JP2021145608A - Method for inspecting refractory hypertension - Google Patents

Abstract

To provide a method for accurately inspecting the risk and/or presence of refractory hypertension and an inspection reagent used for the method.SOLUTION: A method for determining the risk and/or presence of refractory hypertension includes analyzing a single nucleotide polymorphism in DLGAP1 gene in the 18 chromosome short-arm 11.3 region (18p11.3) and inspecting hypertension on the basis of the results of analysis.SELECTED DRAWING: None

Description

The present invention relates to a method for testing the risk and presence of treatment-resistant hypertension, and a reagent for the test.

Hypertension is the most common cardiovascular disease. Since most hypertension has no clear cause and is a complex factor, antihypertensive treatment is used as symptomatic treatment.
However, there are some pathological conditions in which blood pressure does not decrease even after administration of three appropriate antihypertensive drugs ((i) calcium antagonist, (ii) ACE inhibitor, angiotensin receptor blocker (ARB), and (iii) diuretic). It is called treatment-resistant hypertension.

Possible causes of treatment-resistant hypertension include white coat hypertension, non-compliance with medication, excessive salt intake, obesity, excessive drinking, and side effects of other medications. However, in patients who are not due to them and / or who are still hypertensive after removing those causes, the factors of treatment-resistant hypertension are unknown, and no tests or treatments have been available so far. There is a problem that treatment is given by trial and error, and it is delayed to receive appropriate treatment by a specialist. Therefore, in order to enable presymptomatic diagnosis (risk diagnosis) and early diagnosis of treatment-resistant hypertension, and to enable treatment of the cause, identification of genes and single nucleotide polymorphisms (SNPs) related to treatment-resistant hypertension can be performed. It is desired.

The DLGAP1 protein encoded by the disks large-associated protein 1 (DLGAP1) gene located in the short arm 11.3 region (18p11.3) on chromosome 18 is localized in postsynaptic thickening and is associated with the postsynaptic density protein 95 (PSD95) protein. It interacts and is involved in maintaining normal brain function and development (Non-Patent Document 1). However, the association between DLGAP1 and hypertension is unknown.

Kawashima N, Takamiya K, Sun J, Kitabatake A, Sobue K. Differential expression of isoforms of PSD-95 binding protein (GKAP / SAPAP1) during rat brain development. FEBS Lett. Dec 1 1997; 418 (3): 301-304 ..

An object of the present invention is to provide a method for accurately testing the risk and presence of treatment-resistant hypertension, and a test reagent used for the method.

As a result of diligent studies to solve the above problems, the present inventors significantly associated SNP located in the intron region of the DLGAP1 gene located in the short arm 11.3 region (18p11.3) of chromosome 18 with treatment-resistant hypertension. Identified to do. Then, they have found that the risk of treatment-resistant hypertension can be predicted and whether or not the patient's hypertension pathology is treatment-resistant can be accurately determined by examining this SNP, and the present invention has been completed.
That is, the gist of the present invention relates to the following.

[1] Includes analysis of single nucleotide polymorphisms present in the DLGAP1 gene in the short arm 11.3 region (18p11.3) of chromosome 18 and testing for treatment-resistant hypertension based on the analysis results.
How to determine the risk and / or presence of treatment-resistant hypertension.
[2] The single nucleotide polymorphism is a single nucleotide polymorphism in a base corresponding to the 51st base of the base sequence of the base sequence shown in SEQ ID NO: 1 or a base having a linkage disequilibrium relationship with the base [1]. ] The method described in.
[3] A probe for testing treatment-resistant hypertension, which has a sequence of 10 or more bases including the 51st base of the base number in the base sequence shown in SEQ ID NO: 1 or a complementary sequence thereof.
[4] A primer for testing treatment-resistant hypertension, which can amplify a region containing the 51st base of the base number in the base sequence shown in SEQ ID NO: 1.

According to the present invention, it is possible to predict the risk of treatment-resistant hypertension, which has been difficult to predict, and it is possible to accurately and easily determine the presence or absence of treatment-resistant hypertension, that is, whether or not the patient's hypertension condition is treatment-resistant. Can be determined. Therefore, the present invention contributes to the prevention and early treatment of treatment-resistant hypertension.

FIG. 1 shows the results of a genome-wide association study of Japanese patients with refractory hypertension. The horizontal axis shows the position on the chromosome, and the vertical axis shows the strength of SNP association (-log ₁₀ P value of each SNP). The statistically relevant gene regions are above the upper line.

In order to identify a novel locus associated with treatment-resistant hypertension, the present inventors conducted a genome-wide association study using 2,705 cases of treatment-resistant hypertension and 21,296 cases of mild hypertension (control) in a Japanese population. , Rs1442386 on chromosome 18p11.3 (DLGAP1) was identified as a novel susceptibility locus achieved at the significance level (P <5 × 10 ^-8). That is, it was identified that DLGAP1 on chromosome 18 is a novel susceptibility locus for treatment-resistant hypertension in the Japanese population. The present invention has been made based on the same findings.

Hereinafter, the present invention will be described.
(Judgment method)
One aspect of the present invention comprises analyzing a single nucleotide polymorphism present in the DLGAP1 gene of the short arm 11.3 region (18p11.3) of chromosome 18 and examining hypertension based on the analysis result. The present invention relates to a method for determining the risk and / or presence / absence of sexual hypertension (hereinafter, may be referred to as “determination method of the present invention”).
In the present invention, the "test" includes a test for the risk of treatment-resistant hypertension and a test for the presence or absence of treatment-resistant hypertension. In the methods of the invention, the results of SNP analysis are associated with the risk and / or presence or absence of treatment-resistant hypertension. The determination method of the present invention may further include a step of obtaining a sample to be inspected for SNP from the person to be inspected.

The treatment-resistant hypertension examined by the determination method of the present invention is not particularly limited as long as it is a hypertension for which drug treatment is ineffective. The condition in which the target blood pressure does not decrease even if the antihypertensive drug ((i) calcium channel blocker, (ii) ACE inhibitor, angiotensin receptor blocker (ARB), (iii) diuretic) is administered.

The determination method of the present invention can be used for subjects of any race. The method of the present invention is not particularly limited, but can be suitably used for, for example, Asian subjects such as Japanese and Chinese, and Caucasian subjects. In particular, it can be suitably used for Japanese subjects. In addition, the method of the present invention can be used for subjects of any gender.

As a specific SNP present in the short arm 11.3 region of chromosome 18, human rs1442386 can be mentioned.
Here, the rs number indicates the registration number of the dbSNP database (http //www.ncbi.nlm.nih.gov/projects/SNP/) of the National Center for Biotechnology Information. rs1442386 is located in the linkage disequilibrium block containing the 18p11.3 DLGAP1 gene. Therefore, hypertension can be tested, in particular, by analyzing SNPs present in the linkage disequilibrium block, such as SNPs present on the DLGAP1 gene. Specific examples of the human DLGAP1 gene include the region of 3496032..4455441 (complementary strand) of GenBank Accession No. NC_000018.10.

rs1442386 means adenine (A) / guanine (G) polymorphism at base 3938439 of GenBank Accession No. NC_000018.10, and if this base is risk allergic G, the possibility or risk of treatment-resistant hypertension Can be determined to be high. In addition, when analyzed in consideration of genotype, hypertension may be treatment-resistant or the risk of treatment-resistant hypertension is high in the order of GG> GA> AA. Therefore, for subjects whose base is G, it is conceivable to discontinue drug treatment and treat other hypertension.

For rs1442386, a sequence having a total length of 101 bp including the SNP base and a region of 50 bp before and after it is shown in SEQ ID NO: 1. If the 51st base in SEQ ID NO: 1 has a polymorphism and this base is G (risk allele), it can be determined that hypertension may be treatment-resistant or the risk of treatment resistance is high.

In the present invention, a base corresponding to the above base is analyzed. The "base corresponding to the above base" means the corresponding base in the above region. That is, in order to "analyze the base corresponding to the above base", even if the above sequence changes slightly at a position other than SNP due to individual differences or the like, the base recognized as the above SNP is analyzed in view of the preceding and following sequences. For example.

Further, the bases to be analyzed in the present invention are not limited to those described above, and polymorphisms of bases having a linkage disequilibrium with the above bases may be analyzed. Here, the "base in linkage disequilibrium with the above base" means a base ^{satisfying the relationship of r 2} > 0.5, preferably r ² > 0.8, and more preferably r ² > 0.9 with the above base. r ² is the linkage disequilibrium coefficient. Bases that are in linkage disequilibrium with the above bases can be identified using, for example, the HapMap database (http://www.hapmap.org/index.html.ja). Alternatively, it can be identified by analyzing the base sequence of DNA collected from a plurality of persons (usually about 20 to 40 persons) and searching for SNPs having linkage disequilibrium. Bases that are in linkage disequilibrium with the above bases are homozygotes of risk alleles> heterozygotes of risk alleles and non-risk alleles> homozygotes of non-risk alleles when analyzed in consideration of genotype. In order, treatment-resistant hypertension is likely or at high risk.

Treatment-resistant hypertension can be tested by examining the type of SNP base and associating the obtained results with treatment-resistant hypertension based on the above criteria. The SNP may be analyzed alone, or a plurality of SNPs including the SNP may be collectively analyzed (haplotype analysis). For example, the above SNP may be analyzed in combination with a known SNP associated with hypertension or an SNP in linkage disequilibrium with the known SNP. Collective analysis of multiple SNPs associated with hypertension may improve the accuracy of hypertension testing. For any SNP, either strand of double-stranded DNA may be analyzed. For example, the sense strand of the DLGAP1 gene sequence may be analyzed, or the antisense strand may be analyzed.

The sample used for SNP analysis is not particularly limited as long as it is a sample containing chromosomal DNA. Examples of the sample used for SNP analysis include body fluids such as blood, urine and cerebrospinal fluid, cells such as oral mucosa, and body hair such as hair. Although these samples can be used directly for SNP analysis, it is preferable to isolate chromosomal DNA from these samples by a conventional method and analyze using the same.

Analysis of SNP can be performed by a conventional gene polymorphism analysis method. For example, sequence analysis, PCR, hybridization, invader method and the like can be mentioned, but the present invention is not limited thereto.

Sequence analysis can be performed by a conventional method. Specifically, a sequence reaction is performed using a primer set at a position tens of bases 5'from the SNP, and the analysis result is used to determine what kind of base the corresponding position is. Can be done. Before the sequence reaction, it is preferable to amplify the fragment containing the SNP site by PCR or the like in advance.

In addition, SNP analysis can be performed by examining the presence or absence of amplification by PCR. For example, a primer having a sequence corresponding to a region containing an SNP and having a 3'end corresponding to each SNP is prepared. PCR can be performed using each primer to determine which type of polymorphism is present depending on the presence or absence of amplification products. In addition, the LAMP method (Patent No. 3313358), NASBA method (Nucleic Acid Sequence-Based Amplification; Patent No. 2843586), ICAN method (Japanese Patent Laid-Open No. 2002-233379), SmartAmp method (Patent No. 3897805). It is also possible to check the presence or absence of amplification by means of a book) or the like. In addition, a single chain amplification method may be used.

It is also possible to amplify a DNA fragment containing an SNP site and determine which type of polymorphism it is based on the difference in mobility of the amplification product in electrophoresis. Such a method includes, for example, the PCR-SSCP (single-strand conformation polymorphism) method (Genomics. 1992 Jan 1;
12 (1): 139-146.). Specifically, first, the DNA containing the target SNP is amplified, and the amplified DNA is dissociated into a single-stranded DNA. The dissociated single-stranded DNA can then be separated on a non-denatured gel and the type of polymorphism can be determined by the difference in mobility of the separated single-stranded DNA on the gel.

Furthermore, when a base showing a polymorphism is included in the restriction enzyme recognition sequence, it can be analyzed by the presence or absence of cleavage by the restriction enzyme (RFLP method). In this case, first, the DNA sample is cleaved with a restriction enzyme. The DNA fragment can then be separated and the size of the detected DNA fragment can be used to determine what type of polymorphism it is.

It is also possible to analyze the type of polymorphism by examining the presence or absence of hybridization using a DNA chip or the like. That is, it is also possible to investigate which base the SNP is by preparing a probe corresponding to each base and examining which probe it hybridizes to.

By determining which base the SNP is in this way, data for testing hypertension can be obtained.

As an example of a specific aspect of the method of the present invention, first, the base corresponding to the above SNP is analyzed to determine the type of base of the subject. Next, when the type of the base is a risk allergic, it is determined that hypertension may be treatment-resistant or the risk of treatment-resistant hypertension is high, and when it is a non-risk allergic, it is determined that the test result is low. Information can be provided directly to the subject or to a doctor or the like. The determination is mechanically sorted according to whether the subject's base type is a risk allele or a non-risk allele. Therefore, the determination of risk and the provision of information based on the method of the present invention do not require professional judgment by a doctor or the like.

The test results are provided to a doctor or the like as needed, and the doctor or the like can treat the subject. For example, for a subject who is judged to be at high risk of treatment-resistant hypertension, first confirm whether or not he / she already has hypertension by performing a more detailed test such as a clinical test. is important. The initial symptoms may be mild and the patient may not complain of subjective symptoms. However, even if there is no subjective symptom, it is preferable to perform a more detailed examination, follow-up, and early treatment. If hypertension cannot be confirmed even by detailed examination, then regular examinations can be performed to confirm the presence or absence of hypertension. In addition, if it can be determined that hypertension is likely to be treatment-resistant hypertension, it is possible to apply more appropriate treatment for treatment-resistant hypertension at an early stage. On the other hand, for subjects who are judged to have a low risk of hypertension, the frequency and frequency of consultations / tests can be reduced. In this way, by performing the method of the present invention, the number of consultations and the method of examination are appropriately set, and the medical and financial burden and labor of hypertensive patients, patients suspected of having hypertension, medical staff, etc. are reduced. can do.

(Test reagent)
A further aspect of the present invention is capable of analyzing single nucleotide polymorphisms present in the DLGAP1 gene of the short arm 11.3 region (18p11.3) of chromosome 18 and / or bases having a linkage disequilibrium relationship with the same base. The present invention relates to a test reagent for testing treatment-resistant hypertension (hereinafter, may be referred to as “test reagent of the present invention”) containing a polynucleotide such as a probe or a primer.
The matters described in the determination method of the present invention are all applied to the description of the test reagent of the present invention.
The polynucleotide includes, but is not limited to, probes, primers and the like. Further, a polynucleotide capable of detecting one SNP may be included alone in the test reagent, or a plurality of the polynucleotides may be included. In order to improve the accuracy of the test, it is preferable to test a plurality of SNPs with a plurality of polynucleotides. In addition, polynucleotides that can detect non-risk alleles may be included.
Examples of such a probe include a probe that includes the above SNP site and can determine the type of base of the SNP site depending on the presence or absence of hybridization. Specifically, it includes a sequence containing the 51st base of the base sequence shown in SEQ ID NO: 1, a probe having a length of 10 bases or more having a complementary sequence thereof, and a base having a chain imbalance with the base. Examples thereof include probes having a sequence or a complementary sequence thereof and a length of 10 bases or more. For the base sequences of "bases having a linkage disequilibrium relationship with the bases" and the regions before and after them, for example, the dbSNP database of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/projects) It can be obtained from / SNP /). The length of the probe is preferably 15-35 bases, more preferably 20-35 bases. It may be a set including a plurality of probes.

Examples of the primer include a primer that can be used for PCR for amplifying the SNP site, and a primer that can be used for sequence analysis (sequencing) of the SNP site. Specifically, a primer capable of amplifying or sequencing the region containing the 51st base of the base sequence shown in SEQ ID NO: 1 and a region containing a base having a linkage disequilibrium relationship with the base are included. Primers that can be amplified and sequenced can be mentioned. The length of such a primer is preferably 10 to 50 bases, more preferably 15 to 35 bases, even more preferably 20 to 35 bases. The primer may be a single primer, or may be a primer set containing a primer in the 5'side region of the SNP site and a primer in the 3'side region of the SNP site. It may be a set including a plurality of primer sets.

Examples of the primer for sequencing the SNP site include a primer having a sequence 5'-side region of the base, preferably 30 to 100 bases upstream, and a 3'-side region of the base, preferably 30 to 100 bases downstream. Primers having a sequence complementary to the region of are exemplified. Primers used to determine polymorphisms based on the presence or absence of amplification by PCR include a sequence containing the above base, a primer containing the base on the 3'side, and a complementary sequence of the sequence containing the base. Examples include a primer containing a complementary base of the above base on the 3'side.

In addition to these primers and probes, the test reagent of the present invention may contain a polymerase or buffer for PCR, a reagent for hybridization, a fluorescent dye, or the like.

Hereinafter, the present invention will be specifically described with reference to Examples, but these are examples of the present invention, and the scope of the present invention is not limited thereto.

2,705 patients who used 4 or more antihypertensive drugs using clinical information and SNP information of the Japanese population registered in BioBank Japan (https://biobankjp.org/index.html) A genome-wide association study was performed by identifying 21,296 patients with mild hypertension (control group) who used only one antihypertensive drug (treatment-resistant hypertension group). We performed whole-genome imputation using 1000 Genome Projects Phase 3 data as a reference, and examined about 6 million SNPs distributed throughout the genome. The DLGAP1 gene on chromosome 18p11.3 [rs1442386, P = 3.75 × 10 ^-8 , Odds ratio = 0.85], identified a new disease susceptibility locus (Fig. 1).

SNPs (rs1442386) are located within the DLGAP1 gene, and the DLGAP1 protein is involved in maintaining normal brain function and development. Genetic variation of DLGAP1 has been reported to be associated with several psychiatric disorders such as obsessive-compulsive disorder, schizophrenia, and major depressive disorder, but the association with the disease is still poorly understood.
In addition, the SNP-related protein identified this time is obsessive-compulsive disorder, Reduction in Gain of Function.
The association with autism and bipolar disorder has been reported in the of Function.
The present invention has revealed a correlation between rs1442386 and treatment-resistant hypertension. In addition, the above-mentioned findings suggest that psychological stress may be involved in the pathophysiology of treatment-resistant hypertension.

The present invention contributes to the prevention and treatment of treatment-resistant hypertensive patients by early diagnosis.

<Explanation of sequence listing>
SEQ ID NO: 1: Nucleotide sequence containing 50 bp before and after rs1442386

Claims (4)

Includes analysis of single nucleotide polymorphisms present in the DLGAP1 gene of the short arm 11.3 region (18p11.3) of chromosome 18 and testing for hypertension based on the analysis results.
How to determine the risk and / or presence of treatment-resistant hypertension. The single nucleotide polymorphism according to claim 1, wherein the single nucleotide polymorphism is a single nucleotide polymorphism in a base corresponding to the 51st base of the base number of the base sequence shown in SEQ ID NO: 1 or a base having a linkage disequilibrium relationship with the base. the method of. A probe for testing treatment-resistant hypertension, which has a sequence of 10 or more bases including the 51st base of base number 1 or a complementary sequence thereof in the base sequence shown in SEQ ID NO: 1. A primer for testing treatment-resistant hypertension, which can amplify a region containing the 51st base of the base number in the base sequence shown in SEQ ID NO: 1.

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