A genomic biomarker panel for diagnosing keratoconus

Abstract

The present invention discloses a genomic biomarker combination for diagnosing keratoconus. Based on the whole-genome sequencing data of keratoconus, the present invention conducts association analysis, screens significant variant sites related to keratoconus, and performs polygenic risk score modeling and susceptibility risk prediction. The results show that the genetic variant risk sites screened by the present invention can effectively distinguish between normal populations and keratoconus populations.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a genomic biomarker combination for diagnosing keratoconus. Background Art

[0002] In patients with keratoconus, due to reduced corneal rigidity, corneal distortion and focal thinning, vision becomes blurred, and sensitivity to light and glare is caused. Some patients even need to undergo corneal transplantation to restore vision. Keratoconus is a major risk factor for refractive surgery. If keratoconus is not detected or overlooked before surgery, refractive surgery may lead to serious adverse consequences such as severe vision damage.

[0003] The formation of keratoconus is the combined effect of environmental factors and genetic factors, but genetic factors play an obvious dominant role in the pathogenesis of keratoconus. In families with early onset, severe disease, dominant or recessive inheritance, genetic factors may account for almost all risks. Developing an in vitro genetic detection kit suitable for early screening of keratoconus and improving the early intervention rate of related disease-susceptible populations is of great significance for reducing the probability of refractive surgery complications and improving the prognosis of keratoconus. Summary of the Invention

[0004] To make up for the deficiencies of the prior art, the present invention provides a genomic biomarker combination for diagnosing keratoconus.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides the use of a reagent for detecting the expression level of SNP markers in a sample in the preparation of a product for diagnosing keratoconus, and the SNP markers include one or more of rs1243147514, rs3132306, rs3118518, rs1536483, rs1536482, rs3118519, rs11901979, rs1438506149, rs1299510077, rs4894535, rs4894414, rs10245148, rs6445054, rs6445055, rs13094143, rs1994961, rs4718997, rs2293500, rs4894415, rs4630912, rs4894413, rs17766647.

[0007] Further, the reagent includes reagents used for detecting the genotype of the SNP marker by 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, size analysis, and single-strand conformation polymorphism method.

[0008] Further, the reagent includes reagents used for detecting the genotype of the SNP marker by sequencing.

[0009] Further, the sequencing method includes pyrosequencing, microsequencing, next-generation sequencing, Taqman method, cycle sequencing, semiconductor sequencing, amino acid sequencing, and whole-genome sequencing.

[0010] Further, the sequencing method is selected from whole-genome sequencing.

[0011] Further, the sample includes blood and tissue.

[0012] Further, the sample is selected from blood.

[0013] Further, the blood is selected from peripheral blood.

[0014] Further, the product further includes reagents for processing the sample.

[0015] The second aspect of the present invention provides a product for diagnosing keratoconus, the product including a reagent for detecting the SNP marker described in the first aspect of the present invention in a sample.

[0016] Further, the reagent includes a nucleic acid affinity ligand for the SNP marker.

[0017] Further, the nucleic acid affinity ligand includes a primer for specifically amplifying a polynucleotide of the SNP marker or a probe that can specifically bind to the SNP marker.

[0018] Further, the reagent further includes a detectable label.

[0019] Further, the product includes a kit, test strip, or chip.

[0020] Further, the kit further includes a container, reaction buffer, deoxynucleotides, enzymes, DNase, and RNAse inhibitor.

[0021] Further, the kit further includes an instruction manual.

[0022] The third aspect of the present invention provides the application of the SNP marker described in the first aspect of the present invention in constructing a computer model for diagnosing keratoconus.

[0023] The fourth aspect of the present invention provides a system / apparatus for diagnosing keratoconus, the system / apparatus comprising:

[0024] 1) An analysis unit: The analysis unit is used to detect the SNP markers described in the first aspect of the present invention in a subject sample;

[0025] 2) An evaluation unit: The evaluation unit includes a stored reference and a data processor, and the data processor has implemented a method for comparing the biomarker detected by the analysis unit, thereby diagnosing keratoconus.

[0026] Advantages and beneficial effects of the present invention:

[0027] Based on the whole-genome sequencing data of keratoconus, the present invention conducts association analysis, screens significant variant sites related to keratoconus, and performs multi-gene risk score modeling and susceptibility risk prediction. The results show that the genetic variant risk sites screened by the present invention can effectively distinguish between normal populations and keratoconus populations. Description of the Drawings

[0028] Figure 1 is a map of genetic variants related to keratoconus with a large difference in frequency between keratoconus populations and control populations;

[0029] Figure 2 is a predictive effect diagram for distinguishing keratoconus populations and control populations. Detailed Embodiments

[0030] The following provides definitions of some terms used in this specification. Unless otherwise specified, all technical and scientific terms used herein generally have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0031] The present invention provides the use of a reagent for detecting the expression level of SNP markers in a sample in the preparation of a product for diagnosing keratoconus, and the SNP markers include one or more of rs1243147514, rs3132306, rs3118518, rs1536483, rs1536482, rs3118519, rs11901979, rs1438506149, rs1299510077, rs4894535, rs4894414, rs10245148, rs6445054, rs6445055, rs13094143, rs1994961, rs4718997, rs2293500, rs4894415, rs4630912, rs4894413, rs17766647.

[0032] In the present invention, SNP (single nucleotide polymorphism) refers to single-base polymorphism. Due to general mutations where one of the multiple DNA bases appears at a single site on the chromosome, SNPs are highly frequent and stable and are distributed throughout the genome. Thus, genetic polymorphisms occur in the human body. The SNP loci of the present invention are named in the rs- manner, and those skilled in the art can determine their exact positions and nucleotide sequences from suitable databases and relevant information systems such as the Single Nucleotide Polymorphism Database (dbSNP) according to the rs- naming above. In the present invention, the SNP markers or SNP loci can be used interchangeably with SNPs.

[0033] In the present invention, a marker means a biomarker that can detect changes in a living organism and objectively detect the normal or pathological state of the living organism, drug responsiveness, etc.

[0034] In the present invention, the product for diagnosing keratoconus can diagnose keratoconus or predict the risk of occurrence of keratoconus, but is not limited thereto.

[0035] The reagent includes reagents for detecting the genotypes of the SNP markers using methods such as sequencing, single-base extension, allele-specific probe hybridization, allele-specific primer extension, allele-specific amplification (allele-specific PCR, AS-PCR), allele-specific nucleotide incorporation, 5'-nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, and single-strand conformation polymorphism (SSCP).

[0036] In the present invention, single-strand conformation polymorphism (SSCP) refers to the fact that single-stranded DNA can cause conformational differences due to different base sequences, and these differences will lead to different electrophoretic mobilities of single-stranded DNA with the same or similar lengths, so that it can be effectively detected by non-denaturing polyacrylamide gel electrophoresis. PCR-SSCP is a method for detecting gene mutations in PCR amplification products using SSCP. The DNA fragments amplified by PCR are denatured by high-temperature treatment under denaturing agent conditions to unwind the double-stranded DNA amplification fragments and maintain the single-stranded state, and then non-denaturing polyacrylamide gel electrophoresis is further carried out. At present, the PCR-SSCP technique is widely used in various fields of molecular biology.

[0037] 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 of the SNP locus, the amplification reaction can occur; when the base at the 3'-end of the primer is not complementary to the allele of the SNP locus, the amplification reaction cannot occur. Currently, some methods improved based on AS-PCR have emerged, such as tetra-primer amplification refractory mutation system PCR (Tetra-primer ARMS-PCR), fragment length discrepant allele specific PCR (FLDAS-PCR), PCR amplification of multiple specific alleles (PMASA), etc.

[0038] Sequencing methods include pyrosequencing, SNaPshot, next-generation sequencing, Taqman method, cycle sequencing, semiconductor sequencing, amino acid sequencing, whole genome sequencing.

[0039] Those skilled in the art can select any one or several methods (not limited to the above methods) to detect SNP loci, as long as the detection of SNP loci can be achieved.

[0040] In a specific embodiment of the present invention, the sequencing method is selected from whole genome sequencing.

[0041] The present invention provides a product for diagnosing keratoconus, and the product includes a reagent for detecting the above SNP markers in a sample.

[0042] The reagent includes a nucleic acid affinity ligand for the SNP marker.

[0043] In the present invention, a nucleic acid affinity ligand refers to a nucleic acid molecule that can bind to an SNP marker as defined above or a sequence near it. As a non-limiting embodiment, the nucleic acid affinity ligand can be, for example, an RNA, DNA, PNA, CAN, HNA, LNA or ANA molecule or any other suitable nucleic acid form known to those skilled in the art.

[0044] In the present invention, nucleic acid means both DNA and RNA, both of which exist in any possible configuration, namely in the form of double-stranded (ds) nucleic acid, or in the form of single-stranded (ss) nucleic acid, or in a combined form (partially ds or ss). Such nucleic acids correspond to at least two consecutive deoxyribonucleotides or ribonucleotides optionally containing at least one modified nucleotide.

[0045] The nucleic acid affinity ligand includes a primer for a polynucleotide that specifically amplifies the SNP marker or a probe that can specifically bind to the SNP marker.

[0046] In the present invention, specifically amplifying the SNP marker can be under appropriate conditions in an appropriate buffer (e.g., 4 different nucleoside triphosphates, DNA, RNA polymerase or reverse transcriptase, etc., as polymerization agents), and at an appropriate temperature, a single-stranded oligonucleotide that can act as a starting point for DNA synthesis in a columnar display. Depending on the purpose of use, the appropriate length of the primer will vary, but is usually 15 to 30 nucleotides. Short primer molecules usually require a lower temperature to form a columnar and stable hybrid. The primer sequence does not need to form complete complementarity with the polynucleotide including the SNP, but its complementarity needs to reach the extent that it can hybridize with the polynucleotide including the SNP.

[0047] A primer refers to a short sequence with a base sequence having a short-chain free 3'hydroxyl group that can form base pairs with a complementary template and function as a starting point for columnar chain replication. The primer can be used as a sample for a polymerization reaction (i.e., DNA polymerase or reverse transcriptase) under appropriate buffer solvent and temperature conditions, and can initiate DNA synthesis in the presence of 4 different nucleoside triphosphates. At this time, the PCR conditions, sensitivity, and the length of the antisense primer can be modified according to the well-known techniques in the art.

[0048] The probe that specifically binds to the SNP marker can be a hybridization probe or an oligonucleotide that binds to a nucleic acid complementary chain according to a specific sequence. The hybridization conditions need to show a significant difference in the hybridization intensity between alleles to be strictly controlled so that it hybridizes only to one of the alleles. The probe can be applied to test kits such as detecting alleles, diagnosing keratoconus microarrays, or prediction methods, etc.

[0049] The reagent further includes a detectable label.

[0050] In the present invention, a detectable label refers to a composition capable of generating a detectable signal indicating the presence of a target polynucleotide in a sample to be assayed. Suitable labels include, but are not limited to, radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties. Thus, a label is any composition capable of being detected by a device or method, including but not limited to spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical detection devices, or any other suitable device. In some embodiments, the label can be visually detected without the aid of a device.

[0051] Among them, radioisotopes include, but are not limited to 3 H, 14 C, 35 S, 125 I, 131 I.

[0052] Enzymes include, but are not limited to, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase.

[0053] Fluorescent molecules include, but are not limited to, FITC, rhodamine, lanthanide phosphors.

[0054] The primers or probes of the present invention can be chemically synthesized by the phosphoramidite solid support method or other well-known methods. The nucleic acid sequences can be modified by a variety of technical means well-known in the art. Non-limiting examples of such modifications include: substitution with methylation, adsorption, one or more homologues of natural nucleotides, and modifications between nucleotides, such as modification to an uncharged linker (e.g., methylphosphonate, phosphotriester, phosphoramidite, carbamate, etc.) or a charged linker (e.g., phosphorothioate, dithiophosphonate, etc.).

[0055] The products include kits, test strips or chips.

[0056] In the present invention, a kit contains a set of oligonucleotide primers sufficient to detect and / or quantify the genotype of the SNP markers described in the present invention. The oligonucleotide primers can be provided in a lyophilized or reconstituted form, or can be provided as a set of nucleotide sequences. In one embodiment, the primers are provided in the form of a microplate, where each set of primers occupies one well (or multiple wells, such as in the case of replicates) in the microplate. The microplate can further contain primers sufficient to detect one or more housekeeping genes as described below.

[0057] The kit also includes a container, reaction buffer, deoxynucleotides (dNTPs), enzymes, DNase, RNAse inhibitor.

[0058] Suitable containers in the kit generally include at least one vial, test tube, flask, bottle, syringe or other container, in which one component can be placed, and preferably, appropriate aliquots can be made. When there are more than one component in the kit, the kit will usually also include a second, third or other additional containers, in which the additional components are placed separately. However, different combinations of components can be included in one vial. The kit of the present invention will usually also include a container for holding reactants, sealed for commercial sale. Such a container can include injection-molded or blow-molded plastic containers, in which the required vials can be retained.

[0059] The reaction buffer in the kit includes, but is not limited to, various pH values, and the enzymes include, but are not limited to, Taq-polymerase and reverse transcriptase.

[0060] In the present invention, the chip can be used interchangeably with the array and the biochip, and the chip also includes a solid-phase carrier. The solid-phase carrier can adopt various commonly used materials in the field of gene chips, including but not limited to plastic products, microparticles, membrane carriers, etc. The most commonly used plastic products are small test tubes, beads and microtiter plates made of polystyrene; the microparticles are microspheres or particles polymerized from high-molecular monomers, and their diameters are mostly in micrometers. Since they carry functional groups that can bind to proteins, they are easy to form chemical conjugates with antibodies (antigens) and have a large binding capacity; the membrane carriers include microporous filter membranes such as nitrocellulose membranes, glass fiber membranes and nylon membranes.

[0061] The present invention provides the application of the above SNP markers in constructing a computer model for diagnosing keratoconus.

[0062] In some embodiments, the computer model uses the above SNP markers as input variables.

[0063] In some embodiments, the computer model also uses other markers related to keratoconus as input variables.

[0064] In the present invention, the computational model can be constructed by using methods such as Logistic Regression, Random Forest, Extreme Gradient Boosting (XGBoosting) and Support Vector Machine (SVM).

[0065] The present invention provides a system / device for diagnosing keratoconus, and the system / device includes:

[0066] 1) Analysis unit: The analysis unit is used to detect the above SNP markers in the sample of the subject;

[0067] 2) Evaluation unit: The evaluation unit includes a stored reference and a data processor, and the data processor has implemented a comparison for analyzing the biomarkers detected by the analysis unit, thereby diagnosing keratoconus.

[0068] In the present invention, the system / apparatus may include a digital processing device or the use of a digital processing device. In additional embodiments, the digital processing device includes one or more hardware central processing units (CPUs) that perform the functions of the device. In other embodiments, the digital processing device further includes an operating system configured to execute executable instructions. In some embodiments, the digital processing device is optionally connected to a computer network. In additional embodiments, the digital processing device is optionally connected to the Internet such that it can access the World Wide Web. In other embodiments, the digital processing device is optionally connected to a cloud computing infrastructure. In other embodiments, the digital processing device is optionally connected to an intranet. In other embodiments, the digital processing device is optionally connected to a data storage device.

[0069] According to the description of the present invention, by way of non-limiting example, suitable digital processing devices include server computers, desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, netbook tablet computers, set-top computers, handheld computers, Internet devices, mobile smart phones, tablet computers, personal digital assistants, video game consoles, and vehicles. Those skilled in the art will recognize that many smart phones are suitable for the systems described herein. Those skilled in the art will also recognize that selected televisions, video players, and digital music players with optional computer network connectivity are suitable for the systems of the present invention. Suitable tablet computers include those known to those skilled in the art with booklet, tablet, and convertible configurations.

[0070] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the present invention. Without departing from the scope of the present invention, the main features of the present invention can be used in various embodiments.

[0071] Embodiment

[0072] 1. Experimental materials

[0073] 649 cases of keratoconus patients and 748 cases of control subjects. Peripheral blood samples were taken from 1397 people.

[0074] 2. Experimental methods

[0075] Genetic material DNA was extracted from the peripheral blood samples and whole genome sequencing was performed. The average sequencing depth for keratoconus was 10X, and the average sequencing depth for the control group was 20X.

[0076] 3. Experimental Results

[0077] 1) Quality control, alignment, variant detection, and filtering were performed on the sequencing data, and a total of 3,350,611 common single nucleotide variants were detected (minor allele frequency > 0.01, excluding the sex chromosomes and the highly heritable HLA region).

[0078] 2) 649 keratoconus patients and 748 controls were divided into a discovery cohort and a validation cohort (Table 1).

[0079] Table 1 Sample Grouping

[0080]

[0081] 3) Association analysis was performed on the discovery cohort to identify keratoconus gene-related variant sites with large frequency differences between the keratoconus population and the control population ( Figure 1 ). After removing strongly linked sites in terms of position, 22 sites with p < 10e-4 were selected for polygenic risk score (PRS) to test the discrimination effect of these site combinations on the validation cohort.

[0082] The 22 sites and their P-values in the discovery cohort association analysis are shown in Table 2.

[0083] Table 2 The 22 Sites and Their P-values in the Discovery Cohort Association Analysis

[0084]

[0085]

[0086] The prediction effects of the 22 sites individually calculated to distinguish the keratoconus population and the control population in the validation cohort are shown in Table 3.

[0087] Table 3 The Prediction Effects of the 22 Sites in Distinguishing the Keratoconus Population and the Control Population

[0088] Marker number Cutoff AUC SNP1 0.23535175 0.610119048 SNP2 0.15502725 0.578762755 SNP3 0.151661 0.576796344 SNP4 0.1512835 0.575226003 SNP5 0.151052 0.575007086 SNP6 0.14942125 0.570365646 SNP7 -0.12670625 0.567159155 SNP8 0.2813925 0.56547619 SNP9 0.2746225 0.55952381 SNP10 0.14313225 0.55677792 SNP11 0.14210575 0.556653912 SNP12 0.1222885 0.556636196 SNP13 0.145492 0.555077239 SNP14 0.14534025 0.555077239 SNP15 0.12103725 0.553748583 SNP16 -0.11752325 0.551922756 SNP17 0.129398 0.550878685 SNP18 0.12054575 0.54919572 SNP19 -0.12358125 0.547140731 SNP20 -0.172842 0.545209751 SNP21 -0.12358125 0.544855442 SNP22 -0.1672065 0.542304422

[0089] The prediction effects of the 22-site combinations calculated to distinguish the keratoconus population and the control population in the validation cohort are shown in Table 4.

[0090] Table 4 The Prediction Effects of the 22-Site Combinations in Distinguishing the Keratoconus Population and the Control Population

[0091]

[0092]

[0093] 4) Modeling was performed on 22 loci and polygenic risk scores, and the predictive performance (AUC value), receiver operating characteristic curve (ROC), sensitivity, and specificity for differentiating keratoconus patients from the control population were calculated. The results showed that the most significant 22 loci screened in the discovery cohort demonstrated high efficiency in the diagnosis of the keratoconus phenotype, with an AUC reaching 0.713. The sensitivity (true positive rate) and specificity (1 - false positive rate) were 0.738 and 0.583, respectively ( Figure 2 ).

[0094] The description of the above embodiments is only for understanding the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Use of a reagent for detecting the combined expression level of SNP markers in a sample in the preparation of a product for diagnosing keratoconus, characterized in that, The SNP marker combination described above includes rs1243147514, rs3132306, rs3118518, rs1536483, rs1536482, rs3118519, rs11901979, and rs1438506149, or a combination formed by sequentially adding rs1299510077, rs4894535, rs4894414, rs10245148, rs6445054, rs6445055, rs13094143, rs1994961, rs4718997, rs2293500, rs4894415, rs4630912, rs4894413, and rs17766647 on the basis of the above combination; The reagent described above includes primers that specifically amplify polynucleotides of the SNP marker combination or probes that can specifically bind to the SNP marker combination.

2. The application according to claim 1, wherein The reagent described above includes reagents used for detecting the genotypes of the SNP markers by 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, size analysis, and single-strand conformation polymorphism method.

3. The application according to claim 2, characterized in that, The reagent described above includes reagents used for detecting the genotypes of the SNP markers by sequencing.

4. The application according to claim 3, wherein The sequencing methods described above include pyrosequencing, microsequencing, next-generation sequencing, Taqman method, cycle sequencing, semiconductor sequencing, amino acid sequencing, and whole-genome sequencing.

5. The application according to claim 4, characterized in that The sequencing method described above is selected from whole-genome sequencing.

6. The application according to claim 1, characterized in that, The sample described above includes blood and tissue.

7. The application according to claim 6, characterized in that, The sample described above is selected from blood.

8. The application according to claim 7, characterized in that, The blood described above is selected from peripheral blood.

9. The application according to claim 1, wherein The product described above further includes a reagent for processing the sample.

10. The application according to claim 1, characterized in that, The reagent described above further includes a detectable label.

11. The application according to claim 1, wherein The product described above includes a kit, test strip, or chip.

12. The application according to claim 11, wherein, The kit described above further includes a container, reaction buffer, and enzyme.

13. The application according to claim 11, characterized in that, The kit described above further includes an instruction manual.

14. Application of the SNP marker combination described in claim 1 in constructing a computer model for diagnosing keratoconus.

15. A system / device for diagnosing keratoconus, characterized in that, The system / device described above includes: 1) Analysis unit: The analysis unit is used for detecting the SNP marker combination described in claim 1 in a subject sample; 2) Evaluation unit: The evaluation unit includes a stored reference and a data processor, and the data processor has implemented a method for comparing the SNP marker combination detected by the analysis unit to thereby diagnose keratoconus.

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