CN114574574A

CN114574574A - SNP markers related to quantitative traits of right-eye equivalent spherical lens and application thereof

Info

Publication number: CN114574574A
Application number: CN202210386981.2A
Authority: CN
Inventors: 徐良德; 王宏
Original assignee: Wenzhou Optometry International Innovation Center; Eye Hospital of Wenzhou Medical University
Current assignee: Wenzhou Optometry International Innovation Center; Eye Hospital of Wenzhou Medical University
Priority date: 2022-04-13
Filing date: 2022-04-13
Publication date: 2022-06-03

Abstract

The invention belongs to the technical field of biomedicine and gene detection, and particularly relates to a group of SNP markers related to quantitative traits of right-eye equivalent sphero-lenses and application thereof. The invention provides an application of a reagent for detecting rs2547319 in judging an equivalent sphere lens of a right eye.

Description

SNP markers related to quantitative traits of right-eye equivalent spherical lens and application thereof

Technical Field

The invention belongs to the technical field of biomedicine and gene detection, and particularly relates to a group of SNP markers related to quantitative traits of right-eye equivalent sphero-lenses and application thereof.

Background

Ophthalmic diseases refer to diseases occurring in the ocular region, and common ophthalmic diseases include myopia, astigmatism, cataract, glaucoma, central serous retinopathy, dry eye, sympathetic ophthalmia, nyctalopia, amblyopia, trachoma, diabetic retinopathy, conjunctivitis, presbyopia, achromatopsia, retinitis pigmentosa, central retinal artery occlusion, retinal detachment, hypermetropia, pinkeye, snow blindness, aragonioma, muscae volitantes, and the like. In the state of accommodation relaxation, parallel rays pass through the eye dioptric system and are focused in front of the retina, which is called myopia.

Diopter refers to the degree of power generated in the ametropic state, and when diopter occurs, it usually represents clinical performances such as myopia, astigmatism, hyperopia or amblyopia, the myopia and hyperopia are usually Spherical lenses (S), astigmatism is cylindrical lenses (C), and currently, the Spherical Equivalent power (SE) is adopted to evaluate diopter, and the Spherical Equivalent power is Spherical power + astigmatism power 1/2. Diopter is less than or equal to-0.50D and can be used as the evidence-based consensus threshold value for myopia diagnosis. More specifically, myopia is defined as the condition where the eye has an equivalent spherical refractive error of ≦ -0.5D when accommodating relaxation. High myopia is defined as the condition where the equivalent spherical refractive error of the eye is ≦ -6.00D when accommodation is relaxed. Low myopia is defined as the condition where the eye has a sphero-equivalent refractive error of ≦ 0.5 and > -6.00D when accommodation is relaxed.

Myopia has recently received increasing attention both at home and abroad as a global high-grade disease, and it is expected that by 2050, myopia affects nearly 50% of the global population, while high myopia affects nearly 10% of the global population.

Meanwhile, myopia is also the main eye disease affecting the visual health of young people in China at present, wherein high myopia is one of the main reasons causing eye blindness and low vision of eye disease patients in China, and can bring serious economic burden to the patients and families thereof. For the vision problems of children, the prevention is important, and the early discovery, early intervention and early treatment are the keys for preventing the myopic degree from being deepened. Eye diseases in the juvenile stage are extremely harmful to vision development, early discovery and diagnosis of non-ametropia eye diseases affecting vision are very important, and if a plurality of eye diseases cannot be discovered and treated in time, the eyes can be disabled for the lifetime. Although the rate of progression can be partially slowed by drugs, optical treatments and behavioral modification, there is a long way we have to reverse the trend over the past decades. This makes myopia and its associated complications a significant research concern. The occurrence of high myopia involves a multifactorial and complicated process, the pathogenesis is still unclear, and the current various optical correction means and surgical treatment methods, such as refractive surgery and scleral reinforcement surgery, cannot fundamentally prevent and delay the development of fundus lesions with high myopia, so that the effective treatment measures for high myopia are lacked at present. Therefore, finding a more effective method has important clinical significance for early detection, risk prediction and early intervention of high-myopia patients and high-risk groups.

Single Nucleotide Polymorphisms (snps) refer to DNA sequence Polymorphisms caused by Single base transitions, transversions or Nucleotide variations caused by base insertions and deletions on the genome level, are the most common variations among human heritable variations, account for more than 80% of all known Polymorphisms, and have variation frequency higher than 1% in the population, which is also an important factor distinguished from point mutations. In genetic analysis, SNPs have the characteristics of high frequency, stability, easy analysis and the like. The research finds that the SNPs have strong correlation with the occurrence and the development of myopia. The SNPs detection method mainly comprises a time-of-flight mass spectrometry (MALDI-TOFMS) technology, a fluorescence quantitative PCR technology, a gene chip technology, a deformed high performance liquid chromatography and the like.

With the advent of high-throughput SNP detection technology, as the most abundant and easily-detectable polymorphic markers in batches, SNPs will play an increasingly important role in linkage analysis and gene localization, including gene localization and association analysis of complex diseases, and research of individuals and populations on environmental pathogenic factors and drugs. With the development of technology, the cost of SNP detection becomes more and more economical, and SNP markers become a new generation of molecular markers.

Disclosure of Invention

The invention brings a large number of test population of 6-18 years old, collects oral mucosa samples meeting the standard through a Standard Operation Program (SOP), extracts DNA, types single nucleotide polymorphism, screens and finds SNP with high specificity and sensitivity highly related to the right eye equivalent spherical lens, further can develop a corresponding kit convenient for clinical application, provides data support for judging the right eye equivalent spherical lens of a test subject, can intervene in the early stage of the occurrence of ophthalmic problems, reduces the possibility of ophthalmic disease deterioration, and improves the life quality.

In order to achieve the technical purpose of the invention, the invention provides the following technical scheme:

in a first aspect, the invention provides the use of a reagent for detecting rs2547319 in the determination of equivalent sphericity lenses for the right eye.

In another aspect, the present invention provides a set of right eye equivalent sphere related SNP site combinations comprising any one of the following:

(1)rs10889315、rs75144949、rs149213974、rs754615、rs45583335、rs2529438、rs2072236、rs2240403、rs78155900、rs114097201、rs2811736、rs2297722、rs2297723、rs117519669、rs7157977、rs10143899、rs3759779、rs143477571、rs2303221、rs34693726、rs3803752、rs142921938、rs3746295、rs2547319、rs2547318、rs4911402(26)；

(2) rs4648600, rs1061624, rs2275365, rs35909785, rs3103778, rs 247829, rs10889315, rs78587889, rs12568050, rs12564283, rs528123098, rs139842833, rs74953908, rs1077827, rs12492484, rs75144949, rs3732755, rs3135115, rs16837029, rs149213974, rs17001890, rs12651031, rs2736100, rs7724759, rs 75465369, rs150157174, rs130068, rs45583335, rs 783434342561, rs2529438, rs 104949402, rs 2072232236, rs2527878, rs 224040900, rs 1145501, rs 26097297563756375637563756375637563756375637563756375637569, rs 3756375637563756375637563756375637563756375637569, rs 375637563756375637563756375637563756375637563756375637563756375637569, rs 3756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637569, rs 3756375637563756375637563756375637563756375637569, rs 3756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637569, rs 375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637569, rs 375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637569, rs 3756375637563756375637563756375637563756375637563756375637569, rs 375637563756375637563756375637563756375637569, rs 3756375637563756375637563756375637563756375637563756375637569, rs 3756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637569, rs 375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637569, rs 37563756375637563756375637569, rs 37563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637569, rs 375637569, rs 3756375637563756375637563756375637569, rs 375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637569, rs 375637563756375637569, rs 3756375637563756375637563756375637563756375637563756375637569, rs 375637563745, rs 37563745, rs 3756375637563756;

(3) rs 1684885, rs4648600, rs17033266, rs1061624, rs202140107, rs2291804, rs2275365, rs139241751, rs35909785, rs6676052, rs3103778, rs116893711, rs 11686159599, rs 24247829, rs10889315, rs 1785505078, rs78587889, rs 140141141141141141141141141416, rs4656197, rs 455497, rs 355122, rs 68125050 050, rs 64283, rs10482, rs 104989861, rs75401237, rs 28747774947494747708, rs 26840, rs188480146, rs 5213498, rs 6173799, rs 36367909, rs 139848433, rs 357992779, rs 949494949492779, rs 33779, rs 3292777992779, rs 3292779, rs 3277567756779, rs 3277563277798, rs 32775677798, rs 30056775677798, rs 300567756777997798, rs 30056777997798, rs 300563277798, rs 300563277563277798, rs 300563277798, rs 300563272798, rs 3005637798, rs 33798, rs 3356300 56300, rs 33798, rs 3356300 56300, rs 72798, rs 33798, rs 72798, rs 72849, rs 3356300 56300, rs 72849, rs 33849, rs 72849, rs 3356300 56300, rs 72849, rs 72798, rs 72849, rs 72798, rs 72849, rs 3256300, rs117676215, rs115507207, rs369806130, rs78240711, rs 59612812812871, rs117169590, rs3739523, rs145293869, rs139212809, rs3818584, rs 169909677, rs141549766, rs147026086, rs 188626, rs13321, rs10983755, rs7038042, rs11849, rs2275161, rs 8022477, rs2811736, rs2297722, rs 229797723, rs9987876, rs7904554, rs 148225875, rs3740168, rs149647444, rs7100474, rs7079648, rs 2020909090909420182, rs 60420420182, rs36104328, rs 709863, rs2297785, rs 3736936924, rs 3779435743574357799, rs 794354794354799, rs 43547756375637563756375637563756375637569, rs 375637563756375637563756375637563756375637563756375637563756375637569, rs 3756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637569, rs 37563756375637563756375637563756375637563756375637563756375637563756375637563756375637569, rs 3756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637569, rs 37563756375637563756375637563756375637563756375637569, rs 37563756375637563756375637563756375637563756375637563756375637563756375637569, rs 3756375637563756375637563756375637563756375637567, rs 37563756375637563756375637563756375637563756375637563756375637569, rs 729, rs 375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637569, rs 37563756375637563756375637563756375637563756375637563756375637569, rs 375637563756375637563756375637563756375637569, rs 375637563756375637567, rs 3756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637567, rs 375637563756375637569, rs 375637569, rs 3756375637567, rs 37567, rs 375637563756375637563756375637563756375637563756375637563756375637569, rs 37563756375637563756375637563756375637563756375637563756375637563756375637569, rs 37563756375637567, rs 3756375637563756375637563756375637563756375637563756375637567, rs 3756375637563756375637569, rs 3756375637563756375637567, rs 37563756375637563756375637563756375637563756375637567, rs 3756375637567, rs 375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637569, rs 375637563756375637563756375637563756375637563756375637567, rs 375637563756375637563756375637567, rs2303221, rs2304483, rs149645127, rs11550470, rs17232091, rs150889000, rs16954357, rs371756524_ rs 557990, rs192103504, rs115314145, rs34693726, rs2277651, rs2277652, rs71360269, rs71360270, rs3744566, rs3744568, rs9907420, rs 37564456565656567, rs 57628460, rs 380373803752, rs17822735, rs17222523, rs34818467, rs12946397, rs 9729724, rs 354709 _ rs 39779787, rs3744214, rs 1819696969664, rs4789773, rs 2856171791, rs 369797979797979745, rs 616199234, rs 105915637563756375637569, rs 563756375637563756375637563756375637569, rs 3756375637563756375637563756375637563756375637569, rs 3756375637563756375637563756375637563756375637563756375637563756375637569, rs 725637563756375637563756375637563756375637563756375637563756375637563756375637563756375637569, rs 375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637569, rs 3756375637563756375637563756375637563756375637563756375637563756375637563756375637563745, rs 37563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563745, rs 3756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563745, rs 3756375637563756375637563745, rs 375637563756375637563756375637563756375637563756375637563756375637563745, rs 3756375637563756375637563756375637563756375637563745, rs 37563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563745, rs 3756375637563745, rs3745, rs 37563756375637563756375637563756375637563756375637563745, rs 3756375637563745, rs3745, rs 37563745, rs 3756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563745, rs 375637563745.

In the present invention, SNP (single nucleotide polymorphism) refers to a single base position in DNA, and a subject may be homozygous or heterozygous. The SNP sites of the invention are named "rs-" and the person skilled in the art is able to determine their exact position, nucleotide sequence from a suitable database and related information systems, such as the single nucleotide polymorphism database (dbSNP), based on the rs-naming above. For SNPs, there are more than two alleles at each site, e.g., there are two alleles.

Preferably, the SNP locus combination is detected according to a sample of a subject, and then the equivalent spherical lens of the right eye of the subject is judged according to the detection result, and the risk of the ophthalmic problem is predicted.

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

preferably, the sample is a buccal swab (buccal mucosa sample);

on the other hand, the invention also provides a kit for detecting the right eye equivalent spherical lens, and the kit comprises a reagent for detecting the SNP locus combination.

Preferably, the reagent for detecting the combination of SNP sites includes, but is not limited to, the reagents used in the following methods for detecting SNPs: TaqMan probe method, sequencing method, chip method, flight mass spectrometer (MALDI-TOFMS) detection, restriction fragment length polymorphism (PCR-RFLP), single strand conformation polymorphism (PCR-SSCP), allele-specific PCR (AS-PCR), SNaPshot method, SNPlex method, Denaturing High Performance Liquid Chromatography (DHPLC), Denaturing Gradient Gel Electrophoresis (DGGE). One skilled in the art can select any one or several methods for detecting the SNP site as long as the detection of the SNP site can be achieved.

In the present invention, "an agent for detecting SNP" refers to an agent that specifically binds to a SNP marker or SNP included in the SNP marker composition so as to be recognized, or can detect the SNP and amplify, and "an agent that can amplify a SNP marker" refers to an agent that repeatedly replicates a SNP marker or SNP included in the SNP marker composition and increases the number thereof, for example, a primer that specifically amplifies a polynucleotide including the SNP or a probe that can specifically bind, but is not limited thereto.

The primers used for the SNP amplification may be appropriate conditions in an appropriate buffer (for example, 4 different nucleoside triphosphates, a polymerizing agent such as DNA, RNA polymerase or reverse transcriptase) and single-stranded oligonucleotides that function as a column indicating the origin of DNA synthesis at an appropriate temperature, and the appropriate length of the primers may vary depending on the purpose of use, but is usually 15 to 30 nucleotides. Short primer molecules generally require lower temperatures in order to form a mixture of columnar and stable molecules. The primer sequence need not form complete complementarity with the polynucleotide including the SNP, but its complementarity needs to be such that it can be mixed with the polynucleotide including the SNP.

In one embodiment, the reagent for detecting a combination of SNP sites is a primer set and/or a probe set for amplifying the aforementioned combination of SNP sites.

In one embodiment, the kit may further comprise common reagents required for corresponding PCR techniques, such as dNTPs, MgCl2, double distilled water, fluorescent probes, etc., which are well known to those skilled in the art, in addition to standards and controls (e.g., genotype standards and blank controls, etc.).

In another aspect, the present invention provides a system for detecting equivalent sphere lens for right eye, which comprises a computing device for determining equivalent sphere lens for right eye of a subject using the detection result of the above-mentioned SNP site combination.

Preferably, the diagnostic system may further include a detection device for the SNP sites, such as a sequencer or the like.

On the other hand, the invention provides a method for constructing an equivalent sphere lens model for detecting the right eye by using the SNP locus combination.

Preferably, the method uses a logistic regression method.

Preferably, the present invention further comprises a step of 10-fold cross-validation. The 10-fold cross validation specifically refers to selecting 10 random numbers from 0 to 100 as random seeds by using a sklern model _ selection function, dividing samples into 10 training sets and validation sets respectively, performing 10-fold cross validation, and taking the average of 10 results as a model result.

On the other hand, the invention also provides a model for detecting the equivalent spherical lens of the right eye, and the model judges the lower equivalent spherical lens of the right eye of the subject according to the detection result of the SNP locus combination.

Preferably, the model is constructed by the method.

Preferably, the model may be a formula, nomogram, or other manner of facilitating manipulation by the subject; according to the model, the right eye equivalent sphere lens of the subject can be directly calculated.

On the other hand, the invention provides a reagent for detecting the SNP locus combination, and application of the kit, the system and the model in preparation of products for detecting right-eye equivalent spheroscopes.

Preferably, the reagent for detecting the combination of the SNP sites includes, but is not limited to, reagents used in the detection of SNPs by the following methods: TaqMan probe method, sequencing method, chip method, flight mass spectrometer (MALDI-TOFMS) detection, restriction fragment length polymorphism (PCR-RFLP), single strand conformation polymorphism (PCR-SSCP), allele-specific PCR (AS-PCR), SNaPshot method, SNPlex method, Denaturing High Performance Liquid Chromatography (DHPLC), Denaturing Gradient Gel Electrophoresis (DGGE). One skilled in the art can select any one or several methods for detecting the SNP site as long as the detection of the SNP site can be achieved.

Specifically, the reagents include, but are not limited to, primers, probes, chips, and the like.

In another aspect, the present invention provides a method for detecting a right-eye equivalent sphere lens, the method comprising the step of judging a right-eye equivalent sphere lens of a subject based on the detection result of the SNP site combination according to the present invention,

in particular, the method may comprise the steps of:

1) collecting a subject sample, preferably said sample is an oral swab (oral mucosal sample);

2) carrying out SNP detection on the sample, wherein the detection also comprises a step of extracting DNA;

3) judging the right eye equivalent sphere lens of the subject according to the detection result of 2).

The determination may be manually, automatically, or a combination thereof to perform or complete the selected task; the result can be calculated manually according to the detection result or automatically by inputting the detection result into the system.

The "Spherical Equivalent Refraction (SE)" of the present invention is equal to the Spherical power + the astigmatism power 1/2. Diopter is less than or equal to-0.50D and can be used as the evidence-based consensus threshold value for myopia diagnosis. More specifically, myopia is defined as the condition where the eye has an equivalent spherical refractive error of ≦ -0.5D when accommodating relaxation. High myopia is defined as the condition where the equivalent spherical refractive error of the eye is ≦ -6.00D when accommodating relaxation. Low myopia is defined as the condition where the eye has a sphero-equivalent refractive error of ≦ 0.5 and > -6.00D when accommodation is relaxed.

The Single Nucleotide Polymorphisms (SNPs) refer to genetic markers formed by variation of Single Nucleotide on a genome, including conversion, transversion, deletion and insertion, and are large in quantity and rich in polymorphism. There is one SNP in about every 1000 bases in the human genome, and the total amount of SNPs in the human genome is about 3X 10⁶And (4) respectively.Thus, SNPs become third generation genetic markers, and many phenotypic differences in humans, susceptibility to drugs or diseases, and the like may be associated with SNPs.

The term "detecting" as used throughout the specification of the present invention means determining the right eye equivalent sphere lens of the subject as it is.

Specifically, the technical solution of the present invention to solve the problem includes:

(1) establishing a unified specimen library and a database: collecting blood samples meeting the standard by a Standard Operating Procedure (SOP), and collecting complete demographic data and clinical data by a system;

(2) and (3) genotype detection: bringing in a sample which meets the regulation, and finding out SNP related to the right eye equivalent spherical lens in the whole gene range by utilizing a high-density SNP chip;

(3) further detecting the screened positive associated SNP in another sample to verify the repeatability and stability of the SNP applied to clinical diagnosis;

(4) development of a right eye equivalent sphere detection kit: and developing a detection kit according to the SNP which is obviously associated with the right eye equivalent spherical lens.

Drawings

FIG. 1 is a curve showing the variation of AUC values of the selected SNP combinations of the present invention in determining the right eye spheroscope at different thresholds.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

Example 1 sample Collection, sequencing and data analysis

Inclusion criteria for samples:

(1) the equivalent spherical lens of the worst eye is less than or equal to-6.00D;

(2) age 6-18 years;

(3) no other ocular diseases, congenital genetic diseases, other systemic diseases and physical abnormalities;

(4) no history of ocular surgery;

(5) chinese people;

(6) voluntarily attend and sign an informed consent.

The invention arranges sample information of myopia screening and intervention projects of middle and primary school students in the whole Wenzhou city, which are developed by cooperation of an eye vision hospital affiliated to the Wenzhou medical university and the Wenzhou city government in 2019-2020, collects 10348 (9852-8961) samples meeting the inclusion standard to sequence the whole exome, records the sphero-lenses (Sph) and the column lenses (Cyl) of two eyes of the students, and calculates the equivalent sphero-lenses (SE ═ Sph +0.5Cyl) of the two eyes. Meanwhile, the 10348 cases of high myopia patients were subjected to whole exome sequencing to obtain SNPs of whole exome, and SNPs related to traits were obtained by whole exome association analysis.

The specific experimental steps are as follows:

in the embodiment, oral mucosa samples meeting the standard are collected by a Standard Operating Program (SOP), a system collects and visits patients meeting the inclusion standard, an Illumina Novaseq6000 sequencing system is used for sequencing all exons, SNP in the range of all exons is detected, SNP markers and combinations thereof related to the equivalent Spheroscope (SE) of a high-myopia population are mined, and a model for predicting the progress risk of the equivalent spheroscope is constructed.

1. Collecting an oral mucosa swab:food and water intake was stopped half an hour before sample collection to ensure that the sample was not contaminated. The collected oral mucosa swab is put into a sterilized 1.5ml EP tube, filled with a check-in registry and transported to a company at normal temperature for handing in professional personnel for extracting the genomic DNA of the oral mucosa swab.

2. DNA extraction:extracting genomic DNA from an oral mucosa Swab by using a TIANAmp Swab DNA Kit, and specifically comprising the following steps:

(1) the buccal mucosal swab was transferred to a 2ml centrifuge tube, the swab section was cut from its shaft with scissors, and 400. mu.l of buffer GA was added.

(2) Add 20. mu.l of Proteinase K solution, vortex for 10 seconds and mix, stand at 56 ℃ for 60 minutes, vortex for several times every 15 minutes.

(3) 400. mu.l of buffer GB was added thereto, the mixture was thoroughly mixed by inversion, and the mixture was left at 70 ℃ for 10 minutes. At this point the solution was strained to clear, centrifuged briefly to remove the droplets on the inner wall of the tube cap, then the swab was squeezed off and as much lysate as possible was transferred to a new centrifuge tube.

(4) Adding 200 μ l of anhydrous ethanol, mixing thoroughly, centrifuging briefly to remove the liquid drop on the inner wall of the tube cover

(5) The solution and flocculent precipitate obtained in the previous step were added to an adsorption column CR2 (adsorption column CR2 was placed in the collection tube), centrifuged at 12,000rpm (13,400 Xg) for 30 seconds, the waste liquid in the collection tube was decanted, and adsorption column CR2 was returned to the collection tube.

(6) 500. mu.l of buffer GB (before use, whether or not absolute ethanol was added) was added to the adsorption column CR2, and the mixture was centrifuged at 12,000rpm (. about.13,400 Xg) for 30 seconds, and the waste solution in the collection tube was discarded to return the adsorption column CR2 to the collection tube.

(7) 700. mu.l of the rinsing solution PW (previously used to confirm whether or not absolute ethanol was added) was added to the adsorption column CR2, and the tube was centrifuged at 12,000rpm (. about.13,400 Xg) for 30 seconds to discard the waste liquid in the collection tube and return the adsorption column CR2 to the collection tube.

(8) Operation 7 is repeated.

(9) The solution was centrifuged at 12,000rpm (. about.13,400 Xg) for 2 minutes, the waste solution was decanted, and the adsorption column CR2 was left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.

(10) The adsorption column CR2 was transferred to a clean centrifuge tube, 20-50. mu.l of elution buffer TB was added dropwise to the middle of the adsorption membrane, left at room temperature for 2-5 minutes, and centrifuged at 12,000rpm (13,400 Xg) for 2 minutes.

(11) The concentration and purity of the DNA fragment were checked by agarose gel electrophoresis and UV spectrophotometer. The DNA has a significant absorption peak at OD260, and an OD260 value of 1 is equivalent to 50. mu.g/ml of double-stranded DNA and 40. mu.g/ml of single-stranded DNA. The purity (OD 260/280) was 1.7-1.9. A single sample typically gives 0.5-3.5. mu.g of DNA.

3. Constructing a pre-library:50ng of genome DNA is broken into small fragments of about 200bp by an enzymology method, then terminal repair and 3' end A addition operation are carried out, then the DNA fragments are connected with a sequencing adaptor containing a barcode sequence, fragments of about 320bp are selected and recovered, and a pre-library is obtained after PCR amplification.

4. Liquid phase hybridization capture operation:the pre-library was subjected to a liquid phase hybridization capture operation, referred to the standard protocol of XGen Exome Research Panel V1.0(Integrated DNA Technologies, San Diego, USA) of IDT.

5. Obtaining an exon library:and after eluting and recovering the captured product, carrying out PCR amplification and purification to obtain the exon library. The library was quantified by qPCR and the band size was measured using Agilent 2100.

6. Illumina Novaseq6000 sequencing:using an Illumina Novaseq6000 sequencing system to perform 150PE sequencing on the exon library, and using CASAVA v1.82 software to perform base recognition on an original image to produce original sequencing data.

7. Aligning the human reference genome:the sequenced fragments were aligned to the human reference genome (UCSC hg19) using the Burrows-Wheeler Aligner (BWA) tool and PCR repeats were removed using Picard v 1.57. And (3) performing mutation detection by adopting software GATK, and performing statistics on sequencing depth, coverage depth, uniformity and the like.

8. Data quality control

For the samples: the sampling amount of the oral swab is insufficient, the detection rate is less than 90%, the average coverage is less than 10, the average genotype existence probability is less than 65%, the average value of the genotype heterozygosity rate deviates from plus or minus 4 Standard Deviations (SD), the chromosome abnormality and the gender abnormality have genetic relationship and are not samples of east Asian population. For SNPs: and (3) detecting the full exome detection result which does not pass through the quality control of the VQSR, wherein the genotype detection rate is less than 90%, the Ha-Renberg P value is less than 1e-06, and the variation Allole number (AC) is 0 SNP.

9. Statistical analysis method

The research on the quantitative character of the full-exome of the high myopia is carried out through Bayesian test (Bayesian Tests) in SNPtest, and the potential SNP and gene which are obviously related to the clinical state of the high myopia are identified. The Bayesian Tests of quantitative traits were performed by using a conjugate prior formula of a linear model of the expected genotype (-method expected). For an additive model, the formula is:

y_i＝βG_i+e_i,e_i～N(0,σ²)。

where yi is the residual phenotype of the ith individual. The residual phenotype is calculated by subtracting a baseline term and estimating any specified covariates. Gi is an additive code for the expected genotype of the ith individual, with wild homozygous type being "0", heterozygous type being "1", and mutant homozygous type being "2".

σ 2 is the error variance of the model. And σ²～IG(a,β),β～N(m_β,V_βσ²) Default a is 3, β is 2, m_β＝0,V_β0.02. Age and gender were corrected as covariates.

Results of the experiment

1. snps selects:

genome-wide associations of 89095 common variations out of 8961 high myopia samples with quantitative right eye equivalent spheroids were obtained using SNPTEST.

Modeling was performed by screening snps separately through 4 thresholds: log10(BF) > 3; log10(BF) > 2; log10(BF) > 1.5; log10(BF) >1, yielding 342 snps in total.

2. Raw snps genotype profiling:

extracting the corresponding required snps (342 snps in total) in the vcf by using python, selecting an additive model (unmutated is set to be 0, one allele is mutated to be 1, and two alleles are mutated to be 2), and obtaining an original snp genotype spectrum.

3. snps genotype profiling complement deletion:

calculating the number of snps deletions in each sample in the original snps genotype profile, and if the deletions are > 5% (N _ deletions/N _ allosamples), removing the corresponding samples. For samples with deletion condition < 5%, the artificial neural network is used for filling the deletion.

The artificial neural network has three layers including an input layer, a hidden layer and an output layer: the number of nodes in the input layer is len (label _ data), that is, the snp without missing completely; the hidden layer is provided with 20 nodes; the output layer is provided with 3 nodes; the learning rate was set to 0.003 and the number of training iterations was set to 20.

4. Classification model: logistic Regression

1) And according to the quantitative character right eye equivalent sphere lens, taking different thresholds, respectively establishing models, investigating AUC values, and taking the threshold of the model with the maximum AUC as the threshold of the final classification model.

The rules are as follows:

traversing all values of the right-eye equivalent spherical mirror, and dividing the samples into two groups respectively as threshold values;

② the number of samples in each group must not be less than 100

2) Samples were classified using logistic regression with the following parameters:

although its name is logistic regression, it is a linear model used for classification rather than regression.

By default, the logistic regression applies regularization. This is common in machine learning, and one advantage is that the stability of the values is improved, with no regularization being equivalent to setting C to a very high value.

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

similarly, the L1 regularized logistic regression solves the following optimization problem:

a sklern model _ selection function is utilized, 10 random numbers from 0 to 100 are selected as random seeds, samples are divided into 10 groups of training sets and verification sets respectively, 10 times of cross verification is carried out, and the average of 10 results is taken as a model result so as to ensure the objectivity of the model.

The cutoff values and their AUC values for each model are as follows:

TABLE 1 AUC Change under different cutoff values for each model

By logbf>The SNP screened for criteria 3 is rs 2547319;

by logbf>The SNPs screened for criteria 2 were the following 26:

rs10889315、rs75144949、rs149213974、rs754615、rs45583335、rs2529438、rs2072236、rs2240403、rs78155900、rs114097201、rs2811736、rs2297722、rs2297723、rs117519669、rs7157977、rs10143899、rs3759779、rs143477571、rs2303221、rs34693726、rs3803752、rs142921938、rs3746295、rs2547319、rs2547318、rs4911402；

by logbf>1.5 the SNPs screened for the criteria were the following 102:

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；

by logbf>The SNPs screened for criteria 1 were 342 as follows:

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_rs397978887、rs3744214、rs181969864、rs4789773、rs28561791、rs7236574、rs76597875、rs61749945、rs118033234、rs1052696、rs1052692、rs148018996、rs34488963、rs3745379、rs3745385、rs142921938、rs3746295、rs151309111、rs139271842、rs140566569、rs533816037、rs78237760、rs79621739、rs75990051、rs62107869、rs305974、rs1055099、rs41290128、rs76151738、rs16980091、rs2302948、rs182509214、rs2547319、rs2547318、rs16982743、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。

then, the models are optimized, and an optimum threshold (cutoff) and a ratio are screened to draw an ROC curve (as shown in table 2 and figure 1)

TABLE 2 optimal threshold values and AUC values for each model

Model (model)	Threshold value	AUC	Number of SNPs
				Model 1	-11.125	0.59	By logbf>3 is 1 screened from the standard
Model 2	-11.125	0.67	By logbf>2 are 26 screened for the standard
				Model 3	-11.25	0.77	By logbf>1.5 102 screened for Standard
Model 4	-11.125	0.80	By logbf>342 screened for 1 standard

That is, when 1 SNP site screened by logbf >3 as a standard is used as a marker, whether the right eye equivalent sphere lens of the subject is above-11.125 can be effectively judged;

when 26 screened by using logbf >2 as a standard are taken as markers, whether the right eye equivalent sphere lens of the subject is above-11.125 can be effectively judged;

when 102 screened by taking logbf >1.5 as a standard are taken as markers, whether the right eye equivalent sphere lens of the subject is above-11.25 can be effectively judged;

when 342 screened by using logbf >1 as a standard is used as a marker, whether the right eye equivalent sphere lens of the subject is above-11.125 can be effectively judged.

Claims

1. The application of the reagent for detecting rs2547319 in judging the equivalent sphere lens of the right eye.

2. The use of claim 1, wherein the reagent for detecting rs2547319 comprises, but is not limited to, reagents used in the detection of SNPs by: TaqMan probe method, sequencing method, chip method, flight mass spectrometer detection, restriction fragment length polymorphism method, single-strand conformation polymorphism method, allele specific PCR, SNaPshot method, SNPlex method, denaturing high performance liquid chromatography, denaturing gradient gel electrophoresis method.

3. A set of right eye equivalance sphere related SNP site combinations, the SNP site combinations comprising any one of the following sets:

(1)rs10889315、rs75144949、rs149213974、rs754615、rs45583335、rs2529438、rs2072236、rs2240403、rs78155900、rs114097201、rs2811736、rs2297722、rs2297723、rs117519669、rs7157977、rs10143899、rs3759779、rs143477571、rs2303221、rs34693726、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、rs2158460、rs3803752、rs17822735、rs17222523、rs34818467、rs12946397、rs3809724、rs35476409_rs397978887、rs3744214、rs181969864、rs4789773、rs28561791、rs7236574、rs76597875、rs61749945、rs118033234、rs1052696、rs1052692、rs148018996、rs34488963、rs3745379、rs3745385、rs142921938、rs3746295、rs151309111、rs139271842、rs140566569、rs533816037、rs78237760、rs79621739、rs75990051、rs62107869、rs305974、rs1055099、rs41290128、rs76151738、rs16980091、rs2302948、rs182509214、rs2547319、rs2547318、rs16982743、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。

4. a kit for detecting a right-eye Equivalence Lenticular lens, which comprises a reagent for detecting the combination of SNP sites according to claim 3.

5. A model for detecting right eye equivalent spherical lens, which judges the right eye equivalent spherical lens of a subject according to the detection result of the SNP locus combination of claim 3.

6. A method of constructing the model of claim 5.

7. The method of claim 6, comprising using logistic regression.

8. A system for detecting a right-eye equivalent sphere lens, comprising a computing means for determining a right-eye equivalent sphere lens of a subject using a detection result of the SNP site combination according to claim 3.

9. The detection system of claim 8, further comprising means for detecting, collecting or storing the results of the detection of the combination of SNP sites of claim 3.

10. The use of the SNP site combination according to claim 3, the kit according to claim 4, the model according to claim 5 and the detection system according to claim 8 in the preparation of products for detecting right-eye equivalent spherical lenses.