KR20030065205A - Method for measurement of osteoporosis risk factor by measuring polymorphism of genes involved in osteoporotic process and kit for diagnosis of genetic oseoporosis risk factor using the same - Google Patents

Abstract

PURPOSE: A method for measurement of an osteoporosis risk factor by measuring polymorphism of genes involved in osteoporotic process and a kit for diagnosis of a genetic risk factor of osteoporosis are provided, thereby easily measuring the possibility of osteoporosis generation. CONSTITUTION: A method for measurement of an osteoporosis risk factor by measuring polymorphism of genes involved in osteoporotic process comprises the steps of: (1) measuring the polymorphism regions of genes relating to osteoporosis from DNA extracted from a sample; and (2) obtaining relationship between the polymorphism regions obtained from the step (1) and the amount of bone density, wherein the gene is selected from LEPR, ER, vitamin D receptor (VDR) and transforming growth factor-beta1(TGF-β1); the polymorphism region of LEFR is selected from Gln223Arg, Ser492Thr, Ala976Asp and Pro1019Pro; the polymorphism region of ER is PvuII and XbaI recognition sites; and the polymorphism is determined by using DNA sequencing, PCR-SSCP analysis, RFLP analysis, allyl specific hybridization or DNA chip method.

Description

Method for measurement of osteoporosis risk factor by measuring polymorphism of genes involved in osteoporotic process and kit for diagnosis of genetic oseoporosis risk factor using the same }

The present invention relates to a method for predicting the risk of the occurrence of osteoporosis and a diagnostic kit for the genetic risk factors of osteoporosis using the same, in particular, a method for predicting the risk of osteoporosis by measuring the polymorphism of the LEPR and ER gene A diagnostic kit for the genetic risk factors of osteoporosis used.

Osteoporosis is generally regarded as a disease that occurs mainly in women, but it is also a disease that occurs in men. In men, the incidence of osteoporosis fractures is about half that in women (Cooper C, Atkinson EJ, O'Fallon WM, Melton LJ III, J. Bone Miner. Res. , 1992 , 7, 221-227). In addition, in older men, mortality from fractures of the pelvis is significantly higher than in women (Myers AH, Robinson EG, Van Natta ML, Michelson JD, Collins K, Baker SP, Am. J.). Epidemiol. , 1991 , 134, 1128-1137). However, little is known about the cellular and molecular basis on which osteoporosis occurs in men. In general, bone mass at a given age peaks between 20 and 34 years and gradually decreases at later ages (Byers RJ, Hoyland JA, Braidman IP, J. Endocrinol. , 2001 , 168). , 353-362).

On the other hand, peak bone mass is considered to be an important factor in determining the occurrence of osteoporosis (Newton-John HF, Morgan DB, Clin. Orthop. , 1970 , 71, 229-252). And environmental factors such as physical activity may affect, but in fact, genetic factors are known to determine most of the maximum bone mass (Heaney RP, Abrams S, Hughes DB, Looker A, Marcus R, Matkovic V, et al, Osteoporos. Int. , 2000 , 11, 985-1009). Some of these genetic factors, such as vitamin D receptors, estrogen receptors, type I collagen and TGF-β, have been studied for their association with their genetic polymorphism and bone mass (Stewart TL, Ralston SH., J.) . Endocrinology , 2000 , 166, 235-245), in most studies, postmenopausal women whose bone mass stops increasing and bone begins to decrease. In addition, these data were inconsistent and showed that gene-to-gene and gene-to-environment interactions could be important confounding factors in genetic research.

Leptin, a product of the ob gene, is a hormone produced by adipocytes and is known to play a pivotal role in maintaining homeostasis, neurosecretory function and fertility of body weight (Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM, Nature, 1994, 372, 425-432; Barash IA, Cheung CC, Weigle DS, Kabigting EB, Kuijper JL, Clifton DK, Endocrinology , 1996 , 137, 3144-3147). There is also controversy about the mechanism by which leptin plays a role in the regulation of bone mass, but several documents have been reported to show that leptin plays an important role in the regulation of bone mass (Ducy P, Amling M, Takeda S, Priemel M, Schilling AF, Beil FT, et al., Cell , 2000 , 100, 197-207; Steppan CM, Crawford DT, Chidsey-Frink KL, Ke H, Swick AG, Regul.Pept . , 2000 , 92, 73-78) .

The biological action of leptin on target tissues is achieved by binding to leptin-specific receptors, which are members of the cytokine receptor family (Tartaglia LAm, Dembski M, Weng X, Deng N, Culpepper J, Devos R, et al., Cell , 1995 , 83, 1263-1271). Extensive studies have been conducted on the association of this leptin receptor gene polymorphism with obesity (Yiannakouris N, Yannakoulia M, Melistas L, Chan JL, Klimis-Zacas D, Mantzoros CS, J. Clin). Endocrinol. Metab. , 2001 , 86, 4434-4439; Wauters M, Mertens I, Chagnon M, Rankinen T, Considine RV, Chagnon YC, et al., Int. J. Obes.Relat. Metab.Disord. , 2001 . , 25, 714-720; Silver K, Walston J, Chung WK, Yao F, Parikh VV, Anderson R, et al., Diabetes , 1997 , 46, 1898-1900; Matsuoka N, Ogawa Y, Hosoda K, Matsuda J , Masuzaki H, Miyawaki T, et al., Diabetologia , 1997 , 40, 1204-1210), which have not been reported to be associated with bone mass.

In men, estrogen is considered more important than testosterone in maintaining bone mass (Carani C, Qin K, Simoni M, Faustini-Fustini M, Serpente S, Boyd J, et al. , N. Engl. J. Med. , 1997 , 337, 91-95; Szulc P, Munoz F, Claustrat B, Garnero P, Marchand F, Duboeuf F, et al., J. Clin.Endocrinol . Metab. , 2001 , 86, 192-199) Recently, polymorphisms of the ER gene were associated with bone mineral density (BMD) in young men (Lorentzon M, Lorentzon R, Backstrom T, Nordstrom). P., J. Clin. Endocrinol. Metab. , 1999 , 84, 4597-4601). The results suggest the possibility that the polymorphism of the ER gene can affect the accumulation of bone mass. However, no further studies supporting this correlation in men have been reported.

Interestingly, estrogens interact with leptin from a variety of perspectives, and reports have shown that estrogens reduce the expression of the LEPR gene, change the balance between long and short form of LEPR, and sequentially regulate susceptibility to leptin. (Bennett PA, Lindell K, Karlsson C, Robinson IC, Carlsson LM, Carlsson B, Neuroendocrinology , 1998 , 67, 29-36), and also reported to increase the expression of leptin mRNA in cultured rat adipocytes. (Machinal F, Dieudonne MN, Leneveu MC, Pecquery R, Gidicelli Y, Endocrinology , 1999 , 140, 1567-1574). In human studies, hormone replacement therapy significantly increases plasma leptin levels in postmenopausal women (Konukoglu D, Serin O, Ercan M, Maturitas , 2000 , 36, 203-). 207). All these results indicate that estrogen can alter the effect of the leptin system on bone metabolism. However, no studies on the interaction of these genes with respect to bone mass have been reported.

Therefore, the present inventors have studied the effects of the polymorphisms of the LEPR and ER genes and the possible interactions between these genes on BMD and found that they are closely related to BMD, and the method for predicting the risk of osteoporosis using the same And the present invention was completed by inventing a diagnostic kit for the genetic risk factors of osteoporosis using the same.

An object of the present invention to provide a method for predicting the risk of osteoporosis by measuring the polymorphism of the gene involved in osteoporosis, and to provide a diagnostic kit for the genetic risk factors of osteoporosis using the same.

In order to achieve the above object, the present invention provides a method for predicting the risk of developing osteoporosis.

The present invention also provides a diagnostic kit for the genetic risk factors of osteoporosis using the method for measuring the polymorphism of the gene involved in osteoporosis.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for predicting the risk of osteoporosis by measuring the polymorphism of a gene involved in osteoporosis.

The subject of the method for predicting the risk of developing osteoporosis of the present invention can be any mammal, among which human beings are preferred and males are more preferred.

The method of predicting the risk of developing osteoporosis of the present invention

1) measuring a polymorphic site of a gene involved in osteoporosis from DNA extracted from a sample; And

2) obtaining a correlation with the bone mineral density from the measurement result of step 1.

In step 1, the sample is preferably selected from secretions consisting of blood, saliva, and urine, more preferably blood, but not necessarily limited thereto, and any part of the body from which DNA can be extracted will be subjected. Can be. Preferably, the sample is collected by a general serum separation method after blood collection.

In step 2, specific measuring methods for measuring polymorphism include DNA sequencing, single stranded conformation polymorphism (PCR-SSCP) analysis, RFLP analysis, allyl specific hybridization method, and DNA chip method. The present invention is not limited thereto, and genotyping may be performed using restriction enzymes that recognize specific sequences. In addition, all assays based on an antigen-antibody reaction that specifically binds to each protein in which polymorphism occurs can be used, and among them, an enzyme-linked immunosorbent assay (ELISA) commonly used for the analysis of antigen-antibody binding. Methods and immunoblot methods are preferred.

In the above, the gene involved in osteoporosis may be selected from the group consisting of LEPR, ER, Vitamin D receptor (VDR), and Transforming growth factor-β1 (TGF-β1), preferably LEPR or ER, LEPR More preferably.

In the case of the LEPR gene, the polymorphic site may be selected from the group consisting of Gln223Arg, Ser492Thr, Ala976Asp and Pro1019Pro, of which the Gln223Arg position is preferred. In addition, in the case of the ER gene, polymorphism may be investigated by determining the genotype of the DNA sequence variant at the Pvu II and Xba I cleavage sites, but restriction enzymes that may be used for the investigation of polymorphism may be used in addition to the Pvu II and Xba I restriction enzymes. Any restriction enzyme can be used.

The present inventors exemplified the case where the polymorphisms of the LEPR and ER proteins were examined as specific examples of the measurement method of the present invention for measuring the polymorphism of the genes involved in osteoporosis to predict the risk of osteoporosis.

To this end, the serum leptin concentration and bone mineral density were first measured from the subjects. The mean age of the subjects was 25.3 years (SD 3.7, range 20-34). The BMI (Body Mass Index) was found to be related. In addition, no correlation was found between calcium intake, smoking, alcohol consumption, exercise time or serum leptin levels and BMD (see Table 1 ).

Next, genotypes of DNA sequence variants at the Gln223Arg, Ser492Thr, Ala976Asp and Pro1019Pro sites of the LEPR gene and the Pvu II and Xba I cleavage sites of the ER gene showed that all polymorphisms of the LEPR and ER genes were Hardy- Weinberg was in equilibrium, and the frequency of informing about the Gln223Arg, Ser492Thr, and Pro1019Pro polymorphisms was similar to those previously reported in Japan (Matsuoka N, Ogawa Y, Hosoda K, Matsuda J, Masuzaki H, Miyawaki T, et al. , Diabetologia , 1997 , 40, 1204-1210). The absence of polymorphism at Ala976Asp was also the same as reported in Japan. The allyl frequency of ER in Pvu II and Xba I polymorphisms was not different from previous cases observed in Korean women (Han Ko, Moon IG, Kang YS, Chung HY, Min HK, Han IK, J. Clin. Endocrinol. Metab. , 1997 , 82, 991-995) (see Table 2 ).

The frequency of genotype combinations defined by the Pvu II and Xba I polymorphisms was PPXX, 8 (5%); PPXx, 12 (8%); PPxx, 10 (6%); PpXx, 38 (24%); Ppxx, 30 (19%); ppXx, 7 (5%); And ppxx, 52 (33%). Absence of PpXX or ppXX genotypes is consistent with previous reports (Kobayahi S, Inoue S, Hosoi T, Ouchi Y, Shiraki M, Orimo H, J. Bone Miner. Res. , 1996 , 11, 306-311; Albagha OME, Mcguigan FEA, Reid DM, Ralston SH, J. Bone Miner.Res . , 2001 , 16, 128-134). The distribution of the combination genotype indicates that the Pvu II and Xba I polymorphisms are in linkage disequilibrium, while the PPXX genotype was present five times more frequently, while the PpXx and ppxx genotypes were higher than expected. 1.5 times higher frequency.

Next, we analyzed the association between BMD and LEPR genotypes in the lumbar spine or femur after adaptation for BMI. As a result, there was no association between BMD and Pro1019Pro polymorphism of LEPR in the lumbar spine or femur, and the frequency of genotype variants in Ser492Thr and Ala976Asp was too low to be used for analysis of association with BMD. In addition, the number of subjects representing Gln223Gln is so small that the inventors divided the case with Gln223 allyl (Gln223Gln or Gln223Arg) and the case without (Arg223Arg). No difference was observed between the two groups, with a higher BMD in the lumbar spine compared to the non-subject with Gln223 allyl. The association of Gln223Arg polymorphism with BMD was not observed in the femur neck.

Further analysis of the association of BMD with Gln223Arg genotype of LEPR according to BMI revealed that the overweight group (Ko GT, Tang J, Chang JC, Sung R, Wu MM, Wai HP, et al., Br. J. Nutr) ., in the case of 2001, 85, 239-242) is associated with Gln223Arg polymorphism and BMD in both the lumbar spine and femur neck sides were observed (see Table 3). Subjects with Gln223 allyl showed a BMD of about 10% higher in the lumbar spine compared to subjects without Gln223 allyl. Associations were also observed in the femur neck, ward triangles, electrons and stems. However, this association did not appear in the non-overweight group. Leptin levels did not differ between subjects with and without Gln223 allyl.

In addition, the inventors have described the Px haplotype (Kobayahi S, Inoue S, Hosoi T, Ouchi Y, Shiraki M, Orimo H, J. Bone Miner. Res. , 1996 , 11, 306-311) as previously described . haplotype) and BMD were examined, but Px haplotype was not associated with BMD in the lumbar spine as well as the femur neck.

In addition, the inventors determined the difference in BMD in the LEPR genotypes in association with the ER genotypes to investigate the existence of a correlation between the LEPR and ER genes. As a result, the association of LEPR Gln223Arg polymorphism with lumbar BMD was evident only in subjects with P or X allyl of ER. Even in overweight subjects where the effect of LEPR polymorphism was more pronounced, the association disappeared in the absence of P or X allyl in the ER (see Tables 4 and 5 ).

From the above results, the inventors confirmed that the polymorphism at the Gln223Arg region of the LEPR gene correlated closely with the BMD in the lumbar spine.

The Gln223Arg polymorphic region is present in the region encoding the extracellular domain of LEPR, and it has been reported that substitution of Gln with Arg causes a change in charge (Chung WK, Power-Kehoe L, Chua M, Chu). F, Aronne L, Huma Z, et al., Diabetes , 1997 , 46, 1509-1511). Thus, the Gln223Arg polymorphism may affect the functional properties of the receptor. Previous studies have reported that leptin-binding affinity is lower in the carrier of Arg223 allyl (Quinton ND, Lee AJ, Ross RJ, Eastell R, Blakemore AI, Hum. Genet. , 2001 , 108, 233-236). . From these results it can be inferred that the association of Gln223Arg polymorphism with BMD will result from a change in leptin-binding affinity. However, as can be seen from the present invention and other studies, the possibility of linkage disequilibrium cannot be ruled out, given the fact that there is no difference in leptin concentration in serum between genotypes (Rosmond R, Chagnon YC, Holm G, Chagnon M, Perusse L, Lindell K, et al., J. Clin. Endocrinol. Metab. , 2000 , 85, 3126-3131).

In the present invention, the association between the Gln223Arg genotype of LEPR and BMD was observed in overweight subjects (see Table 3 ). There was a 10% difference in lumbar BMD between genotypes. None of the gene polymorphism studies so far showed a significant difference in BMD as shown in the present invention.

To date, the Gln223Arg polymorphism has been studied in association with obesity, although some of them have been found to be associated with obesity (Yiannakouris N, Yannakoulia M, Melistas L, Chan JL, Klimis-Zacas D, Mantzoros CS, J. Clin. Endocrinol. Metab. , 2001 , 86, 4434-4439; Chagnon YC, Chung WK, Perusse L, Changnon M, Leibel RL, Bouchard C, Int. J. Obes.Relat. Metab. Disord. , 1999 , 23, 278-286) , No other associations were found (Silver K, Walston J, Chung WK, Yao F, Parikh VV, Anderson R, et al., Diabetes, 1997, 46, 1898-1900; Matsuoka N, Ogawa Y, Hosoda K , Matsuda J, Masuzaki H, Miyawaki T, et al., Diabetologia , 1997 , 40, 1204-1210). Considering the polygenic nature of human obesity, these results are not surprising. In addition, we did not observe any association between BMI and LEPR genotypes even in the presence of differences in BMD. From the above results, it can be seen that higher BMD in subjects with Gln223 allyl is not simply due to higher BMI itself.

ER is one of the most frequently investigated genes in women because of the possible association in polymorphism with BMD. There is evidence showing such a connection, but it appears to depend on the site of measurement, age and ethnicity of the subject group. Thus, there is no consensus on the role of ER polymorphism in women's BMD (Stewart TL, Ralston SH, J. Endocrinology , 2000 , 166, 235-245). Polymorphism of ER may be interesting for men in terms of evidence of the importance of estrogen on the male skeleton (Byers RJ, Hoyland JA, Braidman IP, J. Endocrinol. , 2001 , 168, 353-362), but only in a few reports Its role in association with male BMD has been studied (Lorentzon M, Lorentzon R, Backstrom T, Nordstrom P, J. Clin. Endocrinol. Metab. , 1999 , 84, 4597-4601; Ongphiphadhanakul B, Rajatanavin R, Chanprasertyothin S, Piaseu N, Chailurkit L, Clin.Endocrinol . , 1998 , 49, 803-809). Two studies have reported that ERα polymorphism is associated with BMD in men (Lorentzon M, Lorentzon R, Backstrom T, Nordstrom P, J. Clin. Endocrinol. Metab. , 1999 , 84, 4597-4601; Ongphiphadhanakul B , Rajatanavin R, Chanprasertyothin S, Piaseu N, Chailurkit L, Clin.Endocrinol . , 1998 , 49, 803-809). We did not find any association between BMD and ER polymorphism in the present invention, but found that ER polymorphism interacts with LEPR polymorphism to control peak bone mass (see Table 4 and Table 5 ). . In addition, the association of LEPR Gln223Arg polymorphism with lumbar BMD was only observed in the presence of P or X allyl of ER. Even in overweight subjects with greater effect of LEPR polymorphism, the association between LEPR genotype and lumbar BMD did not appear in the absence of P or X allyl of ER. These results indicate the possibility that ER genotype can alter the effect of the LEPR gene on bone metabolism. Pvu II and Xba I polymorphisms are approximately 50 bp apart and are inconsistent association. In the present invention, P and X allyl showed a similar effect on the Gln223Arg polymorphism.

In conclusion, in the present invention it was confirmed that the interaction between the LEPR and ER gene is an important factor for determining the maximum bone mass. Variants of the leptin receptor gene may be used as targets for the development of tailored therapeutic interventions for the regulation of bone mass in the future. In particular, the present invention shows that the Gln223Arg polymorphism of the LEPR gene is associated with BMD in overweight young men and this association depends on the Pvu II and Xba I genotypes of ER.

The present invention also provides a diagnostic kit for predicting the genetic risk factors of osteoporosis using the method for measuring the polymorphism of the gene involved in osteoporosis.

Diagnostic kits for the genetic risk factors of osteoporosis of the invention include primer sets, molecular weight markers, amplification enzymes, nucleotides and buffers that can amplify the polymorphic sites of genes involved in osteoporosis.

In the above, the gene involved in osteoporosis may be selected from the group consisting of LEPR, ER, Vitamin D receptor (VDR), and Transforming growth factor-β1 (TGF-β1), preferably LEPR or ER, LEPR More preferably.

The diagnostic kit of the present invention analyzes the polymorphism of the gene, and for this purpose comprises a container containing primers capable of amplifying DNA or RNA. It may also include molecular weight markers consisting of standard allele DNA, and additional components of amplification reactions such as buffers, amplification enzymes, and nucleotides may be added to create suitable environments for PCR amplification. The subject DNA in the present invention may be used a site capable of measuring the polymorphism of the LEPR or ER gene. In the diagnostic kit of the present invention, primers capable of selectively amplifying the polymorphic sites used for the test are used.

Standard allelic DNAs included as molecular weight markers include those obtained by PCR amplification of genomic DNA having each allele, or by cleavage or PCR of cloned alleles with restriction enzymes or the preparation and use of artificial nucleic acids.

In addition, the diagnostic kit of the present invention may be used to attach a signaling material to the primer, and the signaling material includes one of deoxygenin, biotin, radioisotope, fluorescent material, enzyme, and antibody. It may also include antigenic proteins for antibodies, suitable buffers, standard antibodies, secondary antibodies labeled with a chromophore or fluorophore, chromogenic substrates, DNA restriction enzymes and the like. At this time, the enzyme may be used peroxidase (peroxidase), alkaline phosphatase (alkaline phosphatase), luciferase (luciferase) and the like, the fluorescent material may be used FITC, TRITC, etc., the coloring agent 4CN (4- chloro-1-naphtol), diaminobenzidine (DAB), aminoethyl carbazole (AEC), ABTS [2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], OPD (o-phenylenediamine) or TMB (tetramethyl) benzidine) and the like.

The kit of the present invention can be used to specifically amplify the polymorphic sites of the genes involved in osteoporosis, after which DNA sequencing, PCR-SSCP analysis, RFLP analysis, allyl specific hybridization method, DNA chip method, etc. Polymorphism can be determined using.

The DNA sequencing method includes the Maxam-Gilbert method and the Sanger (didioxy) method. Currently, the didioxy method is mainly used. After amplifying a region to be analyzed for human genes by PCR, a sequence is determined using a primer used in PCR or a primer set in amplified DNA, and a gene sequence within the region is determined. This manipulation can be performed using other sample DNA to determine the polymorphism of the gene.

The PCR-SSCP method amplifies the region to be analyzed in the human gene by PCR, and then heat-denatures it into a row of chains, which is subjected to electrophoresis in an unmodified polyacrylamide gel. Double-stranded chains Form secondary structures (hydrogen bonds in molecules) in each chain. Since the secondary structure (hydrogen bond in the molecule) is different according to the difference of the monobasic arrangement, the polymorphism of the gene can be determined according to the difference of electrophoretic distance.

In the allyl specific hybridization method, the region to be analyzed is amplified by PCR, and then a PCR product or oligonucleotide probe of about 20 bases is formed in the region of the membrane (nylon filter), and the radioisotope ³² P, etc. It is to hybridize the sample DNA (detected DNA) shown by the following. The polymorphism of a gene can be determined by adjusting hybridization conditions, such as temperature in that case.

In principle, the DNA chip method is almost the same as the allyl specific hybridization method, but the PCR product or oligonucleotide probe of about 20 bases is arranged in a stationary phase (fluorescence). Sample DNA (detected DNA) is hybridized. By controlling hybridization conditions such as temperature, polymorphism of human genes can be determined by the difference in fluorescence intensity.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Selection of a Test Subject and Statistical Analysis

The subjects of the study were 158 healthy young men who were students of the University of Ulsan College of Medicine or majored in University Hospital (Seoul Central Hospital, AMC), all of whom volunteered for this study. The study was approved by the AMC Ethic Review Committee and received written informed consent from all participants. All subjects wrote questions about demographic characteristics, general health status, duration and severity of smoking, drinking habits, milk product intake and drug treatment. Since all subjects lived in dormitories and consumed the same menus offered at restaurants every meal, they assumed that they consumed similar amounts of calcium. The amount of elemental calcium provided in restaurants was estimated to be 480 mg per day, in addition to the total calcium intake that the individual consumed from milk products. Of the 158 subjects, one who received the finasteride drug for the treatment of alopecia was excluded. All others had no drugs and diseases that affected bone metabolism.

Descriptive characteristics are expressed as mean and standard deviation. Genotype distribution of the polymorphism was tested by Hardy-Weinberg equilibrium by χ ² test. Statistical differences in BMD between groups were assessed by covariance analysis to adjust confounding factors, and p values below 0.05 were considered significant. The statistical method of the present invention used the SPSS 10.0 package (SPSS Inc., Chicago, IL, USA).

Example 2 Measurement of Leptin Concentration and Bone Mineral Density

We collected venous blood from subjects who had not consumed food for one day using EDTA tubes and normal tubes and immediately centrifuged to separate serum and store at −76 ° C. Serum leptin concentrations were measured using a commercially available RIA kit (Mediagnost, Aspenhaustrasse, Reutlingen, Germany). In addition, BMD is a dual-energy X-ray absorptiometry at the lumbar spine (L2-L4, AP), femoral neck, word's triangle, femoral trochanter and shaft. energy X-ray absorptiometry, Lunar, Madison, Wi, USA). The in vivo precision of the machine was 0.82% for the lumbar spine and 1.12% for the femur neck.

As a result, the clinical characteristics and serum leptin concentration of the studied subjects are shown in Table 1 below.

Correlation between Clinical Characteristics and BMD in Subjects Using Multiple Linear Regression factor Average (SD) Correlation with Lumbar BMD Correlation with Femoral Neck BMD β t p β t p BMI (㎏ / m ² ) 22.9 (2.6) 0.312 2.553 0.012 0.077 0.679 0.498 Calcium Intake (mg / day) 571 (67) -0.086 -1.056 0.293 0.073 0.961 0.338 Degree of smoking (the former / year) 1.69 (3.25) 0.042 0.459 0.647 -0.033 -0.379 0.705 Alcohol level (g / day) 12.7 (24.0) 0.056 0.695 0.483 0.087 1.160 0.248 Exercise time (hours / day) 1.6 (1.4) 0.009 0.038 0.970 -0.221 1.471 0.328 Serum Leptin Concentration (ng / ml) 4.01 (2.30) -0.086 -1.056 0.293 0.007 0.108 0.321

The mean age was 25.3 years (SD 3.7, range 20-34). Multiple linear regression analysis revealed that BMD and BMI (body mass index) of lumbar spine and femur neck were related. In addition, no correlation was found between calcium intake, smoking, alcohol consumption, exercise time or serum leptin levels and BMD.

Example 3 Genotyping and Restriction Fragment Length Polymorphism Analysis Using PCR

In order to perform genotyping and RFRP analysis, we first extracted genomic DNA from peripheral blood leukocytes using a commercial kit (Wizard Genomic DNA purification kit, Promega, Madison, Wis., USA). Determination of the genotype of DNA sequence variants at the Gln223Arg, Ser492Thr, Ala976Asp and Pro1019Pro sites of the LEPR gene and the Pvu II and Xba I cleavage sites of the ER gene was performed using PCR-specific pairs of previously described oligonucleotide primers. By restriction enzyme analysis of DNA amplified by (Matsuoka N, Ogawa Y, Hosoda K, Matsuda J, Masuzaki H, Miyawaki T, et al., Diabetologia , 1997 , 40, 1204-1210; Kobayahi S, Inoue S , Hosoi T, Ouchi Y, Shiraki M, Orimo H, J. Bone Miner.Res . , 1996 , 11, 306-311). Amplification of the LEPR and ER genes was performed using thermocyclers (thermocycler, Perin-Elmer, Boston, Mass., USA) with a slight modification of previously reported methods (MatsuokaN, Ogawa Y, Hosoda K, Matsuda J, Masuzaki H). , Miyawaki T, et al., Diabetologia , 1997 , 40, 1204-1210; Kobayahi S, Inoue S, Hosoi T, Ouchi Y, Shiraki M, Orimo H, J. Bone Miner.Res . , 1996 , 11, 306- 311). Specifically, in order to investigate the Gln223Arg, Ser492Thr, Ala976Asp, and Pro1019Pro polymorphisms of the LEPR gene, the present inventors identified PCR products with restriction enzymes Msp I, Pst I, Hae III and Hinc II (New England Biolabs Inc., Beverly, MA). , USA). For the ER gene, Pvu II and Xba I (New England Biolabs Inc., Beverly, MA, USA) restriction enzymes were used. Next, DNA fragments were electrophoresed on 3% agarose gel.

As a result, genotype distribution and allele frequency for polymorphism of LEPR and ER are shown in Table 2 .

Genotype Distribution and Allyl Frequency of Leptin Receptor and Estrogen Receptor Gene Polymorphism Leptin receptor Genotype frequency ^* Allyl frequency Gln223Arg Gln / Gln Gln / Arg Arg / Arg Gln Arg 2 (1%) 40 (26%) 115 (73%) 0.14 0.86 Ser492Thr Ser / Ser Ser / Thr Thr / Thr Ser Thr 156 (99%) 1 (1%) 0 (0%) 0.997 0.003 Ala976Asp Ala / Ala Ala / Asp Asp / Asp Ala Asp 0 (0%) 0 (0%) 157 (100%) 0 One Pro1019Pro G / G ^† G / A ^† A / A ^† G ^† A ^† 5 (3%) 26 (17%) 126 (80%) 0.11 0.89 Estrogen receptor Genotype frequency ^* Frequency PvuII P / P P / p p / p P p 30 (19%) 68 (43%) 59 (38%) 0.41 0.59 XbaI X / X X / x x / x X x 8 (5%) 57 (36%) 92 (59%) 0.23 0.77

*: The numerical value represents a number (%).

†: G and A converted at nucleotide 3057 (codon 1019) without amino acid substitution.

All polymorphisms of the LEPR and ER genes were in the Hardy-Weinberg equilibrium. The frequency of allylation for the Gln223Arg, Ser492Thr, and Pro1019Pro polymorphisms was similar to those reported previously in Japan (Matsuoka N, Ogawa Y, Hosoda K, Matsuda J, Masuzaki H, Miyawaki T, et al., Diabetologia , 1997 , 40, 1204-1210). The absence of polymorphism at Ala976Asp was also the same as reported in Japan. The allyl frequency of ER in Pvu II and Xba I polymorphisms was not different from previous cases observed in Korean women (Han Ko, Moon IG, Kang YS, Chung HY, Min HK, Han IK, J. Clin. Endocrinol. Metab. , 1997 , 82, 991-995.

The frequency of genotype combinations defined by the Pvu II and Xba I polymorphisms was PPXX, 8 (5%); PPXx, 12 (8%); PPxx, 10 (6%); PpXx, 38 (24%); Ppxx, 30 (19%); ppXx, 7 (5%); And ppxx, 52 (33%). Absence of PpXX or ppXX genotypes is consistent with previous reports (Kobayahi S, Inoue S, Hosoi T, Ouchi Y, Shiraki M, Orimo H, J. Bone Miner. Res. , 1996 , 11, 306-311; Albagha OME, Mcguigan FEA, Reid DM, Ralston SH, J. Bone Miner.Res . , 2001 , 16, 128-134). The distribution of the combination genotype indicates that the Pvu II and Xba I polymorphisms are in linkage disequilibrium (χ2 = 65.351, p <0.001), while the PPXX genotype was present five times more frequently, while PpXx And ppxx genotypes showed 1.5 times higher frequency than expected.

<Example 4> Examination of the association between the polymorphism of the LEPR gene and BMD

After correcting BMI, we analyzed the correlation between BMD and LEPR genotypes in the lumbar spine and femur, respectively, in the entire subject group, the overweight group, and the normal weight group. Specifically, Pro1019Pro polymorphism could not be observed between genotype and BMD at each site. Ser492Thr genotype and Ala976Asp genotype were difficult to compare because of small dsjan. Gln223Arg genotype was analyzed by classifying Gln223Gln and Gln223Arg as Gln223 allyl and Gln223Arg as Gln223 allyl.

As a result, the serum leptin concentration and the correlation with BMD according to the Gln223Arg genotype of LEPR are shown in Table 3 below.

Serum Leptin Concentration and BMD According to Gln223Arg Genotype of LEPR Gln223Gln, Gln223Arg Arg223Arg p ^* all n = 42 n = 115 Lumbar spine BMD (g / cm ² ) 1.243 (0.172) 1.191 (0.141) 0.023 Femur Neck BMD (g / cm ² ) 1.093 (0.157) 1.053 (0.156) 0.340 Serum Leptin Concentration (ng / ml) 3.74 (2.03) 4.11 (2.39) 0.741 Overweight ^† n = 14 n = 55 Lumbar spine BMD (g / cm ² ) 1.352 (0.148) 1.212 (0.126) 0.001 Femur Neck BMD (g / cm ² ) 1.157 (0.171) 1.049 (0.135) 0.017 Serum Leptin Concentration (ng / ml) 5.9 (2.14) 5.57 (2.46) 0.705 normal n = 28 n = 60 Lumbar spine BMD (g / cm ² ) 1.188 (0.159) 1.172 (0.152) 0.635 Femur Neck BMD (g / cm ² ) 1.061 (0.142) 1.057 (0.173) 0.800 Serum Leptin Concentration (ng / ml) 3.06 (1.63) 2.77 (1.32) 0.275

All figures represent the mean (standard deviation).

* Corrected for BMI.

† Overweight was based on the BMI 23.0 kg / m ² or more.

The inventors selected 23.0 kg / m ² as the boundary of overweight as suggested by Asian (Ko GT, Tang J, Chang JC, Sung R, Wu MM, Wai HP, et al., Br. J. Nutr. , 2001 , 85, 239-242).

As can be seen in Table 3 above, there was no correlation between BMD and Pro1019Pro polymorphism of LEPR in the lumbar spine or femur, and the frequency of genotype variants in Ser492Thr and Ala976Asp was too low to be used for analysis of association with BMD. In addition, the number of subjects representing Gln223Gln was so small (n = 2) that we compared Gln223 allyl (Gln223Gln or Gln223Arg) and non-Arg223Arg. No differences were observed between the two groups in age, BMI, calcium intake, smoking or alcohol consumption, and BMD in the lumbar spine was higher (1.243 g / cm ² ) when compared to the non-group with Gln223 allyl. [SD 0.172] vs 1.191 g / cm ² [0.141], p = 0.023). The association of Gln223Arg polymorphism with BMD was not observed in the femur neck (p = 0.340).

Further analysis of the association between BMD and GPR223Arg genotypes of LEPR according to BMI revealed that the overweight group (BMI> 23.0 kg / m ² , Ko GT, Tang J, Chang JC, Sung R, Wu MM, Wai HP, et al., Br. J. Nutr. , 2001 , 85, 239-242), the association of Gln223Arg polymorphism with BMD was observed in both lumbar and femoral neck regions (p = 0.001 and p = 0.017, respectively, Table 3). ). Subjects with Gln223 allyl showed a BMD of about 10% higher in the lumbar spine compared to subjects without Gln223 allyl (1.352 g / cm ² [0.148] vs 1.212 g / cm ² [0.126]). Associations were also observed in the femoral neck, word tripod, femoral trochanter, and interfemur (p = 0.017, 0.04, 0.027 and 0.039, respectively). However, this association did not appear in the non-overweight group. Leptin levels did not differ between subjects with and without Gln223 allyl.

Example 5 Investigation of the Relationship between Polymorphism of ER Gene and BMD

We investigated the association between the polymorphism of the ER gene and BMD. To this end, first, the association between Pvu II and Xba I genotypes of ER and BMD was examined, and the results are shown in Table 4 below.

Correlation between Pvu II and Xba I Genotypes of ER and BMD Pvu II PP (n = 30) Pp (n = 68) pp (n = 59) valence L2-4 1.233 ± 0.156 1.199 ± 0.160 1.198 ± 0.138 NS * Femur Neck 1.029 ± 0.138 1.076 ± 0.166 1.067 ± 0.154 NS Word tripod 0.946 ± 0.147 1.004 ± 0.190 1.001 ± 0.164 NS Femur Electronic Part 0.826 ± 0.161 0.854 ± 0.175 0.860 ± 0.186 NS Femur 1.328 ± 0.164 1.344 ± 0.182 1.390 ± 0.202 NS Xba I XX (n = 8) Xx (n = 57) xx (n = 92) valence L2-4 1.188 ± 0.139 1.216 ± 0.160 1.200 ± 0.148 NS Femur Neck 0.975 ± 0.117 1.085 ± 0.174 1.058 ± 0.146 NS Word tripod 0.881 ± 0.119 1.016 ± 0.179 0.992 ± 0.172 NS Femur Electronic Part 0.740 ± 0.120 0.874 ± 0.179 0.846 ± 0.176 NS Femur 1.230 ± 0.144 1.373 ± 0.177 1.360 ± 0.194 NS

* NS: not significant (p> 0.05 when using ANCOVA)

From the above results, it was confirmed that no significant difference in BMD was observed between the Pvu II and Xba I genotypes of ER.

In addition, the inventors also found that Px haplotype, as previously described (Kobayahi S, Inoue S, Hosoi T, Ouchi Y, Shiraki M, Orimo H, J. Bone Miner. Res. , 1996 , 11, 306-311) . (haplotype) and the correlation between the BMD was investigated, the results are shown in Table 5 below.

Correlation between Px Haplotype of ER and BMD Px Haplotype 11 (n = 10) 10 (n = 42) 00 (n = 67) valence L2-4 1.214 ± 0.146 1.235 ± 0.176 1.197 ± 0.136 NS * Femur Neck 1.004 ± 0.116 1.076 ± 0.155 1.056 ± 0.152 NS Word tripod 0.925 ± 0.116 0.998 ± 0.190 0.994 ± 0.164 NS Femur Electronic Part 0.811 ± 0.152 0.864 ± 0.170 0.845 ± 0.183 NS Femur 1.277 ± 0.141 1.364 ± 0.182 1.371 ± 0.202 NS

* NS: not significant (p> 0.05 when using ANCOVA)

From these results, the Px haplotype predicted BMD free in the lumbar spine as well as the femur neck.

Example 6 Investigation of the Effect of the Interaction Between LEPR and ER Genotypes on BMD

In order to examine whether there is a correlation between the LEPR and ER genes, we analyzed the difference of BMDs in the LEPR genotypes and whether the correlation of LEPR genotypes and BMDs varies according to the ER genotypes. Specifically, the correlation between LEPR genotype and BMD in each group was obtained by dividing into two groups with and without P or X allyl among ER Pvu II and Xba I polymorphisms.

As a result, the association of LEPR Gln223Arg polymorphism with lumbar BMD was evident only in subjects with P or X allyl of ER ( Table 6 and Table 7 ). Even in overweight subjects where the effect of LEPR polymorphism is more pronounced, the association disappears in the absence of P or X allyl in the ER.

BMD (g / cm ² ) according to Gln223Arg genotype of LEPR in subgroup of Pvu II genotype of ER Lumbar spine Femur Neck PvuII Genotype of the ER Gene Gln223Gln ^* Gln223ARG Arg223Arg ^* p ^† Gln223Gln ^* Gln223ARG Arg223Arg ^* p ^† all PP 1.335 (0.200, 9) 1.189 (0.111, 21) 0.011 1.076 (0.166) 1.008 (0.124) 0.242 Pp 1.223 (0.189,20) 1.189 (0.148,48) 0.309 1.118 (0.166) 1.059 (0.165) 0.546 pp 1.209 (0.099,13) 1.195 (0.148,46) 0.771 1.065 (0.143) 1.067 (0.159) 0.902 Overweight PP 1.411 (0.082, 4) 1.214 (0.097,12) 0.003 1.114 (0.119) 1.044 (0.134) 0.457 Pp 1.441 (0.198, 4) 1.202 (0.121, 25) 0.005 1.242 (0.221) 1.034 (0.122) 0.011 pp 1.252 (0.092, 6) 1.225 (0.153,18) 0.641 1.128 (0.173) 1.074 (0.156) 0.276 normal PP 1.274 (0.255, 5) 1.156 (0.127, 9) 0.402 1.046 (0.204) 0.961 (0.093) 0.160 Pp 1.169 (0.147, 16) 1.174 (0.147,23) 0.940 1.087 (0.141) 1.086 (0.200) 0.643 pp 1.172 (0.094, 7) 1.176 (0.145,28) 0.976 1.011 (0.092) 1.063 (0.163) 0.337

* The figures represent the mean (SD, number).

† was corrected with BMI.

BMD (g / cm ² ) according to Gln223Arg genotype of LEPR in subgroup of Xba I genotype of ER Lumbar spine Femur Neck XbaI genotype of the ER gene Gln223Gln ^* Gln223ARG Arg223Arg ^* p ^† Gln223Gln ^* Gln223ARG Arg223Arg ^* p ^† all XX / Xx 1.304 (0.193, 19) 1.174 (0.121,46) 0.001 1.091 (0.141) 1.063 (0.183) 0.478 xx 1.192 (0.137,23) 1.203 (0.153,69) 0.942 1.094 (0.173) 1.047 (0.135) 0.152 Overweight XX / Xx 1.447 (0.143, 5) 1.201 (0.118,22) 0.001 1.140 (0.125) 1.054 (0.170) 0.534 xx 1.298 (0.129, 9) 1.220 (0.132,33) 0.128 1.166 (0.199) 1.146 (0.109) 0.238 normal XX / Xx 1.253 (0.186,14) 1.150 (0.121, 24) 0.063 1.074 (0.146) 1.071 (0.198) 0.563 xx 1.124 (0.094,14) 1.187 (0.169,36) 0.238 1.048 (0.143) 1.047 (0.157) 0.910

* The figures represent the mean (SD, number).

† was corrected with BMI.

As discussed above, the method for predicting the risk of developing osteoporosis of the present invention and the diagnostic kit for the genetic risk factor of osteoporosis using the same are not only for postmenopausal women but also for men, especially young men who have not started osteoporosis. The risk of developing osteoporosis can be measured in a simple way, and variant allyls of the leptin receptor gene can be usefully targeted for the development of customized therapeutics for the control of bone mass in the future.

Claims (9)

1) measuring a polymorphic site of a gene involved in osteoporosis from DNA extracted from a sample; And 2) A method for predicting the risk factor of osteoporosis by measuring the polymorphism of the gene involved in osteoporosis comprising the step of obtaining a correlation with the bone mineral density from the measurement result of step 1. The method of claim 1, wherein the gene is selected from the group consisting of LEPR, ER, Vitamin D receptor (VDR), and Transforming growth factor-β1 (TGF-β1). The method of claim 2, wherein the gene is LEPR or ER. The method of claim 3, wherein the polymorphic site of LEPR is selected from the group consisting of Gln223Arg, Ser492Thr, Ala976Asp, and Pro1019Pro. The method of claim 4, wherein the site is Gln223Arg. 4. The method of claim 3, wherein the polymorphic sites of the ER are Pvu II and Xba I cleavage sites. The method of claim 1, wherein the polymorphism is measured using DNA sequencing, PCR-SSCP analysis, RFLP analysis, allyl specific hybridization method or DNA chip method. A diagnostic kit for the genetic risk factors of osteoporosis comprising a primer set, molecular weight markers, amplification enzymes, nucleotides and buffers that can amplify the polymorphic sites of genes involved in osteoporosis. The diagnostic kit of claim 8, wherein the gene is selected from the group consisting of LEPR, ER, Vitamin D receptor (VDR), and Transforming growth factor-β1 (TGF-β1).