CN117417997A - Genome biomarker combination for diagnosing keratoconus - Google Patents

Abstract

The invention discloses a genome biomarker combination for diagnosing keratoconus, which is based on the whole genome sequencing data of the keratoconus, performs correlation analysis, screens obvious variation sites related to the keratoconus, and performs multi-gene risk score modeling and susceptibility risk prediction. The result shows that the genetic variation risk site screened by the invention can effectively distinguish normal population from keratoconus population.

Description

Genome biomarker combination for diagnosing keratoconus

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a genome biomarker combination for diagnosing keratoconus.

Background

Keratoconus patients have a reduced corneal rigidity, a distorted cornea and a focal thinning, resulting in blurred vision, and a sensitivity to light and glare, and some patients even need to receive a corneal transplant to restore vision. Keratoconus is a large risk factor for refractive surgery, which can lead to serious vision impairment or other adverse consequences if keratoconus is not found or ignored prior to surgery.

Keratoconus formation is a combination of environmental and genetic factors, but genetic factors dominate the keratoconus pathogenesis, and genetic factors may occupy almost all risks in families with early onset, severe disease, dominant or recessive inheritance. The development of the in vitro genetic detection kit suitable for the keratoconus early screening improves the early intervention rate of related disease susceptible people, and has great significance for reducing the probability of refractive surgery complications and improving the prognosis level of the keratoconus.

Disclosure of Invention

To remedy the deficiencies of the prior art, the present invention provides a genomic biomarker combination for diagnosing keratoconus.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the first aspect of the invention provides application of a reagent for detecting the expression level of SNP markers in a sample in preparing a product for diagnosing keratoconus, wherein the SNP markers comprise 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 and rs 17766647.

Further, the reagents include reagents for detecting the SNP marker genotype 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 conformational polymorphism methods.

Further, the reagent includes a reagent used for detecting the genotype of the SNP marker by a sequencing method.

Further, the sequencing method comprises pyrophosphoric acid sequencing, micro-sequencing, second generation sequencing, taqman method, cycle sequencing, semiconductor sequencing, amino acid sequencing, whole genome sequencing.

Further, the sequencing method is selected from whole genome sequencing methods.

Further, the sample includes blood, tissue.

Further, the sample is selected from blood.

Further, the blood is selected from peripheral blood.

Further, the product also includes reagents for processing the sample.

In a second aspect the invention provides a product for diagnosing keratoconus comprising reagents for detecting in a sample a SNP marker as described in the first aspect of the invention.

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

Further, the nucleic acid affinity ligand includes a primer that specifically amplifies a polynucleotide of the SNP marker or a probe that can specifically bind to the SNP marker.

Further, the reagent further comprises a detectable label.

Further, the product comprises a kit, a test paper or a chip.

Further, the kit also comprises a container, a reaction buffer, deoxynucleotides, enzymes, DNase and RNAse inhibitors.

Further, the kit also includes instructions.

A third aspect of the invention provides the use of a SNP marker as set forth in the first aspect of the invention in the construction of a computer model for diagnosing keratoconus.

A fourth aspect of the present invention provides a system/device for diagnosing keratoconus, the system/device comprising:

1) Analysis unit: the analysis unit is used for detecting the SNP markers described in the first aspect of the invention in a subject sample;

2) An evaluation unit: the evaluation unit contains a stored reference and a data processor which has been implemented for comparing the biomarkers detected by the analysis unit, thereby diagnosing keratoconus.

The invention has the advantages and beneficial effects that:

based on the whole genome sequencing data of the keratoconus, the invention performs association analysis, screens significant variation sites related to the keratoconus, and performs multi-gene risk score modeling and susceptibility risk prediction. The result shows that the genetic variation risk site screened by the invention can effectively distinguish normal population from keratoconus population.

Drawings

FIG. 1 is a chart of orthokeratology gene-related changes in the frequency of keratoconus population and control population with large differences;

fig. 2 is a graph of predictive effects distinguishing cone-angle membrane populations from control populations.

Detailed Description

The following provides definitions of some of the terms used in this specification. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The invention provides application of a reagent for detecting the expression level of SNP markers in a sample in preparation of a product for diagnosing keratoconus, wherein the SNP markers comprise 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 and rs 17766647.

In the present invention, SNP (single nucleotide polymorphism) refers to a single nucleotide polymorphism, and since a single site on a chromosome causes a general abrupt variation in one of a plurality of DNA bases, SNP is frequently and stably distributed over the entire genome, and thus a genetic polymorphism occurs in a human body. The SNP sites of the invention are named in rs-manner, and the skilled person is able to determine their exact position, nucleotide sequence from a suitable database and related information system, such as the single nucleotide polymorphism database (dbSNP), according to the rs-nomenclature above. In the present invention, the SNP marker or SNP site can be used in combination with SNP.

In the present invention, a marker means a biomarker, which can detect a change in a living body, objectively detect a normal or pathological state of the living body, drug responsiveness, etc.

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

The reagents include reagents for detecting the SNP marker genotype by sequencing, single base extension, allele-specific probe hybridization, allele-specific primer extension, allele-specific amplification (AS-PCR), allele-specific nucleotide incorporation, 5' nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, and Single Strand Conformation Polymorphism (SSCP) methods.

In the present invention, single-strand conformation polymorphism (SSCP) means that single-strand DNA may cause a spatial conformation difference due to a difference in base sequence, which may cause a difference in electrophoretic mobility of single-strand DNA of the same or similar length, so that it can be effectively detected by non-denaturing polyacrylamide gel electrophoresis. PCR-SSCP is a method of using SSCP for gene mutation detection of PCR amplified products, and PCR amplified DNA fragments are subjected to non-denaturing polyacrylamide gel electrophoresis under denaturing conditions by subjecting double-stranded DNA amplified fragments to high temperature treatment to unwind and maintain a single-stranded state. Currently, PCR-SSCP technology is widely used in various fields of molecular biology.

The principle of allele-specific amplification (AS-PCR) is that because the mismatch of a single base at the 3 '-end of a primer cannot be repaired by Taq DNA polymerase, an amplification reaction can only occur when the base at the 3' -end of the primer is complementarily paired with the allele of a SNP site; when the base at the 3' -end of the primer is not complementarily paired with the allele at the SNP site, the amplification reaction cannot occur. Currently, methods based on AS-PCR improvement have emerged, such AS four-primer amplified blocked mutation system PCR (Tetra-primer amplification refractory mutation system PCR, tetra-primer ARMS-PCR), fragment length differential allele specific PCR (fragment length discrepant allele specific PCR, FLDAS-PCR), multiallelic specific amplification (PCR amplification of multiple specific alleles, PMASA), and the like.

Sequencing methods include Pyrosequencing (Pyrosequencing), microsequencing (SNaPshot), second generation sequencing, taqman, cyclic sequencing, semiconductor sequencing, amino acid sequencing, and whole genome sequencing.

Any one or several methods (not limited to the above methods) may be selected by those skilled in the art to detect the SNP site, as long as the detection of the SNP site can be achieved.

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

The present invention provides a product for diagnosing keratoconus comprising reagents for detecting the above-mentioned SNP markers in a sample.

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

In the present invention, a nucleic acid affinity ligand refers to a nucleic acid molecule capable of binding to a SNP marker as defined above or a sequence in the vicinity thereof. As non-limiting embodiments, the nucleic acid affinity ligand may be, for example, a RNA, DNA, PNA, CAN, HNA, LNA or ANA molecule or any other suitable nucleic acid form known to those of skill in the art.

In the present invention, nucleic acid means both DNA and RNA, both in any possible configuration, i.e. in the form of double stranded (ds) nucleic acid, or in the form of (ss) nucleic acid, or in combination thereof (partial ds or ss). Such nucleic acids correspond to at least two consecutive deoxyribonucleotides or ribonucleotides optionally comprising at least one modified nucleotide.

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

In the present invention, the SNP markers may be appropriately amplified in a suitable buffer (e.g., polymerization agent such as 4 different nucleoside triphosphates, DNA, RNA polymerase or reverse transcriptase) and at appropriate temperature, a single-stranded oligonucleotide which can act as a column to show the origin of DNA synthesis, and the appropriate length of the primer varies depending on the purpose of use, but is usually 15 to 30 nucleotides. Short primer molecules typically require lower temperatures in order to form a columnar and stable mixture. The primer sequence need not form complete complementarity with the polynucleotide comprising the SNP, but its complementarity needs to be such that it can be mixed with the polynucleotide comprising the SNP.

The primer is a short sequence that can form a base pair with a complementary template (template) using a base sequence having a short chain free 3'hydroxyl group (free 3' hydroxyl group) and functions as a replication origin of a columnar chain. The primer can be used as a sample for polymerization (i.e., DNA polymerase or reverse transcriptase) in a proper buffer solvent and temperature, and DNA synthesis can be started in the presence of 4 different nucleoside triphosphates. At this time, PCR conditions, sensitivity, and length of the antisense primer may be modified according to known techniques in the art.

The probe specifically binding to the SNP marker may be a mixed probe or an oligonucleotide binding to a nucleic acid complementary strand according to a specific sequence. The mixing conditions are such that they exhibit a significant difference in the intensity of mixing between alleles, so that they are tightly controlled to mix into only one of the alleles. The probe can be used in a sample box such as a microarray for detecting an allele or diagnosing keratoconus, a prediction method, or the like.

The reagent further comprises a detectable label.

In the present invention, a detectable label refers to a composition capable of generating a detectable signal indicative of the presence of a target polynucleotide in an assay sample. 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 that can be detected by a device or method, including but not limited to spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical detection means or any other suitable means. In some embodiments, the indicia may be visually detected without the aid of a device.

Wherein the radioisotope includes, but is not limited to ³ H、 ¹⁴ C、 ³⁵ S、 ¹²⁵ I、 ¹³¹ I。

Enzymes include, but are not limited to, horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase.

Fluorescent molecules include, but are not limited to, FITC, rhodamine, lanthanide phosphors (lanthanide phosphors).

The primer or probe of the present invention may be chemically synthesized by a phosphoramidite solid support method or other known methods. The nucleic acid sequence may be deformed using a variety of techniques well known in the art. Non-limiting examples of such deformations are: substitution is methylation, adsorption, and deformation between nucleotides, such as deformation into uncharged linkers (e.g., methylphosphonate, phosphotriester, phosphoramidite, carbamate, etc.), or charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.).

The product comprises a kit, test paper or chip.

In the present invention, the kit comprises a set of oligonucleotide primers sufficient to detect and/or quantify the genotype of the SNP markers according to the invention. The oligonucleotide primers may be provided in lyophilized or reconstituted form, or may be provided as a set of nucleotide sequences. In one embodiment, the primers are provided in the form of microwells (microplates), wherein each primer set occupies a well (or multiple wells, as in the case of repetition) in the microwell plate. The microplate may further comprise primers sufficient to detect one or more housekeeping genes as described below.

The kit also comprises a container, a reaction buffer solution, deoxynucleotides (dNTPs), enzymes, DNase and RNAse inhibitors.

Suitable containers in the kit typically include at least one vial, test tube, flask, baud bottle, syringe, or other container in which one component may be placed, and preferably, an appropriate aliquot may be performed. Where more than one component is present in the kit, the kit will also typically contain a second, third or other additional container in which the additional components are placed separately. However, different combinations of components may be contained in one vial. The kits of the invention will also typically include a container for holding the reagents, sealed for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials may be retained.

The reaction buffer in the kit includes, but is not limited to, a variety of pH, and the enzymes include, but are not limited to, taq-polymerase and reverse transcriptase.

In the present invention, the chip is used interchangeably with array and biochip, and the chip further comprises a solid support. The solid phase carrier can be made of various common materials in the field of gene chips, including but not limited to plastic products, microparticles, membrane carriers and the like. The most commonly used plastic products are small test tubes, beads and microplates made of polystyrene; the microparticles are microspheres or particles polymerized by high molecular monomers, have the diameter of micrometer, are easy to form chemical coupling with antibodies (antigens) due to the functional groups capable of being combined with proteins, and have large combining capacity; the membrane carrier comprises microporous filter membranes such as nitrocellulose membranes, glass cellulose membranes and nylon membranes.

The invention provides application of the SNP marker in constructing a computer model for diagnosing keratoconus.

In some embodiments, the computer model has the above SNP markers as input variables.

In some embodiments, the computer model also has other keratoconus related markers as input variables.

In the present invention, the computational model may be constructed by employing logistic regression (Logtistic Regression), random forest (random forest), polar gradient lifting (eXtreme Gradient Boosting, XGBoosting) and support vector machine (Support Vector Machine, SVM) methods.

The present invention provides a system/device for diagnosing keratoconus, the system/device comprising:

1) Analysis unit: the analysis unit is used for detecting the SNP markers in a subject sample;

2) An evaluation unit: the evaluation unit contains a stored reference and a data processor which has been implemented for comparing the biomarkers detected by the analysis unit, thereby diagnosing keratoconus.

In the present invention, the system/apparatus may include a digital processing device or the use of a digital processing device. In further 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 the executable instructions. In some embodiments, the digital processing device is optionally connected to a computer network. In further embodiments, the digital processing device is optionally connected to the Internet such that it accesses 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.

Suitable digital processing devices include, by way of non-limiting example, server computers, desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook tablet computers, set-top computers, handheld computers, internet devices, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles, in accordance with the description of the invention. Those skilled in the art will recognize that many smartphones are suitable for use in the systems described herein. Those skilled in the art will also recognize that selected televisions, video players, and digital music players having alternative computer network connectivity are suitable for use in the system of the present invention. Suitable tablet computers include those known to those skilled in the art as having booklets, tablets, and convertible configurations.

The invention is further illustrated below in connection with specific embodiments. It should be understood that the particular embodiments described herein are presented by way of example and not limitation. The principal features of the invention may be used in various embodiments without departing from the scope of the invention.

Examples

1. Experimental materials

649 keratoconus population and 748 control population. Peripheral blood samples were taken from 1397 individuals.

2. Experimental method

Genetic material DNA extraction was performed on the peripheral blood samples and whole genome sequencing was performed. The average sequencing depth of keratoconus was 10X, and the average sequencing depth of the control group was 20X.

3. Experimental results

1) The sequencing data were quality controlled, aligned, mutation detected and filtered, and a total of 3,350,611 common single nucleotide variants (minimal allele frequency >0.01, excluding sex chromosome and high genetic HLA regions) were detected.

2) 649 keratoconus populations and 748 control individuals were divided into discovery and validation queues (Table 1).

Table 1 sample grouping

3) And (3) carrying out association analysis on the found queue, finding out related variation sites of keratoconus genes with large frequency difference between keratoconus population and control population (figure 1), selecting 22 sites of p <10e-4 after removing sites with strong linkage on the positions, carrying out multi-gene risk scoring (Polygenic Risk Score, PRS), and checking the distinguishing effect of the combinations of the sites on the verification queue.

The P values for the 22 sites and their associated analysis in the discovery queue are shown in table 2.

TABLE 2P values for 22 sites and their associated analysis in discovery queue

The predicted effects of distinguishing the verification-queued keratoconus population from the control population are shown in table 3 for the 22-site individual calculation.

TABLE 3 predictive efficacy at 22 sites for differentiating keratoconus population from control population

Number of marks	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

The predicted effects of distinguishing the verification-queued keratoconus population from the control population for the 22-site combination calculation are shown in table 4.

TABLE 4 prediction effect of 22 site combination calculation to distinguish cone-shaped cornea population from control population

4) Modeling 22 sites and polygenic risk scores, calculating the prediction effect (AUC value), the working curve (ROC) of a subject and the sensitivity and the specificity of a cone cornea crowd and a control crowd, and finding that the most obvious 22 sites screened by a queue show higher efficacy on keratoconus phenotype diagnosis, wherein the AUC reaches 0.713. Sensitivity (true positive rate) and specificity (1-false positive rate) were 0.738 and 0.583, respectively (FIG. 2).

The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims (10)

1. The application of the reagent for detecting the expression level of the SNP marker in the sample in the preparation of a product for diagnosing keratoconus is characterized in that the SNP marker comprises 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 and rs 17766647.

2. The use according to claim 1, wherein the reagents comprise reagents for detecting the SNP marker genotype 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 conformational polymorphism method;

preferably, the reagent comprises a reagent for detecting the SNP marker genotype by a sequencing method;

preferably, the sequencing method comprises pyrophosphoric acid sequencing, micro-sequencing, second generation sequencing, taqman method, cyclic sequencing, semiconductor sequencing, amino acid sequencing, whole genome sequencing;

preferably, the sequencing method is selected from whole genome sequencing methods.

3. The use according to claim 1, wherein the sample comprises blood, tissue;

preferably, the sample is selected from blood;

preferably, the blood is selected from peripheral blood.

4. The use of claim 1, wherein the product further comprises a reagent for processing the sample.

5. A product for diagnosing keratoconus, characterized in that it comprises a reagent for detecting the SNP marker as set forth in claim 1 in a sample.

6. The product of claim 5, wherein the reagent comprises a nucleic acid affinity ligand for the SNP marker;

preferably, the nucleic acid affinity ligand comprises a primer that specifically amplifies a polynucleotide of the SNP marker or a probe that can specifically bind to the SNP marker;

preferably, the reagent further comprises a detectable label.

7. The product of claim 5, wherein the product comprises a kit, a test strip, or a chip.

8. The product of claim 7, wherein the kit further comprises a container, a reaction buffer, deoxynucleotides, enzymes, DNase, RNAse inhibitors;

preferably, the kit further comprises instructions.

9. Use of the SNP marker of claim 1 in constructing a computer model for diagnosing keratoconus.

10. A system/device for diagnosing keratoconus, the system/device comprising:

1) Analysis unit: the analysis unit is used for detecting the SNP marker according to claim 1 in a subject sample;

2) An evaluation unit: the evaluation unit contains a stored reference and a data processor which has been implemented for comparing the biomarkers detected by the analysis unit, thereby diagnosing keratoconus.