Biomarker for predicting premature delivery risk and application thereof

Abstract

The invention discloses a biomarker for predicting premature delivery risk and application of the biomarker. The biomarker is an HLA (human leukocyte antigen) mismatched Eplet: HLA-DRB1 (human leukocyte antigen) Eplet 73A; eplet 73A mismatch information is obtained based on HLA typing of a mother and a filial generation, premature delivery prediction can be performed by using the Eplet, and the premature delivery risk level of a pregnant woman to be detected is judged, so that premature prediction and premature prevention of the mother and the child with the premature delivery risk in the gestation period are facilitated; the marker disclosed by the invention is high in sensitivity and high in specificity, and has a better clinical application prospect compared with a traditional method.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a biomarker for predicting the risk of premature birth and its application. Background Art

[0002] Premature birth is a major challenge in the fields of obstetrics and gynecology, pediatrics and public health worldwide. Premature birth is the leading cause of death in children under 5 years old, and surviving premature babies are prone to a variety of adverse health problems and sequelae, such as breathing difficulties, feeding difficulties, cerebral palsy, neurodevelopmental retardation, etc., and are at a higher risk of chronic diseases such as type 2 diabetes, coronary heart disease, and hypertension in adulthood, which seriously threatens children's health and even life safety. It can be seen that preventing the occurrence of premature birth is of great significance to improving the health level of the population and reducing the disease burden and social burden.

[0003] Premature birth is the ultimate result of the combined effects of many intrauterine causes that threaten fetal development. At present, there is a lack of effective prediction and prevention methods for premature birth. The inventor's previous work found that the use of conventional clinical feature data cannot effectively predict the occurrence of premature birth. At the same time, intervention measures such as vaginal progesterone and cervical cerclage for ordinary pregnant women have failed to effectively reduce the risk of premature birth. Therefore, understanding the cause and mechanism of premature birth is the key to preventing premature birth.

[0004] Destruction of maternal-fetal immune tolerance is one of the important causes of spontaneous preterm birth. The fetus shares half of its genome with its parents and is essentially an allogeneic "half-phase" transplant. Therefore, the dynamic changes in the maternal immune system during pregnancy are crucial to the survival and development of the fetus. The occurrence of spontaneous preterm birth is closely related to the early transition from the maternal-fetal immune tolerance phase to the inflammatory phase, but the genetic and pathological mechanisms between the destruction of maternal-fetal immune tolerance and spontaneous preterm birth are still unclear. Therefore, clarifying the genetic and pathophysiological mechanisms of immune tolerance disturbances that affect spontaneous preterm birth can provide new targets for the early prediction and prevention of premature birth.

[0005] HLA molecules encoded by human leucocyte antigen (HLA) genes have antigen presentation function, directly participate in the activation and differentiation of T cells, participate in regulating the body's adaptive immune response, and their core function is to identify foreign bodies. They are mainly divided into HLA type I and type II. Classic HLA class I molecules (including HLA-A, HLA-B, and HLA-C) present antigens in nucleated cells to the cell surface and are recognized by CD8+T cells; HLA class II molecules (including HLA-DP, HLA-DM, HLA-DO, HLA-DQ, and HLA-DR) are only expressed on specific cell surfaces, presenting extracellular antigens to CD4+T cells, and activating the body's cellular and humoral immunity. In terms of maternal-fetal immune tolerance, the extrachorionic trophoblast cells of the placenta, which act as a physical barrier, can express a limited number of antigens, including classical class I HLA-C and non-classical HLA-E, HLA-F, and HLA-G molecules, which can inhibit NK cell- and T cell-mediated cytotoxicity, thereby preventing maternal immune rejection of the fetus.

[0006] According to the classic immune rejection theory, the more similar the maternal and fetal HLA genotypes are, the less likely the maternal immune system will produce an immune attack on the fetus. Since HLA molecules are the product of HLA gene complexes, the genotype cannot directly reflect the molecular structure and immunogenicity of HLA. In recent years, the concept of donor-recipient Eplet mismatch has been introduced in the field of transplantation research. Eplet functional epitopes are polypeptide amino acid configurations on the surface of HLA molecules defined based on sequencing data, some of which have been proven to bind specifically to antibodies. These epitopes are short, continuous and discontinuous amino acid sequences, and various HLA molecules may express the same Eplet or combination. The mismatch load indicates the number of Eplet mismatches on the donor-recipient HLA. The higher the mismatch load, the less match there is between the HLA functional epitopes of the two. The Eplet mismatch load has been widely used in the transplantation field to predict the organ survival time of transplant recipients and to find matching donors for recipients.

[0007] Since the mother and the fetus are allogeneic, in addition to considering HLA genotype matching, the impact of the mother-fetal Eplet mismatch on premature birth should also be considered, but similar research is still lacking. Summary of the invention

[0008] The purpose of the present invention is to provide a biomarker HLA-DRB1 Eplet 73A that can be used for premature birth diagnosis; the risk of premature birth is predicted by detecting the mismatch frequency of HLA-DRB1 Eplet 73A between mother and child.

[0009] The technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides the use of a reagent for detecting HLA functional epitope mismatch in the preparation of a product for predicting the risk of premature birth, wherein the HLA functional epitope is inconsistent between the mother and the offspring, which is called mismatch.

[0011] Preferably, the HLA functional epitope is HLA-DRB1 Eplet 73A.

[0012] Preferably, the reagent for detecting HLA functional epitope mismatch comprises a reagent for detecting HLA functional epitope mismatch by at least one of nucleic acid hybridization technology, nucleic acid amplification technology, sequencing technology, chromatography technology, mass spectrometry technology, and chip technology; specifically, a reagent for detecting HLA functional epitope genotype.

[0013] Preferably, the reagents include primers and probes.

[0014] Preferably, the product includes reagents, test kits, test strips, chips, and detection systems.

[0015] Preferably, the subjects of the detection are pregnant women and fetuses.

[0016] A second aspect of the present invention provides a system for predicting the risk of premature birth, comprising:

[0017] Acquisition module: used to detect HLA functional epitope mismatch information in samples of pregnant women and fetuses to be tested;

[0018] Analysis module: used to obtain the above test results and predict the risk of premature birth of pregnant women based on the test results.

[0019] Preferably, if HLA functional epitope mismatch is detected in the samples of the pregnant woman to be tested and the fetus, the risk of premature birth of the pregnant woman is higher; otherwise, the risk of premature birth of the pregnant woman is low.

[0020] Preferably, the HLA functional epitope is HLA-DRB1 Eplet 73A.

[0021] According to a third aspect of the present invention, a computer-readable storage medium is provided, which stores a computer program. When the computer program is executed by a processor, the functions of the system described in the second aspect of the present invention can be realized.

[0022] According to a fourth aspect of the present invention, there is provided an electronic device comprising a memory and a processor, wherein the memory stores a computer program, and the processor is configured to execute the computer program to implement the functions of the system according to the second aspect of the present invention.

[0023] The present invention also provides a method for predicting the risk of premature birth, by collecting DNA samples from the mother and the offspring, sequencing the DNA samples to obtain the HLA typing of the mother and the offspring, and obtaining the eplet mismatch information based on the HLA typing of the mother and the offspring, so as to perform early prediction and prevention for the mother and the offspring at risk of premature birth.

[0024] The beneficial effects of the present invention are:

[0025] The present invention provides an HLA mismatch Eplet that can significantly assess the risk of premature birth: HLA-DRB1 Eplet 73A. Based on the HLA typing of the mother and the offspring, the Eplet 73A mismatch information is obtained, and the Eplet is used to predict premature birth and determine the risk level of premature birth of the pregnant woman to be tested, thereby facilitating early prediction and early prevention of mothers and children at risk of premature birth during pregnancy. By identifying the impact of the Eplet mismatch characteristics of mothers and children on spontaneous premature birth, the occurrence of premature birth can be predicted, reducing the disease burden and social burden. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the 3D structure of Eplet 73A.

[0027] Figure 2 This is the ROC curve of Eplet 73A. DETAILED DESCRIPTION

[0028] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0029] HLA: Human Leukocyte Antigen. HLA is a group of highly polymorphic cell surface proteins that are mainly found on the surface of human white blood cells and are also distributed on the surface of other tissue cells. HLA molecules play a key role in the immune system. They are responsible for identifying and displaying foreign antigens and regulating and coordinating immune responses. In immunology, HLA molecules are considered to be the main tissue compatibility markers of the immune system. Their polymorphism makes each person's HLA molecules have unique immune characteristics, which determines the individual's immune response, organ transplant compatibility, and susceptibility to autoimmune diseases. HLA molecules are usually encoded by a group of highly polymorphic genes and are divided into two categories: class I and class II. Class I includes HLA-A, HLA-B and HLA-C, which mainly display endogenous antigens on the cell surface, while class II includes HLA-DR, HLA-DP and HLA-DQ, which mainly display exogenous antigens on the surface of immune cells.

[0030] Eplet: refers to an epitope or epitope fragment on the human leukocyte antigen (HLA) molecule, usually composed of one to several amino acid residues. These epitopes can be recognized by antigen-presenting cells in the immune system and trigger corresponding immune responses. HLA Eplet is particularly important for organ transplantation because they determine the tissue compatibility between the donor and the recipient. When the recipient's immune system recognizes an epitope that does not match its own HLA Eplet, it may trigger a rejection reaction. Therefore, understanding the characteristics and pairing patterns of HLA Eplet is crucial to predicting the risk of transplant rejection and optimizing the success rate of organ transplantation. Analysis of HLA Eplet usually involves identifying specific amino acid variations on the HLA molecule sequence and correlating them with immunology-related data to evaluate their role in the immune response.

[0031] Example 1

[0032] A method for screening Eplet mismatch for high risk of premature birth comprises the following steps:

[0033] 1) Obtain DNA sequencing data of the mother-child pairs;

[0034] 2) Perform data analysis and variant detection on DNA sequencing data;

[0035] 3) Use snp2hla software to analyze the HLA types of mother and child;

[0036] 4) HLAMatchmaker software was used to identify Eplet mismatches between maternal and fetal HLA gene pairs;

[0037] 5) Analyze the different Eplet mismatches between the preterm and non-preterm groups and identify Eplet mismatches with a high risk of preterm birth;

[0038] The details are as follows:

[0039] 1. Experimental methods

[0040] Study Design: Cohort study.

[0041] Study subjects: Mothers and children of the Guangzhou Birth Cohort;

[0042] Inclusion criteria: mother-child pairs who were enrolled in the Guangzhou Birth Cohort and completed whole genome sequencing;

[0043] Exclusion criteria: 1) The newborn was born with obvious birth defects; 2) The pregnant woman had a history of organ or bone marrow transplantation before pregnancy.

[0044] Determination of premature birth characteristics: Spontaneous premature birth is diagnosed according to the Chinese Clinical Diagnosis and Treatment Guidelines for Premature Birth, which is defined as spontaneous delivery at 28 weeks of gestation but less than 37 weeks of gestation, including premature rupture of membranes or premature cervical dilatation. The gestational age is checked based on the last menstrual period and ultrasound examination. When the difference between the last menstrual period and the ultrasound-calculated gestational age in the first trimester is greater than 7 days or the difference between the last menstrual period and the ultrasound-calculated gestational age in the second trimester is greater than 10 to 14 days, the ultrasound-calculated gestational age shall prevail.

[0045] Data Analysis:

[0046] 1) Birth cohort genome-wide data analysis and variant detection

[0047] Using BWA, GATK and other software, we independently developed an analysis process ilus (https: / / github.com / ShujiaHuang / ilus) suitable for large-scale population genome data. This process can complete the mutation detection of all samples in this example. This example will strictly control the quality of samples and mutation data;

[0048] Specifically include:

[0049] (1) Samples without clinical record information were discarded, and samples with high contamination rate (1%), high sequencing error rate (1%), low average sequencing depth (<3x), and abnormal number of individual variants compared with the average (more than 3 standard deviations from the mean) were filtered out;

[0050] (2) The theoretical sex of each sample is estimated using the difference in coverage depth between X and Y chromosomes and autosomes, and samples whose sex information is inconsistent with the theoretical results are eliminated;

[0051] (3) The kinship between each sample is calculated using bioinformatics methods, and mother-infant pairs and families with inconsistent kinship are eliminated. Next, the genotypes of each sample are rechecked using accurate family information, and variant sites that do not conform to the Mendelian inheritance law of the family are discarded.

[0052] In addition, Beagle and the 1000 Genomes haploid set will be used to phase and fill in the genotypes of low sequencing depth variant detection. The specific process suitable for this step has also been independently developed and implemented by the inventor's team (https: / / github.com / BIGCS-Lab / phimtools).

[0053] 2) HLA typing

[0054] The whole genome data analysis process is used to detect and filter the high-quality SNPs sites, and then the snp2hla software is used to infer the maternal and fetal HLA genotypes of each sample based on reliable high-quality SNPs sites. snp2hla uses the association between SNP data and HLA genotypes to predict HLA genotypes through statistical models and algorithms. The basic principle is to use the association between known SNPs and HLA genotypes to convert SNP data into predicted results of HLA genotypes.

[0055] 3) Identification of maternal-fetal HLA gene Eplet mismatch

[0056] Based on the common HLA genotype maternal-fetal pairing pattern, HLAMatchmaker software was used to identify the Eplet differences and mismatch loads between HLA gene pairs. This tool is based on the specific ability of HLA molecules to evaluate the similarity between two given HLA molecules and evaluate the suitability of pairing based on this similarity. The smaller the Eplet difference between two HLA molecules, the more compatible the pairing between them. HLAMatchmaker was used to compare the sequences of maternal and fetal HLA molecules, identify and calculate the Eplet of each mother and fetus, and evaluate the degree of match between HLA genotypes based on the number and nature of Eplet differences, indicating the degree of compatibility of maternal and fetal HLA gene pairing.

[0057] 4) Relationship between maternal-fetal HLA gene Eplet mismatch and premature birth

[0058] By calculating the relative risk ratio (RR value, RR = incidence rate in the exposed group / incidence rate in the non-exposed group) of the Eplet type and using the chi-square test, all possible potential risk Eplet types with distribution differences between the preterm group and the term group were obtained. The HLA types with a lower limit of RR value greater than 1 or an upper limit of RR value less than 1 were screened - if RR = 1, it means there is no evidence of an association between exposure and the outcome, and the chi-square test p value after Bonferroni correction is less than 0.05.

[0059] 2. Research results:

[0060] Excluding mother-child pairs with offspring death and abnormal pregnancy, 444 pairs of maternal-fetal genomic data were obtained, of which 144 pairs were premature mother-child pairs and 300 pairs were full-term mother-child pairs. The whole genome data analysis process was used to detect and filter high-quality SNPs sites, and then the snp2hla software was used to infer the classical class I and class II HLA types of each sample based on reliable high-quality SNPs sites.

[0061] The HLA matchMaker computational tool for HLA molecular pairing was further used to calculate the HLA mismatch of 444 pairs of maternal-fetal HLA. Through analysis, a total of 124 Eplets of HLA Class I genes were identified in the study population, of which 65 were related to antibody recognition; a total of 256 Eplets of HLA Class II genes, of which 97 were related to antibody recognition.

[0062] From the perspective of immune tolerance, the inventors' team focused on analyzing the relationship between antibody-related Eplet and premature birth, and selected 60% of the samples as the discovery cohort, including 86 premature births and 180 full-term births. It was found that Eplet 73A in class II HLA (HLA-DRB1 gene maternal-fetal mismatch, representing the 73rd position of the HLA molecule is alanine, Figure 1 ) was associated with an increased risk of premature birth (P=0.0144, RR=10.47). Using 40% of the samples as a validation set, it was shown that Eplet 73A was associated with an increased risk of premature birth (P=0.0105). The results are shown in Table 1.

[0063] Table 1 Correlation between maternal and fetal eplet 73A and preterm birth

[0064]

[0065] Example 2

[0066] 100 mother-child pairs were randomly selected from the verification cohort of Example 1, and the mother-child HLA carrying eplet 73A situation was obtained according to the aforementioned method. Preterm birth was predicted based on the matching of eplet 73A. If a mismatch of the HLA functional epitope HLA-DRB1 Eplet 73A was detected in the samples of the pregnant woman to be tested and the fetus, the risk of premature birth of the pregnant woman was high; otherwise, the risk of premature birth of the pregnant woman was low.

[0067] Draw the ROC curve based on the test results; see Figure 2 ,The results showed that the AUC value was 0.750, which had a high diagnostic value.

[0068] From this study, it can be seen that maternal-fetal HLA mismatch load and Eplet 73A can increase the risk of premature birth. By detecting maternal and fetal HLA types and identifying Eplet mismatch, the occurrence of premature birth can be predicted.

[0069] The above specific implementations have been described in detail for the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Application of reagents for detecting HLA functional epitope mismatch in the preparation of products for predicting the risk of premature birth.

2. The use according to claim 1, characterized in that: The HLA functional epitope is HLA-DRB1 Eplet73A.

3. The use according to claim 1, characterized in that: The reagent for detecting HLA functional epitope mismatch includes a reagent for detecting HLA functional epitope mismatch through at least one of nucleic acid hybridization technology, nucleic acid amplification technology, sequencing technology, chromatography technology, mass spectrometry technology, and chip technology.

4. The use according to claim 1, characterized in that: The reagents include primers and probes.

5. The use according to claim 1, characterized in that: The products include reagents, test kits, test strips, chips, and detection systems.

6. A system for predicting the risk of premature birth, comprising: Acquisition module: used to detect HLA functional epitope mismatch information in samples of pregnant women and fetuses to be tested; Analysis module: used to obtain the above test results and predict the risk of premature birth of pregnant women based on the test results.

7. The system according to claim 6, characterized in that If HLA functional epitope mismatch is detected in the samples of the pregnant woman and the fetus, the pregnant woman has a higher risk of premature birth; otherwise, the pregnant woman has a low risk of premature birth.

8. The system according to claim 6 or 7, characterized in that: The HLA functional epitope is HLA-DRB1Eplet73A.

9. A computer-readable storage medium storing a computer program, wherein when the computer program is executed by a processor, the function of the system according to any one of claims 6 to 8 can be realized.

10. An electronic device comprising a memory and a processor, wherein the memory stores a computer program, wherein: The processor is used to execute the computer program to implement the function of the system according to any one of claims 6 to 8.

Images