Use of a substance for detecting the rs532223 polymorphism in the manufacture of a product for predicting the severity of pmo

Abstract

The application discloses application of a substance for detecting rs532223 polymorphism in preparation of a product for predicting PMO severity. The application also discloses application of the substance for detecting rs532223 polymorphism in any one of 1) to 5) as follows: 1) predicting or assisting in predicting bone loss degree of a PMO patient; 2) screening or assisting in screening a PMO severe patient or an individual carrying a PMO severe genotype; 3) screening or assisting in screening a PMO mild patient or an individual carrying a PMO mild genotype; 4) evaluating or assisting in evaluating PMO disease risk of a to-be-tested person; and 5) screening or assisting in screening a PMO susceptible individual or an individual carrying a PMO susceptible genotype.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the application of a substance for detecting rs532223 polymorphism in the preparation of a product for predicting the severity of PMO. BACKGROUND

[0002] Postmenopausal Osteoporosis (PMO) is the most common bone disease, and the bone density decrease and fracture caused by PMO seriously affect human health. The statistical research in 2016 shows that the PMO prevalence rate of the female population over 50 years old in China is as high as 34.65%. The symptoms of PMO are not easy to be detected in the normal state, and the patients often realize the existence of PMO after fracture, and the treatment of the patients at this time will produce great economic pressure.

[0003] A series of researches at home and abroad at present show that the occurrence and bone density decrease degree of PMO are closely related to the genetic susceptibility of individuals, and the discovered susceptible genes can be used as an important molecular marker for preventing and diagnosing diseases, evaluating the severity of diseases, and providing effective treatment methods. SUMMARY

[0004] The technical problem to be solved by the present application is how to predict the bone loss degree of PMO patients or the severity of PMO or how to evaluate the risk of PMO or how to screen PMO severe patients or PMO susceptible individuals.

[0005] In order to solve the above technical problem, the present application provides a new use of a substance for detecting the polymorphism or genotype of rs532223 site in human genome.

[0006] The present application provides the application of the substance for detecting the polymorphism or genotype of rs532223 site in human genome in the preparation of a product for predicting or assisting in predicting the bone loss degree of PMO patients or the severity of PMO.

[0007] The present application also provides the application of the substance for detecting the polymorphism or genotype of rs532223 site in human genome in predicting or assisting in predicting the bone loss degree of PMO patients or the severity of PMO.

[0008] The present application also provides the application of the substance for detecting the polymorphism or genotype of rs532223 site in human genome in the preparation of a product for screening or assisting in screening PMO severe patients or individuals carrying PMO severe genotype.

[0009] The present application also provides the application of the substance for detecting the polymorphism or genotype of rs532223 site in human genome in screening or assisting in screening PMO severe patients or individuals carrying PMO severe genotype.

[0010] The present application also provides use of a substance for detecting the polymorphism or genotype of the rs532223 site in the human genome in the preparation of a product for screening or assisting in screening PMO mild patients or individuals carrying a PMO mild genotype.

[0011] The present application also provides use of a substance for detecting the polymorphism or genotype of the rs532223 site in the human genome in screening or assisting in screening PMO mild patients or individuals carrying a PMO mild genotype.

[0012] The present application also provides use of a substance for detecting the polymorphism or genotype of the rs532223 site in the human genome in the preparation of a product for evaluating or assisting in evaluating the PMO disease risk of a testee.

[0013] The present application also provides use of a substance for detecting the polymorphism or genotype of the rs532223 site in the human genome in evaluating or assisting in evaluating the PMO disease risk of a testee.

[0014] The present application also provides use of a substance for detecting the polymorphism or genotype of the rs532223 site in the human genome in the preparation of a product for screening or assisting in screening PMO susceptible individuals or individuals carrying a PMO susceptible genotype.

[0015] The present application also provides use of a substance for detecting the polymorphism or genotype of the rs532223 site in the human genome in screening or assisting in screening PMO susceptible individuals or individuals carrying a PMO susceptible genotype.

[0016] In order to solve the above technical problems, the present application also provides a product; the function of the product is any one of the following 1)-5):

[0017] 1) predicting or assisting in predicting the bone loss degree of a PMO patient or the severity of PMO;

[0018] 2) screening or assisting in screening PMO severe patients or individuals carrying a PMO severe genotype;

[0019] 3) screening or assisting in screening PMO mild patients or individuals carrying a PMO mild genotype;

[0020] 4) evaluating or assisting in evaluating the PMO disease risk of a testee;

[0021] 5) screening or assisting in screening PMO susceptible individuals or individuals carrying a PMO susceptible genotype.

[0022] The product provided by the present application comprises a substance for detecting the polymorphism or genotype of the rs532223 site in the human genome.

[0023] In any of the above-mentioned uses or products, the degree of bone loss can be predicted by detecting the genotype of the rs532223 locus in the genomic DNA of the PMO patient, and judging the degree of bone loss of the PMO patient according to the genotype of the rs532223 locus: the degree of bone loss of the PMO patient with AA genotype of the rs532223 locus is more severe than that of the PMO patient with GA genotype of the rs532223 locus, and the degree of bone loss of the PMO patient with GA genotype of the rs532223 locus is more severe than that of the PMO patient with GG genotype of the rs532223 locus.

[0024] The severity of PMO can be predicted by detecting the genotype of the rs532223 locus in the genomic DNA of the PMO patient, and judging the severity of PMO of the PMO patient according to the genotype of the rs532223 locus: the severity of PMO of the PMO patient with AA genotype of the rs532223 locus is higher than that of the PMO patient with GA genotype of the rs532223 locus, and the severity of PMO of the PMO patient with GA genotype of the rs532223 locus is higher than that of the PMO patient with GG genotype of the rs532223 locus.

[0025] The risk of PMO can be evaluated by detecting the genotype of the rs532223 locus in the genomic DNA of the subject to be tested, and evaluating the risk of PMO of the subject to be tested according to the genotype of the rs532223 locus: the risk of PMO of the subject to be tested with AA genotype or GA genotype of the rs532223 locus is higher than that of the subject to be tested with GG genotype of the rs532223 locus.

[0026] The PMO severe patient is a patient carrying a PMO severe genotype. The PMO severe genotype is AA genotype; the AA genotype is a homozygote of A at the rs532223 locus.

[0027] The PMO mild patient is a patient carrying a PMO mild genotype. The PMO mild genotype is GG genotype; the GG genotype is a homozygote of G at the rs532223 locus.

[0028] The PMO susceptible individual is an individual carrying a PMO susceptible genotype. The PMO susceptible genotype is AA genotype or GA genotype; the AA genotype is a homozygote of A at the rs532223 locus; and the GA genotype is a heterozygote of G and A at the rs532223 locus.

[0029] In any of the above-mentioned uses or products, the means for detecting the polymorphism or genotype of the rs532223 site in the human genome comprises reagents and / or instruments for detecting the polymorphism or genotype of the rs532223 site in the human genome; the reagents and / or instruments for detecting the polymorphism or genotype of the rs532223 site in the human genome can comprise reagents and / or instruments required for determining the polymorphism or genotype of the rs532223 site by at least one of the following methods: DNA sequencing, restriction enzyme digestion fragment length polymorphism, single strand conformation polymorphism, denaturing high performance liquid chromatography, and SNP chip. The SNP chip comprises a chip based on nucleic acid hybridization reaction, a chip based on single base extension reaction, a chip based on allele-specific primer extension reaction, a chip based on "one-step" reaction, a chip based on primer ligation reaction, a chip based on restriction enzyme reaction, a chip based on protein-DNA binding reaction, and a chip based on fluorescence molecule-DNA binding reaction.

[0030] Further, the reagents for detecting the polymorphism or genotype of the rs532223 site in the human genome comprise a specific primer pair for amplifying the rs532223 site and / or a single base extension primer. The specific primer pair for amplifying the rs532223 site has no special requirements in sequence, as long as it can amplify a human genomic DNA fragment containing the rs532223 site. The single base extension primer can be designed according to the sequence upstream or downstream of the rs532223 site in the human genome (excluding the SNP site). The 3' terminal nucleotide of the single base extension primer corresponds to the adjacent nucleotide of the rs532223 site in the human genome (i.e. the nucleotide before the rs532223 site or the nucleotide after the rs532223 site).

[0031] The instruments for detecting the polymorphism or genotype of the rs532223 site in the human genome comprise one or more of the following instruments: a PCR instrument, a DNA sequencer, and a mass spectrometer.

[0032] Further, the specific primer pair consists of a single-stranded DNA molecule represented by SEQ ID NO: 1 and a single-stranded DNA molecule represented by SEQ ID NO: 2.

[0033] The single base extension primer is a single-stranded DNA molecule represented by SEQ ID NO: 3.

[0034] In any of the above-mentioned uses or products, the product can be a reagent or a kit or a system, and the system can comprise a combination of reagents or kits and instruments, such as a product consisting of a primer pair for PCR amplification of the rs532223 site, a single base extension primer, and a mass spectrometer, or a product consisting of a primer pair for PCR amplification of the rs532223 site, a single base extension primer, and a DNA sequencer.

[0035] In practical applications, in order to improve the accuracy, the substance for detecting the polymorphism or genotype of rs532223 site can be combined with other substances (such as a substance for detecting other single nucleotide polymorphisms or genotypes related to PMO) to prepare a product for screening individuals susceptible to PMO.

[0036] In any of the above applications or products, the product is suitable for women, and in particular, can be suitable for Chinese Han women.

[0037] In any of the above applications or products, the rs532223 site is a deoxyribonucleotide at position 7016792 of chromosome 6 of the human genome.

[0038] In any of the above applications or products, the subject is a woman, and in particular, a Chinese Han woman.

[0039] In any of the above applications or products, the PMO patient is a postmenopausal osteoporosis patient.

[0040] The present application takes two groups of unrelated female PMO patients of Chinese Han descent as the research object to conduct experiments and verification, and finds that the genotype of rs532223 site is significantly related to the bone density reduction level of the PMO patient group, and after linear regression analysis and correction of age and body mass index, it is proved that the rs532223 polymorphism site is a risk factor for the severity of PMO patients (bone density T-score decreases, BETA = -0.69, p = 5.65 x 10 -6 ). The present application first discovers that the rs532223 site is a single nucleotide polymorphism site related to the severity of PMO, the bone loss degree of PMO patients with AA genotype at rs532223 site is more severe than that of PMO patients with GA genotype at rs532223 site, and the bone loss degree of PMO patients with GA genotype at rs532223 site is more severe than that of PMO patients with GG genotype at rs532223 site. The substance for detecting the polymorphism or genotype of rs532223 site can be used for screening individuals susceptible to PMO, predicting the severity of PMO, screening individuals carrying severe genotypes of PMO, and evaluating the risk of PMO. DETAILED DESCRIPTION

[0041] The present application will be further described in detail below in conjunction with the specific embodiments, and the examples given are only for illustrating the present application, but not for limiting the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application in any way.

[0042] The experimental methods in the following examples are all conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0043] Example 1, establishment of a method for detecting the genotype of the rs532223 site

[0044] The rs532223 site in the present application is located at position 7016792 of chromosome 6 of the human genome (reference human reference sequence version 38, GRCh38), and the polymorphism of the site is G / A, and there are three genotypes as follows: GG, GA or AA. The method for detecting the genotype of the rs532223 site is as follows:

[0045] 1. Extraction of genomic DNA

[0046] The genomic DNA of the sample to be tested is extracted, and it is used as a template for genotype detection.

[0047] 2. Design and synthesis of primers

[0048] The specific primer pair (consisting of a forward primer and a reverse primer) for PCR amplification of the rs532223 site and the single base extension primer for single base amplification reaction are designed using Sequenom Genotyping Tools and MassARRAY Assay Design software, and are synthesized by a biological company. The specific sequences of the primers are as follows:

[0049] Forward primer: 5'-ACGTTGGATGCCTCTATCCATTAGTGGGTC-3' (Sequence 1);

[0050] Reverse primer: 5'-ACGTTGGATGCTGATTATCATGTGTGCGAC-3' (Sequence 2);

[0051] Single base extension primer: 5'-GGGTCTTTTCCCTATGC-3'.

[0052] 3. Genotype detection of the rs532223 site

[0053] 1) PCR amplification

[0054] The PCR reaction system is prepared according to Table 1, and the PCR amplification is performed according to the following program: 94℃ for 4 minutes; 94℃ for 20 seconds, 56℃ for 30 seconds, 72℃ for 1 minute, 45 cycles; 72℃ for 3 minutes; 4℃ holding. The PCR product is obtained after the PCR amplification is completed.

[0055] Table 1, components of each PCR reaction system

[0056]

[0057] 2) PCR product alkaline phosphatase treatment

[0058] After the PCR reaction, the PCR product obtained in 1) above was treated with SAP (shrimp alkaline phosphatase) to remove free dNTPs in the system, to obtain an alkaline phosphatase-treated product.

[0059] The SAP reaction system is shown in Table 2.

[0060] The SAP reaction conditions were as follows: 37°C for 40 minutes; 85°C for 5 minutes; 4°C for maintenance.

[0061] Table 2, SAP reaction system

[0062]

[0063] 3) Single base extension

[0064] After the alkaline phosphatase treatment, a single base extension reaction was performed, to obtain a single base extension product.

[0065] The single base extension reaction system is shown in Table 3.

[0066] The single base extension reaction conditions were as follows:

[0067] I. 94°C for 30 seconds;

[0068] II. 94°C for 5 seconds;

[0069] III. 52°C for 5 seconds;

[0070] IV. 80°C for 5 seconds;

[0071] V. Return to III, 4 cycles;

[0072] VI. Return to II, 39 cycles;

[0073] VII. 72°C for 3 minutes;

[0074] VII. 4°C.

[0075] Table 3, single base extension reaction system

[0076]

[0077] 4) Resin purification

[0078] The Clean Resin resin (Sequenom, USA) was spread into a 6 mg resin plate; then 16 μL water was added to the corresponding hole of the single base extension product obtained in the above 3); the dried resin was poured into the single base extension product plate, sealed, and vertically rotated at low speed for 30 minutes to allow the resin to fully contact the single base extension product; finally, the resin was centrifuged to the bottom of the hole to obtain the resin-purified extension product.

[0079] 5) Chip spotting

[0080] The MassARRAY Nanodispenser RS1000 spotting instrument (SEQUENOM company) was started, and the resin-purified extension product was moved to a 384-well SpectroCHIP (Sequenom) chip (SEQUENOM company).

[0081] 6) Mass spectrometry detection

[0082] The SpectroCHIP chip after spotting was detected by MALDI-TOF, and the detection results were typed and output by TYPER 4.0 software (sequenom), to determine the genotype of the rs532223 site in the sample to be tested.

[0083] Example 2, correlation analysis of rs532223 site genotype and PMO severity

[0084] I. Research subjects

[0085] The research subjects were peripheral blood samples of the first group of 150 PMO patients and the second group of 191 PMO patients obtained by Beijing Jishuitan Hospital from September 2019 to April 2021. All PMO patients underwent qCT (quantitative CT) examination, and the average bone mineral density q value of the lumbar vertebral cancellous bone of all 341 patients was <80, meeting the diagnostic criteria for osteoporosis. All PMO patients had no autoimmune-related diseases (such as vitiligo, psoriasis, type I diabetes, etc.) and tumors. All PMO patients were Chinese female Han people who were not related by blood, with an age range of 54-92 years old and an average age of 68.24±8.09 years old, of which 2% of PMO patients had a history of smoking, 0.3% of PMO patients had a history of drinking, 47.2% of PMO patients had hypertension, and 20% of PMO patients had type 2 diabetes. Each of the above research subjects signed an informed consent form, and this study was approved by the Beijing Jishuitan Hospital Medical Ethics Committee.

[0086] II. Correlation analysis of rs532223 site genotype and PMO disease risk and severity

[0087] 1. Correlation analysis of rs532223 locus genotype with PMO risk and severity based on the first group of PMO patients.

[0088] Following the rs532223 locus genotyping method established in Example 1, genomic DNA was extracted from peripheral blood samples of 150 PMO patients in the first group for PCR amplification, single-base extension reaction, and genotyping to determine the rs532223 locus genotype of PMO patients' DNA. Then, a correlation analysis was performed between the rs532223 locus genotype and the risk and severity of PMO. All experimental data were processed and statistically analyzed using R 4.0 software. In the correlation analysis between the rs532223 locus genotype and the risk of PMO, the normal population bone mineral density reference database (“normal population bone mineral density reference database” is described in the literature “Chinese population quantitative CT vertebral bone mineral density normal reference database and correlation with BMI and age: a cohort study based on Chinese healthy quantitative CT big data”) was used as the benchmark.

[0089] In the first group of 150 PMO patients, 106 individuals had the AA genotype at the rs532223 locus, 39 individuals had the GA genotype at the rs532223 locus, and 5 individuals had the GG genotype at the rs532223 locus. After adjustment by logistic regression analysis, the significance p-value for the variation at the rs532223 locus compared to the population was 5.65 × 10⁻⁶. -6 The Beta (contribution of the mutation phenotype) was -0.69, with a 95% confidence interval of 0.40–0.98. These results indicate that the rs532223 locus is a single nucleotide polymorphism associated with the risk of PMO, and individuals with the AA and GA genotypes at the rs532223 locus have a higher risk of PMO than individuals with the GG genotype at the rs532223 locus.

[0090] The genotype frequency distribution and mean T-score of the rs532223 locus in the DNA of the first group of 150 PMO patients are shown in Table 4. The results showed that among the 150 PMO patients, 106 individuals had the AA genotype at the rs532223 locus, with a genotype frequency of 70.7% and a mean T-score of -4.11; 39 individuals had the GA genotype at the rs532223 locus, with a genotype frequency of 26% and a mean T-score of -3.38; and 5 individuals had the GG genotype at the rs532223 locus, with a genotype frequency of 3.3% and a mean T-score of -3.01. The above results indicate that the rs532223 site is a single nucleotide polymorphism site associated with the severity of PMO. PMO patients with the AA genotype at the rs532223 site have more severe bone loss than PMO patients with the GA genotype at the rs532223 site, and PMO patients with the GA genotype at the rs532223 site have more severe bone loss than PMO patients with the GG genotype at the rs532223 site.

[0091] Table 4. Genotype frequency and severity score of rs532223 polymorphism locus in osteoporosis patients.

[0092] PMO patients (n=150) Mean T-score AA 106(70.7％) -4.11 GA 39(26％) -3.38 GG 5(3.3％) -3.01

[0093] Note: The mean T-score represents the average T-score of all patients in this group, where T-score is the q value measured by qCT.

[0094] 2. Correlation analysis of rs532223 locus genotype with PMO risk and severity based on the second group of PMO patients.

[0095] To verify the correlation between the rs532223 locus genotype and the risk and severity of PMO, a second group of 191 PMO patients was used as a sample for further correlation analysis of the rs532223 locus genotype and the risk and severity of PMO, following the same principles and methods. Data were processed and analyzed using R 4.0.1 statistical software. The bone mineral density reference database for the normal population (“Bone mineral density reference database for the normal population” is described in the literature “Normal Reference Database of Vertebral Bone Mineral ...

[0096] In the second group of 191 PMO patients, 141 individuals had the AA genotype at the rs532223 locus, 47 individuals had the GA genotype at the rs532223 locus, and 3 individuals had the GG genotype at the rs532223 locus. After adjustment by logistic regression analysis, the significance p-value of the rs532223 locus variation with the population was 0.033, the Beta (contribution of the mutation phenotype) was 0.32, and the 95% confidence interval was 0.028–0.613. These results indicate that the rs532223 locus is a single nucleotide polymorphism associated with the risk of PMO, consistent with the results obtained in step 1.

[0097] The genotype frequency distribution and mean T-score of the rs532223 locus in the DNA of the second group of 191 PMO patients are shown in Table 5. The results showed that among the 191 PMO patients, 141 individuals had the AA genotype at the rs532223 locus, with a genotype frequency of 73.83% and a mean T-score of -3.68; 47 individuals had the GA genotype at the rs532223 locus, with a genotype frequency of 24.6% and a mean T-score of -3.39; and 3 individuals had the GG genotype at the rs532223 locus, with a genotype frequency of 1.57% and a mean T-score of -2.96. The above results indicate that the rs532223 locus genotype is also significantly associated with the degree of bone loss in this population, consistent with the results obtained in step 1.

[0098] Table 5. Genotype frequencies and severity scores of the rs532223 polymorphism locus in osteoporosis patients.

[0099]

[0100]

[0101] Note: The mean T-score represents the average T-score of all patients in this group, where T-score is the q value measured by qCT.

[0102] In summary, the rs532223 locus is a risk locus associated with the risk and severity of PMO, and can be used to screen individuals with severe PMO, predict the degree of bone loss in PMO patients, screen individuals carrying PMO susceptibility genotypes, and evaluate the risk of PMO.

[0103] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The application of substances that detect rs532223 site polymorphisms or genotypes in the human genome in the preparation of products that predict or assist in predicting the degree of bone loss or the severity of PMO in patients.

2. Application of substances that detect rs532223 polymorphisms or genotypes in the human genome in the preparation of products for screening or assisting in the screening of patients with severe PMO or individuals carrying the severe PMO genotype.

3. Application of substances that detect rs532223 polymorphisms or genotypes in the human genome in the preparation of products for screening or assisting in the screening of patients with mild PMO or individuals carrying the mild PMO genotype.

4. The application according to any one of claims 1-3, characterized in that: The substance used to detect the rs532223 site polymorphism or genotype in the human genome is a reagent and / or instrument for detecting the rs532223 site polymorphism or genotype in the human genome.

5. The application according to claim 4, characterized in that: The reagents for detecting polymorphism or genotype at the rs532223 locus in the human genome include specific primer pairs and / or single-base extension primers for amplifying the rs532223 locus.

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