CN114736960A - Molecular marker and kit for predicting sudden cardiac death based on METTL16 gene polymorphism sites - Google Patents
Molecular marker and kit for predicting sudden cardiac death based on METTL16 gene polymorphism sites Download PDFInfo
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Abstract
The invention relates to a molecular marker and a kit for predicting sudden cardiac death based on a polymorphism site of METTL16 gene, wherein the molecular marker is an rs58928048 site of METTL16 gene, a base TGTCTG is inserted or deleted in the site, and the insertion type indicates that the risk of sudden cardiac death is high. According to the invention, through the research on the correlation between the insertion deletion polymorphism of the rs58928048 site of the METTL16 gene and the sudden cardiac death phenotype, a polymorphism molecular marker related to sudden cardiac death is provided, and a sudden cardiac death detection kit is further provided, so that the sudden cardiac death risk of an individual is detected.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a molecular marker and a kit for predicting sudden cardiac death based on METTL16 gene polymorphism sites.
Background
Sudden Cardiac Death (SCD) refers to sudden unexpected death caused by cardiac causes, manifested by sudden loss of consciousness and circulatory and respiratory arrest occurring in a short period of time. "short-term" is defined as less than 1 hour for witnesses and 24 hours for no witnesses. Numerous previous studies have shown that the cause of death of SCD is usually coronary atherosclerotic heart disease or fatal arrhythmia. The former is often found in the elderly, while the latter is often found in young people. Considering that the precise cause of death is difficult to find in autopsy and histological examination after death, it is urgent to uncover the molecular mechanism of SCD occurrence and to find the genetic marker of SCD in order to accurately diagnose SCD.
The past research aiming at the SCD diagnosis field mainly focuses on the correlation analysis of coding region mutation and sudden death of important protein coding genes, however, the related mutations are not only limited in number, but the relatively high sudden death incidence rate is difficult to completely explain by the extremely low gene frequency of the general population. With the development of genome-wide association studies and second-generation sequencing technologies, more and more genetic markers are found to be involved in the regulation of SCD. The identification of the SCD genetic marker can further understand the potential mechanism of the SCD genetic marker, optimize the risk stratification of the SCD and provide a powerful theoretical basis for molecular diagnosis and prevention. Considering that the 3'UTR plays an important role in regulating and controlling gene expression, the attention is paid to genetic polymorphism in the region, and the correlation between the genetic polymorphism of the 3' UTR in related genes and SCD susceptibility is analyzed through case contrast research, so that the possibility is provided for establishing a gene polymorphism detection system for SCD risk assessment.
N6-methyladenosine (m6A) is the most prevalent and abundant type of internal post-transcriptional RNA modification in eukaryotic cells, reversibly and dynamically regulated by its effector proteins methyltransferases (METTL16, METTL3, METTL14, etc.), demethylases (FTO, ALKBH5, etc.), and associated binding proteins (YTHDF 1-3). m6A plays an important role in gene expression regulation, mRNA stability and homeostasis, various diseases, and the like. With the development of scientific technology, more and more researches show that m 6A-related protein-based abnormality is a sign of the development of cardiovascular diseases, including cardiac hypertrophy, heart failure, myocardial infarction, ischemic heart disease, aortic aneurysm, vascular calcification, pulmonary hypertension and the like. A number of m6A-SNPs associated with cardiovascular disease have also been identified using genome-wide association studies (GWAS) together, and genetic functional mechanisms have been explored. For example, rs9847953 and rs197922 have been identified as potential functional variants which contribute to the regulation of blood pressure. Further studies have shown that rs12286 may mechanistically affect the methylation level of m6A and is significantly associated with SCD. In general, m6A methylation regulation is crucial to maintaining cardiac physiological functions, and m6A modification may help to further understand the molecular mechanisms of cardiovascular pathogenesis and may become a potential biomarker in the future.
METTL16 is a novel m6A methyltransferase that comprises a Rossmann-like fold of class I methyltransferases involved in m6A modification by modulation of SAM synthetase or other factors. The research shows that METTL16 plays an important role of a cancer promotion gene in the occurrence and development of tumors (such as liver cancer). Low expression of MetTLL16 has also been identified in Ovarian Cancer (OC), and METTL 16-mediated methylation of RNA structures is an important participant in mouse embryonic development, and it is also hypothesized that MetT16/Malat1 interaction may be involved in angiogenesis.
In the prior art, no research report on the correlation between rs58928048 insertion deletion polymorphism and SCD is reported, and no correlation report on predicting SCD susceptibility by detecting insertion deletion polymorphic sites of 3' UTR of METTL16 gene is reported.
Disclosure of Invention
In order to solve the technical problems, the invention provides a polymorphic molecular marker related to sudden cardiac death through researching the correlation between the insertion deletion polymorphism of the rs58928048 site of the METTL16 gene and the phenotype of sudden cardiac death, and further provides a sudden cardiac death detection kit, thereby obtaining the risk of sudden cardiac death of an individual.
The first purpose of the invention is to provide an insertion deletion polymorphism molecular marker for detecting the onset risk of Sudden Cardiac Death (SCD), which is an rs58928048 site of METTL16 gene, wherein the site has insertion deletion polymorphism of 6 bases, specifically, insertion or deletion base TGTCTG.
Further, the inserted base TGTCTG (insertion type) indicates that the sudden cardiac death risk is high, and the deleted base TGTCTG (deletion type) indicates that the sudden cardiac death risk is low.
The second purpose of the invention is to provide an application of the insertion deletion polymorphism site of the METTL16 gene in the preparation of a sudden cardiac death detection kit, wherein the kit is used for detecting the genotype of the METTL16 gene rs58928048 site.
Further, the sudden cardiac death detection kit comprises a primer pair for amplifying the rs58928048 site of the METTL16 gene.
Further, the primer pair for amplifying the rs58928048 site of the METTL16 gene comprises a sense primer and an antisense primer, wherein the nucleotide sequence of the sense primer is shown as SEQ ID NO.1, the nucleotide sequence of the antisense primer is shown as SEQ ID NO.2, and the specific sequence information is as follows:
SEQ ID NO.1：5′-CACATGAATGCTTGGACTAGAAT-3′，
SEQ ID NO.2：5′-GTGTATTTTTTTTCCTGGAGAGA-3′。
further, the primer pair is provided with an identification label, such as a fluorescent label, for identification.
The primer pair is designed aiming at the insertion deletion site rs58928048 on the METTL16 gene, can specifically amplify a fragment containing the insertion deletion, labels a fluorescent dye at the 5' end of the oligonucleotide primer by a fluorescent labeling technology, and one strand of a PCR amplified product carries the fluorescent dye for labeling the primer. It will be appreciated by those skilled in the art that specific primer pairs can be synthesized using conventional synthetic techniques, and that the primers of the present invention are not limited to the pair of primers SEQ ID NO.1 and SEQ ID NO. 2.
Further, the Tm value of the sense primer was 57 ℃ and that of the antisense primer was 59 ℃.
Further, the sudden cardiac death detection kit also comprises components for PCR amplification and capillary electrophoresis.
Further, the components for PCR amplification and capillary electrophoresis include Taq DNA polymerase, dNTP mixed solution, MgCl2Solution, PCR reaction buffer and deionized water. The PCR amplified product is used for analyzing the genotype of the site by a capillary electrophoresis separation technology.
Further, the storage temperature of the kit was-20 ℃.
The principle of the invention is as follows: the inventor finds that different allelic types of one insertion deletion polymorphism rs58928048 in 3' UTR of METTL16 gene can influence the stability of METTL16 gene mRNA structure through molecular biological research, and can be used as a regulatory element to interact with promoters of adjacent genes to remotely regulate the transcriptional activity of the adjacent genes; in addition, it was found from the SCD case control study of a certain scale population that the deletion allele of the insertion deletion polymorphism is positively correlated with the occurrence of SCD, and the correlation still exists even after adjusting factors such as age and sex. This indel polymorphism is therefore considered useful for assessing an individual's susceptibility to SCD.
The third purpose of the invention is to provide application of a primer for amplifying the rs58928048 locus of the METTL16 gene in preparation of a sudden cardiac death detection kit.
Further, primers for amplifying rs58928048 site of the METTL16 gene comprise a sense primer and an antisense primer, wherein the nucleotide sequence of the sense primer is shown as SEQ ID NO.1, the nucleotide sequence of the antisense primer is shown as SEQ ID NO.2, and the specific sequence information is as follows:
SEQ ID NO.1：5′-CACATGAATGCTTGGACTAGAAT-3′，
SEQ ID NO.2：5′-GTGTATTTTTTTTCCTGGAGAGA-3′。
the fourth purpose of the invention is to provide a sudden cardiac death detection kit based on insertion deletion polymorphism of METTL16 gene, which is used for detecting the genotype of the rs58928048 locus of the METTL16 gene and comprises a primer for amplifying the rs58928048 locus of the METTL16 gene; the primers comprise a sense primer and an antisense primer, the nucleotide sequence of the sense primer is shown as SEQ ID NO.1, the nucleotide sequence of the antisense primer is shown as SEQ ID NO.2, and the specific sequence information is as follows:
SEQ ID NO.1：5′-CACATGAATGCTTGGACTAGAAT-3′，
SEQ ID NO.2：5′-GTGTATTTTTTTTCCTGGAGAGA-3′。
by the scheme, the invention at least has the following advantages:
the specific primer pair contained in the kit provided by the invention is designed aiming at the insertion deletion site No. rs58928048 on the METTL16 gene, can specifically amplify a DNA fragment containing the site and identify different genotypes by detecting the mobility of the fragments with different lengths in capillary electrophoresis, and combined with the discovery of the case contrast research, the person with the insertion allele carried by the insertion deletion site No. rs58928048 on the METTL16 gene of the detected DNA is considered to be SCD susceptible. Therefore, the susceptibility of individual SCD can be predicted by detecting the genotype of the rs58928048 inserted deletion site on the METTL16 gene of the individual.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to make the technical solutions of the present invention practical in accordance with the contents of the specification, the following description is made with reference to the preferred embodiments of the present invention and the accompanying drawings.
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In order that the present disclosure may be more readily and clearly understood, reference will now be made in detail to the present disclosure, examples of which are illustrated in the accompanying drawings.
FIG. 1 is a diagram of gene sequencing and a diagram of SDS-PAGE gel electrophoresis typing.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
EXAMPLE 1 use of the detection kit
Step 1: extraction of DNA template
Genomic DNA of peripheral blood was extracted using a blood genomic DNA extraction system (non-centrifugal column type).
Step 2: PCR reaction-replication of the fragment of interest
A PCR detection kit capable of detecting SCD susceptibility is used, wherein the kit comprises the following primers:
SEQ ID NO. 1: 5'-CACATGAATGCTTGGACTAGAAT-3', Tm value 57 ℃;
SEQ ID NO. 2: 5'-GTGTATTTTTTTTCCTGGAGAGA-3', Tm value of 59 ℃;
the primer pair can specifically amplify a fragment containing the polymorphism of the insertion deletion site No. rs58928048 in the METTL16 gene.
The total volume of the PCR reaction system is 10uL, which comprises: mu.L of DNA template, 0.04 mu.L of each of two 50 mu M specific primer pairs, and 0.08 mu.L of 2.5U/. mu.L Taq DNA polymerase; 0.2. mu.L of 2.5mM dNTP mixture; 25mM MgCl2Solution 0.6 μ L; 10 XPCR reaction buffer 1 uL; supplementing deionized water;
the reaction was carried out on an Eppendorf Mastercycler nexus PCR amplification apparatus under the following conditions: 3min at 94 ℃; another 30 PCR cycles were performed: 30s at 94 ℃, 30s at 59 ℃ and 1min at 72 ℃; finally 5min at 72 ℃.
And 3, step 3: indel genotype analysis
After the amplification is finished, products are subjected to capillary electrophoresis separation by using an ABI 3500 gene sequencer to obtain the genotype of the detected individual, and the genotype is explained by professional staff.
The experimental results of the invention are as follows:
the gene frequency difference and the disease risk OR value at the site between the control group and the SCD case group are shown in Table 1.
TABLE 1 Association between polymorphic site rs58928048 and SCD risk
Note: a CI confidence interval; OR ratio;acorrection according to age and gender
As shown in table 1, in the co-dominant model, individuals with genotype deletion/deletion were 0.45 times as high as individuals with insertion/insertion and individuals with genotype insertion/deletion were 0.69 times as high as individuals with insertion/insertion within the 95% confidence interval; in the recessive model, individuals with genotype of deletion/deletion are 0.56 times as high at risk of SCD as individuals with insertion/insertion or insertion/deletion; in the additive model, individuals carrying the deletion allele were 0.69-fold more at risk for SCD than individuals carrying the insertion allele.
The gene sequencing and SDS-PAGE gel electrophoresis profiles are shown in FIG. 1. Wherein FIG. 1A is an example of the sequencing result of a template strand, the six-base insertion deletion at rs58928048 of the coding strand is underlined; FIG. 1B is a schematic diagram of electrophoresis of 14 DNA samples from different individuals using the PCR amplification system of the present invention, wherein 2, 3, 11, and 12 are insertion homozygotes, 1, 4, 7, and 13 are deletion homozygotes, and the remainder are heterozygotes; ", DNA Marker.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Various other modifications and alterations will occur to those skilled in the art upon reading the foregoing description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.
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Claims (10)
1. An indel polymorphic molecular marker for detecting the risk of sudden cardiac death, characterized in that: the insertion deletion polymorphism molecule is marked as rs58928048 site of METTL16 gene, and the site is inserted or deleted with base TGTCTG.
2. The indel polymorphic molecular marker according to claim 1, characterized in that: the insertion of the base TGTCTG indicates a high risk of sudden cardiac death.
The application of the METTL16 gene insertion deletion polymorphism site in the preparation of a sudden cardiac death detection kit is characterized in that: the sudden cardiac death detection kit is used for detecting the genotype of the site rs58928048 of the METTL16 gene.
4. Use according to claim 3, characterized in that: the sudden cardiac death detection kit comprises a primer for amplifying the rs58928048 site of the METTL16 gene.
5. Use according to claim 4, characterized in that: the primers for amplifying the rs58928048 locus of the METTL16 gene comprise a sense primer and an antisense primer, wherein the nucleotide sequence of the sense primer is shown as SEQ ID NO.1, and the nucleotide sequence of the antisense primer is shown as SEQ ID NO. 2.
6. Use according to claim 3, characterized in that: the sudden cardiac death detection kit also comprises components for PCR amplification and capillary electrophoresis.
7. Application of a primer for amplifying the rs58928048 site of the METTL16 gene in preparation of a sudden cardiac death detection kit.
8. Use according to claim 7, characterized in that: the primers for amplifying the rs58928048 locus of the METTL16 gene comprise a sense primer and an antisense primer, wherein the nucleotide sequence of the sense primer is shown as SEQ ID NO.1, and the nucleotide sequence of the antisense primer is shown as SEQ ID NO. 2.
9. A sudden cardiac death detection kit based on METTL16 gene insertion deletion polymorphism is characterized in that: the sudden cardiac death detection kit is used for detecting the genotype of the gene rs58928048 site of the METTL16 gene and comprises a primer for amplifying the gene rs58928048 site of the METTL16 gene.
10. The sudden cardiac death detection kit of claim 9, wherein: the primer comprises a sense primer and an antisense primer, wherein the nucleotide sequence of the sense primer is shown as SEQ ID NO.1, and the nucleotide sequence of the antisense primer is shown as SEQ ID NO. 2.
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| CN115433776A (en) * | 2022-09-30 | 2022-12-06 | 中国医学科学院阜外医院 | Application of CCN3 in regulating vascular smooth muscle cell calcification |
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| CN112111572A (en) * | 2020-10-12 | 2020-12-22 | 苏州大学 | STAT5A gene insertion deletion polymorphism site-based sudden cardiac death susceptibility detection kit |
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| CN112111572A (en) * | 2020-10-12 | 2020-12-22 | 苏州大学 | STAT5A gene insertion deletion polymorphism site-based sudden cardiac death susceptibility detection kit |
| WO2022077539A1 (en) * | 2020-10-12 | 2022-04-21 | 苏州大学 | Susceptibility detection kit for sudden cardiac death based on insertion-deletion polymorphic site of stat5a gene |
Cited By (2)
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| CN115433776A (en) * | 2022-09-30 | 2022-12-06 | 中国医学科学院阜外医院 | Application of CCN3 in regulating vascular smooth muscle cell calcification |
| CN115433776B (en) * | 2022-09-30 | 2023-12-22 | 中国医学科学院阜外医院 | Application of CCN3 in regulating vascular smooth muscle cell calcification |
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