阅读说明：本技术 一种与单纯性先天性心脏病相关的snp标志物及其应用 (SNP marker related to simple congenital heart disease and application thereof ) 是由 王凤羽 王海丽 耿尧 孙蕾 于 2020-08-26 设计创作，主要内容包括：本发明提供了一种可用于先天性心脏病易感者筛选的基因标志物,即DNMT3A基因的rs1550117位点。通过检测样本DNMT3A基因的rs1550117位点中的GG、AG、AA基因型,从而判断样本是否与先心病易感性相关,其中,rs1550117位点的AA基因型患单纯性先天性心脏病的风险显著高于该位点其他基因型患单纯性先天性心脏病的风险。本发明进一步提供了可以用于检测DNMT3A基因的rs1550117位点多态性的特异性扩增引物。本发明提供了一种新的先天性心脏病易感者的方法,具有较高的准确性,为先天性心脏病的诊断提供更客观准确的依据,有利于我国先天性心脏病高危人群的早期筛查、及早治疗和预防。(The invention provides a gene marker for screening patients susceptible to congenital heart disease, namely rs1550117 locus of DNMT3A gene. And (3) detecting GG, AG and AA genotypes in the rs1550117 locus of the DNMT3A gene of the sample to judge whether the sample is related to susceptibility to congenital heart disease, wherein the risk of the AA genotype of the rs1550117 locus suffering from the simple congenital heart disease is obviously higher than the risk of other genotypes of the locus suffering from the simple congenital heart disease. The invention further provides a specific amplification primer for detecting the rs1550117 site polymorphism of the DNMT3A gene. The invention provides a new method for patients susceptible to congenital heart disease, which has higher accuracy, provides more objective and accurate basis for the diagnosis of congenital heart disease, and is beneficial to early screening, early treatment and prevention of high risk groups of congenital heart disease in China.)

Use of the DNMT3A gene in the preparation of a reagent for detecting a susceptible patient of simple congenital heart disease.

2. The use according to claim 1, characterized in that it is a polymorphism at the rs1550117 site of the DNMT3A gene for detection.

3. The use according to claim 2, wherein the risk of simple congenital heart disease at the AA genotype of site rs1550117 is significantly higher than at other genotypes at that site.

4. A specific primer pair for amplifying rs1550117 site of DNMT3A gene is characterized in that the sequences of the primer pair are shown as SEQ ID NO. 4 and SEQ ID NO. 5.

5. A specific single-base extension primer for amplifying rs1550117 site of DNMT3A gene is characterized in that the sequence of the primer is shown as SEQ ID NO. 6.

6. Use of a specific primer for amplifying the rs1550117 site of the DNMT3A gene in preparation of a reagent for detecting a susceptible person of simple congenital heart disease.

7. The use according to claim 6, wherein the primers are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively.

8. The use according to claim 6, wherein the primer is as set forth in SEQ ID NO 6.

9. A kit for detecting susceptibility to congenital heart disease is characterized in that the kit comprises a specific primer for amplifying rs1550117 site of DNMT3A gene.

10. The kit according to claim 9, wherein the kit comprises specific primers as shown in SEQ ID NO 4 and SEQ ID NO 5 and/or SEQ ID NO 6, respectively.

Technical Field

The invention relates to the technical field of biology, in particular to an SNP marker related to simple congenital heart disease.

Background

Congenital Heart Disease (CHD), abbreviated as congenital heart disease, refers to the structural and functional abnormalities of the heart and blood vessels that are present at birth. The clinical consequences are extremely severe, often leading to abortion, stillbirth, neonatal death, and disability in children, adolescents, and adults. At present, the congenital heart disease accounts for the major birth defect disease 1/3, becomes a major public health problem affecting physical and mental health of children and quality of population life, and causes serious economic and mental burdens on society and families.

About 80-90% of congenital heart diseases only manifest as cardiac malformations, but are not accompanied by congenital abnormalities of other systems, and are called simple congenital heart diseases. The research for many years has proved that it is a polygenic genetic disease, mainly caused by abnormal development of heart blood vessels due to the combined action of genetic factors and environmental factors in embryonic period, with the inheritance degree of 55-65%. It is now considered that pure CHD not only involves multiple genes, but also is involved in the sequential expression and interaction of these genes at different times and in different spaces, where qualitative or quantitative abnormalities in the expression of any gene may affect cardiac development. Such as: at the genome level, the transcription factors NKX2-5, GATA4, TBX5, a signal path, certain gene mutations involved in cardiac development, gene polymorphisms such as Single Nucleotide Polymorphism (SNP) and gene Copy Number Variation (CNV), epigenetic aspects including DNA methylation, MicroRNA and histone modification and the like can be related to the occurrence and development of CHD.

DNA methylation is the most deeply studied mechanism in epigenetics and is involved in the development of various diseases in humans. Current research indicates that three DNA methyltransferases (DNMTs) are mainly involved in DNA methylation in mammalian CpG islands: DNA methyltransferases that maintain methylation (DNMT1) and de novo methylation (DNMT3A, DNMT 3B). DNMT1 is the most abundant DNMT in cells, primarily responsible for the maintenance of genomic methylation after cell division, and serves to maintain the same specific methylation pattern in daughter cells as in mother cells. DNMT3A and DNMT3B are de novo methyltransferases, which are primarily responsible for new methylation processes of DNA in germ cells, embryonic stem cells, and during embryonic development.

The conventional DNMTs gene polymorphism research mainly focuses on the aspect of tumors, and the research report on the aspect of heart disease is not found. SNP studies of DNMTs gene have involved relatively few studies of promoter region, particularly DNMT1 gene, and promoter region plays an important role in regulation of gene expression.

One important reason for affecting gene expression levels is gene polymorphism, which is also an important factor affecting differences in individual disease susceptibility, phenotype and response to treatment, with the most important and common manifestation being SNPs. At present, a plurality of documents report that the SNP site of the DNMTs gene has relevance with diseases in the aspects of tumor, psychosis, infection, growth and development and the like, and the expression or translation process of the DNMTs gene is regulated. However, the DNMTs genes currently studied in children with simple heart attack disease only have four sites of rs 1699593, rs2228612, rs10420321 and rs2288349 in DNMT1, and are still the work content in the laboratory, and other SNP sites, especially in promoter regions having important influence on gene expression, have not been reported. Therefore, the correlation between the polymorphism of the three genes DNMT1, DNMT3A and DNMT3B and the simple congenital heart disease is researched, a clinical detection method is established, SNP information of DNMTs genes of Henan populations is summarized, more effective and more specific molecular diagnosis targets or specific biomarkers are searched, a new thought is provided for predicting or evaluating the potential risk of the birth congenital heart disease of the population, and the method has very important significance for reducing the birth of children patients to the maximum extent and improving the comprehensive quality of the population in China.

Studies have reported that the incidence of Congenital Heart Disease (CHD) in newborns is about 1% o, and is higher in newborns with spontaneous abortion or death. Therefore, the prenatal screening and detecting method for the congenital heart disease is provided, the accuracy of detection is improved, a more objective and accurate basis is provided for diagnosis of the congenital heart disease, and early screening, early treatment and prevention of high risk groups of the congenital heart disease in China are facilitated.

Disclosure of Invention

In order to overcome the defects of the detection method of the congenital heart disease of the newborn in the prior art and the current situation of low detection accuracy, the invention provides an SNP marker related to the simple congenital heart disease.

The research aims to perform comprehensive and systematic research on SNP sites, particularly promoter regions, of three genes, namely DNMT1, DNMT3A and DNMT3B in congenital heart disease patients of Han nationality people in Henan province by using a nucleic acid mass spectrometry Massarray technology, and analyzes whether DNMTs gene polymorphism is associated with congenital heart disease, which genotypes are risk factors of the disease and whether interaction of DNMTs related genes influences the occurrence of the congenital heart disease compared with normal children. And establishing a specific detection method for the screened specific sites aiming at the screened specific sites to form a simple congenital heart disease susceptibility gene detection kit.

The invention discloses an application of DNMT3A gene in preparing a detection reagent for detecting a person susceptible to simple congenital heart disease.

Preferably, the invention further discloses application of the rs1550117 site polymorphism of the DNMT3A gene in preparing a detection reagent for detecting the simple congenital heart disease.

Preferably, the risk of simple congenital heart disease in the AA genotype of the rs1550117 site of the DNMT3A gene is obviously higher than the risk of simple congenital heart disease in other genotypes of the site.

The invention discloses a specific primer pair for amplifying rs1550117 site of DNMT3A gene.

Preferably, the nucleotide sequences of the primer pair are ACGTTGGATGCTGCTGGAGGAGTGAGATG and ACGTTGGATGCACCTCTGTCTAATTCCACC, which are shown as SEQ ID NO. 4 and SEQ ID NO. 5 respectively.

The invention discloses a specific single-base extension primer for amplifying an rs1550117 site of a DNMT3A gene.

Preferably, the sequence of the single-base extension primer is GCACAGCCACTCACT, as shown in SEQ ID NO. 6.

The invention discloses application of a specific primer for amplifying an rs1550117 site of a DNMT3A gene in preparation of a detection reagent for detecting a simple congenital heart disease.

Preferably, the primers are shown as SEQ ID NO. 4 and SEQ ID NO. 5 respectively.

Preferably, the primer is shown as SEQ ID NO. 6.

The invention discloses a kit for detecting congenital heart disease.

Preferably, the kit comprises a specific primer for amplifying the rs1550117 site of the DNMT3A gene.

Preferably, the specific primers included in the kit are respectively shown as SEQ ID NO. 4 and SEQ ID NO. 5 and/or SEQ ID NO. 6.

The invention provides a gene marker for screening patients susceptible to congenital heart disease, namely rs1550117 locus of DNMT3A gene. And (3) detecting GG, AG and AA genotypes in the rs1550117 locus of the DNMT3A gene of the sample to judge whether the sample is related to susceptibility to congenital heart disease, wherein the risk of the AA genotype of the rs1550117 locus suffering from the simple congenital heart disease is obviously higher than the risk of other genotypes of the locus suffering from the simple congenital heart disease.

Drawings

FIG. 1 shows a W1-UEP mass spectrum.

FIG. 2 shows a W2-UEP mass spectrum.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.