WO2006038265A1

WO2006038265A1 - Medicinal composition for treating otospongiosis

Info

Publication number: WO2006038265A1
Application number: PCT/JP2004/014390
Authority: WO
Inventors: Yutaka Imauchi
Original assignee: Yutaka Imauchi
Priority date: 2004-09-30
Filing date: 2004-09-30
Publication date: 2006-04-13

Abstract

It is intended to provide a medicinal composition for treating otospongiosis, a method of determining its dose, a method of administering the same and a diagnostic marker for otospongiosis. As a diagnostic marker for otospongiosis, DNA is isolated from a human subject and the genotype of the allele at the M235T polymorphism in angiotensinogen gene is examined by PCR. Based on the results thus obtained, the cause of otospongiosis is estimated. In the case where otospongiosis is seemingly caused by the hyperactivity of an angiotensinogen receptor, a drug compound capable of lowering the activity is administered to the patient.

Description

Specification

Pharmaceutical composition for the treatment of otosclerosis

Technical field

TECHNICAL FIELD [0001] The present invention relates to a method for treating otosclerosis, a pharmaceutical thread composition for otosclerosis treatment, a method for its administration, and a diagnostic marker for otosclerosis.

Background art

[0002] Otosclerosis is a degenerative disease in which abnormal spongy bone growth occurs in the middle ear, and the bottom of the stapes sticks. As a result, the stapes are prevented from vibrating in response to sound and progressive hearing loss occurs.

[0003] Although there are few otosclerosis patients in Japanese, it occurs in 0.2-1% of adults with white race. Some countries are said to have an impact on up to 10% of the population. In addition, there are gender differences in morbidity, and the morbidity rate of women is about twice that of men. The true cause of the suggestion of genetic factors in the development of otosclerosis is unknown (Chole RA and McKenna M., “Pathophysiology of otosclerosis. Otol Neurotol. Vol. 22,

2001: 249-57).

Disclosure of the invention

Problems to be solved by the invention

[0004] As described above, despite the cause of deafness that is common in white races, at present, the only cure for ear sclerosis is the stapes surgery.

[0005] Therefore, the present invention was made for the purpose of providing a method for treating otosclerosis, a pharmaceutical thread and composition for otosclerosis treatment, a method for its administration, and a diagnostic marker for otosclerosis. . Means for solving the problem

[0006] As a result of investigating the relationship between otosclerosis and genetic polymorphism, the present inventor has found that the relationship between the M235T polymorphism in the angiotensinogen gene and the onset of otosclerosis that have been found to be related to hypertension. (Example 1). This M235T polymorphism consists of a M235 allele that encodes wild-type angiotensin 含む containing methionine as the 235th amino acid, and a T235 allele that encodes mutant angiotensin II containing threonine. And power. This T235 allele is thought to enhance the activity of the angiotensin II receptor signaling system by increasing the cytoplasmic concentration of angiotensinogen, and by increasing the tissue concentration of angiotensin II. In fact, the present inventor has revealed that the activity of angiotensin II receptor signaling system is enhanced when stimulated with IL 6 in primary bone cultured cells of otosclerosis patients. (Example 2).

[0007] Therefore, for example, by increasing the activity of angiotensin II and Z or angiotensin Π receptor, an increase in the tissue concentration of angiotensin 、 can be suppressed, and angiotensin II receptor The activity of the signal transduction system can be returned to normal. Many pharmaceutical compositions effective in the treatment of hypertension have been found in accordance with such principles. Power These pharmaceutical compositions are considered to have an effect on otosclerosis as well.

[0008] Based on such a principle, the pharmaceutical composition for the treatment of otosclerosis, which is based on the present invention, contains an angiotensin II receptor signaling system activity reducing agent. The angiotensin II receptor signaling system activity reducing agent may comprise an angiotensin II activity reducing agent or an angiotensin II receptor activity reducing agent. In particular, angiotensin IV activity-reducing ability Angiotensin I inhibitor, angiotensin II inhibitor, angiotensin converting enzyme (Angiotensin Converting Enzyme) inhibitor, or angiotensinogen gene expression inhibitor Is preferred. In addition, the angiotensin converting enzyme inhibitor is a group force that also includes ramipril, arasperil, captopril, quinapril, enalapril, trandolapril, imidapril, cilazapril, perindopril elpmine, temocapril, and their salt power It is preferable to include an agent. In addition, an angiotensin II receptor activity-reducing ability is preferably an angiotensin II receptor inhibitor or an angiotensin receptor gene expression inhibitor. Furthermore, it is preferable to include an inhibitor selected from the group consisting of angiotensin Π receptor inhibitory power salalaracin, oral sultan, telmisartan, palsartan, candesartan cilexetil, and salts thereof.

[0009] The therapeutic method according to the present invention is characterized in that the activity of the angiotensin II receptor signaling system is reduced. Specifically, angiotensin II and Z or angiotensin II receptor activity reducing agent may be administered. Saraco, low activity of angiotensin II Laxatives include activity-reducing agents selected from ramipril, alaspril, captopril, quinapril, enalapril, trandolapril, imidapril, cilazapril, perindopril elpmine, temocapril, and their salts. However, angiotensin II receptor activity-reducing ability may be included. The activity-reducing agent selected from the group consisting of salaracin, oral sultan, telmisartan, valsartan, candesartan cilexetil, and salts thereof may be included.

[0010] The method for administering a pharmaceutical composition for the treatment of otosclerosis comprising an angiotensin II receptor signaling system activity reducing agent according to the present invention comprises the M235T polymorphism of the angiotensinogen gene It is characterized in that it is determined based on the genotypes of alleles.

[0011] The method for selecting a pharmaceutical composition for the treatment of otosclerosis comprising an angiotensin II receptor signaling system activity reducing agent according to the present invention comprises the M235T polymorphism of the angiotensinogen gene The pharmaceutical composition is selected based on the genotypes of alleles.

[0012] A method for determining the dosage of a pharmaceutical composition for the treatment of otosclerosis comprising an angiotensin II and / or an angiotensin II receptor active depressant according to the present invention is an angiotensinogen. The dosage of the pharmaceutical composition is determined based on the genotype of the allele in the M235T polymorphism of the gene.

[0013] The diagnostic marker for otosclerosis according to the present invention contains genetic material for determining the genotype of the allele in the M23 5T polymorphism of the angiotensinogen gene. Here, genetic material refers to a substance that occurs in all stages from the gene-bearing DNA to the expression of the protein, which is the final product of the gene, such as DNA, hnRNA, mRNA, polypeptide, protein, etc. Point to. Therefore, this genetic material may be a peptide having the 235th amino acid sequence of angiotensin II or a DNA having a base pair in the M235T polymorphism of the angiotensinogen gene.

[0014] The estimation method for estimating the possibility of developing otosclerosis according to the present invention is performed by determining the genotype of the allele in the M235T polymorphism of the angiotensinogen gene in healthy individuals. The estimation method for estimating the cause of otosclerosis according to the present invention is performed by determining the genotype of the allele in the M235T polymorphism of the angiotensinogen gene in otosclerosis patients.

[0016] The otosclerosis diagnostic kit useful for the present invention includes a PCR primer for detecting a base pair in the M235T polymorphism of the angiotensinogen gene.

Brief Description of Drawings

[0017] FIG. 1 is a view showing the base sequence of a DNA fragment amplified by PCR performed in Example 1 of the present invention to examine the genotype of an allele in the M235T polymorphism. Primers used for PCR are underlined. (T / C) is a base showing the M235T polymorphism.

[0018] FIG. 2 shows the activity of angiotensin II receptor signal transduction system when angiotensin II is administered using primary bone cultured cells in Example 2 of the present invention. It is a figure which shows the result measured using 6 as a parameter | index.

BEST MODE FOR CARRYING OUT THE INVENTION

[0019] There is no particular explanation for the embodiments and examples! In the case of J. Sambrook, E. F. Fritsch &

T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold bpnng Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, A method described in a standard protocol collection such as JA Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd., or a modified or modified method thereof is used. Also, when using commercially available reagent kits and measuring devices, use the protocols attached to them unless otherwise specified.

[0020] The object, features, advantages, and idea of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily read the description from the description of the present specification. The invention can be reproduced. The embodiments and specific examples of the invention described below show preferred and embodiments of the present invention, and are shown for illustration or explanation. It is not limited to them. Within the spirit and scope of the invention disclosed herein, various changes and modifications may be made based on the description herein. It will be apparent to those skilled in the art that this can be done.

[0021] = = Otosclerosis diagnostic marker = =

When the allelic genotype and the frequency of each genotype in the M235T polymorphism of the angiotensinogen gene were examined in otosclerosis patients and healthy individuals, There is a significant genotyping bias, and many patients have the T235 allele and many healthy individuals have the M235 allele. In patients with otosclerosis who have the T235 allele, angiotensin II force is also transmitted to the angiotensin II receptor. This is thought to have caused otosclerosis.

[0022] Therefore, it is possible to estimate the possibility of future development of otosclerosis in healthy individuals by determining the genotype of an allele for the M235T polymorphism in the angiotensinogen gene. On the other hand, it is possible to estimate the cause of otosclerosis in otosclerosis patients by determining the allelic genotype for the M235T polymorphism.

[0023] As an otosclerosis diagnostic marker for determining the genotype of an allele for such M235T polymorphism, inheritance for determining the genotype of the allele for the M235T polymorphism in the angiotensinogen gene Use substances. For example, the genetic material may be a peptide having the 235th amino acid sequence of angiotensin II, or DNA having a base pair for the M235T polymorphism in the angiotensinogen gene. When the marker is a peptide, a monoclonal antibody that specifically recognizes only one of the M235T polymorphisms can be used to identify the peptide having each genotype. A monoclonal antibody having such specificity can be prepared by a conventional method using an oligopeptide containing either T235 or M235 at the 235th amino acid of angiotensin II. If the marker is DNA, genomic DNA is extracted from human cells to be examined, and the base sequence encoding the amino acid of M235T is examined. In some cases, use the power of PCR or RELP (restriction enzyme length polymorphism).

[0024] In this way, the genotype of the M235T polymorphism of the angiotensinogen gene was determined. For this purpose, if an otosclerosis diagnostic kit containing a primer for PCR is provided, it will be possible to easily test and diagnose.

[0025] = = Suppression of angiotensin II receptor signaling system = =

As described above, the M235T polymorphism in the angiotensinogen gene has a significant bias between otosclerosis patients and healthy individuals. Increased activity of the otensin II receptor signaling system is thought to have caused otosclerosis. Therefore, for the treatment of otosclerosis, the activity of this signal transmission system is preferably suppressed to a normal level. In this case, in order to specifically inhibit the angiotensin Π receptor signaling system, it is preferable to reduce the activity of the ligand angiotensin II and the activity of the receptor angiotensin II receptor.

[0026] However, even if it does not have the T235 allele, it becomes clear by some other method that the cause of otosclerosis is the enhancement of the angiotensin II receptor signaling system. For the treatment of otosclerosis, a pharmaceutical composition that suppresses the activity of this signal transduction system can be used.

[0027] In order to reduce the activity of angiotensin II, for example, an angiotensin I inhibitor that binds to angiotensin I and inhibits conversion to angiotensin II, angiotensin II binds to angiotensin II Angiotensin II inhibitor that inhibits binding to the otensin II receptor, Angiotensin Converting Enzyme inhibitor that inhibits the angiotensin II-converting enzyme to angiotensin II, or An angiotensin II activity-reducing agent, including an angiotensinogen gene expression inhibitor that suppresses angiotensinogen gene expression, may be administered to otosclerosis patients. The activity reducing agent to be used is not limited to these.

[0028] Examples of angiotensin I inhibitors include compounds that bind to angiotensin I and anti-angiotensin I antibodies. Angiotensin II inhibitors include compounds that bind to angiotensin II, anti-angiotensin II antibodies, mutant angiotensin II receptors that bind to angiotensin II but cannot transmit signals (e.g., angiotensin II). Such as shortened angiotensin II receptor, which also has the ability of the extra-membrane domain of the II receptor) It is done. Examples of the angiotensin converting enzyme inhibitor include ramipril, alaspril, captopril, quinapril, enalapril, trandolapril, imidapril, cilazapril, perindopril elpmine, temocapril, and salts thereof. As the angiotensinogen gene expression inhibitor, antisense RNA or RNAi for the angiotensinogen gene is preferable in order to specifically recognize the expression of the angiotensinogen gene.

[0029] In order to reduce the activity of angiotensin II receptor, an angiotensin II receptor activity-reducing agent containing an angiotensin II receptor inhibitor or an angiotensin receptor gene expression inhibitor is used. Although it may be administered to a sclerosis patient, the method for reducing the activity and the activity reducing agent to be administered are not limited to these.

[0030] Angiotensin II receptor inhibitor is a compound that binds to angiotensin II receptor and inhibits angiotensin II receptor activity, or antagonizes angiotensin II receptor and angiotensin II receptor. And an angiotensin II receptor antagonist that inhibits the activity of. Specifically, powers including, but not limited to, salaracin, oral sultan, telmisartan, valsartan, candesartan cilexetil, and salts thereof. As an angiotensin II receptor antagonist, a force signal that strongly or temporarily binds to angiotensin II receptor is not transmitted, and mutant angiotensin II is preferable.

. As an angiotensin receptor gene expression inhibitor, antisense RNA or RNAi against the angiotensin receptor gene is preferred to specifically suppress the expression of the angiotensin receptor gene! /.

[0031] = = Method of administering a pharmaceutical thread and compound for the treatment of otosclerosis = =

The content of the pharmaceutical composition for the treatment of otosclerosis in the preparation can vary between 1 and 90% by weight. For example, when taking the form of tablets, capsules, granules, powders, etc., it is preferable to contain 5-80% by weight of the active ingredient. In the case of liquids such as syrups, it is preferable to contain 1 to 30% by weight of the active ingredient. Furthermore, in the case of injections administered parenterally, it is preferable to contain 10% by weight of the active ingredient.

[0032] Formulation of a pharmaceutical composition for the treatment of otosclerosis involves the use of excipients (sugars such as lactose, sucrose, glucose and man-tol, starch such as potato, wheat and corn, calcium carbonate, , Inorganic substances such as calcium sulfate and sodium bicarbonate, crystalline cellulose, etc.), binders (starch paste, gum arabic, gelatin, sodium alginate, methyl cellulose, ethyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, hydroxypropylene Cellulose, carmellose, etc.), lubricant (magnesium stearate, talc, hydrogenated vegetable oil, macrogol, silicone oil), disintegrant (starch, agar, gelatin powder, crystalline cellulose, CMC 'Na, CMC' Ca, Calcium carbonate, sodium bicarbonate, sodium alginate, etc.), flavoring agents (lactose, sucrose, glucose, mannitol, aromatic essential oils, etc.), solvent (water for injection, sterile purified water, sesame oil, soybean oil, corn oil) , Olive oil, cottonseed oil, etc.), stabilizer (nitrogen, Inert gases such as carbon dioxide, chelating agents such as EDTA and thioglycolic acid, reducing substances such as sodium hydrogen sulfite, sodium thiosulfate, L-ascorbic acid and Rongalite), preservatives (paraoxybenzoic acid esters, chlorobutanol, Benzyl alcohol, phenol, benzalkonium chloride, etc.), surfactant (hydrogenated castor oil, polysorbate 80, 20, etc.), buffer (taenoic acid, acetic acid, sodium phosphate salt, boric acid, etc.), diluent It is carried out by a known method using formulation additives such as

[0033] The pharmaceutical composition for the treatment of otosclerosis as described above may be orally administered in the form of tablets, capsules, granules, powders, syrups, etc., or injections, suppositories. And may be administered parenterally by intraperitoneal, intravenous, intraarterial, intramuscular, subcutaneous injection, direct injection into the ear, or intratympanic injection. It can also be applied in liquid form and applied to the skin and mucous membranes.

[0034] The above-described pharmaceutical yarn for treating otosclerosis and the dosage thereof may be determined according to the genotype of the angiotensinogen gene M235T polymorphism. As shown in Example 1, the T235 mutation shows incomplete dominance, with a greater proportion of homozygotes having more heterozygotes than normal in otosclerosis patients. Therefore, for example, a pharmaceutical composition having a more potent effect is selected and administered to a homozygous otosclerosis patient than a heterozygous otosclerosis patient with a genotype in the M235T polymorphism. More efficient treatment can be achieved by administering a pharmaceutical composition.

Example The embodiment of the present invention described above will be described in more detail with reference to examples.

First, the M235T polymorphism of the angiotensinogen gene in 97 patients with otosclerosis who had been operated at Beaujon Hospital from 1997 to 2002 and 63 normal French patients who visited the hospital at the same time. The genotypes of alleles and the frequency of each gene type were examined. Judgment of otosclerosis is based on the following findings: 1. Progressive hearing loss or mixed hearing loss associated with normal eardrum 2. Loss of stapes reflex 3. Abnormal findings of vestibular window circumference in CT image Therefore, the diagnosis was confirmed based on the stapes findings during surgery and loss of mobility of the stapes.

[0037] Genomic DNA was extracted from the peripheral blood cells of each person, and PCR was performed using the following primers. As conditions, genomic DNA was treated at 94 ° C for 3 minutes, followed by 38 cycles of 94 ° C for 15 seconds, 60 ° C for 45 seconds to 72 ° C for 45 seconds.

[0038] S primer: 5,-CCGTTTGTGCAGGGCCTGGCTCTCT-3, (SEQ ID NO: 1) AS primer: 5,-CAGGGTGCTGTCCACACTGGacCCC -3, (SEQ ID NO: 2)

Since the AS primer has 2 bases (base sequence shown in lower case in the above sequence) that mismatch with genomic DNA, if the amplified DNA has the T235 allele, a restriction enzyme site Tthllll that does not exist in genomic DNA is generated ( Hum Mol Genet, 1993, Vol. 2, No. 5, 609-610). Therefore, the amplified DNA fragment (shown in Figure 1 shows the base sequences of M235 allele <SEQ ID NO: 3> and T235 allele SEQ ID NO: 4>) with restriction enzyme Tthl 1 II and electrophoresed. The M235 allele is a single band of 165 bp, and the T235 allele is detected in two bands of 141 bp and 24 bp. Table 1 shows the results of examining the genotype of each person.

[table 1] MM MT T

TT allele

Frequency of healthy people 22 (35%) 34 (54%) 7 (1 1%) 0.38

(n = 63) Otosclerosis patients 20 (21%) 49 (51%) 28 (29%) 0.54 †

(n = 97)

Genotype distribution vs T + TT vs MT + TT

vs MT + TT

Significance test) f = 4.036, X ^z = 7.045,

ns

(X ² test) dx = 1, p = 0.045 df = 1, p = 0.008 odds 1 1.59 4.40

95%

Confidential 0.75-3.34 1.58-12.28

Interval

[0039] Statistical processing was performed on the obtained results by% 2 analysis. There is a significant bias in genotypes between otosclerosis patients and healthy individuals, with many healthy people having the T235 allele and many having the M235 allele. The odds ratio of each genotype is 1.59 for the 235M235T heterozygote and 4.40 for the T235 homozygote compared to the M235 homozygote. People who have are becoming more susceptible to otosclerosis. Thus, by determining the genotype of the allele in the M235T polymorphism of the angiotensinogen gene, it is possible to estimate the high possibility of developing otosclerosis, and in patients with otosclerosis, The cause can be estimated and treatments can be selected accordingly.

[0040] <Example 2>

Bone cells were primarily cultured from the patients and healthy subjects targeted in Example 1, and the activity of the angiotensin receptor signaling system was examined.

[0041] Bone fragments were cultured at 37 ° C and 5% CO using D MEM containing 4.5% glucose, 12.5 mg / rencomycin, 30% FCS.

2

1. Nodular calcined deposits due to Von Kossa staining

2. Doing osteocalcin minutes ¾¾

3. Morphologically consistent with bone cells As an index, it was confirmed that the isolated primary cultured cells were bone cells.

[0042] In the following experiments, operation was performed using confluent cultured cells in each of the three passages.

[0043] First, when the primary bone cells were seeded in a 48-well culture dish and became confluent, the cells were washed three times with a medium not containing FCS, and angiotensin II ( 2 mu 1, the final concentration 10- ⁷ Micromax) or solvent (2 mu 1) was added, and cultured for 24 hours. Thereafter, the medium is recovered, protease inhibitor cocktail (Roche) is added, the supernatant is recovered by centrifugation, and the concentration of IL 6 secreted into the medium (pg / 1) is determined by ELISA kit (Amersham). ). Figure 2 shows the results.

[0044] In the bone cells of otosclerosis patients, it is clear that IL 6 secretion to angiotensin II is significantly increased and the activity of angiotensin II receptor signaling system is enhanced. Became.

Industrial applicability

[0045] The present invention relates to a method for treating otosclerosis, a pharmaceutical thread composition for the treatment of otosclerosis, a method for its administration, a method for determining its method of administration, a method of determining its dose, and the possibility of developing otosclerosis. It is possible to provide an estimation method for estimating sex, an estimation method for estimating the cause of otosclerosis, and a diagnostic tool for otosclerosis.

Claims

The scope of the claims

[1] A pharmaceutical composition for treating otosclerosis comprising an angiotensin II receptor signaling system activity reducing agent.

[2] The angiotensin II receptor signaling system activity reducing agent comprises an angiotensin II activity reducing agent or angiotensin Π receptor activity reducing agent. A pharmaceutical composition for the treatment of otosclerosis.

[3] Angiotensin II activity-reducing ability including angiotensin I inhibitor, angiotensin II inhibitor, angiotensin converting enzyme inhibitor, or angiotensinogen gene expression inhibitor The pharmaceutical composition according to claim 2.

[4] Angiotensin II activity-reducing ability Ramipril, Alasepril, Captopril, Quinapril, Enalapril, Trandolapril, Imidapril, Cilazapril, Perindoprilhelbumin, Temocapril, and their group power are also selected 1 3. The pharmaceutical composition according to claim 2, comprising the above activity reducing agent.

[5] The pharmaceutical composition according to claim 2, further comprising an angiotensin II receptor inhibitor or an angiotensin receptor gene expression inhibitor.

[6] Activity reducing activity of the angiotensin II receptor comprising one or more activity reducing agents selected from the group consisting of salaracin, oral sultan, telmisartan, valsartan, candesartan cilexetil, and salts thereof The pharmaceutical composition according to claim 2, wherein

[7] A method for treating otosclerosis, comprising reducing the activity of angiotensin II receptor signaling system.

[8] The method for treating otosclerosis according to [7], wherein an angiotensin II and Z or angiotensin Π receptor activity reducing agent is administered.

[9] Angiotensin II activity-reducing ability Ramipril, Alasepril, Captopril, Quinapril, Enalapril, Trandolapril, Imidapril, Cilazapril, Perindoprilrubmine, Temocapril, and their group power are also selected 1 Above life The method for treating otosclerosis according to claim 8, further comprising a hypotensive agent.

[10] Activity reducing activity of the angiotensin II receptor comprising at least one activity reducing agent selected from the group consisting of salaracin, oral sultan, telmisartan, valsartan, candesartan cilexetil, and salts thereof 9. The method of treating otosclerosis according to claim 8, wherein

[11] A method for administering a pharmaceutical thread and composition for the treatment of otosclerosis comprising angiotensin II and Z or angiotensin 活性 receptor activity reducing agent,

A method of administration, comprising determining based on the genotype of an allele in the M235T polymorphism of the angiotensinogen gene.

[12] A method for selecting a pharmaceutical composition for treating otosclerosis comprising an angiotensin II receptor signaling system activity-reducing agent,

A selection method comprising selecting the pharmaceutical composition based on the genotype of an allele in the M235T polymorphism of the angiotensinogen gene.

[13] A method for determining the dosage of a pharmaceutical composition for treating otosclerosis comprising an angiotensin II receptor signaling system activity reducing agent,

A determination method comprising determining the dose of the pharmaceutical composition based on the genotype of the allele in the M235T polymorphism of the angiotensinogen gene.

[14] An otosclerosis diagnostic marker containing genetic material for determining the allelic genotype in the M235T polymorphism of the angiotensinogen gene.

15. The diagnostic marker for otosclerosis according to claim 13, wherein the genetic material is a peptide having an amino acid sequence of 235 of angiotensin II.

16. The otosclerosis diagnostic marker according to claim 13, wherein the genetic material is DNA having a base pair in the M235T polymorphism of the angiotensinogen gene.

[17] An estimation method for estimating the likelihood of developing otosclerosis in healthy individuals by determining the genotype of an allele in the M235T polymorphism of the angiotensinogen gene.

[18] An estimation method for estimating the cause of otosclerosis in patients with otosclerosis by determining the allelic genotype of the M235T polymorphism of the angiotensinogen gene. An otosclerosis diagnostic kit comprising a PCR primer for detecting base pairs in the M235T polymorphism of the angiotensinogen gene.