A functional genomic region biomarker combination for diagnosing high myopia

Abstract

The application belongs to the field of biomedicine and particularly relates to a functional genomic region biomarker combination for diagnosing high myopia. Specifically, according to whole-exome sequencing data, correlation analysis is performed, the SNP sites with the most significant p values are screened, and the susceptibility risk prediction of two independent groups is performed, and the results show that these sites can significantly and effectively distinguish normal people and high myopia people.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a combination of functional genomic region biomarkers for diagnosing high myopia. Background Technology

[0002] Patients with high myopia are prone to structural changes in the fundus due to axial elongation, leading to pathological myopia, including posterior staphyloma, myopic macular degeneration, traction macular degeneration, and glaucoma-like optic neuropathy. High myopia and its complications have become a serious cause of blindness. Most patients exhibit a trend towards younger age of onset and a longer disease course, making early detection and intervention crucial for treatment. However, the current clinical system for ophthalmology lacks key early diagnostic and treatment technologies, hindering early detection and treatment. Therefore, developing in vitro genetic testing kits suitable for early screening of high myopia in children and improving early intervention rates in susceptible populations is of great significance for reducing blindness and disability rates and improving the prognosis of high myopia.

[0003] Currently, kits for genetic testing of high myopia in the Chinese population primarily identify susceptibility genes from genetic and genomic studies of myopia or refractive errors in European and American populations. Probes are then designed for these genes, and detection is performed using microarray hybridization or time-of-flight mass spectrometry. However, the data from European and American studies mainly comes from microarray data with sparse locus density, with most loci located in intergenic regions where no functional reports have been published. Furthermore, susceptibility genes for myopia from European and American populations have poor applicability to the Chinese population. The genetic susceptibility mechanisms for myopia and high myopia differ significantly, meaning that myopia susceptibility genes cannot represent high myopia susceptibility genes. Additionally, a few kits obtain pathogenic genes from small Chinese family studies; however, candidate genes from family studies often represent rare variants and have poor representativeness for high myopia populations. Summary of the Invention

[0004] To address the aforementioned technical challenges, this invention presents a cohort study of 10,000 people with high myopia in China, focusing on the genetic and developmental mechanisms of the condition. Based on whole-exome sequencing results, the identified mutation sites are primarily located in functional genes, offering greater representativeness and interpretability for the high myopia population and ensuring effective risk prediction.

[0005] This invention uses whole-exome sequencing data to perform association analysis, screen 88 sites with the most significant P-values, and predict susceptibility risk in two independent cohorts. The results show that these sites can significantly and effectively distinguish between normal populations and people with high myopia.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of this invention provides a set of SNP loci combinations susceptible to high myopia and their application in products for screening individuals susceptible to high myopia. The SNP loci combinations are selected from at least nine of the following SNP loci: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 3 7, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 (i.e., all):

[0008] rs210162 rs2302767 rs2071391 rs28362580 rs11150813 rs1614887 rs12363226 rs533280354 rs35730227 rs3797026 rs4907588 rs72839911 rs2806034 rs6007500 rs2292663 rs72633372 rs79423227 rs2244930 rs2074888 rs909530 rs11553951 rs7100474 rs72780891 rs75057869 rs57041981 rs3785790 rs2234075 rs3758526 rs75372916 rs8177371 rs555754 rs139186286 rs11732887 rs3138141 rs17051276 rs12119390 20:2291164:AG:A rs17740607 rs143175221 rs11114581 rs76994934 rs11544072 rs17138865 rs1288159678 rs16898116 rs17883958 rs113652806 rs10947600 rs3806263 rs552017077 rs191479100 rs556216016 rs185358392 rs74887188 rs149264410 rs3814205 rs148823241 rs767763626 rs192039756 rs7156201 rs116672033 rs181691910 rs80155214 rs372216878 rs373634470 rs537819778 rs142367919 rs63750114 rs201045148 rs60185525 rs200364229 rs116585138 rs201556819 rs193008240 rs777399434 rs558627389 rs66881636 rs571451752 rs771802917 rs527414552 rs774696817 rs76609278 rs561125301

[0009] In one specific implementation, the screening of individuals susceptible to high myopia can be used to diagnose whether the current subject is a person with high myopia, or it can be used to predict the probability that the subject will develop high myopia in the future (i.e., the risk of developing high myopia).

[0010] As verified in the specific embodiments of the present invention, combining the above 1-80 SNP sites can obtain an AUC value of over 0.7 in both validation cohorts, which demonstrates the effectiveness of the above 1-80 SNP site combination in distinguishing between highly myopic individuals and control groups.

[0011] In one specific implementation, the SNP site combination is a combination of all the following SNP sites:

[0012] rs210162 rs2302767 rs2071391 rs28362580 rs11150813 rs1614887 rs12363226 rs533280354 rs35730227 rs3797026 rs4907588 rs72839911 rs2806034 rs6007500 rs2292663 rs72633372 rs79423227 rs2244930 rs2074888 rs909530 rs11553951 rs7100474 rs72780891 rs75057869 rs57041981 rs3785790 rs2234075 rs3758526 rs75372916 rs8177371 rs555754 rs139186286 rs11732887 rs3138141 rs17051276 rs12119390 20:2291164:AG:A rs17740607 rs143175221 rs11114581 rs76994934 rs11544072 rs17138865 rs1288159678 rs16898116 rs17883958 rs113652806 rs10947600 rs3806263 rs552017077 rs191479100 rs556216016 rs185358392 rs74887188 rs149264410 rs3814205 rs148823241 rs767763626 rs192039756 rs7156201 rs116672033 rs181691910 rs80155214 rs372216878 rs373634470 rs537819778 rs142367919 rs63750114 rs201045148 rs60185525 rs200364229 rs116585138 rs201556819 rs193008240 rs777399434 rs558627389 rs66881636 rs571451752 rs771802917 rs527414552 rs774696817 rs76609278 rs561125301

[0013] The terms "SNP" and "SNP site" used in this invention have the meanings known to those skilled in the art, referring to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level, occurring at a frequency of not less than 1% in the population, including single base transitions, transversions, insertions, and deletions. The SNP sites in this invention are named using the "rs-" format. Those skilled in the art can determine their exact location and nucleotide sequence from suitable databases and related information systems, such as the Single Nucleotide Polymorphism Database (dbSNP), based on the rs- nomenclature used above.

[0014] More specifically, the present invention provides the use of reagents for detecting the SNP locus combinations and apparatus for calculating the risk of developing high myopia based on the detection results of the SNP locus combinations in the preparation of genetic testing products for high myopia (early screening of susceptible populations for high myopia).

[0015] More specifically, the reagent for detecting SNP site combinations is a reagent used to detect SNPs in samples collected from subjects;

[0016] More specifically, the SNP site combination detection result refers to the result obtained by SNP detection (genotyping) from samples from the subject.

[0017] In another aspect, the present invention provides a system for genetic testing of high myopia, the system comprising a calculation device for calculating the risk of developing high myopia based on the detection results of the SNP locus combination.

[0018] In one specific embodiment, the system includes:

[0019] 1) Storage device: used to store the patient's sequencing results at the aforementioned SNP loci combination;

[0020] 2) Calculation device: Calculates the risk of developing high myopia based on the detection results of the SNP site combination;

[0021] 3) Risk acquisition device: used to acquire the calculation results of the computing device to obtain the risk of high myopia.

[0022] The calculation can be performed manually, automatically, or in combination thereof to complete the selected task; the result can be calculated manually based on the detection result, or the result can be automatically obtained by inputting it into a computer.

[0023] In another aspect, the present invention provides a kit for genetic testing of high myopia, the kit comprising reagents for detecting the aforementioned SNP locus combinations.

[0024] More specifically, the step of detecting SNP sites refers to detecting the genotype of SNP sites. Based on the genotyping results, it can be determined whether it is a risk genotype. After detecting all SNP sites, the risk of high myopia in the subject can be determined based on the results.

[0025] In some implementations, the kit may specifically include primers, probes, chips, test strips, gels, etc.

[0026] In a preferred embodiment, the reagents for detecting the aforementioned SNP site combinations include, but are not limited to, reagents used in the following methods for detecting SNP sites: TaqMan probe assay, sequencing, microarray assay, MALDI-TOFMS, restriction fragment length polymorphism (PCR-RFLP), single-strand conformation polymorphism (PCR-SSCP), allele-specific PCR (AS-PCR), SNaPshot assay, SNP genotyping system, SNPStream analysis system, Sequenom genotyping system, denaturing high-performance liquid chromatography (DHPLC), and denaturing gradient gel electrophoresis (DGGE). Those skilled in the art can choose any one or more methods to detect SNP sites, as long as the detection of SNP sites can be achieved.

[0027] More specifically, suitable containers in the kit typically include at least one vial, test tube, long-necked flask, PET bottle, syringe, or other container for holding one component. When more than one component is present in the kit, the kit will also typically include a second, third, or other additional container for separately holding the additional components. The kit of the present invention will also typically include a container for containing the reactants, sealed for commercial sale.

[0028] In another aspect, the present invention provides a method for genetic testing of high myopia, the method comprising determining the subject’s current high myopia status or predicting the risk probability of developing high myopia in the future based on the test results of the subject at the aforementioned SNP locus combination.

[0029] The present invention has the following beneficial effects:

[0030] The SNP biomarker provided by this invention is a novel genetic biomarker that is stable, minimally invasive, and easy to detect. It can detect results at an early stage, improving the early intervention rate for people susceptible to high myopia. This is of great significance for reducing the rate of blindness and disability and improving the prognosis of high myopia. Attached Figure Description

[0031] Figure 1 This is a graph showing the results of correlation analysis on the discovery queue.

[0032] Figure 2 This is a receiver operating curve for a combination of 88 loci. Detailed Implementation

[0033] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0034] Example 1: Whole exome sequencing and variant site screening

[0035] Experimental methods: Ten thousand individuals with high myopia and ten thousand control individuals were included. Oral swabs were collected from 20,000 individuals, and DNA was extracted for whole-exome sequencing, with an average sequencing depth of 111.2X.

[0036] Experimental results:

[0037] 1) Quality control, alignment and variant detection were performed on the sequencing data, and a total of 3,386,821 variant sites were detected (excluding HLA regions with high genetic heterozygosity).

[0038] 2) Randomly divide the 20,000 people into three independent queues, as shown in the table below:

[0039] Discovery queue 6233 6098 Verification queue 1 (cohort1) 2996 3627 Verification queue 2 (cohort2) 623 1650 Total number of samples 9852 11375

[0040] 3) Perform association analysis on the discovery cohort to identify variant sites with significant frequency differences between the highly myopic population and the control group, such as... Figure 1 .

[0041] 4) After removing strongly linked sites, the 88 most significant sites in the association analysis of the discovery cohort were selected, and a polygenic risk score (PRS) was performed to examine the discriminative effect of these site combinations on the validation cohort. The 88 sites and their p-values ​​in the association analysis of the discovery cohort are shown in the table below:

[0042]

[0043]

[0044]

[0045] The predictive performance of each of the 88 loci in distinguishing between highly myopic individuals and controls is calculated individually as follows (ranked by the distinguishing performance of cohort1, which has the largest number of individuals):

[0046]

[0047]

[0048]

[0049] The predictive performance of the 88-locus combination in distinguishing between highly myopic individuals and a control group is as follows:

[0050]

[0051]

[0052]

[0053] Receiver operating curves (ROCs) for all 88 site combinations in two independent validation cohorts were analyzed to determine AUC values, sensitivity, and specificity. Figure 2 .

[0054] The 88 most significant loci identified in the cohort screening showed high efficacy in diagnosing high myopia phenotypes in two independent validation cohorts, with AUCs of 0.70 and 0.73 for cohort1 and cohort2, respectively. Sensitivity (true positive rate) and specificity (1-false positive rate) were 0.604 and 0.702, respectively.

[0055] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A system for detecting high myopia, the system comprising a calculation device for calculating the risk of developing high myopia based on the detection results of a combination of SNP loci, wherein the combination of SNP loci is a combination of the following 88 SNP loci:

2. The system as described in claim 1, characterized in that, The system comprises the following devices in sequence: 1) A storage device for storing sequencing results of a patient at the SNP locus combination; 2) A calculation device for calculating the risk of developing high myopia based on the results of the SNP site combination detection; 3) A device for obtaining the calculation results of the computing device to obtain the risk of high myopia.

3. A kit for detecting the risk of developing high myopia, the kit comprising reagents for detecting combinations of SNP sites, said SNP site combinations being combinations of the following 88 SNP sites:

4. The kit according to claim 3, characterized in that, The reagents include one or more of the following methods: TaqMan probe method, sequencing method, microarray method, mass spectrometry of flight detection, restriction fragment length polymorphism method, single strand conformation polymorphism method, allele-specific PCR, SNaPshot method, SNPlex genotyping system, SNPStream analysis system, Sequenom genotyping system, denaturing high performance liquid chromatography, and denaturing gradient gel electrophoresis.

5. The reagent kit as described in claim 3, characterized in that, The reagents include specific primers and / or probes designed for the combination of the SNP sites.

6. The application of reagents for detecting SNP locus combinations in the preparation of products for early screening of individuals susceptible to high myopia, wherein the SNP locus combination is a combination of the following 88 SNP loci:

7. The use of a reagent for detecting SNP locus combinations, an apparatus for calculating the risk of high myopia based on the detection results of SNP locus combinations, the system of claim 1 or 2, and the kit of any one of claims 3-5 in the preparation of a high myopia detection product, wherein the SNP locus combination is a combination of the following 88 SNP loci:

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