Application of variation site group in congenital megacolon

Abstract

The invention relates to the technical field of molecular genetics, in particular to application of a variation site group in genetic diagnosis of congenital megacolon. According to the application, a variation site group comprises at least one RET variation site and / or at least one PLK5 variation site; the RET variation site comprises rs1800858 and / or rs1800861, and the RET variation site comprises rs1800858 and / or And the PLK5 variation site comprises at least one of the following sites: rs10853954, rs265282 and rs12984316. The variation site group used by the application can be used as a biomarker of the congenital megacolon, so that the high-risk congenital megacolon can be effectively predicted.

Description

Technical Field

[0001] The present application relates to the technical field of molecular genetics, and in particular to the application of a mutation site group in congenital megacolon. Background Art

[0002] Hirschsprung's disease (HSCR) is a congenital disease characterized by the lack of ganglion cells in the distal rectum. The current prevalence worldwide is about 1 / 5000. Current studies have shown that defects in the proliferation or migration of intestinal neural crest cells can lead to the occurrence of HSCR, and HSCR patients usually experience severe constipation and abdominal distension, and may still face long-term complications even after surgery. Gene mutation is the main cause of HSCR. At present, more than 20 HSCR pathogenic genes have been identified at home and abroad, such as RET, EDNRB, etc., but these known HSCR pathogenic genes can only explain less than 30% of HSCR cases. Each gene contains thousands of site information. Therefore, if the site information of these genes is tested to evaluate their association with HSCR, it will cost a lot of manpower and material resources, and can still only explain 30% of HSCR cases.

[0003] At the same time, because HSCR is a complex multi-gene disease, and there are also differences in the genetic background of different populations, these reasons will lead to different compositions of HSCR risk genes in different populations. However, most genetic studies on HSCR are conducted in European HSCR patient cohorts. Although there are reports on the genetic risk score of congenital megacolon in the Chinese population, the SNP sites involved in the genetic risk score are screened using chips, and its SNP sites will largely miss a large number of potential risk sites, resulting in the risk of inaccurate detection of HSCR through SNP sites. Therefore, it is urgent to optimize the existing genetic risk score in terms of sequencing technology and analysis methods to obtain accurate SNP sites. Summary of the invention

[0004] The present application provides an application of a variation site group in congenital megacolon to solve the technical problem of inaccuracy in the SNP sites of congenital megacolon in the prior art.

[0005] In a first aspect, the present application provides an application of a variant site group as a biomarker for Hirschsprung's disease, the application comprising: using the variant site group as a biomarker for Hirschsprung's disease, wherein the variant site group comprises at least one RET variant site and / or at least one PLK5 variant site;

[0006] The RET variant sites include rs1800858 and / or rs1800861;

[0007] The PLK5 variant site includes at least one of the following:

[0008] rs10853954, rs265282 and rs12984316.

[0009] Optionally, the mutation site group includes two RET mutation sites and one PLK5 mutation site.

[0010] Optionally, the mutation site group includes RET mutation site rs1800858, RET mutation site rs1800861 and PLK5 mutation site rs10853954.

[0011] Optionally, when the variant site group includes two variant sites, the maximum OR risk value of the variant site group is ≥9; and / or,

[0012] When the variant site group includes three variant sites, the maximum OR risk value of the variant site group is ≥36.

[0013] In a second aspect, the present application provides a method for screening the variable site group in the application of the first aspect for non-diagnostic purposes, the method comprising:

[0014] Collect peripheral blood samples from patients;

[0015] Performing whole exome sequencing on the peripheral blood sample, and then performing quality control on the whole exome sequencing data to obtain principal component data;

[0016] Healthy control group data were screened from public databases;

[0017] Performing genetic association analysis using the principal component data and the control group data to obtain genetic risk score data of the peripheral blood sample;

[0018] Screening is performed according to the genetic risk score data to obtain a primary variable site group;

[0019] The primary variant site group is verified and evaluated according to the genetic risk score data to obtain a final variant site group.

[0020] Optionally, the quality control includes performing quality control using PLINK software.

[0021] Optionally, the genetic association analysis includes quality control using PLINK software.

[0022] In a third aspect, the present application provides a detection reagent for detecting congenital megacolon, wherein the detection reagent includes a detection reagent for detecting two RET variant sites and a detection reagent for detecting a PLK5 variant site; the RET variant site includes rs1800858 and / or rs1800861;

[0023] The PLK5 variation site includes rs10853954.

[0024] In a fourth aspect, the present application provides a detection kit, which includes the detection reagent described in the third aspect.

[0025] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0026] The present application provides an application of a variation site group as a biomarker for congenital megacolon. By collecting peripheral blood samples from different patients, and then performing whole exome sequencing and quality control on the collected peripheral blood samples in turn, principal component data of the peripheral blood samples can be obtained. Then, based on the healthy control group data in the public database, comparison and genetic association analysis can be performed, most of the overlapping data and interference data can be eliminated from the principal component data, and genetic risk score data of the peripheral blood samples can be obtained. Finally, the genetic risk score data is screened and verified, so that accurate SNP site variation site groups with a high correlation with congenital megacolon can be finally obtained. These variation site groups can be used as biomarkers for congenital megacolon to effectively predict high-risk congenital megacolon. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0028] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0029] Figure 1 A schematic flow chart of a method for screening a set of variant sites in the application for non-diagnostic purposes provided in an embodiment of the present application;

[0030] Figure 2 A detailed flow chart of a method for screening a variable site group in the application for non-diagnostic purposes is provided for the embodiment of the present application;

[0031] Figure 3A schematic diagram of the PCA analysis results provided in the embodiment of the present application, wherein: Figure 3 a is the outlier result graph of the selected 107 cases. Figure 3 b is a comparison chart of the selected 107 cases and healthy controls;

[0032] Figure 4 The observed and expected P-value QQ graph of the logistic regression of the common variant sites provided in the embodiments of the present application;

[0033] Figure 5 This is a graph of the whole exome association results in the genetic risk score provided in the embodiment of the present application, wherein: Figure 5 a is the Manhattan plot of the association of common variants in the whole exome. Figure 5 b is the result diagram of common variant association analysis;

[0034] Figure 6 Schematic diagram of the HSCR genetic risk scoring model for the three variant sites, namely, RET variant site rs1800858, RET variant site rs1800861 and PLK5 variant site rs10853954, provided in the embodiments of the present application. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0036] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0037] It should be noted that the current genetic cause of HSCR is mainly mutations in exon regions, and whole exome sequencing (WES) technology can detect mutations in all exon regions, and the range covered by WES is very wide.

[0038] Therefore, a comprehensive genetic association analysis was conducted based on the genetic information of the Hirschsprung's disease cohort in the Chinese population. After verification by new cohorts and experiments, a stable and personalized genetic risk score for Hirschsprung's disease in the Chinese population was constructed. This can better determine the genetic association of Hirschsprung's disease patients, and ultimately screen out the variant sites with the greatest association with the Hirschsprung's disease gene.

[0039] like Figure 1 As shown, an embodiment of the present application provides an application of a variation site group as a biomarker for congenital megacolon, the application comprising: using the variation site group as a biomarker for congenital megacolon, wherein the variation site group comprises at least one RET variation site and / or at least one PLK5 variation site;

[0040] The RET variant sites include rs1800858 and / or rs1800861;

[0041] The PLK5 variant site includes at least one of the following:

[0042] rs10853954, rs265282 and rs12984316.

[0043] In some optional embodiments, the variant site group includes two RET variant sites and one PLK5 variant site; in these embodiments, certain variant sites of the RET gene and the PLK5 gene discovered based on the genetic risk score are significantly associated with congenital megacolon, so that the variant site group can accurately reflect whether there is a risk of congenital megacolon in the body.

[0044] In some optional embodiments, the variable site group includes RET variable site rs1800858, RET variable site rs1800861 and PLK5 variable site rs10853954.

[0045] In some optional embodiments, when the variant site group includes two variant sites, the maximum OR risk value of the variant site group is ≥9; and / or,

[0046] When the variant site group includes three variant sites, the maximum OR risk value of the variant site group is ≥36; in these embodiments, based on the fact that the variant site group may include RET variant site rs1800858, RET variant site rs1800861 and PLK5 variant site rs10853954, meta-analysis combined with the genetic risk scoring model can clarify the following situations in the genetic risk scoring models of different variant site groups:

[0047] Compared with patients without the variant site group or with one variant site risk allele, patients with four risk alleles (two variant sites) had a 9.80-fold increased risk of HSCR (the highest OR was 9.8, and the 95% CI was 2.61-64.38), while patients with six risk alleles (three variant sites) had a 9.80-fold increased risk of HSCR (the highest OR was 36.61, and the 95% CI was 9.23-248.09), which indicated that this variant site group was significantly associated with congenital megacolon.

[0048] like Figure 1 As shown, based on a general inventive concept, the embodiment of the present application provides a method for screening a variable site group in the application for non-diagnostic purposes, the method comprising:

[0049] S1. Collect peripheral blood samples from patients;

[0050] S2. performing whole exome sequencing on the peripheral blood sample, and then performing quality control on the whole exome sequencing data to obtain principal component data;

[0051] S3. Filter healthy control group data from public databases;

[0052] S4. performing genetic association analysis using the principal component data and the control group data to obtain genetic risk score data of the peripheral blood sample;

[0053] S5. Screening according to the genetic risk score data to obtain a primary variable site group;

[0054] S6. Verify the primary variant site group and evaluate it according to the genetic risk score data to obtain the final variant site group.

[0055] The method is implemented for the above-mentioned application. The specific principles of the application can refer to the above-mentioned embodiments. Since the method adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be described one by one here.

[0056] It should be noted that this validation can be performed using new HSCR cases.

[0057] It should be noted that the public database may be the 1000 Genomes Project public database, and the screened healthy control data may be genome data of 505 healthy people from East Asia.

[0058] It should be noted that the experimental group used for validation of this primary variant site group was 124 new HSCR patients, while the control group used was genetic data of 10,588 Chinese people from ChinaMAP (www.mBiobank.com).

[0059] In some optional embodiments, the quality control includes quality control using PLINK software; in these embodiments, the quality control may include quality control using PLINK software, which may exclude variant sites with missing genotype>0.05, variant sites with Hardy-Weinberg equilibrium (HWE) test P<0.0001, and variant sites located on chromosomes X / Y.

[0060] In some optional embodiments, the genetic association analysis includes quality control using PLINK software; in these embodiments, the genetic association analysis may include quality control using PLINK software, and logistic regression may be performed on the principal component data to adjust the different genders in the principal component data and the first five principal component data, thereby improving the accuracy of the association analysis.

[0061] Based on a general inventive concept, an embodiment of the present application provides a detection reagent for detecting congenital megacolon, wherein the detection reagent includes a detection reagent for detecting two RET variation sites and a detection reagent for detecting one PLK5 variation site, wherein the RET variation site includes rs1800858 and / or rs1800861, and the PLK5 variation site includes rs10853954.

[0062] The detection reagent is implemented based on the above application. The specific principle of the application can refer to the above embodiment. Since the application adopts part or all of the technical solutions of the above embodiment, it at least has all the beneficial effects brought by the technical solutions of the above embodiment, which will not be repeated here.

[0063] Based on a general inventive concept, an embodiment of the present application provides a detection kit, which includes the detection reagent.

[0064] The detection kit is implemented based on the above detection reagent. The specific composition of the detection reagent can refer to the above embodiment. Since the detection kit adopts part or all of the technical solutions of the above embodiment, it has at least all the beneficial effects brought by the technical solutions of the above embodiment, which will not be repeated here.

[0065] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are intended only to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are usually measured according to industry standards. If there is no corresponding industry standard, then the conditions recommended by the manufacturer are followed.

[0066] Example 1

[0067] Experimental steps for variant loci as biomarkers for Hirschsprung's disease:

[0068] 1. Sample collection:

[0069] All patients were from Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, and signed written informed consent. This project has been approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (Ethics Approval No.: 2020-S226). First, 107 patients with HSCR were selected for whole exome sequencing. After quality control of the DNA samples, whole exome sequencing was performed on these 107 cases. The diagnostic criteria for HSCR are: postoperative pathology shows a lack of ganglion cells in the distal rectum.

[0070] 2. Whole exome sequencing:

[0071] DNA was extracted from peripheral blood samples of patients, captured using SureSelectXT Human All Exon Kit V6 kit (Agilent), and sequenced with 150 bp double-end sequencing on the Illumina Hiseq / NovaSeq sequencing platform. Variant site detection was performed using the GATK best practice method.

[0072] The detected sequence reads were then aligned using Burrows–Wheeler Aligner (BWAv0.7.17) and combined with the human reference genome GRCh38 to remove duplicate reads and simultaneously perform variation detection of single nucleotide variant sites and small insertion and deletion variant sites.

[0073] Finally, ANNOVAR was used to annotate the variant sites to exclude non-target variant sites that were not in the exon region.

[0074] 3. Common variations and sample quality control:

[0075] PLINK software was used for quality control to exclude variants with missing genotypes>0.05, variant sites with Hardy-Weinberg equilibrium (HWE) test P<0.0001, and variant sites located on chromosomes X / Y. In addition, this application also examined samples with gender differences and missing genotypes, and removed one sample that failed the heterozygosity test (greater than or less than the mean ± 3 standard deviations).

[0076] Then, principal component analysis (PCA) was performed on the patient cases, and two outliers were removed. PCA was further performed on individuals in the 1000genome project, and the results showed that the cases studied in this application were all East Asians.

[0077] 4. Genetic association studies of common variants:

[0078] In the association analysis of the discovery set, this application used 505 healthy control individuals of East Asian descent from the 1000genome project. The variants in this 1000genome project include Filtered SNV, INDEL, Structure variants (SV), Insertions (INS), Duplications (DUP) and Inversions variant (INV).

[0079] PLINK software was used for logistic regression, adjusting for sex and the first five principal component data of each group of principal component data, and then genetic association analysis was performed on 24,417 common variants (MAF>0.1).

[0080] 5. Construction of a genetic risk score based on common variants:

[0081] The MassARRAY platform was used to sequence the 30 variant sites screened by the above genetic association analysis in 124 HSCR cases. First, DNA was extracted from the peripheral blood samples of newly collected HSCR patients. Subsequently, Sequenom's Genotyping Tools and Massarray Assay Design software were used to design PCR primers and single-base extension primers for the target sites. Then, the extracted DNA was PCR amplified, alkaline phosphatase treated, and then mass spectrometry was performed using MassARRAY AnalyzerCompact. Finally, TYPER software was used to analyze the experimental results and provide genotype data. The genotypes of the 30 variant sites of 124 HSCR were obtained.

[0082] 10,588 healthy control individuals from ChinaMAP were used in the validation set. These healthy control individuals were randomly selected from 8 ethnic groups in 27 provinces in China without biased selection or penetration, so they can represent the genetic background of Chinese people. The allele frequency of each variant site was retrieved from ChinaMAP (www.mBiobank.com).

[0083] Furthermore, based on the results of association analysis and meta-analysis of the variant sites in the discovery set and validation set, a total of 5 variant sites from RET (rs1800861 and rs1800858) and PLK5 (rs12984316, rs265282 and rs10853954) were selected.

[0084] Finally, a genetic risk scoring model was constructed based on the above five variant sites.

[0085] 6. Results description:

[0086] 1. Experimental process and quality control:

[0087] A total of 107 peripheral blood specimens were collected from HSCR patients and whole exome sequencing was performed. The demographic and clinical characteristics of HSCR patients are shown in Table 1.

[0088] Table 1 Demographic and clinical characteristics of HSCR patients

[0089]

[0090] After quality control, the results are as follows Figure 3 As shown, this application retains 104 cases of data through PCA analysis, such as Figure 3 As shown in a, there are 163906 variant sites. Figure 3 As shown in b, PCA software analyzed and confirmed that all cases were East Asians.

[0091] 2. Preliminary screening of variant sites for genetic risk scoring:

[0092] This application first performs logistic regression of 24417 common variants {minor allele frequency (MAF)>0.1} on common variant sites and displays the observed and expected P values ​​using QQ plots, such as Figure 4 As shown, there is a significant deviation in the low P-value region, indicating that there may be a genetic signal related to the HSCR trait in this data set;

[0093] Then, 505 East Asians from the 1000 Genomes Project were used as healthy controls to conduct genetic association analysis on 24,417 common variants, such as Figure 5 As shown in a;

[0094] It should be noted that Figure 5 The P values ​​in a are two-sided and were calculated using logistic regression analysis with an additive model adjusted for sex and the first five principal components; the associations [–log 10 (P) values, y axis] are plotted according to genomic position (x axis according to chromosome and chromosomal position of GRCh38), and the horizontal line in the figure indicates the significance threshold (1×10 -4 ).

[0095] The results showed that two synonymous mutation sites (rs1800858 and rs1800861) were located in the RET gene and were significantly associated with HSCR (P < 5 × 10 -8 ). By using a relaxed P threshold of (1×10 -3 ), a total of 30 variant sites (located on 23 genes) were associated with HSCR, including five variants on the PLK5 gene. The 30 variant sites were rs1800858, rs1800861, 17_4933822_AAGCCCGACCACCCCAGAGCCCACCTCA.., rs11084897, rs10853954, 19_1535194_GCCC_G, rs12984316, rs265282, 6_32746314_A_G, 19_50667931_G_A, rs1480615, 19_971949_G_A, 19_966693_C_T, rs17124220, rs11549806, rs3733082, 10_87234508_TCTC_T, rs3783185, rs2270590, rs11651537, rs11868065, 8_14329 6014_C_G, rs140524, rs1366272, 6_49492265_A_G, rs2305908, 22_50525807_G_A, rs56144153, rs4801844, rs17032893.

[0096] 3. Further screening of variant sites for genetic risk scoring:

[0097] The MassARRAY platform was used to sequence the 30 variant sites screened by the above genetic association analysis in 124 HSCR cases. First, DNA was extracted from the peripheral blood samples of newly collected HSCR patients. Subsequently, Sequenom's Genotyping Tools and Massarray Assay Design software were used to design PCR primers and single-base extension primers for the target sites. Then, the extracted DNA was PCR amplified, alkaline phosphatase treated, and then mass spectrometry was performed using MassARRAY AnalyzerCompact. Finally, TYPER software was used to analyze the experimental results and provide genotype data. The genotypes of the 30 variant sites of 124 HSCR were obtained.

[0098] 10,588 healthy control individuals from ChinaMAP were used in the validation set. These healthy control individuals were randomly selected from 8 ethnic groups in 27 provinces in China without biased selection or penetration, so they can represent the genetic background of Chinese people. The allele frequency of each variant site was retrieved from ChinaMAP (www.mBiobank.com).

[0099] Furthermore, the results of the association analysis and Meta-analysis of the variant sites based on the discovery set and validation set are shown in Table 2.

[0100] Table 2 Results of association analysis and meta-analysis of variant sites in the discovery set and validation set

[0101]

[0102]

[0103] As shown in Table 2, two RET variants (rs1800858 and rs1800861) and three PLK5 variants (rs10853954, rs265282, and rs12984316) were found to be closely associated with an increased risk of HSCR, reaching 2×10 -6 The exome-wide significance level (e.g. Figure 5 b).

[0104] It should be noted that Figure 5 In b, OR is odds ratio, Chr is chromosome, R is reference allele, and A is variant allele.

[0105] 4. Constructing a genetic risk model:

[0106] Based on two RET variants (rs1800858 and rs1800861) and one PLK5 variant (rs10853954), the present application further constructs a genetic risk scoring model for HSCR, which can be detected using the PCR primers and single-base extension primers designed in step 2. These PCR primers and single-base extension primers are specifically shown in Table 3.

[0107] Table 3 PCR primers and single base extension primers designed for three variant sites

[0108]

[0109] In addition, Figure 6 As shown, the genetic risk scoring model showed that compared with normal people without risk alleles and patients with one risk allele, the risk of HSCR in patients with four risk alleles (two variant sites) increased by 9.80 times (OR=9.8, 95% CI value was 2.61-64.38), while the risk of patients with six alleles (three variant sites) increased by 36.61 times (OR=36.61, 95% CI value was 9.23-248.09).

[0110] In summary, the application of a variation site group as a biomarker for congenital megacolon provided in an embodiment of the present application, by collecting peripheral blood samples from domestic patients, and then performing whole exome sequencing and quality control on the collected peripheral blood samples in turn, the principal component data of the peripheral blood samples can be obtained, and then compared and genetic association analysis is performed based on the healthy control group data in the public database, most of the overlapping data and interference data can be eliminated from the principal component data to obtain the genetic risk score data of the peripheral blood samples, and then the genetic risk score data is screened to screen out the primary variation site group, and finally verified by new HSCR cases, and finally an accurate SNP site variation site group with a high correlation with congenital megacolon can be obtained: RET variation site rs1800858, RET variation site rs1800861 and PLK5 variation site rs10853954, these variation site groups can effectively predict high-risk congenital megacolon.

[0111] At the same time, the application of a variation site group provided in the embodiment of the present application as a biomarker of congenital megacolon can also provide an important judgment basis for eugenics and genetic counseling for patients with congenital megacolon.

[0112] At the same time, an embodiment of the present application provides an application of a variant site group as a biomarker for congenital megacolon. During the application process, a genetic risk scoring model was designed. The genetic risk scoring model has been verified by GWAS association analysis and a new cohort, and can effectively predict high-risk congenital megacolon patients. At the same time, the genetic risk scoring model reduces the number of sites included in the traditional gene scoring model, which is beneficial to the development of gene panel kits and can also simplify the prediction process to save prediction costs.

[0113] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0114] In the present application, in the absence of any contrary description, the directional words used, such as "upper" and "lower", are specifically the directions of the drawings in the accompanying drawings. In addition, in the description of the present specification, the terms "including", "comprising", etc. refer to "including but not limited to". In this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. In this article, "and / or" describes the association relationship of the associated objects, indicating that there may be three relationships, for example, A and / or B, which can represent: A exists alone, A and B exist at the same time, and B exists alone. Wherein A, B can be singular or plural. In this article, "at least one" refers to one or more, and "plural" refers to two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of singular or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or plural, respectively.

[0115] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features applied for herein.

Claims

1. Application of a set of variant sites as a biomarker for Hirschsprung's disease, characterized in that: The application includes: using a mutation site group as a biomarker for Hirschsprung's disease, wherein the mutation site group includes at least one RET mutation site and / or at least one PLK5 mutation site; The RET variant sites include rs1800858 and / or rs1800861; The PLK5 variant site includes at least one of the following: rs10853954, rs265282 and rs12984316.

2. The use according to claim 1, characterized in that: The mutation site group includes two RET mutation sites and one PLK5 mutation site.

3. The use according to claim 2, characterized in that: The variable site group includes RET variable site rs1800858, RET variable site rs1800861 and PLK5 variable site rs10853954.

4. The use according to claim 1, characterized in that: When the variant site group includes two variant sites, the maximum OR risk value of the variant site group is ≥9; and / or, When the variant site group includes three variant sites, the maximum OR risk value of the variant site group is ≥36.

5. A method for screening a set of variant sites in the application of any one of claims 1 to 4 for purposes other than diagnosis and treatment, characterized in that: The method comprises: Collect peripheral blood samples from patients; Performing whole exome sequencing on the peripheral blood sample, and then performing quality control on the whole exome sequencing data to obtain principal component data; Healthy control group data were screened from public databases; Performing genetic association analysis using the principal component data and the control group data to obtain genetic risk score data of the peripheral blood sample; Screening is performed according to the genetic risk score data to obtain a primary variable site group; The primary variant site group is verified and evaluated based on the genetic risk score data to obtain a final variant site group.

6. The method according to claim 5, characterized in that The quality control includes using PLINK software to perform quality control.

7. The method according to claim 5, characterized in that The genetic association analysis included quality control using PLINK software.

8. A detection reagent for detecting Hirschsprung's disease, characterized in that: The detection reagent includes a detection reagent for detecting a RET variation site and a detection reagent for detecting a PLK5 variation site, the RET variation site includes rs1800858 and rs1800861, and the PLK5 variation site includes rs10853954.

9. A detection kit, characterized in that: The detection kit comprises the detection reagent as claimed in claim 8.

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