CN1661067A - Correlation between insulin receptor gene SNP and essential hypertension - Google Patents

Abstract

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

Description

Correlation between insulin receptor gene SNP and essential hypertension

technical field

The present invention relates to the fields of molecular biology and medicine. More specifically, it relates to the single nucleotide polymorphism (single nucleotide polymorphism, SNP) of the insulin receptor gene (Insulinreceptor, INSR) 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 on the correlation between INSR gene and essential hypertension, and there is no confirmation that the SNP of INSR 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 INSR gene, transcript and/or protein, and comparing it with normal INSR 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 INSR, and compares the difference with the normal INSR nucleotide sequence.

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

1408 bit G-T;

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

In a second aspect of the present invention, there is provided a method for detecting whether a single nucleotide polymorphism of the insulin receptor gene INSR exists in a sample, comprising the steps of:

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

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

1408 bits G→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 1408th 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 INSR gene or transcript, more preferably, the amplified length of the primers is 100-3000bp, And contain the amplified product at position 1408 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 1408 in SEQ ID NO: 1;

(b) Recognition of the mutant restriction enzyme at position 1408 in SEQ ID NO:1.

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

1408 bits G→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 INSR 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 part of the SNP of the INSR gene is closely related to hypertension, and its new function was discovered: the change of INSR 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.

INSR gene

The detailed sequence of the insulin receptor gene (Insulin receptor, INSR) can refer to the nucleotide sequence whose accession number is M32822 (see website http://www.ncbi.nlm.nih.gov/), as shown in SEQ ID NO: 1 Show.

The INSR gene belongs to a hormone receptor whose structure and function have been relatively clear. The receptor gene is located at the end of the short arm of the 19th pair of human chromosomes, located at 19P13.2-13.3. In 1993, Trask et al. located INSR at 19p13.3. At present, three transcripts have been identified, which are distributed at 276, 282, and 283 base sites upstream of the translation initiation site, respectively, and have genetic heterogeneity in insulin receptors in different tissues. Insulin receptors have typical allosteric properties, and insulin binding to them has negative cooperativity, so each insulin receptor can only bind one insulin molecule with high affinity, and even so, it is enough to fully trigger the corresponding cellular response.

Insulin receptor (IR) is a transmembrane glycoprotein composed of two α subunits (molecular mass 135Kd) and two β subunits (molecular mass 95Kd) linked by disulfide bonds. The α subunit consists of 719 amino acid residues and is located on the outer surface of the cell; the β subunit consists of 620 amino acid residues and is divided into three structural regions: the N-terminal region composed of 194 amino acid residues is exposed on the cell surface, through The disulfide bonds are connected to the α subunit; the transmembrane region consists of 23-26 highly hydrophobic amino acid residues and the C-terminal region consists of 403 amino acid residues. The N-terminus of the α-subunit peptide chain and the domain rich in cysteine residues are the binding sites for insulin. The tyrosine kinase active domain is located in the 996-1263 amino acid residue region of the β subunit, and has important functions in the insulin receptor. The amino acid residue sequence in this region is highly conserved, among which 1003Gly-Gln-Gly -Ser-Phe-Gly1008 is the ATP binding site, followed by 15-25 amino acid residues is a conserved Lys, which is involved in the transfer of phosphate groups and subsequent receptor activation. The catalytic domain of the kinase is located at the C-terminal 100-150 amino acid residues of the ATP binding site, including the main site of autophosphorylation ( ¹¹⁵⁸ Tyr-Glu-Thr-Asp-Tyr-Tyr ¹¹⁶³ ). The two highly conserved sequences ¹¹³¹ AArg-Asp-Leu-Ala-ala-Arg-Asn ¹¹³⁷ and ¹¹⁷² Pro-Val-Arg-Try-Met-Ala-Pro-Glu ¹¹⁷⁹ on both sides of it are insulin receptor kinase activity required. In addition, there are two autophosphorylation sites Tyr ¹³¹⁶ and Tyr ¹³²² at the C-terminus of the β subunit.

The coding region of the insulin receptor gene consists of 22 exons and 11 introns. Exons 1 to 11 encode the α subunit, located outside the cell membrane, and function to bind insulin; exons 12 to 22 encode the β subunit, which is a transmembrane structure, contains 13 tyrosine residues, and has a tyrosine kinase The activity is also called the catalytic subunit. Among them, exons 17 to 22 encode the main active region of β subunit tyrosine kinase. When insulin binds to the α subunit, the β subunit undergoes autophosphorylation and activates tyrosine kinase, and then activates a series of intermediate molecules in the cell, such as SH-2, IRS-1 and PI-3 kinase (Phosphatidylinasital-3 Kinase ), etc., regulate the synthesis and metabolism of sugar, fat, protein, ribonucleic acid and other important substances.

Insulin receptor primary transcript processing or structural gene abnormalities will lead to insulin receptor defects, leading to insulin resistance and a series of metabolic disorders such as fat metabolism and glucose metabolism, resulting in hyperinsulinemia or diabetes, which eventually lead to vascular and Damage to organs such as other tissues and cause atherosclerosis. Existing studies have found that most of the gene mutations occur in exons. For example, some studies have found that in patients with insulin resistance characteristics, a mutation from arginine to L-cysteine at position 252 of the receptor is found, which inhibits the internal function of the receptor. Swallow (internalisation) but does not prevent the delivery of the signal.

The inventors sequenced almost the entire region of the INSR gene and found many SNPs, most of which are not associated with susceptibility to hypertension, but association studies have shown that 1408 G→T is associated with susceptibility to hypertension Very high SNP. The SNP is located in the promoter region of INSR, suggesting that it has an impact on the transcription level of INSR, which in turn affects the expression level of insulin 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 INSR protein or polypeptide has many new uses. These uses include (but are not limited to): direct use as drugs to treat diseases caused by low or loss of INSR protein function (such as hypertension), and for screening substances that promote the function of INSR protein, such as antibodies, polypeptides or other ligands. Screening the polypeptide library with expressed recombinant human INSR protein can be used to find therapeutically valuable polypeptide molecules that can stimulate the function of human INSR protein.

On the other hand, the present invention also includes polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies, specific for human INSR DNA or polypeptides encoded by fragments thereof. Here, "specificity" means that the antibody can bind to human INSR gene product or fragment. Preferably, it refers to those antibodies that can bind to human INSR gene products or fragments but do not recognize and bind to other irrelevant antigenic molecules. Antibodies in the present invention include those molecules that can bind to and inhibit human INSR protein, as well as those that do not affect the function of human INSR 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 INSR gene products, or antigenic fragments thereof, can be administered to animals to induce polyclonal antibody production. Similarly, cells expressing human INSR 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 INSR protein and antibodies that do not affect the function of human INSR protein. Various antibodies of the present invention can be obtained by conventional immunization techniques using fragments or functional regions of human INSR 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 human INSR gene products 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).

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

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

When the INSR 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 INSR polypeptides can be used directly in the treatment of diseases, for example, in the treatment of hypertension. When using the INSR 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 INSR 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, a safe and effective amount of INSR protein or its antagonist, agonist is administered to the mammal, 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 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 INSR protein may 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 INSR protein or expression of abnormal/inactive INSR protein. The method for constructing a recombinant viral vector carrying the INSR gene can be found in existing literature (Sambrook, et al.). In addition, the recombinant human INSR 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 present invention also relates to a diagnostic test method for quantitative and localized detection of human INSR protein level. These assays are well known in the art and include ELISA and the like.

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

The polynucleotide of INSR protein can be used for the diagnosis and treatment of diseases related to INSR protein. In terms of diagnosis, the polynucleotide of INSR protein can be used to detect the expression of INSR protein or the abnormal expression of INSR protein in a disease state. For example, the INSR DNA sequence can be used for hybridization of biopsy specimens to determine the abnormal expression of INSR 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 INSR protein-specific primers can also detect INSR protein transcripts.

Detection of mutations in the INSR gene can also be used to diagnose hypertension. Detection can be against cDNA or genomic DNA. The forms of INSR protein mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared with the normal wild-type INSR 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 INSR gene of the sample with INSR gene-specific primers to obtain the amplified product; then detect whether the following single nucleotide polymorphism exists in the amplified product: 1408 G → 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 INSR 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 It is determined whether 1408 bits G→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 1408th 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 INSR 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 aagctttccctccctctcct 2 antisense primer gggactgtctctcggctct 3

1.2.3 PCR amplification of INSR 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 275 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: G→T at position 1408.

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 T-type SNP at position 1408 in SEQ ID NO: 1 in hypertensive individuals was significantly higher than that in hypotensive normal population, and the difference between the frequency of T-type SNP in hypotensive population was about 10% , confirmed that there is a correlation between the T/G type SNP at position 1408 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 G→T at position 1408 in SEQ ID NO: 1 is closely related to hypertension. Therefore, INSR gene-specific primers can be designed based on this mutation and detected by amplification 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/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 amplified product and the normal control are subjected to chromatographic analysis with a denaturing high-performance liquid chromatograph (DHPLC), and the 1408-position G→T can also be detected.

As a result of the test, the susceptibility of the test subjects including 1408 G→T to high blood pressure 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> Correlation between insulin receptor gene SNP and essential hypertension

<130>040143

<160>3

<170>PatentIn version 3.1

<210>1

<211>2085

<212>DNA

<213> Homo sapiens

<400>1

agatctggcc attgcactcc agcctgggca acagagaaaa actccatcta aaaaaaaaaa 60

aaaaaaaaaa aaaaaacaga gagagagaga gagagagaga gaaggaaacg gaactggggg 120

gaggatttgc aaaaatatgg ttagggatgg cacttcagag atgaagccat cctggagtgt 180

tacgggcaag ggaaatgctg gggcaaagcc ccagaggcag gaataggttt ggcctgttgc 240

atgaacagtg ggtccagctc ctagcaaact gtttattgaa tgaaagaaga atgaatgcct 300

tgggtctagg gttgtgctgg gcgctttctt aagttttctt tcccgggtac ctccccagaa 360

ctggcatgca ggtattatta aacccattac acaagtgaaa ctggcccaga gacagaaaag 420

tccctggtcc aagaccacac aggagtgagg ggtggaggaa ccctcctccc attgagttct 480

ggctttccta tactgaaagc cccttcctct cctgcagtaa ggtaggtgga accgctgtcc 540

cgccttgttg gtgaatgtcg ttgctagact tcagacacat acaggctggt ctgctgaaaa 600

tcagagatgt ccacctgcgc cctattcgag gtctccggcg tcttctttgg cgtcgtcttt 660

gccctttcag aagcgtctgc aatttttcc aggtgtcatt tctccaactt gaacacaggg 720

agcgcactgg gcacgcgggc acgtggctgt ccccaggggc ctggcttggg tctcgcccct 780

gggccggggc gcacgcgcgg gcgggacatc tggggggcgcc cacgcgctct gggacgagtg 840

tcgctggcca ggcccggact gaggaaaggc gagtgagaca ctactcgcct ggggtgcaaa 900

atttaaggga gtgaaaaaaa aaaaaaaaga aagaaaccaa aaccacctcg agtcaccaaa 960

ataaacattt taatgcagta ttttttaaaa aatcaacagg aatcctccaa agcccactat 1020

gaacaaaata gcaaaatggt agagaaagga tctgtgccgc tgcgtcgggc ctgtggggcg 1080

cctccggggg tctgaaactg gaggagactc ggggctgtag ggcgcgcgga tctggggcgc 1140

gccctcggtc ccggcgcgcc cagggcctcc cgcgcggggc ccggcacagg gaggcgggga 1200

ggcgggcggg gcggggcggg accgggcggc acctccctcc cctgcaagct ttccctccct 1260

ctcctgggcc tctcccgggc gcagagtccc ttcctaggcc agatccgcgc cgccttttcc 1320

cgcggcccgc acggggccca gctgacgggc cgcgttgttt acgggccgga gcagccctct 1380

ctcccgccgc ccgcccgcca cccgccagcc caggtgcccg cccgccagtc agctagtccg 1440

tcggtccgcg cgtccctctg tcccggagcc cgcagatcgc gacccagagc gcgcggggcc 1500

gagagccgag agacagtccc gggcgcagcg cggagctccg ggccccgaga tcctgggacg 1560

gggcccgggc cgcagcggcc gggggtcgg ggccaccacc gcaagggcct ccgctcagta 1620

tttgtagctg gcgaagccgc gcgcgccctt cccggggctg cctctgggcc ctccccggca 1680

ggggggctgc ggcccgcggg tcgcgggcgt ggaagagaag gacgcgcggc ccccagcgcc 1740

tcttgggtgg ccgcctcgga gcatgacccc cgcgggccag cgccgcgcgc tctgatccga 1800

ggagaccccg cgctcccgca gccatgggca ccgggggccg gcggggagcg gcggccgcgc 1860

cgctgctggt ggcggtggcc gcgctgctac tgggcgccgc gggccacctg taccccggag 1920

agggtgagtc tgggggcgcg ggcgtgggcg gggagcgccg cgatggggag aggacccac 1980

ccaagccaaa atcgatcccc cgcttgtgga ctgagaaccc tccccagggg cggggggcgg 2040

tggccaggac ggtagctcct gcatcgcgta gggggagcgg gaagc 2085

<210>2

<211>20

<212> DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>2

aagctttccc tccctctcct 20

<210>3

<211>19

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> primer

<400>3

gggactgtct ctcggctct 19

Claims (10)

1. a method for in vitro detection of whether there is a single nucleotide polymorphism of the insulin receptor gene INSR in a sample, characterized in that it comprises the steps of: (a) amplifying the INSR gene of the sample with INSR gene-specific primers to obtain an amplified product; and (b) Detect whether the following single nucleotide polymorphisms exist in the amplification product: 1408 bits G→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 1408th position in SEQ ID NO:1. 4. A test kit for detecting hypertension, characterized in that it includes primers for specific amplification of INSR genes or transcripts, and the amplified length of the primers is 100-3000bp, and contains SEQ ID NO: 1 The amplification product at position 1408. 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 1408 in SEQ ID NO: 1; (b) Recognition of the mutant restriction enzyme at position 1408 in SEQ ID NO:1. 6. test kit as claimed in claim 5, is characterized in that, described mutation is following single nucleotide polymorphism: 1408 bits G → 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: detecting the individual's INSR gene, transcript and/or protein, and comparing it with normal INSR 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 INSR is detected, and the difference is compared with the normal INSR nucleotide sequence. 10. The method of claim 9, wherein said difference is the following single nucleotide polymorphism: 1408 bits G→T; Wherein, the nucleotide position numbers are based on SEQ ID NO:1.