CN101892298B - Method for detecting mononucleotide polymorphism rs2236055 locus of hypertension susceptibility gene and kit thereof - Google Patents

Abstract

The invention belongs to the field of molecular biology and medicine, relating to a method for detecting primary hypertension susceptibility and a kit thereof. The method for detecting primary hypertension susceptibility comprises the following step of: detecting the genotype of the rs2236055 locus of a mitochondrion fusion gene 2(Mfn2)/a hyperplasia suppressor gene (HSG) of an individual. The hypertension susceptibility of the rs2236055 individual with an genotype A is remarkably higher than that of the general public. The invention also discloses the corresponding detection kit. The kit contains a primer for amplifying a primer of a region in the second intron of the Mfn2 gene. The genotype of the rs2236055 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

The detection method of mononucleotide polymorphism rs 2236055 locus of hypertension susceptibility gene and test kit thereof

Technical field

The present invention relates to molecular biology and medical field.Relate more specifically to human mitochondrion fusion gene 2 (Mfn2 gene) single nucleotide polymorphism (single nucleotide polymorphism, SNP) rs 2236055 locus 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] .NatlMed 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 suppressor gene, HSG).It includes the base pair of 4160bp, 661 amino acid of encoding altogether (NM_014874, GeneBank Access U41803).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 rs2236055 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

Purpose of the present invention just provides a kind of method and detection kit of new detection hypertension susceptible gene.

Particularly, the invention provides a kind of detection method of hypertension susceptibility gene mononucleotide polymorphism site, it comprises the genotype that detects Mfn2 gene rs2236055 site; Rs2236055 obtains the dependency of described gene and essential hypertension with the genotypic individuality of A.

Detected result of the present invention shows that its hypertensive susceptibility of individuality that carries said gene is significantly higher than the general population.

Rs2236055 of the present invention is arranged in No. 2 intron zone of Mfn2.Better, described rs2236055 is positioned at 6575958 positions of fragment contig NT_021937.18.

The invention provides the genotypic method of single nucleotide polymorphism in No. 2 intron zone of a kind of Mfn2 of detection gene, the method may further comprise the steps:

(a) genomic dna of the primer amplified sample in No. 2 intron zone of usefulness Mfn2 gene obtains amplified production;

(b) genotype of mononucleotide polymorphism rs 2236055 locus in the detection amplified production.

In the aforesaid method, the sequence of the primer in No. 2 intron zone of amplification Mfn2 gene is shown in SEQ ID NO:2 and SEQ ID NO:3.The sequence of the probe of detection rs2236055 is shown in SEQ ID NO:4 or SEQ ID NO:5.

Accordingly, the invention provides a kind of test kit that detects hypertension susceptible gene, it comprises the primer in No. 2 intron zone of specific amplification Mfn2 gene, and its sequence is shown in SEQ ID NO:2 and SEQ ID NO:3.It can also contain the probe of being combined with rs2236055, and its sequence is shown in SEQ ID NO:4 or SEQ ID NO:5.This test kit can also comprise used conventional damping fluid, dNTP and the specification sheets of PCR reaction, etc.

In the present invention, provide a kind of method that the hypertension susceptibility of individuality is diagnosed, it comprises step:

Detect the genotype in this individual Mfn2 gene rs2236055 site, judge that with this this individuality suffers from hypertensive onset risk and whether be higher than the general population.

In another preference, described difference is to be selected from following single nucleotide polymorphism:

Rs2236055 is positioned at Mfn2, in the intron 2 (fragment contig contig:NT_021937.18 position 6579628 A/G)

Wherein, thymus nucleic acid (DNA) sequence numbering: the rs2236055 position is based on SEQ ID NO:1.

In a second aspect of the present invention, provide a kind of test sample whether to have the method for the single nucleotide polymorphism 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 rs 2236055 locus in the detection amplified production:

Rs2236055 is positioned at Mfn2, in the intron 2 (fragment contig contig:NT_021937.18 position 6579628 A/G);

Wherein, thymus nucleic acid (DNA) sequence numbering: primer 1 is based on SEQ ID NO:2; Primer 2 is based on SEQ ID NO:3; Probe 1 is based on SEQ ID NO:4; Probe 2 is based on SEQ ID NO:5; Amplified production is based on SEQ ID NO:6.

In another preference, described single nucleotide polymorphism is that rs2236055 is positioned at Mfn2, in the intron 2 (fragment contig contig:NT_021937.18 position 6579628 A/G)

Wherein, thymus nucleic acid (DNA) sequence numbering: the rs2236055 position is based on SEQ ID NO:1.

In another preference, described test kit also contains and is selected from following reagent: Mfn2 intron 2 Auele Specific Primers, the probe of being combined with mononucleotide polymorphism rs 2236055 locus.

Wherein, thymus nucleic acid (DNA) sequence numbering: primer 1 is based on SEQ ID NO:2; Primer 2 is based on SEQ ID NO:3; Probe 1 is based on SEQ ID NO:4; Probe 2 is based on SEQ ID NO:5; Amplified production is based on SEQ ID NO:6.

In another preference, described Mfn2 intron 2 mononucleotide polymorphism sites:

Rs2236055 is positioned at Mfn2, in the intron 2 (fragment contig contig:NT_021937.18 position 6579628 A/G)

Wherein, thymus nucleic acid (DNA) sequence numbering: the rs2236055 position is based on SEQ ID NO:1.

The present invention is through for many years research, find first and proved that Mfn2 gene mononucleotide polymorphism rs 2236055 locus (is arranged in Mfn2 intron 2, fragment contig contig:NT_021937.18 position 6579628A/G) closely related with hypertension, and found its new function: Mfn2 gene mononucleotide polymorphism rs 2236055 locus is positioned at Mfn2, (fragment contig contig:NT_021937.18 position 6579628 A/G) 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 rs2236055G → A in case and control group, this SNP polymorphism can change the transcription factor binding site point.Finished on this basis the present invention.

The detailed sequence of Mfn2 gene can be the nucleotide sequence (can referring to network address http://www.ncbi.nlm.nih.gov/) of rs2236055 referring to accession number.

The inventor checks order to (fragment contig contig:NT_021937.18 position 6579628) zone in the intron 2 in the Mfn2 gene.

The polymorphism in Mfn2 gene rs2236055 site can be directly used in hypertensive personalized treatment.When using the polymorphism in Mfn2 gene rs2236055 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 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, 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 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 rs2236055 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 rs2236055 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 susceptibility, it comprises the genotype that detects individual mitochondrial fusion gene 2 (Mfn2)/hyperplasia suppressor gene (HSG) rs2236055 site, rs2236055 is with the genotypic individuality of A, and hypertensive susceptibility is significantly higher than the general population.The invention also discloses corresponding detection kit, this test kit contains the primer in No. 2 intron zone of amplification Mfn2 gene.Utilize the present invention to detect the genotype in rs2236055 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 has shown that Mfn2 rs2236055 is positioned at Mfn2, the genotypic sequencing result of a kind of SNP of (fragment contig contig:NT_021937.18 position 6579628 A/G) in the intron 2.

Fig. 2 is the detected result of No. 766 sample gene group DNA extracting.

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:ColdSpring 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 (TaqManEXPress Master Mix) is synthetic by Applied biosystems (ABI) customization.

Two, primer and probe design and synthetic:

Take the partial sequence of MFn2 gene intron 2 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 rs2236055 upstream primer sequence: 5 '-TGTGCTCTCCTGGACCTCTG-3 '

MFn2 gene rs2236055 downstream primer sequence: 5 '-GAAGAGCCAGCACAGCAGAGA-3 '

Fluorescent probe:

MFn2 gene rs2236055 fluorescent probe 1:5 '-VIC-CAAGTCTCTTGAATG-TAMRA-3 '

MFn2 gene rs2236055 fluorescent probe 2:5 '-FAM-CAAGTCTCTTGGATG-TAMRA-3 '

Three, pattern detection:

Experiment detects 918 routine hypertension cases and 499 routine normal control crowds altogether, and every example is collected the about 2ml of blood sample, and with phenol/chloroform drawer genomic dna, the extracting result detects with micro-ultraviolet/visible light spectrophotometer (INFINIGEN company).

By carry out the fluorescent PCR amplification such as the system of following table 1, at last with SDS 2.3 scanning and cluster analyses.

Table 1

	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 genomic dna of all samples meets testing requirement (260/280＞1.8, concentration＞10ng/ul, the detected result that No. 766, sample as shown below)

The detected result of genome DNA extraction is as shown in table 2:

The detected result of table 2MFn2 gene rs2236055 loci gene type

All documents of mentioning in the present invention are all quoted in this application and are made reference, just as each piece document is quoted separately as a reference.Should be understood that in addition read of the present invention above-mentioned tell about content after, those skilled in the art can make various changes or modification to the present invention, these forms fall within the application's appended claims limited range equally.

Embodiment 2

Detect the rs2236055 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 rs2236055.

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 rs2236055 genotype and hypertension susceptible

The relatively employing RxC χ2-test,chi-square test that MFn2 gene rs2236055 distributes in hypertensive patient and contrast. carry out statistical study with SPSS software, detected result and SPSS software analysis result are as shown in table 3.

Table 3

SEQUENCE LISTING

＜110〉loyal hospital is pacified in the attached Beijing of the Capital University of Medical Sciences

Fudan University

＜120〉detection method of mononucleotide polymorphism rs 2236055 locus of hypertension susceptibility gene and test kit thereof

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Claims (4)

1. the purposes of probe in the test kit of preparation detection hypertension susceptible gene of the primer in No. 2 intron zone of amplification Mfn2 gene and detection rs2236055 is characterized in that it comprises the genotype that detects Mfn2 gene rs2236055 site; Described detection comprises step:

(a) genomic dna of the primer amplified sample in No. 2 intron zone of usefulness Mfn2 gene obtains amplified production;

(b) genotype of mononucleotide polymorphism rs 2236055 locus in the detection amplified production;

The sequence of the primer in No. 2 intron zone of described amplification Mfn2 gene is shown in SEQ ID NO:2 and SEQ ID NO:3;

The sequence of the probe of described detection rs2236055 is shown in SEQ ID NO:4 or SEQ ID NO:5.

2. purposes as claimed in claim 1 is characterized in that, described rs2236055 is positioned at 6579628 positions of fragment contig NT_021937.18.

3. a test kit that detects hypertension susceptible gene is characterized in that, it comprises the primer in No. 2 intron zone of specific amplification Mfn2 gene, and the sequence of this primer is shown in SEQ ID NO:2 and SEQ IDNO:3.

4. test kit as claimed in claim 3 is characterized in that, it also contains the probe of being combined with rs2236055, and the sequence of this probe is shown in SEQ ID NO:4 or SEQ ID NO:5.

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