Primer set, kit and application for screening patients with autism spectrum disorder

Abstract

The application discloses a primer group, a kit and application for screening autism spectrum disorder patients, and belongs to the field of disease diagnosis. The technical problem solved by the application is how to screen autism spectrum disorder patients in Chinese Han population. The primer group for screening autism spectrum disorder patients disclosed by the application is directed to 132 autism susceptible gene sites, and is composed of 264 single-stranded DNAs shown in SEQ ID No. 1 to SEQ ID No. 264. According to the 132 autism susceptible gene sites, a data model is established by SVM, autism spectrum disorder patients can be screened, and the data model has good accuracy, precision, recall rate, F1 score and AUC value, can provide stable and efficient prediction performance between balanced precision and recall rate, and has strong ability in distinguishing autism spectrum disorder patients and healthy individuals.

Description

Technical Field

[0001] This invention belongs to the field of disease diagnosis, specifically relating to primer sets, reagent kits, and applications for screening patients with autism spectrum disorder. Background Technology

[0002] Autism spectrum disorder (ASD) is a neurodevelopmental disorder that typically begins around age 3 and lasts a lifetime, posing a significant threat to families and society. ASD is characterized by clear social impairments, repetitive behaviors, narrow interests, and language and communication difficulties. Currently, there is no effective treatment for ASD; treatment primarily relies on behavioral therapy. Therefore, early screening and diagnosis are a shared goal of both patients and doctors, and can significantly improve the prognosis for children with autism. However, in practice, the lack of readily available objective markers for ASD presents considerable challenges to diagnosis.

[0003] Autism spectrum disorder has a high heritability, and studies have shown a close correlation between genetic factors causing neurodevelopmental abnormalities and the onset of autism. Epidemiological data show that the concordance rate of autism is as high as 92% in monozygotic twins, while it is only 10% in dizygotic twins. The concordance rate among siblings of affected children is 2-8%, which is 20-80 times higher than in the general population. While the heritability of autism is as high as 80-90%, its inheritance pattern is complex and does not conform to Mendelian inheritance laws; it is a polygenic inherited disease. Therefore, combined testing of multiple risk genes holds promise for early screening of the risk of developing autism spectrum disorder.

[0004] Currently, there are no specialized early screening devices for the genetic risk of autism on the market. Current genetic testing for individuals mainly relies on genotyping methods based on single loci. For example, DNA molecules are extracted from peripheral blood or saliva samples from patients, and then genotyping is performed using methods such as quantitative real-time PCR or nucleic acid mass spectrometry to analyze different detection loci. Furthermore, most of these methods target rare autism-related variants. While this approach is advantageous for detecting mutations at single loci, testing only a few loci has the drawback of being incomplete and unable to accurately determine the actual function of the gene. Invention Overview

[0006] The technical problem to be solved by this invention is how to screen patients with autism spectrum disorder in the Han Chinese population, and provides susceptibility loci for early clinical screening of autism spectrum disorder in the Han Chinese population.

[0007] To address the aforementioned technical problems, the present invention first provides a primer set, named Primer Set 1, which consists of 264 single-stranded DNA strands as shown in SEQ ID No. 1 to SEQ ID No. 264.

[0008] The primers with sequence numbers 2n-1 and 2n in primer set 1 form a primer pair for amplifying the same DNA fragment, where n is a natural number between 1 and 66.

[0009] The molar ratio of each single-stranded DNA in primer set 1 can be equal.

[0010] The present invention also provides another primer set, named primer set 2, which is obtained by adding fixed sequence 1 to the 5′ end of primers with odd sequence numbers in primer set 1 and fixed sequence 2 to the 5′ end of primers with even sequence numbers in primer set 1; fixed sequence 1 is different from fixed sequence 2, and both are used for further amplification of the PCR product of primer set 1.

[0011] The purpose of the fixed sequence 1 and the fixed sequence 2 is to allow the addition of the same fragment upstream and downstream of the PCR product of primer set 1 during PCR amplification using primer set 2. This facilitates further amplification of the PCR product of primer set 2 using the same primers (i.e., the universal primer pair hereinafter referred to as primers). There are no special requirements for the sequences of fixed sequence 1 and fixed sequence 2, as long as they meet the above objectives.

[0012] In one embodiment of the present invention, the sequence of fixed sequence 1 is SEQ ID No. 265 in the sequence list, and the sequence of fixed sequence 2 is SEQ ID No. 266 in the sequence list.

[0013] The molar ratio of each single-stranded DNA in primer set 2 can be equal.

[0014] Both primer set 1 and primer set 2 can be used to diagnose or assist in the diagnosis of autism spectrum disorder, screen or assist in the screening of patients with autism spectrum disorder, detect the severity of autism spectrum disorder, or detect the genetic risk of autism spectrum disorder.

[0015] The present invention also provides a kit, named kit 1, which contains the primer set 1.

[0016] The present invention also provides another kit, named kit 2, which contains the primer set 2.

[0017] The above-mentioned kit 2 may also contain a universal primer pair, which is used to identify the fixed sequence 1 and the fixed sequence 2 in order to further amplify the PCR product of the primer set 2.

[0018] The primers of the universal primer pair may contain tag sequences to facilitate the differentiation of different samples.

[0019] In one embodiment of the present invention, the universal primer pair consists of two single-stranded DNAs as shown in SEQ ID No. 267 and SEQ ID No. 268.

[0020] The above-mentioned kits 1 and 2 may also contain a data conversion device, which is used to convert the detection results of the SNP combination into an autism spectrum disorder score, which is used for the diagnosis, screening, disease severity assessment or genetic risk assessment of autism spectrum disorder.

[0021] The SNP combination refers to the following SNP sites in the human genome: rs2181419, rs67980110, rs66512, rs1222064, rs12130426, rs2391769, rs6701243, rs12778231, rs4750990, rs671154, rs492768, rs523871, rs61925919, rs11160145, rs7215239, rs17565025, rs242559, rs3785884, rs62062271, rs62073098, rs2532424, rs199526, rs1994. 98. rs72837297, rs11872054, rs1597749, rs12477455, rs145741754, rs6730686, rs2883872, rs1548635, rs6079536, rs6074798, rs6042917, rs3767 11. rs6082289, rs6047225, rs6047270, rs6132406, rs12625304, rs1000177, rs6035821, rs112777342, rs3787500, rs6035868, rs1090226, rs2424362 , rs57080033, rs8182800, rs6027989, rs2249666, rs405182, rs1452075, rs2139816, rs1522603, rs4916723, rs2635182, rs325501, rs13188074, rs6 63338、rs9366877、rs16879023、rs407238、rs9375188、rs72560713、rs35971735、rs56189730、rs6945821、rs143762771、rs111931861、rs7794285、r s73186025, rs35851667, rs17148231, rs2979184, rs2921036, rs11992186, rs6985207, rs693109, rs2048419, rs12545499, rs7013471, rs13270070, rs36059156, rs6992534, rs35305751, rs35694744, rs7005133, rs7387360, rs10081437, rs393155, rs12547955, rs11249905, rs2929453, rs1466555,rs6982308, rs7005363, rs7001281, rs11775702, rs10096777, rs4841410, rs7823200, rs7461950, rs10099100, rs 4841432, rs1991651, rs877116, rs755856, rs10090444, rs12542037, rs6989105, rs7834248, rs10086521, rs78434 70, rs12679529, rs7000276, rs7004551, rs11783418, rs6601560, rs35499486, rs11250130, rs2736305, rs12156009, rs6980884, rs2729940, rs2409784, rs2898290, rs60527016, rs366249, rs77495304, rs76569799, and rs73503170.

[0022] The data conversion device can use a classification model to convert the detection results of the SNP combination into autism spectrum disorder score values. The classification model can be a Logistic Regression, Support Vector Machine (SVM), Random Forest, Extreme Gradient Boosting (XGBoost), Gradient Boosting, Multilayer Perceptron (MLP) model, etc.

[0023] The classification model can obtain a score by analyzing the characteristics of the SNP combination in patients with autism spectrum disorder, and then further determine the diagnosis, screening, disease severity assessment or genetic risk assessment results of the subject's autism spectrum disorder.

[0024] In one embodiment of the present invention, the classification model is a support vector machine model.

[0025] The kit 1 may consist only of the primer set 1, or it may consist of the primer set 1 and other reagents required for PCR amplification besides the primers, or it may consist of the primer set 1 and the data processing device, or it may consist of the primer set 1, other reagents required for PCR amplification besides the primers, and the data processing device.

[0026] The reagent kit 2 may consist solely of primer set 2, or it may consist of primer set 2 and the universal primer pair, or it may consist of primer set 2 and other reagents required for PCR amplification besides primers, or it may consist of primer set 2, the universal primer pair and other reagents required for PCR amplification besides primers, or it may consist of primer set 2 and the data processing device, or it may consist of primer set 2, the universal primer pair and the data processing device, or it may consist of primer set 2, other reagents required for PCR amplification besides primers and the data processing device, or it may consist of primer set 2, the universal primer pair, other reagents required for PCR amplification besides primers and the data processing device.

[0027] Both kit 1 and kit 2 can be used to diagnose or assist in the diagnosis of autism spectrum disorder, screen or assist in the screening of patients with autism spectrum disorder, detect the severity of autism spectrum disorder, or detect the genetic risk of autism spectrum disorder.

[0028] The present invention also provides any of the following applications of primer set 1 or primer set 2:

[0029] X1) Prepare products for diagnosing or assisting in the diagnosis of autism spectrum disorder;

[0030] X2) Products for screening or assisting in the screening of patients with autism spectrum disorder;

[0031] X3) Prepare products for detecting or assisting in the detection of the severity of autism spectrum disorder in patients;

[0032] X4) Prepare products for detecting or assisting in the detection of genetic risk of autism spectrum disorder;

[0033] X5) Diagnostic or auxiliary diagnosis of autism spectrum disorder;

[0034] X6) Screening or auxiliary screening for patients with autism spectrum disorder;

[0035] X7) Detection or auxiliary detection of the severity of autism spectrum disorder in patients;

[0036] X8) testing or auxiliary testing for the genetic risk of autism spectrum disorder.

[0037] The present invention also provides for any of the following applications of reagent kit 1 or reagent kit 2:

[0038] X1) Prepare products for diagnosing or assisting in the diagnosis of autism spectrum disorder;

[0039] X2) Products for screening or assisting in the screening of patients with autism spectrum disorder;

[0040] X3) Prepare products for detecting or assisting in the detection of the severity of autism spectrum disorder in patients;

[0041] X4) Prepare products for detecting or assisting in the detection of genetic risk of autism spectrum disorder;

[0042] X5) Diagnostic or auxiliary diagnosis of autism spectrum disorder;

[0043] X6) Screening or auxiliary screening for patients with autism spectrum disorder;

[0044] X7) Detection or auxiliary detection of the severity of autism spectrum disorder in patients;

[0045] X8) testing or auxiliary testing for the genetic risk of autism spectrum disorder.

[0046] The present invention also provides the application of the primer set 1, the primer set 2, the kit 1 or the kit 2 in the preparation of products for detecting or assisting in the detection of autism-related genetic risk gene polymorphisms, or in the detection or assisting in the detection of autism-related genetic risk gene polymorphisms;

[0047] The detection of autism-related genetic risk gene polymorphisms involves detecting nucleotide combinations of the aforementioned SNPs in the human genome.

[0048] This invention also provides any of the following applications of a substance for detecting gene polymorphisms associated with autism:

[0049] X1) Prepare products for diagnosing or assisting in the diagnosis of autism spectrum disorder;

[0050] X2) Products for screening or assisting in the screening of patients with autism spectrum disorder;

[0051] X3) Prepare products for detecting or assisting in the detection of the severity of autism spectrum disorder in patients;

[0052] X4) Prepare products for detecting or assisting in the detection of genetic risk of autism spectrum disorder;

[0053] X5) Diagnostic or auxiliary diagnosis of autism spectrum disorder;

[0054] X6) Screening or auxiliary screening for patients with autism spectrum disorder;

[0055] X7) Detection or auxiliary detection of the severity of autism spectrum disorder in patients;

[0056] X8) Testing or auxiliary testing for the genetic risk of autism spectrum disorder;

[0057] The autism-related genetic risk gene polymorphisms are the polymorphisms of the SNP combinations in the human genome.

[0058] The present invention also provides any of the following methods:

[0059] Y1) A method for diagnosing or assisting in the diagnosis of autism spectrum disorder, including: receiving nucleotides from a genetic risk locus related to autism in a subject to be tested, and obtaining a diagnostic result of autism spectrum disorder in the subject to be tested;

[0060] Y2) Screening or assisted screening for autism spectrum disorder, including: receiving nucleotides from autism-related genetic risk gene loci of the subject to be tested, and obtaining the screening results for autism spectrum disorder of the subject to be tested;

[0061] Y3) Detection or auxiliary detection of the severity of autism spectrum disorder in patients, including: receiving nucleotides from autism-related genetic risk gene loci in a patient with autism spectrum disorder to be tested, and obtaining the result of the severity of autism spectrum disorder in the patient with autism spectrum disorder to be tested;

[0062] Y4) Detection or auxiliary detection of genetic risk of autism spectrum disorder, including: receiving nucleotides of autism-related genetic risk gene loci from a patient with autism spectrum disorder to be tested, and obtaining the genetic risk of autism spectrum disorder in the patient with autism spectrum disorder to be tested;

[0063] The autism-related genetic risk loci are the SNP combinations in the human genome.

[0064] In the above method, the diagnostic results of autism spectrum disorder of the test subject, the screening results of autism spectrum disorder of the test subject, the severity results of autism spectrum disorder of the test subject, and the genetic risk of autism spectrum disorder of the test subject can all be obtained using models such as Logistic Regression, Support Vector Machine (SVM), Random Forest, Extreme Gradient Boosting (XGBoost), Gradient Boosting, and Multilayer Perceptron (MLP).

[0065] In one embodiment of the present invention, the classification model is a support vector machine model.

[0066] The present invention also provides a data processing apparatus / device / system, including a memory, a processor, and a computer program stored in the memory, wherein the processor executes the computer program to implement the steps of the method described in Y1), Y2), Y3), or Y4).

[0067] The present invention also provides a computer-readable storage medium having a computer program / instructions stored thereon, wherein the computer program / instructions, when executed by a processor, implement the steps of the method described in Y1), Y2), Y3), or Y4).

[0068] The present invention also provides a computer program product, including a computer program / instruction, characterized in that: when the computer program / instruction is executed by a processor, it implements the steps of the method described in Y1), Y2), Y3), or Y4).

[0069] The computer program product includes a non-transitory computer-readable medium, wherein the non-transitory computer-readable medium stores computer program code for data processing, wherein the computer program code is executable by a processor in a device containing the computer program product to perform the method according to Y1), Y2), Y3), or Y4).

[0070] In one specific embodiment, the data processing device is a device for diagnosing autism spectrum disorder, screening for autism spectrum disorder, detecting the severity of autism spectrum disorder, or detecting the genetic risk of autism spectrum disorder.

[0071] In one specific embodiment, the data processing device is a computer device.

[0072] The subjects in this study (test subjects or patients with autism spectrum disorder) were individuals with at least one of the following characteristics: diagnosed with autism spectrum disorder according to the criteria defined in the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-V), meeting the following criteria: an Autism Behavior Checklist (ABC) score ≥53 or a Childhood Autism Rating Scale (CARS) score >36. Subjects in this study may also have any of the following characteristics: diagnosed with autism; excluded individuals with Asperger syndrome and pervasive developmental disorder-not otherwise specified (PDD-NOS); excluded individuals with phenylketonuria, fragile X syndrome, tuberous sclerosis, or other neurological disorders, as well as those with severe physical illnesses and chromosomal abnormalities.

[0073] In some specific embodiments of the present invention, the subject is of Asian ethnicity. Further, the Asian ethnicity is East Asian. Even further, the subject is Chinese.

[0074] The autism spectrum disorder in this invention can be autism.

[0075] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. Attached Figure Description

[0076] Figure 1 A bar chart comparing the performance of each model.

[0077] Figure 2 Receiver operating characteristic curves for each model.

[0078] Figure 3 Precision-recall curves for each model.

[0079] Embodiments of the present invention

[0080] Those skilled in the art will understand that the computer programs / instructions or steps of the present invention described above can be implemented using general-purpose computing devices. They can be centralized on a single computing device or distributed across a network of multiple computing devices. Optionally, they can be implemented using computer-executable program code, thereby storing them in a storage device for execution by a computing device. In some cases, the steps can be executed in a different order than described here, or they can be fabricated as separate integrated circuit modules, or multiple modules or steps can be fabricated as a single integrated circuit module. Thus, the present invention is not limited to any particular combination of hardware and software.

[0081] The general-purpose computing device mentioned here typically includes a processor and a memory. The memory is used to store instructions, which, when executed by the processor, cause the computing device to perform "the various steps or modules of the present invention".

[0082] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available.

[0083] In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5′ terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA / RNA.

[0084] Example 1: Preparation of a kit for screening patients with autism spectrum disorder

[0085] Step 1: Initial screening of single nucleotide polymorphisms (SNPs). Using existing SNPs from the European population autism genome-wide association database (PGC, https: / / pgc.unc.edu / ), a significance p-value of less than 5 x 10^E6 was used as the screening criterion. Combined with the results of whole-genome sequencing of autism in the Han Chinese population (The 1000 Genomes Project Consortium. A global reference for human genetic variation. Nature 526, 68–74 (2015). https: / / doi.org / 10.1038 / nature15393), 3582 SNPs were identified with identical polymorphic sites (minimum allele frequency MAF > 0.05).

[0086] Step 2: Determination of single nucleotide polymorphism sites. The 3582 single nucleotide polymorphism sites obtained in the initial screening in Step 1 were sorted from smallest to largest according to the significance p value. The linkage disequilibrium coefficient between different sites was calculated based on the linkage disequilibrium data of the Han Chinese population genome. Sites with high linkage disequilibrium were removed according to the coefficient LD>0.5, and the final number of sites was determined to be 134.

[0087] Step 3: Design primers. Amplify the capture region by designing amplification primers upstream and downstream of each SNP site, including at least 50 bp fragments upstream and downstream of each single nucleotide polymorphism site. Primer length is between 18-35 bp, and amplicon size is 100-200 bp. The final number of successfully designed primers is 132 sites.

[0088] The specific information for each SNP site and its specific primers is as follows:

[0089] SNP1: The site name is rs2181419, located on chr1. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No. 1 and SEQ ID No. 2, and the amplification product is 155 bp in length.

[0090] SNP2: The site name is rs67980110, located on chr1. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 3 and SEQ ID No. 4, and the amplification product is 165 bp in length.

[0091] SNP3: The site name is rs66512, located on chr1. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 5 and SEQ ID No. 6, and the amplification product is 164 bp in length.

[0092] SNP4: The site name is rs1222064, located on chr1. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No. 7 and SEQ ID No. 8, and the amplification product is 160 bp in length.

[0093] SNP5: The site name is rs12130426, located on chr1. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No. 9 and SEQ ID No. 10, and the amplification product is 157 bp in length.

[0094] SNP6: The site name is rs2391769, located on chr1. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No.11 and SEQ ID No.12, and the amplification product is 151 bp in length.

[0095] SNP7: The site name is rs6701243, located on chr1. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.13 and SEQ ID No.14, and the amplification product is 160 bp in length.

[0096] SNP8: The site name is rs12778231, located on chr10. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No.15 and SEQ ID No.16, and the amplification product is 162 bp in length.

[0097] SNP9: The site name is rs4750990, located on chr10. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No.17 and SEQ ID No.18, and the amplification product is 164 bp in length.

[0098] SNP10: The site name is rs671154, located on chr11. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.19 and SEQ ID No.20, and the amplification product is 165 bp in length.

[0099] SNP11: The site name is rs492768, located on chr11. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No. 21 and SEQ ID No. 22, and the amplification product is 163 bp in length.

[0100] SNP12: The site name is rs523871, located on chr11. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No.23 and SEQ ID No.24, and the amplification product is 165 bp in length.

[0101] SNP13: The site name is rs61925919, located on chr12. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No. 25 and SEQ ID No. 26, and the amplification product is 149 bp in length.

[0102] SNP14: The site name is rs11160145, located on chr14. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 27 and SEQ ID No. 28, and the amplification product is 151 bp in length.

[0103] SNP15: The site name is rs7215239, located on chr17. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No. 29 and SEQ ID No. 30, and the amplification product is 159 bp in length.

[0104] SNP16: The site name is rs17565025, located on chr17. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.31 and SEQ ID No.32, and the amplification product is 165 bp in length.

[0105] SNP17: The site name is rs242559, located on chr17. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.33 and SEQ ID No.34, and the amplification product is 163 bp in length.

[0106] SNP18: The site name is rs3785884, located on chr17. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.35 and SEQ ID No.36, and the amplification product is 161 bp in length.

[0107] SNP19: The site name is rs62062271, located on chr17. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No.37 and SEQ ID No.38, and the amplification product is 145 bp in length.

[0108] SNP20: The site name is rs62073098, located on chr17. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 39 and SEQ ID No. 40, and the amplification product is 145 bp in length.

[0109] SNP21: The site name is rs2532424, located on chr17. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No.41 and SEQ ID No.42, and the amplification product is 165 bp in length.

[0110] SNP22: The site name is rs199526, located on chr17. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No.43 and SEQ ID No.44, and the amplification product is 157 bp in length.

[0111] SNP23: The site name is rs199498, located on chr17. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No.45 and SEQ ID No.46, and the amplification product is 158 bp in length.

[0112] SNP24: The site name is rs72837297, located on chr17. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.47 and SEQ ID No.48, and the amplification product is 147 bp in length.

[0113] SNP25: The site name is rs11872054, located on chr17. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.49 and SEQ ID No.50, and the amplification product is 165 bp in length.

[0114] SNP26: The site name is rs1597749, located on chr2. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 51 and SEQ ID No. 52, and the amplification product is 165 bp in length.

[0115] SNP27: The site name is rs12477455, located on chr2. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 53 and SEQ ID No. 54, and the amplification product is 150 bp in length.

[0116] SNP28: The site name is rs145741754, located on chr2. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 55 and SEQ ID No. 56, and the amplification product is 159 bp in length.

[0117] SNP29: The site name is rs6730686, located on chr2. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No. 57 and SEQ ID No. 58, and the amplification product is 165 bp in length.

[0118] SNP30: The site name is rs2883872, located on chr2. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 59 and SEQ ID No. 60, and the amplification product is 161 bp in length.

[0119] SNP31: The site name is rs1548635, located on chr2. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 61 and SEQ ID No. 62, and the amplification product is 165 bp in length.

[0120] SNP32: The site name is rs6079536, located on chr20. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 63 and SEQ ID No. 64, and the amplification product is 157 bp in length.

[0121] SNP33: The site name is rs6074798, located on chr20. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 65 and SEQ ID No. 66, and the amplification product is 158 bp in length.

[0122] SNP34: The site name is rs6042917, located on chr20. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 67 and SEQ ID No. 68, and the amplification product is 160 bp in length.

[0123] SNP35: The site name is rs376711, located on chr20. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 69 and SEQ ID No. 70, and the amplification product is 161 bp in length.

[0124] SNP36: The site name is rs6082289, located on chr20. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.71 and SEQ ID No.72, and the amplification product is 165 bp in length.

[0125] SNP37: The site name is rs6047225, located on chr20. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.73 and SEQ ID No.74, and the amplification product is 165 bp in length.

[0126] SNP38: The site name is rs6047270, located on chr20. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.75 and SEQ ID No.76, and the amplification product is 145 bp in length.

[0127] SNP39: The site name is rs6132406, located on chr20. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.77 and SEQ ID No.78, and the amplification product is 165 bp in length.

[0128] SNP40: The site name is rs12625304, located on chr20. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.79 and SEQ ID No.80, and the amplification product is 155 bp in length.

[0129] SNP41: The site name is rs1000177, located on chr20. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 81 and SEQ ID No. 82, and the amplification product is 148 bp in length.

[0130] SNP42: The site name is rs6035821, located on chr20. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 83 and SEQ ID No. 84, and the amplification product is 147 bp in length.

[0131] SNP43: The site name is rs112777342, located on chr20. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 85 and SEQ ID No. 86, and the amplification product is 165 bp in length.

[0132] SNP44: The site name is rs3787500, located on chr20. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 87 and SEQ ID No. 88, and the amplification product is 149 bp in length.

[0133] SNP45: The site name is rs6035868, located on chr20. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 89 and SEQ ID No. 90, and the amplification product is 147 bp in length.

[0134] SNP46: The site name is rs1090226, located on chr20. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 91 and SEQ ID No. 92, and the amplification product is 161 bp in length.

[0135] SNP47: The site name is rs2424362, located on chr20. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 93 and SEQ ID No. 94, and the amplification product is 145 bp in length.

[0136] SNP48: The site name is rs57080033, located on chr20. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No. 95 and SEQ ID No. 96, and the amplification product is 154 bp in length.

[0137] SNP49: The site name is rs8182800, located on chr20. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No. 97 and SEQ ID No. 98, and the amplification product is 154 bp in length.

[0138] SNP50: The site name is rs6027989, located on chr20. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 99 and SEQ ID No. 100, and the amplification product is 155 bp in length.

[0139] SNP51: The site name is rs2249666, located on chr21. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 101 and SEQ ID No. 102, and the amplification product is 158 bp in length.

[0140] SNP52: The site name is rs405182, located on chr3. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 103 and SEQ ID No. 104, and the amplification product is 146 bp in length.

[0141] SNP53: The site name is rs1452075, located on chr3. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 105 and SEQ ID No. 106, and the amplification product is 165 bp in length.

[0142] SNP54: The site name is rs2139816, located on chr4. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 107 and SEQ ID No. 108, and the amplification product is 158 bp in length.

[0143] SNP55: The site name is rs1522603, located on chr4. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 109 and SEQ ID No. 110, and the amplification product is 152 bp in length.

[0144] SNP56: The site name is rs4916723, located on chr5. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No. 111 and SEQ ID No. 112, and the amplification product is 165 bp in length.

[0145] SNP57: The site name is rs2635182, located on chr5. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 113 and SEQ ID No. 114, and the amplification product is 165 bp in length.

[0146] SNP58: The site name is rs325501, located on chr5. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 115 and SEQ ID No. 116, and the amplification product is 165 bp in length.

[0147] SNP59: The site name is rs13188074, located on chr5. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 117 and SEQ ID No. 118, and the amplification product is 145 bp in length.

[0148] SNP60: The site name is rs663338, located on chr5. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 119 and SEQ ID No. 120, and the amplification product is 165 bp in length.

[0149] SNP61: The site name is rs9366877, located on chr6. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 121 and SEQ ID No. 122, and the amplification product is 151 bp in length.

[0150] SNP62: The site name is rs16879023, located on chr6. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.123 and SEQ ID No.124, and the amplification product is 165 bp in length.

[0151] SNP63: The site name is rs407238, located on chr6. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.125 and SEQ ID No.126, and the amplification product is 164 bp in length.

[0152] SNP64: The site name is rs9375188, located on chr6. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No. 127 and SEQ ID No. 128, and the amplification product is 149 bp in length.

[0153] SNP65: The site name is rs72560713, located on chr6. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 129 and SEQ ID No. 130, and the amplification product is 160 bp in length.

[0154] SNP66: The site name is rs35971735, located on chr7. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No. 131 and SEQ ID No. 132, and the amplification product is 155 bp in length.

[0155] SNP67: The site name is rs56189730, located on chr7. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.133 and SEQ ID No.134, and the amplification product is 165 bp in length.

[0156] SNP68: The site name is rs6945821, located on chr7. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 135 and SEQ ID No. 136, and the amplification product is 161 bp in length.

[0157] SNP69: The site name is rs143762771, located on chr7. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 137 and SEQ ID No. 138, and the amplification product is 158 bp in length.

[0158] SNP70: The site name is rs111931861, located on chr7. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 139 and SEQ ID No. 140, and the amplification product is 145 bp in length.

[0159] SNP71: The site name is rs7794285, located on chr7. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 141 and SEQ ID No. 142, and the amplification product is 164 bp in length.

[0160] SNP72: The site name is rs73186025, located on chr7. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 143 and SEQ ID No. 144, and the amplification product is 155 bp in length.

[0161] SNP73: The site name is rs35851667, located on chr7. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.145 and SEQ ID No.146, and the amplification product is 162 bp in length.

[0162] SNP74: The site name is rs17148231, located on chr7. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.147 and SEQ ID No.148, and the amplification product is 159 bp in length.

[0163] SNP75: The site name is rs2979184, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 149 and SEQ ID No. 150, and the amplification product is 165 bp in length.

[0164] SNP76: The site name is rs2921036, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 151 and SEQ ID No. 152, and the amplification product is 153 bp in length.

[0165] SNP77: The site name is rs11992186, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 153 and SEQ ID No. 154, and the amplification product is 164 bp in length.

[0166] SNP78: The site name is rs6985207, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 155 and SEQ ID No. 156, and the amplification product is 163 bp in length.

[0167] SNP79: The site name is rs693109, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 157 and SEQ ID No. 158, and the amplification product is 147 bp in length.

[0168] SNP80: The site name is rs2048419, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 159 and SEQ ID No. 160, and the amplification product is 165 bp in length.

[0169] SNP81: The site name is rs12545499, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 161 and SEQ ID No. 162, and the amplification product is 165 bp in length.

[0170] SNP82: The site name is rs7013471, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 163 and SEQ ID No. 164, and the amplification product is 145 bp in length.

[0171] SNP83: The site name is rs13270070, located on chr8. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No. 165 and SEQ ID No. 166, and the amplification product is 165 bp in length.

[0172] SNP84: The site name is rs36059156, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.167 and SEQ ID No.168, and the amplification product is 156 bp in length.

[0173] SNP85: The site name is rs6992534, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.169 and SEQ ID No.170, and the amplification product is 165 bp in length.

[0174] SNP86: The site name is rs35305751, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.171 and SEQ ID No.172, and the amplification product is 165 bp in length.

[0175] SNP87: The site name is rs35694744, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.173 and SEQ ID No.174, and the amplification product is 146 bp in length.

[0176] SNP88: The site name is rs7005133, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.175 and SEQ ID No.176, and the amplification product is 165 bp in length.

[0177] SNP89: The site name is rs7387360, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 177 and SEQ ID No. 178, and the amplification product is 157 bp in length.

[0178] SNP90: The site name is rs10081437, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.179 and SEQ ID No.180, and the amplification product is 161 bp in length.

[0179] SNP91: The site name is rs393155, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.181 and SEQ ID No.182, and the amplification product is 155 bp in length.

[0180] SNP92: The site name is rs12547955, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.183 and SEQ ID No.184, and the amplification product is 165 bp in length.

[0181] SNP93: The site name is rs11249905, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 185 and SEQ ID No. 186, and the amplification product is 155 bp in length.

[0182] SNP94: The site name is rs2929453, located on chr8. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No. 187 and SEQ ID No. 188, and the amplification product is 162 bp in length.

[0183] SNP95: The site name is rs1466555, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 189 and SEQ ID No. 190, and the amplification product is 165 bp in length.

[0184] SNP96: The site name is rs6982308, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.191 and SEQ ID No.192, and the amplification product is 146 bp in length.

[0185] SNP97: The site name is rs7005363, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.193 and SEQ ID No.194, and the amplification product is 155 bp in length.

[0186] SNP98: The site name is rs7001281, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 195 and SEQ ID No. 196, and the amplification product is 148 bp in length.

[0187] SNP99: The site name is rs11775702, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No.197 and SEQ ID No.198, and the amplification product is 149 bp in length.

[0188] SNP100: The site name is rs10096777, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 199 and SEQ ID No. 200, and the amplification product is 145 bp in length.

[0189] SNP101: The site name is rs4841410, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 201 and SEQ ID No. 202, and the amplification product is 148 bp in length.

[0190] SNP102: The site name is rs7823200, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 203 and SEQ ID No. 204, and the amplification product is 149 bp in length.

[0191] SNP103: The site name is rs7461950, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 205 and SEQ ID No. 206, and the amplification product is 165 bp in length.

[0192] SNP104: The site name is rs10099100, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 207 and SEQ ID No. 208, and the amplification product is 151 bp in length.

[0193] SNP105: The site name is rs4841432, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 209 and SEQ ID No. 210, and the amplification product is 162 bp in length.

[0194] SNP106: The site name is rs1991651, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 211 and SEQ ID No. 212, and the amplification product is 165 bp in length.

[0195] SNP107: The site name is rs877116, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 213 and SEQ ID No. 214, and the amplification product is 159 bp in length.

[0196] SNP108: The site name is rs755856, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 215 and SEQ ID No. 216, and the amplification product is 147 bp in length.

[0197] SNP109: The site name is rs10090444, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 217 and SEQ ID No. 218, and the amplification product is 165 bp in length.

[0198] SNP110: The site name is rs12542037, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 219 and SEQ ID No. 220, and the amplification product is 158 bp in length.

[0199] SNP111: The site name is rs6989105, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 221 and SEQ ID No. 222, and the amplification product is 151 bp in length.

[0200] SNP112: The site name is rs7834248, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 223 and SEQ ID No. 224, and the amplification product is 164 bp in length.

[0201] SNP113: The site name is rs10086521, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 225 and SEQ ID No. 226, and the amplification product is 158 bp in length.

[0202] SNP114: The site name is rs7843470, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 227 and SEQ ID No. 228, and the amplification product is 157 bp in length.

[0203] SNP115: The site name is rs12679529, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 229 and SEQ ID No. 230, and the amplification product is 150 bp in length.

[0204] SNP116: The site name is rs7000276, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 231 and SEQ ID No. 232, and the amplification product is 165 bp in length.

[0205] SNP117: The site name is rs7004551, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 233 and SEQ ID No. 234, and the amplification product is 162 bp in length.

[0206] SNP118: The site name is rs11783418, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 235 and SEQ ID No. 236, and the amplification product is 163 bp in length.

[0207] SNP119: The site name is rs6601560, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 237 and SEQ ID No. 238, and the amplification product is 146 bp in length.

[0208] SNP120: The site name is rs35499486, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 239 and SEQ ID No. 240, and the amplification product is 165 bp in length.

[0209] SNP121: The site name is rs11250130, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 241 and SEQ ID No. 242, and the amplification product is 163 bp in length.

[0210] SNP122: The site name is rs2736305, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 243 and SEQ ID No. 244, and the amplification product is 160 bp in length.

[0211] SNP123: The site name is rs12156009, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 245 and SEQ ID No. 246, and the amplification product is 161 bp in length.

[0212] SNP124: The site name is rs6980884, located on chr8. Its specific primers consist of two single-stranded DNA molecules shown in SEQ ID No. 247 and SEQ ID No. 248, and the amplification product is 165 bp in length.

[0213] SNP125: The site name is rs2729940, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 249 and SEQ ID No. 250, and the amplification product is 151 bp in length.

[0214] SNP126: The site name is rs2409784, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 251 and SEQ ID No. 252, and the amplification product is 145 bp in length.

[0215] SNP127: The site name is rs2898290, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 253 and SEQ ID No. 254, and the amplification product is 154 bp in length.

[0216] SNP128: The site name is rs60527016, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 255 and SEQ ID No. 256, and the amplification product is 163 bp in length.

[0217] SNP129: The site name is rs366249, located on chr8. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 257 and SEQ ID No. 258, and the amplification product is 158 bp in length.

[0218] SNP130: The site name is rs77495304, located on chr9. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 259 and SEQ ID No. 260, and the amplification product is 165 bp in length.

[0219] SNP131: The site name is rs76569799, located on chr9. Its specific primers consist of two single-stranded DNAs shown in SEQ ID No. 261 and SEQ ID No. 262, and the amplification product is 156 bp in length.

[0220] SNP132: The site name is rs73503170, located on chr9. Its specific primers consist of two single-stranded DNA molecules as shown in SEQ ID No. 263 and SEQ ID No. 264, and the amplification product is 150 bp in length.

[0221] The 264 primers are designated as primer set 1.

[0222] Primer set 2: Among the 264 specific primers in primer set 1 above, a fixed sequence 1 (5′-CCGCTTGGCCTCCGACTT-3′, SEQ ID No. 265) is added to the 5′ end of primers with odd sequence numbers. The resulting primers are obtained by directly connecting the 3′ end of the fixed sequence 1 to the 5′ end of the specific primers. A fixed sequence 2 (5′-ACATGGCTACGATCCGACTT-3′, SEQ ID No. 266) is added to the 5′ end of primers with even sequence numbers. The resulting primers are obtained by directly connecting the 3′ end of the fixed sequence 2 to the 5′ end of the specific primers. The 264 primers containing the fixed sequences are referred to as primer set 2.

[0223] Universal primer pair: Consists of universal primer 1 and universal primer 2, with the following sequence information:

[0224] Universal primer 1: 5′-TGTGAGCCAAGGAGTTGNNNNNNNNNNTTGTCTTCCTAAGACCGCTTGGCCTCCGACTT-3′-3′ (SEQ ID No. 267);

[0225] Universal primer 2: 5′-phos-GAACGACATGGCTACGATCCGACTT-3′ (SEQ ID No. 268, the 5′ terminal nucleotide of this primer is phosphorylated).

[0226] In the universal primer 1, the 10 "N" sequences are barcode tags, where N represents A, T, C, or G, and include at least the following 8: 5′-ATCGGACCTA-3′ (SEQ ID No. 269), 5′-GATTCCGTCC-3′ (SEQ ID No. 270), 5′-CGGCAGTAAG-3′ (SEQ ID No. 271), 5′-TCAATTAGGT-3′ (SEQ ID No. 272), 5′-CGGATACGAA-3′ (SEQ ID No. 273), 5′-GCTCGTTACC-3′ (SEQ ID No. 274), 5′-TTATACGTTG-3′ (SEQ ID No. 275), and 5′-AACGCGACGT-3′ (SEQ ID No. 276).

[0227] The kit prepared in this embodiment for screening patients with autism spectrum disorder contains primer set 2 and universal primer pairs.

[0228] Example 2: Detection of the severity of autism spectrum disorder in the test subjects

[0229] The participants included 737 individuals with autism spectrum disorder and 2,870 healthy individuals, and were matched by gender.

[0230] Inclusion criteria for patients with autism spectrum disorder: A diagnosis of autism spectrum disorder by at least two experienced psychiatrists based on the criteria defined in the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-V), and simultaneously meeting the following criteria: an Autism Behavior Checklist (ABC) score ≥53 and a Childhood Autism Rating Scale (CARS) score >36. To ensure homogeneity of the study participants, only individuals diagnosed with autism were included, excluding those with Asperger syndrome and pervasive developmental disorder-not otherwise specified (PDD-NOS); those with phenylketonuria, fragile X syndrome, tuberous sclerosis or other neurological disorders, and those with severe physical illnesses or chromosomal abnormalities were also excluded.

[0231] Inclusion criteria for healthy individuals: those who do not meet the diagnostic criteria for autism as defined in DSM-V.

[0232] The following steps were taken to test the subjects using the kit for screening patients with autism spectrum disorder as described in Example 1:

[0233] Step 1: Building the library.

[0234] Venous blood was collected from the subjects to be tested, and then total DNA was extracted from peripheral blood mononuclear cells to obtain DNA samples. The first round of target-specific amplification was performed using primer set 2 from the kit in Example 1, followed by a second round of universal amplification using universal primer pairs to complete target site enrichment and high-throughput sequencing library construction. The obtained products were purified, electrophoresed, and subjected to concentration quality control before proceeding to the next step.

[0235] The first round of target-specific amplification system consisted of 10 μL of the DNA sample to be tested, 12.5 μL of PCR Enzyme Mix, 0.5 μL of PCRClean Enzyme, and 2 μL of Custom Panel PCR Primer Pool. The Custom Panel PCR Primer Pool was prepared by equal molar amounts of the 264 primers from primer set 2.

[0236] First-round target-specific amplification conditions: 105℃ heat capping; 37℃ for 5 min, 95℃ for 10 min, 1 cycle; 95℃ for 20 s, 62℃ for 1 min, 58℃ for 1 min, 72℃ for 30 s, 10 cycles; 4℃ hold.

[0237] The second-round universal amplification system consisted of: 11.5 μL of purified first-round target-specific amplification product, 4 μL of PCR Barcode Primer Mix, 12.5 μL of PCR Enzyme Mix, 0.5 μL of PCR Clean Enzyme, 0.5 μL of PCR Additive, and 2 μL of Custom Panel PCR Block. The PCR Barcode Primer Mix was prepared from universal primers 1 and 2, each with different barcode tags, in equimolar amounts. The barcode tags in universal primer 1 varied depending on the target organism.

[0238] Second round of general amplification conditions: 105℃ hot cap; 37℃ for 5 min, 95℃ for 10 min, 1 cycle; 95℃ for 20 s, 62℃ for 1 min, 58℃ for 1 min, 72℃ for 30 s, 27 cycles; 4℃ hold.

[0239] Among them, PCR Enzyme Mix, PCR Clean Enzyme, PCR Additive, and Custom Panel PCRBlock are all products of MGI Tech (MGI) ATOPlex.

[0240] Step 2: Sequencing.

[0241] All samples were mixed in equal molar amounts to prepare DNB, which was then loaded onto a sequencing chip for paired-end PE150 sequencing.

[0242] Step 3: Data reading.

[0243] Sequencing data (FASTQ file) is obtained, and the sequencing data is compared with a reference genome to analyze single nucleotide polymorphisms (SNPs). Specifically, the FASTQ file is compared with a human GRCh38 version reference genome to obtain a BAM file. Low-quality sequencing data is filtered out, and then the BAM file is loaded and analyzed using an IGV genome visualization browser to obtain the occurrence of gene polymorphisms and structural variations. If different bases or sequences appear in the sequenced gene sequence set compared to the reference genome, the polymorphism is interpreted as occurring at the specific site.

[0244] Genotyping was performed on 132 SNP loci in the subjects to be tested, and the correlation between each SNP locus and autism spectrum disorder was analyzed. The results are shown in Table 1. In Table 1, PP represents the physical position of the SNP locus.

[0245] Table 1. Correlation analysis between each SNP locus and autism spectrum disorder.

[0246]

[0247]

[0248]

[0249] Step 4: Genetic risk assessment.

[0250] For feature selection, all SNP genotyping features were used to maximize the utilization of available information. During data standardization, StandardScaler was employed to standardize the SNP feature data, eliminating dimensional differences between different features and ensuring convergence speed and stability during model training. To address class imbalance, Synthetic Minority Oversampling Technique (SMOTE) was used to increase the number of minority class samples by generating new samples, thereby balancing the class distribution in the dataset and improving the model's predictive ability for the minority class. After data preprocessing, the dataset was divided into training and test sets in a 7:3 ratio to ensure that a standardized dataset was used for model training and evaluation.

[0251] Referring to the polygenic risk scoring method, various classification models were designed based on the significance of the association between different susceptibility risk loci and the disease (Table 1), including Logistic Regression, Support Vector Machine (SVM), Random Forest, Extreme Gradient Boosting (XGBoost), Gradient Boosting, and Multilayer Perceptron (MLP). By using multiple models to classify the data from different perspectives, model selection bias was reduced. During model training and evaluation, the performance of each model was evaluated using metrics such as accuracy, precision, recall, F1 score, area under the curve (AUC), and cross-validation F1. These evaluation metrics reflect the model's performance on unknown data, ensuring a comprehensive understanding of each model's capabilities. To avoid overfitting, 5-fold cross-validation was performed, and the average F1 score was calculated to further assess the model's robustness. The evaluation metrics and cross-validation results of each model were compiled and a performance comparison bar chart was drawn. Figure 1 These charts, based on the test set results, visually demonstrate the performance of different models on various metrics.

[0252] The six learning models performed differently across multiple evaluation metrics. Precision represents the proportion of correctly classified cases; SVM performed best with a score of 0.94, indicating very strong overall classification performance. Precision measures the proportion of samples predicted as autism spectrum disorder (ASD) that were actually diagnosed with ASD; SVM's precision of 0.94 demonstrates its high accuracy in predicting ASD. Recall reflects the model's ability to identify all samples that actually have ASD; SVM's recall of 0.94 indicates it can accurately identify ASD samples. The F1 score is the harmonic mean of precision and recall; SVM's F1 score of 0.94 shows a good balance between precision and recall. AUC measures the model's ability to distinguish between ASD and healthy individuals; MLP had the highest AUC at 0.98, indicating strong discriminative ability. Cross-validation F1 score is used to evaluate the stability of the model under different data partitions. MLP's cross-validation F1 score is 0.94, demonstrating high stability and generalization ability. Overall, SVM performs well across all metrics, especially in accuracy and AUC, while MLP excels in stability and generalization ability.

[0253] In addition, receiver operating characteristic (ROC) curves were plotted. Figure 2 ) and precision-recall curve (PR curve) ( Figure 3 The performance of each model was further evaluated at different thresholds to help select the optimal model.

[0254] Receiver Operating Characteristic (ROC) curves illustrate the trade-off between the False Positive Rate (FPR) and True Positive Rate (TPR) of multiple classification models, aiming to evaluate the classification performance of different models. The X-axis represents the false positive rate, i.e., the proportion of healthy individuals incorrectly predicted as having autism spectrum disorder, and the Y-axis represents the true positive rate, i.e., the proportion of samples correctly identified as having autism spectrum disorder. Ideally, the model curve should be as close as possible to the upper left corner, indicating that the model has a high true positive rate and a low false positive rate. The diagonal line represents the baseline for random guessing; any curve above this line indicates that the model has a certain discriminative ability. By comparing the AUC values ​​of each model, the performance of the model can be visually evaluated; the closer the AUC value is to 1, the better the model's classification performance. According to the performance in the figure, MLP and SVM both achieved an AUC value of 0.98, showing the most outstanding performance and demonstrating their excellent performance in the autism spectrum disorder classification task. Both Random Forest and Gradient Boosting achieved an AUC of 0.96, demonstrating stable and excellent performance with strong generalization ability. XGBoost, with an AUC of 0.95, still exhibited high classification efficiency. In contrast, Logistic Regression, with an AUC of 0.88, performed weaker, indicating limited classification ability in this task. Overall, the ROC curves provided a reliable basis for model selection, supporting the choice of the most suitable classification model in practical applications.

[0255] The precision-recall curve illustrates the trade-off between precision and recall for various classification models, aiming to evaluate their classification performance at different thresholds. The X-axis represents recall, the proportion of correctly identified autism spectrum disorder (ASPD) samples, while the Y-axis represents precision, the proportion of correctly predicted ASPD out of all predicted ASPD. Ideally, a model should maintain a balance between high precision and high recall. The graph shows that MLP and SVM perform better overall, maintaining high precision across a wider recall range. RandomForest, XGBoost, and Gradient Boosting also perform well, but their precision decreases at high recall. Logistic Regression performs relatively poorly, especially with a significant drop in precision at high recall. This graph provides important guidance for selecting a model suitable for specific needs, particularly in ASPD prediction applications where a balance between precision and recall is required.

[0256] In summary, among all six models, SVM performed the best, demonstrating superior classification capabilities, particularly in several key performance metrics. SVM excelled in accuracy (0.94), precision (0.94), recall (0.94), and F1 score (0.94), indicating its ability to provide stable and efficient predictive performance while balancing precision and recall. Furthermore, SVM's AUC (0.98) approached 1, demonstrating its strong ability to distinguish between individuals with autism spectrum disorder and healthy individuals, making it an ideal choice for practical applications. Although MLP slightly outperformed SVM in AUC and cross-validation F1 score, exhibiting stronger discriminative ability and better stability, SVM's balance between precision and recall made it more suitable for autism spectrum disorder prediction tasks. Early diagnosis of autism spectrum disorder is crucial for patient intervention, and SVM, while ensuring high recall, also maintains high precision, avoiding excessive false positives. Therefore, SVM provides an ideal solution to effectively identify patients with autism spectrum disorder while reducing unnecessary misdiagnosis.

[0257] In this invention, SVM has been proven to be the optimal classification model, particularly suitable for practical applications requiring high precision and recall, and can provide strong support for early screening and risk assessment of autism spectrum disorder. Therefore, by establishing an SVM fitting model, different weight levels are set for different susceptibility risk sites and assigned corresponding score values ​​to estimate the severity of the disease in each individual. This invention can help individuals with autism spectrum disorder understand their risk of developing the disorder and take appropriate preventative interventions, such as early behavioral interventions to improve disease prognosis.

[0258] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

[0259] Industrial applicability

[0260] This invention, based on large-scale genome-wide association studies and combined with single nucleotide polymorphism (SNP) information from the Han Chinese population, uses molecular genetic methods to screen 132 high-risk autism susceptibility gene loci and designs primer sets targeting these 132 loci. The primer sets and kits of this invention can accurately detect polymorphic loci on high-risk autism susceptibility genes. The results are consistent with existing PCR sequencing methods and the gold standard first-generation sequencing. In the same sample, gene polymorphisms are verified by two or more methods, and the genotypes are all consistent. The average read length range of the DNA sample is PE 150 bp. Using the detection method of this invention, the coverage is broad, and 132 autism-related polymorphic loci can be detected simultaneously. The data model established using SVM based on these 132 autism susceptibility gene loci can screen patients with autism spectrum disorder, and has good accuracy (0.94), precision (0.94), recall (0.94), and F1 score (0.94). It provides stable and efficient predictive performance while balancing precision and recall, and has an AUC value of 0.98. This invention has a strong ability to distinguish patients with autism spectrum disorder from healthy individuals.

Claims

1. A primer set, characterized in that: The primer set is named Primer Set 1, and Primer Set 1 consists of 264 single-stranded DNA molecules as shown in SEQ ID No. 1 to SEQ ID No.

264.

2. A reagent kit, characterized in that: The kit contains the primer set as described in claim 1.

3. The reagent kit according to claim 2, characterized in that: The kit also includes a data conversion device for converting the test results of SNP combinations into autism spectrum disorder scores, which are used for the diagnosis, screening, severity assessment, or genetic risk assessment of autism spectrum disorder. The SNP combination refers to the following SNP sites in the human genome: rs2181419, rs67980110, rs66512, rs1222064, rs12130426, rs2391769, rs6701243, rs12778231, rs4750990, rs671154, rs492768, rs523871, rs61925919, rs11160145, rs7215239, rs17565025, rs242559, rs3785884, rs62062271, rs62073098, rs2532424, rs199526, rs1994.

98. rs72837297, rs11872054, rs1597749, rs12477455, rs145741754, rs6730686, rs2883872, rs1548635, rs6079536, rs6074798, rs6042917, rs3767 11. rs6082289, rs6047225, rs6047270, rs6132406, rs12625304, rs1000177, rs6035821, rs112777342, rs3787500, rs6035868, rs1090226, rs2424362 , rs57080033, rs8182800, rs6027989, rs2249666, rs405182, rs1452075, rs2139816, rs1522603, rs4916723, rs2635182, rs325501, rs13188074, rs6 63338、rs9366877、rs16879023、rs407238、rs9375188、rs72560713、rs35971735、rs56189730、rs6945821、rs143762771、rs111931861、rs7794285、r s73186025, rs35851667, rs17148231, rs2979184, rs2921036, rs11992186, rs6985207, rs693109, rs2048419, rs12545499, rs7013471, rs13270070, rs36059156, rs6992534, rs35305751, rs35694744, rs7005133, rs7387360, rs10081437, rs393155, rs12547955, rs11249905, rs2929453, rs1466555,rs6982308, rs7005363, rs7001281, rs11775702, rs10096777, rs4841410, rs7823200, rs7461950, rs10099100, rs 4841432, rs1991651, rs877116, rs755856, rs10090444, rs12542037, rs6989105, rs7834248, rs10086521, rs78434 70, rs12679529, rs7000276, rs7004551, rs11783418, rs6601560, rs35499486, rs11250130, rs2736305, rs12156009, rs6980884, rs2729940, rs2409784, rs2898290, rs60527016, rs366249, rs77495304, rs76569799, and rs73503170.

4. A primer set, characterized in that: The primer set is named primer set 2. Primer set 2 is obtained by adding fixed sequence 1 to the 5′ end of primers with odd sequence numbers in primer set 1 of claim 1 and fixed sequence 2 to the 5′ end of primers with even sequence numbers in primer set 1. Fixed sequence 1 is different from fixed sequence 2, and both are used for further amplification of the PCR product of primer set 1.

5. A reagent kit, characterized in that: The kit contains the primer set as described in claim 4.

6. The reagent kit according to claim 5, characterized in that: The kit also contains a universal primer pair for identifying the fixed sequence 1 and the fixed sequence 2.

7. The kit according to claim 5 or 6, characterized in that: The kit also includes a data conversion device for converting the test results of SNP combinations into autism spectrum disorder scores, which are used for the diagnosis, screening, severity assessment, or genetic risk assessment of autism spectrum disorder. The SNP combination refers to the following SNP sites in the human genome: rs2181419, rs67980110, rs66512, rs1222064, rs12130426, rs2391769, rs6701243, rs12778231, rs4750990, rs671154, rs492768, rs523871, rs61925919, rs11160145, rs7215239, rs17565025, rs242559, rs3785884, rs62062271, rs62073098, rs2532424, rs199526, rs1994.

98. rs72837297, rs11872054, rs1597749, rs12477455, rs145741754, rs6730686, rs2883872, rs1548635, rs6079536, rs6074798, rs6042917, rs3767 11. rs6082289, rs6047225, rs6047270, rs6132406, rs12625304, rs1000177, rs6035821, rs112777342, rs3787500, rs6035868, rs1090226, rs2424362 , rs57080033, rs8182800, rs6027989, rs2249666, rs405182, rs1452075, rs2139816, rs1522603, rs4916723, rs2635182, rs325501, rs13188074, rs6 63338、rs9366877、rs16879023、rs407238、rs9375188、rs72560713、rs35971735、rs56189730、rs6945821、rs143762771、rs111931861、rs7794285、r s73186025, rs35851667, rs17148231, rs2979184, rs2921036, rs11992186, rs6985207, rs693109, rs2048419, rs12545499, rs7013471, rs13270070, rs36059156, rs6992534, rs35305751, rs35694744, rs7005133, rs7387360, rs10081437, rs393155, rs12547955, rs11249905, rs2929453, rs1466555,rs6982308, rs7005363, rs7001281, rs11775702, rs10096777, rs4841410, rs7823200, rs7461950, rs10099100, rs 4841432, rs1991651, rs877116, rs755856, rs10090444, rs12542037, rs6989105, rs7834248, rs10086521, rs78434 70, rs12679529, rs7000276, rs7004551, rs11783418, rs6601560, rs35499486, rs11250130, rs2736305, rs12156009, rs6980884, rs2729940, rs2409784, rs2898290, rs60527016, rs366249, rs77495304, rs76569799, and rs73503170.

8. Any of the following applications of the primer set described in claim 1 or 4: X1) Prepare products for diagnosing or assisting in the diagnosis of autism spectrum disorder; X2) Products for screening or assisting in the screening of patients with autism spectrum disorder; X3) Prepare products for detecting or assisting in the detection of the severity of autism spectrum disorder in patients; X4) Prepare products for detecting or assisting in the detection of genetic risk of autism spectrum disorder; X5) Diagnostic or auxiliary diagnosis of autism spectrum disorder; X6) Screening or auxiliary screening for patients with autism spectrum disorder; X7) Detection or auxiliary detection of the severity of autism spectrum disorder in patients; X8) testing or auxiliary testing for the genetic risk of autism spectrum disorder.

9. Any of the following applications of the kit described in claims 2, 3, or any of claims 5-7: X1) Prepare products for diagnosing or assisting in the diagnosis of autism spectrum disorder; X2) Products for screening or assisting in the screening of patients with autism spectrum disorder; X3) Prepare products for detecting or assisting in the detection of the severity of autism spectrum disorder in patients; X4) Prepare products for detecting or assisting in the detection of genetic risk of autism spectrum disorder; X5) Diagnostic or auxiliary diagnosis of autism spectrum disorder; X6) Screening or auxiliary screening for patients with autism spectrum disorder; X7) Detection or auxiliary detection of the severity of autism spectrum disorder in patients; X8) testing or auxiliary testing for the genetic risk of autism spectrum disorder.

10. The use of the primer set of claim 1 or 4, or the kit of any one of claims 2, 3, 5-7, in the preparation of products for detecting or assisting in the detection of autism-related genetic risk gene polymorphisms, or in the detection or assisting in the detection of autism-related genetic risk gene polymorphisms; The detection of autism-related genetic risk gene polymorphisms involves detecting rs2181419, rs67980110, rs66512, rs1222064, rs12130426, rs2391769, rs6701243, rs12778231, rs4750990, rs671154, rs492768, rs523871, rs61925919, rs11160145, rs7215239, rs17565025, rs242559, rs3785884, rs62062271, rs62073098, rs2532424, rs199526, and rs in the human genome. 199498, rs72837297, rs11872054, rs1597749, rs12477455, rs145741754, rs6730686, rs2883872, rs1548635, rs6079536, rs6074798, rs6042917, rs3 76711, rs6082289, rs6047225, rs6047270, rs6132406, rs12625304, rs1000177, rs6035821, rs112777342, rs3787500, rs6035868, rs1090226, rs2424 362、rs57080033、rs8182800、rs6027989、rs2249666、rs405182、rs1452075、rs2139816、rs1522603、rs4916723、rs2635182、rs325501、rs13188074、r s663338, rs9366877, rs16879023, rs407238, rs9375188, rs72560713, rs35971735, rs56189730, rs6945821, rs143762771, rs111931861, rs7794285, rs73186025, rs35851667, rs17148231, rs2979184, rs2921036, rs11992186, rs6985207, rs693109, rs2048419, rs12545499, rs7013471, rs13270070, rs36059156, rs6992534, rs35305751, rs35694744, rs7005133, rs7387360, rs10081437, rs393155, rs12547955, rs11249905, rs2929453, rs1466555,rs6982308, rs7005363, rs7001281, rs11775702, rs10096777, rs4841410, rs7823200, rs7461950, rs10099100, rs4 841432, rs1991651, rs877116, rs755856, rs10090444, rs12542037, rs6989105, rs7834248, rs10086521, rs7843470 Nucleotides of rs12679529, rs7000276, rs7004551, rs11783418, rs6601560, rs35499486, rs11250130, rs2736305, rs12156009, rs6980884, rs2729940, rs2409784, rs2898290, rs60527016, rs366249, rs77495304, rs76569799, and rs73503170.

11. Any of the following applications of substances used to detect autism-related genetic risk gene polymorphisms: X1) Prepare products for diagnosing or assisting in the diagnosis of autism spectrum disorder; X2) Products for screening or assisting in the screening of patients with autism spectrum disorder; X3) Prepare products for detecting or assisting in the detection of the severity of autism spectrum disorder in patients; X4) Prepare products for detecting or assisting in the detection of genetic risk of autism spectrum disorder; X5) Diagnostic or auxiliary diagnosis of autism spectrum disorder; X6) Screening or auxiliary screening for patients with autism spectrum disorder; X7) Detection or auxiliary detection of the severity of autism spectrum disorder in patients; X8) Testing or auxiliary testing for the genetic risk of autism spectrum disorder; The autism-related genetic risk gene polymorphisms mentioned are the polymorphisms of the following SNP sites in the human genome: rs2181419, rs67980110, rs66512, rs1222064, rs12130426, rs2391769, rs6701243, rs12778231, rs4750990, rs671154, rs492768, rs523871, rs61925919, rs11160145, rs7215239, rs17565025, rs242559, rs3785884, rs62062271, rs62073098, rs2532424, rs1 99526, rs199498, rs72837297, rs11872054, rs1597749, rs12477455, rs145741754, rs6730686, rs2883872, rs1548635, rs6079536, rs6074798, rs60 42917, rs376711, rs6082289, rs6047225, rs6047270, rs6132406, rs12625304, rs1000177, rs6035821, rs112777342, rs3787500, rs6035868, rs10902 26. rs2424362, rs57080033, rs8182800, rs6027989, rs2249666, rs405182, rs1452075, rs2139816, rs1522603, rs4916723, rs2635182, rs325501, rs 13188074, rs663338, rs9366877, rs16879023, rs407238, rs9375188, rs72560713, rs35971735, rs56189730, rs6945821, rs143762771, rs111931861, rs7794285, rs73186025, rs35851667, rs17148231, rs2979184, rs2921036, rs11992186, r s13270070, rs36059156, rs6992534, rs35305751, rs35694744, rs7005133, rs7387360, rs10081437, rs393155, rs12547955, rs11249905, rs2929453,rs1466555, rs6982308, rs7005363, rs7001281, rs11775702, rs10096777, rs4841410, rs7823200, rs7461950, rs1009 9100, rs4841432, rs1991651, rs877116, rs755856, rs10090444, rs12542037, rs6989105, rs7834248, rs10086521, rs 7843470, rs12679529, rs7000276, rs7004551, rs11783418, rs6601560, rs35499486, rs11250130, rs2736305, rs12156009, rs6980884, rs2729940, rs2409784, rs2898290, rs60527016, rs366249, rs77495304, rs76569799, and rs73503170.

12. Any of the following methods: Y1) Methods for diagnosing or assisting in the diagnosis of autism spectrum disorder include: Nucleotides from autism-related genetic risk gene loci in the subject of the test are received to obtain a diagnosis of autism spectrum disorder in the subject of the test. Y2) Screening or assisted screening for autism spectrum disorder, including: receiving nucleotides from autism-related genetic risk gene loci of the subject to be tested, and obtaining the screening results for autism spectrum disorder of the subject to be tested; Y3) Detection or auxiliary detection of the severity of autism spectrum disorder in patients, including: receiving nucleotides from autism-related genetic risk gene loci in a patient with autism spectrum disorder to be tested, and obtaining the result of the severity of autism spectrum disorder in the patient with autism spectrum disorder to be tested; Y4) Detection or auxiliary detection of genetic risk of autism spectrum disorder, including: receiving nucleotides of autism-related genetic risk gene loci from a patient with autism spectrum disorder to be tested, and obtaining the genetic risk of autism spectrum disorder in the patient with autism spectrum disorder to be tested; The autism-related genetic risk loci are the following SNP loci in the human genome: rs2181419, rs67980110, rs66512, rs1222064, rs12130426, rs2391769, rs6701243, rs12778231, rs4750990, rs671154, rs492768, rs523871, rs61925919, rs11160145, rs7215239, rs17565025, rs242559, rs3785884, rs62062271, rs62073098, rs2532424, rs19952.

6. rs199498, rs72837297, rs11872054, rs1597749, rs12477455, rs145741754, rs6730686, rs2883872, rs1548635, rs6079536, rs6074798, rs604291 7. rs376711, rs6082289, rs6047225, rs6047270, rs6132406, rs12625304, rs1000177, rs6035821, rs112777342, rs3787500, rs6035868, rs1090226, rs2424362, rs57080033, rs8182800, rs6027989, rs2249666, rs405182, rs1452075, rs2139816, rs1522603, rs4916723, rs2635182, rs325501, rs131 88074, rs663338, rs9366877, rs16879023, rs407238, rs9375188, rs72560713, rs35971735, rs56189730, rs6945821, rs143762771, rs111931861, rs 7794285, rs73186025, rs35851667, rs17148231, rs2979184, rs2921036, rs11992186, rs6985207, rs693109, rs2048419, rs12545499, rs7013471, rs 13270070, rs36059156, rs6992534, rs35305751, rs35694744, rs7005133, rs7387360, rs10081437, rs393155, rs12547955, rs11249905, rs2929453,rs1466555, rs6982308, rs7005363, rs7001281, rs11775702, rs10096777, rs4841410, rs7823200, rs7461950, rs1009 9100, rs4841432, rs1991651, rs877116, rs755856, rs10090444, rs12542037, rs6989105, rs7834248, rs10086521, rs 7843470, rs12679529, rs7000276, rs7004551, rs11783418, rs6601560, rs35499486, rs11250130, rs2736305, rs12156009, rs6980884, rs2729940, rs2409784, rs2898290, rs60527016, rs366249, rs77495304, rs76569799, and rs73503170.

13. A data processing apparatus / device / system, comprising a memory, a processor, and a computer program stored in the memory, characterized in that: The processor executes the computer program to implement the steps of the method of claim 12.

14. A computer-readable storage medium having a computer program / instructions stored thereon, characterized in that: When the computer program / instructions are executed by the processor, they implement the steps of the method of claim 12.

15. A computer program product comprising a computer program / instructions, characterized in that: When the computer program / instructions are executed by the processor, they implement the steps of the method of claim 12.

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