CN1661072A - The relationship between arginine vasopressin receptor 1A gene and essential hypertension - Google Patents

Abstract

A process for testing the susceptibility of primary hypertension includes detecting if there are variations in arginine-vasopressin receptor 1A gene AVPR1A, transcript and/or protein of an individual, and determining its high susceptibility if there are. Its reagent kit is also disclosed.

Description

The relationship between arginine vasopressin receptor 1A 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 (singlenucleotide polymorphism, SNP) of the arginine vasopressin receptor 1A gene (arginine vasopressin receptor 1A, AVPR1A) 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 palpitation, shortness of breath, chest tightness, and precordial pain occur, it indicates that the heart has been involved. When frequent urination, polyuria, and light urine occur at night, 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 becomes inconvenient, numb or even paralyzed gradually, 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 cause 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 on the correlation between AVPR1A gene and essential hypertension, and there is no confirmation that the SNP of AVPR1A 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:

detection of the individual's AVPR1A gene, transcript and/or protein, and comparison with normal AVPR1A 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 AVPR1A, and compares the difference with the normal AVPR1A nucleotide sequence.

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

2408 bits 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 a single nucleotide polymorphism of the arginine vasopressin receptor 1A gene AVPR1A exists in a sample, comprising the steps of:

(a) amplifying the AVPR1A gene of the sample with AVPR1A gene-specific primers to obtain an amplified product; and

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

2408 bits 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 2408th 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 AVPR1A gene or transcripts, more preferably, the amplified length of the primers is 100-3000bp, And contain the amplified product at position 2408 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 2408 in SEQ ID NO: 1;

(b) Recognition of the mutant restriction enzyme at position 2408 in SEQ ID NO:1.

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

2408 bits 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 AVPR1A 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 AVPR1A gene are closely related to hypertension, and its new function was discovered: the change of AVPR1A will lead to hypertension. 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.

AVPR1A gene

The detailed sequence of the arginine vasopressin receptor 1A gene (arginine vasopressin receptor 1A, AVPR1A) can be found in the nucleotide sequence with the accession number U19906 (see the website http://www.ncbi.nlm.nih.gov/ ), as shown in SEQ ID NO:1.

The protein encoded by the AVPR1A gene is a vasopressin receptor protein. This protein belongs to a family of receptor proteins associated with G proteins, which also includes receptor proteins such as AVPR1B, V2R and OXT. When activated, the G protein coupled to this protein will initiate a biochemical process using calcium ions as the second messenger. The AVPR1A gene is involved in many cellular regulatory responses, including cell contraction, cell proliferation, platelet aggregation, lectin release, and glycogen breakdown.

Antidiuretic hormone, also known as vasopressin, is synthesized in a part of neurons in the supraoptic nucleus and paraventricular nucleus of the hypothalamus. The axons of these neurons travel in the hypothalamus-pituitary tract and enter the posterior pituitary gland, and the vasopressin released from their terminals enters the blood circulation as a pituitary hormone. Vasopressin can promote the reabsorption of water in the renal collecting duct, and when it acts on the corresponding receptor of vascular smooth muscle, it will cause the contraction of vascular smooth muscle, which is one of the strongest known vasoconstrictor substances. Vasopressin plays an important role in the regulation of extracellular fluid volume in vivo. In the case of water deprivation, dehydration, blood loss, etc., the release of vasopressin increases, which not only plays an important role in retaining the amount of fluid in the body, but also in maintaining arterial blood pressure.

Under normal circumstances, when the concentration of vasopressin in plasma increases, the antidiuretic effect first appears; only when its plasma concentration is significantly higher than normal, does it cause blood pressure to increase. However, when the antidiuretic hormone receptor is mutated and loses its normal regulatory function, the regulation of blood pressure will be affected. Because the blood pressure will be difficult to increase in time after the gene is mutated, the frequency of the mutant type is higher than that of the original type in people with low blood pressure.

The inventors sequenced almost the entire region of the AVPR1A gene and found many SNPs, most of which were not associated with susceptibility to hypertension. However, association studies showed that 2408 positions C→T were associated with susceptibility to hypertension Very high SNP. The SNP is located in exon 1 of AVPR1A. Although it does not cause amino acid mutations, it may affect the transcription level due to codon preference, which in turn affects the activity of AVPR1A, 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 AVPR1A protein or polypeptide has many new uses. These uses include (but are not limited to): direct use as drugs to treat diseases caused by AVPR1A protein function hypofunction or loss (such as hypertension), and for screening substances that promote AVPR1A protein function, such as antibodies, polypeptides or other ligands. Screening the polypeptide library with the expressed recombinant human AVPR1A protein can be used to find therapeutically valuable polypeptide molecules that can stimulate the function of the human AVPR1A protein.

On the other hand, the present invention also includes polyclonal antibodies and monoclonal antibodies specific to human AVPR1A DNA or polypeptides encoded by its fragments, especially monoclonal antibodies. Here, "specificity" means that the antibody can bind to human AVPR1A gene product or fragment. Preferably, it refers to those antibodies that can bind to human AVPR1A gene products or fragments but do not recognize and bind to other irrelevant antigen molecules. Antibodies in the present invention include those molecules that can bind to and inhibit human AVPR1A protein, and those that do not affect the function of human AVPR1A 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 AVPR1A gene product, or an antigenic fragment thereof, can be administered to an animal to induce polyclonal antibody production. Similarly, cells expressing human AVPR1A 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 AVPR1A protein and antibodies that do not affect the function of human AVPR1A protein. Various antibodies of the present invention can be obtained by conventional immunization techniques using fragments or functional regions of human AVPR1A 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 AVPR1A 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 AVPR1A protein can be used in immunohistochemical techniques to detect the amount and/or mutation of human AVPR1A protein in biopsy specimens. A preferred anti-AVPR1A antibody is an antibody that does not recognize normal AVPR1A but recognizes mutant AVPR1A, or an antibody that recognizes normal AVPR1A but not mutant AVPR1A. Using these antibodies, the detection of hypertension susceptibility at the protein level can be conveniently performed.

Using the AVPR1A protein of the present invention, substances that interact with the AVPR1A protein, such as inhibitors, agonists or antagonists, can be screened out through various conventional screening methods.

When the AVPR1A 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.

Normal AVPR1A can be directly used for disease treatment, for example, for hypertension treatment. When using the AVPR1A protein of the present invention, other agents for treating hypertension can also be used simultaneously.

The present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the AVPR1A 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 safe and effective amount of AVPR1A protein or its antagonist, agonist is administered to mammals, wherein the safe and effective amount is usually at least about 0.1 μg/kg body weight, and in most cases no more than about 10 mg/kg body weight, preferably the dosage is about 0.1 μg/kg body weight to about 100 μg/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 AVPR1A protein can also be used for various therapeutic purposes. Gene therapy technology can be used to treat abnormalities in cell proliferation, development or metabolism due to non-expression of AVPR1A protein or expression of abnormal/inactive AVPR1A protein. The method for constructing a recombinant viral vector carrying the AVPR1A gene can be found in existing literature (Sambrook, et al.). In addition, the recombinant human AVPR1A 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 AVPR1A protein level. These assays are well known in the art and include ELISA and the like.

A method for detecting the presence of AVPR1A protein in a sample is to use a specific antibody for the AVPR1A protein to detect, which includes: contacting the sample with an AVPR1A protein-specific antibody; observing whether an antibody complex is formed, which means that the antibody complex is formed AVPR1A protein is present in the sample.

The polynucleotide of AVPR1A protein can be used for the diagnosis and treatment of diseases related to AVPR1A protein. In terms of diagnosis, the polynucleotide of AVPR1A protein can be used to detect the expression of AVPR1A protein or the abnormal expression of AVPR1A protein in a disease state. For example, AVPR1A DNA sequence can be used for hybridization of biopsy specimens to determine the abnormal expression of AVPR1A 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. RNA-polymerase chain reaction (RT-PCR) in vitro amplification with AVPR1A protein-specific primers can also detect the transcripts of AVPR1A protein.

Detection of mutations in the AVPR1A gene can also be used to diagnose high blood pressure. Detection can be against cDNA or genomic DNA. The mutated forms of AVPR1A protein include point mutations, translocations, deletions, recombinations, and any other abnormalities compared with the normal wild-type AVPR1A 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 AVPR1A gene of the sample with AVPR1A gene-specific primers to obtain the amplified product; then detect whether there is the following single nucleotide polymorphism in the amplified product: 2408 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 AVPR1A 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 there is 2408 bits C→T. 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 2408th 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 AVPR1A 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 cacctcttcatccgacacct 2 antisense primer tgacattgttcacctcgatca 3

1.2.3 PCR amplification of AVPR1A 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 amplified product of 334 bp was obtained.

1.2.4 SNP discovery and detection

After the PCR products were purified by Resin resin, the ABI-PRISM ^TM 377 DNA sequencer (appliedbiosystems (ABI)) was used for bidirectional sequencing with fluorescent labeling end 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: C→T at position 2408.

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.

After the allele frequencies are counted, chi-square test is carried out. By comparing the individual data of the highest 11% with the lowest blood pressure of 7%, it is preliminarily determined whether the allele frequencies are linked with the blood pressure level.

It was found that the frequency of the 2408-type C SNP in SEQ ID NO: 1 was 39% in hypertensive individuals and 50% in hypotensive individuals. The frequency difference between the two is 11%, confirming that there is a correlation between the 2408 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 2408 in SEQ ID NO: 1 is closely related to hypertension. Therefore, based on this mutation, AVPR1A gene-specific primers can be designed to amplify and detect using the patient's DNA as a template.

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/μ, 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 2408-position C→T can also be detected.

The test results showed that the susceptibility to hypertension of 2408 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 arginine vasopressin receptor 1A gene and essential hypertension

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<170>PatentIn version 3.1

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gccagagcaa gggtgcagag caagcgggtg tggccttcca aaaggggttc ctgctcgcac 2820

cctgtgtcag cagcgtgaag tccatttccc gggccaagat ccgcacggtg aagatgactt 2880

ttgtgatcgt gacggcttac atcgtctgct gggcgccttt cttcatcatc cagatgtggt 2940

ctgtctggga tcccatgtcc gtctggaccg gtacgtgccg ggaaaataga ggaaagtgca 3000

gggataggag tgtgtgtgtg tgtgtgtgtg tgtgtgtgag agagagag agagagag 3060

agaaaaaaaa atgagaatct agcaattttc ttcatagtat cttctagggc agtagttttt 3120

aaacttctat gtgtacataa gagtcgcacc ttggaggaac actgtttaaa aaaaaaaaaa 3180

gcatattcct ggattcccca tgcagacatt ctaattaaga atgtctggaa tggggctcag 3240

gactctgcat ttgtagtgtg caatgatcct aagaccacaa ttccagaaac tgtgtcctag 3300

agtcagccaa gcaactacaa agccgaagaa tgacttttgc taaatcacta gctactgtat 3360

taacaccatt tcaggtctat tggattcagg gaaatatttt tactgtcaca actgcttttt 3420

gttggattgt gaaaagtatt acaattaatt ttaaaaatgt gtagaaatgc ctcaggggca 3480

aggatagaaa cagatatata tttctaaaga aagctggaga aaaattcttt agaaggtgag 3540

taattccaat ttggcattta gctaataatg ttcctttctc gttataatct atttttttct 3600

aacaatgatt aatagttttt tcttggtatt tcaaagcaga attattgata aactaaattc 3660

attagactaa ttcattagtc tttcaccact catttgacca cacggctcta catctctcca 3720

cccaactctt acagtagttt actggaaatg ttctctgcca cctactattg tgtttgattt 3780

taattccttc ccaaactaga aaaactaact tcacagtgac ttgcaaaaaa aattatttaa 3840

ttttgcatct tgaaaatatt ttcttctagt aaagacaaaa actcaaacta aataaattcc 3900

agtgcttgtc ggaactcaaa ccaaataaat tcaatggtac agttgttacc agaatgtttc 3960

tggtaacagg aaataggtat ctgggagtag ctgttctcct tttgcttttt ggaattggag 4020

tagggggaggt atgtaatatg ctttggaagt tatatttgca aataaaacat ttcagtatga 4080

atttaactta aatattctta ctgactataa tactagcgat aatgaaaaat acaatataaa 4140

cactttatt ttggtttgct atttcttatc ttgcttgatc ttagaagcct cttcatattg 4200

tccatcaaat aaagaaattc agtctaatta ttgctttagc agaatttaca ctcaagtaat 4260

aaaaacttca attgtgcata gatatgttgg taattttcat tctttgtgaa taccatctta 4320

cccatggctc ctgatcacct ttgatagcag catcttagca ctaagtatga ttaaataata 4380

acctgtaatt gttttctggc ataacaagag tgagaagatc caagtttata tttaataatc 4440

aaggaaaagt cagtgtttat tgattattct tatttttaga aaaggtatat tatcagcact 4500

gtagctccac tgtgaaaggt tataatattt atgcagttta ccagtgctaa ttatcataaa 4560

atattttaga atcctgttgg aatttcctaa ttctactgtt cttcttaata taatttgttt 4620

gaaccacaac cacagatggt tttccaaatt tctaaccaaa gaaaaacaac taaagcttat 4680

atcatccagg gacttcttct gtatggtttt catattataa gaatattaa aactactaaa 4740

cttgatccct aatgcaatat tttttcctga gttattagga taaatacaat ttggtataca 4800

tggttatta aaattatctt aaaatttcat tacaattgta gccattctgt aactgctgtg 4860

tcattagcac atgctagttc gagtatagaa gatagagatt ttttaaatca attacttaat 4920

agtcttaacc tcgtaaaatt cccactcaat tctatttaaa tattttgata gtgttttaaa 4980

aatacttgaa ttaattttaa ggcatcttgc ttacaaaaat attttatagt caagcaattt 5040

tcaaacactc cccatttccc tgattgataa acaaaatagt tcattttcta tgataatcca 5100

gaagtttatg ccttcttaat tagttaatag aaaaatgagt ttatccatgg ttcacttaca 5160

ttacatgatt tccctttata tttttcatgc agaatcggaa aaccctacca tcaccatcac 5220

tgcattactg ggttcccttga atagctgctg taatccctgg atatacatgt tttttagtgg 5280

ccatctcctt caagactgtg ttcaaagctt cccatgctgc caaaacatga aggaaaaatt 5340

caacaaagaa gatactgaca gtatgagcag aagacagact ttttattcta acaatcgaag 5400

cccaacaaac agtacgggta tgtggaagga ctcgcctaaa tcttccaagt ccatcaaatt 5460

cattcctgtt tcaacttgag ccttgcattc atgcaacttg attcttgtga ttgacttttt 5520

ggctcattag ctgaattgag ctagaaatca caagaacaaa tacactttat taatataacc 5580

ataaatcaat tcattgtgta tgagactgtg tttctagttg cattttcata ttgctaccaa 5640

aaactagaca ttattttgta tggaatatta atggaaacat gctgtactaa aatatgcagg 5700

tctgattccc agaaatacaa cagaagttat attttaaag gaaaaatcat aaccaccta 5760

gctttatatt ttgttgttag tttcttttat tttcatttct aacataagta agacttgatt 5820

ggtttaaaag tcacataaaa tgcggcacta tttctgaaca aagagagctc atcatcagtc 5880

ttaatattca gagaaaactt cagagaaatt atgttttcat ccattaaaat taatttgtgc 5940

atcagaaaat gcagccttaa acagtgtcca ggagatggga tggtacctcc taggagtaca 6000

agtgcctggg gtgtaatgag ctcctgctca ttgtggccag tttagagttc tattagaagc 6060

tatcaatcac cttgcatttc aaaatggtaa ctttacaact ggcagtggcc tccttttggt 6120

tcctcacata ttattggtca agaaaagcat gaaaactgag atgctgaagg tgagaggaaa 6180

tgttgactgg ccaaaaatat cttttttccc ccactgcaag gttgttttaa agtcagattt 6240

gtataaggaa agccaaattt tattaaaaga gtagaaagg attgcttaag gtactctgga 6300

ctttctcttg gacattgtaa acgtattttg atcagtatta caagggtatc ctgtgctatg 6360

ctggacatta acaagatcat tatcttcatg tttggggaat tc 6402

<210>2

<211>20

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>2

cacctcttca tccgacacct 20

<210>3

<211>21

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> primer

<400>3

tgacattgtt cacctcgatc a 21

Claims (10)

1. A method for in vitro detection of whether a single nucleotide polymorphism of the arginine vasopressin receptor 1A gene AVPR1A exists in a sample, characterized in that it comprises the steps of: (a) amplifying the AVPR1A gene of the sample with AVPR1A gene-specific primers to obtain an amplified product; and (b) Detect whether the following single nucleotide polymorphisms exist in the amplification product: 2408 bits C → T; Wherein, the nucleotide position numbers are based on SEQ ID NO:1. 2. The method of claim 1, wherein the gene-specific primer has the sequences of SEQ ID NO: 2 and 3. 3. The method according to claim 1, wherein the length of the amplified product is 100-3000bp, and contains the 2408th position in SEQ ID NO:1. 4. A test kit for detecting hypertension, characterized in that it includes primers for specific amplification of the AVPR1A gene or transcripts, and the amplified length of the primers is 100-3000bp, and contains SEQ ID NO: 1 Amplification product at position 2408. 5. The kit of claim 4, further comprising reagents selected from the group consisting of: (a) a probe that binds to the mutation at position 2408 in SEQ ID NO: 1; (b) Recognition of the mutant restriction enzyme at position 2408 in SEQ ID NO:1. 6. test kit as claimed in claim 5, is characterized in that, described mutation is following single nucleotide polymorphism: 2408 bits C → T; Wherein, the nucleotide position numbers are 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. A method for diagnosing an individual's susceptibility to hypertension, characterized in that it comprises the steps of: detection of the individual's AVPR1A gene, transcript and/or protein, and comparison with normal AVPR1A gene, transcript and/or protein, A difference indicates that the individual is more likely to have high blood pressure than the normal population. 9. The method according to claim 8, characterized in that the gene or transcript of AVPR1A is detected, and the difference is compared with the normal AVPR1A nucleotide sequence. 10. The method of claim 9, wherein said difference is the following single nucleotide polymorphism: 2408 bits C → T; Wherein, the nucleotide position numbers are based on SEQ ID NO:1.