Polycystic ovarian syndrome related SNP (Single Nucleotide Polymorphism) molecular marker based on all-exon sequencing technology and application thereof

Abstract

The invention discloses a polycystic ovarian syndrome related SNP (Single Nucleotide Polymorphism) molecular marker based on an all-exon sequencing technology and application thereof, and relates to the technical field of molecular biology and reproductive medicine. The 32 SNP loci provided by the invention have significant differences from the total population frequency and the Asian population frequency in the polycystic ovarian syndrome population, and when the 32 SNP loci are used as the marker of the polycystic ovarian syndrome, a new possibility is provided for individualized precise diagnosis and treatment of the polycystic ovarian syndrome patient.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and reproductive medicine, and more specifically to SNP markers for predicting the risk of polycystic ovary syndrome and applications thereof. Background Art

[0002] Polycystic Ovary Syndrome (PCOS) is a common, complex and heterogeneous reproductive endocrine and metabolic disorder characterized by ovulation disorders, high androgen levels and polycystic ovary-like symptoms. PCOS may lead to fertility problems, metabolic disorders and other health problems.

[0003] At present, there are three consensuses or standards for the diagnosis of PCOS at home and abroad, namely the NIH standard, the Rotterdam standard, and the domestic standard of my country. Since the cause of PCOS is still unclear and the clinical manifestations are highly heterogeneous, there are still large differences between the above diagnostic consensuses or standards, which leads to difficulties in clinical diagnosis and treatment.

[0004] Therefore, providing a product or method for accurately diagnosing PCOS is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides a polycystic ovary syndrome-related SNP molecular marker based on full-penetrance sequencing technology and its application.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A polycystic ovary syndrome-related SNP molecular marker based on full-penetrance sequencing technology, the site physical information of the SNP molecular marker is shown in Table 2, and the site physical position information in Table 2 is determined based on the reference genome GRCh37 / hg19 (GCF_000001405.13) sequence alignment.

[0008] Another object of the present invention is to provide the use of 32 SNP molecular markers in the preparation of polycystic ovary syndrome risk prediction reagents, the site physical information of the SNP molecular markers is shown in Table 2, and the site physical position information in Table 2 is determined based on the reference genome GRCh37 / hg19 (GCF_000001405.13) sequence comparison.

[0009] Another object of the present invention is to provide a molecular probe combination for identifying SNP molecular markers, wherein the molecular probe combination is used to detect the SNP molecular markers shown in Table 2.

[0010] Another object of the present invention is to provide the use of the above molecular probe combination in the preparation of a risk prediction reagent for polycystic ovary syndrome.

[0011] Another object of the present invention is to provide a polycystic ovary syndrome risk prediction reagent, comprising the above-mentioned molecular probe combination.

[0012] Preferably, the prediction reagent is a kit.

[0013] More preferably, the kit further comprises DNA polymerase, dNTPs, and PCR reaction buffer.

[0014] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses a polycystic ovary syndrome-related SNP molecular marker based on full-penetrance sequencing technology and its application. The 32 SNP sites screened by the present invention have significant differences in frequency between the polycystic ovary syndrome population and the general population and the Asian population. Using them as markers for polycystic ovary syndrome provides new possibilities for individualized precision diagnosis and treatment of polycystic ovary syndrome patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] 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 or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0016] Figure 1 This is a schematic diagram of the process of Example 2 of the present invention. DETAILED DESCRIPTION

[0017] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] Example 1

[0019] 1. Determine the experimental sample.

[0020] 2. Extract peripheral blood genomic DNA from samples (Manufacturer: Tiangen; Product No.: DP348-03):

[0021] Add 400 μL of whole blood into a centrifuge tube (if the amount of blood is insufficient, add GS to make up to 400 μL).

[0022] Add 800 μL CL, vortex to mix, centrifuge at 10,000 rpm for 1 min, and discard the supernatant.

[0023] Add 200 μL GS and vortex to resuspend the nuclear pellet;

[0024] Add 200 μL of buffer GB and 20 μL of Proteinase K into the above centrifuge tube and vortex briefly.

[0025] Incubate in a 56°C water bath for 10 minutes, inverting several times to mix.

[0026] After incubation, leave at room temperature for 2-5 minutes.

[0027] Add 350 μL of buffer BD to the above centrifuge tube and vortex briefly.

[0028] Add the solution and flocculent precipitate obtained in the previous step into adsorption column CG2, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid in the collection tube.

[0029] Add 500 μL of buffer GDB to the above adsorption column CG2, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid in the collection tube.

[0030] Add 600 μL of rinsing solution PWB (new kits need to be mixed with 200 mL of anhydrous ethanol before use) to the above adsorption column CG2, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid in the collection tube. Repeat this step once.

[0031] Centrifuge at 12000rmp for 2 minutes, discard the waste liquid, and blow air on the clean bench at level 3 for 3-5 minutes.

[0032] Place the adsorption column CG2 in a 1.5 mL centrifuge tube, suspend it vertically, add 50 μL of nuclease-free water, incubate at room temperature for 5 minutes, and centrifuge at 12,000 rpm for 2 minutes.

[0033] The liquid in the 1.5 mL centrifuge tube was re-aspirated and vertically suspended and dropped onto the silicon matrix membrane in the middle of the adsorption column. The mixture was reacted at room temperature for 5 minutes, centrifuged at 12,000 rpm for 2 minutes, and the DNA extract was recovered in the 1.5 mL centrifuge tube.

[0034] The DNA concentration was measured using Qubit 2.0 / 3.0 / 4.0 Fluorometer (Thermo, USA), and the DNA extract was stored at -20°C for later use.

[0035] 3. Construction of whole exome library (Manufacturer: Suzhou Beikang Medical; whole exome detection kit v1.0 (Cat. No.: J000078 / J000125 / J000126); double-ended index primer 97-192 (Cat. No.: J000127)):

[0036] Step 1: Pre-capture library construction

[0037] (1) Genomic DNA fragmentation and end repair with A tailing:

[0038] Reaction system: 35-X μL nuclease-free water, X μL genomic DNA, 10 μL fragmentation enzyme mixture, 5 μL fragmentation buffer, total volume 50 μL.

[0039] Reaction program: 4°C for 1 min, 37°C for 20 min, 65°C for 30 min, and hold at 4°C.

[0040] (2) Adapter ligation reaction: Prepare the reaction system in an ice box in the following order: (50 μL of the product from the previous step, 5 μL of adapter D, 25 μL of DNA ligation buffer, 5 μL of DNA ligase, 15 μL of nuclease-free water, total volume 100 μL). Vortex the prepared adapter ligation system and place it on an ice box for later use (Note: Adapter D needs to be added separately to the PCR product from the previous step and does not participate in the Mix configuration).

[0041] Take the prepared PCR tube out of the ice box and place it on a gene amplification instrument, incubate at 20°C for 15 minutes, and store at 4°C.

[0042] (3) Purification of the connector ligation product:

[0043] Add 80 μL of DNA purification magnetic beads to the PCR tube containing the ligation product, mix well, let stand at room temperature for 5 minutes, and discard the supernatant.

[0044] Add 150 μL of 80% ethanol solution to the PCR tube in the previous step, let stand at room temperature for 30 seconds, and discard the supernatant. Repeat twice.

[0045] Open the PCR tube cap and let it stand at room temperature to allow the ethanol to evaporate completely and the magnetic beads to dry.

[0046] Add 22.6 μL of nuclease-free water, vortex thoroughly to mix, and let stand at room temperature for 5 minutes.

[0047] Place the PCR tube that has been left to stand in the previous step on a magnetic rack, wait for the solution to become clear, and pipette 20 μL of the supernatant into a new PCR tube.

[0048] (4) PCR amplification before capture:

[0049] Pre-capture PCR reaction system: 20 μL of the product from the previous step, 25 μL of amplification enzyme mixture, 5 μL of double-ended label primer X, and a total volume of 50 μL.

[0050] Pre-capture PCR amplification program (when the genomic DNA input amount ≥ 200 ng, N = 5; when the genomic DNA input amount < 200 ng, N = 6): 95°C for 3 minutes; 98°C for 20 seconds, 60°C for 15 seconds, 72°C for 30 seconds (N cycles); 72°C for 5 minutes; hold at 4°C.

[0051] (5) Purification of PCR products before capture:

[0052] After the previous step, add 55 μL of nuclease-free water to each PCR tube, mix by pipetting, transfer 100 μL of liquid to a new PCR tube, add 60 μL of DNA purification magnetic beads to the 100 μL liquid, mix thoroughly and let stand at room temperature for 5 minutes.

[0053] Place the PCR tube from the previous step on a magnetic rack, take 145 μL of supernatant into a new PCR tube, add 30 μL of DNA purification magnetic beads, mix well, let stand for 5 minutes, and discard the supernatant.

[0054] Add 150 μL of 80% ethanol solution to the above PCR tube, let stand for 30 seconds, discard the supernatant, and repeat 2 times.

[0055] Open the cap of the PCR tube, evaporate the ethanol on the surface of the magnetic beads at room temperature until the beads are dry, add 30 μL of nuclease-free water, remove the PCR tube, mix well, and let stand for 5 minutes.

[0056] Place the PCR tube from the previous step on a magnetic rack, wait for the solution to become clear, and pipette 27 μL of the supernatant into a new centrifuge tube with the corresponding number.

[0057] (6) Quality inspection of the library before capture:

[0058] Take 1 μL of library and use Qubit fluorometer to quantify the library. The library concentration should be ≥20 ng / μL, the library yield should be ≥500 ng, and both conditions are met to be qualified.

[0059] Step 2: Hybridization capture

[0060] (1) Probe hybridization: prepare the hybridization reaction system in the following order: (hybridization buffer 13 μL, blocking solution 15 μL, blocking solution 35 μL, nuclease-free water 3 μL, total volume 26 μL).

[0061] 500 ng of each of the four libraries with different double-end index primers were placed in the same PCR tube for mixed hybridization.

[0062] Add DNA purification magnetic beads (total library volume: DNA purification magnetic beads volume = 1:1.8) into the above PCR tube, mix well, and let stand for 5 minutes.

[0063] Place the above PCR tube on a magnetic rack, wait for the solution to clarify, and discard the supernatant.

[0064] Add 150 μL of 80% ethanol solution to the above PCR tube, let stand for 30 seconds, discard the supernatant, and repeat 2 times.

[0065] Open the PCR tube cap and evaporate the ethanol on the surface of the magnetic beads at room temperature until the beads are dry.

[0066] Add 26 μL of the prepared hybridization reaction system into the above PCR tube, remove the PCR tube, mix well, and let stand for 3 minutes.

[0067] Place the above PCR tube on a magnetic rack and pipette 26 μL of supernatant into a new PCR tube.

[0068] Add 2 μL of blocking solution 2 and 2 μL of probe into the new PCR tube, mix well, and centrifuge.

[0069] Place the above PCR tube on a gene amplification instrument and run the program according to (80°C for 5 minutes, hold at 50°C).

[0070] Incubate at 0°C overnight (hybridization 12 to 18 hours).

[0071] Wash capture beads: Add 100 μL of capture beads to a PCR tube.

[0072] Place the above PCR tube on a magnetic rack, wait for the solution to clarify, and discard the supernatant.

[0073] Remove the above PCR tube, add 180 μL of washing buffer 1, and mix by pipetting. Place the PCR tube on a magnetic rack, wait for the solution to become clear, and discard the supernatant. Repeat 3 times.

[0074] Remove the above PCR tube, add 180 μL of washing buffer 1, mix well by pipetting and set aside.

[0075] (2) Enrichment of the target region DNA library. Keep the hybridization product on the gene amplification instrument, add 180 μL of the resuspended capture magnetic beads to the hybridization product, and mix by pipetting.

[0076] Place the above PCR tubes on a silent mixer at a speed no greater than 10 rpm and combine at room temperature for 30 minutes.

[0077] After 30 minutes, remove the PCR tube, centrifuge, place the PCR tube on a magnetic rack, wait for the solution to clarify, and discard the supernatant.

[0078] Add 150 μL of washing buffer 2 into the above PCR tube and mix well by pipetting.

[0079] The PCR tubes were washed on a silent mixer at room temperature for 15 minutes at a speed not exceeding 10 rpm.

[0080] Add 150 μL of 50°C preheated washing buffer 3 to the above PCR tube, pipette to mix, and incubate at 50°C for 10 minutes. Place the PCR tube on a magnetic rack, wait for the solution to clarify, and discard the supernatant. Repeat this step twice.

[0081] Add 150 μL of 80% ethanol solution into the above PCR tube, let it stand for 30 seconds, discard the supernatant, and dry it at room temperature.

[0082] Add 48 μL of nuclease-free water into the above PCR tube and mix by pipetting.

[0083] (3) Post-capture PCR amplification: Prepare the post-capture PCR amplification system in an ice box in the following order: (24 μL of the magnetic bead product after the previous step, 25 μL of post-capture amplification enzyme mixture, 1 μL of post-capture primer mixture, total volume 50 μL).

[0084] PCR program: 95°C for 1 minute; 98°C for 20 seconds, 60°C for 30 seconds, 72°C for 30 seconds (9 cycles); 72°C for 5 minutes; hold at 4°C.

[0085] (4) Purification of PCR products after capture: After the PCR reaction is completed, centrifuge immediately, place on a magnetic rack, let stand at room temperature for 5 minutes, and take 50 μL of supernatant into a new PCR tube.

[0086] Add 55 μL of DNA purification magnetic beads, mix well, and let stand for 5 minutes.

[0087] Add 150 μL of 80% ethanol solution, let stand at room temperature for 30 seconds, and discard the supernatant. Repeat twice.

[0088] Dry at room temperature, add 25 μL of nuclease-free plum water, mix well, and let stand for 5 minutes.

[0089] Pipette 23 μL of supernatant into a new 1.5 mL centrifuge tube.

[0090] (5) Post-capture library quality control: Take 1 μL of library and use Qubit fluorometer to quantify the library. The library concentration should be ≥ 4 ng / μL.

[0091] 4. Perform sequencing on the machine (Manufacturer: Suzhou Beikang Medical; select Universal Sequencing Reaction Kit (Combined Probe Anchor Polymer Sequencing Method DA5000-PE150-FCL) (Cat. No.: J000104) or Universal Sequencing Reaction Kit (Combined Probe Anchor Polymer Sequencing Method DA5000-PE150-FCS) (Cat. No.: J000106)).

[0092] 5. Data analysis and SNP identification

[0093] (1) Use BWA software and MEM algorithm to align the raw data (Fsatq) to the reference genome GRCh37 / hg19 (GCF_000001405.13) and output a bam format file.

[0094] (2) Use GATK-HaplotypeCaller for variant calling, with bam file as input and VCF file as output. The basic workflow includes four main steps: identifying active regions, determining haplotypes by reassembling active regions, determining the likelihood value of the haplotype of each read, and determining the genotype.

[0095] (3) Quality control system is shown in Table 1.

[0096] Table 1

[0097] Quality parameters standard Rawbases(G) >＝12 Q30(％) >＝85 Capture efficiency (%) >＝60 Comparison rate (%) >99 Average sequencing depth of target region (×) >＝100 Uniformity (%) [= 0.2*mean_dp coverage] >＝90 Fold80 <2.5 30×Coverage(%) >＝90 20×Coverage(%) >＝95 10×Coverage(%) >＝98 1×Coverage(%) >＝99 Average mitochondrial sequencing depth (×) >＝10000 Mitochondria 1000×Coverage(%) >＝100

[0098] Example 2

[0099] A total of 318 test samples were collected in the present invention, including 159 PCOS case group (from patients visiting the Reproductive Center of Xinjiang Jiayin Hospital) and 159 control group (from the Physical Examination Center of Xinjiang Jiayin Hospital), and all of them signed the informed consent for sample acquisition.

[0100] The method of Example 1 was used to obtain sites with high site quality (GQ value ≥ 99) in the VCF file of the full-external sequencing data of the PCOS case group (159 cases) and the control group (159 cases). The SNP sites screened between the two groups were batched and the chi-square test was performed to calculate the significant difference P value, and the significant difference sites (P < 0.05) were screened. A total of 8513 sites were screened from the BAM file of the WES sequencing data. 8513 sites were screened in 318 samples. 4815 sites with a coverage depth of ≥ 10X in 318 samples. A group of 40 sites with a frequency of ≤ 0.01 and extremely significant differences between the two groups of data (P < 0.01) were screened from the 4815 sites, and the total population frequency and Asian population frequency of these sites in the gnomAD database were searched. 32 sites in the PCOS population were screened out, which were significantly different from the total population frequency and the Asian population frequency (see Table 2). They were used as markers for polycystic ovary syndrome, providing new possibilities for individualized precision diagnosis and treatment of patients with polycystic ovary syndrome.

[0101] Table 2

[0102]

[0103]

[0104] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0105] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A polycystic ovary syndrome-related SNP molecular marker based on full-penetrance sequencing technology, characterized in that: The site physical information of the SNP molecular marker is shown in Table 2. The site physical position information in Table 2 is determined based on the reference genome GRCh37 / hg19 (GCF_000001405.13) sequence alignment.

2. The use of 32 SNP molecular markers in the preparation of a risk prediction reagent for polycystic ovary syndrome, characterized in that: The site physical information of the SNP molecular marker is shown in Table 2. The site physical position information in Table 2 is determined based on the reference genome GRCh37 / hg19 (GCF_000001405.13) sequence alignment.

3. The molecular probe combination for identifying the SNP molecular marker according to claim 1, characterized in that: The molecular probe combination is used to detect the SNP molecular markers shown in Table 2.

4. Use of the molecular probe combination according to claim 3 in the preparation of a risk prediction reagent for polycystic ovary syndrome.

5. A polycystic ovary syndrome risk prediction reagent, characterized in that: Comprising the molecular probe combination according to claim 4.

6. The polycystic ovary syndrome risk prediction reagent according to claim 5, characterized in that: The prediction reagent is a kit.

7. The polycystic ovary syndrome risk prediction reagent according to claim 6, characterized in that: The kit also includes DNA polymerase, dNTPs, and PCR reaction buffer.

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