JP2005210991A - Metabolic alkalosis marker - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for evading from side effects caused by a medicament when administering a thiazide-based diuretic by regulating the dose. <P>SOLUTION: The marker for detecting the variation of an amino acid residue of a thiazide-sensitive Na-Cl cotransporter (SLC12A3) is provided. The marker can be used for diagnosing the metabolic alkalosis, and for detecting the sensitivity of a patient to the thiazide-based diuretic. The method for detecting the metabolic alkalosis or a diathesis of the metabolic alkalosis based on the variation of the amino acid residue of the SLC12A3 is also provided. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a marker for detecting a mutation of an amino acid residue of a thiazide sensitive Na-Cl cotransporter (SLC12A3). The markers of the present invention can be used to diagnose metabolic alkalosis and to examine a patient's sensitivity to thiazide diuretics. The present invention further relates to a method for detecting metabolic alkalosis or a predisposition to metabolic alkalosis based on mutations in amino acid residues of SLC12A3.

The acid-base balance of body fluids is regulated by the lungs and kidneys. A state in which the acid-base balance of a body fluid, particularly blood, is inclined toward the alkali side is called alkalosis. The alkalosis caused by the decrease in [H ₂ CO ₃ ] is called respiratory alkalosis (or CO ₂ or gas alkalosis), and the one caused by the increase in [HCO ₃ ⁻ ] is called metabolic alkalosis. Metabolic alkalosis is caused by an increase in [HCO ₃ ⁻ ] due to an increase in the overall base in the body. Causes include vomiting, loss of HCl due to gastric fluid aspiration, etc., and large doses of sodium bicarbonate (sodium bicarbonate).

Thiazide diuretics, the distal tubule near tubular end of the renal Na ⁺ / Cl ^- acts on cotransporter, Na ^+, K ⁺ and Cl ^- facilitate excretion (diuresis) of such ions To do. As a result, the amount of body fluid (blood) is reduced, and blood pressure is expected to decrease, so that it is used as a hypotensive drug. Side effects such as hypokalemia have been reported for thiazide diuretics.

Ditelman syndrome (GS) is an autosomal recessive inherited disease of renal tubules caused by a functional deletion mutation in the thiazide-sensitive Na-Cl cotransporter (SLC12A3) gene (Simon DB et al., Nature Genet. (1996) 12: 24-30). The clinical features of GS were similar to the effects of thiazide administration, and the decrease in salt reabsorption in GS was thought to be related to hypertension resistance. Cruz et al. Reported that individuals with homozygous variants and members of the young (under 18 years) heterozygous GS Amish family had lower blood pressure levels compared to wild-type relatives. (Non-Patent Document 1). They further report that both homo and hetero subjects had higher 24-hour urinary Na ⁺ reflecting higher self-selection (compensatory) salt intake than wild-type subjects (non-patent literature). 1). These observations suggest that the hetero-GS mutant group is not harmless and may contribute to hypertension resistance. GS is considered a rare disease. However, because it is an autosomal recessive mutation, the GS mutation may significantly affect blood pressure levels in the general population, assuming that the frequency of heterozygous individuals is higher than estimated. . For the Japanese, 8 types of GS mutations have been reported (Non-Patent Documents 2 to 5).

Cruz DN et al., Hypertension (2001) 37: 1458-64 Takeuchi K et al., J. Clin. Endocrinol. Metab. (1996) 81: 4496-9 Yahata K et al., Am. J. Kid. Dis. (1999) 34: 845-53 Monkawa T et al., J. Am. Soc. Nephrol. (2000) 11: 65-70 Tajima T et al., Endocr. J. (2002) 49: 91-6

  Ditelman syndrome (GS) is a congenital renal tubular disease caused by a loss-of-function mutation in the thiazide-sensitive Na-Cl cotransporter gene (SLC12A3) (Simon DB et al., Nature Genet. (1996) 12 : 24-30). It is known that patients with GS develop hypomagnesemia and urinary Ca excretion decreases. GS is considered a rare disease, but because it is an autosomal recessive genetic disease, the frequency of people with heterozygous mutations may be higher than expected. The researchers therefore assessed the prevalence of GS in Japanese and examined the effects of heterogeneous GS mutations on blood pressure levels.

  Heterogeneous GS mutation did not affect blood pressure levels in Japanese, but Cruz et al. (Non-patent Document 1) and subjects with complex heterozygous mutations (180K and 849H) showed low blood pressure levels As suggested by this observation, homo-GS mutations are expected to lower blood pressure levels. The total frequency of all eight GS mutations reported for Japanese is (56 + 14 + 1 + 1 + 47/1880 x 2) = 0.0321. The number of people with heterogeneous GS mutations was calculated as 1 in 15.6. This value is higher than the estimated value. Calculated from this result, the presence of 10.3 GS patients is estimated in 10,000 Japanese. GS can be a major factor in hypotension in Japanese.

  Although heterozygous GS mutations did not have a significant effect on blood pressure, as reported by Cruz et al., Subjects with heterozygous GS mutations seemed to complement the deficiency of SLC12A3 protein by increasing salt consumption. Yes (Non-Patent Document 1). In this study, higher urine pH was also observed in subjects with GS mutations. This may suggest that GS is associated with metabolic alkalosis. That is, it was considered that subjects with hetero GS mutations were sensitive to the side effects of thiazide diuretics. Furthermore, subjects with heterogeneous GS mutations may be prone to dehydration due to salt loss due to solar radiation or diarrhea. Accordingly, an object of the present invention is to provide a method for avoiding side effects caused by a drug by adjusting the dose when a thiazide diuretic is administered.

  According to the present invention, it was suggested that metabolic alkalosis was accompanied by GS, and those having hetero GS mutations were considered to be sensitive to the side effects of thiazide diuretics. Further, according to the present invention, a method capable of easily detecting a GS mutation has been established. Therefore, it was considered that diagnosis of metabolic alkalosis and susceptibility to side effects of thiazide diuretics could be made by testing using polymorphism of thiazide sensitive Na-Cl cotransporter (SLC12A3) gene. .

That is, the present invention
(1) Metabolic alkalosis marker for detecting mutation of amino acid residue of thiazide sensitive Na-Cl cotransporter (SLC12A3) (2) Mutation of amino acid residue of SLC12A3 is 180, 242, 569, 623, 846 The marker described in (1) above, which is a mutation of the 849th or 955th amino acid residue (3) 1) change of cytosine (C) at position 545 to adenine (A) in the SLC12A3 gene, 2) at position 1712 Change from cytosine (C) to thymine (T), 3) Change from thymine (T) at position 1874 to cytosine (C), 4) Change from thymine (T) at position 2579 to adenine (A), 5 ) Deletion of thymine (T) at position 731 and guanine (G) at position 732, 6) deletion of thymine (T) at positions 2543 and 2544, 7) guanine (G) at position 2897 to adenine (A) And 8) the marker according to (2) above, which detects the presence or absence of one or more changes selected from each corresponding change in their complementary strands (4) SEQ ID NOs: 1-12 and 17-3 The marker according to any one of (1) to (4) above, which is used as a probe or a primer in the detection of metabolic alkalosis (5), which has a base sequence selected from 2 (5) ) A metabolic alkalosis marker set comprising a primer having the base sequence of SEQ ID NOs: 1 and 2 and a probe having the base sequence of SEQ ID NOS: 3 and 4; and a primer having the base sequence of SEQ ID NOS: 5 and 6; A metabolic alkalosis marker set comprising probes having base sequences of Nos. 7 and 8 (8) a primer having base sequences of SEQ ID Nos. 9 and 10, and a metabolic product comprising probes having the base sequences of SEQ ID Nos. 11 and 12 Alkalosis marker set (9) having primers having base sequences of SEQ ID NOs: 17 and 18, and base sequences of SEQ ID NOs: 19 and 20 A metabolic alkalosis marker set (10), a primer having the nucleotide sequences of SEQ ID NOs: 21 and 22, and a metabolic alkalosis marker set (11) SEQ ID NO: including the probes having the nucleotide sequences of SEQ ID NOs: 23 and 24 A primer having a nucleotide sequence of SEQ ID NOS: 29 and 30, and a primer having a nucleotide sequence of SEQ ID NOS: 29 and 30, and a primer having a nucleotide sequence of SEQ ID NOS: 27 and 28; A method for detecting metabolic alkalosis or a predisposition to metabolic alkalosis comprising detecting a mutation in an amino acid residue of a metabolic alkalosis marker set (13) SLC12A3 comprising a probe having a base sequence of 32 from a biological sample of a subject (14 ) SLC12A3 amino acid residue mutations are 180, 242, 569, 623, 846 The detection method according to the above (13), which is a mutation of the 849th or 955th amino acid residue, (15) mutation of the amino acid residue of SLC12A3, wherein 1 of the SLC12A3 gene in a nucleic acid sample obtained from a biological sample of a subject ) Change from cytosine (C) at position 545 to adenine (A), 2) Change from cytosine (C) to thymine (T) at position 1712, 3) From thymine (T) at position 1874 to cytosine (C) 4) Change from thymine (T) at position 2579 to adenine (A), 5) Deletion of thymine (T) at position 731 and guanine (G) at position 732, 6) Positions at positions 2543 and 2544 Deletion of thymine (T), 7) Change from guanine (G) at position 2897 to adenine (A), and 8) Presence or absence of one or more changes selected from each corresponding change in their complementary strands The detection method according to (14), wherein the detection is performed.
(16) The detection method according to any of (13) to (15) above, wherein a change in the SLC12A3 gene is detected by determining a base sequence. (17) A change in the SLC12A3 gene is detected using a probe. The detection method according to any one of (13) to (15) above, which is detected by hybridization (18) The SLC12A3 gene fragment, wherein the probe comprises the 545, 731, 732, 1712, 1874, 2543, 2544, 2579 or 2897 bases The detection method according to the above (17), which is a polynucleotide having a nucleotide sequence of (19) any one of the above (13) to (15), wherein a change in the SLC12A3 gene is detected by performing an amplification reaction of a site containing a mutation (20) The detection method according to (19) above, wherein an amplification reaction is carried out on the region containing the nucleotides 545, 731, 732, 1712, 1874, 2543, 2544, 2579 or 2897 of the SLC12A3 gene ( 21) The change in the SLC12A3 gene is determined according to any one of (5) to (12) above. The detection method according to the above (15), which is detected using a sex alkalosis marker set and Taq polymerase (22) The sensitivity to the thiazide diuretic is examined by the detection method according to any one of the above (13) to (21) Provide a way to do it.

  According to the present invention, based on genetic factors, metabolic alkalosis and predisposition to metabolic alkalosis (preliminary diagnosis of metabolic alkalosis; easiness of getting illness, severity of illness, responsiveness to treatment, etc.), prognosis Etc. can be determined. In addition, since patients with heterogeneous GS mutations may have side effects on thiazide diuretics, it is clinically beneficial to screen for GS mutations before diuretic treatment.

[Metabolic alkalosis marker]
According to the present invention, it was suggested that a heterozygous mutation of thiazide-sensitive Na-Cl cotransporter (SLC12A3), which is a causative gene of ditelman syndrome (GS), is associated with metabolic alkalosis. Thus, the present invention relates to a metabolic alkalosis marker for detecting a mutation in the amino acid residue of SLC12A3. Such amino acid residue mutations may be caused by polymorphisms in the SLC12A3 gene. That is, the metabolic alkalosis marker of the present invention includes, but is not limited to, a polynucleotide that enables detection of a polymorphism of the SLC12A3 gene.

  “Polymorphism” is defined genetically as a change in the base present in a gene that occurs at a frequency of 1% or more of the population. However, “polymorphism” in the present specification includes changes in bases of less than 1% in the population, as long as the base changes cause a decrease in the function of SLC12A3 that causes metabolic alkalosis. The position, number, etc. are not particularly limited. Examples of the polymorphism include polymorphisms in which deletion, substitution or insertion of several tens of bases has occurred from a single nucleotide polymorphism (SNP). Several polymorphisms have been reported for SLC12A3, and polynucleotides for detecting these known mutations are included in the metabolic alkalosis marker of the present invention. Particularly useful markers are those that detect mutations in amino acid residues 180, 242, 569, 623, 846, 849, or 955 of SLC12A3, such as cytosine (C) at position 545 of the SLC12A3 gene (a) Change to A), 2) Change from cytosine (C) at position 1712 to thymine (T), 3) Change from thymine (T) at position 1874 to cytosine (C), 4) Thymine at position 2579 (T) ) To adenine (A), 5) deletion of thymine (T) at position 731 and guanine (G) at position 732, 6) deletion of thymine (T) at positions 2543 and 2544, 7) 2897 And a marker capable of detecting the presence or absence of one or more changes selected from the corresponding changes in their complementary strands, and the change from guanine (G) to adenine (A).

  The mutation of the base constituting the gene can be detected using a probe containing the mutation site or a primer constructed so as to sandwich the mutation site. Accordingly, such probes and primers are included in the metabolic alkalosis marker of the present invention. When a mutation of the SLC12A3 gene is detected by combining a plurality of probes and / or a plurality of primers, the individual probes and primers included in this combination are also referred to as “metabolic alkalosis markers”. For example, polynucleotides having the nucleotide sequences set forth in SEQ ID NOs: 1 to 12 and SEQ ID NOs: 17 to 32 can be used as preferred metabolic alkalosis markers of the present invention.

Here, the `` polynucleotide '' is not limited as long as it can detect the polymorphism of the SLC12A3 gene, and consists of a plurality of bases such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a base pair. It is a single chain or double chain polymer. A polynucleotide may contain one or more modified bases, for example, a non-naturally occurring base such as inosine, or a tritylated base. The length of the polynucleotide used as the metabolic alkalosis marker of the present invention is not particularly limited. However, when used as a primer, the chain length is usually 3 to 100 bp, preferably 10 to 50 bp, more preferably about 15 to 35 bp. The 3 ′ region needs to be complementary to the target sequence, but a restriction enzyme recognition sequence, a tag, etc. can be added to the 5 ′ region. On the other hand, when used as a probe, the base to be detected (base expected to be mutated) is designed to be near the middle of the base constituting the probe, and the total chain length is 10 bp or more, preferably 12 bp or more. More preferably, it is 13 bp or more. If necessary, it may be labeled with a label component for facilitating detection. A label component that can be detected by spectroscopic means, optical means, biochemical means, immunological means, enzymatic chemistry means, radiochemical means, or the like can be used. Examples thereof include enzymes such as peroxidase and alkaline phosphatase, radioactive labels such as ³² P, isotopes, biotin, fluorescent dyes, luminescent substances, and coloring substances. Furthermore, for example, for the purpose of use in TaqMan ^™ 5 ′ exonuclease analysis, a probe in which the 5 ′ end of the probe is labeled with a fluorescent dye and a quencher is modified at the 3 ′ end is also used in the metabolism of the present invention. Included in sex alkalosis markers.

  The polynucleotide used as the marker of the present invention can be chemically synthesized by a known method. For example, it can be chemically synthesized by a method such as triester method, phosphite method, phosphoamidite method, phosphonate method (see Agnew. Chem. Int. Ed. Engl. (1989) 28: 716-34). In general, synthesis of polynucleotides can be performed on a modified solid support. It can also be performed using an automated synthesizer. Such a synthesizer is commercially available.

  As shown in the Examples, mutations in the SLC12A3 gene could be easily detected using primers and probes having the nucleotide sequences of SEQ ID NOs: 1-32. Since subjects with detected mutations in amino acid residues 180, 242, 569, 623, 846, 849, or 955 of SLC12A3 showed high urine pH, markers for detecting these mutations were metabolic apoptosis. It is considered to be particularly useful for diagnosis. The change from cytosine (C) at position 545 causing a mutation from threonine (T) to lysine (K) at position 180 of SLC12A3 is a primer having the sequences of SEQ ID NOS: 1 and 2, respectively. In addition, detection was performed using probes each having the sequences shown in SEQ ID NOs: 3 and 4. Similarly, the change from cytosine (C) at position 1712 causing mutation from 569th alanine (A) to valine (V) of SLC12A3 is the sequence of SEQ ID NO: 5 and 6, respectively. And a probe having the sequences described in SEQ ID NOs: 7 and 8, respectively. A change from thymine (T) at position 1874 to cytosine (C) causing mutation from 623 leucine (L) to proline (P) of SLC12A3 is a primer having the sequences of SEQ ID NOS: 9 and 10, respectively. And it detected using the probe which has the arrangement | sequence of sequence number 11 and 12, respectively. A change from thymine (T) at position 2579 causing a mutation from leucine (L) to histidine (H) at position 849 of SLC12A3 to adenine (A) is a primer having the sequences of SEQ ID NOS: 17 and 18, respectively. And it detected using the probe which has the arrangement | sequence of sequence number 19 and 20, respectively. Deletions of thymine (T) and guanine (G) at positions 731 and 732 causing a mutation from 242nd alanine (A) to valine (V) in SLC12A3 have the sequences described in SEQ ID NOs: 21 and 22, respectively. Detection was carried out using primers and probes having the sequences shown in SEQ ID NOs: 23 and 24, respectively. Deletion of two thymines (T) at positions 2543 and 2544 causing a mutation from the codon of the 846th phenylalanine (F) to the stop codon of SLC12A3, a primer having the sequences of SEQ ID NO: 25 and 26, respectively, and Detection was carried out using probes each having the sequences shown in SEQ ID NOs: 27 and 28. The change from guanine at position 2897 (G) to adenine (A) causing a mutation from 955th arginine (R) to glutamine (Q) of SLC12A3 is a primer having the sequences of SEQ ID NOS: 29 and 30, respectively. And it detected using the probe which has the arrangement | sequence of sequence number 31 and 32, respectively.

  Therefore, the present invention provides 1) a metabolic alkalosis marker set comprising a primer having the nucleotide sequences of SEQ ID NOS: 1 and 2, and a probe having the nucleotide sequences of SEQ ID NOS: 3 and 4, 2) the nucleotide sequences of SEQ ID NOS: 5 and 6 A metabolic alkalosis marker set comprising a primer having the nucleotide sequences of SEQ ID NOs: 7 and 8, 3) a primer having the nucleotide sequences of SEQ ID NOs: 9 and 10, and a probe having the nucleotide sequences of SEQ ID NOs: 11 and 12 4) a metabolic alkalosis marker set comprising 4) a primer having the nucleotide sequence of SEQ ID NOS: 17 and 18, and a probe having a nucleotide sequence of SEQ ID NOS: 19 and 20, 5) of SEQ ID NOS: 21 and 22 A metabolic alkalo comprising a primer having a base sequence and a probe having the base sequences of SEQ ID NOs: 23 and 24 A marker set, 6) a metabolic alkalosis marker set comprising a primer having the nucleotide sequences of SEQ ID NOs: 25 and 26, and a probe having the nucleotide sequences of SEQ ID NOs: 27 and 28, and 7) a nucleotide sequence of SEQ ID NOs: 29 and 30 And a metabolic alkalosis marker set including a probe having the nucleotide sequences of SEQ ID NOS: 31 and 32.

  If necessary, these primer sets can be further combined to form a metabolic alkalosis marker set. For example, the most common genotype of a complex heterozygous mutation in Japanese is a type in which amino acid residues 180 and 849 are mutated. The subject having this complex heterozygous mutation was found to have a low blood pressure level in the present invention, suggesting the possibility that the GS mutation is a major factor in hypotension in Japanese. Therefore, the combination of the metabolic alkalosis marker set of 1) and 4) above, which detects mutations at the 180th and 849th amino acid residues, can be particularly useful in the diagnosis of hypotension.

[Diagnosis of metabolic alkalosis]
The present invention suggests a relationship between heterozygous mutation of SLC12A3 which is a causative gene of GS and metabolic alkalosis. Therefore, the present invention comprises detecting a mutation in the amino acid residue of SLC12A3 from a biological sample of a subject, metabolic alkalosis or metabolic alkalosis predisposition (ease of becoming ill, severity of illness, treatment And the like. Since patients with metabolic alkalosis may have side effects on thiazide diuretics, it is useful to test for metabolic alkalosis or a predisposition to metabolic alkalosis, particularly prior to administration of thiazide diuretics.

  Such amino acid residue mutations may be caused by polymorphisms in the SLC12A3 gene. Therefore, in the present invention, metabolic alkalosis or a predisposition to metabolic alkalosis is not limited to this, but is performed by detecting a polymorphism of the SLC12A3 gene in a nucleic acid sample obtained from a biological sample of a subject. . The position, number, etc. of the polymorphism detected in the method of the present invention is not particularly limited as long as it is a base change that causes a decrease in the function of SLC12A3 that causes metabolic alkalosis. Examples include polymorphisms in which deletion, substitution or insertion of several tens of bases occurs from (SNP). Several polymorphisms have been reported for SLC12A3, and those known mutations can be detected in the method of the present invention. For example, the mutation of the 180th, 24th, 569th, 623th, 846th, 849th, or 955th amino acid residues of SLC12A3, which has been shown to be associated with metabolic alkalosis in Japanese, can be detected by the method of the present invention . These amino acid residue mutations are, for example, 1) a change from cytosine (C) at position 545 to adenine (A) in the SLC12A3 gene, 2) a change from cytosine (C) at position 1712 to thymine (T), 3) 1874 position thymine (T) to cytosine (C) 4) 2579 position thymine (T) to adenine (A) 5) 731 position thymine (T) and 732 position guanine (G) deletion, 6) deletion of thymine (T) at positions 2543 and 2544, 7) change of guanine (G) to adenine (A) at position 2897, and 8) correspondence in their complementary strands It can be detected by determining the presence or absence of one or more changes selected from each change.

The biological sample of the subject used for the test includes peripheral blood leukocytes, liver, kidney, adrenal gland, brain, uterus, and other tissues or cells, with peripheral blood leukocytes being particularly preferred. The nucleic acid sample extracted from these tissues or cells is not particularly limited as long as it has a nucleotide sequence encoding SLC12A3. Examples include nucleic acids derived from appropriate cells or tissues (including total genomic DNA and cDNA transcribed from total cellular RNA), such as genomic DNA, cDNA, and mRNA. Known methods of preparing nucleic acid samples, for example,. "Molecular Cloning: A Laboratory Manual, 2 nd edition" (Sambrook J, Fritsch F and Maniatis T ed, Cold Spring Harbor Press, Cold Spring Harbor, the New York (1989) In particular, methods for obtaining cDNA include the Okayama-Berg method (Okayama H and Berg P, Mol. Cell. Biol. (1982) 2: 161-70) and the Gubler-Hoffman method (Gubler U and Hoffman J, Gene (1983) 25: 263-9), etc. When a full-length clone is required, a capping method (Kato S et al., Gene ( 1994) 150: 243-250).

  Various methods are known as methods for detecting gene mutations, and can be used in the method of the present invention. For example, methods for examining genes with mutations include direct sequencing, Southern blotting, heteroduplex gel mobility, denaturing HPLC, single strand conformational polymorphism (SSCP) analysis, Denaturing gradient gel electrophoresis (DGGE), dideoxy fingerprint, mismatch chemical or enzymatic cleavage methods, protein shortening test (PTT), oligonucleotide arrays using hybridization chips or mini-sequence chips, fluorescence in situ Hybridization (FISH) method, RLGS (restriction landmark genomic scanning method) method, polymerase chain reaction (PCR) method, oligonucleotide ligation assay (OLA) method, allele-specific oligonucleotide (ASO) hybridization method, allele Specific PCR amplification method (ARMS), ligase method (LMGD method, LCR method, etc.), restriction fragment length polymorphism (RFLP) method and the like can be mentioned.

  Specifically, for example, a change in the SLC12A3 gene can be detected by determining the nucleotide sequence and comparing it with a control (normal (wild type) or sequence having a mutation). For the base sequence of the control, the sequence registered in the database (GenBank Accession No. NM — 000339; Unigene Hs. 158462) may be referred to, and if necessary, the sequence may be determined together during the test. The determination of the base sequence can be performed by a method known to those skilled in the art. For example, it can be carried out by a chemical decomposition method (Maxam-Gilbert method), a chain terminator method (Sanger dideoxy method), or the like. The base sequence may be determined by determining the sequence of the full-length gene, but when the base for detecting the mutation is specified, the base sequence may be determined only for the region including the mutation site. If necessary, the nucleotide sequence can be determined after amplifying the region containing the mutation site by PCR or the like. PCR methods: "PCR protocols: A guide to methods and applitaion" (Innis MA, Gelfaud DM, Snindky JJ and White TJ ed., Academic Press Inc., New York (1990)), "PCR: A practical approach" ( McPherson MJ, Quirke P and Taylor GR ed., IRL Press, Oxford (1991)), Saiki RK et al., Science (1988) 239: 487-91, Frohman MA et al., Proc. Natl. Acad. Sci. USA (1988) 85: 8998-9002 and the like. The PCR conditions are not particularly limited, and may be known conditions that are usually performed. For example, one cycle consisting of heat denaturation of DNA strands, primer annealing, and synthesis of complementary strands by polymerase is repeated, for example, 10 to 50 times, preferably 20 to 35 times, more preferably 25 to 30 times.

  For detection of mutation, a known mutation detection method such as a hybridization method using an appropriate DNA fragment containing a mutation site as a probe can be used. As a suitable DNA fragment containing a mutation site, a polynucleotide having the base sequence of SLC12A3 gene fragment containing the 545, 731, 732, 1712, 1874, 2543, 2544, 2579, or 2897th base can be mentioned.

As a method using such hybridization, Southern blot can be exemplified. In Southern blotting, a nucleic acid sample, preferably DNA, obtained from a biological sample of a subject is treated with a restriction enzyme, developed by electrophoresis, and hybridized with a probe. Genomic Southern method, PCR Southern method and the like are known, and both can be used in the method of the present invention. If necessary, the probe for facilitating detection may be labeled with a label component. A label component that can be detected by spectroscopic means, optical means, biochemical means, immunological means, enzymatic chemistry means, radiochemical means, or the like can be used. Examples thereof include enzymes such as peroxidase and alkaline phosphatase, radioactive labels such as ³² P, isotopes, biotin, fluorescent dyes, luminescent substances, and coloring substances. Further, for example, for the purpose of use in TaqMan ^™ 5 ′ exonuclease analysis, a probe in which the 5 ′ end of the probe is labeled with a fluorescent dye and a quencher is modified at the 3 ′ end may be used. .

  Further, detection may be performed by fixing the probe on the substrate as necessary. An example of such a method is a DNA array method (see Kenichi Matsubara and Yoshiyuki Tsuji "SNP gene polymorphism strategy", Nakayama Shoten, p128-135). There are no particular limitations on the shape, material, etc. of the substrate, as long as it can immobilize the polynucleotide that serves as the probe and does not interfere with the hybridization between the polynucleotide and the target nucleic acid in the nucleic acid sample. For example, the probe can be fixed to a plate-like material made of glass or a permeable membrane (such as a nitrocellulose membrane). The immobilization method is not particularly limited. For example, a polynucleotide that serves as a probe can be synthesized in situ by a photolithographic technique developed by Affymetrix, or an inkjet technique for immobilizing a chemical of Rosetta Inpharmatics. . Generally, when immobilized on a substrate, the probe is about 10-100 bp, preferably 10-50 bp, more preferably 15-25 bp.

  Still another method using hybridization for detecting a mutation in a base sequence is an allele-specific oligonucleotide (ASO) hybridization method. This method is a general method for identifying point mutations. First, a polynucleotide of about 18 mer containing a base that is considered to have a mutation is prepared. If necessary, the polynucleotide may be labeled. Hybridization is performed on a nucleic acid sample by PCR or the like from a biological sample obtained from a subject. At this time, the stringency of hybridization is appropriately adjusted so that only a sequence completely complementary to the polynucleotide hybridizes. Then, if there is a mutation, the efficiency of hybridization decreases, so by measuring the hybridization efficiency by Southern blotting or a method using a special fluorescent reagent that is quenched by intercalation, etc. Mutations can be detected.

  Yet another method is a mismatch cutting method. As the mismatch cleavage method, a method using an enzyme (RNase) and a chemical cleavage method are known. In either method, first, a target region containing a base that is considered to have a mutation in the SLC12A3 gene is amplified by a PCR method or the like. Next, hybridization with a labeled probe (labeled RNA when RNase is used) is performed. The labeled probe is designed so that the hybrid takes a single-stranded structure only in the part where the mutation exists. Subsequently, the single-stranded structure is cleaved using RNase. This utilizes the property that RNase does not degrade double-stranded RNA and RNA / DNA complexes, but only single-stranded RNA. On the other hand, in the chemical cleavage method, cytosine (C) in the single-stranded structure is modified with hydroxylamine and thymine (T) with osmium tetraoxide, and the nucleotide chain is cleaved by piperidine treatment. After performing the cleavage treatment, the presence or absence of a base mutation can be determined by confirming the number of bands detected by electrophoresis (two if a mutation is present).

  Furthermore, in the detection method of the present invention, a change in the SLC12A3 gene may be detected by performing an amplification reaction (PCR) of a site containing a mutation. Examples of suitable sites containing mutations include a region containing the 545, 731, 732, 1712, 1874, 2543, 2544, 2579, or 2897th base of the SLC12A3 gene. PCR-based methods include restriction fragment length polymorphism (RFLP), single-stranded conformation polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), oligonucleotide ligation assay (OLA), allele Examples include gene-specific PCR amplification method (ARMS). Particularly, RFLP method, ARMS, and OLA method are exemplified as methods suitable for examining a specific mutation.

  Although it is not always necessary to perform PCR in the method using RFLP, detection with higher sensitivity is possible by performing PCR. By the RFLP method, it is possible to detect a mutation in which a base sequence mutation creates or eliminates a recognition site for a restriction enzyme. Specifically, when there is a mutation at a restriction enzyme recognition site in a gene, or when a base insertion or deletion is present in a fragment generated by the restriction enzyme treatment, the gene is obtained by restriction enzyme treatment. By comparing the size of the obtained DNA fragment between that derived from the subject and that derived from the control, mutations in the base sequence can be detected. For example, a gene region containing this mutation is amplified, the amplified product is treated with a restriction enzyme, and then subjected to electrophoresis on a polyacrylamide or agarose gel, thereby detecting a base mutation as a difference in band mobility. be able to. Alternatively, the presence or absence of mutation may be detected by Southern blotting using a probe containing a mutation site after electrophoresis. In this method, in addition to genomic DNA, a cDNA prepared using reverse transcriptase or the like based on RNA obtained from a subject, an amplification product amplified by PCR using the cDNA as a template, or the like may be used as a sample. . Further, when the mutation to be detected does not cause a change in the restriction enzyme recognition site, the restriction enzyme recognition site can be introduced as follows, and the mutation of the base by RFLP can be detected. First, a primer containing a mismatch located immediately before the mutated base is constructed. The mismatched base substitution site is a site that is not important for the hybridization reaction and amplification reaction of the normal sequence and the mutant sequence. The primer is designed so that a restriction enzyme recognition site is introduced into a PCR product produced by a transcription reaction using the primer only when it has a mutated base or a normal base. Thereafter, by using the primer and performing steps such as PCR reaction and electrophoresis in the same manner as described above, the mutation of the base sequence can be detected.

  As another method for detecting mutations in the base sequence, DNA containing the SLC12A3 gene is amplified based on a nucleic acid sample obtained from a subject, the amplified DNA is dissociated into single strands, and the mobility on a non-denaturing gel is used as a control. The method of comparing is mentioned. This is a method utilizing the property that each single strand forms a higher order structure depending on its base sequence when the double-stranded DNA is dissociated into single strands. It is known that single-stranded substitution also changes the higher-order structure of single-stranded DNA and exhibits different mobility in gel electrophoresis. Examples of such methods include the SSCP method (Genomics (1992) 12: 139-46; Oncogene (1991) 6: 1313-8; PCR Methods Appl. (1995) 4: 275-82). This method is particularly suitable for testing a large number of samples because of the ease of operation and the ability to carry out with a small amount of test samples. In SSCP, first, DNA containing the SLC12A3 gene is amplified by PCR or the like. Usually, a DNA fragment having a length of about 200 to 400 bp containing the target mutant base is amplified. A DNA fragment may be amplified as a reverse transcription PCR product. If necessary, the amplified DNA fragment may be labeled using a primer or substrate base labeled with an isotope, a fluorescent dye, biotin or the like during PCR. Next, the obtained DNA fragment is denatured. Denaturation is preferably performed by applying heat. Subsequently, the denatured DNA fragment is electrophoresed on a polyacrylamide gel containing no denaturant. In order to increase the ability to separate the DNA fragments of the polyacrylamide gel, about 5 to 10% glycerol may be added. The conditions for electrophoresis are not particularly limited, but if preferable separation is not obtained by performing at room temperature of 20-25 ° C, the temperature that gives the optimum mobility at temperatures of 4-30 ° C may be examined. it can. After electrophoresis, the mobility of the DNA fragment can be measured by autoradiography, fluorescence detection, or the like depending on the substance used to label the DNA fragment. In addition, even when labeled DNA is not used, it is possible to detect the band of a DNA fragment and detect the presence or absence of a base mutation by staining the gel after electrophoresis with ethidium bromide staining, silver staining, etc. it can. Furthermore, if necessary, a band having a difference in mobility can be excised from the gel, amplified by PCR, and sequenced to confirm a specific mutation.

  Yet another method is the DGGE method. This method takes advantage of the fact that heteroduplexes with mismatches in PCR products are easier to dissociate than homoduplexes. Since the mobility of gel electrophoresis decreases as dissociation progresses, adding a density gradient of urea and formamide to the developing polyacrylamide gel further emphasizes the difference in mobility, and the presence of double-stranded DNA containing mismatches That is, the presence of the mutation can be detected.

  Other methods for detecting base mutations using PCR include allele-specific PCR amplification (ARMS) and the like. In PCR, after a primer anneals to a template DNA, a complementary strand DNA is synthesized from 5 'to 3' by DNA polymerase. It is known that when the primer has a mismatch at the 3 'terminal base, the efficiency of PCR is reduced. That is, it becomes impossible to observe the PCR product by electrophoresis. ARMS utilizes this principle, and is performed by performing PCR using a primer complementary to a mutated base whose 3 'terminal base is to be detected, and detecting the presence or absence of an amplification product. Basically, two PCR reactions are performed. One primer (common primer) is the same for both reaction systems. The other primer should be slightly different, one specific for the normal base sequence and the other specific for the mutant sequence. Furthermore, if necessary, a primer for amplifying an irrelevant sequence can be added as a control primer to check whether the PCR reaction is working well. The position of the common primer is set so that the size of the PCR product is different from that of the normal base sequence as a control if there is a mutation. When PCR products using these primers are electrophoresed, multiple products appear as ladders on the gel. At this time, by attaching a different label to each primer so that the PCR products obtained using each mutation-specific primer can be distinguished, by detecting which primer the amplified PCR product was amplified, Base mutation in sample DNA can be confirmed.

The TaqMan ^TM method (also called fluorescence fading method) is an example of a method applied so that the principle of ARMS can be tracked in real time. This method is particularly suitable for the metabolic alkalosis and metabolic alkalosis predisposition detection methods of the present invention. Is the method. For specific methods, reference can be made to the examples. Preferably, 1) a primer having the nucleotide sequences of SEQ ID NOS: 1 and 2, and a metabolic alkalosis marker set comprising a probe having the nucleotide sequences of SEQ ID NOS: 3 and 4, 2) a primer having the nucleotide sequences of SEQ ID NOS: 5 and 6 And a metabolic alkalosis marker set comprising a probe having the base sequences of SEQ ID NOs: 7 and 8, 3) a metabolism comprising a primer having the base sequences of SEQ ID NOs: 9 and 10, and a probe having the base sequences of SEQ ID NOs: 11 and 12 Alkalosis marker set, 4) a metabolic alkalosis marker set comprising a primer having the nucleotide sequences of SEQ ID NOs: 17 and 18, and a probe having the nucleotide sequences of SEQ ID NOs: 19 and 20, 5) the nucleotide sequences of SEQ ID NOs: 21 and 22 Metabolic alkalosis comprising a primer having a nucleotide sequence and probes having the nucleotide sequences of SEQ ID NOS: 23 and 24 Carset, 6) a metabolic alkalosis marker set comprising a primer having the nucleotide sequence of SEQ ID NO: 25 and 26, and a probe having the nucleotide sequence of SEQ ID NO: 27 and 28, or 7) having the nucleotide sequence of SEQ ID NO: 29 and 30 The reaction is performed using a primer and a metabolic alkalosis marker set including a probe having the nucleotide sequences of SEQ ID NOS: 31 and 32, and Taq polymerase, and 1) a change from cytosine (C) at position 545 to adenine (A). Or a corresponding change in its complementary strand, 2) a change from cytosine (C) at position 1712 to thymine (T) or a corresponding change in its complementary strand, 3) from thymine (T) at position 1874 to cytosine (C) Or a corresponding change in its complementary strand, 4) a change from thymine (T) at position 2579 to adenine (A), 5) a deletion of thymine (T) at position 731 and guanine (G) at position 732, or Its complementary strand 6) Deletion of thymine (T) at positions 2543 and 2544 or corresponding changes in its complementary strand, or 7) Change from guanine (G) to adenine (A) at position 2897 or complement thereof Detect the corresponding change in the strand.

The ligase method is mentioned as another method for detecting a mutated base. The ligase method is also called oligonucleotide ligation assay (OLA). This method utilizes the fact that when two oligonucleotides are linked by DNA ligase, they cannot be bound if there is a mismatch between the template DNA and the binding position. Examples of OLA include LMGD (ligase-mediated gene detection) method and LCR (ligase chain reaction). In the LMGD method, one oligo DNA is labeled with a radioactive label such as ³² P, and the other is labeled with biotin. After ligation, it is recovered by adsorption of streptavidin. If they are linked (ie not mismatched), the radiation dose of ³² P is high and can be detected. On the other hand, in LCR, the ligation reaction is repeated using a heat-resistant ligase. As with PCR, oligo DNA anneals to DNA strands, so that mutation can be detected with high sensitivity.

[Detection of side effect sensitivity to thiazide diuretics]
As described above, by detecting metabolic alkalosis and a predisposition to metabolic alkalosis in a subject, the subject's sensitivity to side effects of thiazide diuretics can be determined. Patients with metabolic alkalosis can have side effects on thiazide diuretics. Therefore, the present invention provides a method for testing sensitivity to thiazide diuretics by detecting metabolic alkalosis and a predisposition to metabolic alkalosis. According to the method of the present invention, before administering a thiazide diuretic, by examining the sensitivity of the patient to the drug, the dosage can be adjusted and side effects due to the drug can be avoided.

BMI;ボディマス指数(kg/m²)、SBP;最大血圧(mmHg)、DBP;最小血圧(mmHg)、HTN;高血圧有り(%)、AHT;抗高血圧処置有り(％) 1． Study population: Selection criteria and plans for Suita studies have been previously reported (Mannami T et al., Stroke (1997) 28: 518-25; Iwai N et al., J. Am. Soc. Nephrol. (2002 ) 13: 80-5). During the period from April 2002 to March 2003, the genotypes of 1852 subjects who submitted written informed consent for genetic analysis were determined. The Ethics Committee of the National Cardiovascular Center approved the experimental plan. The characteristics of the subjects analyzed in this study are summarized in Table 1. At this time, plasma K ⁺ levels were not measured. High blood pressure = 140 mmHg, minimum blood pressure = 90 mmHg, or subjects currently using antihypertensive drugs were defined as hypertension.

BMI; body mass index (kg / m ² ), SBP; maximal blood pressure (mmHg), DBP; diastolic blood pressure (mmHg), HTN; hypertension (%), AHT; antihypertensive treatment (%)

2． Selection of candidate mutations in SLC12A3: Eight GS mutations were selected from previous studies on Japanese (see Non-Patent Documents 1 to 4) (FIG. 1). Based on the reported sequence, a TaqMan ^™ system was designed to detect mutations (Table 2). The validity of this method was confirmed by sequencing subjects identified as heterozygous (Figure 2).

  3. Statistical analysis: Numerical values are expressed as mean ± SE. All statistical analyzes were performed using the JMP statistical package (SAS Institute, Inc.). The error of the blood pressure value was calculated by correcting by age and BMI. The error represents the difference between each observed actual blood pressure value and the predicted value based on age and BMI.

  4). Results: 56, 14, 1, 1 and 47 T180K, A569V, L623P, R642C and L849H heterogenotypes were detected from 1852 subjects, respectively. No 846X, 242V and 955Q alleles were detected. The validity of these genotypes was confirmed by sequencing (Figure 2). The frequencies of the 180K, 569V, 623P, 642C and 849H alleles were 1.51%, 0.38%, 0.03%, 0.03% and 1.27%, respectively. The phenotypes associated with these mutations are summarized in Table 3. We found subjects with the potential for multiple heterozygous mutations (180K and 849H genotypes) that showed relatively low blood pressure levels.

数値は平均(SE)で表される。1人の被験者は複合へテロ遺伝子型(K180+H849)であった。BMI;ボディマス指数(kg/m²)、HTN;高血圧有り(%)、AHT;抗高血圧処置有り(%)、SBP;最大血圧(mmHg)、DBP;最小血圧(mmHg)、R-SBP;年齢及びBMIにより補正したSBP誤差、R-DBP;BMIにより補正したDBP誤差、U-pH;尿ｐH、Gitelman;GS変異を持つ被験者を一つの群にまとめた、WT；野生型の遺伝子を持つ被験者群。p値は、GS変異を持たない被験者(WT)に対して算出した。 Blood pressure levels of subjects with T180K, A569V or L849H heterogenotype were not significantly different from wild type subjects. The prevalence of hypertension in subjects with GS mutations was not significantly different from wild-type subjects (Table 3). Thus, SLC12A3 heterozygous mutation does not appear to reduce blood pressure levels in Japanese. However, subjects with GS mutations showed higher urine pH than wild type subjects.

Numbers are expressed as mean (SE). One subject had a composite heterogenotype (K180 + H849). BMI: Body mass index (kg / m ² ), HTN: Hypertension (%), AHT: Antihypertensive treatment (%), SBP: Maximum blood pressure (mmHg), DBP: Minimum blood pressure (mmHg), R-SBP; Age And SBP error corrected by BMI, R-DBP; DBP error corrected by BMI, U-pH; urine pH, Gitelman; subjects with GS mutations combined into one group, WT; subjects with wild-type genes group. The p value was calculated for a subject (WT) who did not have a GS mutation.

As mentioned above, the frequency of 180K, 569V and 849H alleles measured by TaqMan ^™ system was unexpectedly high, 1.51, 0.38 and 1.27%, respectively. Blood pressure levels in heterozygous subjects with GS mutations were not significantly different from wild-type subjects. This result is consistent with a report by Cruz et al. However, because the population studied this time was composed of subjects over 30 years of age, the observation that cruz et al. Young (under 18 years) GS mutant heterozygous subjects showed low blood pressure levels cannot be retraced. It was.

Heterogeneous GS mutations did not affect blood pressure levels in Japanese, but Cruz et al. (Non-Patent Document 1) and subjects who may have multiple heterozygous mutations (180K and 849H) had low blood pressure Homo GS mutations are expected to lower blood pressure levels, as suggested by this observation that they were levels. Recognizing all eight GS mutations reported for Japanese, the combined frequency of these mutations is (56 + 14 + 1 + 1 + 47/1880 × 2) = 0.0321. That is, the existence of 10.3 GS patients per 10000 is predicted. GS can be a major factor in hypotension in Japanese. The most common genotype is a 180K and 849H complex heterozygote, which can be easily detected by a method utilizing the TaqMan ^™ method established in this study.

  The heterozygous GS mutation did not have a significant effect on blood pressure, but does not appear to be completely harmless. As reported by Cruz et al., Subjects with heterogeneous GS mutations seem to complement the defective SLC12A3 protein by increasing salt consumption (Non-Patent Document 1). In this study, higher urine pH was also observed in subjects with GS mutations. This may suggest that GS is associated with metabolic alkalosis. Thus, heterogeneous GS mutations may not be harmless. That is, subjects with heterogeneous GS mutations may be susceptible to side effects of thiazide diuretics. Furthermore, subjects with heterogeneous GS mutations may be prone to dehydration due to salt loss due to solar radiation or diarrhea. The number of subjects with heterogeneous GS mutations was calculated as 1 in 15.6. This value is higher than the estimated value. Therefore, it is considered clinically beneficial to screen for GS mutations before diuretic treatment.

It is a schematic diagram which shows the position on the protein of the SLC12A3 mutation (GS mutation) which causes Ditelman syndrome in Japanese. T180K, A569V, L623P, R642C and F849H heterogenotypes were found in 56, 14, 1, 1 and 47 subjects, respectively. The 846X, 242V and 955Q alleles found by one subject with multiple heterozygous mutations (K180 + H849) were not found in this population. To examine the validity of the TaqMan ^TM method is a diagram showing the results of sequencing. The mutation R642C ( C GC to T GC) is present at the junction between exons 15 and 16. "C GC" and third nucleotide "C" of the "T GC" is derived from exon 16.

Claims (22)

A metabolic alkalosis marker for detecting mutations in amino acid residues of thiazide-sensitive Na-Cl cotransporter (SLC12A3). 2. The marker according to claim 1, wherein the mutation of the amino acid residue of SLC12A3 is a mutation of the 180th, 242, 569, 623, 846, 849 or 955th amino acid residue. SLC12A3 gene 1) Change from cytosine (C) at position 545 to adenine (A), 2) Change from cytosine (C) to thymine (T) at position 1712, 3) Thymine (T) at position 1874 to cytosine Change to (C), 4) Change from thymine (T) at position 2579 to adenine (A), 5) Deletion of thymine (T) at position 731 and guanine (G) at position 732, 6) Position 2543 And a deletion of thymine (T) at position 2544, 7) a change from guanine (G) to adenine (A) at position 2897, and 8) one or more selected from the corresponding respective changes in their complementary strands The marker according to claim 2, wherein the presence or absence of a change is detected. The marker according to claim 3, which has a base sequence selected from SEQ ID NOs: 1 to 12 and 17 to 32. The marker according to any one of claims 1 to 4, which is used as a probe or a primer in detection of metabolic alkalosis. A metabolic alkalosis marker set comprising a primer having the nucleotide sequences of SEQ ID NOs: 1 and 2, and a probe having the nucleotide sequences of SEQ ID NOs: 3 and 4. A metabolic alkalosis marker set comprising a primer having the nucleotide sequences of SEQ ID NOs: 5 and 6, and a probe having the nucleotide sequences of SEQ ID NOs: 7 and 8. A metabolic alkalosis marker set comprising a primer having the nucleotide sequences of SEQ ID NOs: 9 and 10, and a probe having the nucleotide sequences of SEQ ID NOs: 11 and 12. A metabolic alkalosis marker set comprising a primer having the nucleotide sequences of SEQ ID NOs: 17 and 18, and a probe having the nucleotide sequences of SEQ ID NOs: 19 and 20. A metabolic alkalosis marker set comprising a primer having the nucleotide sequences of SEQ ID NOs: 21 and 22, and a probe having the nucleotide sequences of SEQ ID NOs: 23 and 24. A metabolic alkalosis marker set comprising a primer having the nucleotide sequences of SEQ ID NOs: 25 and 26 and a probe having the nucleotide sequences of SEQ ID NOs: 27 and 28. A metabolic alkalosis marker set comprising a primer having the nucleotide sequences of SEQ ID NOs: 29 and 30, and a probe having the nucleotide sequences of SEQ ID NOs: 31 and 32. A method for detecting metabolic alkalosis or a predisposition to metabolic alkalosis, comprising detecting a mutation of an amino acid residue of SLC12A3 from a biological sample of a subject. 14. The detection method according to claim 13, wherein the mutation of the amino acid residue of SLC12A3 is a mutation of the amino acid residue at position 180, 242, 569, 623, 846, 849 or 955. Mutation of the amino acid residue of SLC12A3 was determined by changing 1) 545 cytosine (C) to adenine (A) of the SLC12A3 gene in the nucleic acid sample obtained from the biological sample of the subject, 2) 1712 cytosine (C ) To thymine (T), 3) 1874 position thymine (T) to cytosine (C), 4) 2579 position thymine (T) to adenine (A), 5) 731 position Deletion of thymine (T) and guanine (G) at position 732, 6) deletion of thymine (T) at positions 2543 and 2544, 7) guanine (G) at position 2897 to adenine (A) And 8) the detection method according to claim 14, wherein the detection is performed based on the presence or absence of one or more changes selected from corresponding changes in their complementary strands. The detection method according to any one of claims 13 to 15, wherein a change in the SLC12A3 gene is detected by determining a base sequence. The detection method according to any one of claims 13 to 15, wherein a change in the SLC12A3 gene is detected by hybridization using a probe. 18. The detection method according to claim 17, wherein the probe is a polynucleotide having a base sequence of an SLC12A3 gene fragment containing the 545, 731, 732, 1712, 1874, 2543, 2544, 2579 or 2897th base. The detection method according to any one of claims 13 to 15, wherein a change in the SLC12A3 gene is detected by performing an amplification reaction of a site containing a mutation. 20. The detection method according to claim 19, wherein an amplification reaction is performed on a region containing the 545, 731, 732, 1712, 1874, 2543, 2544, 2579, or 2897th base of the SLC12A3 gene. 16. The detection method according to claim 15, wherein a change in the SLC12A3 gene is detected using the metabolic alkalosis marker set according to any one of claims 5 to 12 and Taq polymerase. A method for examining sensitivity to a thiazide diuretic by the detection method according to claim 13.

Images