Application of CHRM3 gene polymorphism in diagnosis or screening of atropine susceptible myopia population

Abstract

The invention discloses an application of CHRM3 gene polymorphism in diagnosis or screening of atropine susceptible myopia crowds, and finds that an SNP site rs60234066 is related to diagnosis of the atropine susceptible myopia crowds for the first time, and whether a tested crowd is the atropine susceptible myopia crowd or not is judged by detecting an SNP genotype. The SNP site rs60234066 has relatively high accuracy in diagnosis of atropine susceptible myopia people, can be used for early diagnosis or screening of the atropine susceptible myopia people to improve the early intervention rate of related disease people, so that the clinical guidance effect of the related disease people is improved, the myopia progress is reduced, and the SNP site rs60234066 has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the application of CHRM3 gene polymorphism in the diagnosis or screening of atropine-susceptible myopia population, and more specifically, the SNP involved is rs60234066. Background Art

[0002] Myopia is not only the leading cause of correctable visual impairment, but is also associated with a range of vision disorders, such as myopic retinopathy, retinal detachment, and choroidal neovascularization. Therefore, there is an urgent need to slow the progression of myopia to prevent these potential complications. Currently, 0.01% atropine eye drops have been widely studied and evaluated as the most effective drug for slowing the progression of myopia in children. Despite the widespread use of this drug, there is evidence that a subset of pediatric patients respond poorly to 0.01% atropine eye drops. Approximately half of children experience myopia progression of more than 0.50D per year after treatment with 0.01% atropine. There is individual variability in the response to 0.01% atropine eye drops. The mechanisms that lead to poor response to 0.01% atropine eye drops have not been fully elucidated. While some studies have linked poor response to treatment with younger age, a family history of myopia, and higher initial myopia levels, other studies have contradicted these findings. The lack of consensus on predictors of poor response to treatment highlights the need for a more targeted approach. Developing an in vitro genetic testing kit suitable for early screening of the therapeutic effects of atropine and increasing the early intervention rate of people with related diseases is of great significance for improving clinical guidance and reducing the progression of myopia.

[0003] The genomic association analysis method can more effectively identify genetic variations associated with the effect of atropine by limiting the relevant gene set and detecting the association between single nucleotide polymorphisms (SNPs) and phenotypes within the genome. EMMAX (Efficient Mixed-Model Association eXpedited), as a GWAS method based on a mixed model, has high analysis accuracy and computational efficiency because it takes into account the influence of population structure and genetic background. At present, traditional methods for evaluating the therapeutic effect of atropine mainly rely on the observation of clinical phenotypes, such as changes in visual acuity and changes in axial length. However, these methods often have difficulty in accurately predicting individual treatment responses. In addition, methods based on statistical models, such as linear regression models, can analyze the association between genes and phenotypes to a certain extent, but they are insufficient in dealing with complex gene-environment interactions. In addition, there is currently no evaluation of the effect of atropine on individuals from a genetic perspective for myopic people to screen susceptible populations. Summary of the invention

[0004] In view of this, in order to overcome the technical problems existing in the above-mentioned prior art, the purpose of the present invention is to provide an application of CHRM3 gene polymorphism in the diagnosis or screening of atropine-susceptible myopia population, the specific SNP involved is rs60234066, and the SNP genotype is detected to determine whether the subject population is an atropine-susceptible myopia population.

[0005] The above-mentioned object of the present invention is achieved through the following technical solutions: The first aspect of the present invention provides the use of a reagent for detecting SNP markers in a sample of a subject in the preparation of a product for diagnosing or screening atropine-susceptible myopia population.

[0006] Furthermore, the SNP marker is rs60234066.

[0007] Further, the reagents include reagents for detecting the rs60234066 genotype by direct sequencing, single base extension, allele-specific probe hybridization, allele-specific primer extension, allele-specific nucleotide incorporation, 5' nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, MALDI-TOF / MS, RCA, imprint hybridization, dot hybridization or single-stranded conformation polymorphism method.

[0008] Furthermore, the atropine-susceptible myopic population carries the rs60234066 mutation (rs60234066 C>CT).

[0009] Further, the sample is selected from blood or tissue; Optionally, the reagent includes a specific primer, a specific probe or a specific antibody for detecting rs60234066; Optionally, the reagents further include a reagent for processing the subject sample to obtain nucleic acid; Optionally, the product includes a kit or a chip.

[0010] In some embodiments, the SNP (single nucleotide polymorphism) refers to a single nucleotide polymorphism, i.e., a nucleic acid sequence polymorphism caused by a change in a single nucleotide. This variation is very common in the human genome, and it is estimated that there is about one SNP per thousand bases.

[0011] In some embodiments, the SNP marker rs60234066 is a SNP site on the CHRM3 gene. The CHRM3 (cholinergic receptor muscarinic 3 [Homo sapiens (human)]) gene has a Gene ID of 1131 in NCBI, and its corresponding detailed information can be obtained at https: / / www.ncbi.nlm.nih.gov / .

[0012] In some embodiments, the subject refers to any animal, and also refers to humans and non-human animals. The non-human animals include all vertebrates, for example, mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; and non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human, and in a more preferred embodiment, the subject is a myopic person.

[0013] In some embodiments, the sample refers to any composition containing or assumed to contain a target nucleic acid. This includes samples of tissues or fluids isolated from individuals, for example, skin, plasma, serum, spinal fluid, lymph, synovial fluid, urine, tears, blood cells, organs, and also represents samples of in vitro cultures established from cells taken from individuals, including formalin-fixed paraffin-embedded tissues (FFPET) and nucleic acids isolated therefrom. The sample may also include cell-free material, such as a fraction containing cell-free DNA (cfDNA). The sample may also refer to a processed tissue or biological fluid, for example, a purified or partially purified nucleic acid.

[0014] In some embodiments, the genotype refers to the identity of the alleles present in an individual or sample. Typically, it refers to the genotype of an individual associated with a particular gene of interest; in a polyploid individual, it refers to what combination of gene alleles an individual carries.

[0015] In some embodiments, the reagent for detecting SNP sites refers to a reagent that specifically binds to the SNP marker or SNP to facilitate identification, or can detect and amplify the SNP, and the reagent that can amplify the SNP marker refers to a reagent 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.

[0016] The primers used for the SNP amplification can be appropriate conditions in a suitable buffer (e.g., 4 different nucleoside triphosphates, DNA, RNA polymerase or reverse transcriptase and other polymerizing agents) and single-stranded oligonucleotides that can be used as the starting point of columnar DNA synthesis at an appropriate temperature. Depending on the purpose of use, the appropriate length of the primers will be different, but is usually 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 be able to mix with the polynucleotide comprising the SNP.

[0017] In some embodiments, the primer is the same as an amplification primer, which refers to an oligonucleotide that can be used in an amplification method (such as polymerase chain reaction (PCR)) to amplify a nucleotide sequence based on a polynucleotide sequence corresponding to a target gene. The primer generally refers to a nucleic acid fragment containing 5-100 nucleotides. In a preferred embodiment, the primer or amplification primer contains 15-30 nucleotides that can initiate an enzymatic reaction (e.g., an enzymatic amplification reaction).

[0018] In some embodiments, the probe refers to a molecule that can bind to a specific sequence or subsequence or other part of another molecule. In other embodiments, the probe refers to any molecule that can be indirectly or directly, covalently or non-covalently bound to any substrate and / or reaction product and / or protease disclosed herein or associated with it, and its association or binding can be detected using the methods disclosed herein. In other embodiments, the probe is a fluorescent probe, an antibody, or a probe based on absorbance. If it is an absorbance-based probe, the chromophore pNA (p-nitroaniline) can be used as a probe for detecting and / or quantifying the target nucleic acid sequence disclosed herein. In some embodiments, the probe can be a nucleic acid sequence comprising a fluorescent molecule or substrate that becomes fluorescent when exposed to an enzyme, and the nucleic acid sequence is complementary to a fragment of a nucleic acid sequence.

[0019] Unless otherwise indicated, in the present invention, a probe generally refers to a polynucleotide probe that can bind to another polynucleotide (often referred to as a target polynucleotide) through complementary base pairing. Depending on the stringency of the hybridization conditions, the probe can bind to a target polynucleotide that lacks complete sequence complementarity with the probe. Hybridization methods include, but are not limited to, solution phase, solid phase, mixed phase, or in situ hybridization assays. Exemplary probes in the present invention include gene-specific DNA oligonucleotide probes, such as microarray probes fixed on a microarray substrate, quantitative nuclease protection test probes, probes connected to molecular barcodes, and probes fixed on beads.

[0020] In some embodiments, the probe used for the SNP site detection can be a mixed probe or an oligonucleotide that can bind to a complementary nucleic acid chain based on a specific sequence. The mixing conditions should show significant differences in the mixing intensity between alleles so as to be strictly controlled so that it is only mixed into one of the alleles. Optimally, the central portion of the probe described in the present invention is arranged with the diversity portion of the diversity sequence. Therefore, optimized mixing differences between mutually different allelic morphologies may be produced. The probe can be used to detect alleles. In addition, the probe can be labeled with a radioactive isotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelate or an enzyme. Properly labeling the probe is a well-known technique in the art and can be implemented using conventional methods.

[0021] In some embodiments, the antibody refers to a specific immunoglobulin for an antigenic site, which is well known in the art. The antibody described in the present invention can be manufactured according to conventional methods in the art. The form of the antibody includes polyclonal antibodies or monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2 and Fv fragments), single-chain Fv (scFv) antibodies, multispecific antibodies (such as bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising the antigen binding site of the antibody, and any other modified immunoglobulin molecules comprising the antigen binding site, as long as the antibody exhibits the desired biological binding activity.

[0022] In some embodiments, the detection of rs60234066 genotype can be performed by any one or more of the following methods: direct sequencing, single base extension, allele-specific probe hybridization, allele-specific primer extension, allele-specific amplification, allele-specific nucleotide incorporation, 5' nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, MALDI-TOF / MS, RCA, imprint hybridization, dot hybridization or single-stranded conformation polymorphism method. It should be noted that those skilled in the art can select any one or more of the above methods (not limited to the above methods) to detect the SNP site described in the present invention, as long as the method can detect the genotype of the SNP site or detect whether the SNP site has a mutation, it can be applied to the present invention.

[0023] In some embodiments, the direct sequencing method is the most direct and reliable method for detecting SNPs, with a detection rate of up to 100%. Representative sequencing technologies include pyrosequencing, Taqman technology, and microsequencing (SNaPshot). This method detects SNPs by comparing the sequencing results of PCR amplification products of the same gene or gene fragment in different samples, or by resequencing and analyzing the located sequence tag sites (STS) and expressed sequence tags (EST). The PCR product can be purified and recovered and then connected to a vector for sequencing, or the PCR product can be sequenced directly. By comparing the sequences, the mutation type and position of the SNP can be accurately detected.

[0024] In some embodiments, the principle of allele-specific PCR (AS-PCR) is that Taq DNA polymerase cannot repair the mismatch of a single base at the 3' end of the primer. When the base at the 3' end of the primer is complementary to the allele at the SNP site, the amplification reaction can only occur; when the base at the 3' end of the primer is not complementary to the allele at the SNP site, the amplification reaction cannot occur. At present, some methods based on the improvement of AS-PCR have emerged, such as tetra-primer amplification refractory mutation system PCR (tetra-primer ARMS-PCR), fragment length discrepant allele specific PCR (fragment length discrepant allele specific PCR, FLDAS-PCR), PCR amplification of multiple specific alleles (PMASA), etc.

[0025] A second aspect of the present invention provides a product for diagnosing or screening atropine-susceptible myopic population.

[0026] Furthermore, the product includes a reagent for detecting the genotype of the SNP site rs60234066 in a subject's sample.

[0027] Further, the product includes specific primers, specific probes or specific antibodies for detecting rs60234066; Optionally, the product further comprises a reagent for processing the subject sample to obtain nucleic acid; Optionally, the product includes a kit or a chip.

[0028] In some embodiments, the kit includes, but is not limited to, a PCR kit, a RT-PCR kit, or a DNA chip kit.

[0029] In some embodiments, in addition to the specific polynucleotide, primer or probe of the SNP, the PCR kit may also include: test tubes or other appropriate containers, reaction buffer (various pH and magnesium concentrations), deoxyribonucleic acid (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitor, DEPC-water (DEPC-water) and sterile water, etc.

[0030] In some embodiments, in addition to the various primers specific to the SNP, the RT-PCR kit may also include: test tubes or other appropriate containers, reaction buffer (with various pH values ​​and magnesium concentrations), deoxynucleotides (dNTPs), dideoxynucleotides (ddNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitor, DEPC-water (DEPC-water) and sterile water, etc.

[0031] In some embodiments, the DNA chip kit may be a kit including the necessary components for running a DNA chip, and the DNA chip kit includes: a substrate to which the specific polynucleotide, primer or probe of the SNP is attached, and the substrate may include a nucleic acid corresponding to a quantitative comparison area or a fragment thereof.

[0032] In some embodiments, the chip includes: a solid phase carrier; and oligonucleotide probes fixed in order on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to the sequence where the SNP site rs60234066 is located.

[0033] In some embodiments, the solid phase carrier may be made of various materials commonly used in the field of gene chips, such as but not limited to: plastic products, microparticles, membrane carriers, etc. The plastic products can be combined with antibodies or protein antigens through non-covalent or physical adsorption mechanisms. The most commonly used plastic products are small test tubes, beads and micro-reaction plates made of polystyrene; the microparticles are microspheres or particles polymerized from high molecular monomers, and their diameters are mostly micrometers. Since they carry functional groups that can bind to proteins, they are easy to form chemical couplings with antibodies (antigens) and have large binding capacity; the membrane carriers include microporous filter membranes such as nitrocellulose membranes, glass cellulose membranes and nylon membranes.

[0034] The third aspect of the present invention provides a method for screening or predicting atropine-susceptible myopia population based on the SNP site rs60234066 on the CHRM3 gene.

[0035] Further, the method comprises: Obtaining SNP site data on the CHRM3 gene in the sample to be tested; Extracting the genotype data of the SNP site rs60234066 on the CHRM3 gene; Classification prediction is performed based on the genotype data of the SNP site rs60234066 to obtain a classification result of whether the sample to be tested is a sample of atropine-susceptible myopia population.

[0036] Further, the classification result is obtained by classification prediction based on whether the genotype of the SNP site rs60234066 is mutated; If the genotype of the SNP site rs60234066 mutates, the sample to be tested is a sample of atropine-susceptible myopia population; If the genotype of the SNP site rs60234066 has not mutated, the sample to be tested is a sample of a non-atropine susceptible myopia population.

[0037] The fourth aspect of the present invention provides a system for screening or predicting atropine-susceptible myopia population based on the SNP site rs60234066 on the CHRM3 gene.

[0038] Further, the system comprises: Obtaining a data unit, obtaining SNP site data on the CHRM3 gene in the sample to be tested; Extracting data unit, extracting genotype data of SNP site rs60234066 on the CHRM3 gene; A prediction result unit performs classification prediction based on the genotype data of the SNP site rs60234066 to obtain a classification result of whether the sample to be tested is a sample of atropine susceptible myopia population; The classification result is obtained by classification prediction based on whether the genotype of the SNP site rs60234066 is mutated; If the genotype of the SNP site rs60234066 mutates, the sample to be tested is a sample of atropine-susceptible myopia population; If the genotype of the SNP site rs60234066 has not mutated, the sample to be tested is a sample of a non-atropine susceptible myopia population.

[0039] A fifth aspect of the present invention provides a device for screening or predicting atropine-susceptible myopia population based on the SNP site rs60234066 on the CHRM3 gene.

[0040] Furthermore, the device includes: a memory and a processor, the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the method for screening or predicting atropine-susceptible myopia population based on the SNP site rs60234066 on the CHRM3 gene described in the third aspect of the present invention is implemented.

[0041] A sixth aspect of the present invention provides a computer-readable storage medium.

[0042] Furthermore, the computer-readable storage medium stores a computer program, and when the computer program is executed by the processor, it implements the method for screening or predicting atropine-susceptible myopia population based on the SNP site rs60234066 on the CHRM3 gene described in the third aspect of the present invention.

[0043] Compared with the prior art, the present invention has the following advantages and beneficial effects: The present invention screened and verified for the first time from the whole genome sequencing data of myopic people using atropine in China that a SNP site rs60234066 can be used as an effective detection marker for atropine-related receptors in myopic people. The present invention discovered for the first time that the SNP site rs60234066 is related to the diagnosis of atropine-susceptible myopic people. By detecting the SNP genotype, it is determined whether the subject population is an atropine-susceptible myopic population. The SNP site rs60234066 has a high accuracy in diagnosing atropine-susceptible myopic population, and can be used in the early diagnosis or screening of atropine-susceptible myopic people to improve the early intervention rate of people with related diseases, thereby improving the clinical guidance effect of people with related diseases and reducing the progression of myopia. The clinical application prospect is broad. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 : In the discovery cohort, candidate gene set association analysis was performed to find the result graphs corresponding to the receptor gene-related variant sites that had significant differences between fast and slow axial progression after the use of atropine; Figure 2 : In the discovery cohort, there was a very significant difference in the progression of axial length in patients carrying the rs60234066 mutation (P<0.001), and there was a significant difference in the progression of axial length in patients carrying the rs55887984 mutation (P<0.05); Figure 3 : In the validation cohort, patients carrying the rs60234066 mutation had a highly significant difference in axial length progression (P<0.001); Figure 4 :In the discovery cohort, the SNP site rs60234066 was used to diagnose and distinguish between people with faster and slower axial length progression after using atropine; Figure 5 :In the validation cohort, the SNP site rs60234066 was used to diagnose and distinguish the fast and slow axial progression of people after using atropine; Figure 6 : A schematic flow chart of a method for screening or predicting atropine-susceptible myopia population based on the SNP site rs60234066 on the CHRM3 gene provided by an embodiment of the present invention; Figure 7 : Schematic diagram of a system for screening or predicting atropine-susceptible myopia population based on the SNP site rs60234066 on the CHRM3 gene provided by an embodiment of the present invention; Figure 8 : Schematic diagram of a device for screening or predicting atropine-susceptible myopia population based on the SNP site rs60234066 on the CHRM3 gene provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with specific examples. The specific examples are only used to explain the present invention and cannot be construed as limiting the present invention. It will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purposes of the present invention, and the scope of the present invention is defined by the claims and their equivalents. The reagents and raw materials used in the present invention are easily available to those of ordinary skill in the art, and can be obtained from commercial sources unless otherwise specified. The experimental methods for which specific conditions are not specified in the present invention are usually implemented and detected under conventional conditions or according to the conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way.

[0046] Example Screening and verification of SNP sites related to diagnosis of atropine susceptible myopia population

[0047] 1. Sample source Children aged 6 to 12 years with a spherical equivalent (SE) refractive error ranging from -1.00 D to -6.00 D in both eyes who visited Tongren Hospital in Beijing, China, between April and July 2018 were recruited for this study. Children with other comorbid eye diseases (such as amblyopia, strabismus, corneal scarring, cataract, glaucoma, or ocular tumors) were excluded. A total of 220 myopic children were randomly assigned to receive 0.01% placebo or atropine eye drops once daily in a 1:1 ratio for 1 year.

[0048] The subjects in this study were from the 0.01% atropine eye drops group. In order to completely divide the data into two groups, fast and slow, the samples with an annual axial length progression of 0.32-0.36 mm were removed. If the axial length progression is less than 0.32 mm within 1 year, the child's axial length progression is considered slow, and greater than 0.36 mm is considered rapid axial length progression. Among them, the discovery cohort and the validation cohort were 51 and 50 people who used atropine for one year, respectively, and peripheral blood samples were taken from the population.

[0049] The people who used atropine for one year were divided into groups according to the axial length, as shown in Table 1 below.

[0050]

[0051] 2. Experimental methods Genetic material DNA was extracted from peripheral blood samples and whole genome sequencing was performed. The average sequencing depth was 20X, and the specific sequencing method was as follows: (1) DNA extraction The DNA in cells is extracted using chemical or physical methods to ensure high quality and concentration of DNA.

[0052] (2) DNA quality testing Quantitative detection: DNA concentration is measured using spectrophotometer, fluorescence measurement and other methods.

[0053] Quality check: Check the integrity and contamination of DNA by agarose gel electrophoresis or bioanalyzer.

[0054] (3) Library construction-fragmentation Library construction-fragmentation: The extracted DNA is randomly cut into small fragments, generally ranging from a few hundred to a few thousand base pairs in length.

[0055] Adapter ligation: Specific adapter sequences are connected to both ends of each DNA fragment to facilitate subsequent amplification and sequencing.

[0056] Select target fragments: Select fragments of appropriate length by selective amplification or purification. Common methods are polymerase chain reaction (PCR) or magnetic bead purification.

[0057] (4) Sequencing Select sequencing platform: high-throughput sequencing platform (BGI DNBSEQ-T7).

[0058] Sequencing on the machine: Load the constructed library into the sequencer, perform sequencing reaction, and generate raw sequencing data.

[0059] (5) Data processing Data cleaning: remove low-quality reads, adapter sequences, and other contaminating information.

[0060] Sequence alignment: Align the cleaned reads to the reference genome using alignment software.

[0061] Variant calling: Identify variant points (such as single nucleotide variants, insertions and deletions, etc.) and use corresponding algorithms.

[0062] 3. Experimental results (1) The sequencing data were quality controlled, aligned, detected, and filtered. Sites with a depth of ≤15, a sample missing rate of ≤0.1, multiple alleles, and a Hardy-Weinberg equilibrium (HWE) test P value ≥10-6 were excluded based on sample grouping. A total of 8,500,039 common single nucleotide variants were detected.

[0063] (2) The study recruited children aged 6 to 12 years who visited Tongren Hospital in Beijing, China between April and July 2018. Their spherical equivalent (SE) refractive errors ranged from -1.00 D to -6.00 D in both eyes. Children with other comorbid eye diseases (such as amblyopia, strabismus, corneal scars, cataracts, glaucoma, or ocular tumors) were excluded. A total of 220 myopic children were randomly assigned to receive 0.01% placebo or atropine eye drops once a day in a 1:1 ratio for 1 year. The subjects in this study were from the 0.01% atropine eye drops group. In order to completely divide the data into two groups with rapid and slow axial length progression, samples with an annual axial length progression of 0.32-0.36 mm were removed. If the axial length progression was less than 0.32 mm within 1 year, the child was considered to have slow axial length progression, and more than 0.36 mm was considered to have rapid axial length progression. Among them, 51 cases in the discovery cohort underwent whole genome sequencing, and 50 cases in the validation cohort underwent first-generation sequencing of target sites. The grouping results of the people who used atropine for one year according to the axial length are shown in Table 1.

[0064] (3) By searching literature and databases related to atropine metabolic receptors, a total of 34 acetylcholinesterase, acetyltransferase, adrenergic receptor, dopamine receptor, nicotinic acetylcholine receptor and muscarinic acetylcholine receptor genes were obtained. Omics analysis was performed on these 34 genes, and the expression distribution data of ocular tissues and cells were collected from the Single Cell PORTAL database (https: / / singlecell.broadinstitute.org / single_cell), and the z-score was used to further adjust and standardize the data. DRD3, CHRNA9, CHRM4, CHRNA1 and CHRNG were not expressed in ocular tissues and cells, and the remaining 29 genes were used as candidate gene sets.

[0065] (4) Candidate gene set association analysis was performed on the discovery cohort to identify receptor gene-related variant sites that had significant differences in fast and slow axial progression after atropine use. The experimental results are as follows: Figure 1 As shown, the results showed that there was a significant difference in expression between the SNP site rs60234066 on the CHRM3 gene between people with faster axial length progression after using atropine and people with slower axial length progression after using atropine.

[0066] The sites with P<0.001 were selected for phenotypic association analysis, and the distinguishing effect of these sites on the validation cohort was further tested. The relevant information of the SNP sites and their P values ​​in the association analysis of the discovery cohort are shown in Table 2 below.

[0067] Table 2 Relevant information of the SNP loci and their P values ​​in the association analysis of the discovery cohort

[0068] (5) Linkage disequilibrium analysis of the loci revealed that the R-squared of rs12117089 and rs60234066 was 0.737457, indicating that the alleles of these two loci have a high genetic correlation. Therefore, the more significant rs60234066 locus was retained for phenotypic association analysis.

[0069] In the discovery cohort, the results of phenotypic association analysis of the SNP loci are as follows Figure 2 As shown, the results showed that in the discovery cohort, there were differences in the progression of axial length in patients carrying rs55887984 and rs60234066 (P<0.05), among which rs60234066 showed extremely significant differences (P<0.001).

[0070] In the validation cohort, the results of phenotypic association analysis of the highly significant locus rs60234066 (P < 0.001) were as follows: Figure 3 As shown, the results showed that in the validation cohort, patients carrying the rs60234066 mutation (rs60234066 C>CT) had significant differences in axial length progression (P<0.05), and the axial length progression was slower.

[0071] (6) The predictive effect (AUC value), receiver operating characteristic (ROC), sensitivity, and specificity of the rs60234066 locus in distinguishing between people with faster and slower axial length progression after the use of atropine were calculated.

[0072] In the discovery cohort, the prediction effect of the rs60234066 locus on differentiating the fast and slow progression of axial length after atropine use was calculated and the corresponding results are shown in the figure below. Figure 4As shown, the results showed that in the discovery cohort, the rs60234066 site had a high diagnostic efficacy in distinguishing between people with fast and slow axial progression after using atropine, and showed a high correlation with the effect of atropine, with an AUC value of 0.762, a sensitivity of 92.3%, and a specificity of 60.0%.

[0073] In the validation cohort, the predictive effect of the rs60234066 locus on differentiating the fast and slow progression of axial length after atropine use was calculated, as shown in the figure below. Figure 5 As shown, the results showed that in the validation cohort, the rs60234066 locus also had a high diagnostic efficacy in distinguishing between people with fast and slow axial progression after using atropine, and showed a high correlation with the effect of atropine, with an AUC value of 0.720, a sensitivity of 100%, and a specificity of 44.0%.

[0074] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0075] In some of the processes described in the specification and claims of the present invention and the above-mentioned drawings, multiple operations appearing in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this document or may be executed in parallel. The serial numbers of the operations, such as S101, S102, etc., are only used to distinguish different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel.

[0076] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0077] Figure 6 The present invention provides a method for screening or predicting atropine-susceptible myopia population based on the SNP site rs60234066 on the CHRM3 gene, specifically, the method comprises the following steps: S101: Obtaining SNP site data on the CHRM3 gene in the sample to be tested; In one embodiment, the sample to be tested is a sample to be tested from a myopic population.

[0078] In one embodiment, the sample to be tested is selected from blood or tissue.

[0079] In one embodiment, the CHRM3 (cholinergic receptor muscarinic 3 [Homosapiens (human)]) gene has a Gene ID of 1131 in NCBI, and its corresponding detailed information can be obtained at https: / / www.ncbi.nlm.nih.gov / .

[0080] In one embodiment, the SNP site (Single Nucleotide Polymorphism) refers to the variation of a single nucleotide on the genome, including conversion, transversion, deletion and insertion, forming a genetic marker. The SNP site is a nucleic acid sequence polymorphism caused by a single nucleotide change. Theoretically, each SNP site can have four different forms of variation, but in fact only two occur, namely conversion and transversion, and the ratio of the two is 2:1.

[0081] In one embodiment, any one or more of the following detection methods can be used to obtain the SNP site data on the CHRM3 gene in the sample to be tested: Sanger sequencing method, TaqMan probe method, Amplification Refractory Mutation System PCR (ARMS-PCR), molecular beacon method, high-resolution melting analysis (HRM), Cleaved Amplified Polymorphic Sequence (CAPS), SNaPshot method, KASP (Kompetitive Allele-Specific PCR, KASP), gene chip method, mass spectrometry detection method based on matrix-assisted laser desorption / ionization time-of-flight massspectrometry (MALDI-TOF MS).

[0082] In one embodiment, the present invention performs quality control, alignment, variation detection and filtering on sequencing data, excludes sites with depth ≤15, sample missing rate ≤0.1, multi-allelic and Hardy-Weinberg equilibrium (HWE) test P value ≥10-6 according to sample grouping, and detects a total of 8,500,039 common single nucleotide variations.

[0083] S102: extracting the genotype data of the SNP site rs60234066 on the CHRM3 gene; In one embodiment, this study recruited children aged 6 to 12 years who visited Tongren Hospital in Beijing, China between April and July 2018, with a spherical equivalent (SE) refractive error ranging from -1.00 D to -6.00 D in both eyes, and children with other comorbid eye diseases (such as amblyopia, strabismus, corneal scars, cataracts, glaucoma, or ocular tumors) were excluded. A total of 220 myopic children were randomly assigned to receive 0.01% placebo or atropine eye drops once a day in a 1:1 allocation ratio for 1 year. The subjects in this study were from the 0.01% atropine eye drops group, and in order to completely divide the data into two groups with rapid and slow axial length progression, the samples with an annual axial length progression of 0.32-0.36 mm were removed. If the axial length progression was less than 0.32 mm in 1 year, the child's axial length progression was considered to be slow, and greater than 0.36 mm was considered to be rapid axial length progression. Among them, 51 cases in the discovery cohort underwent whole genome sequencing, and 50 cases in the validation cohort underwent first-generation sequencing of target sites. The grouping results of the people who used atropine for one year according to the axial length are shown in Table 1 above.

[0084] In one embodiment, a total of 34 acetylcholinesterase, acetyltransferase, adrenergic receptor, dopamine receptor, nicotinic acetylcholine receptor and muscarinic acetylcholine receptor genes were obtained by searching literature and databases related to atropine metabolic receptors. Omics analysis was performed on these 34 genes, and expression distribution data of ocular tissues and cells were collected from the Single Cell PORTAL database (https: / / singlecell.broadinstitute.org / single_cell), and z-score was used for further adjustment to standardize the data. DRD3, CHRNA9, CHRM4, CHRNA1 and CHRNG were not expressed in ocular tissues and cells, and the remaining 29 genes were used as candidate gene sets.

[0085] In one embodiment, candidate gene set association analysis was performed on the discovery cohort to find receptor gene-related variant sites that had significant differences in fast and slow axial progression after the use of atropine. The experimental results were as follows: Figure 1 As shown, the results showed that there was a significant difference in expression between the SNP site rs60234066 on the CHRM3 gene between people with faster axial length progression after using atropine and people with slower axial length progression after using atropine.

[0086] In one embodiment, sites with P<0.001 are selected for phenotypic association analysis, and the distinguishing effect of these sites on the validation cohort is further tested. The relevant information of the SNP sites and their P values ​​in the association analysis of the discovery cohort are shown in Table 2 above.

[0087] In one embodiment, linkage disequilibrium analysis was performed on the site, and the R square of rs12117089 and rs60234066 was found to be 0.737457, indicating that the alleles at these two sites have a high genetic correlation. Therefore, the more significant rs60234066 site was retained for phenotypic association analysis.

[0088] In the discovery cohort, the results of phenotypic association analysis of the SNP loci are as follows Figure 2 As shown, the results showed that in the discovery cohort, there were differences in the progression of axial length in patients carrying rs55887984 and rs60234066 (P<0.05), among which rs60234066 showed extremely significant differences (P<0.001).

[0089] In the validation cohort, the results of phenotypic association analysis of the highly significant locus rs60234066 (P < 0.001) were as follows: Figure 3 As shown, the results showed that in the validation cohort, there was a significant difference in the progression of axial length in patients carrying rs60234066 (P<0.05), and the axial length progression was slower.

[0090] In one embodiment, the prediction effect (AUC value), receiver operating curve (ROC), sensitivity and specificity of the rs60234066 site for distinguishing between people with fast and slow axial length progression after using atropine are calculated.

[0091] In the discovery cohort, the prediction effect of the rs60234066 locus on differentiating the fast and slow progression of axial length after atropine use was calculated and the corresponding results are shown in the figure below. Figure 4 As shown, the results showed that in the discovery cohort, the rs60234066 site had a high diagnostic efficacy in distinguishing between people with fast and slow axial progression after using atropine, and showed a high correlation with the effect of atropine, with an AUC value of 0.762, a sensitivity of 92.3%, and a specificity of 60.0%.

[0092] In the validation cohort, the predictive effect of the rs60234066 locus on differentiating the fast and slow progression of axial length after atropine use was calculated, as shown in the figure below. Figure 5As shown, the results showed that in the validation cohort, the rs60234066 locus also had a high diagnostic efficacy in distinguishing between people with fast and slow axial progression after using atropine, and showed a high correlation with the effect of atropine, with an AUC value of 0.720, a sensitivity of 100%, and a specificity of 44.0%.

[0093] The above results indicate that the SNP site rs60234066 provided by the present invention can be used to effectively distinguish between people with fast and slow axial progression after using atropine, that is, the SNP site rs60234066 provided by the present invention can be used to effectively screen or predict people with atropine-susceptible myopia.

[0094] S103: performing classification prediction based on the genotype data of the SNP site rs60234066 to obtain a classification result of whether the sample to be tested is a sample of atropine susceptible myopia population; In one embodiment, the classification result is obtained by classification prediction based on whether the genotype of the SNP site rs60234066 is mutated; If the genotype of the SNP site rs60234066 mutates, the sample to be tested is a sample of atropine-susceptible myopia population; If the genotype of the SNP site rs60234066 has not mutated, the sample to be tested is a sample of a non-atropine susceptible myopia population.

[0095] Figure 7 The present invention provides a schematic flow chart of a system for screening or predicting atropine-susceptible myopia population based on the SNP site rs60234066 on the CHRM3 gene. Specifically, the system includes: Obtaining a data unit, obtaining SNP site data on the CHRM3 gene in the sample to be tested; Extracting data unit, extracting genotype data of SNP site rs60234066 on the CHRM3 gene; A prediction result unit performs classification prediction based on the genotype data of the SNP site rs60234066 to obtain a classification result of whether the sample to be tested is a sample of atropine susceptible myopia population; The classification result is obtained by classification prediction based on whether the genotype of the SNP site rs60234066 is mutated; If the genotype of the SNP site rs60234066 mutates, the sample to be tested is a sample of atropine-susceptible myopia population; If the genotype of the SNP site rs60234066 has not mutated, the sample to be tested is a sample of a non-atropine susceptible myopia population.

[0096] Figure 8 The present invention provides a device for screening or predicting atropine-susceptible myopia population based on the SNP site rs60234066 on the CHRM3 gene. Specifically, the device includes: A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the method for screening or predicting atropine-susceptible myopia population based on the SNP site rs60234066 on the CHRM3 gene as described above is implemented.

[0097] An embodiment of the present invention provides a computer-readable storage medium. Specifically, a computer program is stored on the computer-readable storage medium. When the computer program is executed by a processor, the method for screening or predicting atropine-susceptible myopia population based on the SNP site rs60234066 on the CHRM3 gene as described above is implemented.

[0098] The validation results of this validation example show that assigning intrinsic weights to indications can moderately improve the performance of the method relative to the default setting.

[0099] Those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working processes of the systems, devices and units described above can refer to the corresponding processes in the aforementioned method embodiments and will not be repeated here.

[0100] In the several embodiments provided in the present application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interfaces, devices or units, which can be electrical, mechanical or other forms.

[0101] The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0102] In addition, each functional unit in each embodiment of the present invention may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit. The above-mentioned integrated unit may be implemented in the form of hardware or in the form of software functional units.

[0103] A person skilled in the art may understand that all or part of the steps in the various methods of the above embodiments may be completed by instructing related hardware through a program, and the program may be stored in a computer-readable storage medium, and the storage medium may include: a read-only memory (ROM), a random access memory (RAM), a disk or an optical disk, etc.

[0104] A person skilled in the art can understand that all or part of the steps in the above-mentioned embodiment method can be completed by instructing related hardware through a program, and the program can be stored in a computer-readable storage medium. The above-mentioned storage medium can be a read-only memory, a disk or an optical disk, etc.

[0105] The above is a detailed introduction to a computer device provided by the present invention. For a person skilled in the art, according to the concept of the embodiments of the present invention, there may be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. Use of a reagent for detecting SNP markers in a sample of a subject in the preparation of a product for diagnosing or screening atropine-susceptible myopia population, characterized in that: The SNP marker is rs60234066.

2. The use according to claim 1, characterized in that: The reagents include reagents for detecting the rs60234066 genotype by direct sequencing, single base extension, allele-specific probe hybridization, allele-specific primer extension, allele-specific nucleotide incorporation, 5' nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, MALDI-TOF / MS, RCA, imprint hybridization, dot hybridization or single-stranded conformation polymorphism method.

3. The use according to claim 1, characterized in that: The sample is selected from blood or tissue; Optionally, the reagent includes a specific primer, a specific probe or a specific antibody for detecting rs60234066; Optionally, the reagents further include a reagent for processing the subject sample to obtain nucleic acid; Optionally, the product includes a kit or a chip.

4. A product for diagnosing or screening atropine-susceptible myopia population, characterized in that: The product includes a reagent for detecting the genotype of the SNP site rs60234066 in a subject's sample.

5. The product according to claim 4, characterized in that The product includes specific primers, specific probes or specific antibodies for detecting rs60234066; Optionally, the product further comprises a reagent for processing the subject sample to obtain nucleic acid; Optionally, the product includes a kit or a chip.

6. A method for screening or predicting atropine-susceptible myopia population based on the SNP site rs60234066 on the CHRM3 gene, characterized in that: The method comprises: Obtaining SNP site data on the CHRM3 gene in the sample to be tested; Extracting the genotype data of the SNP site rs60234066 on the CHRM3 gene; Classification prediction is performed based on the genotype data of the SNP site rs60234066 to obtain a classification result of whether the sample to be tested is a sample of atropine-susceptible myopia population.

7. The method for screening or predicting atropine-susceptible myopia population based on the SNP site rs60234066 on the CHRM3 gene according to claim 6, characterized in that: The classification result is obtained by classification prediction based on whether the genotype of the SNP site rs60234066 is mutated; If the genotype of the SNP site rs60234066 mutates, the sample to be tested is a sample of atropine-susceptible myopia population; If the genotype of the SNP site rs60234066 has not mutated, the sample to be tested is a sample of a non-atropine susceptible myopia population.

8. A system for screening or predicting atropine-susceptible myopia population based on the SNP site rs60234066 on the CHRM3 gene, characterized in that: The system comprises: Obtaining a data unit, obtaining SNP site data on the CHRM3 gene in the sample to be tested; Extracting data unit, extracting genotype data of SNP site rs60234066 on the CHRM3 gene; A prediction result unit performs classification prediction based on the genotype data of the SNP site rs60234066 to obtain a classification result of whether the sample to be tested is a sample of atropine susceptible myopia population; The classification result is obtained by classification prediction based on whether the genotype of the SNP site rs60234066 is mutated; If the genotype of the SNP site rs60234066 mutates, the sample to be tested is a sample of atropine-susceptible myopia population; If the genotype of the SNP site rs60234066 has not mutated, the sample to be tested is a sample of a non-atropine susceptible myopia population.

9. A device for screening or predicting atropine-susceptible myopia population based on the SNP site rs60234066 on the CHRM3 gene, characterized in that: The device comprises: a memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the method for screening or predicting atropine-susceptible myopia population based on the SNP site rs60234066 on the CHRM3 gene as described in claim 6 or 7 is implemented.

10. A computer-readable storage medium, characterized in that: The computer-readable storage medium stores a computer program, and when the computer program is executed by the processor, the method for screening or predicting atropine-susceptible myopia population based on the SNP site rs60234066 on the CHRM3 gene as described in claim 6 or 7 is implemented.

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