CN105349632A - Gene variant of primary hypertension susceptibility gene Mfn2 and detection method thereof - Google Patents
Gene variant of primary hypertension susceptibility gene Mfn2 and detection method thereof Download PDFInfo
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Abstract
The invention discloses a gene variant of primary hypertension susceptibility gene Mfn2 and a detection method thereof, discloses a single nucleotide locus related to primary hypertension susceptibility and discloses a method for detecting single nucleotide polymorphism of primary hypertension related gene-mitochondrial fusion gene 2 (Mfn2) or hyperplasia suppressor gene (HSG). The method includes: detecting genotype of a region between a -2824 locus and a -2828 locus in a 5' end non-coding region of the mitochondrial fusion gene 2 (Mfn2) or hyperplasia suppressor gene (HSG) of an individual. The invention further discloses a corresponding detection kit.
Description
Technical field
The present invention relates to molecular biology and medical field.More particularly, relate to human mitochondrion fusion gene 2(Mitofusin2, Mfn2gene) single nucleotide polymorphism (Singlenucleotidepolymorphism, SNP) in region and the detection with essential hypertension dependency thereof between 5 ' end non-coding region-2824 to-2828 sites.The invention still further relates to the test kit detecting this SNP site.
Background technology
Essential hypertension (Essentialhypertension, EH) be a kind of common and multiple, by multifactor, the polygene disease of environment and heredity factor common pathogenetic, great impact is caused on human health.Along with the development of molecular medicine, the hypertension relative gene had been found that at present has 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 completely.EH is the coefficient result of gene and environment, and the change 30%-60% of blood pressure is owing to heredity.Because environmental factors can control and confirm, and be changeless to hypertension genetic factor.Therefore, to the control of variable factor, 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 but be drastically influence to a certain extent to hypertension incidence, Diagnosis and Treat.Therefore to the research of hypertension genetic very necessary (Wang Zuoguang, Wen Shaojun, Wu Zhaosu. hypertension, tumor susceptibility gene and single nucleotide polymorphism [J]. hypertension magazine, 2001,9:259-264).
Nearly more than two decades comes, and the research about hypertension therapeutic concentrates on the control to blood pressure and the protection to target organ more, and obtains the development of advancing by leaps and bounds.But, these means fundamentally can not control blood pressure, also little to the research of mankind's endogenous hypertension gene at present, therefore, Study on Endogenous hypertension mechanism is one of Main way of hypertension prevention and control research from now on, has important researching value and application prospect, if by regulating endogenous hypertension mechanism Hypertension, the development of China's bio-medical technology will be promoted, and save huge medical expense every year for China.
The hypertension relative gene found at present, sum up and get up may be summarized to be short shr gene and hypertension gene, Mfn2 belongs to the latter.The predecessor of Mfn2 is the intelligent grade of Chinese scholar Chen Guang [ChenGH, ZhangCH, ZhuYQ, etal.Expressionofanovelgenerelatedtohypertension [J] .NatlMedJChina, 1997,77:823-828.] in 1997 to the spontaneous hypertensive rats (SHR) cultivated and normal Wistar Kyoto(WKY) vascular smooth muscle cell of rat carries out differential disply, clone a new gene---hyperplasia suppressor gene (hyperplasiasuppressorgene, HSG).It includes the base pair of 4160bp, 661 amino acid (NM_014874, GeneBankAccessU41803) of encoding altogether.[ChenKH is found by a series of In vitroandin vivotrial, GuoXM, DalongMa, etal.DysregulationofHSGtriggersvascularproliferativediso rders [J] .NatureCellBiol, 2004,6,872-883.], HSG can suppress Ras-Raf albumen-mitogen-activated kinase genetic expression significantly, and the expression of antioncogene can be activated, the propagation of suppression of cell cycle and suppression various kinds of cell effectively, and this restraining effect is realized by apoptotic mode.Therefore, HSG gene is one of candidate gene of essential hypertension, shows by gene functional research, and HSG gene participates in mitochondrial fusion, and therefore definite designation is mitochondrial fusion gene 2 (Mitofusin2, Mfn2).
Because Mfn2 gene main feature in primary hypertension patient and spontaneous hypertensive rat blood vessel and white corpuscle occurs expressing declining, this should be that exception has appearred in its expression regulation, but less to the research of Mfn2 gene transcription regulation aspect at present.5 ' end non-coding region is in gene transcription start site upstream, promotor and central transcription factor Present site, 5 ' end non-coding region-2824bp, to the region of-2828bp(from " A " of initiator codon upwards the 2824th site to 2828 sites, is between the 18th and 19 Nucleotide in SEQIDNO:1) making a variation just in time is in this position.We find that when carrying out the bioinformatic analysis of sequence this site is positioned at Mfn2 gene 5 ' end non-coding region, and are in relatively conservative region.In addition, this variant sites is the potential combining site of some very important transcriptional regulator Dfd etc., from transcriptional control angle, has very important value, thus may have very important significance.
In sum, in order to finally realize effecting a radical cure essential hypertension, this area is in the urgent need to furtheing investigate the pathogenesis of essential hypertension, find essential hypertension tumor susceptibility gene, filter out and there is high risk primary hypertension patient, exploitation detects method, the test kit of essential hypertension tumor susceptibility gene, and relevant medicine etc.
Summary of the invention
The object of this invention is to provide a kind of method and the detection kit that detect hypertension susceptible gene Mfn2 pleomorphism site.
The invention provides a kind of can by the method for the detection prediction of hypertension onset risk of hypertension susceptibility gene, namely detect the genotype between site, Mfn2 gene 5 ' end non-coding region-2824 to-2828.For its hypertensive onset risk of genotypic individuality of delAAC variation is far above general population between 5 ' end non-coding region-2824 to-2828 sites.
5 ' described end non-coding region-2824, to-2828 sites, is arranged in 5 ' end non-coding region (between 11977357 to 11977361 sites in Chromosome1-NC_000001.11 region) of Mfn2.Wherein, thymus nucleic acid (DNA) sequence numbering: the region between 5 ' end non-coding region-2824 to-2828 sites is based on SEQIDNO:1; Primer 1 is based on SEQIDNO:2; Primer 2 is based on SEQIDNO:3; Amplified production is based on SEQIDNO:4.
Specifically, the method comprising the steps of: the genomic dna of (a) extracting sample, and amplification obtains Mfn2 gene 5 ' end non-coding region; B () comprises the region between 5 ' end non-coding region-2824 to-2828 sites by specific primer amplification acquisition Mfn2 gene, amplified production is SEQIDNO:4; C in () detecting step (b) product, mononucleotide polymorphism site 5 ' holds the genotype in region between non-coding region-2824 to-2828 sites.
The primer sequence of described amplification Mfn2 gene 5 ' end non-coding region is as shown in SEQIDNO2 and SEQIDNO3.
The technology such as the order-checking related in aforesaid method, amplification, extracting genomic dna all can adopt the conventional practices of this area.
The invention provides a kind of test kit detecting hypertension susceptible gene, it contains the primer that can comprise and comprise region between-2824 to-2828 sites in specific amplification Mfn2 gene 5 ' end non-coding region.
In one embodiment of the invention, and comprise 5 ' in specific amplification Mfn2 gene 5 ' end non-coding region and hold the sequence of the primer in region between non-coding region-2824 to-2828 sites as shown in SEQIDNO:2 and SEQIDNO:3.
The present invention shows after deliberation, demonstrate region between end non-coding region-2824 to-2828 sites, Mfn2 gene mononucleotide polymorphism site 5 ' first (to be arranged in Mfn25 ' and to hold non-coding region, 11977357 to 11977361 sites in Chromosome1-NC_000001.11 region m-/AAC) closely related with hypertension, and having found its New function: between end non-coding region-2824 to-2828 sites, Mfn2 gene mononucleotide polymorphism site 5 ', region (between 11977357 to 11977361 sites in Chromosome1-NC_000001.11 region) genotypic change will cause hypertensive onset risk to raise.Wherein association study result display, site, Mfn2 gene 5 ' end non-coding region-2825 to-2827-there is significant difference (p<0.05) in the distribution of/AAC in case and control group, namely in primary hypertension patient, this genotype main manifestations is delAAC variation, and this genotype is mainly AAC in Normal group, this SNP change can change Binding site for transcription factor.Complete the present invention on this basis.
The invention provides a kind of method that the hypertension susceptible of individuality is diagnosed, it comprises step: the genotype in region between the site, Mfn2 gene 5 ' end non-coding region-2824 to-2828 detecting this individuality, judges that whether this individuality suffers from hypertensive onset risk higher than general population with this.
In a preference, described difference is the single nucleotide polymorphism being selected from 5 ' end site, non-coding region-2824.Region between 5 ' end non-coding region-2824 to-2828 sites is arranged in Mfn2 gene 5 ' end non-coding region (between 11977357 to 11977361 sites in Chromosome1-NC_000001.11 region).Wherein, the region between 5 ' end non-coding region-2824 to-2828 sites is based on SEQIDNO:1.
The invention provides and a kind ofly detect the method whether sample exists the single nucleotide polymorphism of Mfn2 gene, comprise step:
A (), with the genomic dna of Mfn2 gene 5 ' end non-coding region primer amplified sample, obtains amplified production; With
B () is detected mononucleotide polymorphism site 5 ' in amplified production and is held the genotype in region between non-coding region-2824 to-2828 sites.
The detailed sequence of Mfn2 gene can be see accession number: NG_007945.1, and between 5 ' end non-coding region-2824 to-2828 sites, the nucleotide sequence in region can see network address: http://www.ncbi.nlm.nih.gov/.
The present inventor checks order to (11977357 to 11977361 sites in Chromosome1-NC_000001.11 region m-/AAC) region in 5 ' end non-coding region in Mfn2 gene.
Between 5 ' end non-coding region-2824 to-2828 sites of Mfn2 gene, the genotype in region can by existing technology as Southern blotting, DNA sequence analysis, PCR and in situ hybridization detect sudden change.
The invention discloses a site relevant to essential hypertension susceptibility.Provide a kind of method detecting essential hypertension susceptible gene Mfn2 polymorphism, it comprises detection line mitochondrial fusion gene 2(Mfn2) 5 ' end non-coding region-2824 to-2828 sites between the genotype in region.The invention also discloses corresponding detection kit, this test kit contains the primer that region between non-coding region-2824 to-2828 sites is held in amplification 5 ', can also comprise the primer of amplification Mfn2 gene 5 ' end non-coding region.The present invention is utilized to detect the genotype in region between 5 ' end non-coding region-2824 to-2828 sites, method is simple, rapidly and efficiently, with low cost, for the detection of this gene polymorphism sites and the prediction of Prevalence of Essential Hypertension risk provide a simple and direct new way.
Accompanying drawing explanation
Fig. 1 is through and uses the primer of this patent by products therefrom sequencing result sectional drawing after pcr amplification.Show the genotypic sequencing result of a kind of SNP in region between 5 ' end non-coding region-2824 to-2828 sites.Region between 5 ' end non-coding region-2824 to-2828 sites is arranged in Mfn2 gene 5 ' end non-coding region (between 11977357 to 11977361 sites in Chromosome1-NC_000001.11 region).Wherein A is Normal group forward sequencer map, and B is primary hypertension patient group forward sequencer map.As can be seen from forward sequencer map, the AAC base of Normal group lacks in essential hypertension group.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition as people such as Sambrook, molecular cloning; Condition described in laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989), or according to the condition that manufacturer advises.
Embodiment 1
The region between non-coding region-2824 to-2828 sites is held with 5 ' of sequencing detection essential hypertension tumor susceptibility gene MFn2 gene.Select each 30 examples of above-mentioned hypertension case-control group sample and carry out the genotype that region between non-coding region-2824 to-2828 sites is held in order-checking judgement 5 '.
One, experimental technique
PCR sequencing primer still adopts above-mentioned PCR primer, the purified rear direct Sequencing of product of amplification.The instrument of order-checking is the 3130xl genetic analyzer of ABI company, and analyze with sequenceanalysis5.2 analysis software, result chromas also can check.
Two, experimental result
Sequencing result sectional drawing as shown in Figure 1.
The all documents mentioned in the present invention are quoted all in this application and are made reference, and are just quoted separately as a reference as each section of document.In addition should be understood that read of the present invention above-mentioned tell about content after, those skilled in the art can make various change or amendment to the present invention, these forms fall within the application's appended claims limited range equally.
Claims (9)
1., with the closely-related DNA fragmentation of essential hypertension, it is characterized in that described DNA fragmentation comprises the sequence as shown in SEQIDNO.1.
2. mitochondrial fusion gene 2(Mfn2) 5 ' end non-coding region-2824 to-2828 sites between region polymorphism preparation detect hypertension susceptible reagent in purposes.
3. purposes as claimed in claim 2, it is characterized in that region between site, described Mfn2 gene 5 ' end non-coding region-2824 to-2828 be positioned at 11977357 to 11977361 sites in Chromosome1-NC_000001.11 region m-/AAC.
4. purposes as claimed in claim 2 or claim 3, is characterized in that the delAAC sudden change in region between site, described Mfn2 gene 5 ' end non-coding region-2824 to-2828 increases the risk of essential hypertension.
5., for detecting a test kit for hypertension susceptible, it is characterized in that described test kit comprises and can comprise the primer that 5 ' holds region between non-coding region-2824 to-2828 sites by specific amplification Mfn2 gene.
6. test kit as claimed in claim 5, the region that it is characterized in that between site, described Mfn2 gene 5 ' end non-coding region-2824 to-2828 be positioned at 11977357 to 11977361 sites in Chromosome1-NC_000001.11 region m-/AAC.
7. the test kit as described in claim 5 or 6, is characterized in that described primer sequence is as shown in SEQIDNO:2 and SEQIDNO:3.
8. the test kit as described in claim 5 or 6, is characterized in that obtaining Mfn2 gene by described primer specificity amplification comprises the DNA fragmentation that 5 ' holds region between non-coding region-2824 to-2828 sites; And detecting the Nucleotide in region between 5 ' end non-coding region-2824 to-2828 sites in the DNA fragmentation obtained, the delAAC sudden change in described site increases the risk of essential hypertension.
9. test kit as claimed in claim 8, is characterized in that the method for the Nucleotide detecting region between site, Mfn2 gene 5 ' end non-coding region-2824 to-2828 is selected from: Southern blotting, DNA sequence analysis, PCR and in situ hybridization.
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| CN107058537A (en) * | 2017-04-18 | 2017-08-18 | 首都医科大学附属北京安贞医院 | Primary hypertriglyceridemia tumor susceptibility gene ANGPTL8 variant sites and its detection method and purposes |
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