JP6041338B2 - Method for examining aldosterone-producing adenoma using KCNJ5 gene and screening method for aldosterone-producing adenoma - Google Patents

Description

The present invention relates to a method for testing aldosterone-producing adenoma using genetic analysis, and to probes and primers used therefor.
The present invention also relates to a screening method for a therapeutic agent for aldosterone-producing adenoma.

Hypertension is a typical lifestyle-related disease in Japan, and the number of patients has been reported to be 40 million. Many causes of hypertension are considered to be essential hypertension of unknown cause, but part of the cause is secondary hypertension with clear underlying disease, especially primary aldosteronism that produces excessive aldosterone in adrenal tumors Have been shown to account for 5-10% of total hypertension. In Japan, the diagnosis of aldosterone-producing adenoma, which accounts for about 80% of the cause, is based on preoperative blood test findings, and no definitive diagnosis method by genetic diagnosis has been reported.
Until now, there were no reports of genetic mutations related to aldosterone-producing adenomas.
In January, mutations in the KCNJ5 gene (KCNJ5 G151R (G / A), KCNJ5 L168R and KCNJ5 T158A) in aldosterone-producing adenoma were reported for the first time (Non-patent Document 1). However, the G151R mutation was only the 451st base G mutation to A, and no other mutation was reported.
The relationship between the KCNJ5 gene and the onset and severity of aldosterone-producing adenoma was not clear.

Science 331,768, 2011

  An object of the present invention is to provide a test method for diagnosing aldosterone-producing adenoma and accurately predicting the severity.

The present inventors have made extensive studies to solve the above problems. As a result, it was found that a single base mutation in a specific base on the KCNJ5 gene is related to aldosterone-producing adenoma. Furthermore, the present inventors have found that the expression level of the KCNJ5 gene is highly expressed in aldosterone-producing adenomas and is closely related to the severity of aldosterone-producing adenomas, thereby completing the present invention.

That is, the present invention is as follows.
(1) A method for examining aldosterone-producing adenoma, comprising a step of measuring the expression level of mRNA or protein of KCNJ5 gene.
(2) a step of adding a drug candidate substance to a cell expressing a reporter gene linked to a promoter of KCNJ5 gene or KCNJ5 gene, a step of measuring the expression level of KCNJ5 gene or reporter gene, and a substance that decreases the expression level A method of screening for a therapeutic agent for aldosterone-producing adenoma, which comprises the step of:
(3) A single base mutation in the base corresponding to the base number 451 in the base sequence of SEQ ID NO: 1 on the KCNJ5 gene is analyzed, and the aldosterone-producing adenoma is based on whether or not the base is cytosine How to inspect.
(4) An aldosterone-producing adenoma test probe having a sequence of 10 bases or more including the base at position 451 in the base sequence of SEQ ID NO: 1 or a complementary sequence thereof.
(5) A primer for testing an aldosterone-producing adenoma capable of amplifying a region containing the base at position 451 in the base sequence of SEQ ID NO: 1.

According to the test method of the present invention, aldosterone-producing adenoma can be accurately diagnosed, the severity etc. can be clarified, and prognosis can be predicted and an appropriate treatment method can be selected.
In some severe cases of aldosterone-producing adenomas, the prognosis is poor due to complications after glandectomy, so genetic diagnosis of KCNJ5 mRNA levels and mutations in adenomas should be used to determine surgical indications and postoperative management It is very useful if you can.

The graph which shows the comparison of the KCNJ5 gene expression level in the aldosterone production adenoma patient (MUT) which has a variation | mutation in a KCNJ5 gene, and the aldosterone production adenoma patient (WT) which does not have a variation | mutation. The graph which shows the comparison of a KCNJ5 gene expression level in an aldosterone production adenoma patient (APA), a subclinical Cushing syndrome patient (SCS), a Cushing syndrome patient (CS), and a pheochromocytoma patient (PHEO). The graph which shows the relationship between the blood aldosterone level (A), the urinary aldosterone level (B), the diastolic blood pressure (C), and the expression level of a KCNJ5 gene in a patient with aldosterone-producing adenoma. The graph which shows the relationship between the amount of aldosterone synthase CYP11B2 mRNA (A) and the amount of steroid dehydrogenase HSD3B2 mRNA (B) and KCNJ5 gene expression in patients with aldosterone-producing adenoma. The graph which shows the relationship between the presence or absence of KCNJ5 gene mutation and the mRNA expression level of KCNJ5 in aldosterone-producing adenoma patients.

<1> Test method for aldosterone-producing adenoma based on single nucleotide polymorphism of KCNJ5 gene The test method of the present invention analyzes a single-base mutation of a base present on KCNJ5 gene, and tests aldosterone-producing adenoma based on the corresponding analysis. It is a method to do. Aldosterone-producing adenoma refers to a disease state diagnosed according to the criteria described in J Clin Endocrinol Metab 93: 3266-3281, 2008, for example. In the present invention, the “test” includes a test for determining whether the aldosterone-producing adenoma is present and a test for predicting the severity of the aldosterone-producing adenoma.

The KCNJ5 gene encodes a G protein-sensitive inward rectifier potassium channel and is also called Kir3.4 or GIRK4. The KCNJ5 gene is preferably a human KCNJ5 gene, and examples thereof include a gene having the base sequence described in SEQ ID NO: 1. However, the gene of interest is not limited to the gene of the above sequence because there is a possibility of substitution or deletion in one or more bases depending on the race or the like.

As a single nucleotide mutation of the KCNJ5 gene related to aldosterone-producing adenoma, a single nucleotide mutation in the base corresponding to the base number 451 in the nucleotide sequence of SEQ ID NO: 1 is analyzed. The base corresponding to the base number 451 in the base sequence of SEQ ID NO: 1 is a mutation present in the coding region of the KCNJ5 gene (SEQ ID NO: 1), and the base in the base number 451 is guanine ( Whereas G), the mutant type is thymine (C) or adenine (A), and the 151st amino acid Gly of SEQ ID NO: 2 is substituted with Arg.
In the present invention, when the base is C, the test is performed based on the criterion of aldosterone-producing adenoma.

In the present invention, a base corresponding to this base is analyzed. Here, “corresponding” means a corresponding base in the region having the sequence on the human KCNJ5 gene, and even if the sequence is slightly changed at a position other than the SNP due to a difference in race, It also includes analyzing the corresponding base in it. For example, the human KCNJ5 gene may not completely match SEQ ID NO: 1, and may have substitutions, deletions, insertions, etc. other than the 451st base. If there is an insertion or deletion of a single base before the 451st base in SEQ ID NO: 1, the “451st base” may be mixed. The analysis target is a base corresponding to the “451st base” in contrast to SEQ ID NO: 1.

The aldosterone-producing adenoma can be examined by examining the type of the base of the mutation. The number of mutations to be examined may be one of the above, but may be combined with mutations at other positions of the KCNJ5 gene, or may be combined with mutations of other genes that correlate with aldosterone-producing adenomas. The sequence of the KCNJ5 gene may be analyzed for the sense strand or the antisense strand.

The sample used for the analysis of the single nucleotide mutation of the KCNJ5 gene is not particularly limited as long as it is a sample containing DNA or mRNA of the subject's aldosterone-producing adenoma.

Analysis of a single nucleotide mutation in the KCNJ5 gene can be performed by a usual single nucleotide mutation analysis method. Examples include, but are not limited to, rapid nucleic acid amplification methods (SMAP method (Nature Methods (2007), Vol. 4, No. 3, p257-262 and Japanese Patent No. 3942627), sequence analysis, PCR, hybridization, and the like.

The sequence can be performed by a usual method. Specifically, a sequence reaction is performed using a primer set at a position of several tens of bases on the 5 ′ side of the base showing mutation, and from the analysis result, it is determined which type of base the corresponding position is. be able to. In addition, when performing a sequence, it is preferable to amplify the fragment containing a mutation beforehand by PCR.

  It can also be analyzed by examining the presence or absence of amplification by PCR. For example, a primer having a sequence corresponding to a region containing a base showing a mutation and corresponding to each mutation is prepared. PCR can be performed using each primer, and the type of mutation can be determined based on the presence or absence of an amplification product.

It is also possible to amplify a DNA fragment containing a mutation and determine which type of mutation is based on the difference in mobility in electrophoresis of the amplified product. Examples of such a method include a PCR-SSCP (single-strand conformation polymorphism) method (Genomics. 1992 Jan 1; 12 (1): 139-146.). Specifically, first, DNA containing the mutation site of the KCNJ5 gene is amplified, and the amplified DNA is dissociated into single-stranded DNA. Next, the dissociated single-stranded DNA is separated on a non-denaturing gel, and the type of mutation can be determined by the difference in mobility of the separated single-stranded DNA on the gel.

  Furthermore, when a base showing a mutation is included in the restriction enzyme recognition sequence, it can be analyzed by the presence or absence of cleavage by a restriction enzyme (RFLP method). In this case, first, a DNA sample is amplified by PCR and cut with a restriction enzyme. The DNA fragments can then be separated and the type of mutation determined by the size of the detected DNA fragment.

  It is also possible to analyze the type of mutation by examining the presence or absence of hybridization. That is, it is possible to determine which base a mutation is by preparing probes corresponding to each base and examining which probe hybridizes.

<2> Method for examining aldosterone-producing adenoma based on the expression level of KCNJ5 gene In the present invention, the expression level of KCNJ5 is increased in patients with aldosterone-producing adenoma, and the high level of KCNJ5 defines the severity of aldosteronism It was found that blood and urinary aldosterone levels and diastolic blood pressure increased. Therefore, the present invention provides a method for examining aldosterone-producing adenoma, which comprises the step of measuring the expression level of KCNJ5 gene mRNA or protein. Examples of the sample to be used include adrenal tissue isolated from a test subject.

If the expression level of KCNJ5 gene mRNA or protein in the adrenal tissue of the test subject is increased compared to the value (control value) in a person who does not suffer from aldosterone-producing adenoma, such as a healthy person, aldosterone-producing adenoma It can be determined that the affected or aldosterone-producing adenoma is more severe. In addition, if the expression level of KCNJ5 gene mRNA or protein in the adrenal tissue of the test subject is equal to or higher than the value in aldosterone-producing adenoma patients (control value), the patient is suffering from aldosterone-producing adenoma. Alternatively, it can be determined that the severity of aldosterone-producing adenoma is high.

The expression level of the KCNJ5 gene is increased in aldosterone-producing adenomas, but not in Cushing syndrome patients and pheochromocytoma patients. Therefore, it is possible to distinguish these diseases from aldosterone-producing adenomas.

The expression level of the KCNJ5 gene can be examined by RT-PCR, Northern blot, microarray method and the like. The expression level of the KCNJ5 gene product can be examined by ELISA, Western blot, or the like. As the antibody, a commercially available antibody can be used, and an antibody prepared by using a part of the amino acid sequence of the KCNJ5 protein (for example, SEQ ID NO: 2) as an antigen can also be used. Examples of the base sequence of the coding region of KCNJ5 include (SEQ ID NO: 1), and primers or probes for expression analysis can be designed or obtained using this sequence or the like. Samples used for the examination include adrenal tissue.

<3> Method for screening therapeutic agent for aldosterone-producing adenoma In the present invention, the amount of mRNA of aldosterone synthase CYP11B2 and steroid dehydrogenase HSD11B2 derived from the adrenal cortex related to aldosterone synthesis and the amount of KCNJ5 mRNA show a positive correlation. It was also found that the KCNJ5 gene is involved in aldosterone synthesis. Therefore, a substance that can be a therapeutic agent for aldosterone-producing adenoma can be obtained by screening a drug using the activity of decreasing the expression of KCNJ5 as an index.
That is, the screening method of the present invention includes a step of adding a drug candidate substance to a cell expressing a reporter gene linked to a KCNJ5 gene or a promoter of KCNJ5 gene, a step of measuring the expression level of KCNJ5 gene or reporter gene, and The screening method of the therapeutic agent of aldosterone production adenoma including the process of selecting the substance which reduces the said expression level is mentioned.
For example, when the expression level of the KCNJ5 gene or reporter gene is suppressed in the presence of a drug candidate substance compared to the absence of the drug candidate substance, the drug candidate substance is selected as a candidate for a therapeutic drug for aldosterone-producing adenoma be able to. As cells expressing the KCNJ5 gene, cells derived from the adrenal cortex and the like can be used.

When a reporter gene linked to the promoter of the KCNJ5 gene is used, the KCNJ5 gene promoter is preferably a region containing about 1 kbp upstream of the transcription start site, and more preferably a region containing about 2 kbp upstream. The sequence information of the promoter can be obtained from Proc Natl Acad Sci US A. 2008 June 10; 105 (23): 8148-8153.
Examples of reporter genes include luciferase gene, GFP gene, chloramphenicol acetyltransferase gene and the like. These reporter genes are linked to the promoter of the KCNJ5 gene, incorporated into a plasmid used for introducing the gene into mammalian cells, and transfected into cells by a conventional method such as lipofection.
A drug candidate substance is added to a cell expressing the KCNJ5 gene as described above or a cell into which the reporter gene has been introduced, and the expression level of the KCNJ5 gene or the reporter gene is measured.

There is no restriction | limiting in particular as a pharmaceutical candidate substance, For example, a low molecular synthetic compound may be sufficient and the compound contained in a natural product may be sufficient. Further, it may be a peptide or a nucleic acid. Although individual test substances may be used for screening, a compound library containing these substances may be used. By selecting a candidate substance that lowers the expression level of the KCNJ5 gene or reporter gene (compared to when it is not added), a substance that can be a therapeutic drug for aldosterone-producing adenoma can be obtained.

The expression level of the KCNJ5 gene can be measured by methods such as RT-PCR, quantitative PCR, Northern blot, ELISA, Western blotting, In situ hybridization, and immunohistochemical staining.
Although the expression level of the reporter gene depends on the type of reporter gene, it can be measured by fluorescence intensity, luminescence intensity, radioactivity intensity, and the like.

In the present invention, the expression level of the KCNJ5 gene can be compared between the presence and absence of an aldosterone-producing adenoma therapeutic agent to determine the effect of the aldosterone-producing adenoma therapeutic agent.

<4> Reagent for test | inspection of this invention This invention also provides test reagents, such as a primer and a probe for test | inspecting aldosterone production adenoma. Examples of such a probe include a probe that includes the mutation site in the KCNJ5 gene and can determine the type of base at the mutation site based on the presence or absence of hybridization. Specifically, a probe having a length of 10 bases or more having a sequence containing the 451st base of the base sequence of SEQ ID NO: 1 or a complementary sequence thereof can be mentioned. The length of the probe is preferably 15 to 35 bases, more preferably 20 to 35 bases.

Examples of the primer include a primer that can be used for PCR for amplifying the mutation site in the KCNJ5 gene, or a primer that can be used for sequence analysis (sequencing) of the mutation site. Specifically, a primer that can amplify or sequence a region containing the 451st base of the base sequence of SEQ ID NO: 1 can be mentioned. The length of such a primer is preferably 15 to 50 bases, more preferably 20 to 35 bases. Such a primer can be set, for example, at a position upstream or downstream of the mutation site in SEQ ID NO: 1.
As a primer for sequencing the mutation site, a primer having a sequence 5′-side of the mutant base, preferably 30 to 100 bases upstream, or 3 ′ of the mutant base is used.
Examples include a primer having a sequence complementary to a side region, preferably a region downstream of 30 to 100 bases. Primers used for judging mutations based on the presence or absence of amplification by PCR have a sequence containing the above-mentioned mutant base, a primer containing the above-mentioned mutant base on the 3 ′ side, and a complementary sequence of the sequence containing the above-mentioned mutant base. Examples thereof include a primer containing a complementary base of the mutant base on the 3 ′ side.

The test reagent of the present invention may contain PCR polymerases, buffers, hybridization reagents, etc. in addition to these primers and probes.

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

<Patient and clinical data>
We analyzed 23 patients with aldosterone-producing adenoma who underwent surgery at Gunma University Hospital between 2007 and 2011 after obtaining informed consent. The diagnosis of aldosterone-producing adenoma was according to J Clin Endocrinol Metab 93: 3266-3281, 2008 and J Clin Endocrinol Metab 96: 2512-2518, 2011. The clinical data for each patient is shown in Table 1.

<Mutation analysis>
Total RNA was isolated from frozen aldosterone-producing adenoma samples from patients, cDNA was synthesized, and sequence analysis was performed by direct sequencing. The primer sequences used are as follows.
Forward 5-taagggacaccccagaagttagcat-3 (SEQ ID NO: 3)
Reverse 5-aaaacatgagggtctccgctctctt-3 (SEQ ID NO: 4)

a: no data b: 30 minutes resting after 1 to 3 months after surgery or sitting value c: no data for transfer
HT: duration of hypertension
C (calcium channel inhibitor), R (angiotensin II receptor antagonist), β (β inhibitor), S (spironolactone), K (potassium supplement), E (angiotensin converting enzyme inhibitor)
DM / IGT: Diabetes mellitus / glucose intolerance
HL: Hyperlipidemia
PAC: Blood aldosterone concentration
PRA: blood renin concentration
eGFR: Estimated glomerular filtration rate
DST: Dexamethasone suppression test

<Result>
Table 1 shows the mutation of the KCNJ5 gene in each patient. As a result, KCNJ5 gene mutation was not found in 8 people, but mutation was seen in 15 people, 3 of which were L16
8R mutations, 8 were G151R G / A mutations, and 4 were novel G151R G / C mutations.
Further, as a result of examining the mRNA amount of the KCNJ5 gene in 23 patients, it was found that the amount of mRNA of the KCNJ5 gene was significantly higher in patients having mutations in the KCNJ5 gene (FIG. 1).

In addition, the mRNA level of the KCNJ5 gene in 24 patients (22 cases other than No. 5 and 23 and 3 new cases in Table 1) and 3 patients with aldosterone-producing adenomas (APA) was measured in patients with subclinical Cushing syndrome (SCS: 7), Cushing's syndrome patients (CS: 5), and pheochromocytoma patients (PHEO: 10), KCNJ5 gene mRNA levels were found to be significantly higher in aldosterone-producing adenoma patients (Fig. 2). ). In FIG. 2, the amount of KCNJ5 mRNA in the normal adrenal is 1.0 (N = 3), and the amount of KCNJ5 mRNA is displayed.
The mRNA level of KCNJ5 gene was standardized by GAPDH. Each was measured using a Taqman probe.
TaqMan probes for KCNJ5 (Hs00168476_m1), GAPDH (Hs99999905_g1)

Next, in the above 24 aldosterone-producing adenoma patients, blood aldosterone levels, urinary aldosterone levels, and diastolic blood pressure were measured and plotted together with the expression level of KCNJ5 mRNA. As shown in FIG. Positive correlations were observed between blood and urinary aldosterone levels (FIGS. 3A and 3B) and diastolic blood pressure (FIG. 3C).

Next, in the above 24 aldosterone-producing adenoma patients, the amount of aldosterone synthase CYP11B2 mRNA and the amount of steroid dehydrogenase HSD3B2 mRNA were measured and plotted together with the KCNJ5 mRNA expression level, as shown in FIG. It was found that the amount of KCNJ5 mRNA and the amount of aldosterone synthase CYP11B2 mRNA showed a positive correlation, and the amount of steroid dehydrogenase HSD3B2 mRNA showed a positive correlation.

  Of the 24 aldosterone-producing adenoma patients, 16 patients had mutations in the KCNJ5 gene (L168R or G151R) and 8 patients had no mutation in the KCNJ5 gene, but the amount of KCNJ5 mRNA was mutated As shown in FIG. 5, the amount of KCNJ5 mRNA was found to be significantly high in the mutant cases. There were no mutations in the KCNJ5 gene in patients with subclinical Cushing syndrome, patients with Cushing syndrome, and patients with pheochromocytoma.

Based on the above, it was discovered that blood and urinary aldosterone levels that define the severity of aldosteronism and diastolic blood pressure increase in patients with high levels of KCNJ5 mRNA in aldosterone-producing adenomas. KCNJ5 gene mutation cases showed significantly higher KCNJ5 mRNA levels than those without mutations. Furthermore, the mRNA levels of adrenal cortex-derived aldosterone synthase CYP11B2 and steroid dehydrogenase HSD11B2, which are related to aldosterone synthesis, were positively correlated with the KCNJ5 mRNA level, and it was elucidated that the KCNJ5 gene was involved in aldosterone synthesis.

Claims (3)

A method for examining aldosterone-producing adenoma, comprising the step of measuring the expression level of KCNJ5 gene mRNA in adrenal tissue isolated from a test subject , wherein aldosterone-producing adenoma is examined according to the following criteria: Examination method of producing adenoma .
If the expression level of KCNJ5 gene mRNA in the adrenal gland of the test subject is increased compared to the value in those who are not affected by aldosterone-producing adenoma, or if the patient is affected by aldosterone-producing adenoma Judge that the severity is high, or
If the expression level of KCNJ5 mRNA in the adrenal gland of the subject is equal to or greater than that in patients with aldosterone-producing adenoma, the patient is suffering from aldosterone-producing adenoma, or the severity of aldosterone-producing adenoma Judge as high . Adding a drug candidate substance to a cell derived from the adrenal cortex that expresses a reporter gene linked to the KCNJ5 gene or the KCNJ5 gene promoter (excluding the in vivo process) , the mRNA level of the KCNJ5 gene or the expression level of the reporter gene A method for screening a therapeutic agent for aldosterone-producing adenoma, comprising a step of measuring (excluding a step in vivo in human ) and a step of selecting a substance that decreases the mRNA level of the KCNJ5 gene or the expression level of a reporter gene . Based on the criteria that a single base mutation in the base corresponding to the base number 451 in the base sequence of SEQ ID NO: 1 on the KCNJ5 gene is cytosine, and the affected base is affected by an aldosterone-producing adenoma. To test for aldosterone-producing adenoma.