CN1661052A - Relationship between endothelin 1 gene and essential hypertension - Google Patents

Abstract

A process for testing the susceptibility of primary hypertension includes detecting if there are variations in endotheline (gene EDN1, transcript and protein of an individual and determining its high susceptibility if there are. Its reagent kit is also disclosed.

Description

Relationship between endothelin 1 gene and essential hypertension

technical field

The present invention relates to the fields of molecular biology and medicine. More specifically, it involves the single nucleotide polymorphism (single nucleotide polymorphism, SNP) of the endothelin 1 gene (Endothelin 1, EDN1) and its correlation with essential hypertension. The present invention also relates to methods and kits for detecting these SNPs.

Background technique

Hypertension refers to a group of clinical syndromes with elevated systolic or diastolic blood pressure. There is an obvious causal relationship between the increase of blood pressure and the occurrence of coronary heart disease, renal dysfunction, hypertensive heart disease and hypertension complicated with stroke. The latest diagnostic criteria for hypertension are systolic blood pressure ≥ 19kpa (140mmHg) or diastolic blood pressure ≥ 12kpa (90mmHg), and those who meet one of them can be diagnosed as hypertension.

Most hypertensive patients only have mild subjective symptoms in the early stage of high blood pressure, such as headache, dizziness, insomnia, tinnitus, irritability, difficulty in concentrating work and study, and fatigue. With the development of the disease, especially when complications occur, the symptoms gradually increase and become more obvious, such as numbness and stiffness of fingers, pain in the lower limbs when walking more, or muscle pain and tension in the neck and back. When there is palpitation, shortness of breath, chest tightness, and precordial pain, it indicates that the heart has been involved. When there is nocturnal frequent urination, polyuria, and light urine, it indicates that the kidneys are involved and the renal arterioles are hardened. If a hypertensive patient suddenly develops confusion, deep and irregular breathing, and incontinence, etc., it may indicate cerebral hemorrhage. If one side of the body gradually becomes inconvenient, numb or even paralyzed, it should be suspected whether there is a cerebral thrombosis.

There were no obvious abnormal signs in the early stage of hypertension. Abnormal signs may appear when the brain, heart, kidney and other vital organs are slightly damaged. Common heart abnormalities include leftward shift of the apical beat, lifting-like pulsation sensation in the precordial area, enhanced first heart sound in the apical area, enhanced second heart sound in the aortic valve area, and systolic murmurs and diastolic murmurs. Arteriosclerosis and left ventricular hypertrophy, and a galloping rhythm in the apex may indicate heart failure. In addition, earlobe creases, capillary pulsation, hard or pulseless radial artery, and intermittent claudication of the lower extremities are also common.

In addition, due to certain predisposing factors or the development of hypertension itself, some hypertensive patients may have a significant or sudden increase in blood pressure, accompanied by damage to the brain, heart, kidney, retina and other important organs, which is seriously life-threatening, and a series of Special clinical signs are called hypertensive emergencies. The incidence of hypertensive emergencies accounts for 5% of the hypertensive population, common hypertensive encephalopathy, cerebral hemorrhage, acute left heart failure, clonidine acute withdrawal syndrome, acute myocardial infarction, rapidly progressive malignant hypertension, etc.

About 5% of hypertensive patients have no symptoms, do not know when blood pressure rises, and do not know when complications of blood vessel and organ damage have occurred. have high blood pressure. Therefore, finding out the genetic causes of hypertension, early prevention and detection can effectively control the incidence of hypertension and minimize the damage of the disease to the human body.

Essential hypertension (essential hypertension, EH), also called hypertension, is an independent disease with its own etiology, occurrence, development, outcome, and clinical manifestations. Clinically, it is mainly manifested as an increase in arterial blood pressure. Accounting for more than 90% of hypertensive patients in the population, the pathogenesis is not fully understood at present, and it is mainly diagnosed as essential hypertension (hypertension) only after the hypertension caused by other diseases has been ruled out. The increase in arterial blood pressure is mainly due to the increase in the resistance of peripheral small arteries, and at the same time there are varying degrees of increase in blood volume and cardiac output. In the late stage, the heart, brain, kidney and other organs are often involved, and serious complications such as hypertension, heart disease, heart failure, renal dysfunction, and cerebral hemorrhage occur. The treatment of essential hypertension is mainly to lower blood pressure while preventing the occurrence of complications. The causes of death in patients with essential hypertension are cerebrovascular accident, cardiovascular accident and renal insufficiency. In my country, cerebrovascular accident is more common, followed by heart failure and uremia. In European and American countries, heart failure is more common, and cerebrovascular accident is more common. and uremia followed.

As the most common cardiovascular disease, the incidence of essential hypertension is increasing year by year, and it can cause serious heart, brain, and kidney complications. It is a major risk factor for stroke and coronary heart disease. EH is a polygenic disease caused by the interaction of genetic factors and environmental factors. Finding EH-related genes and clarifying the genetic mechanism of hypertension has become a research hotspot.

Although there have been many studies on the relationship between various gene polymorphisms and essential hypertension, there is no report confirming the correlation between the EDN1 gene and essential hypertension, and there is no confirmation that the SNP of the EDN1 gene is associated with essential hypertension sex reports.

To sum up, in order to finally realize the treatment of hypertension, there is an urgent need in this field to find essential hypertension susceptibility genes, and to develop methods, kits, and related therapeutic drugs for detecting essential hypertension.

Contents of the invention

The object of the present invention is to provide a method and detection kit for diagnosing (especially early diagnosis) hypertension.

Another object of the present invention is to provide a new method for treating hypertension.

Another object of the present invention is to provide a pharmaceutical composition for treating hypertension.

In a first aspect of the present invention, a method of diagnosing susceptibility to hypertension in an individual is provided, comprising the steps of:

detecting the individual's EDN1 gene, transcript and/or protein, and comparing it with normal EDN1 gene, transcript and/or protein,

A difference indicates that the individual is more likely to have high blood pressure than the normal population.

In another preferred example, the method detects the gene or transcript of EDN1, and compares the difference with the normal EDN1 nucleotide sequence.

In another preferred example, the difference is the following single nucleotide polymorphism:

10868 bit C→T;

Wherein, the nucleotide position numbers are based on SEQ ID NO:1.

In the second aspect of the present invention, there is provided a method for detecting whether there is a single nucleotide polymorphism of the endothelin 1 gene EDN1 in a sample, comprising the steps of:

(a) amplifying the EDN1 gene of the sample with EDN1 gene-specific primers to obtain an amplified product; and

(b) Detect whether the following single nucleotide polymorphisms exist in the amplification product:

10868 bit C → T;

Wherein, the nucleotide position numbers are based on SEQ ID NO:1.

In another preferred example, the gene-specific primers have the sequences of SEQ ID NO: 2 and 3.

In another preferred example, the length of the amplified product is 100-3000bp, and contains the 10868th position in SEQ ID NO:1.

In the third aspect of the present invention, a kit for detecting hypertension is provided, which includes primers for specifically amplifying the EDN1 gene or transcripts, more preferably, the amplified length of the primers is 100-3000bp, And contain the amplified product at position 10868 in SEQ ID NO:1.

In another preferred embodiment, the kit also contains reagents selected from the following group:

(a) a probe that binds to the mutation at position 10868 in SEQ ID NO: 1;

(b) Recognition of the mutant restriction enzyme at position 10868 in SEQ ID NO:1.

In another preferred example, the mutation is selected from the following single nucleotide polymorphisms:

10868 bit C → T;

Wherein, the nucleotide position numbers are based on SEQ ID NO:1.

In the fourth aspect of the present invention, a pharmaceutical composition is provided, which contains a safe and effective amount of EDN1 protein and a pharmaceutically acceptable carrier.

Detailed ways

After intensive and extensive research, the inventors have determined and analyzed the SNPs of a large number of candidate genes. For the first time, it was discovered and proved that some SNPs of the EDN1 gene are closely related to high blood pressure, and its new function was discovered: the change of EDN1 will lead to high blood pressure. There is a significant difference (P<0.05) in the distribution of T) between the case and the control group, so it can be used as a specific SNP for detecting hypertension. The present invention has been accomplished on this basis.

EDN1 gene

The detailed sequence of the endothelin 1 gene (Endothelin 1, EDN1) can refer to the nucleotide sequence that the accession number is J05008 (can refer to the website http://www.ncbi.nlm.nih.gov/), such as SEQ ID NO: 1.

The endothelin (also known as "endothelin") gene belongs to a family of polypeptides whose structure and function are relatively well understood. In 1989, Inoue et al. identified this peptide family in humans and sequenced it. There are three members in the family, respectively named endothelin 1, 2, 3 (endothelin1, 2, 3).

Endothelin 1 is a polypeptide composed of 21 amino acid residues secreted by vascular endothelial cells. It has a strong vasoconstrictive effect (Inoue et al. Proc. Net. Acad. Sci. 86: 2863-2867, 1989). This polypeptide was first isolated from pig vascular endothelial cells and proved to have a strong vasoconstrictive effect. In subsequent work, the vascular endothelial cells in the human body also obtained the peptide, and confirmed that it was identical to the peptide found in pigs. In addition, in addition to the effect of constricting blood vessels, the EDN1 polypeptide also has an effect on the central nervous system and the excitation of nerve cells.

In 1989, Giaid et al. found the expression of endothelin 1 gene mRNA in spinal cord and posterior root nerve cells (Giaid et al. Proc. Nat. Acad. Sci. 86: 7634-7638, 1989). In 1996, Maemura et al. also detected the mRNA of the endothelin 1 gene in the lungs of adult mice (Maemura et al. Genomics 31: 177-184, 1996).

Since endothelin 1 has a strong and long-lasting effect of constricting blood vessels, endothelin 1 has a strong effect on the cardiovascular system. The effect of endothelin 1 on blood vessels is to maintain basic vascular tension and regulate vascular caliber; the effect on the heart is positive intropic effects, chronotropic effects, and active potential duration , APD), influence on coronary circulation, influence on cardiac output, and influence on signal transduction in cardiomyocytes, etc. In addition, endothelin 1 also plays a role in the regulation of water balance, as well as the respiratory, digestive and reproductive systems.

Endothelin 1 has a strong regulating effect on blood pressure, mainly manifested in: it can strongly contract blood vessels, significantly increase peripheral resistance; promote smooth muscle cell proliferation, increase blood vessel tension; form myocardial hypertrophy and have positive inotropic force on the myocardium effect; enhance the effect of vasoconstrictor substances; and weaken the effect of vasodilation caused by endothelin 1 receptor activation. Through these specific physiological regulation effects, endothelin 1 can cause the increase of blood pressure.

The inventors sequenced almost the entire region of the EDN1 gene and found many SNPs, most of which were not associated with susceptibility to hypertension, but association studies showed that position 10868 C→T was associated with susceptibility to hypertension Very high SNP. The SNP is located at the 5′ terminal region of EDN1, suggesting that it may have a certain impact on the transcription level of EDN1, which in turn affects the interaction between prostaglandin E and receptors, and ultimately leads to a significantly higher susceptibility to hypertension in carriers than in normal populations.

Based on the new discovery of the present invention, the EDN1 protein or polypeptide has many new uses. These uses include (but are not limited to): screening substances that promote the function of EDN1 protein, such as antibodies, polypeptides or other ligands. Screening the polypeptide library with expressed recombinant human EDN1 protein can be used to find therapeutically valuable polypeptide molecules that can stimulate the function of human EDN1 protein.

On the other hand, the present invention also includes polyclonal antibodies and monoclonal antibodies specific to human EDN1 DNA or polypeptides encoded by its fragments, especially monoclonal antibodies. Here, "specificity" means that the antibody can bind to human EDN1 gene product or fragment. Preferably, it refers to those antibodies that can bind to human EDN1 gene products or fragments but do not recognize and bind to other irrelevant antigen molecules. Antibodies in the present invention include those molecules capable of binding and inhibiting human EDN1 protein, as well as those antibodies that do not affect the function of human EDN1 protein.

The present invention includes not only complete monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) ₂ fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules; or chimeric antibodies.

Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, purified human EDN1 gene product, or antigenic fragments thereof, can be administered to animals to induce polyclonal antibody production. Similarly, cells expressing human EDN1 protein or antigenic fragments thereof can be used to immunize animals to produce antibodies. Various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant and the like.

Antibodies of the invention may also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology. The antibodies of the present invention include antibodies capable of blocking the function of human EDN1 protein and antibodies that do not affect the function of human EDN1 protein. Various types of antibodies of the present invention can be obtained by conventional immunization techniques using fragments or functional regions of human EDN1 gene products. These fragments or functional regions can be prepared using recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to unmodified forms of the human EDN1 gene product can be produced by immunizing animals with gene products produced in prokaryotic cells (e.g., E. coli); antibodies that bind to post-translationally modified forms (such as glycosylated or phosphorylated Proteins or polypeptides), which can be obtained by immunizing animals with gene products produced in eukaryotic cells (such as yeast or insect cells).

The antibody against human EDN1 protein can be used in immunohistochemical techniques to detect the amount and/or mutation of human EDN1 protein in biopsy specimens. A preferred anti-EDN1 antibody is an antibody that does not recognize normal EDN1 but recognizes mutant EDN1, or an antibody that recognizes normal EDN1 but not mutant EDN1. Using these antibodies, the detection of hypertension susceptibility at the protein level can be conveniently performed.

Using the EDN1 protein of the present invention, substances that interact with the EDN1 protein, such as inhibitors, agonists or antagonists, can be screened out through various conventional screening methods.

When the EDN1 protein of the present invention and its antibody, inhibitor, agonist, antagonist, etc. are administered (administered) therapeutically, various effects can be provided. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intravenous, or subcutaneous administration.

Antagonists of EDN1 can be used directly in the treatment of diseases, for example, in the treatment of hypertension. When using the antagonist of the EDN1 protein of the present invention, other drugs for treating hypertension can also be used at the same time.

The present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the antagonist of the EDN1 protein of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 0.1 microgram/kg body weight to about 10 mg/kg body weight per day. In addition, the polypeptides of the invention can also be used with other therapeutic agents.

When using the pharmaceutical composition, the antagonist of the EDN1 protein is administered to the mammal in a safe and effective amount, wherein the safe and effective amount is usually at least about 0.1 microgram/kg body weight, and in most cases no more than about 10 mg/kg body weight , preferably the dosage is about 0.1 microgram/kg body weight to about 100 microgram/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

Polynucleotides of human EDN1 protein can also be used for various therapeutic purposes. Gene therapy technology can be used to treat cell proliferation, development or metabolic abnormalities caused by non-expression of EDN1 protein or expression of abnormal/inactive EDN1 protein. The method for constructing a recombinant viral vector carrying the EDN1 gene can be found in existing literature (Sambrook, et al.). In addition, the recombinant human EDN1 gene can be packaged into liposomes and then transferred into cells.

The methods for introducing polynucleotides into tissues or cells include: directly injecting polynucleotides into tissues in the body; or first introducing polynucleotides into cells in vitro through vectors (such as viruses, phages, or plasmids, etc.) , and then transplant the cells into the body, etc.

The invention also relates to a diagnostic test method for quantitative and localized detection of human EDN1 protein level. These assays are well known in the art and include ELISA and the like.

A method for detecting whether there is an EDN1 protein in a sample is to use a specific antibody for the EDN1 protein to detect, which includes: contacting the sample with an EDN1 protein specific antibody; observing whether an antibody complex is formed, and the formation of an antibody complex means that EDN1 protein is present in the sample.

The polynucleotide of EDN1 protein can be used for the diagnosis and treatment of EDN1 protein-related diseases. In terms of diagnosis, the polynucleotide of EDN1 protein can be used to detect the expression of EDN1 protein or the abnormal expression of EDN1 protein in a disease state. For example, EDN1 DNA sequence can be used for hybridization of biopsy specimens to determine the abnormal expression of EDN1 protein. Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These technical methods are all open and mature technologies, and relevant kits are available from commercial sources. Part or all of the polynucleotides of the present invention can be immobilized as probes on microarrays or DNA chips (also known as "gene chips") for analysis of differential expression of genes in tissues and gene diagnosis. EDN1 protein transcripts can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification with EDN1 protein-specific primers.

Detection of mutations in the EDN1 gene can also be used to diagnose high blood pressure. Detection can be against cDNA or genomic DNA. The mutated forms of EDN1 protein include point mutations, translocations, deletions, recombinations, and any other abnormalities compared with the normal wild-type EDN1 DNA sequence. Mutations can be detected using established techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene has a mutation.

The most convenient method for detecting the SNP of the present invention is to amplify the EDN1 gene of the sample with EDN1 gene-specific primers to obtain the amplified product; then detect whether the following single nucleotide polymorphism exists in the amplified product: 10868 position C → T, wherein the nucleotide position numbering is based on SEQ ID NO:1.

It should be understood that after the present invention first revealed the correlation between the SNP of the EDN1 gene and hypertension, those skilled in the art can easily design an amplification product that can specifically amplify the position of the SNP, and then through methods such as sequencing Determine if 10868 bits C→T exist. Usually, the length of the primer is 15-50bp, preferably 20-30bp. Although it is preferred that the primer is completely complementary to the template sequence, those skilled in the art know that it can also be specifically amplified (that is, only amplify the desired fragment). Kits containing these primers and methods using these primers are within the scope of the present invention, as long as the amplified product amplified by the primers contains the corresponding position of the SNP of the present invention. A preferred primer pair has the sequences of SEQ ID NO: 2 and 3.

Although the length of the amplified product is not particularly limited, generally the length of the amplified product is 100-3000 bp, preferably 150-2000 bp, more preferably 200-1000 bp. These amplified products should all contain the 10868th position in SEQ ID NO:1.

Because the SNP of the present invention has a very high correlation with hypertension, it can not only be used for early and more accurate diagnosis of essential hypertension, but also can take precautions to make the carrier take reasonable preventive measures before the onset of the disease (such as in the Therefore, it has extremely important application value and social benefits.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions suggested conditions.

Example 1

1.1 Research object

Since blood pressure is a quantitative trait that is affected by multiple genes and is affected by various environmental factors, it is necessary to consider the issue of genetic heterogeneity when analyzing related genes. Selecting isolated populations with a relatively single genetic background as materials for the study of polygenic diseases will help reduce the impact of genetic heterogeneity. reduce. Selecting such a group, whether it is used for linkage disequilibrium analysis or selecting families for family analysis, will help to improve the detection sensitivity of hypertension susceptibility loci. Therefore, in this embodiment, the isolated population is selected as the research object (the isolated population has the advantages of relative consistency of genetic background and relatively small number of founders).

On the basis of informed consent, 324 individuals of Han nationality over the age of 50 were randomly collected from Xiangchang Town, Dabie Mountain, Yuexi County, Anhui Province. 80% of individuals have only 6 surnames, the number of founders should be relatively small, and individuals of the same gender come from the same founder. The selected hypertensive samples require that the systolic blood pressure is not lower than 140mmHg, and the diastolic blood pressure is not lower than 90mmHg, and the blood pressure is measured twice to take the average value.

11% of them are located at the top of the blood pressure distribution (37 individuals; SBP>178mmHg) and 7% are located at the bottom of the blood pressure distribution (22 individuals; SBP<104mmHg), a total of 59 individuals were selected by direct sequencing method Perform SNP detection.

1.2 Experimental methods and results

1.2.1 DNA extraction

DNA was extracted from human blood by the conventional phenol-chloroform method, and the concentration was corrected to 20ng/ul for conventional PCR amplification.

1.2.2 Design of PCR and sequencing primers

According to the genome sequence of EDN1 in GenBank, the following primers were designed and synthesized. The specific primers are shown in Table 1 below.

Table 1 Primer sequence list Primer name Sequence (5'-3') SEQ ID NO: sense primer tcctgcttctccttcacctg 2 antisense primer ggcaaagtggagaggaagtg 3

1.2.3 PCR amplification of EDN1 gene

Using the extracted DNA as a template, PCR amplification was performed on a GeneAmp 9700 PCR instrument with the Touchdown program using Taq enzyme. The reaction conditions are: pre-denaturation at 94°C for 2 minutes, denaturation at 94°C for 30 seconds, annealing at 63°C for 40 seconds, extension at 72°C for 40 seconds, a total of 10 cycles, and the annealing temperature decreases by 0.5°C for each cycle; after denaturation at 94°C for 30 seconds, Anneal at 58°C for 40 seconds, extend at 72°C for 40 seconds, a total of 30 cycles; finally extend at 72°C for 7 minutes. PCR amplification products were verified by agarose gel electrophoresis.

As a result, an amplification product of 400 bp was obtained.

1.2.4 SNP discovery and detection

After the PCR products were purified by Resin resin, ABI-PRISM ^TM 377 DNA sequencer (appliedbiosystems (ABI) of the United States) was used for bidirectional sequencing of fluorescently labeled terminal termination method, and Polyphred software (University of Washington, USA http://droog. mbt.washington.edu/Polyphred.html) for sequence interpretation and SNP confirmation.

As a result, the following SNP was found to exist: 10868 position C→T.

1.2.5 SNP genotyping and association analysis

SNP genotyping by direct one-way sequencing. That is, typing and correlation analysis were carried out in hypertensive patients and normotensive control groups.

The chi-square test was performed after the allele frequency was calculated. By comparing the individual data of the highest 11% with the lowest blood pressure of 7%, it was preliminarily determined whether the allele frequency was linked to the blood pressure level.

It was found that the frequency of 10868 T-type SNPs in SEQ ID NO: 1 was 27% in hypertensive individuals and 13.6% in hypotensive individuals. The frequency difference between the two is 13.4%, which confirms that there is a correlation between the 10868 C/T type SNP in SEQ ID NO: 1 and the occurrence of hypertension.

Example 2

Essential Hypertension Susceptibility Detection Kit

As described in Example 1, the mutation of C→T at position 10868 in SEQ ID NO: 1 is closely related to hypertension. Therefore, specific primers for the EDN1 gene can be designed based on this mutation and then amplified using the patient's DNA as a template for detection.

Prepare a test kit (100 person-times), which contains: name sequence concentration sense primer SEQ ID NO: 2 100pmol antisense primer SEQ ID NO: 3 100pmol PCR reaction solution Containing Taq Enzyme dNTP Magnesium Ion PCR Reaction Buffer

Take 3ml of blood from the male patient to be tested, and use a conventional method (or use a specific kit) to extract DNA from the blood. Dilute the PCR primers in the high blood pressure detection kit to 1μmol/μl, and use the extracted DNA as a template to perform a PCR reaction with the provided primers. After the PCR products were purified, ABI-PRISM ^TM 377 DNA sequencer was used for bidirectional sequencing by fluorescence-labeled end termination method, and Polyphred software was used for sequence interpretation and SNP confirmation.

Alternatively, the chromatographic analysis of the amplified product and the normal control is performed with a denaturing high-performance liquid chromatograph (DHPLC), and the 10868 position C→T can also be detected.

As a result of the test, the susceptibility to hypertension of the subjects including 10868 C→T subjects was higher than that of the normal population.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Sequence Listing

<110> Fudan University

  Shanghai Human Genome Research Center

<120>Relationship between endothelin 1 gene and essential hypertension

<130>040173

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<170>PatentIn version 3.1

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gccacagcaa actttctatt ggggaaaaaa aaaaatcctc ctctttacat ctctcccttc 480

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ctgaggggtg gggtggggct atgaagctat ccttcatatt cactcctttg tccagctctt 600

ttcacctcta gttcttctcc ccgcatctct gtctagcagt gccttaagtg gaggaggggt 660

gggggcatca agcttgtaaa actggtttgt tggggttctc cttctcccct catttcttga 720

ttcttgggaa aatgtcttgc tgggaggctg cctggcgagt gccctagctg ccttctgtgg 780

gcttgaatgg ggcttccctc tgcccctaca ggaggaaaag ggagctgctg ccagaggggag 840

aaatggagag atggacagag aaggcaggtg ccaccccctcg cccctgacac acaaagaaaa 900

agacacggaa attctctctc tctcttctct tctcctatct ctctctctct ccctctctct 960

ctctctctct ctctctctca cacacacaca cacacacaca cacacacaca caggcgcgcg 1020

ccgcgcgcgc aggcacacgt cttgcaaatt caggattcaa agagacaggg gcaccattat 1080

atttggcacg gtggggcctt ccaggtctga aatcctgcat tcttccttac tatttacttt 1140

ccccgagctc gagaagggcc aggtgtgggc ggatggctgg ccacgttttg tgtttccaat 1200

tcatattcac gggatgacac agacggggcg tggtgagtgc tgttggaggc gcttgggcag 1260

tttcattttg ccccacttct ccacctgaag gctgggcgtt gctggaacct gcaggggcag 1320

cctcagcaag gtggggtggc gtggagtggg gtgggagaag ggactccagc tgaagtagaa 1380

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tgaggacatg ttggtcctga ctgttgtcag tgtttggtca aagttgccaa aaggttaaaa 1500

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gagtgttggg ggggaggcgc tgcttggaac ctgtgaatgt gacatcagct ctcctctcct 1620

ctcccaaggt cggctttgga gagggaggtc agggcaccct tgcctggcac aggcacgctg 1680

gcttccggct cagtgccgcc tgctctccgg gagctgtgcg ctccctgggc cccggggcta 1740

ggctgaggta agcgcacagc ggaggccagg cgcgccggca gaggcctggg ggatagggtg 1800

gaggcatctc tgggtgtggg tgtgggtgtg ggtgtgggag ggagagttct tgcctctctc 1860

tctcccatct ccaactcttg cttcagtggc tcttttagag gatgcatgtc attatggacc 1920

tgtcgctgcc actgtccctg ttcccccagc tgtgacttcg aggggaggtct ggggatctga 1980

gtctgtccaa accacggct ttgctgttgg gataaaaact gtccttttga ttttagaagg 2040

aggagggaaa aaaggtttcc cagcatgtgt gttgtgccag tcttggaaat tcatccgtgc 2100

ttgaattcca ccctccatcc ccagaaaaac tggagtaaaa caaaaagagg agatggacaa 2160

agtgtgtatt tgatggcatc ccctgggaag agactctaaa tttatcccat aggtcttact 2220

gggccactgt gagcgctttg gtggagaaca aacaaaaatt ctgggtgctc agttgtctaa 2280

cctgaaaaat gggactagcg gaaaaagcca atgtgttcca tgcacctttt gctttcttta 2340

ttaaggcatg atgtcacctg tacagtaact gccctgtgtg tacttcaggg ggggatttca 2400

aggttagata gacaggaaat tgttttgaaa atgtaaacac atttattaaat gtgaagtatt 2460

atctgattcc ttgttcgaat ggcatttcct tctcagcacc accttccttg catattcact 2520

taaccttgta caagaacacc tttttgccct aaatgaagac acccccccaa aaaaaagagt 2580

cccagaaaat atgtccctgc ttgtgcggga ataaatagaa tattctgagg tgcattcctc 2640

cttcctatgt taggcaacat tccttgaccc tcctcggccc ccaagccagg ttgcgttttt 2700

ttctgccatt tagaagggtt ttcctttttg tcctagtaaa acatcagccc ctgtagctct 2760

tcatctcccc ctggtgttct tctcccgcca tgtcttaaga ttggtggcac cgaccaatct 2820

taagattaa gttctgtgtg aaaaacacct ttgcttttca atcagtttat cagcctcctc 2880

cgcaggggaa tgtggcacacaaaagaact tatcggggct tctcatcagt gatagggaaa 2940

agactggcat gtgcctaaac gagctctgat gttattttta agctcccttt cttgccaatc 3000

cctcacggat ctttctccga tagatgcaaa gaacttcagc aaaaaagacc cgcaggaagg 3060

ggcttgaaga gaaaagtacg ttgatctgcc aaaatagtct gacccccagt agtgggcagt 3120

gacgaggggag agcattccct tgtttgactg agactagaat cggagagaca taaaaggaaa 3180

atgaagcgag caacaattaa aaaaaattcc ccgcacacaa caatacaatc tattaaact 3240

gtggctcata cttttcatac caatggtatg actttttttc tggagtcccc tcttctgatt 3300

cttgaactcc ggggctggca gcttgcaaag gggaagcgga ctccagcact gcacgggcag 3360

gtttagcaaa ggtctctaat gggtattttc tttttcttag ccctgccccc gaattgtcag 3420

acggcgggcg tctgcttctg aagttagcag tgatttcctt tcgggcctgg cttatctccg 3480

gctgcacgtt gcctgttggt gactaataac acaataacat tgtctggggc tggaataaag 3540

tcggagctgt ttaccccccac tctaataggg gttcaatata aaaagccggc agagagctgt 3600

ccaagtcaga cgcgcctctg catctgcgcc aggcgaacgg gtcctgcgcc tcctgcagtc 3660

ccagctctcc accgccgcgt gcgcctgcag acgctccgct cgctgccttc tctcctggca 3720

ggcgctgcct tttctccccg ttaaagggca cttgggctga aggatcgctt tgagatctga 3780

ggaacccgca gcgctttgag ggacctgaag ctgtttttct tcgttttcct ttgggttcag 3840

tttgaacggg aggtttttga tccctttttt tcagaatgga ttatttgctc atgattttct 3900

ctctgctgtt tgtggcttgc caaggagctc cagaaacagg taggcacgct cgttgacttg 3960

taagtctcgg aattacaagt taggtgtgttc ttatccacct tcatgctttt cttgcttcta 4020

tttttccccg ttctttttat gactgcagct tagagagcaa gtgtctgaga attattgctg 4080

aaacgtactt taagtcttct agtgtaaaat gtaaaattcc tctactgaat acaattaggt 4140

gcaattgact ataacatgac attaaaataa cttatcgttt tattattatt attccattat 4200

gtgtttcctt ggcttttaaa aaatgagaag agtatggaca tatacaattt agtcaaatgt 4260

atgtttgtaa tatatgtgtt tatacaggta cacaggccat ataggaactt aaatcttatt 4320

taaacactat tttaatagtg tgttaacgtg taaaatattt aagcattcca gcttgaagcc 4380

aaggaattgt atccagtcgt tcaagcaatg tatgttcagt aaaatcacct gcagagcaaa 4440

agtctgttga ctaactaccg cctcccccgc ccccccacca ccccccgcag gcggtttctg 4500

ggtgaagcag atgttttctt taaaatttgt catcattgac tttaggtttc ttttggcagg 4560

tttttggcac ccaaaacagt gtgagctctc ttttcagctt tattcacctg tgctgggagg 4620

ggagctagga taattcttgg ctgccgaagg atttaggcag tgcgtgtgca tctgcccggg 4680

tcccccccgt ttttagggtc agtgcacttt ttttgtcttt tcgtgaccct gactaaagag 4740

aaaggatgtc aagggaatga aaatcctgga atgtgtctga tcatttgaaa tgtacaaaat 4800

tgggcagata agctgcatgg ctaaattgtt aggaggaaga ggcaaggcag tagtggagaa 4860

gggggaggca gtggatccca cacaagcctg atgcccaggg attcggaatt caaaatcccc 4920

ccagcctacc ttcagtcccc tgacctgctt ctcagcccca ccttaggtca ctggtttcta 4980

tggagttacc ctactgaatt gaatattgaa tagttaattt ctctctccaa tcattttccc 5040

cacctaattt tgaaagatat acatcatctg gggtaccctg tgccctacac agcatgtgaa 5100

gtggatgggt acccccctaaa gagaggtca tcctgaatgg ggaagtggcc ccaaagctag 5160

gaataactgt gatttcttgt ctttagtcat gtgccaatgt taagtaagct tcagtggata 5220

gtgctgtcct accaagttcc ttgtagaagc cagccggatt ttcaacaggc agcattccac 5280

agcatttccc tgagcctgct tcaagagggg tgggggaagt cccttttcag gtgtttatct 5340

cctctgcatt tgtgtaatct ccctgaaggt ggataagcca agggcatgag ggggaggcaa 5400

aaggtgaact catgttaagg agggaaaaaa ataaagagcc cttttttctg tgtttcttgc 5460

tgatggcagg ctgtgtgctt catctgcttt tatctgctct gctagctctg actctactgt 5520

gatccagcat gtctctcggc gtttgaggag acatccccca ctgacctgct ctttctctcc 5580

ccagcagtct taggcgctga gctcagcgcg gtgggtgaga acggcgggga gaaacccact 5640

cccagtccac cctggcggct ccgccggtcc aagcgctgct cctgctcgtc cctgatggat 5700

aaagagtgtg tctacttctg ccacctggac atcatttggg tcaacactcc cgagtaagtc 5760

tctagagggc attgtaaccc tattcattca ttagcgctgg ctccactgga gcccagtttt 5820

agagtttctt ttctagggac tctgaaggta gtccttctaa caccatccaa gtgcctcagt 5880

ggggacagtt tccctctatt cctgaaaata acgacagctt cgttcttagc aaccaagggg 5940

agggtcttct gaggccccgt agctcaggct actcatgatg ggacaagcag gaggccactg 6000

cacgtttcaa atgaggaact ttcagtgaga gggcctcagg gggacactct cacagtggca 6060

tctgatgggg tttcgggaat aattgccgag gtcagatgtg ggttagtgca acctgtgctt 6120

ctcatgggag ggtggagact gagaggcaga agtgatgata tagagggtta gaatcactta 6180

attttagtta cagaaaaacc taggctcaaa gtgttgaagc catttgtgca ggagtgagtt 6240

tgtagcagag ctagaactgg agcccggatt tcctttgctg ctatattttc cctttagaaa 6300

tgcccatttc agaactgaaa tagaaatact gtccataggc ttctctttca cctacagaga 6360

agaaaagcag atttcctcct tctgccctgg acactagttc atcatctgtc ggaagcagtc 6420

ataaacaagc acacatttac tatgcataca atgtaccgtt atgacaaagg aggaccaaaa 6480

tccaaacaat atcaaaccac accaaaaacc acaaggagcc taataattac taaggtgata 6540

cttccaaagg gaggacttta tttcttagat gagaatgaaa atggacacat tggaaattat 6600

tggagagccc tctggctatg agtccttcca caaccatatg gtaccaccga ctggcaggag 6660

aaatgtgtga acatgtgcct cctctcccca accactgggg ccggtggggt gacggtggca 6720

cttttagcag tatcctccgt ggtttgagtt gaaaataagt tttaaaaatc ctgtgagtca 6780

tggttttgca ttgaaacctc ttcccactgt gtacccacaa atagttaact aaatagacca 6840

ttagaaaagg aagaaaatat aaagcagatg ccaagcagag atgtcctaat ttttgacaaa 6900

aaagcaatgt tgcttgtgtc aagaagaaac tgaactttgt gaagagttga aatggaattc 6960

cactgaatta gaaaaacttg ttttctcctg cctggataca tacagtcagg gccattgatg 7020

cacaggtgtt cctggctgtt gttacacttt accctctgaa atgatgctcc caagtgctat 7080

gtgatgagct ccttgtgtgc ccagtggaat aggtgtgtcc atgtgtcatt ttaaagacta 7140

ttaattacac taatatagtt tctttctctc tttggataat aggcacgttg ttccgtatgg 7200

acttggaagc cctaggtcca agagagcctt ggagaattta cttcccacaa aggcaacaga 7260

ccgtgagaat agatgccaat gtgctagcca aaaagacaag aagtgctgga atttttgcca 7320

agcaggaaaa gaactcaggt gagcagaaac acctttgctt ttcaatcagt ttaacagcct 7380

cctgaactcc ttcctatcat ggtactgcct tcctgtttta gagagactaa cagagacatt 7440

gaaagtcagg gtaaagctga atataacatt gctgaaatgt ttttccttgt gtattttaac 7500

agggctgaag acattatgga gaaagactgg aataatcata agaaaggaaa agactgttcc 7560

aagcttggga aaaagtgtat ttatcagcag ttagtgagag gaagaaaaat cagaagaagt 7620

tcagaggaac acctaagaca aaccaggtaa gagggaagga agaaaaatta ggtaagaggt 7680

tcacaagaac aactagcccc agtcagtgat gccagcagcc tgttcctcca gcccttctta 7740

cccgggcagg tgaaagactt agaaaacagt agcagaggag atctatgcat cttatagatt 7800

aaaaggagca aaagaatccc tcttaaatat ttccatgaag ctctggaatg caaaccgatg 7860

tcctctgtac ctttagcaca taccatttca tctacaggta gatttcccaa ccaaaatata 7920

tccagagatg cctttgtcat tgggttatat acagcctttg cctctctgag tcaatgtatt 7980

taccactttc cctgagaaat cgaaaatcat tttggggagc ggacatttag aaaaagaatc 8040

aaagtgtcat ggataatcaa attcttcaat aagttgcagt tattcagatg gccaaaggaa 8100

aaataaagtc attagatagg gttggtagaa tttagaacat gctgtttttc aggtttatgg 8160

tctttttttt tttttttttt tttttttaaa tagggaaatg tgtttggtgc agagccaatg 8220

tcattccaaa aagctctctc ttttcctggt cagtcatgtg ctgggacaga gaagggatct 8280

ggattaggca acatcataga gttgctctga gctgctcttt ggtgataacc cttccaaatc 8340

ctaaactttt tggaattcac aagctcaaag gaggaaacct actctctgat ctaccacatg 8400

ttctgcattt ttctatcatg gtctatggaa acttctctta gaaatccagt ggcaagaagt 8460

tctatgatta aagtgttctg agctcaggcc aggcagtcat gaactacttc tgagttgttt 8520

actactgatt tgtggggcag cctcagctat cggtttcttc acacctgctt atgagagtat 8580

ccatattt ggtcgcaggc agtaatgctc cccacgagat cagtttctga actaacctgg 8640

aattttttat gggtttttat tatgccaact attaaatcaa cattacagtt cttccctctg 8700

tatttctcct gtaaaacatt aggcctgcaa aaaaaaaaaa tctttttaaa aataattgcc 8760

ataaagtat ttgctctgggc ctactgtatg cttcttttyt ttttctctct tttcaactaa 8820

gtcaccgtca atttattaag atggccataa ctattcaaaa cctatgctga gttcctcaag 8880

gcagggtcgc atagtgatga aggttgggat ggggctacgg aagaaaccag aacaactcta 8940

gtttattaa aacctgtatt tactgcccac ttccccttag acttgaccat atgacccctt 9000

gctccccatt ctaagcatag gggcaggctt tatttttaca atggtaatag atgatatcac 9060

ttgaggtttt atcaaagagt tgcggcgggt ggtgaaagtt cacaaccaga ttcaggtttt 9120

gtttgtgcca gattctaatt ttacatgttt cttttgccaa agggtgattt ttttaaaata 9180

acatttgttt tctcttatct tgctttatta ggtcggagac catgagaaac agcgtcaaat 9240

catcttttca tgatcccaag ctgaaaggca agccctccag agagcgttat gtgacccaca 9300

accgagcaca ttggtgacag accttcgggg cctgtctgaa gccatagcct ccacggagag 9360

ccctgtggcc gactctgcac tctccaccct ggctgggatc agagcaggag catcctctgc 9420

tggttcctga ctggcaaagg accagcgtcc tcgttcaaaa cattccaaga aaggttaagg 9480

agttccccca accatcttca ctggcttcca tcagtggtaa ctgctttggt ctcttctttc 9540

atctggggat gacaatggac ctctcagcag aaacacacag tcacattcga attcgggtgg 9600

catcctccgg agagagagag aggaaggaga ttccacacag gggtggagtt tctgacgaag 9660

gtcctaaggg agtgtttgtg tctgactcag gcgcctggca catttcaggg agaaactcca 9720

aagtccacac aaagattttc taaggaatgc acaaattgaa aacacactca aaagacaaac 9780

atgcaagtaa agaaaaaaaa aagaaagact tttgtttaaa tttgtaaaat gcaaaactga 9840

atgaaactgt tactaccata aatcaggata tgtttcatga atatgagtct acctcaccta 9900

tattgcactc tggcagaagt atttcccaca tttaattatt gcctccccaa actcttccca 9960

cccctgctgc cccttcctcc atcccccata ctaaatccta gcctcgtaga agtctggtct 10020

aatgtgtcag cagtagatat aatattttca tggtaatcta ctagctctga tccataagaa 10080

aaaaaagatc attaaatcag gagattccct gtccttgatt tttggagaca caatggtata 10140

gggttgttta tgaaatatat tgaaaagtaa gtgtttgtta cgctttaaag cagtaaaatt 10200

attttccttt atataaccgg ctaatgaaag aggttggatt gaattttgat gtacttattt 10260

ttttatagat atttatattc aaacaattta ttccttatat ttaccatgtt aaatatctgt 10320

ttgggcaggc catattggtc tatgtatttt taaaatatgt atttctaaat gaaattgaga 10380

acatgctttg ttttgcctgt caaggtaatg actttagaaa ataaatattt ttttccttac 10440

tgtactgatt tggaatcatt actgaaattt gtaaggagtg ggccaacgtg attaagtacc 10500

ataaaggcaa ataaatggtt aaagacggtt tcatagaaaa gtgacaatta gaaggatatt 10560

acggtctaag ctaattatat aaagaatttt atctgtatct taaatgttga ttttatactg 10620

cattgaggta aaaacacaaa acaaaaaagc agctttaaca cctctgtctt ctcttgggta 10680

gcagcctcct gcttctcctt cacctgaaaa attctccagg gacttcatcc attaacttgg 10740

ctcaggctat tggcaggatt cacagtttaa gctgatggtg tggtgagaga tgctttatcc 10800

atattaatgg actgaaggaa gtaatggcaa gacaaccccc caaaacatac ctaattatac 10860

aaagttacat accaaagttg cttttagaaa atggcctgct cagagcaagt agaggtttcc 10920

aatggctttt tattttctca cattaaggat gttgtttctt aaggaacatt gagtaccat 10980

gcttcttcgt gatagcctag gactgccgtg tgcccatgga ggtagagaca ccaggtactg 11040

attctaggtc ctctgccaca aagcaccact tcctctccac tttgccttgg ctggccttgt 11100

cagctcactg gagagcacag tattgcaatt gcagtattgc aaatggtcac tactaactga 11160

attctctaag agcttgatta gccctcgaga atcttccttg cccttctcta atagtgtctg 11220

aaggaattcc tggcatttaa caaatattag catgtagtga tcactgtcgt cctaacagtg 11280

acacatcaga aggatttcaa ataacagtct tcaggcatgc gtaatcaatg tcctgtgcag 11340

agtctccgtc ctcattgatc ctcatttttc tctttaaggc acagtccaat gtctttgggg 11400

aattgtttat aaagcttact ttatccataa actgtttctc agtgcgtgac tctgaagaaa 11460

attttgaagt tttgcccatg ttgacaaggt gcttggtctg aacttggcca gtatttaatc 11520

ttgagcaaac gattcaattt ccttctatcg tgagttttct catctatgaa acaagggagt 11580

tgaggggagt ttctttcata cctctgagaa agagtttgag attacataaa gaagttgaag 11640

tggcatgaaa aaaaataaag atctgagctt agaagacatg gatctaatac atttaagagg 11700

aagtcagaat cagagaagcc actgaacaaa acagtccaaa cggagcatag taagtcagat 11760

tgatgagttt tggttgggtt tttcatcagt caaacccttg agcccccctt tcccatgctt 11820

cctgcttcag tatccagtag gaaaaatgaa agggatgatg tagacactct agggcatgag 11880

gatttgcagt aaataagttg ggagactcac agaaaattaa tatttttcaa acatgaagac 11940

gaaacattca attatattac agtccacatc agcttgaagg gtaaactgat gggatgatct 12000

gtcacatttc ttgctctgtt tccagtaaaa gcatggtttc tggaaaccca cttagcaag 12060

ctttctctct ttacactgat agcccaggca agctttgatc tcagaactcc agaaaccaga 12120

gaactctagg tggaatgtgg taacttttgc cagggcagag ggaacaccta ctaataggta 12180

cttcatttgc accaccagag attggcatct tttttgatgg atccactggc tttgatactg 12240

cctgtactcc cccaaaacac agcttgggta ttggactaat ctagagctcc ctcaggagaa 12300

ctcttgctga cattaagaaa gagcaacatt ttgtctttcc aggtgaaaat ccaaggccaa 12360

aaagggagtg actcacctaa gatcacagaa ggagctgtag catctctgga gcctgaacac 12420

ttaagttaag cacgactatt tcacgcagag ggcatgaatt c 12461

<210>2

<211>20

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>2

tcctgcttct ccttcacctg 20

<210>3

<211>20

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>3

ggcaaagtgg agaggaagtg 20

Claims (10)

1. whether a vitro detection sample exists the method for the single nucleotide polymorphism of endothelin 1 gene EDN1, it is characterized in that, comprises step:

(a) with the EDN1 gene of EDN1 gene-specific primer amplification sample, obtain amplified production; With

(b) detect whether there is following single nucleotide polymorphism in the amplified production:

10868 C → T;

Wherein, the nucleotide position numbering is based on SEQ ID NO:1.

2. the method for claim 1 is characterized in that, described gene-specific primer has the sequence of SEQID NO:2 and 3.

3. the method for claim 1 is characterized in that, the length of described amplified production is 100-3000bp, and contains among the SEQ ID NO:1 the 10868th.

4. one kind is detected hypertensive test kit, it is characterized in that it comprises the primer of specific amplification EDN1 gene or transcript, and it is 100-3000bp that described primer amplification goes out length, and contains among the SEQ ID NO:1 the 10868th amplified production.

5. test kit as claimed in claim 4 is characterized in that, it also contains the reagent that is selected from down group:

(a) with SEQ ID NO:1 in the 10868th sudden change bonded probe;

(b) the 10868th sudden change restriction enzyme among the identification SEQ ID NO:1.

6. test kit as claimed in claim 5 is characterized in that, described sudden change is following single nucleotide polymorphism:

10868 C → T;

Wherein, the nucleotide position numbering is based on SEQ ID NO:1.

7. test kit as claimed in claim 4 is characterized in that described primer has the sequence of SEQ ID NO:2 and 3.

8. method that the hypertension susceptibility of individuality is diagnosed is characterized in that it comprises step:

Detect this individual EDN1 gene, transcript and/or albumen, and compare with normal EDN1 gene, transcript and/or albumen,

There are differences and just show that this individuality suffers from hypertensive possibility and be higher than normal population.

9. method as claimed in claim 8 is characterized in that, detection be gene or the transcript of EDN1, and with normal EDN1 nucleotide sequence comparing difference.

10. method as claimed in claim 9 is characterized in that, described difference is following single nucleotide polymorphism:

10868 C → T;

Wherein, the nucleotide position numbering is based on SEQ ID NO:1.