CN111534583A

CN111534583A - SNP marker related to preeclampsia diagnosis and application thereof

Info

Publication number: CN111534583A
Application number: CN202010408031.6A
Authority: CN
Inventors: 刘世国; 张璐; 王敬丽; 汤潜
Original assignee: Affiliated Hospital of University of Qingdao
Current assignee: Affiliated Hospital of University of Qingdao
Priority date: 2020-05-14
Filing date: 2020-05-14
Publication date: 2020-08-14

Abstract

The invention provides an SNP marker related to preeclampsia diagnosis and application thereof, and relates to the technical field of biomedicine. The invention is based on the whole exon sequencing technology, and simultaneously utilizes large sample verification and case contrast correlation analysis to successfully screen and identify the susceptibility gene of PE and the susceptibility SNP sites related to the susceptibility gene, namely rs27044 and rs30187, thereby providing a sufficient theoretical basis for revealing the PE generation mechanism and laying a foundation for developing an early disease early warning model and an individualized medical prevention and treatment scheme suitable for the population in China.

Description

SNP marker related to preeclampsia diagnosis and application thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to an SNP marker related to preeclampsia diagnosis and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Preeclampsia (PE) is a common condition specific to pregnant women, is the most severe stage of hypertensive disorders of pregnancy, and is the leading cause of maternal and fetal death due to hypertensive disorders of pregnancy. Epidemiological investigation of China shows that: the incidence of PE is 2.5% -5.5%, and the incidence of PE reported abroad is 6% -8%. PE not only shows symptoms such as hypertension, proteinuria and the like, but also is accompanied by a plurality of serious complications, which seriously affect the life safety and health of mothers and infants, and is one of the main causes of morbidity and mortality of pregnant and lying-in women and perinatal infants. Although wide researchers have conducted extensive studies from the aspects of epidemiology, immunology and the like, the etiology and pathogenesis of the disease are not completely clarified so far, and a definite and effective treatment method is to terminate pregnancy, so that a series of adverse consequences such as premature delivery are brought.

The genetic susceptibility of PE consists of a series of susceptibility genes, a single susceptibility gene is micro-effective to the pathogenesis of PE, and the resultant force of the susceptibility genes constitutes the genetic susceptibility of PE. So far, many studies on PE-screening susceptibility genes have been reported, and genes related to blood pressure and body fluid regulation, oxidative stress, inflammatory immunity, involvement in vascular endothelial functions and the like are likely to become PE genetic susceptibility candidate genes. The inventor finds that because the allele frequencies of a plurality of gene polymorphism sites in different populations are different, the experimental results of the traditional candidate gene association research strategy are often inconsistent.

Disclosure of Invention

In view of the problems in the prior art, the present invention aims to provide an SNP marker related to preeclampsia diagnosis and its application. The invention is based on the whole exon sequencing technology, and simultaneously utilizes large sample verification and case contrast correlation analysis to successfully screen and identify the obtained PE susceptibility gene and the related susceptible SNP locus, thereby providing a sufficient theoretical basis for revealing PE occurrence mechanism and laying a foundation for developing early disease early warning models and individualized medical prevention and treatment schemes suitable for Chinese population.

In order to solve the technical problem, the invention firstly provides any one of the following applications:

a1, and the application of the substance for detecting the polymorphism or genotype of rs27044 and/or rs30187 in human genome in the preparation of products for screening preeclampsia patients;

a2, and the application of the substance for detecting the polymorphism or genotype of rs27044 and/or rs30187 in human genome in the preparation of products for detecting pre-eclampsia susceptibility;

a3, and the application of the substance for detecting the polymorphism or genotype of rs27044 and/or rs30187 in human genome in the preparation of products for detecting the single nucleotide polymorphism related to preeclampsia;

a4, and the application of the substance for detecting the polymorphism or genotype of rs27044 and/or rs30187 in human genome in the preparation of products for identifying or assisting in identifying the single nucleotide polymorphism related to preeclampsia;

B1) use of a polymorphism or genotype of rs27044 and/or rs30187 in a human genome in the preparation of a product for screening patients with preeclampsia;

B2) the application of the polymorphism or genotype of rs27044 and/or rs30187 in human genome in the preparation of a product for detecting pre-eclampsia susceptibility;

B3) the use of a substance for detecting a polymorphism or genotype of rs27044 and/or rs30187 in the human genome for screening a patient suffering from preeclampsia;

B4) the use of a substance for detecting a polymorphism or genotype of rs27044 and/or rs30187 in the human genome for detecting a susceptibility to preeclampsia;

B5) the use of a substance that detects a polymorphism or genotype of rs27044 and/or rs30187 in the human genome for detecting a single nucleotide polymorphism associated with preeclampsia;

B6) the use of a substance that detects a polymorphism or genotype of rs27044 and/or rs30187 in the human genome for identifying or aiding in the identification of a single nucleotide polymorphism associated with preeclampsia;

B7) use of a polymorphism or genotype of rs27044 and/or rs30187 in the human genome for screening a patient for preeclampsia;

B8) use of a polymorphism or genotype of rs27044 and/or rs30187 in the human genome for detecting a susceptibility to preeclampsia.

rs27044 is an SNP site of one biallelic polymorphism on a human chromosome, and the rs27044 genotype is GG, GC or CC. The polymorphism (i.e. allele) or genotype for detecting rs27044 in the human genome can be specifically the nucleotide species for detecting rs 27044.

rs30187 is a SNP site of a biallelic polymorphism on a human chromosome, and the rs30187 genotype is TT, TC or CC. The polymorphism (i.e. allele) or genotype for detecting rs30187 in human genome can be specifically the nucleotide type for detecting rs 30187.

In the above application, the substance for detecting the polymorphism or genotype of rs27044 and/or rs30187 in the human genome may be a PCR primer for amplifying a genomic DNA fragment including rs27044 and/or rs30187 and/or a probe for detecting rs27044 and/or rs 30187.

The product may comprise said PCR primers and/or said probes for detecting rs27044 and/or rs 30187.

In the application, the preeclampsia can be specifically preeclampsia of Chinese people. The preeclampsia can be further preeclampsia of Chinese Han nationality population.

In order to solve the technical problems, the invention also provides a product containing a substance for detecting the polymorphism or genotype of rs27044 and/or rs30187 in human genome, which is any one of the products a) to d):

a) products for detecting single nucleotide polymorphisms or genotypes associated with preeclampsia;

b) identifying or aiding in the identification of products that are related to a single nucleotide polymorphism or genotype associated with preeclampsia;

c) screening pre-eclampsia patient products;

d) and detecting a susceptibility product in preeclampsia.

In the above product, the substance for detecting the polymorphism or genotype of rs27044 and/or rs30187 in the human genome may be a PCR primer for amplifying a genomic DNA fragment including rs27044 and/or rs30187 and/or the probe for detecting rs27044 and/or rs 30187.

In the product, the preeclampsia can be specifically preeclampsia of Chinese people. The preeclampsia can be further preeclampsia of Chinese Han nationality population.

The invention also provides the following methods of M1) or M2):

m1) a method of screening patients for preeclampsia comprising: detecting the genotype of the rs27044 and/or rs30187 locus in the genome of the object to be detected;

m2) a method of detecting a susceptibility to preeclampsia, comprising: and detecting the genotype of the rs27044 and/or rs30187 locus in the genome of the object to be detected.

In the method, the preeclampsia can be specifically preeclampsia of Chinese people. The preeclampsia can be further preeclampsia of Chinese Han nationality population.

In the method, the detection of the genotype of the rs27044 and/or rs30187 site in the genome of the object to be detected can be performed by using the substance for detecting the polymorphism or genotype of the rs27044 and/or rs 30187.

In practical application, the substance for detecting the polymorphism (i.e. allele) or genotype of rs27044 and/or rs30187 and other substances (such as substances for detecting other single nucleotide polymorphism or genotype related to preeclampsia) can be combined together to prepare a product for screening preeclampsia patients.

The beneficial technical effects of one or more technical schemes are as follows:

according to the technical scheme, the rs27044 and/or rs30187 are found in a sample from Chinese population to be related to the single nucleotide polymorphism of preeclampsia, and the method can be used for diagnosing the preeclampsia. Meanwhile, the substance for detecting the polymorphism (i.e. allele) or genotype of the rs27044 and/or the rs30187 and other substances (such as substances for detecting other single nucleotide polymorphism (i.e. allele) or genotype related to preeclampsia) can be combined together to prepare a product for screening preeclampsia patients or prepare a product for detecting preeclampsia susceptibility.

The technical scheme provides a sufficient theoretical basis for revealing the preeclampsia generation mechanism and lays a foundation for developing early disease early warning models and individualized medical prevention and treatment schemes suitable for people in China, so that the method has a good practical application value.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In an exemplary embodiment of the invention, any of the following applications is provided:

In another embodiment of the present invention, in the above use, the substance for detecting polymorphism or genotype of rs27044 and/or rs30187 in human genome may be a PCR primer for amplifying a genomic DNA fragment including rs27044 and/or rs30187 and/or a probe for detecting rs27044 and/or rs 30187.

In yet another embodiment of the present invention, said product may comprise said PCR primer and/or said probe for detecting rs27044 and/or rs 30187.

In another embodiment of the present invention, in the above application, the preeclampsia may be preeclampsia of Chinese population. The preeclampsia can be further preeclampsia of Chinese Han nationality population.

In another embodiment of the present invention, there is also provided a product containing a substance for detecting a polymorphism or genotype of rs27044 and/or rs30187 in the human genome, which is any one of the products of a) to d):

c) screening pre-eclampsia patient products;

d) and detecting a susceptibility product in preeclampsia.

In another embodiment of the present invention, in the above product, the substance for detecting rs27044 and/or rs30187 polymorphism or genotype in human genome may be a PCR primer for amplifying a genomic DNA fragment including rs27044 and/or rs30187 and/or the probe for detecting rs27044 and/or rs 30187.

In another embodiment of the present invention, in the above product, the preeclampsia may be preeclampsia of chinese population. The preeclampsia can be further preeclampsia of Chinese Han nationality population.

In still another embodiment of the present invention, the present invention further provides the following method of M1) or M2):

In another embodiment of the present invention, in the above method, the preeclampsia may be preeclampsia of the chinese population. The preeclampsia can be further preeclampsia of Chinese Han nationality population.

In another embodiment of the present invention, in the method, the detection of the genotype of the rs27044 and/or rs30187 site in the genome of the subject to be tested can be performed by using the substance for detecting the polymorphism or genotype of the rs27044 and/or rs 30187.

Wherein, the substance for detecting the polymorphism or genotype of rs27044 and/or rs30187 in human genome can be a reagent and/or an instrument required for determining the polymorphism or genotype of rs27044 and/or rs30187 by at least one of the following methods: DNA sequencing, restriction enzyme fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography, SNP chip, microfluidic chip technology, TaqMan probe technology and Sequenom MassArray technology. Wherein, the reagents and/or instruments required for determining the polymorphism or genotype of rs27044 and/or rs30187 by utilizing the Sequenom MassArray technology comprise PCR primer pairs, extension primers based on single-base extension reaction, phosphatase, resin, chips, MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) and/or other reagents and instruments required by the Sequenom MassArray technology; reagents and/or instruments required for determining the polymorphism or genotype of rs27044 and/or rs30187 by using the TaqMan probe technology comprise a TaqMan probe, a PCR primer pair, a quantitative PCR instrument, a genotyping module and/or other reagents required by the TaqMan probe technology; the SNP chip includes a chip based on nucleic acid hybridization reaction, a chip based on single base extension reaction, a chip based on allele-specific primer extension reaction, a chip based on "one-step" reaction, a chip based on primer ligation reaction, a chip based on restriction enzyme reaction, a chip based on protein DNA binding reaction and/or a chip based on fluorescent molecule DNA binding reaction. Reagents and/or instruments required for determining the polymorphism or genotype of rs27044 and/or rs30187 by the microfluidic chip technology comprise a DNA extraction microfluidic module and reagents, a DNA amplification module and PCR primer pair, a nucleic acid labeling module and related reagents, and an SNP chip and related hybridization, elution and scanning microfluidic modules and reagents.

The product can be a reagent or a kit, and can also be a system consisting of the reagent or the kit and an instrument, such as a system consisting of a primer and a DNA sequencer, a system consisting of a PCR reagent and a DNA sequencer, a system consisting of a TaqMan probe, a PCR primer pair, a quantitative PCR instrument and a module for genotyping and other reagents required by TaqMan probe technology, a system consisting of a probe, a PCR primer pair and other reagents and instruments required by Ligase Detection Reaction (LDR), and a system consisting of a PCR primer pair, a single base extension primer, a chip, a PCR instrument, a module for genotyping and/or other reagents and instruments required by Sequenom MassArray technology.

And amplifying a genome DNA fragment including rs27044 and/or rs30187 by using a PCR primer, taking an obtained PCR amplification product as a template, detecting the sequence of the obtained extension product by using the probe for detecting rs27044 or rs30187, and determining the polymorphism (namely allele) and genotype of rs27044 or rs 30187. The PCR primer has no special requirement on the sequence as long as the genomic DNA segment including rs27044 or rs30187 can be amplified. The probe for detecting rs27044 or rs30187 can be designed according to the upstream and downstream of rs27044 or rs30187 in the human genome, and the sequence of the probe covers the nucleotides of rs27044 or rs30187 in the human genome.

In another embodiment of the present invention, the PCR primers are any one or two of:

1) rs27044-F and rs 27044-R;

2) rs30187-F and rs 30187-R;

rs27044-F is any one of the following single-stranded DNA from a1) to a 4):

a1) single-stranded DNA shown in a sequence 1 in a sequence table;

a2) single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of a 1);

a3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined in a1) or a 2);

a4) single-stranded DNA which hybridizes with the single-stranded DNA defined in a1) or a2) under stringent conditions;

rs27044-R is any one of the following single-stranded DNA from b1) to b 4):

b1) single-stranded DNA shown in a sequence 2 in a sequence table;

b2) single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of b 1);

b3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined in b1) or b 2);

b4) a single-stranded DNA which hybridizes with the single-stranded DNA defined in b1) or b2) under stringent conditions.

The rs30187-F is any one of the following single-stranded DNA from c1) to c 4):

c1) single-stranded DNA shown in a sequence 3 in a sequence table;

c2) single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of c 1);

c3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined in c1) or c 2);

c4) single-stranded DNA which hybridizes with the single-stranded DNA defined in c1) or c2) under stringent conditions;

the rs30187-R is any one of the following single-stranded DNA from d1) to d 4):

d1) single-stranded DNA shown in a sequence 4 in a sequence table;

d2) single-stranded DNA obtained by adding one or several nucleotides to the 5 '-end and/or 3' -end of d 1);

d3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined by d1) or d 2);

d4) a single-stranded DNA which hybridizes with the single-stranded DNA defined by d1) or d2) under stringent conditions.

The probe can be a probe 1 and/or a probe 2, wherein the probe 1 is any one of the following single-stranded DNA from e1) to e 4):

e1) single-stranded DNA shown in a sequence 5 in a sequence table;

e2) single-stranded DNA obtained by adding one or several nucleotides to the 5 '-end and/or 3' -end of e 1);

e3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined by e1) or e 2);

e4) single-stranded DNA which hybridizes under stringent conditions with the single-stranded DNA defined in e1) or e 2);

the probe 2 is any one of the following single-stranded DNA from f1) to f 4):

f1) single-stranded DNA shown in sequence 6 in the sequence table;

f2) single-stranded DNA obtained by adding one or several nucleotides to the 5 '-end and/or 3' -end of f 1);

f3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined by f1) or f 2);

f4) a single-stranded DNA which hybridizes with the single-stranded DNA defined in f1) or f2) under stringent conditions.

The addition of one or several nucleotides may be an addition of one to ten nucleotides.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, or SEQ ID No. 4 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The stringent conditions are hybridization and washing of the membrane 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

In another embodiment of the present invention, the identity of 85% or more may be 85%, 90% or 95% or more.

In another embodiment of the present invention, the 5 'end of each of the probe 1 and the probe 2 may have FAM or VIC modification, and the 3' end may have MGB modification.

In yet another embodiment of the present invention, rs27044 and/or rs30187 are typed using a taqman (thermofisher) genotyping platform. The DNA template containing the SNP site region is amplified by PCR technology and then detected by using a probe.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Examples

1. Exon sequencing:

exome capture was performed using the Agilent SureSelect Human All Exon (V5) capture platform with an effective average sequencing depth of 100X-fold for each sample. The data are compared with a UCSChg19 database through BWA to carry out data yield statistical analysis, sequencing depth analysis and coverage uniformity analysis. The SNP database analysis comprises bioinformatics analysis such as database annotation analysis of dbSNP, thousand-person genome data, ESP exome database and Yanhuang genome (Asia Pacific region only) data, PCA analysis, single-sample SNP conservative prediction, pathogenicity analysis (software: SIFT, Polyphen-2, GERP scenes, mutationssosser, Condel and FATHMM) distribution statistics of SNP on each gene functional element, and the like. Linker and low quality reads were removed using soaponike; alignment was performed using BWA mem, then duplicate removal with Picard, then call variants with GATK Haplotpypecaller, and finally annotation with annovar. After quality control of sequencing, the high quality mutations were then integrated together for association analysis of common and gene-burden analysis of rare variations.

2. Verification of 2 candidate SNP sites in Shandong population in North China

(1) 960 unrelated PE case samples and 1248 age and gender matched unrelated normal control samples in Shandong area are selected to carry out case control correlation analysis. A questionnaire is designed, and the survey contents comprise demographic data, hypertension history, family history, menstruation and fertility history, and the like. Selecting obstetric outpatient clinic and in-patient preeclampsia patients in a tri-hospital in Shandong areas including tobacco terrace, jujube village, Jinnan, chat, Linyi, Taian, Jinning and Zibo and the like, and randomly selecting a normal late pregnant woman control group at the same time. The diagnostic criteria for PE refer to "gynecological science", seventh edition. All cases had no history of hypertension, heart disease, nephropathy, diabetes, hyperthyroidism, etc., and no history of blood transfusion and immunotherapy. The selection criteria of pregnant women with normal pregnancy are as follows: the pregnancy is normal, the fetus develops normally, no complication or complication occurs, no bad pregnancy history exists, and no history of diabetes, kidney disease, immune disease, cancer and the like exists. Exclusion criteria for normal pregnant women: the normal pregnant women with gestational week less than 20 weeks are complicated with twins and multiple fetuses, endocrine diseases, heart diseases, kidney diseases, hypoproteinemia, hydrothorax and ascites, diseases affecting heart and kidney such as systemic lupus erythematosus, etc. The age, the initial rate and the body mass index of the pregnant women in each group are compared, and the difference has no statistical significance (P is more than 0.05). The patients who were admitted to the study and the normal controls were informed, indicated to be willing to participate in the study, and signed the written informed consent and approved by the ethical committee of the affiliated hospital of the medical college of Qingdao university. Venous blood was drawn at 5ml and stored at-70 ℃ until NDA extraction.

(2) Designing candidate sensitive SNP site primers of an ERAP-1 gene (https:// www.mysequenom.com/Tools is designed by Sequenom on-line special design software), carrying out PCR on 2 missense sites one by one, detecting the correctness of primer synthesis, mixing the primers in the same group according to the grouping of primer design, carrying out PCR, and detecting the independence between the primers. After Sap digestion and base extension, the machine is used for mass spectrum detection and data are collected.

The primer sequence is as follows:

TaqMan allele-discriminating real-time polymerase chain reaction including 12.5. mu.L of 2 × PCR Master Mix, 10.25. mu.L of DNA enzyme-free water, 1.25. mu.L of 20 × SNP probe and 1. mu.L of sample DNA at C1000^TMThermal cycler and CFX96^TMAmplification was performed in a real-time system (BIO-RAD, Hercules, Calif.) under the following conditions: the treatment is carried out for 3min at 95 ℃, for 15s at 95 ℃ and for 1min at 60 ℃ for 45 cycles. In each cycle, fluorescent signals of FAM and VIC labeled probes were collected, respectively. Genotype determination was performed using Bio-RadCFX manager3.0 software.

(3) The balance of the SNP Hardy-Weinberg of the research object is tested by using Haploview software, and the randomness of the selection of the research object and the linkage relationship between every two SNP sites are confirmed.

(4) Data were processed using SPSS statistical software. The genetic distribution of each genotype and allele frequency in case and control groups was first chi-square tested to determine the SNP sites associated with PE.

(5) Haplotype analysis is constructed by Phase software to determine the susceptibility haplotype of PE.

(6) And establishing a logistic regression equation by taking the polymorphic site data with significant difference distributed between the PE group and the control group as independent variables and clinical expression as dependent variables, and analyzing the influence of the positive SNP sites on the PE morbidity. Age and the exact phenotypic data obtained above are also simultaneously entered into the regression equation as covariates to provide analytical accuracy.

The experimental results are as follows:

1. exon sequencing results

490 PE patients and 441 female normal controls were selected as candidate genes in the preliminary stage of the subject group. As a result, common mutation analysis finds that rs27044 site (C/G mutation) (OR 0.6941, P0.0004172) of 2 missense variant 13 exons of the ERAP1 gene and rs30187(a/C/T mutation) (OR 0.7107, P0.000999) of 9 exons are significantly associated with PE (table 1), which lays a foundation for further candidate gene verification and identification of the final pathogenic gene of PE. The ERAP-1 gene is related to genetic susceptibility of diseases such as immune diseases, tumors, hypertension and the like. So far, the correlation research of the ERAP-1 gene and PE at home and abroad is rarely reported.

TABLE 1.490 PE patients and female Normal controls 441 who were analyzed by Whole exon resequencing and found SNP sites of the ERAP1 gene positively associated with PE

2. Case control correlation analysis results

The PE group and the control group have no significant difference in age, pregnancy and abortion frequency (P is more than 0.05); the difference between the two groups of pregnant women after admission and the pregnant women after delivery has statistical significance (P is less than 0.001); the systolic pressure and diastolic pressure of the preeclampsia group are obviously increased compared with the control group, and the difference has statistical significance (P is less than 0.001) (see table 2).

TABLE 2 comparison of preeclamptic patients with normal controls for clinical and biochemical indices

Note: p < 0.05

The frequency of each genotype of the gene polymorphism of the rs27044 site is as follows: the case groups comprise 21.82 percent of CC genotype, 47.39 percent of CG genotype and 30.80 percent of GG genotype; the control group comprises 13.08% of CC genotype, 60.41% of CG genotype and 26.51% of GG genotype. The distribution of the rs27044 locus genotype is obviously different between a case group and a control group, (chi)²＝36.683，P<0.001) but no significant difference in allele frequency. (χ²＝0.373，P＝0.541，OR[95％CI]＝0.954[0.820-1.110])). Meanwhile, the frequency of each genotype of the gene polymorphism of the rs30187 site in a case group is CC: 29.93%, CT: 58.16%, TT: 11.90 percent; the frequency of each genotype in the control group was CC: 24.22%, CT: 67.25%, TT: 8.53 percent. The distribution of each genotype differed between the two groups (X)²12.422, P0.002), there was no significant difference in allele frequency. (χ²＝0.226，P＝0.635，OR[95％CI]＝1.042[0.880-1.233]). See tables 3 and 4.

TABLE 3 distribution of rs27044 alleles and genotypes in preeclamptic patients and healthy populations

TABLE 4 distribution of rs30187 alleles and genotypes in preeclamptic patients and healthy populations

The invention discovers that the genotype frequencies of the polymorphisms of the rs27044 and rs30187 loci of the ERAP-1 gene in PE patients and control groups are obviously different by utilizing large sample verification and case control correlation analysis. The risk allele of rs27044 is C, the frequency of the CC genotype in PE patients is 0.2182, the frequency of the CC genotype in normal controls is 0.1308, and the genotype is increased by 66.82% in PE patients compared to normal controls; the risk allele of rs30817 is C, the allele has a gene frequency of 0.2993 in PE patients and 0.2422 in normal controls, and the allele is increased by 23.56% in PE patients compared to normal controls.

According to the invention, through a genetic research means, PE is taken as a research object, the existing research foundation of a subject group is fully utilized, the susceptible gene of the PE of Chinese Han nationality and the susceptible SNP site related to the susceptible gene are screened and identified, a full theoretical basis is provided for revealing the generation mechanism of PE, a foundation is laid for developing an early disease early warning model and an individualized medical prevention and treatment scheme suitable for the population in China, the PE is treated fundamentally, the generation of PE is reduced, the health of mother and infant is ensured, and the method has important clinical significance and social significance.

It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.

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Claims

1. Application of a substance for detecting polymorphism or genotype of rs27044 and/or rs30187 in human genome in preparation of products for screening preeclampsia patients.

2. Application of a substance for detecting polymorphism or genotype of rs27044 and/or rs30187 in human genome in preparation of a product for detecting pre-eclampsia susceptibility.

3. The use of a substance that detects a polymorphism or genotype of rs27044 and/or rs30187 in the human genome in the preparation of a product for detecting a single nucleotide polymorphism associated with preeclampsia.

4. The use of a substance that detects a polymorphism or genotype of rs27044 and/or rs30187 in the human genome in the preparation of a product for identifying or assisting in identifying a single nucleotide polymorphism associated with preeclampsia.

5. Any of the following applications:

6. A product containing a substance for detecting a polymorphism or genotype of rs27044 and/or rs30187 in the human genome, being any one of a) to d):

c) screening pre-eclampsia patient products;

d) and detecting a susceptibility product in preeclampsia.

7. The product of claim 6, wherein: the substance for detecting the polymorphism or genotype of rs27044 and/or rs30187 in the human genome is a PCR primer and/or probe for amplifying a genomic DNA fragment including rs27044 and/or rs 30187.

8. The product of claim 7, wherein: the PCR primer is any one or two of the following primers:

1) rs27044-F and rs 27044-R;

2) rs30187-F and rs 30187-R;

rs27044-F is any one of the following single-stranded DNA from a1) to a 4):

a1) single-stranded DNA shown in a sequence 1 in a sequence table;

rs27044-R is any one of the following single-stranded DNA from b1) to b 4):

b1) single-stranded DNA shown in a sequence 2 in a sequence table;

b4) single-stranded DNA which hybridizes with the single-stranded DNA defined in b1) or b2) under stringent conditions;

the rs30187-F is any one of the following single-stranded DNA from c1) to c 4):

c1) single-stranded DNA shown in a sequence 3 in a sequence table;

the rs30187-R is any one of the following single-stranded DNA from d1) to d 4):

d1) single-stranded DNA shown in a sequence 4 in a sequence table;

9. The product according to claim 7 or 8, wherein the probe is probe 1 and/or probe 2, and the probe 1 is any one of the following single-stranded DNAs e1) to e 4):

e1) single-stranded DNA shown in a sequence 5 in a sequence table;

the probe 2 is any one of the following single-stranded DNA from f1) to f 4):

f1) single-stranded DNA shown in sequence 6 in the sequence table;

10. The following M1) or M2):