JP2007053995A - Method for judging essential hypertension - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for judging essential hypertension, by which the presence or absence of a genetic factor of the essential hypertension can simply be judged in a highly reliable state reducing pseudopositive results. <P>SOLUTION: This method for judging the essential hypertension in the present invention is characterized by relating a gene polymorphism of human follicle stimulating hormone receptor gene and/or a haplo-type composed from the gene polymorphism to the presence or absence of individual hypertension symptom. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for determining essential hypertension. Specifically, the present invention relates to a method for determining the presence or absence of a genetic factor of essential hypertension using a gene polymorphism.

  Essential hypertension (EH) is hypertension that occurs without a known cause, and is broadly classified as secondary hypertension with obvious causes. Essential hypertension accounts for approximately 80-90% of all cases of hypertension and can probably be caused by various disorders in various normal blood pressure regulation mechanisms, but many are genetically conditioned. It is considered to belong to a so-called multifactor hereditary disease.

  Since essential hypertension is a multifactor hereditary disease, determination of the causal gene and disease susceptibility gene has been extremely difficult.

  The reason is that clinical symptoms and characteristic clinical data (these are called phenotypes) of the disease are not constant, and the genetic form in the family is often not constant. One of the reasons is that phenotypes usually appear after adult age, and the patient's parents' genotype is often not already alive. .

As a method proposed to solve such a problem, a diseased sibling pair method focusing on an angiotensinogen gene is known (see Non-Patent Document 1). However, this method has not yet obtained enough data to recognize that it is effective, and it can be very time consuming and laborious for preparation and analysis. Was low.
Jeunemaitre X, et.al., Molecular basis of human hypertension: role of angiotensinogen, Cell, 71 (1), p.169-180, 1992

  Therefore, the problem to be solved by the present invention is to provide a method for the above determination that is simple and highly reliable with reduced false positive results in determining the presence or absence of a genetic factor of essential hypertension. It is in.

  The present inventor has intensively studied to solve the above problems. As a result, the human follicle stimulating hormone receptor (FSHR) gene, which is one of the pituitary hormone receptors, is one of the genes responsible for essential hypertension and disease susceptibility genes. Focused on. Then, the present inventors have found that a gene polymorphism present in the FSHR gene and a haplotype constituted by the gene polymorphism (especially SNP) are very useful as genetic markers for essential hypertension, thereby completing the present invention. .

  That is, the present invention is as follows.

 (1) Determination of essential hypertension characterized by associating a gene polymorphism of the human follicle-stimulating hormone receptor gene and / or a haplotype composed of the gene polymorphism with the presence or absence of hypertension in an individual Method.

  In the determination method of the present invention, examples of the gene polymorphism include at least one selected from the group consisting of a single nucleotide polymorphism, an insertion polymorphism, a deletion polymorphism, and a base repeat polymorphism. More specific examples of the gene polymorphism include at least one selected from those shown in Table 1 shown below.

  In the determination method of the present invention, examples of the haplotype include at least one selected from those shown in Table 2 shown later.

 (2) An oligonucleotide prepared so as to include a gene polymorphic site present in a gene sequence encoding a follicle stimulating hormone receptor or a complementary sequence thereof.

 (3) A microarray in which the oligonucleotide according to (2) is fixed to a support.

 (4) A kit for determining essential hypertension, comprising the oligonucleotide according to (2) above and / or the microarray according to (3) above.

  ADVANTAGE OF THE INVENTION According to this invention, when determining the presence or absence of the genetic factor of essential hypertension, the novel determination method with high reliability which is very simple and the false positive result was reduced significantly can be provided. Moreover, the oligonucleotide, microarray, and determination kit which can be used for the said determination method can also be provided.

Hereinafter, the present invention will be described in detail.
1. SUMMARY OF THE INVENTION The present invention is a method for determining the presence or absence of a genetic factor of essential hypertension (EH), and the method focuses on a gene polymorphism in the human follicle stimulating hormone receptor (FSHR) gene. The above determination is performed by associating the detection result with the presence or absence of a hypertension symptom.

  The present invention focuses on a specific gene called FSHR gene among many genes that are candidates for essential hypertension causal genes and disease susceptibility genes, gene polymorphisms present in this FSHR gene, and the gene A haplotype composed of polymorphisms has been completed based on the novel finding that it is very useful as a genetic marker for essential hypertension. This makes it possible for the first time to determine the presence or absence of genetic factors that do not depend on the conventional affected sib-pair method. Furthermore, in the present invention, since it is sufficient to focus only on the desired polymorphism among gene polymorphisms associated with essential hypertension, the method of the present invention makes determination results much easier and clearer than the conventional method. In addition, it has an excellent effect that the possibility of false positives can be sufficiently reduced (that is, it has high reliability).

By the way, as an analysis method of gene polymorphisms that has been frequently used in recent years, there is a related analysis. Unlike linkage analysis, this does not look at the family line, but collects a large amount of information on the disease group (case) and non-disease group (control), and looks at the difference in the frequency of genetic polymorphism in both groups. . This analysis method is a successful method for selecting and determining causative genes or disease susceptibility genes of multifactor hereditary diseases. This time, the present inventor has used this association analysis to relate the genetic polymorphism in the FSHR gene to essential hypertension, particularly the single nucleotide polymorphism (SNP) in the FSHR gene and essential hypertension. The relationship between was clarified. And it discovered that a certain SNP was especially useful in determining the presence or absence of the genetic factor of essential hypertension. In addition, the present inventors have succeeded in narrowing down particularly useful gene polymorphisms by performing haplotype analysis in order to select the above-mentioned useful gene polymorphisms (especially SNPs). These processes greatly contribute to improving the reliability of the determination method of the present invention.

2. Gene polymorphism of follicle stimulating hormone receptor (FSHR) The FSHR gene polymorphism of the present invention is mainly composed of single nucleotide polymorphism (SNP (or SNPs)), insertion / deletion polymorphism, and base. The polymorphism resulting from the difference in the number of repeating sequences is included, and is not limited.

  SNP means a polymorphism resulting from substitution of one specific base in a gene sequence with another base. An insertion / deletion polymorphism means a polymorphism resulting from deletion / insertion of one or more bases. In addition, polymorphisms (base repeat polymorphisms) caused by differences in the number of repeats of the base sequence include microsatellite polymorphism (number of bases: about 2 to 4 bases) and VNTR (variable number of tandem) depending on the number of repeat bases. repeat) A polymorphism (repetitive base: several to several tens of bases), which means a polymorphism in which the number of repeat bases varies depending on the individual.

  Information on FSHR gene polymorphisms preferably used in the method of the present invention is listed in Table 1 below (SEQ ID NOs: 1 to 5). The polymorphism information shown in Table 1 relates to single nucleotide polymorphism (SNP). Among the gene polymorphisms shown in Table 1, polymorphisms shown in the nucleotide sequences of SEQ ID NOs: 1, 2, 4, and 5 are more preferable, and still more preferable are shown in the nucleotide sequences of SEQ ID NOs: 1, 2, and 5. A polymorphism, particularly preferably a polymorphism shown in the nucleotide sequence of SEQ ID NO: 1.

  “Position” in Table 1 means the position of the FSHR gene on the human genome, and represents Exon and Intron. `` Genetic polymorphism name '' is the SNP name at a position on the genome, both of which are the website of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/), and It is indicated by the accession number (accession number) confirmed using the website of the Celera Discovery System-Applied Biosystems (Applied Biosystems-Celera Discovery System) (the latter) The accession number confirmed by is shown in parentheses.)

In “Sequence” in Table 1, a base sequence consisting of 51 bases (SEQ ID NOs: 1 to 5) is displayed, and the SNP site is displayed at the 26th position. "A / g" means "a" and "g" SNPs, "a / t" means "a" and "t" SNPs To do.
The “26th base” means a base at a polymorphic site, and the base is not necessarily one base. Therefore, if it is a delation type polymorphism, there is no base, so it is not counted as the number of bases. Even if a plurality of bases are repeated, the “26th base” ".

  In the present invention, the method for obtaining information on the FSHR gene polymorphism is not limited, and for example, a method obtained by searching various databases such as dbSNP of GenBank and the method described below can be employed.

  First, genomic DNA is purified from a blood sample collected from a human using the phenol method or the like. At that time, a commercially available genomic DNA extraction kit or apparatus such as GFX Genomic Blood DNA Purification Kit may be used. Next, using the obtained genomic DNA as a template, the genomic DNA is divided into several parts by PCR, and used as a template DNA for sequencing. Since the present invention targets gene polymorphisms, it is desirable to use an enzyme used in the PCR method with as high a fidelity as possible. The 5 'untranslated region (5'UTR and 5'flanking region) of FSHR should be divided into two regions as needed for the region up to 99 bp upstream from the start codon, preferably up to 500 bp. Is preferred. Exons 1, 2, 3,..., 10 and introns 1, 2, 3,..., 9 amplify the entire region by the PCR method at each location. Exon 10 may include about 230 bp downstream from the stop codon. The 3 ′ untranslated region is preferably 230 bp downstream of the stop codon, preferably up to 500 bp, and PCR amplification is preferably performed by dividing it into two regions as desired. The base sequence of the entire region of these PCR fragments can be decoded by a sequencing method using a primer designed based on the sequence information published in GenBank by a sequence method of about 400 bp to obtain the desired gene polymorphism.

In the present invention, oligonucleotides containing all FSHR gene polymorphisms can be provided. Among them, preferably, SEQ ID NOs: 1 to 5 (more preferably SEQ ID NOs: 1, 2, 4, and 5, more preferably, Table 1). Is at least 10 bases, preferably 10 to 100 bases, more preferably 10 including the polymorphic site (the 26th base) in the base sequence shown in SEQ ID NOs: 1, 2 and 5, particularly preferably SEQ ID NO: 1) It is an oligonucleotide selected from a base sequence of -60 bases or a base sequence complementary thereto. Specifically, the nucleotide sequence shown in SEQ ID NO: 1 to 5 (more preferably SEQ ID NO: 1, 2, 4 and 5, more preferably SEQ ID NO: 1, 2 and 5, particularly preferably SEQ ID NO: 1) or complementary thereto. Oligonucleotides selected from typical base sequences are preferred.

3. Haplotype analysis In the method of the present invention, a haplotype can be constructed using SNP among the above gene polymorphisms and used to determine the presence or absence of a genetic factor of essential hypertension. Usually, it is very difficult to clarify an unknown gene polymorphism existing outside the untranslated region. Therefore, the haplotype is very useful for finding the association between genetic polymorphism and essential hypertension.

  A haplotype usually refers to a combination of genes, SNPs, and the like that are one chromosome derived from one parent and are relatively close to each other. For example, if several SNPs are involved in the development of a particular disease, haplotype analysis of the associated SNP may be the key to elucidating the cause of the disease.

  In general, it is preferable to select a SNP subject to haplotype analysis that has a polymorphism frequency of 10% or more, more preferably 20% or more, and still more preferably 30% or more. Further, the SNPs to be subjected to haplotype analysis may be all or a part thereof.

Haplotype analysis can be performed with various computer programs, for example,
SNPAlyze version 3.2 (DYNACOM Co., Ltd. Yokohama, Japan) (available at http://www.dynacom.co.jp/products/package/snpalyze/index.html),
Arlequin program (available at http://anthro.unige.ch/arlequin) (Schneider S, Roessli D, Excoffier L. Arlequin 2000: a software for population genetics data analysis.Ver 2.000. Genetics and Biometry Lab, Dept of Anthropology Univ of Geneva.), And
GeneSpring GT (Varia) (Tommy Digital Biology Co., Ltd.)
Etc. can be used.

  As an example of haplotype analysis, haplotypes are estimated using SNPAlyze version 3.2 for the five SNPs shown in Table 1. The estimated haplotypes in the disease (EH) group are shown in Table 2 below.

  Among the haplotypes shown in Table 2, those that can be preferably used for the determination of the presence or absence of genetic factors of essential hypertension are the haplotypes of No. 1 to 25, more preferably the haplotypes of No. 1 to 16, The haplotypes No. 1 to 13 are preferred, and the haplotypes No. 1 to 9 are particularly preferred.

Next, linkage disequilibrium analysis is performed based on the haplotype frequency. The D ′ value and the r ² value, which are values indicating the scale of linkage disequilibrium, can be calculated based on the following definition explanation. Linkage disequilibrium refers to the case where the polymorphism and polymorphism are linked on the chromosome and thus deviate from the equilibrium state according to Mendel's independent law. Conversely, linkage equilibria refers to two polymorphisms. When the chromosomal relationship between them is independent.
Definition explanation:
Regarding the D ′ value, there is a linkage disequilibrium coefficient (D) as an index representing linkage disequilibrium. When there is no linkage disequilibrium, D = 0, and when D> 0, it is called positive linkage disequilibrium. For example, there are four types of haplotypes formed by SNP1 and SNP2, 1-1, 1-2, 2-1 and 2-2, and if each frequency is p11, p12, p21, p22, the D value is Represented by the formula

D = p11 × p22-p12 × p21
Next, assuming that the maximum value that can be taken by the D value is D _max and the minimum value that can be taken is D _min , the D ′ value is expressed by the following equation, with the D value normalized.

D '= D / D _max (when D value is positive)
D '= D / D _min (when D value is negative)
In general, when the D ′ value is close to 1, each SNP can be said to be located in one haplotype block due to linkage disequilibrium. Although there is no fixed criterion for how close the D ′ value is to 1, each SNP can be said to be located in one haplotype block, the criterion of D ′> 0.5 can be preferably used in the present invention.

For r ² value
The r ² value is also an index of linkage disequilibrium. For information on the differences between the calculated expressions and D 'value of r ² value, following the literature "Devlin, B. and Risch, N .," A comparison of linkage disequilibrium measures for fine-scale mapping. ", Genomics, vol.29, p.311-322 (1995) ”.

In general, when the paired SNPs take r ² values close to 1, it is disadvantageous to use the two for the association analysis using the haplotype at the same time, so it is preferable to estimate the haplotype using one of them. There is no fixed criterion for how close the r ² value is to 1, but in the present invention, the criterion r ² <0.1 can be preferably used.

  An example showing linkage disequilibrium between FSHR gene polymorphisms in the disease (EH) group is shown in FIG.

Focusing on the D ′ value in FIG. 1, significant linkage disequilibrium is confirmed in the SNP combinations of rs1394205 + rs2055571 and rs11692782 + rs1007541 + rs2268361. That is, the first two SNPs (rs1394205 and rs2055571) are located in one haplotype block, and the next three SNPs (rs11692782, rs1007541 and rs2268361) are located in another haplotype block. Further, paying attention to the values of r ^2, Rs1394205 + r ² values in combination with SNP of Rs2055571 is greater than 0.1, it is disadvantageous to use this two SNP (rs1394205 and rs2055571) simultaneously to the relevant analysis using haplotypes I know that there is.

  The haplotype block can be estimated from the result of the haplotype analysis using, for example, SNPAlyze version 3.2 (above) and Arlequin program (above).

By examining a specific SNP in the estimated haplotype block, it is possible to know information on SNPs that are indirectly linked in the same block. That is, when examining the FSHR gene polymorphism, it is not necessary to analyze all SNPs, and only a few specific SNPs need to be typed.

4). Correlation between follicle-stimulating hormone receptor gene polymorphisms and essential hypertension When polymorphisms occur in follicle-stimulating hormone receptor genes, the expression level and function of FSHR are thought to change. Therefore, the FSHR gene polymorphism may be correlated with various phenotypes related to FSHR. In the FSHR gene abnormality as a single gene disease, infertility due to gonadal dysfunction usually appears as a symptom in both men and women, but hypertension is one of the phenotypes. Here, the phenotype can include a phenotype related to the onset of a disease (EH). Examples of the phenotype related to the onset of this disease include hypertension detection by measuring blood pressure in peripheral blood vessels, headache, hot flashes, neck pain, eye pain, nausea and the like.

  The correlation between the FSHR gene polymorphism and the phenotype can be examined as follows. As the FSHR gene polymorphism, a representative polymorphism within the estimated linkage block, for example, SNP, is selected as a result of linkage disequilibrium analysis and haplotype analysis in healthy individuals. Next, the polymorphism frequency about this polymorphism (for example, SNP) in a test (patient) individual is analyzed, and it compares with the polymorphism frequency of a healthy person. In comparison, it is effective to use a statistical method such as chi-square test (chi-square test).

If the phenotype or symptom is hypertension detected by measuring blood pressure in peripheral blood vessels, for example, stage 1: 140-159 / 90-99 (mmHg), stage 2: 160 or more / 100 or more (mmHg) (by the Joint Committee on Hypertension) Blood pressure classification (JNC7, 2003). Then, for each classification, the polymorphism frequency and genotype of the healthy person and the subject are compared. As a result, polymorphisms that had a significant difference in polymorphism frequency from the control group were assessed for whether or not they were affected by a disease (EH), or the likelihood of being affected by the disease. Can be used to determine. However, since it is suggested that the tendency of genetic polymorphism is influenced by race, birthplace, etc., genetic polymorphism similar to the population used to find related polymorphisms (eg SNP) It is desirable to perform the above assessment using the polymorphism within the population shown.

5. Sample Preparation and Detection Method of Gene Polymorphism A genomic sample from a test subject can be extracted from any of blood, saliva, skin, etc., and is not limited to these as long as the genomic sample can be collected. Methods for extracting and purifying genomic DNA are well known. For example, genomic DNA is purified from specimens such as blood, saliva and skin collected from humans using the phenol method or the like. At that time, a commercially available genomic DNA extraction kit or apparatus such as GFX Genomic Blood DNA Purification Kit may be used. If the gene polymorphism (SNP, etc.) to be detected is in the open reading frame, mRNA or total RNA may be extracted instead of genomic DNA. Below, the detection method (outline) of the gene polymorphism from the said test sample is illustrated.

(1) Detection using PCR
In order to amplify a test sample by PCR, it is preferable to use a DNA polymerase having a high fidelity, such as KOD Dash polymerase (TOYOBO). The primer to be used is designed and synthesized so that the gene polymorphism is included at an arbitrary position of the primer so that the target SNP in the test sample can be amplified.

  After completion of the amplification reaction, the amplification product is detected and the presence or absence of the polymorphism is determined.

(2) Detection by nucleotide sequencing method In the present invention, the polymorphism of the present invention can also be detected by nucleotide sequencing method based on dideoxy method. A commercially available ABI series (Amersham Bioscience) etc. can be used for the sequencer used for base sequence determination.

(3) Detection by DNA microarray
The DNA microarray has a nucleotide probe immobilized on a support, and includes a DNA chip, a Gene chip, a microchip, a bead array, and the like.

  First, a polynucleotide of a test sample is isolated, amplified by PCR, and labeled with a fluorescent reporter group. Subsequently, labeled DNA / mRNA and total RNA are incubated with the array. The array is then inserted into a scanner and the hybridization pattern is detected. Hybridization data is collected as luminescence from fluorescent reporter groups attached to the probe array (ie, incorporated into the target sequence). A probe that perfectly matches the target sequence produces a stronger signal than one that has a portion that does not match the target sequence. Since the sequence and position of each probe on the array is known, the sequence of the target polynucleotide reacted with the probe array can be determined by complementation.

(4) Detection by TaqMan PCR
The TaqMan PCR method uses a fluorescently labeled allele-specific oligo (TaqMan probe) and a PCR reaction with Taq DNA polymerase. Specifically, the TaqMan probe first hybridizes with the template DNA, and then an extension reaction from the PCR primer occurs. Then, the fluorescent dye-binding portion is cleaved by the 5 'nuclease activity of Taq DNA polymerase, and the fluorescent dye is liberated, and it develops color without being affected by the quencher. By detecting this fluorescence, a specific polymorphism (SNP) Etc.) and the genome type can be determined. Allele-specific oligos (referred to as TaqMan probes) used in the TaqMan PCR method can be designed based on the gene polymorphism information.

(5) Detection by invader method The invader method is a method for detecting a gene polymorphism by hybridizing an allele-specific oligo and a template. Specifically, when Invader Oligo causes at least one base penetration (invasion) into the duplex between the target DNA and the probe to form a partial triplex structure, a structure-specific 5'-nuclease is probed. Cut the 5 'end. Then, the specific polymorphism (SNP, etc.) is identified by using an invader oligo that generates invasion corresponding to the nucleic acid sequence of the target polymorphism (SNP, etc.), and the genome type is determined. be able to. Kits for performing the invader method are commercially available, and gene polymorphism can be easily detected by this method.

6). Kit In the present invention, an oligonucleotide containing a FSHR gene polymorphism (eg, SNP) site can be included in a kit for determining essential hypertension.

  The kit of the present invention contains one or more components necessary for carrying out the present invention. For example, the kit of the present invention may contain a reaction component necessary for storing or supplying an enzyme and / or performing genetic polymorphism detection. Such components include, but are not limited to, the oligonucleotides of the invention, enzyme buffers, dNTPs, control reagents (eg, tissue samples, target oligonucleotides for positive and negative controls, etc.), labeling and And / or a detection reagent, a solid support, and instructions. Further, the kit of the present invention may be a partial kit containing only a part of necessary components, and in that case, the user can prepare other components.

  The kit of the present invention can also be provided as a microarray in which the oligonucleotide is immobilized on a support. The microarray is obtained by fixing the oligonucleotide of the present invention on a support, and includes a DNA chip, a Gene chip, a microchip, a bead array, and the like. The microarray may be included in the kit of the present invention together with the oligonucleotide.

As described above, the kit of the present invention includes an oligonucleotide containing the FSHR gene polymorphism. Therefore, before or after use of a therapeutic drug for a patient or the like, blood is collected to isolate the FSHR gene, and this gene is reacted with the oligonucleotide in the kit to identify the genotype. By associating the identified genotype with the presence or absence of a hypertension symptom, the presence or absence of a genetic factor of essential hypertension can be determined easily and with a high reliability by greatly reducing the probability of false positives.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Identification of useful genetic markers for essential hypertension In order to identify genetic markers useful for determining the presence of genetic factors for essential hypertension (EH), genetic polymorphisms present in the human FSHR gene ( Paying particular attention to SNP), the following procedure was used for analysis.

(1) Study subjects A total of 235 EH patients (of which 158 were men and 79 were women) were targeted for the disease group (EH group) of essential hypertension. The conditions to be included in the disease group are at least 3 occasions in the examination within 2 months after the first visit, systolic blood pressure (SBP) 160 mmHg or more and / or diastolic blood pressure (DBP) 100 mmHg or more, and antihypertensive agent Was not taken. The condition was that no secondary hypertension had been diagnosed.

  On the other hand, as a control group (NT group), a total of 237 people (including 152 men and 83 women) were normotensive (NT). The conditions included in the NT group were that there was no family history of hypertension, systolic blood pressure was less than 130 mmHg, and diastolic blood pressure was less than 100 mmHg. Hypertension family history was defined as the subject's grandparents, uncles, aunts, parents, or siblings previously diagnosed with hypertension.

(2) DNA extraction
Extraction of DNA from EH group and NT group was performed by phenol-chloroform method followed by ethanol precipitation (Nakayama T, Soma M, Rahmutula D, Ozawa Y, Kanmatsuse K: Isolation of the 5'-flanking region of (See Medical science monitor: international medical journal of experimental and clinical research 7: 345-349, 2001.). The outline is shown below.

  That is, blood was collected into an EDTA2Na blood collection tube (7 ml or 5 ml), centrifuged to remove plasma, and then the leukocyte membrane and the nuclear membrane were destroyed with SDS buffer and Nonidet P40 (NP40). Proreinase K was used as a proteolytic enzyme, then the protein was removed by the phenol / chloroform method, and DNA was extracted by the ethanol precipitation method.

(3) Genotyping
SNPs present in the human FSHR gene were searched from the dbSNP database of GenBank, and five SNPs with a minor allele frequency (the frequency of the allele with the lower occurrence frequency) greater than 20% were selected (see FIG. 2). This is because SNP with a relatively high minor allele frequency is very useful as a genetic marker for gene-related studies. Information on the SNP allelic frequency can be found on the NCBI and Celera Discovery System-Applied Biosystems website (in order, HYPERLINK "http://www.ncbi.nlm.nih.gov" http: //www.ncbi.nlm. nih.gov/; http://www.appliedbiosystems.com/).

  The location, polymorphism information, and accession number (accession number confirmed by the latter in NCBI and Celera Discovery System-Applied Biosystems) are shown in parentheses. As shown in Table 3 below.

  Determination of individual genotypes of EH group and NT group was performed using Assays-on-Demand kit (Applied Biosystems, Branchburg, NJ) using TaqMan PCR method. The PCR primer and TaqMan probe used for Genotyping of each SNP were in a mixed state in the above kit and were used. Each TaqMan probe is labeled with a fluorescent dye VIC or FAM that becomes a reporter dye at the 5 ′ end, and with NFQ that becomes a quencher dye at the 3 ′ end.

The TaqMan PCR uses GeneAmp9700 (Applied Biosystems) with the following reaction solution composition, “thermal denaturation: 95 ° C. (10 minutes) → annealing: 92 ° C. (15 seconds) → extension: 60 ° C. (1 minute) The total number of cycles was 40.

PCR reaction composition
Template DNA: 2ng (0μL for dry up)
2 x TaqMan Universal
Master Mix UNG: 12.5μL
20 × Assays-on-Demand: 0.43μL
TE buffer: 0.82μL
Sterile water: 11.25μL
Total: 25μL

The fluorescence intensity of the PCR product was detected using ABI PRISM7700 Sequence Detector (Applied Biosystems), and the individual genotypes belonging to the EH group and NT group were determined based on the results.

  The overall distribution of genotypes and alleles was analyzed by chi-square test using 2 × 3 or 2 × 2 contingency tables between EH group and NT group. Statistical analysis was performed using StatView 5.0 (manufactured by SAS) and SPSS Dr. II (manufactured by SPSS). The results are shown in Table 4 below. Statistical significance was p <0.05 (marked with *).

  From the above results, among the above five SNPs, SNPs of rs1394205, rs2055571, rs1007541, and rs2268361 are preferable as useful genetic markers that can be used to determine the presence or absence of genetic factors of essential hypertension (EH). Among them, it was found that the SNPs of rs1394205, rs2055571 and rs2268361 are more preferable, and the SNP of rs1394205 is most preferable. In particular, SRS of rs1394205 was found to be a very useful genetic marker in women.

(4) Linkage disequilibrium analysis and related analysis using haplotypes For the five SNPs described above, linkage disequilibrium (LD) analysis and related analysis using haplotypes were performed. Both analyzes were performed using SNPAlyze version 3.2 (DYNACOM Corporation, Yokohama, Japan). The SNPAlyze version 3.2 above is available on the website (HYPERLINK "http://www.dynacom.co.jp/products/package/snpalyze/index.html" http://www.dynacom.co.jp/ products / package / snpalyze / index.html).

  The analysis result is as shown in FIG. As in the above-described Genotyping results, both gene analyzes described above are preferably useful genetic markers that can be used to determine the presence or absence of EH genetic factors, and SNPs of rs1394205, rs2055571, rs1007541, and rs2268361 are preferred. It was found that the SNPs of rs1394205, rs2055571 and rs2268361 were more preferable, and the SNP of rs1394205 was most preferable.

Measurement of transcriptional activity of G allele and A allele of “rs1394205” In Example 1, for the SNP of rs1394205, which is the most useful genetic marker for the determination of the presence or absence of EH genetic factors, this SNP and the upstream region of this SNP Transcriptional activity was measured using a reporter construct containing The procedure is shown below.

(1) Preparation of plasmid containing DNA fragment of G allele or A allele
A plasmid having a base sequence containing the SNP polymorphic site of rs1394205 was constructed. As the plasmid, a plasmid containing a polymorphic site of G allele (wild type) and a plasmid containing a polymorphic site of A allele (mutant) were constructed.

  First, as shown in FIG. 3, using the following two synthetic oligonucleotide primers, PCR was performed using a part of the Exon1 region of the FSHR gene as a template (G allele and A allele), and the SNP polymorphic site of rs1394205 A fragment containing was amplified.

F primer:
5'-AT GGTACC CCTTAGGTCAGGGTGTAAGAAACCC-3 '(SEQ ID NO: 6)
(Counted from start codon of FSHR gene -202 to -226)
R primer:
5'-AT AAGCTT GGCCATAATTATGCATCCATCCACC-3 '(SEQ ID NO: 7)
(-19 to +6 from the start codon of FSHR gene)
The primer is designed so that the region can be analyzed based on the known knowledge that there is a major promoter activity (core promoter activity) in the region up to -250 bases upstream of the FSHR gene. These primers include a Kpn I restriction enzyme cleavage site (F primer) and a Hind III restriction enzyme cleavage site (R primer).

The PCR described above uses GeneAmp 9700 (Applied Biosystems) with the following reaction solution composition: “thermal denaturation: 98 ° C. (25 seconds) → annealing: 63 ° C. (30 seconds) → extension: 72 ° C. (2 minutes)” A total of 35 cycles were carried out.

PCR reaction composition
Template DNA (100ng / μL): 1μL
TaqDNA polymerase: 0.1μL (5unit / μL)
F primer (10 pmol/μL): 0.4μL
R primer (10 pmol/μL): 0.4μL
dNTP (2.5mM): 3.2μL
10x buffer: 2μL
25 mM MgCl ₂ : 2 μL
Sterile water: appropriate amount (10.9μL)
Total: 20μL

Next, the obtained PCR product was digested with Kpn I and Hind III, and the digested fragment was obtained by digesting the luciferase reporter vector pGV-B2 (manufactured by Toyo Ink, Tokyo) with Kpn I and Hind III. By inserting into a fragment (fragment containing a luciferase gene) and subcloning, a plasmid having a G allele (G-plasmid) and a plasmid having an A allele (A-plasmid) were constructed. Whether the plasmid has a G allele or an A allele was confirmed by a known nucleotide sequencing method.

(2) Measurement of transcription activity of G allele and A allele The G-plasmid and A-plasmid obtained in (1) above were respectively introduced into Chinese hamster ovary (CHO) cells. Specifically, it was introduced as follows by a liposome method using a luciferase assay system (PicaGene Dual SeaPansy, manufactured by Toyo Ink).

  First, 60-70% confluent CHO cells were preincubated with OptiMEM (Lipofectamine, Gibco BRL, Gaithersburg MD) at 37 ° C. for 30 minutes.

  Separately, the G-plasmid (1 μg) obtained in (1) above and a pRL plasmid (200 ng, manufactured by Toyo Ink Co.) containing thymidine kinase (TK) were added to a liposome solution (Lipofectamine, Gibco BRL, 6 μl / well). And dissolved for 20 minutes at room temperature to obtain a lipid-coated plasmid (liposome-encapsulated plasmid). Further, a liposome-encapsulated plasmid was obtained in the same manner as above except that A-plasmid was used instead of G-plasmid. The pRL plasmid is used as a control (internal standard) for confirming that the efficiency is normal.

  Next, 0.8 ml of the cell suspension after the preincubation was placed in each well of the multiwell plate, and the liposome-encapsulated plasmid was added to each well, followed by incubation at 37 ° C. for 3 hours. Thereafter, it was replaced with fresh medium, and further incubated at 37 ° C. for 48 hours.

  The CHO cells in each well after the incubation were lysed with a cell lysate (400 μl), and then centrifuged to remove unlysed cells and debris.

  The extract (50 μl) after centrifugation was used for measurement of luciferase activity. The measurement was performed using luminometers (LB-9507, Berthold, Bad Wildbad, Germany), each aliquoting the extract. All detection data were corrected with pRL-TK activity, an internal standard.

  The results of measuring the transcriptional activity of G allele and A allele obtained as described above are shown in the graph of FIG. FIG. 4 shows the transcriptional activity of the A allele as a relative value when the transcriptional activity of the G allele is 1.0. Each data is shown as mean ± SD. For statistical analysis, StatView 5.0 (SAS Corporation) and SPSS Dr. II (SPSS Corporation) were used. To evaluate the quantitative effects of covariates, multiple logistics regression analysis was performed with SPSS II. Statistical significance was p <0.05.

  From the results shown in FIG. 4, the transcription activity of the A allele was 56% lower than that of the G allele. As a result, it was shown that A allele of SNP of rs1394205 present in the Exon1 untranslated region of the human FSHR gene decreases transcriptional activity and is associated with essential hypertension (particularly in women).

It is a figure which shows the linkage disequilibrium between the gene polymorphisms (SNP) in a FSHR gene. It is a figure which shows the position and kind of gene polymorphism (SNP) in a FSHR gene. It is a figure which shows the binding position of the primer used in Example 2. FIG. 4 is a graph showing the results of measurement of transcription activity in Example 2.

Sequence number 6: Synthetic DNA
Sequence number 7: Synthetic DNA

Claims (7)

  A method for determining essential hypertension, comprising associating a gene polymorphism of a human follicle-stimulating hormone receptor gene and / or a haplotype constituted by the gene polymorphism with the presence or absence of hypertension symptoms in an individual.   The method according to claim 1, wherein the gene polymorphism is at least one selected from the group consisting of a single nucleotide polymorphism, an insertion polymorphism, a deletion polymorphism, and a base repeat polymorphism.   The method according to claim 2, wherein the genetic polymorphism is at least one selected from those shown in Table 1.   The method according to any one of claims 1 to 3, wherein the haplotype is at least one selected from those shown in Table 2.   An oligonucleotide prepared so as to include a gene polymorphic site present in a gene sequence encoding a follicle stimulating hormone receptor or a complementary sequence thereof.   A microarray in which the oligonucleotide according to claim 5 is immobilized on a support. A kit for determining essential hypertension, comprising the oligonucleotide according to claim 5 and / or the microarray according to claim 6.