JP2010246424A - Method for diagnosing onset risk of osteoporosis, osteogenesis imperfecta or osteogenesis imperfecta - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for diagnosing the onset risk of osteoporosis, osteogenesis imperfecta or osteogenesis imperfecta, and to provide a method for detecting the base substitution of an expression gene. <P>SOLUTION: The method for diagnosing the onset risk of the osteoporosis, the osteogenesis imperfecta or the osteogenesis imperfecta includes using a female nail as a specimen, and detecting the base substitution at least in one of the expression gene in an estrogen receptor expression gene, an LDL receptor-associated protein 5 expression gene, and a type-I collagen expression gene. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for diagnosing the onset risk of osteoporosis, osteogenesis imperfecta, or osteogenesis imperfecta, and a method for detecting base substitution of an expressed gene.

A disease that develops through the interaction of an environmental factor such as a lifestyle habit and a genetic factor is called a multifactorial disease. Examples of such a disease include osteoporosis. Osteoporosis refers to a symptom in which bone mass is reduced, the structure in the bone is broken, and the bone becomes very brittle and easy to break. This is because the maximum bone mass of women is lower than that of men, and female hormones that affect bone formation decrease with age.
As a genetic factor, it is known that a single nucleotide polymorphism (SNP) increases the risk of developing a specific disease such as a bone disease (Non-Patent Documents 1 to 12). Diseases such as osteoporosis related to bone density reduction are difficult to cure after onset, so knowing the risk of onset before onset, for example, from puberty to reaching the maximum bone mass, around 20 years old, It is very effective in improving and preventing onset.
For the diagnosis of genes such as SNP, DNA extracted from biological samples such as blood, hair, cells in the oral cavity and saliva is mainly used. However, these biological samples are problematic in that they are painful to collect, take a long time to collect, require medical personnel and medical equipment, and give resistance to subjects.

Gentics of Osteoporpsis John A. Eisman: Genetics of Osteroporosis Endocrine Reviewa in Endocrine Society 20 (6) 788-804 (1999) Molecular studies of identification of genes for osteoporosis: the 2002 update.Yao-Zhong Liu, Young-Jun Liu, Robert R Recker, Hong-Wee Deng Journal of Endocrinology 177 147-196 (2003) Japan Medical Function Evaluation Organization HP (Medical Information Service Minds) Osteoporosis Prevention and Treatment GL Committee (2006) Japan Medical Association HP Health Forest http://www.med.or.jp/forest

Omasu F, Kitagawa J, Koyama K, Asakawa K, Yokouchi J, Ando D, Nakahara Y. The influence of VDR genotype andd exercise on ultrasound parameters in young adult Japanese women.J Physiol Anthropol Human Sci 23 (2) 49-55 ( 2004) Morrison NA, QiJC, Tokita A, Kelly PJ, Crofts L, Nguyen TV, Sambrook PN, Eisman JA.Prediction of bone density from vitamin D receptor alleles.Nature Jan 20 367 (6460) 284-287 (1994) Heaney RP Vitamin D in health and disease.Clin J Am Soc Nephrol. 2008 Sep; 3 (5): 1535-41. Epub (2008 Jun 4) Blair D, Byham-Gray L, Lewis E, McCaffrey S Prevalence of vitamin D [25 (OH) D] deficiency and effects of supplementation with ergocalciferol (vitamin D2) in stage 5 chronic kidney disease patients. J Ren Nutr. 2008 Jul; 18 (4): 375-82 Zisman AL, Hristova M, Ho LT, Sprague SM. Impact of ergocalciferol treatment of vitamin D deficiency on serum parathyroid hormone concentrations in chronic kidney disease. Am J Nephrol. 2007; 27 (1): 36-43. Epub 2007 Jan 11 Makino Hidenori, Suzuki Yasunobu, Takayama Masaomi The effects of vitamin D receptor and estrogen receptor gene polymorphisms on the effects of various treatments on bone loss in postmenopausal Japanese women 50 (3) 125-132 (1998) ) Shimizu Shishi Vitamin D binding protein (DBP) gene polymorphism and bone mass and bone metabolism markers in postmenopausal women Saitama Medical University Journal 32 (2) T43-T50 (2005) Kazutoshi Nakamura Interaction between calcium intake and vitamin D receptor gene polymorphism in the acquisition of maximum bone mass in young women-Using the Duplicate Portion Sampling, Research Report on Milk Nutrition Research Committee H16 127-135 Japan Dairy Milk Industry Association (2005)

  An object of the present invention is to provide a method for diagnosing the onset risk of osteoporosis, osteogenesis imperfecta, or osteogenesis imperfecta, which is low in invasiveness and easy to collect a sample, and a method for detecting base substitution of an expressed gene Is to provide.

The present inventor has conducted research to solve the above problems, and as a result of dissolving nails in proteases derived from various animals and plants, DNA can be extracted more efficiently than commercially available kits, contributing to multifactorial diseases. It was found that gene polymorphism can be detected.
That is, the present invention
(1) Osteoporosis characterized by detecting base substitution in at least one expression gene of an estrogen receptor expression gene, an LDL receptor-related protein 5 expression gene, and a type I collagen expression gene using a female nail as a specimen , A method of diagnosing the risk of developing bone dysplasia or bone mineralization dysplasia,
(2) The method according to (1), wherein the estrogen receptor expression gene is ESRX or ESRP,
(3) The method according to (1) or (2), wherein the age of the woman is 12 to 20 years old,
(4) DNA is extracted by dissolving a female nail in an alkaline aqueous solution of an animal or plant-derived protease, and the extracted DNA is subjected to a PCR method to obtain an estrogen receptor expression gene, an LDL receptor-related protein 5 expression gene, and I A method for detecting a base substitution of an expressed gene, comprising amplifying a base substitution site of at least one expressed gene of a type I collagen expressing gene,
(5) The method according to (4), wherein the animal or plant-derived protease is a melon-derived protease.

  According to the present invention, a method for diagnosing the onset risk of osteoporosis, osteogenesis imperfecta, or osteogenesis imperfecta, which is low in invasiveness and easy and easy to collect a specimen sample, and base substitution of an expressed gene. A method of detecting is provided.

It is a figure which shows the determination result of ESRX by real-time PCR. It is a figure which shows the determination result of ESRP by real-time PCR. It is a figure which shows the determination result of LRP5 by real-time PCR. It is a figure which shows the correlation with the gene polymorphism of ESRX, and a bone evaluation value. It is a figure which shows the correlation with the gene polymorphism of ESRP, and a bone evaluation value. It is a figure which shows the correlation with the gene polymorphism of LRP5, and a bone evaluation value.

Hereinafter, the present invention will be described in detail.
In the present invention, the nail used as a specimen may be a nail of an animal's limb, regardless of gender and age, but a female nail is preferable from the viewpoint of preventing a disease associated with bone density reduction, and the maximum bone Women's nails from puberty to around 20 years of age are particularly preferred. The nail may be a toenail or a hand nail. The nail is usually collected with scissors, but the collected nail may be further shredded or crushed.
The base substitution of the gene to be detected may be any base substitution in a gene involved in bone formation, and the gene involved in bone formation is not particularly limited. For example, estrogen receptor expression genes (ESRX, ESRP), LDL reception SNPs in the body-related protein 5 expression gene (LRP5), type I collagen expression gene (CLA1) and the like can be mentioned. Examples of the SNP include ESRX: rs9340799, ESRP: rs2234693, LRP5: rs376228, CLA1: rs1800012, and the like.

In the present invention, extraction of DNA from the nail is performed by dissolving the nail with an alkaline aqueous solution of animal or plant-derived protease. Any plant and animal-derived protease may be used as long as it decomposes keratin, which is the main component of the nail. As a product containing a large amount of such protease, fruit pulp such as papaya, pineapple, kiwi fruit, melon, fig, pear is preferable, and melon pulp is particularly preferable. Further, it is preferable to obtain the pulp by homogenizing and filtering while cooling with ice.
The alkaline aqueous solution is not particularly limited, and a known aqueous solution can be used, but from the viewpoint of keeping the pH constant during DNA extraction, Tris-HCl buffer, Tricine buffer, Glycine buffer, Ammonia Buffers such as ammonium chloride buffer, borate-sodium carbonate buffer, Good buffer (AMPSO, CHES, CAPSO, AMP, CAPS) are preferred, and glycine buffer is particularly preferred.
The pH of the alkaline aqueous solution is preferably about pH 8 to 12, particularly preferably about pH 9 to 11, from the viewpoint of degrading nail keratin, high protease activity, and efficient DNA extraction.
Alkaline aqueous solution includes sodium dodecyl sulfate (SDS), fatty acid salt, alpha sulfo fatty acid ester salt (α-SFE), alkyl benzene sulfonate (ABS), alkyl sulfate, alkyl ether sulfate ester, alkyl sulfate triethanolamine, Surfactants such as fatty acid diethanolamide, polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether, alkyltrimethylammonium salt, dialkyldimethylammonium chloride, alkylpyridinium chloride, alkylcarboxybetaine, ethylenediaminetetraacetic acid disodium (EDTA), ethylene It may further contain a chelating agent such as glycol bis-β-aminoethyl ether (EGTA) or citric acid.
About 9 to 11 parts by weight of protease and about 16 to 20 parts by weight of an alkaline aqueous solution are mixed with 1 part by weight of the nail, and the DNA contained in the nail is transferred to the aqueous solution by stirring as desired. Extraction is performed.

The extraction temperature of DNA is not particularly limited, but is preferably about 60 to 80 ° C., particularly preferably about 65 to 75 ° C. from the viewpoint that protease activity is high and DNA can be extracted efficiently. The extraction time of DNA is not particularly limited, but is preferably about 20 to 40 minutes, and particularly preferably about 25 to 35 minutes from the viewpoint that a sufficient amount for DNA amplification can be obtained.
An organic solvent (for example, a lipophilic organic solvent such as phenol, chloroform, isoamyl alcohol, etc.) is added to the alkaline aqueous solution containing DNA, centrifuged, the aqueous phase is separated, and an aqueous alkaline solution (for example, sodium acetate aqueous solution) is added. The target DNA is obtained by adding an oily organic solvent (isopropyl alcohol), centrifuging, and collecting the precipitate.
The extracted DNA can be amplified by a known PCR (polymerase chain reaction) method. By subjecting the amplified DNA to a known method such as agarose gel electrophoresis, the target base substitution site (SNP described above) can be detected. It is particularly preferable to use a PCR method or a real-time PCR method using a fluorescently labeled probe such as a Taqman probe because the target SNP can be detected simultaneously with the amplification of DNA, and agarose gel electrophoresis is not required. Regarding the risk of developing osteoporosis, osteogenesis imperfecta, or osteogenesis imperfecta, if SNP is detected from the amplified DNA, it is confirmed that the gene is mutant heterozygous or mutant homozygous. It is judged that there is a risk of developing dysplasia or bone density dysplasia in the future, especially after menopause.
According to the present invention, even if it is determined that there is a risk of developing osteoporosis, osteogenesis imperfecta, or osteogenesis imperfecta, yogurt, milk, bean products, for example, which are exercised at least once a week for about 2 hours The risk of developing the above-mentioned diseases can be significantly reduced by improving life such as ingesting foods high in calcium such as fish, refraining from smoking and drinking.

[Preparation of melon-derived protease]
The pulp of Andean melon was homogenized with a Waring blender. The homogenized pulp part was filtered through gauze moistened with 10 mM phosphate buffer (pH 7) while cooling with ice to obtain a melon-derived protease.

[Preparation of DNA solution]
Targeted 175 students from Mukogawa Women's University (2007). Table 1 shows the body data of 175 subjects.

A nail obtained by obtaining informed consent from the subject was used as a specimen. After finely cutting the nail and washing with 100% ethanol, 5 mg is taken into a 1.5 mL microtube, solution (50 mM glycine buffer (pH 10.0), 100 mM EDTA) 80 μL, and 5% (w / w) SDS 20 μL of aqueous solution was added. After lightly tapping, it was allowed to stand for 10 minutes. Melon-derived protease (50 μL) was added and incubated at 70 ° C. for 30 minutes while tapping every 5 minutes. After adding 150 μL of phenol / chloroform / isoamyl alcohol (25: 24: 1 (volume ratio)), the mixture was mixed by inverting for 5 minutes, and then centrifuged at 14000 rpm and 23-24 ° C. for 5 minutes.
The upper layer (aqueous phase) was taken in another 1.5 mL microtube, 10 μL of 3M CH ₃ COONa aqueous solution and 100 μL of isopropyl alcohol were added, and centrifuged at 14000 rpm, 23-24 ° C. for 5 minutes. The supernatant was removed by decantation and the precipitate was incubated at 55 ° C. and dried. 70% (v / v) ethanol stored at −30 ° C. was added and centrifuged at 14000 rpm, 23-24 ° C. for an additional 5 minutes. The upper layer (ethanol) was removed by decantation, and the lower layer was incubated at 55 ° C. and dried. 20 μL of TE buffer (10 mM Tris-HCl buffer (pH 8), 0.5 M EDTA) was added to prepare a DNA solution. The solution was stored refrigerated at 3 ° C.

[Real-time PCR]
A gene fragment containing a polymorphic site was amplified using the following three types of primers. The SNP ID of each gene and the nucleotide sequence of the gene polymorphism are shown below.

  12.5 μL of Master Mix, 1.25 μL of primer, and 1.25 μL of sterilized water were mixed into a 1.5 mL microtube by pipetting. 13.75 μL of each DNA solution was added to Micr Amp. Plate well and 10.25 μL of sterilized water was further added to prepare a reaction solution. However, 11.25 μL of sterilized water was added to NEGATIVE CONTROL instead of DNA solution. The reaction composition is shown in Table 2 below.

  Using these reaction solutions, real-time PCR was performed by Real-Time PCR 7500 (Applied Biosystems). Pre-Read is performed at 25 ° C for 1 minute, reacted at 50 ° C for 2 minutes and 95 ° C for 10 minutes, followed by 40 cycles of reaction at 92 ° C for 15 seconds and 60 ° C for 1 minute, and finally at 25 ° C for 1 minute. I did Post-Read. Each gene polymorphism was determined by the fluorescence intensity of VIC and FAM.

[Genetic polymorphism distribution frequency and ratio]
(i) Estrogen receptor-α (ESRX)
Table 4 below shows the distribution frequency and the ratio of ESRX gene polymorphisms in 175 subjects. Moreover, the determination result by the fluorescence intensity of ESRX by real-time PCR is shown in FIG.

(ii) Estrogen receptor-α (ESRP)
Table 5 below shows the distribution frequency and the ratio of ESRP gene polymorphisms in 175 subjects. Moreover, the determination result by the fluorescence intensity of ESRP by real-time PCR is shown in FIG.

(iii) LDL receptor-related protein 5 (LRP5)
Table 6 below shows the distribution frequency and ratio of LRP5 gene polymorphisms in 175 subjects. Moreover, the determination result by the fluorescence intensity of LRP5 by real-time PCR is shown in FIG.

[Correlation between genetic polymorphism and bone evaluation value]
An ultrasonic bone evaluation value (OSI) was measured by irradiating the ribs of the foot with an ultrasonic wave using an ultrasonic bone evaluation value measurement apparatus (Ultrasonic Bone Evaluation Apparatus AOS-100NW, manufactured by Aloka Co., Ltd.).
(i) Estrogen receptor-α (ESRX)
As shown in Table 8 below, in the ESRX gene polymorphism, the bone evaluation value of the Xx type (hetero) was lower than that of the XX type (wild homo) and the Xx type (mutant homo).

(ii) Estrogen receptor-α (ESRP)
As shown in Table 9 below, in the ESRP gene polymorphism, the bone evaluation value of Pp type (hetero) was lower than that of pp type (wild homo) and PP type (mutant homo).

(iii) LDL receptor-related protein 5 (LRP5)
As shown in Table 10 below, in the gene polymorphism of LRP5, the bone evaluation value of CT type (hetero) and TT type (mutant homo) was lower than that of CC type (wild homo).

  The method for diagnosing the onset risk of osteoporosis, osteogenesis imperfecta, or osteogenesis imperfecta and the method for detecting base substitution of an expressed gene are low in invasiveness and simple.

Claims (5)

Osteoporosis and bone formation characterized by detecting base substitution in at least one expression gene of an estrogen receptor expression gene, an LDL receptor-related protein 5 expression gene, and a type I collagen expression gene using a female nail as a specimen A method for diagnosing the risk of developing insufficiency or osteogenesis imperfecta. The method according to claim 1, wherein the estrogen receptor expression gene is ESRX or ESRP. The method according to claim 1 or 2, characterized in that the age of the female is 12-20 years old. DNA is extracted by dissolving female nails in an alkaline aqueous solution of proteases derived from animals and plants, and the extracted DNA is subjected to a PCR method to express estrogen receptor expression gene, LDL receptor-related protein 5 expression gene, and type I collagen expression A method for detecting a base substitution of an expressed gene, comprising amplifying a base substitution site of at least one expressed gene of the gene. The method according to claim 4, wherein the animal or plant-derived protease is a melon-derived protease.

Images