 | Primary hypertension susceptibility gene variation locus and detection method thereof |
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2010
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