A set of SNP markers related to right eye spherical equivalent quantitative traits and their application

Abstract

The present invention belongs to the fields of biomedicine and gene detection technology, and specifically relates to a group of SNP markers related to quantitative traits of right eye spherical equivalent and their applications. The present invention provides the application of a reagent for detecting rs2547319 in determining right eye spherical equivalent.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and gene detection technology, and specifically discloses a group of SNP markers related to right eye spherical equivalent quantitative traits and applications thereof. Background Art

[0002] Ophthalmic diseases refer to diseases that occur in the eye area. Common ophthalmic diseases include myopia, astigmatism, cataracts, glaucoma, central serous retinopathy, dry eye, sympathetic ophthalmia, night blindness, amblyopia, trachoma, diabetic retinopathy, conjunctivitis, presbyopia, color blindness, retinitis pigmentosa, central retinal artery occlusion, retinal detachment, hyperopia, stye, snow blindness, chalazion, and floaters. Myopia occurs when parallel light rays pass through the eye's refractive system and focus in front of the retina when the eye is relaxed.

[0003] Refraction refers to the degree of refractive error. When refraction occurs, it generally represents clinical manifestations such as myopia, astigmatism, hyperopia, or amblyopia. Myopia and hyperopia are usually spherical (S), and astigmatism is cylindrical (C). Currently, spherical equivalent refraction (SE) is used to evaluate refraction. Spherical equivalent refraction = spherical degree + astigmatism degree * 1 / 2. Refraction ≤ -0.50D can be used as the evidence-based consensus threshold for myopia diagnosis. More specifically, myopia is defined as a situation where the eye's equivalent spherical refractive error is ≤ -0.5D when accommodation is relaxed. High myopia is defined as a situation where the eye's equivalent spherical refractive error is ≤ -6.00D when accommodation is relaxed. Low myopia is defined as a situation where the eye's equivalent spherical refractive error is ≤ -0.5 and > -6.00D when accommodation is relaxed.

[0004] Myopia, as a global high-incidence disease, has received increasing attention at home and abroad in recent years. It is estimated that by 2050, myopia will affect nearly 50% of the world's population, and high myopia will also affect nearly 10% of the world's population.

[0005] Myopia is also a major eye disease affecting the visual health of Chinese adolescents. High myopia is one of the leading causes of blindness and low vision in my country, placing a significant economic burden on patients and their families. Prevention is crucial for children's vision problems, and early detection, intervention, and treatment are key to preventing the progression of myopia. Eye diseases in childhood and adolescence are extremely detrimental to visual development. Early detection and diagnosis of non-refractive eye diseases that affect vision are crucial. Many eye diseases, if not detected and treated promptly, can cause lifelong eye disability. While medication, optical therapy, and behavioral modification can partially slow the progression of myopia, we are still a long way from reversing the trends of the past few decades. This makes myopia and its related complications a top research priority. The development of high myopia involves a complex, multifactorial process, and its pathogenesis remains unclear. Current optical correction methods and surgical treatments, such as refractive surgery and scleral reinforcement, cannot fundamentally prevent or delay the progression of fundus lesions in high myopia. It can be said that there is currently a lack of effective treatments for high myopia. It can be seen that finding more effective methods for early detection, risk prediction and early intervention in patients with high myopia and high-risk groups has important clinical significance.

[0006] Single nucleotide polymorphisms (SNPs) refer to DNA sequence polymorphisms caused by nucleotide variations due to the conversion or transversion of a single base at the genomic level, or the insertion or deletion of a base. They are the most common type of heritable variation in humans, accounting for over 80% of all known polymorphisms and having a frequency of more than 1% in the population, which is an important factor that distinguishes them from point mutations. In genetic analysis, SNPs are characterized by high frequency, stability, and ease of analysis. Studies have found that SNPs are strongly associated with the occurrence and development of myopia. The main methods for SNP detection include time-of-flight mass spectrometry (MALDI-TOFMS), fluorescence quantitative PCR, gene chip technology, and deformable high-performance liquid chromatography.

[0007] With the emergence of high-throughput SNP detection technology, SNPs, as the most numerous and easily detected polymorphic markers, will play an increasingly important role in linkage analysis and gene mapping, including genetic mapping of complex diseases, association analysis, and the study of individual and population responses to environmental pathogens and drugs. As technology advances, SNP detection costs are becoming increasingly affordable, and SNP markers will become the next generation of molecular markers. Summary of the Invention

[0008] The present invention includes a large number of subjects aged 6-18 years old, collects standard oral mucosal samples through standard operating procedures (SOP), extracts DNA, types single nucleotide polymorphisms, and screens to find SNPs with high specificity and sensitivity that are highly correlated with the right eye equivalent spherical lens. Further, a corresponding kit that is convenient for clinical application can be developed to provide data support for determining the right eye equivalent spherical lens of the subjects, so as to intervene in the early stage of ophthalmic problems, reduce the possibility of worsening of ophthalmic diseases, and improve the quality of life.

[0009] In order to achieve the technical purpose of the present invention, the present invention provides the following technical solutions:

[0010] In a first aspect, the present invention provides the use of a reagent for detecting rs2547319 in determining the spherical equivalent of the right eye.

[0011] On the other hand, the present invention provides a group of right eye spherical equivalent related SNP site combinations, wherein the SNP site combination comprises any one of the following groups:

[0012] (1)rs10889315, rs75144949, rs149213974, rs754615, rs45583335, rs2529438, rs2 072236, rs2240403, rs78155900, rs114097201, rs2811736, rs2297722, rs2297723, r s117519669, rs7157977, rs10143899, rs3759779, rs143477571, rs2303221, rs3469 3726, rs3803752, rs142921938, rs3746295, rs2547319, rs2547318, rs4911402(26);

[0013] (2)rs4648600、rs1061624、rs2275365、rs35909785、rs3103778、rs2457829、rs10889315、rs78587889、rs12568050、rs12564283、rs528123098、rs139842833、rs74953908、rs1077827、rs12492484、rs75144949、rs3732755、rs3135115、rs16837029、rs149213974、rs17001890、rs56382032、rs12651031、rs2736100、rs7724759、rs754615、rs5369、rs150157174、rs130068、rs45583335、rs78342561、rs2529438、rs1049402、rs2072236、rs2527878、rs2240403、rs78155900、rs114097201、rs2680903、rs59612871、rs3818584、rs16909677、rs10983755、rs2248077、rs2811736、rs2297722、rs2297723、rs9987876、rs7904554、rs907609、rs117519669、rs3750996、rs3802799、rs2276296、rs139692587、rs904628、rs11551723、rs144796593、rs78648016、rs7157977、rs10143899、rs3759779、rs7142098、rs143477571、rs3736911、rs11849022、rs77481241、rs3212112、rs9282879、rs55958706、rs3212056、rs34100926、rs114770841、rs3742368、rs118146919、rs2289702、rs2303221、rs34693726、rs3744566、rs9907420、rs375644567、rs185576254、rs3803752、rs17822735、rs17222523、rs34818467、rs12946397、rs3809724、rs181969864、rs7236574、rs142921938、rs3746295、rs151309111、rs533816037、rs2302948, rs182509214, rs2547319, rs2547318, rs2281209, rs4911402, rs138894461, rs5756223 (102);

[0014] (3)rs16824585、rs4648600、rs17033266、rs1061624、rs202140107、rs2291804、rs2275365、rs139241751、rs35909785、rs6676052、rs3103778、rs116893711、rs116861599、rs2457829、rs10889315、rs17855078、rs78587889、rs140141416、rs4656197、rs45445497、rs143355122、rs12568050、rs12564283、rs10482、rs1929861、rs75401237、rs28517482、rs1264208208、rs2631840、rs188480146、rs528123098、rs61732269、rs367931009、rs139842833、rs79908358、rs79358798、rs1434087、rs4894048、rs13421990、rs13419085、rs143196218、rs139713392、rs6734083、rs150887565、rs4663795、rs74953908、rs143001518、rs6437368、rs147838565、rs1077827、rs144623840、rs78233496、rs9876921、rs6622、rs2279909、rs547922447、rs300977、rs12492484、rs75144949、rs3732755、rs3135115、rs16837029、rs74787220、rs527903313、rs149213974、rs17001890、rs79769348、rs10025654、rs76996680、rs2306369、rs79482316、rs56382032、rs12651031、rs2736100、rs35130836、rs3749684、rs7724759、rs754615、rs45625534、rs3805625、rs137898880、rs147518598、rs202007699、rs2270900、rs75164992、rs17133512、rs76720430、rs5369、rs150157174、rs7756481、rs117298661、rs117644703、rs3094672、rs4713420、rs130068、rs67196728、rs185990098、rs9349593、rs143580489、rs3757302、rs3218602、rs9495370、rs45583335、rs78342561、rs117358940、rs9689983、rs2529438、rs1049402、rs2072236、rs2527878、rs2240403、rs781980620、rs56083327、rs2245368、rs2430307、rs3748126、rs36028884、rs78155900、rs114097201、rs13438494、rs117211799、rs115734214、rs148474510、rs200506987、rs8190955、rs8178175、rs2680903、rs117676215、rs115507207、rs369806130、rs78240711、rs59612871、rs117169590、rs3739523、rs145293869、rs139212809、rs3818584、rs16909677、rs141549766、rs147026086、rs188197626、rs13321、rs10983755、rs2301576、rs7038042、rs11849、rs2275161、rs2248077、rs2811736、rs2297722、rs2297723、rs9987876、rs7904554、rs148582275、rs3740168、rs149647444、rs7100474、rs7079648、rs202166096、rs60420182、rs36104328、rs7098255、rs3814163、rs2297785、rs3736924、rs3736923、rs141296329、rs117520659、rs57285449、rs112687793、rs73401681、rs79948463、rs10902170、rs10902171、rs867839、rs907608、rs907609、rs117519669、rs3750996、rs214086、rs3802799、rs76215382、rs147852038、rs2276296、rs143930161、rs183113103、rs139692587、rs10792769、rs7102675、rs116878689、rs35466364、rs142908193、rs7966459、rs7135745、rs34181394、rs1921038、rs201814780、rs11068531、rs904628、rs11551723、rs7983667、rs144796593、rs140526815_rs77878407、rs78646508、rs34523940、rs9521906、rs78648016、rs7157977、rs10143899、rs3759779、rs7142098、rs41309252、rs535020964、rs3742770、rs3742768、rs116881452、rs143477571、rs61992274、rs142973720、rs530110551、rs3742464、rs3736911、rs11849022、rs77481241、rs3212112、rs9282879、rs55958706、rs3212056、rs34100926、rs114770841、rs3742368、rs144857261_rs373032548、rs189734192、rs188393827、rs118146919、rs562829514、rs1805025、rs8192352、rs2289702、rs150921132、rs61869621、rs1802752、rs113355794、rs7198064、rs2291407、rs9941128、rs9941210、rs9941215、rs9931527、rs12708649、rs13306653、rs11643517、rs5723、rs7196736、rs2303221、rs2304483、rs149645127、rs11550470、rs17232091、rs150889000、rs16954357、rs371756524_rs557159990、rs192103504、rs115314145、rs34693726、rs2277651、rs2277652、rs71360269、rs71360270、rs3744566、rs3744568、rs9907420、rs375644567、rs185576254、rs2158460, rs3803752, rs17822735, rs17222523, rs34818467, rs12946397, rs3809724, rs35476409_rs39797 8887, rs3744214, rs181969864, rs4789773, rs28561791, rs7236574, rs76597875, rs61749945, rs118033234, rs1052696, rs1052692, rs148018996, rs34488963, rs3745379, rs3745385, rs142921938, rs3746295, rs15130 9111, rs139271842, rs140566569, rs533816037, rs78237760, rs79621739, rs75990051, rs62107869, rs30597 4. rs1055099, rs41290128, rs76151738, rs16980091, rs2302948, rs182509214, rs2547319, rs2547318, rs169 82743, rs2284385, rs2281209, rs4911402, rs80158178, rs7509972, rs150511237, rs2231391, rs2231390, rs7 62228, rs72564613, rs191574093, rs138894461, rs5999924, rs5750146, rs5756223, rs3747168, rs148345118, rs910796, rs910797, rs910798, rs738712, rs16445, rs3922872, rs56248708, rs9628315, rs8137790 (342).

[0015] In the present invention, SNP (single nucleotide polymorphism) refers to a single base position in DNA, and a subject can be homozygous or heterozygous. The SNP sites of the present invention are named in an "rs-" manner. Those skilled in the art can determine their exact position and nucleotide sequence from suitable databases and related information systems such as the Single Nucleotide Polymorphism Database (dbSNP) based on the rs- nomenclature above. For SNPs, each site has two or more alleles, for example, two alleles.

[0016] Preferably, the SNP site combination is tested based on a sample from a subject, and then the subject's current right eye equivalent spherical lens is determined based on the test results, and the risk of developing ophthalmic problems is predicted.

[0017] As used herein, "sample" refers to any sample containing nucleic acids (particularly DNA) from or derived from a human patient, such as body fluids (blood, saliva, urine, etc.), biopsies, tissues and / or waste from the patient. Thus, tissue biopsies, feces, sputum, saliva, blood, lymph fluid, etc. can be easily screened for SNPs, as can essentially any target tissue containing appropriate nucleic acids. In one embodiment, the sample is oral epithelial cells. These samples are typically obtained from the patient by standard medical laboratory methods after informed consent. The sample can be in a form obtained directly from the patient, or can be at least partially processed (purified) to remove at least some non-nucleic acid material;

[0018] Preferably, the sample is a buccal swab (oral mucosal sample);

[0019] On the other hand, the present invention also provides a kit for detecting the right eye spherical equivalent, wherein the kit comprises a reagent for detecting the aforementioned SNP site combination.

[0020] Preferably, the reagents for detecting the SNP site combination include, but are not limited to, the reagents used in the following methods for detecting SNPs: TaqMan probe method, sequencing method, chip method, flight mass spectrometry (MALDI-TOFMS) detection, restriction fragment length polymorphism method (PCR-RFLP), single-strand conformation polymorphism method (PCR-SSCP), allele-specific PCR (AS-PCR), SNaPshot method, SNPlex method, denaturing high performance liquid chromatography (DHPLC), denaturing gradient gel electrophoresis (DGGE). Those skilled in the art can select any one or more methods to detect SNP sites, as long as the detection of SNP sites can be achieved.

[0021] In the present invention, "a preparation for detecting SNP" refers to a preparation that specifically binds to the SNP marker or SNP included in the SNP marker composition to facilitate identification, or can detect the SNP and amplify it, and "a preparation that can amplify the SNP marker" refers to a preparation that repeatedly replicates the SNP marker or SNP included in the SNP marker composition and increases its number, for example, a primer that specifically amplifies the polynucleotide including the SNP or a probe that can specifically bind, but is not limited to this.

[0022] The primers used for the SNP amplification can be appropriate conditions (for example, polymerizing agents such as 4 different nucleoside triphosphates, DNA, RNA polymerase or reverse transcriptase) in a suitable buffer and at an appropriate temperature, can be used as the starting point of columnar display DNA synthesis, a single-stranded oligonucleotide that works, and according to the purpose of use, the appropriate length of the primers will be different, but typically 15 to 30 nucleotides. Short primer molecules usually require lower temperatures in order to form a columnar and stable mixture. The primer sequence does not need to form complete complementarity with the polynucleotide comprising the SNP, but its complementarity needs to reach a degree that can be mixed with the polynucleotide comprising the SNP.

[0023] In one embodiment, the reagent for detecting the SNP site combination is a primer set and / or probe set for amplifying the aforementioned SNP site combination.

[0024] In one embodiment, the kit may further include common reagents required for the corresponding PCR technology, such as dNTPs, MgCl2, double-distilled water, fluorescent probes, etc. These common reagents are well known to those skilled in the art, as well as standards and controls (such as genotype standards and blank controls, etc.).

[0025] On the other hand, the present invention provides a right eye spherical equivalent detection system, which includes a computing device for determining the right eye spherical equivalent of a subject using the detection results of the above-mentioned SNP site combination.

[0026] Preferably, the diagnostic system may further include a device for detecting SNP sites, such as a sequencer.

[0027] On the other hand, the present invention provides a method for constructing a right eye equivalent spherical model using the above SNP site combination.

[0028] Preferably, the method uses a logistic regression method.

[0029] Preferably, the present invention further includes a 10-fold cross-validation step. The "10-fold cross-validation" specifically refers to using the sklearn.model_selection function to select 10 random numbers from 0 to 100 as random seeds, dividing the samples into 10 training sets and validation sets, performing 10-fold cross-validation, and taking the average of the 10 results as the model result.

[0030] On the other hand, the present invention also provides a model for detecting the right eye equivalent spherical lens, which determines the current right eye equivalent spherical lens of the subject based on the detection results of the aforementioned SNP site combination.

[0031] Preferably, the model is constructed by the aforementioned method.

[0032] Preferably, the model can be a formula, a nomogram, or other methods that are convenient for the subject to operate; based on this model, the subject's current right eye equivalent spherical lens can be directly calculated.

[0033] On the other hand, the present invention provides reagents for detecting the aforementioned SNP site combination, and uses of the aforementioned kit, system, and model in preparing products for detecting the right eye spherical equivalent.

[0034] Preferably, the reagents for detecting the above-mentioned SNP site combination include, but are not limited to, the reagents used in the following methods for detecting SNPs: TaqMan probe method, sequencing method, chip method, flight mass spectrometry (MALDI-TOFMS) detection, restriction fragment length polymorphism method (PCR-RFLP), single-strand conformation polymorphism method (PCR-SSCP), allele-specific PCR (AS-PCR), SNaPshot method, SNPlex method, denaturing high performance liquid chromatography (DHPLC), denaturing gradient gel electrophoresis (DGGE). Those skilled in the art can select any one or more methods to detect SNP sites, as long as the detection of SNP sites can be achieved.

[0035] Specifically, the reagents include but are not limited to primers, probes, chips, etc.

[0036] On the other hand, the present invention provides a method for detecting the right eye spherical equivalent, the method comprising the steps of determining the right eye spherical equivalent of a subject based on the detection results of the SNP site combination of the present invention,

[0037] Specifically, the method may include the following steps:

[0038] 1) Collecting a sample from a subject, preferably, the sample is an oral swab (oral mucosal sample);

[0039] 2) performing SNP detection on the sample, preferably, the detection may also include a step of extracting DNA;

[0040] 3) Based on the test results of 2), determine the subject's right eye equivalent spherical lens.

[0041] The judgment may be performed manually, automatically, or a combination thereof to execute or complete the selected task; the result may be calculated manually based on the detection result, or input into the aforementioned system to automatically obtain the calculated result.

[0042] The "spherical equivalent refraction (SE)" mentioned in the present invention is equal to the spherical power + astigmatism power * 1 / 2. Refractive power ≤-0.50D can be used as the evidence-based consensus threshold for the diagnosis of myopia. More specifically, myopia is defined as a situation where the eye's equivalent spherical refractive error is ≤-0.5D when the accommodation is relaxed. High myopia is defined as a situation where the eye's equivalent spherical refractive error is ≤-6.00D when the accommodation is relaxed. Low myopia is defined as a situation where the eye's equivalent spherical refractive error is ≤-0.5 and >-6.00D when the accommodation is relaxed.

[0043] The single nucleotide polymorphisms (SNPs) mentioned in this invention refer to the variation of a single nucleotide in the genome, including transitions, transversions, deletions and insertions, which form genetic markers. There are many SNPs and they are rich in polymorphisms. There is about one SNP per 1000 bases in the human genome, and the total number of SNPs in the human genome is about 3×10 6 Therefore, SNPs have become the third-generation genetic markers, and many phenotypic differences in the human body, susceptibility to drugs or diseases, etc. may be related to SNPs.

[0044] The term "detection" used throughout the specification of the present invention means determining the current spherical equivalent of the right eye of a subject.

[0045] Specifically, the technical solutions to solve the problems of the present invention include:

[0046] (1) Establish a unified standard specimen library and database: collect blood samples that meet the standards according to standard operating procedures (SOPs) and systematically collect complete demographic and clinical data;

[0047] (2) Genotype detection: Samples that meet the requirements are included, and a high-density SNP chip is used to identify SNPs associated with the right eye equivalent spherical lens in the entire gene range;

[0048] (3) The positively associated SNPs screened out are further tested in other samples to verify their repeatability and stability in clinical diagnosis;

[0049] (4) Development of a right eye spherical equivalent detection kit: Develop a detection kit based on SNPs that are significantly associated with right eye spherical equivalent. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 AUC value change curve of the screened SNP combination of the present invention when judging the right eye equivalent spherical lens at different thresholds. DETAILED DESCRIPTION

[0051] The present invention will be further described below with reference to the following embodiments. The following description is merely a preferred embodiment of the present invention and does not limit the present invention in any other form. Any person skilled in the art may utilize the above disclosed technical content to make equivalent embodiments with equivalent variations. Any simple modification or equivalent variation of the following embodiments made in accordance with the technical essence of the present invention without departing from the content of the present invention shall fall within the scope of protection of the present invention.

[0052] Example 1 Sample collection, sequencing and data analysis

[0053] Sample inclusion criteria:

[0054] (1) The spherical equivalent of the worst eye is less than or equal to -6.00D;

[0055] (2) Age 6–18 years old;

[0056] (3) No other eye diseases, congenital genetic diseases, other systemic diseases, or physical abnormalities;

[0057] (4) no history of eye surgery;

[0058] (5) Chinese;

[0059] (6) Participate voluntarily and sign the informed consent form.

[0060] The present invention is based on the sample information of the Wenzhou City Myopia Screening and Intervention Project for Primary and Secondary School Students, which was carried out in cooperation with the Eye and Vision Hospital Affiliated to Wenzhou Medical University and the Wenzhou Municipal Government from 2019 to 2020. 10,348 (9,852-8,961) samples that met the above inclusion criteria were recruited for whole-exome sequencing, and their spherical lens (Sph) and cylindrical lens (Cyl) of both eyes were recorded, and the equivalent spherical lens of both eyes (SE = Sph + 0.5Cyl) was calculated. At the same time, whole-exome sequencing was performed on these 10,348 patients with high myopia to obtain SNPs in the whole exome, and trait-related SNPs were obtained through whole-exome association analysis.

[0061] The specific experimental steps are as follows:

[0062] In this example, oral mucosal samples meeting the criteria were collected according to standard operating procedures (SOPs), and patients meeting the inclusion criteria were systematically collected and followed up. Whole-exome sequencing was performed using the Illumina Novaseq6000 sequencing system to detect SNPs across the exome. SNP markers and their combinations associated with spherical equivalent (SE) in people with high myopia were discovered, and a model for predicting the risk of spherical equivalent progression was constructed.

[0063] 1. Collect oral mucosal swabs:Stop eating and drinking half an hour before sample collection to ensure the sample is not contaminated. Place the collected oral mucosal swab in a sterile 1.5ml EP tube, complete the inspection registration form, and transport it to the company at room temperature for professional personnel to extract genomic DNA from the oral mucosal swab.

[0064] 2. DNA extraction: Genomic DNA was extracted from oral mucosal swabs using the TIANamp Swab DNA Kit. The specific steps are as follows:

[0065] (1) Transfer the oral mucosal swab to a 2 ml centrifuge tube, cut the cotton swab from its stem with scissors, and add 400 μl of buffer GA.

[0066] (2) Add 20 μl of Proteinase K solution, vortex for 10 seconds to mix, and incubate at 56°C for 60 minutes, vortexing several times every 15 minutes to mix.

[0067] (3) Add 400 μl of Buffer GB, mix thoroughly by inversion, and incubate at 70°C for 10 minutes. The solution should be clear at this point. Centrifuge briefly to remove any droplets on the inner wall of the tube cap, then squeeze out the swab and transfer as much lysate as possible to a new centrifuge tube.

[0068] (4) Add 200 μl of anhydrous ethanol, mix thoroughly by inversion, and centrifuge briefly to remove droplets on the inner wall of the tube cap.

[0069] (5) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CR2 (adsorption column CR2 is placed in a collection tube), centrifuge at 12,000 rpm (~13,400 × g) for 30 seconds, pour out the waste liquid in the collection tube, and put the adsorption column CR2 back into the collection tube.

[0070] (6) Add 500 μl of buffer GB to the adsorption column CR2 (please confirm whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (~13,400 × g) for 30 seconds, discard the waste liquid in the collection tube, and return the adsorption column CR2 to the collection tube.

[0071] (7) Add 700 μl of rinse solution PW to the adsorption column CR2 (please confirm whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (~13,400 × g) for 30 seconds, discard the waste liquid in the collection tube, and return the adsorption column CR2 to the collection tube.

[0072] (8) Repeat step 7.

[0073] (9) Centrifuge at 12,000 rpm (~13,400 × g) for 2 minutes, discard the waste liquid, and place the adsorption column CR2 at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material.

[0074] (10) Transfer the adsorption column CR2 to a clean centrifuge tube, add 20-50 μl of elution buffer TB dropwise to the middle of the adsorption membrane, let it stand at room temperature for 2-5 minutes, and centrifuge at 12,000 rpm (~13,400 × g) for 2 minutes.

[0075] (11) DNA fragment concentration and purity were determined using agarose gel electrophoresis and UV spectrophotometry. DNA has a significant absorption peak at OD260, with an OD260 value of 1 corresponding to 50 μg / ml double-stranded DNA and 40 μg / ml single-stranded DNA. The purity (OD260 / 280) was between 1.7 and 1.9. A single sample typically yielded 0.5–3.5 μg of DNA.

[0076] 3. Build a pre-library: 50 ng of genomic DNA was broken into small fragments of about 200 bp using an enzymatic method, followed by end repair and 3' end A addition. The DNA fragments were then connected to sequencing adapters containing barcode sequences, and fragments of about 320 bp were selected and recovered. The pre-library was obtained after PCR amplification.

[0077] 4. Liquid phase hybridization capture operation: The prelibrary was subjected to liquid phase hybridization capture operation according to the standard procedure of IDT's xGen Exome Research Panel V1.0 (Integrated DNA Technologies, San Diego, USA).

[0078] 5. Obtain exon library: After elution and recovery of the captured product, PCR amplification and purification were performed to obtain the exon library. The library was quantified by qPCR, and band size was determined using an Agilent 2100.

[0079] 6. Illumina Novaseq6000 sequencing: The exon library was sequenced with 150 PE using the Illumina Novaseq6000 sequencing system, and the raw images were base-identified using CASAVA v1.82 software to generate raw sequencing data.

[0080] 7. Comparison to the human reference genome: Sequence fragments were aligned to the human reference genome (UCSC hg19) using the Burrows-Wheeler Aligner (BWA) tool, and PCR duplicates were removed using Picard v1.57. GATK software was used for variant detection, and statistics for sequencing depth, coverage depth, and uniformity were analyzed.

[0081] 8. Data quality control

[0082] For samples: Delete samples with insufficient buccal swab sampling volume, detection rate less than 90%, average coverage less than 10, average genotype presence probability less than 65%, average genotype heterozygosity deviation from ±4 standard deviations (SD), chromosomal abnormalities, gender abnormalities, and samples from related non-East Asian populations. For SNPs: Delete SNPs with whole-exome test results that failed VQSR quality control, genotype detection rate less than 90%, Hardy-Weinberg P value less than 1e-06, and variant allele number (AC) of 0.

[0083] 9. Statistical analysis methods

[0084] A full exome-wide quantitative trait study of high myopia was conducted using Bayesian tests in SNPtest to identify potential SNPs and genes significantly associated with the clinical status of high myopia. Bayesian tests for quantitative traits were performed using a conjugate prior formula for a linear model with the expected genotype (-method expected). For the additive model, the formula is:

[0085] y i =βG i +e i ,e i ~N(0,σ 2 ).

[0086] where yi is the residual phenotype for the i-th individual. The residual phenotype is calculated by subtracting a baseline term and estimating any specified covariates. Gi is the additive encoding of the expected genotype for the i-th individual, where wildtype homozygous = "0", heterozygous = "1", and mutant homozygous = "2".

[0087] σ2 is the error variance of the model. And σ 2 ~IG(a,β),β~N(m β ,V β σ 2 ), the default a=3,β=2,m β =0,V β =0.02. Age and sex were used as covariates for adjustment.

[0088] Experimental results

[0089] 1. snps selection:

[0090] SNPTEST was used to obtain the genome-wide association between 89,095 common variants and the quantitative trait right eye equivalent spherical lens in 8,961 high myopia samples.

[0091] SNPs were screened for modeling using four thresholds: log10(BF)>3; log10(BF)>2; log10(BF)>1.5; log10(BF)>1, and a total of 342 SNPs were obtained.

[0092] 2. Obtaining the original SNPs genotype spectrum:

[0093] Python was used to extract the required SNPs (342 SNPs in total) in the VCF, and the additive model was selected (no mutation was set to 0, one mutation allele was set to 1, and two mutation alleles were set to 2) to obtain the original SNP genotype spectrum.

[0094] 3. SNPs genotype spectrum complement missing:

[0095] The number of missing SNPs in each sample in the original SNPs genotype profile was calculated. If the missing number was >5% (N_missing / N_allsamples), the corresponding sample was removed. For samples with missing information <5%, an artificial neural network was used to fill in the missing information.

[0096] The artificial neural network has three layers, including input layer, hidden layer and output layer: the number of nodes in the input layer is len(label_data), that is, there are no missing SNPs; the hidden layer is set to 20 nodes; the output layer is set to 3 nodes; the learning rate is set to 0.003, and the number of training iterations is set to 20.

[0097] 4. Classification Model: Logistic Regression

[0098] 1) According to the quantitative trait right eye equivalent spherical mirror, different thresholds were taken to establish models respectively, and the AUC values ​​were examined. The threshold of the model with the largest AUC was taken as the threshold of the final classification model.

[0099] The rules are as follows:

[0100] ① Traverse all values ​​of the right eye equivalent spherical lens and use them as thresholds to divide the samples into two groups;

[0101] ②The number of samples in each group shall not be less than 100

[0102] 2) Use logistic regression to classify the samples with the following parameters:

[0103]

[0104] Despite its name, logistic regression is a linear model used for classification rather than regression.

[0105] Logistic regression applies regularization by default. This is very common in machine learning, and one of its advantages is that it improves numerical stability. Without regularization, it is equivalent to setting C to a very high value.

[0106] As an optimization problem, binary class L2 penalized logistic regression minimizes the following cost function:

[0107]

[0108] Similarly, L1 regularized logistic regression solves the following optimization problem:

[0109]

[0110] Use the sklearn.model_selection function to select 10 random numbers from 0 to 100 as random seeds, divide the samples into 10 groups of training sets and validation sets, perform 10-fold cross-validation, and take the average of the 10 results as the model result to ensure the objectivity of the model.

[0111] The cutoff values ​​and AUC values ​​of each model are as follows:

[0112] Table 1. AUC changes of each model under different cutoff values

[0113]

[0114]

[0115] The SNP screened using the logbf>3 standard was rs2547319;

[0116] The following 26 SNPs were screened using the logbf>2 standard:

[0117] rs10889315, rs75144949, rs149213974, rs754615, rs45583335, rs2529438, rs20 72236, rs2240403, rs78155900, rs114097201, rs2811736, rs2297722, rs2297723, rs117519669, rs7157977, rs10143899, rs3759779, rs143477571, rs2303221, rs34 693726, rs3803752, rs142921938, rs3746295, rs2547319, rs2547318, rs4911402;

[0118] The following 102 SNPs were screened using the logbf>1.5 standard:

[0119] rs4648600、rs1061624、rs2275365、rs35909785、rs3103778、rs2457829、rs10889315、rs78587889、rs12568050、rs12564283、rs528123098、rs139842833、rs74953908、rs1077827、rs12492484、rs75144949、rs3732755、rs3135115、rs16837029、rs149213974、rs17001890、rs56382032、rs12651031、rs2736100、rs7724759、rs754615、rs5369、rs150157174、rs130068、rs45583335、rs78342561、rs2529438、rs1049402、rs2072236、rs2527878、rs2240403、rs78155900、rs114097201、rs2680903、rs59612871、rs3818584、rs16909677、rs10983755、rs2248077、rs2811736、rs2297722、rs2297723、rs9987876、rs7904554、rs907609、rs117519669、rs3750996、rs3802799、rs2276296、rs139692587、rs904628、rs11551723、rs144796593、rs78648016、rs7157977、rs10143899、rs3759779、rs7142098、rs143477571、rs3736911、rs11849022、rs77481241、rs3212112、rs9282879、rs55958706、rs3212056、rs34100926、rs114770841、rs3742368、rs118146919、rs2289702、rs2303221、rs34693726、rs3744566、rs9907420、rs375644567、rs185576254、rs3803752、rs17822735、rs17222523、rs34818467、rs12946397、rs3809724、rs181969864、rs7236574、rs142921938、rs3746295、rs151309111、rs533816037、rs2302948、rs182509214、rs2547319、rs2547318、rs2281209、rs4911402、rs138894461、rs5756223;、

[0120] The following 342 SNPs were screened using the logbf>1 standard:

[0121] rs16824585、rs4648600、rs17033266、rs1061624、rs202140107、rs2291804、rs2275365、rs139241751、rs35909785、rs6676052、rs3103778、rs116893711、rs116861599、rs2457829、rs10889315、rs17855078、rs78587889、rs140141416、rs4656197、rs45445497、rs143355122、rs12568050、rs12564283、rs10482、rs1929861、rs75401237、rs28517482、rs1264208208、rs2631840、rs188480146、rs528123098、rs61732269、rs367931009、rs139842833、rs79908358、rs79358798、rs1434087、rs4894048、rs13421990、rs13419085、rs143196218、rs139713392、rs6734083、rs150887565、rs4663795、rs74953908、rs143001518、rs6437368、rs147838565、rs1077827、rs144623840、rs78233496、rs9876921、rs6622、rs2279909、rs547922447、rs300977、rs12492484、rs75144949、rs3732755、rs3135115、rs16837029、rs74787220、rs527903313、rs149213974、rs17001890、rs79769348、rs10025654、rs76996680、rs2306369、rs79482316、rs56382032、rs12651031、rs2736100、rs35130836、rs3749684、rs7724759、rs754615、rs45625534、rs3805625、rs137898880、rs147518598、rs202007699、rs2270900、rs75164992、rs17133512、rs76720430、rs5369、rs150157174、rs7756481、rs117298661、rs117644703、rs3094672、rs4713420、rs130068、rs67196728、rs185990098、rs9349593、rs143580489、rs3757302、rs3218602、rs9495370、rs45583335、rs78342561、rs117358940、rs9689983、rs2529438、rs1049402、rs2072236、rs2527878、rs2240403、rs781980620、rs56083327、rs2245368、rs2430307、rs3748126、rs36028884、rs78155900、rs114097201、rs13438494、rs117211799、rs115734214、rs148474510、rs200506987、rs8190955、rs8178175、rs2680903、rs117676215、rs115507207、rs369806130、rs78240711、rs59612871、rs117169590、rs3739523、rs145293869、rs139212809、rs3818584、rs16909677、rs141549766、rs147026086、rs188197626、rs13321、rs10983755、rs2301576、rs7038042、rs11849、rs2275161、rs2248077、rs2811736、rs2297722、rs2297723、rs9987876、rs7904554、rs148582275、rs3740168、rs149647444、rs7100474、rs7079648、rs202166096、rs60420182、rs36104328、rs7098255、rs3814163、rs2297785、rs3736924、rs3736923、rs141296329、rs117520659、rs57285449、rs112687793、rs73401681、rs79948463、rs10902170、rs10902171、rs867839、rs907608、rs907609、rs117519669、rs3750996、rs214086、rs3802799、rs76215382、rs147852038、rs2276296、rs143930161、rs183113103、rs139692587、rs10792769、rs7102675、rs116878689、rs35466364、rs142908193、rs7966459、rs7135745、rs34181394、rs1921038、rs201814780、rs11068531、rs904628、rs11551723、rs7983667、rs144796593、rs140526815_rs77878407、rs78646508、rs34523940、rs9521906、rs78648016、rs7157977、rs10143899、rs3759779、rs7142098、rs41309252、rs535020964、rs3742770、rs3742768、rs116881452、rs143477571、rs61992274、rs142973720、rs530110551、rs3742464、rs3736911、rs11849022、rs77481241、rs3212112、rs9282879、rs55958706、rs3212056、rs34100926、rs114770841、rs3742368、rs144857261_rs373032548、rs189734192、rs188393827、rs118146919、rs562829514、rs1805025、rs8192352、rs2289702、rs150921132、rs61869621、rs1802752、rs113355794、rs7198064、rs2291407、rs9941128、rs9941210、rs9941215、rs9931527、rs12708649、rs13306653、rs11643517、rs5723、rs7196736、rs2303221、rs2304483、rs149645127、rs11550470、rs17232091、rs150889000、rs16954357、rs371756524_rs557159990、rs192103504、rs115314145、rs34693726、rs2277651、rs2277652、rs71360269、rs71360270、rs3744566、rs3744568、rs9907420、rs375644567、rs185576254、rs2158460, rs3803752, rs17822735, rs17222523, rs34818467, rs12946397, rs3809724, rs35476409_rs3979 78887, rs3744214, rs181969864, rs4789773, rs28561791, rs7236574, rs76597875, rs61749945, rs11803323 4. rs1052696, rs1052692, rs148018996, rs34488963, rs3745379, rs3745385, rs142921938, rs3746295, rs15 1309111, rs139271842, rs140566569, rs533816037, rs78237760, rs79621739, rs75990051, rs62107869, rs30 5974, rs1055099, rs41290128, rs76151738, rs16980091, rs2302948, rs182509214, rs2547319, rs2547318, r s16982743, rs2284385, rs2281209, rs4911402, rs80158178, rs7509972, rs150511237, rs2231391, rs223139 0. rs762228, rs72564613, rs191574093, rs138894461, rs5999924, rs5750146, rs5756223, rs3747168, rs148 345118, rs910796, rs910797, rs910798, rs738712, rs16445, rs3922872, rs56248708, rs9628315, rs8137790. ,

[0122] Then optimize the above model, select the best threshold (cutoff) and draw the ROC curve (as shown in Table 2, Figure 1 shown)

[0123] Table 2. Optimal thresholds and AUC values ​​for each model

[0124] Model Threshold AUC Number of SNPs Model 1 -11.125 0.59 1 selected based on logbf>3 Model 2 -11.125 0.67 26 samples were screened based on the standard of logbf>2 Model 3 -11.25 0.77 102 samples were screened based on the standard logbf>1.5 Model 4 -11.125 0.80 342 samples were screened based on the standard logbf>1

[0125] That is, when a SNP site selected based on the logbf>3 standard is used as a marker, it can be effectively determined whether the subject's right eye equivalent spherical lens is above -11.125;

[0126] When the 26 markers screened out with logbf>2 as the standard were used, it was possible to effectively determine whether the subject's right eye spherical equivalent was above -11.125;

[0127] When the 102 markers screened with logbf>1.5 as the standard were used, it was possible to effectively determine whether the subject's right eye spherical equivalent was above -11.25;

[0128] When the 342 markers screened out with logbf>1 as the standard are used, it is possible to effectively determine whether the subject's right eye equivalent spherical lens is above -11.125.

Claims

1. A model for detecting the right eye spherical equivalent, wherein the model determines the subject's current right eye spherical equivalent based on the detection results of SNP locus combinations; The SNP site combination includes any one of the following groups: (1)rs10889315, rs75144949, rs149213974, rs754615, rs45583335, rs2529438, rs 2072236, rs2240403, rs78155900, rs114097201, rs2811736, rs2297722, rs2297723 , rs117519669, rs7157977, rs10143899, rs3759779, rs143477571, rs2303221, rs3 4693726, rs3803752, rs142921938, rs3746295, rs2547319, rs2547318, rs4911402; (2)rs4648600、rs1061624、rs2275365、rs35909785、rs3103778、rs2457829、rs10889315、rs78587889、rs12568050、rs12564283、rs528123098、rs139842833、rs74953908、rs1077827、rs12492484、rs75144949、rs3732755、rs3135115、rs16837029、rs149213974、rs17001890、rs56382032、rs12651031、rs2736100、rs7724759、rs754615、rs5369、rs150157174、rs130068、rs45583335、rs78342561、rs2529438、rs1049402、rs2072236、rs2527878、rs2240403、rs78155900、rs114097201、rs2680903、rs59612871、rs3818584、rs16909677、rs10983755、rs2248077、rs2811736、rs2297722、rs2297723、rs9987876、rs7904554、rs907609、rs117519669、rs3750996、rs3802799、rs2276296、rs139692587、rs904628、rs11551723、rs144796593、rs78648016、rs7157977、rs10143899、rs3759779、rs7142098、rs143477571、rs3736911、rs11849022、rs77481241、rs3212112、rs9282879、rs55958706、rs3212056、rs34100926、rs114770841、rs3742368、rs118146919、rs2289702、rs2303221、rs34693726、rs3744566、rs9907420、rs375644567、rs185576254、rs3803752、rs17822735、rs17222523、rs34818467、rs12946397、rs3809724、rs181969864、rs7236574、rs142921938、rs3746295、rs151309111、rs533816037、rs2302948、rs182509214、rs2547319、rs2547318、rs2281209、rs4911402、rs138894461、rs5756223;、 (3)rs16824585、rs4648600、rs17033266、rs1061624、rs202140107、rs2291804、rs2275365、rs139241751、rs35909785、rs6676052、rs3103778、rs116893711、rs116861599、rs2457829、rs10889315、rs17855078、rs78587889、rs140141416、rs4656197、rs45445497、rs143355122、rs12568050、rs12564283、rs10482、rs1929861、rs75401237、rs28517482、rs1264208208、rs2631840、rs188480146、rs528123098、rs61732269、rs367931009、rs139842833、rs79908358、rs79358798、rs1434087、rs4894048、rs13421990、rs13419085、rs143196218、rs139713392、rs6734083、rs150887565、rs4663795、rs74953908、rs143001518、rs6437368、rs147838565、rs1077827、rs144623840、rs78233496、rs9876921、rs6622、rs2279909、rs547922447、rs300977、rs12492484、rs75144949、rs3732755、rs3135115、rs16837029、rs74787220、rs527903313、rs149213974、rs17001890、rs79769348、rs10025654、rs76996680、rs2306369、rs79482316、rs56382032、rs12651031、rs2736100、rs35130836、rs3749684、rs7724759、rs754615、rs45625534、rs3805625、rs137898880、rs147518598、rs202007699、rs2270900、rs75164992、rs17133512、rs76720430、rs5369、rs150157174、rs7756481、rs117298661、rs117644703、rs3094672、rs4713420、rs130068、rs67196728、rs185990098、rs9349593、rs143580489、rs3757302、rs3218602、rs9495370、rs45583335、rs78342561、rs117358940、rs9689983、rs2529438、rs1049402、rs2072236、rs2527878、rs2240403、rs781980620、rs56083327、rs2245368、rs2430307、rs3748126、rs36028884、rs78155900、rs114097201、rs13438494、rs117211799、rs115734214、rs148474510、rs200506987、rs8190955、rs8178175、rs2680903、rs117676215、rs115507207、rs369806130、rs78240711、rs59612871、rs117169590、rs3739523、rs145293869、rs139212809、rs3818584、rs16909677、rs141549766、rs147026086、rs188197626、rs13321、rs10983755、rs2301576、rs7038042、rs11849、rs2275161、rs2248077、rs2811736、rs2297722、rs2297723、rs9987876、rs7904554、rs148582275、rs3740168、rs149647444、rs7100474、rs7079648、rs202166096、rs60420182、rs36104328、rs7098255、rs3814163、rs2297785、rs3736924、rs3736923、rs141296329、rs117520659、rs57285449、rs112687793、rs73401681、rs79948463、rs10902170、rs10902171、rs867839、rs907608、rs907609、rs117519669、rs3750996、rs214086、rs3802799、rs76215382、rs147852038、rs2276296、rs143930161、rs183113103、rs139692587、rs10792769、rs7102675、rs116878689、rs35466364、rs142908193、rs7966459、rs7135745、rs34181394、rs1921038、rs201814780、rs11068531、rs904628、rs11551723、rs7983667、rs144796593、rs140526815_rs77878407、rs78646508、rs34523940、rs9521906、rs78648016、rs7157977、rs10143899、rs3759779、rs7142098、rs41309252、rs535020964、rs3742770、rs3742768、rs116881452、rs143477571、rs61992274、rs142973720、rs530110551、rs3742464、rs3736911、rs11849022、rs77481241、rs3212112、rs9282879、rs55958706、rs3212056、rs34100926、rs114770841、rs3742368、rs144857261_rs373032548、rs189734192、rs188393827、rs118146919、rs562829514、rs1805025、rs8192352、rs2289702、rs150921132、rs61869621、rs1802752、rs113355794、rs7198064、rs2291407、rs9941128、rs9941210、rs9941215、rs9931527、rs12708649、rs13306653、rs11643517、rs5723、rs7196736、rs2303221、rs2304483、rs149645127、rs11550470、rs17232091、rs150889000、rs16954357、rs371756524_rs557159990、rs192103504、rs115314145、rs34693726、rs2277651、rs2277652、rs71360269、rs71360270、rs3744566、rs3744568、rs9907420、rs375644567、rs185576254、<h2 style=";text-align:left;direction:ltr">rs2158460, rs3803752, rs17822735, rs17222523, rs34818467, rs12946397, rs3809724, rs35476409_rs397978887, rs3744214, rs181969864, rs4789773, rs28561791, rs7236574, rs76597875, rs61749945, rs11803323 4, rs1052696, rs1052692, rs148018996, rs34488963, rs3745379, rs3745385, rs142921938, rs3746295, rs151309111, rs139271842, rs140566569, rs533816037, rs78237760, rs79621739, rs75990051, rs62107869, rs30 5974、rs1055099、rs41290128、rs76151738、rs16980091、rs2302948、rs182509214、rs2547319、rs2547318、r s16982743、rs2284385、rs2281209、rs4911402、rs80158178、rs7509972、rs150511237、rs2231391、rs2231390 rs762228, rs72564613, rs191574093, rs138894461, rs5999924, rs5750146, rs5756223, rs3747168, rs148345118, rs910796, rs910797, rs910798, rs738712, rs16445, rs3922872, rs56248708, rs9628315, rs8137790; The model was constructed using logistic regression.

2. A right eye spherical equivalent detection system, comprising a computing device for determining the right eye spherical equivalent of a subject using the detection results of the SNP site combination described in claim 1.

3. The detection system according to claim 2, wherein: The detection system further comprises a device for detecting, collecting or storing the detection results of the SNP site combination according to claim 1.

4. Use of the SNP site combination described in claim 1, the model described in claim 1, and the detection system described in claim 2 in the preparation of a product for detecting the equivalent spherical lens of the right eye.

Images