SNP (Single Nucleotide Polymorphism) molecular marker related to susceptibility of polycystic ovarian syndrome, detection primer and application of SNP molecular marker

Abstract

The invention discloses an SNP molecular marker related to polycystic ovarian syndrome susceptibility, a detection primer and application of the SNP molecular marker and the detection primer, and belongs to the technical field of biology. The SNP molecular marker comprises an SNP (Single Nucleotide Polymorphism) site rs701848, and further comprises an SNP site rs11202592 and an SNP site rs11202607. According to the invention, the polymorphic sites of the PTEN gene are detected by adopting a # imgabs0 # multiple SNP (Single Nucleotide Polymorphism) typing technology, and the TT genotype of the rs701848 site of the PTEN gene and haplotypes T-C-C of the rs701848 site, the rs11202592 site and the rs11202607 site can increase the onset risk of the polycystic ovarian syndrome. According to the invention, the pathogenesis of the female polycystic ovarian syndrome in China can be further researched, and a basis is provided for auxiliary diagnosis and early warning of the polycystic ovarian syndrome.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a SNP molecular marker associated with polycystic ovary syndrome susceptibility, a detection primer and an application thereof. Background Art

[0002] Polycystic Ovary Syndrome (PCOS) is a common clinical complex endocrine disease that affects female fertility and metabolic health. Epidemiological surveys show that the global incidence rate is as high as 6% to 26%, and it has become one of the main factors of female infertility. PCOS patients often face problems such as irregular menstruation, infertility, hyperandrogenism, insulin resistance and metabolic syndrome. So far, the etiology and pathogenesis of PCOS have not been fully elucidated, and it has always been a hot topic and focus of research in the field of gynecological endocrinology. In recent years, with the continuous development of molecular biology, genetics and other fields, it is generally believed that PCOS is the result of the combined action of multiple genetic, environmental and endocrine factors, among which genetic factors play a vital role.

[0003] The phosphatase and tensin homologue deleted on chromosome ten (PTEN) gene is located on human chromosome 10q23.3. It is about 200,000 bp in length, including 9 exons and 8 introns. It has sequence homology with tensin and accessory proteins at its amino terminus. In recent years, the function of PTEN as a metabolic regulator has attracted great attention. Studies have shown that PTEN, as a negative regulator of the PI3K / PTEN / Akt signaling pathway, can regulate many cellular processes, including cell growth, differentiation, proliferation and invasion. More studies have shown that PTEN may be involved in the pathogenesis of a variety of reproductive endocrine diseases, suggesting that it may be a very valuable biomarker and a potential therapeutic target for related diseases. However, there is no literature report on the genetic susceptibility correlation between the PTEN gene and PCOS. Therefore, in-depth research on the correlation between the PTEN gene and the onset of PCOS is of great significance for the early diagnosis, treatment and prognosis of PCOS. Summary of the invention

[0004] The purpose of the present invention is to provide a SNP molecular marker related to the susceptibility of polycystic ovary syndrome, a detection primer and its application, so as to solve the problems existing in the above-mentioned prior art. The present invention analyzes the relationship between the PTEN gene SNPs mutation and polycystic ovary syndrome, provides the application of the reagent for detecting the relevant sites in the preparation of a kit for auxiliary diagnosis of polycystic ovary syndrome, and provides a basis for auxiliary diagnosis and early warning of polycystic ovary syndrome.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a SNP molecular marker associated with polycystic ovary syndrome susceptibility, comprising a SNP site rs701848; the physical position of the SNP site rs701848 in a genome with a version number of GRCH37 is Chr1089726745; the genotype of the SNP site rs701848 comprises CC, CT and TT genotypes.

[0007] Furthermore, it also includes SNP site rs11202592 and SNP site rs11202607; the physical positions of the SNP site rs11202592 and the SNP site rs11202607 in the genome with version number GRCH37 are Chr1089624218 and Chr1089727414 respectively; the genotype of the SNP site rs11202592 includes GG, CG and CC genotypes; the genotype of the SNP site rs11202607 includes CC, CT and TT genotypes.

[0008] The present invention also provides a primer combination for detecting the SNP molecular marker, wherein the primer combination for detecting the SNP site rs701848 includes the primer group shown in SEQ ID NOs. 1 to 2 and the primer probe group shown in SEQ ID NOs. 7 to 9; the primer combination for detecting the SNP site rs11202592 includes the primer group shown in SEQ ID NOs. 3 to 4 and the primer probe group shown in SEQ ID NOs. 10 to 12; the primer combination for detecting the SNP site rs11202607 includes the primer group shown in SEQ ID NOs. 5 to 6 and the primer probe group shown in SEQ ID NOs. 13 to 15.

[0009] The present invention also provides the use of the primer combination in preparing the detection kit for the SNP molecular marker.

[0010] The present invention also provides a detection kit for the SNP molecular marker, comprising the primer combination.

[0011] The present invention also provides the use of the primer combination or the detection kit in preparing a polycystic ovary syndrome auxiliary diagnosis kit.

[0012] Furthermore, the method for using the polycystic ovary syndrome auxiliary diagnosis kit includes performing multiple PCR amplification on the test sample using the primer combination, performing a ligation reaction after purification of the amplification product, and sequencing the ligation product after purification, and determining the genotype of the SNP site rs701848 or the haplotype of the SNP site rs701848, the SNP site rs11202592 and the SNP site rs11202607 according to the sequencing results.

[0013] Furthermore, individuals whose genotype of the SNP site rs701848 is TT have a higher risk of suffering from polycystic ovary syndrome than individuals whose genotypes are CC and CT.

[0014] Furthermore, individuals whose haplotypes of the SNP site rs701848, the SNP site rs11202592, and the SNP site rs11202607 are TCC have a higher risk of suffering from polycystic ovary syndrome than those with other haplotypes.

[0015] The present invention discloses the following technical effects:

[0016] The present invention adopts the method of case-control study, using The multiple SNP typing technology was used to detect the polymorphic sites of the PTEN gene. The plasma PTEN protein expression levels of the control group and the polycystic ovary syndrome patient group were detected by enzyme-linked immunosorbent assay. The statistical software multi-logistic and non-parametric rank sum tests were used to analyze the correlation between the PTEN site polymorphism and polycystic ovary syndrome and the protein expression. It was proved that the TT genotype of the PTEN gene rs701848 site, the dominant model (CT+TT vs.CC) and the recessive model (TT vs.CC+CT) and the haplotype TCC of the three sites of rs701848, rs11202592 and rs11202607 would increase the risk of polycystic ovary syndrome. The PTEN gene polymorphism may cause the onset of polycystic ovary syndrome by affecting the plasma PTEN protein level. The present invention will help to further study the pathogenesis of polycystic ovary syndrome in Chinese women and provide a basis for auxiliary diagnosis and early warning of polycystic ovary syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1This is the sequencing map of the rs701848 locus, where a represents the CC genotype, b represents the CT genotype, and c represents the TT genotype;

[0019] Figure 2 This is the sequencing map of the rs11202592 locus, where a represents the CC genotype, b represents the CG genotype, and c represents the GG genotype;

[0020] Figure 3 This is the sequencing map of the rs11202607 locus, where a represents the CC genotype, b represents the CT genotype, and c represents the TT genotype;

[0021] Figure 4 Linkage disequilibrium analysis diagram for rs701848, rs11202592 and rs11202607 loci;

[0022] Figure 5 This is a comparison of PTEN protein expression levels between the control group and the PCOS group;

[0023] Figure 6 This is a comparison of PTEN protein expression in different genotypes of rs701848 locus in PCOS patients;

[0024] Figure 7 This is a comparison of PTEN protein expression between the CC genotype and other genotypes at the rs701848 locus in PCOS patients. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Unless otherwise specified, the methods used in the present invention are all conventional experimental methods in the art, and the materials and reagents used can be obtained from commercial channels unless otherwise specified.

[0031] Example

[0032] 1. Research subjects

[0033] 1. Experimental subjects

[0034] Case group (PCOS group): patients with polycystic ovary syndrome diagnosed in the Reproductive Medicine Center of the Affiliated Hospital of Youjiang Medical College for Nationalities from May 2020 to May 2022 were selected. All of them were from Guangxi, aged 18-45 years, with an average age of 29.71±4.76 years. The specific inclusion criteria are as follows: According to the criteria revised by the ESHRE / ASRM Rotterdam Conference in 2003: (1) occasional ovulation and / or anovulation; (2) elevated serum androgen levels and / or clinical manifestations of hyperandrogenism (acne, hirsutism); (3) polycystic ovarian changes (ultrasound examination of at least one ovary with a diameter of 2-9 mm and a small number of follicles ≥12 or an ovarian volume ≥10 ml), and two of the above three items appeared at the same time.

[0035] Control group: Healthy women who were treated for male factors or underwent fertility assessment during the same period were selected as the control group. The inclusion criteria were: (1) age ranged from 18 to 45 years, with an average of 30.23±5.27 years; (2) regular menstrual cycle; (3) basic endocrine and metabolic indicators were within the normal range; (4) ultrasound examination showed normal ovulation, normal ovarian size and shape, and no polycystic manifestations. Women with diabetes, hypertension, cardiovascular disease, thyroid and other endocrine diseases were excluded.

[0036] All subjects were not pregnant or breastfeeding, had not taken hormonal drugs in the past three months, had not used drugs that affect blood sugar and insulin before blood collection, and had not implemented weight loss.

[0037] This invention was approved by the Ethics Committee of the Affiliated Hospital of Youjiang Medical College for Nationalities, and all research subjects gave informed consent.

[0038] 2. Determination of sample content

[0039] Quanto software was used to estimate the sample size, and the statistical power was set at 80%. Combined with the risk value of the site, the minimum allele frequency, and the inclusion and exclusion criteria, 277 cases in the case group and 236 cases in the control group were determined.

[0040] 2. Experimental Methods

[0041] 1. PTEN gene locus information

[0042] The location information of the PTEN gene was searched in the NCBI database, and the information of the PTEN gene SNP sites in the Chinese population was also searched. The screening criteria for SNP sites were as follows: (1) Searching for sites with a minor allele frequency (MAF) greater than 5% in the Chinese Beijing Han population (HapMap-HCB) in the HapMap database; (2) Prioritizing sites located in important functional positions of the gene, such as exons, 5'UTR, 3'UTR, promoter region, etc.; (3) Selecting relevant sites that have been published in the literature as much as possible. Finally, the rs701848, rs11202592, and rs11202607 sites were included. The basic information of the three SNP sites is shown in Table 1. The gene version number is GRCH37.

[0043] Table 1 Basic information of the three SNPs of PTEN gene

[0044]

[0045]

[0046] 2. Collection and storage of whole blood and plasma specimens

[0047] After all the subjects were enrolled, 3 mL of venous blood was collected and injected into EDTA-K2 anticoagulant tubes. Two 1.5 mL EP tubes were prepared, and the whole blood samples were divided into them and stored in a -70°C refrigerator for subsequent DNA extraction and protein expression detection experiments.

[0048] 3. Extraction and preservation of genomic DNA

[0049] The extraction of genomic DNA was completed using the whole blood DNA extraction kit (centrifugal column method) provided by Yaneng Biotechnology (Shenzhen) Co., Ltd.

[0050] (1) Add 500 μL of WB lysis buffer LB into a 1.5 mL centrifuge tube;

[0051] (2) Pipette 100 μL of anticoagulated whole blood sample into a centrifuge tube and vortex vigorously for 20 seconds;

[0052] (3) Briefly centrifuge, add 100 μL of WB precipitation solution PB to the centrifuge tube, and vortex vigorously for 20 seconds;

[0053] (4) The above liquid was centrifuged at 12000 rpm for 5 min.

[0054] (5) Transfer the supernatant in the centrifuge tube to the groove below the WB centrifuge column inserted into the WB collection tube and centrifuge again at the same speed as before, but for 30 seconds.

[0055] (6) Remove the WB collection tube and discard the filtrate. Put the WB centrifuge column back into the WB collection tube, add 500 μL of WB washing solution W1 to the bottom of the groove of the WB centrifuge column, and centrifuge at the same speed and time settings;

[0056] (7) Remove the WB collection tube and discard the filtrate. Put the WB spin column back into the WB collection tube, add 500 μL of WB wash solution W2 to the bottom of the groove of the WB spin column, and centrifuge in the same way as before.

[0057] (8) Repeat step 7 once;

[0058] (9) After centrifugation, remove the WB collection tube and discard the filtrate again. Set the collection tube aside. Prepare a WB centrifuge column, insert it into the tube, and centrifuge again at the same speed for 2 minutes.

[0059] (10) Remove the WB centrifuge column. Prepare a 1.5 mL clean centrifuge tube, insert it, add 100 μL of WB eluent EB (10 mM Tris-HCl, pH 8.5) to the center of the membrane, let it stand for 1 min, and perform it at room temperature. After the timing is completed, centrifuge at the same speed as before, and it takes a total of 1 min.

[0060] (11) Take out the WB centrifuge column, and the DNA solution at the bottom is frozen and stored at -70°C. This is the DNA template.

[0061] 4. Multiplex PCR reaction

[0062] Take 1 μL of DNA sample, evaluate its concentration by 1% agarose electrophoresis, and then dilute it to a concentration of 10-30 ng / μL for subsequent experiments. The PCR primer sequences are shown in Table 2.

[0063] Table 2 PCR primer sequences

[0064]

[0065] The PCR reaction conditions were as follows: a 20 μL reaction system was used, which included a reaction system (20 μL) containing 1× HotStarTaq buffer, 3.0 mM Mg 2+ , 0.3 mM dNTP, 1 U HotStarTaq polymerase (Qiagen Inc.), 1 μL sample DNA and 1 μL multiplex PCR primer, and the remaining volume was made up with strictly autoclaved double distilled water.

[0066] The PCR reaction cycle program is as follows: PCR reaction parameters: 95°C pre-denaturation for 2 min; amplification stage: 94°C pre-denaturation for 20 s, 65°C annealing for 40 s, 72°C extension for 1.5 min, 11 cycles; 94°C pre-denaturation for 20 s, 59°C annealing for 30 s, 72°C extension for 1.5 min, 24 cycles; the final stage is 72°C extension for 2 min.

[0067] 5. PCR product purification

[0068] Purification of multiplex PCR products: Add 5U SAP enzyme and 2U Exonuclease I enzyme to 20μL PCR product, incubate at 37℃ for 1h, and then inactivate at 75℃ for 15min.

[0069] 6. Ligation reaction

[0070] A 6 μL reaction system was used, including 1 μL of 10×ligase buffer, 0.25 μL of Labelp Mix, 0.4 μL of Ligase Primer Mix (1 μM), 1.0 μL of DNA ligase (50 U / μL), and 3.35 μL of ddH2O. The primer sequences are shown in Table 3.

[0071] Table 3 Linking primer sequences

[0072]

[0073]

[0074] Note: The underlined italic part represents the fluorescent linker sequence.

[0075] The PCR reaction cycle program was: 94°C pre-denaturation for 1 min, 56°C for 4 min, and 38 cycles; maintaining at 4°C.

[0076] 7. Sequencing of ligation products

[0077] After diluting the ligation product 10 times, take 1 μL and mix it with 0.5 μL Liz500 SIZE STANDARD and 8.5 μL Hi-Di. Denature at 95°C for 5 minutes and then load it onto the ABI3730XL sequencer.

[0078] 8. Data Analysis

[0079] The raw data collected on the ABI3730XL sequencer were analyzed using the software GeneMapper4.1 (Applied Biosystems, USA).

[0080] 9. ELISA to detect plasma PTEN protein levels

[0081] 9.1. Reagent preparation

[0082] (1) Sample preparation: 2 hours before the experiment, take the sample to be tested out of the refrigerator and equilibrate it in the room.

[0083] (2) Preparation of standards: Dissolve the standards at room temperature for 25-30 min, shaking occasionally. The concentrations of the standards are 20, 10, 5, 2.5, 1.25, and 0 ng / mL, respectively.

[0084] (3) Preparation of ELISA plates: If the ELISA plates are not used up after opening, the remaining plates should be placed in a sealed bag for further storage.

[0085] (4) Preparation of detergent: Dilute the concentrated detergent 20 times with distilled water. Heat it in a water bath to promote dissolution.

[0086] 9.2 Experimental steps

[0087] (1) Addition of standard samples: Add 50 μL of standard samples of different concentrations to each standard sample well.

[0088] (2) Sample addition: Set up blank wells and sample wells. First add 40 μl of sample diluent to the sample well on the enzyme-labeled plate, and then add 10 μl of the sample (the final dilution factor of the sample is 5 times). Try not to touch the well wall when adding the sample, and shake gently to mix.

[0089] (3) Add enzyme: Add 100 μL of enzyme-labeled reagent to each well, except for the blank well.

[0090] (4) Incubation: Seal the plate with a sealing film, gently mix by shaking, and incubate in a constant temperature incubator (37°C) for 60 min.

[0091] (5) Liquid preparation: Dilute the concentrated washing solution 20 times with distilled water and set aside.

[0092] (6) Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution (about 0.35 mL per well), let it stand for 30 seconds and then discard. Repeat this process 5 times and pat dry.

[0093] (7) Color development: First add 50 μL of color developer A to each well, then add 50 μL of color developer B, and gently mix by shaking. Seal the plate with a sealing film and place it in a constant temperature incubator (37°C) in the dark for 15 min for color development.

[0094] (8) Termination: Add 50 μL of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0095] (9) Determination: Use a microplate reader with a wavelength of 450 nm to detect the absorbance (OD value) of each well in sequence. The determination should be carried out within 15 minutes after adding the stop solution.

[0096] (10) Calculation of sample concentration: The OD value of the blank well was subtracted from the OD value of the standard well and the sample well as the correction value. With the PTEN concentration as the horizontal axis and the OD value as the vertical axis, the Origin 2019 software was used to calculate and draw the standard curve and fit the standard curve formula. The corrected OD value of the sample was substituted into the formula to obtain the initial concentration, which was then multiplied by the dilution factor (5 times) to obtain the serum PTEN concentration.

[0097] 10. Statistical analysis

[0098] SPSS17.0 software package was used to analyze the experimental data. The Hardy-Weinberg genetic equilibrium test was used to evaluate the population representativeness of the sample; continuous variables were analyzed using the t test, and categorical variables were analyzed using the χ test. 2 Test; Multivariate Logistic regression method was used to calculate the association between PTEN4 SNP genotypes and PCOS after adjusting for related risk factors. The relative risk was expressed as odds ratio (OR) and 95% confidence intervals (CI). Haplotype joint analysis was detected using SHEsis online software. Nonparametric rank sum test was used to analyze the differences in plasma PTEN protein levels. The test level was α=0.05, and P<0.05 was considered statistically significant.

[0099] 3. Results

[0100] 1. Analysis of general clinical data

[0101] The comparison of general data of the subjects is shown in Table 4. There was no significant difference in age between the PCOS group and the control group (P>0.05), BMI, LH, E2, PRL, T and AMH in the PCOS group were higher than those in the control group (P<0.05), while there was no significant difference in FSH between the two groups (P>0.05).

[0102] Table 4 Comparison of general information of the subjects

[0103]

[0104]

[0105] 2. Genotyping results of three loci of PTEN gene

[0106] The results of imLDRTM multiple SNP typing method showed that there were three genotypes at the three sites of PTEN gene. The rs701848 site had CC, CT and TT genotypes; the rs11202592 site had CC, CG and GG genotypes; the rs11202607 site had CC, CT and TT genotypes. Figure 1 , Figure 2 and Figure 3 shown.

[0107] 3. Association analysis between PTEN gene polymorphism and PCOS susceptibility

[0108] The genotype and allele frequency distribution of the three SNP sites of the PTEN gene in the normal group and the PCOS group are shown in Table 5. Logistic regression analysis showed that the TT genotype at the rs701848 site, the dominant model (CT+TT vs.CC) and the recessive model (TT vs.CC+CT) may increase the risk of PCOS (TT vs.CC: OR=2.19, 95%CI: 1.34-3.57, P=0.003; dominant model: OR=1.60, 95%CI: 1.11-2.31; P=0.009; recessive model: OR=1.83, 95%CI: 1.18-2.84; P=0.015); however, the polymorphisms at the rs11202592 and rs11202607 sites may not be associated with the onset of PCOS (P>0.05).

[0109] Table 5 Correlation between PTEN gene polymorphism and genetic susceptibility to PCOS

[0110]

[0111]

[0112] Note: OR: odds ratio; 95% CI: 95% confidence interval; P*: P value of logistic regression after adjusting for age and BMI.

[0113] 4. Linkage disequilibrium and haplotype analysis of PTEN gene between PCOS group and control group

[0114] The SHEsis online software was used to perform linkage disequilibrium (LD) analysis on the rs701848, rs11202592, and rs11202607 loci. Figure 4 The haplotype distribution of the three SNP sites of the PTEN gene in the PCOS group and the control group is shown in Table 6. The TCC haplotype carriers in the PCOS group had a significantly increased risk of polycystic ovary syndrome compared with the control group (OR = 1.79, 95% CI, 1.34-2.38, P < 0.001).

[0115] Table 6 Correlation analysis between the haplotypes of the three PTEN gene loci and PCOS

[0116]

[0117] Note: OR: odds ratio; 95%CI: 95% confidence interval; PValue: P value.

[0118] 5. Expression of PTEN protein in normal group and PCOS patient group

[0119] The present invention performed ELISA detection on the expression level of PTEN protein in the plasma of 90 controls and 90 PCOS patients. The results showed that the plasma PTEN protein level of the control group was (12.41±1.75) ng / mL, and the plasma PTEN protein level of the PCOS patient group was (7.43±1.54) ng / mL. The plasma PTEN level of the PCOS patient group was significantly higher than that of the control group, and the difference was statistically significant (P<0.001). Figure 5 As shown. Further analysis of the expression level of PTEN gene rs701848 polymorphism in the plasma of the PCOS group showed that the PTEN protein expression level of patients carrying the TT genotype of the rs701848 locus (13.32±1.27) ng / mL was significantly higher than that of the CT genotype (12.12±0.74) ng / mL and the CC genotype (11.10±0.85) ng / mL, and the differences were statistically significant (P<0.001)( Figure 6 In addition, the plasma PTEN protein expression level of PCOS patients carrying the CT / TT genotype of the rs701848 gene was significantly higher than that of those carrying the CC genotype ( Figure 7), suggesting the correlation between rs701848 polymorphism and PTEN protein expression level in PCOS patients.

[0120] The present invention adopts The multiple SNP typing technology was used to detect the genotypes of the three PTEN loci, and the correlation between the polymorphisms of the three PTEN gene loci rs701848, rs11202592 and rs11202607 and PCOS was analyzed. It was concluded that the polymorphism of the PTEN gene locus rs701848 was correlated with the occurrence of PCOS. The rs701848 locus in the PTEN promoter region may change the transcriptional activity and affect the differential expression of serum PTEN protein, thereby increasing the risk of PCOS. That is, the PTEN gene rs701848 can increase the risk of PCOS by upregulating its protein expression, and can be used to prepare reagents or kits for auxiliary diagnosis of PCOS, so as to diagnose PCOS in patients.

[0121] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A SNP molecular marker associated with susceptibility to polycystic ovary syndrome, characterized in that: It includes SNP site rs701848; the physical position of the SNP site rs701848 in the genome with version number GRCH37 is Chr1089726745; the genotype of the SNP site rs701848 includes CC, CT and TT genotypes.

2. The SNP molecular marker according to claim 1, characterized in that: It also includes SNP site rs11202592 and SNP site rs11202607; the physical positions of the SNP site rs11202592 and the SNP site rs11202607 in the genome with version number GRCH37 are Chr1089624218 and Chr1089727414 respectively; the genotype of the SNP site rs11202592 includes GG, CG and CC genotypes; the genotype of the SNP site rs11202607 includes CC, CT and TT genotypes.

3. A primer combination for detecting the SNP molecular marker according to claim 1 or 2, characterized in that: The primer combination for detecting the SNP site rs701848 includes the primer group shown in SEQ ID NOs. 1 to 2 and the primer probe group shown in SEQ ID NOs. 7 to 9; the primer combination for detecting the SNP site rs11202592 includes the primer group shown in SEQ ID NOs. 3 to 4 and the primer probe group shown in SEQ ID NOs. 10 to 12; the primer combination for detecting the SNP site rs11202607 includes the primer group shown in SEQ ID NOs. 5 to 6 and the primer probe group shown in SEQ ID NOs. 13 to 15.

4. Use of the primer combination according to claim 3 in preparing a detection kit for the SNP molecular marker according to claim 1 or 2.

5. A detection kit for SNP molecular markers according to claim 1 or 2, characterized in that: Comprising the primer combination described in claim 3.

6. Use of the primer combination according to claim 3 or the detection kit according to claim 5 in preparing a kit for auxiliary diagnosis of polycystic ovary syndrome.

7. The use according to claim 6, characterized in that: The method for using the polycystic ovary syndrome auxiliary diagnosis kit comprises using the primer combination described in claim 3 to perform multiple PCR amplification on the sample to be tested, purifying the amplified product and performing a ligation reaction, purifying the ligation product and performing sequencing, and determining the genotype of the SNP site rs701848 or the haplotype of the SNP site rs701848, the SNP site rs11202592 and the SNP site rs11202607 according to the sequencing results.

8. The use according to claim 6, characterized in that: Individuals whose genotype of the SNP site rs701848 is TT have a higher risk of suffering from polycystic ovary syndrome than individuals whose genotypes are CC and CT.

9. The use according to claim 6, characterized in that: Individuals whose haplotypes of the SNP site rs701848, the SNP site rs11202592 and the SNP site rs11202607 are TCC have a higher risk of suffering from polycystic ovary syndrome than those with other haplotypes.

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