WO2023068406A1 - Method for providing information for predicting onset of osteoporosis and composition for predicting onset of osteoporosis - Google Patents

Abstract

The present invention relates to a method for providing information for predicting the onset of osteoporosis and a composition for predicting the onset of osteoporosis, by which the possibility of the onset of osteoporosis may be predicted in an accurate and simple manner by measuring the expression level of a vitamin D binding protein, and thus the onset of osteoporosis may be prevented.

Description

Method for providing information for prediction of osteoporosis and composition for predicting osteoporosis

The present invention relates to a method for providing information for predicting the onset of osteoporosis and a composition for predicting the onset of osteoporosis.

BACKGROUND OF THE INVENTION Osteoporosis is one of skeletal diseases in which bones are easily fractured due to weakened strength, and is a state in which minerals (particularly calcium) and substrate constituting bones are reduced. Even if the size and volume of the bone is the same, the mass of the bone itself is very small, and the bone has many small holes like a sponge, making it soft and easily broken.

In addition, fractures caused by osteoporosis or the like generally do not end once, and there is a high possibility of additional fractures.

Accordingly, in order to predict the possibility of osteoporosis in advance, technology using bone regeneration markers has been developed in the past, but osteoporosis does not necessarily occur according to bone regeneration markers, and since there is great variation among individuals, accuracy is low and osteoporosis can be diagnosed only with bone regeneration markers. There is a problem that is difficult to determine whether

Accordingly, there is a need for a method capable of predicting the possibility of developing osteoporosis simply and accurately.

The present invention provides a method for providing information for predicting the onset of osteoporosis.

The present invention provides a composition for predicting the onset of osteoporosis.

1. A method for providing information for predicting the onset of osteoporosis, comprising measuring the expression level of vitamin D binding protein (VDBP) mRNA or the protein in a sample isolated from the subject.

2. In the above 1, if the expression level is lower than the control group, the individual is determined to have a higher possibility of developing osteoporosis than the control group, the information providing method for predicting the onset of osteoporosis.

3. The method of providing information for predicting the onset of osteoporosis in the above 1, wherein the sample is serum.

4. A composition for predicting the onset of osteoporosis comprising a substance specifically binding to vitamin D binding protein (VDBP) mRNA or the protein.

5. The composition for predicting the onset of osteoporosis according to 4 above, wherein the substance is at least one selected from the group consisting of antibodies, aptamers, DNA, RNA, proteins, and polypeptides.

6. A kit for predicting the onset of osteoporosis comprising the composition of 4 or 5 above.

The present invention relates to a method for providing information for predicting the onset of osteoporosis and a composition for predicting the onset of osteoporosis, wherein the possibility of the onset of osteoporosis can be accurately and simply predicted by measuring the expression level of a vitamin D-binding protein, thereby preventing the onset of osteoporosis. .

1 is a comparison of (A) total 25(OH)D (B) bioavailable 25(OH)D (C) free 25(OH)D and (D) VDBP concentrations between a patient suspected of osteoporosis and a control group.

Hereinafter, the present invention will be described in detail.

The present invention provides an information providing method for predicting the onset of osteoporosis.

The present invention includes measuring the expression level of vitamin D binding protein (VDBP) mRNA or protein in a sample isolated from a subject.

The subject may be an animal having experience with osteoporosis, an animal suspected of having osteoporosis, or an animal for which information for predicting the onset of osteoporosis is desired, and the animal may be a mammal including a human.

A sample is isolated from an individual, and examples thereof may include tissue, cells, whole blood, plasma, serum, or blood, preferably serum, but are not limited thereto.

VDBP is present in a sample derived from an individual, and is a vitamin D binding protein of the individual. For example, in the case of humans, it may be a protein consisting of the amino acid sequence of SEQ ID NO: 1.

The measurement may be performed by treating the sample with a substance that detects VDBP.

The material is not limited as long as it can detect VDBP mRNA or its protein, but may be at least one selected from the group consisting of antibodies, aptamers, DNA, RNA, proteins, and polypeptides.

As used herein, "antibody" refers to a protein molecule specific for an antigenic site. For purposes of this application, an antibody refers to an antibody that specifically binds to the marker protein, VDBP, and may include both monoclonal antibodies, polyclonal antibodies and recombinant antibodies.

The monoclonal antibody may be prepared using a hybridoma method well known in the art or a phage antibody library technology, but may not be limited thereto.

The polyclonal antibody may be produced by a method well known in the art, including injecting the protein antigen into an animal and collecting blood from the animal to obtain antibody-containing serum. Such polyclonal antibodies may be produced from any host of animal species such as goat, rabbit, sheep, monkey, horse, pig, cow, dog, etc., but may not be limited thereto.

In addition, the antibodies of the present application may also include special antibodies such as chimeric antibodies and humanized antibodies.

The "peptide" has the advantage of high binding ability to a target substance, and does not undergo denaturation even when subjected to heat/chemical treatment. In addition, because of its small molecular size, it can be attached to other proteins and used as a fusion protein. Specifically, since it can be used by attaching it to a polymer protein chain, it can be used as a diagnostic kit and a drug delivery material.

The "aptamer" is composed of a special kind of single-stranded nucleic acid (DNA, RNA or modified nucleic acid) that has a stable tertiary structure and can bind to a target molecule with high affinity and specificity. A type of polynucleotide. As described above, aptamers can specifically bind to antigenic substances in the same way as antibodies, but are more stable than proteins, have a simple structure, and are composed of polynucleotides that are easy to synthesize, so they can be used instead of antibodies. can

Substances that specifically bind to the mRNA may be sense and antisense primers or probes, but are not limited thereto.

In the present invention, "primer" is a short gene sequence serving as the starting point of DNA synthesis, and refers to an oligonucleotide synthesized for the purpose of diagnosis, DNA sequencing, and the like. The primers may be synthesized and used in a length of 15 to 30 base pairs, but may vary depending on the purpose of use, and may be modified by methylation, capping, or the like by a known method.

In the present invention, "probe" refers to a nucleic acid that can specifically bind to an mRNA having a length of several to several hundred bases prepared through enzymatic chemical separation and purification or synthesis. The presence or absence of mRNA can be confirmed by labeling a radioactive isotope or an enzyme, and it can be designed and modified using a known method.

Since the nucleotide sequence of the gene encoding the VDBP, the sequence complementary to the nucleotide sequence, or the probe or primer that specifically binds to the fragment of the nucleotide is known, the nucleotide sequence of the gene encoding the VDBP is known, so those skilled in the art can use the sequence Based on this, the primer or probe can be designed according to a conventional method in the art.

As a method for measuring the concentration of the mRNA in the sample, reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (Competitive RT-PCR), real-time reverse transcriptase polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA; RNase protection assay), Northern blotting, and DNA chips, but are not limited thereto.

As a method of measuring the concentration of the protein in a sample, the amount of the protein can be confirmed using an antibody that specifically binds to the protein. Analysis methods for this include immuno-rubbing test, sandwich assay, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, and Ouchterlony immunodiffusion method. , rocket immunoelectrophoresis, immunohistochemistry, immunoprecipitation assay, complement fixation assay, fluorescence-activated cell sorting (FACS), western blotting, fluid cytometry ( flow cytometry), enzyme substrate coloring method, antigen-antibody aggregation method, protein chip, etc., preferably ELISA (enzyme linked immunosorbent assay) can be used, but is not limited thereto.

The present invention may further include comparing the expression level with that of a control group.

For example, by comparing the expression level of VDBP mRNA or its protein in a sample of a patient suspected of osteoporosis with the expression level of a control group (unsuspected patient), if the VDBP expression level of the patient suspected of osteoporosis is lower than that of the control group, the control group It can be judged that the possibility of developing osteoporosis is higher than that of In addition, when the expression level of the VDBP mRNA or its protein has no statistically significant difference (ex. p<0.05) or is measured high compared to the control expression level, it is possible to provide information for determining that the possibility of osteoporosis is low. there is.

In addition, by comparing the expression level of VDBP mRNA or its protein in the samples of two patients suspected of osteoporosis, it can be determined that a patient with a lower expression level has a higher possibility of developing osteoporosis than a patient with a higher expression level.

The present invention provides a composition for predicting the onset of osteoporosis.

The present invention includes a substance specifically binding to vitamin D binding protein (VDBP) mRNA or the protein.

The material is not limited as long as it can detect VDBP mRNA or its protein, but may be at least one selected from the group consisting of antibodies, aptamers, DNA, RNA, proteins, and polypeptides.

Details of the material are as described above.

The present invention provides a kit for predicting the onset of osteoporosis comprising the composition.

The kit includes not only a material that specifically binds to VDBP mRNA or protein thereof, but also one or more other components suitable for the analysis method for measuring the expression level of VDBP mRNA or protein used in the kit, including a composition, solution or device. can include

When the kit is a kit for measuring the expression level of VDBP mRNA or protein, it may be a kit including essential elements necessary for performing RT-PCR. The RT-PCR kit contains, in addition to each pair of primers specific for the mRNA of the marker gene, a test tube or other suitable container, reaction buffer, deoxyribonucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC-water, sterile water, and the like. In addition, a pair of "primer" specific to a gene used as a quantitative control may be included.

The kit includes a substrate, a suitable buffer for immunological detection of a nucleotide sequence of a gene encoding VDBP, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide or a substance that specifically binds to a protein encoded by the nucleotide sequence It may include a solution, a chromogenic enzyme or a fluorescently labeled secondary antibody, or a chromogenic substrate. As the substrate, a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, and glass slide glass may be used, and the coloring enzyme may be peroxidase, alkaline phosphatase ( alkaline phosphatase) may be used, FITC, RITC, etc. may be used as the fluorescent substance, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or o- Phenylenediamine (OPD), tetramethyl benzidine (TMB), and the like may be used.

The kit may be a microarray for predicting the occurrence of osteoporosis capable of measuring the expression level of VDBP mRNA. The microarray can be easily prepared by a person skilled in the art according to a method known in the art using the indicator factor, and according to one embodiment, the sequence corresponding to the mRNA of the gene encoding the VDBP protein or a fragment thereof It may be a microarray in which cDNA is attached to a substrate as a probe.

In addition, the kit of the present application includes an antibody that specifically binds to a marker component, a secondary antibody conjugate conjugated with a label that develops color by reaction with a substrate, a solution of a color-developing substrate to react with the label, a washing solution, and an enzyme. It may include a reaction stop solution and the like, and may be manufactured in a number of separate packaging or compartments containing reagent components to be used, but may not be limited thereto.

The kit of the present disclosure may include an agent capable of measuring the expression level of VDBP mRNA or protein thereof in a patient sample, as well as one or more types of compositions, solutions, or devices suitable for expression level analysis. For example, the kit may include, but is not limited to, a substrate, an appropriate buffer solution, a secondary antibody labeled with a detection label, and a chromogenic substrate for immunological detection of the antibody.

As a specific example, the kit may be a kit characterized in that it includes essential elements required to perform ELISA in order to implement various ELISA methods such as an ELISA kit and a sandwich ELISA. These ELISA kits include antibodies specific for the above proteins. The antibody has high specificity and affinity for VDBP and little cross-reactivity to other proteins, and may be a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. ELISA kits may also include antibodies specific for a control protein. Other ELISA kits may include reagents capable of detecting bound antibodies, for example, labeled secondary antibodies, chromophores, enzymes and their substrates, or other substances capable of binding to antibodies, etc. may not be limited thereto.

In addition, the kit may be a kit for implementing Western blot, immunoprecipitation assay, complement fixation assay, flow cytometry, or protein chip, and may further include additional components suitable for each assay method. Through these analysis methods, anticancer drug resistance can be diagnosed by comparing the amount of antigen-antibody complex formation.

Hereinafter, examples will be described in detail to explain the present invention in detail.

Materials and Methods

1. Subject of research

This study included 210 healthy women working in agriculture and 180 healthy women working in other occupations in Jinju City. All participants in agriculture were enrolled in the National Farmer Health Screening Program, a free government-funded program. The occupations of 180 occupations controlled were 55 housewives, 61 office workers, 41 managers, 22 self-employed, and 1 unemployed. All participants visited the Gyeongsang National University Hospital General Medical Service Center for health checkups between May and September 2019.

2. Demographic and Medical Data Collection

This study was conducted retrospectively by analyzing medical records. Menopause was defined as cessation of menstruation for more than 1 year. Women diagnosed with osteoporosis or receiving medications such as calcium or bisphosphonates were excluded. Also excluded were women with hyperthyroidism, hyperparathyroidism, rheumatic joint disease, cancer, kidney disease, long-term corticosteroid use, and women with spinal prostheses inserted due to spinal disease.

A self-diagnosis questionnaire was conducted during the health checkup at the General Health Examination Center to collect medical history and lifestyle information such as medical history, medication history, smoking status, alcohol consumption, and physical activity. Smoking was defined as smoking more than 1 cigarette per day, and drinking was defined as drinking more than once a month. Height and weight were measured using an automatic height-weight-body mass index (BMI) measuring instrument (Genix DS-102; DS Medical Genix, Korea) while standing barefoot after taking off outerwear. BMI was calculated by dividing weight (kg) by the square of height (m ² ). All body measurements are shown to one decimal place.

3. Diagnosis of osteopenia and osteoporosis

BMD (bone mineral density) was measured by dual-energy X-ray absorptiometry (Horizon CI, HOLOGIC, MA) of the lumbar spine (vertebral bodies L1-L4) and femoral neck, read according to international clinical densitometry guidelines. The instrument's accuracy was measured twice in 30 participants. The precision error of the spine was 0.007 g/cm ² , and the smallest change in the 95% confidence interval was measured as 0.019 g/cm ² . Bone mineral density was classified into normal (T-score ≥ -1.0 SD), osteopenia (T-score -1.0 to -2.5 SD) and osteoporosis (T-score ≤ - 2.5 SD).

4. Vitamin D and VDBP measurements

Each serum sample was aliquoted into two tubes and stored at 80°C until total 25(OH)D and VDBP analysis. Serum total 25(OH)D concentrations were analyzed using the Elecsys Vitamin D Total Kit with the Cobas e602 module (Roche Diagnostics, Mannheim, Germany). This electrochemiluminescence assay is based on microparticles coated with VDBP labeled with ruthenium, Vitamin D labeled with biotin and streptavidin. VDBP concentration was measured using the Human Vitamin D BP Quantikine enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol.

5. Calculation of bioavailable 25(OH)D and free 25(OH)D

Bioavailable 25(OH)D and free 25(OH)D concentrations were calculated according to Equation 1 below based on total 25(OH)D, VDBP and albumin concentrations.

[Equation 1]

6. Statistical analysis

All analyzes were performed with R version '4.0.3' and analyzed using significance level P=.05. For the data presented in Tables 1 and 2, the following analyzes were used to compare differences between groups according to distribution: Quantitative data were analyzed by analysis of variance, t test, Kruskal-Wallis test and Mann-Whitney U test (Wilcoxon rank -sum test); Qualitative data were analyzed using Chi-square test and Fisher exact test.

In the case of the data presented in Table 3, a log-linear model was constructed as a model for the frequency of the contingency table to reveal the relationship between variables. In order to fit the optimal model, a variable selection process was performed using the inverse elimination method. The shape of the optimal model is shown in Equation 2 below.

[Equation 2]

The data presented in Table 4 were obtained by performing univariate linear regression analysis considering L1-L4 BMD (g/cm ² ) and femoral neck BMD (g/cm ² ) as the results. For the data presented in Table 5, to fit the optimal model, L1-L4 BMD (g/cm ² ) and femoral neck BMD (g/cm ² ) were considered as results, and LASSO and stepAIC were used for model construction.

result

1. Demographic and Clinical Characteristics

BMI was higher in the farming group (24.7±3.8kg/m ² ) than in the control group (23.6±3.3kg/m ² ; P=.002), but there was a difference between the groups only in height (P=.000) and no difference in weight. (Table 1). There were no significant differences between the two groups in childbirth history, drinking and smoking history, or the presence of menopause (Table 1).

2. Vitamin D Biomarkers and BMD

Serum concentrations of calcium and phosphorus were not different between the two groups. However, albumin concentration was lower in the agricultural group (4.4 ± 0.2 g/dL) than in the control group (4.6 ± 0.2 g/dL; P = .000; Table 2). The albumin concentrations of both groups were within the normal reference range (3.5-5.2 g/dL).

BMD did not differ between groups at L1-L4. However, a significantly lower femoral neck BMD was observed in the agricultural group (0.9±0.2 g/cm ² ) compared to the control group (1.0±0.2 g/cm ² ; P=.012; Table 2).

The concentration of total 25(OH)D was not significantly different between the groups (P=.132; Table 2 and Figure 1). Concentrations of bioavailable 25(OH)D (12.8±3.7 vs 8.7±5.1 ng/mL) and free 25(OH)D (32.4±9.5 vs 21.2±12.5 pg/mL) were significantly higher in the agricultural group than in the control group. higher (both P=.000; Table 2 and Figure 1). In contrast, the VDBP concentration was significantly lower in the agricultural group (201.8 ± 45.0 mg/mL) than in the control group (216.0 ± 68.2 mg/mL; P = .013; Table 2 and Figure 1).

3. Relationship between menopause and osteopenia and osteoporosis in agricultural labor

A log-linear model was used to determine the association between menopause and incidence of osteopenia and osteoporosis in agricultural labor. Postmenopausal women had a 7.1-fold higher odds ratio for osteopenia than non-menopausal women (P=.000; Table 3). Agricultural labor was not associated with osteopenia (P=.684), but the odds ratio increased 4.3 times compared to the non-agricultural group in relation to osteoporosis (P<.001; Table 3).

4. Univariate linear models for general traits influencing bone health and vitamin D status

Univariate linear models showed that all factors such as age, BMI, smoking, agricultural labor, menopause, bioavailability and free 25(OH)D, VDBP, calcium and phosphate were associated with femoral neck BMD (Table 4). . Interestingly, these same factors, excluding agricultural labor, were also associated with lumbar BMD (Table 4).

5. Multivariate linear models for vitamin D biomarkers affecting femoral neck and lumbar spine (L1-L4) BMD

Coefficients of multivariate linear model analysis were used to determine which vitamin D biomarkers affect bone health and to evaluate how agricultural labor affects bone health, menopause, and bioavailable 25(OH)D. VDBP affected femoral neck bone health (P=.000, .006, .000 and .000, respectively), whereas menopause, bioavailable 25(OH)D and VDBP affected lumbar bone health (respectively P=.027, .009 and .000; Table 5).

Claims (6)

1. A method of providing information for predicting the onset of osteoporosis, comprising measuring the expression level of vitamin D binding protein (VDBP) mRNA or the protein in a sample isolated from a subject.
2. The method according to claim 1, wherein if the expression level is lower than that of the control group, it is determined that the subject has a higher possibility of developing osteoporosis than the control group.
3. The method according to claim 1, wherein the sample is serum.
4. A composition for predicting the onset of osteoporosis, comprising a substance specifically binding to vitamin D binding protein (VDBP) mRNA or the protein.
5. The composition for predicting the onset of osteoporosis according to claim 4, wherein the substance is at least one selected from the group consisting of antibodies, aptamers, DNA, RNA, proteins, and polypeptides.
6. A kit for predicting the onset of osteoporosis comprising the composition of claim 4 or 5.

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