CN117587022A - Methylation marker and kit for predicting occurrence risk of drug-induced metabolic syndrome - Google Patents

Methylation marker and kit for predicting occurrence risk of drug-induced metabolic syndrome Download PDF

Info

Publication number
CN117587022A
CN117587022A CN202211000092.4A CN202211000092A CN117587022A CN 117587022 A CN117587022 A CN 117587022A CN 202211000092 A CN202211000092 A CN 202211000092A CN 117587022 A CN117587022 A CN 117587022A
Authority
CN
China
Prior art keywords
methylation
metabolic syndrome
drug
seq
primer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202211000092.4A
Other languages
Chinese (zh)
Inventor
秦胜营
周伟
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Jiaotong University
Original Assignee
Shanghai Jiaotong University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Jiaotong University filed Critical Shanghai Jiaotong University
Priority to CN202211000092.4A priority Critical patent/CN117587022A/en
Publication of CN117587022A publication Critical patent/CN117587022A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a methylation marker and a kit for predicting occurrence risk of drug-induced metabolic syndrome; the nucleotide sequence of the gene is shown as SEQ ID NO.1, and the methylation mark position is cg27243685, chr21:43642366. the method for detecting the methylation level of the nucleotide comprises the following steps: collecting blood samples of patients suffering from schizophrenia taking olanzapine and clozapine, and detecting methylation levels of the sites; analysis was performed at chr21 using the pyrophosphate sequencing methylation detection method: methylation level of 43642366 region site. The invention lays a foundation for researching the relation between the methylation level and the drug-induced metabolic syndrome, and provides a molecular target for predicting the risk of developing the drug-induced metabolic syndrome for patients with schizophrenia taking olanzapine and clozapine.

Description

Methylation marker and kit for predicting occurrence risk of drug-induced metabolic syndrome
Technical Field
The invention belongs to the technical field of molecular diagnosis and gene detection, and relates to a methylation site of drug-induced metabolic syndrome of a patient with schizophrenia and the correlation between the methylation site and the drug-induced metabolic syndrome; in particular to an identification and application method of the methylation level of a site cg27243685 on an ABCG1 gene in the technical field of gene detection, in particular to a methylation marker for predicting the occurrence risk of drug-induced metabolic syndrome and a kit thereof, and also relates to a method for predicting the occurrence risk of drug-induced metabolic syndrome based on the methylation level of the site and application thereof.
Background
Schizophrenia (SCZ) is a high disabling, severe mental disorder with a prevalence of about 1%. Statistics in 2022 by the world health organization indicate that there are over 2400 ten thousand schizophrenic patients worldwide. Antipsychotic therapy is currently the most prominent, most effective way to treat schizophrenia, however clinical practice has found that some schizophrenic patients experience an adverse effect of the metabolic syndrome caused by antipsychotics, namely the drug-induced metabolic syndrome (Atypical Antipsychotic Induced Metabolic Syndrome, API-Mets). The adverse reaction is one of the most common adverse reactions in antipsychotic drug treatment, the incidence rate is about 40 percent, and the adverse reaction is mainly represented by the phenomenon of aggregation of multiple cardiovascular risk factors such as hypertension, diabetes, dyslipidemia, central obesity and the like caused by long-term administration. Among the currently common second-generation antipsychotics, olanzapine and clozapine have the highest risk of causing metabolic syndrome. After short-term administration of antipsychotics, the first patients with schizophrenia can observe more obvious weight gain, and long-term administration can also lead to obesity, so that symptoms related to metabolic syndromes such as glucose tolerance reduction, insulin resistance, blood lipid disorder, diabetes and the like appear. The risk of suffering from coronary heart disease, myocardial infarction and apoplexy of patients with drug induced metabolic syndrome is 3-6 times higher than that of general population, and mortality is 4-6 times higher than that of general population.
Clinical practice shows that the occurrence of drug-induced metabolic syndrome has obvious individual difference and is influenced by various factors such as the genotype of the patient, the age, the type of drug administration, other environmental factors and the like. At present, a trial and error method is generally adopted for making and adjusting a medication scheme in clinic, however, the medication scheme is difficult to predict and manage the risk of the occurrence of the drug-induced metabolic syndrome. Thus, it is an urgent challenge to find effective molecular markers that predict the occurrence of drug-induced metabolic syndrome and take targeted preventive measures.
The drug-induced metabolic syndrome is affected by non-genetic factors such as sex, age, nutritional status, food intake and exercise of the patient, and both these non-genetic factors and antipsychotics themselves can affect the metabolic function of the body by altering the DNA methylation level of the individual. Many studies have reported that DNA methylation is associated with metabolic diseases such as obesity, diabetes, hypertension, etc. Studies in the general population have also indicated that alterations in DNA methylation play an important role in the development and progression of metabolic syndrome, and that DNA methylation molecular markers associated with metabolic syndrome have been found to be mainly involved in fatty acid oxidation, leptin and insulin signaling, cholesterol and phospholipid transport and other related genes, such as IGF2BP1, COL11A2, SOCS3, CPT1A and TOMM20, and the like. In the population of common metabolic syndrome and in the mouse model, researchers have found that local DNA methylation correlates with the occurrence of metabolic syndrome. Currently, there is little research on the discovery of methylation molecular markers for metabolic syndrome based on antipsychotics, and several methylation molecular markers found in the gene regions of cell adhesion protein family members such as CDH22, CCDC8, MAP3K13, etc. suggest that DNA methylation is correlated with the occurrence of drug-induced metabolic syndrome, and that the mechanism of drug-induced metabolic syndrome affected by DNA methylation may be different from that of normal population. Thus, systematic and comprehensive mining of DNA methylation signatures of the occurrence of drug-induced metabolic syndrome provides a new direction for predicting the risk of occurrence of drug-induced metabolic syndrome.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a methylation level detection and application of a methylation site cg27243685 on an ABCG1 gene related to occurrence of drug induced metabolic syndrome.
According to the invention, 141 cases of drug-induced metabolic syndrome samples and 127 cases of drug-induced metabolic syndrome control samples are collected, the difference of genome DNA methylation of case and control population is screened through a Infinium HumanMethylationEPIC (850 k) Beadchip methylation chip, and the methylation level of a methylation site cg27243685 on the ABCG1 gene is found to be highly related to the occurrence of drug-induced metabolic syndrome, and is not reported in the past. The methylation level of this site was significantly higher than that of the control population (cg 27243685, p=2.35×10 -6 Δβ=1.4%). FIG. 1 shows the methylation level difference at cg27243685 site between the case group (Mets) and the control group (Non-Mets).
To further verify the relationship of this site methylation change to the drug-induced metabolic syndrome, the inventors further performed methylation editing of this site in HepG2 cells using CRISPR/dCas9 technology. As a result, the TG and TC contents of the cells are obviously reduced compared with a control after the methylation level of the site is artificially reduced; conversely, after increasing the methylation level of this site, the TG and TC content of the cell increased significantly. Fig. 2 is a graph showing changes in Triglyceride (TG) and Total Cholesterol (TC) levels of HerpG2 cells treated with olanzapine or empty vector 24 hours after CRISPR-based methylation modification.
Therefore, the invention provides the value that the methylation level of the cg27243685 locus has the risk of occurrence of the drug-induced metabolic syndrome for the first time, lays a foundation for researching the relation between the methylation level of the locus and the drug-induced metabolic syndrome, provides a theoretical basis for early intervention of the drug-induced metabolic syndrome, and also provides an important clue for exploring the pathogenesis of the drug-induced metabolic syndrome.
The aim of the invention is achieved by the following technical scheme,
in a first aspect, the invention provides an isolated nucleotide comprising a methylated region, said nucleotide having a sequence as set forth in SEQ ID NO.1, said methylated region being located at chr21:43642366 (hg 19 reference genome).
In a second aspect, the invention provides an isolated nucleotide having a sequence as set forth in SEQ ID NO:1, and bit 4100 is C.
In a third aspect, the present invention provides the use of an isolated nucleotide containing a methylation region as a molecular marker of the drug-induced metabolic syndrome, or in the preparation of a reagent or kit for assisting in the prevention of the drug-induced metabolic syndrome or in assisting in the detection of the risk of occurrence of the drug-induced metabolic syndrome.
As one embodiment of the invention, the reagents include primers, probes or chips.
As an embodiment of the invention, the kit contains one or more reagents selected from the group consisting of:
(a) Specific primers for detecting the methylation level of said region;
(b) A specific probe for detecting the methylation level of said region;
(c) A chip for detecting the methylation level of said region.
As one embodiment of the invention, methylation site cg27243685 is located at chr21:43642366 (hg 19 reference genome), the primers are:
forward primer 5'-CCTATCAATCTAAAATAAACCAACTCTCT-3' SEQ ID No.2,
reverse primer 5'-CTAAAAACACATCCTCC-3' SEQ ID No.3;
the primer 5'-GTGGGGGAAGGTTAAAGATAAT-3' SEQ ID No.4 was extended.
In a fourth aspect, the present invention provides a kit for aiding in the screening or detection of a risk of a drug-induced metabolic syndrome, the kit comprising: according to the sequence set forth in SEQ ID NO:1, and the methylation site cg27243685 of the sequence shown in 1.
As one embodiment of the present invention, the site is located at position 4100, cg27243685 is C, and the primer pair is:
forward primer 5'-CCTATCAATCTAAAATAAACCAACTCTCT-3' SEQ ID No.2,
reverse primer 5'-CTAAAAACACATCCTCC-3' SEQ ID No.3;
the primer 5'-GTGGGGGAAGGTTAAAGATAAT-3' SEQ ID No.4 was extended.
As one embodiment of the present invention, the primer pair is used to detect its methylation level; substituting the site methylation level into the formula yields the risk of the patient developing the primary metabolic syndrome:
the calculation formula is as follows:
y= -21.62+24.34 cg27243685 (methylation level) +2.84 family history (1 no, 2 is, 0 is unknown) + -0.035 course (month);
metabolic syndrome risk p=exp (Y)/(1+exp (Y)).
As an embodiment of the present invention, the kit further comprises: sample DNA/mRNA extraction reagent and amplification reaction system; the total volume of the amplification reaction system was 25. Mu.l, wherein,
10×CoralLoad Concentrate 2.5μl，
2×PyroMark PCR Master Mix 12.5μl，
5×Q solution 5μl，
bisulfite treated genomic DNA 1. Mu.g,
10uM forward primer 0.5. Mu.l,
extension primer of 10uM 0.5. Mu.l,
the double distilled water was made up to 25 μl.
Amplification reaction conditions: pre-denaturation at 95℃for 15min; 45X (94 ℃ C. 30s;56 ℃ C. 30s,72 ℃ C. 30 s); and at 72℃for 10mins.
In a fifth aspect, the present invention provides a method for using the kit for non-diagnostic purposes, comprising the steps of:
s1, extracting DNA of a blood sample to be detected and carrying out bisulfite conversion treatment;
s2, using the genomic DNA treated by S1 as a template, using chr21 according to the sequence shown in SEQ ID No. 1: primer pairs designed in 43642366 area utilize forward primer SEQ ID No.2 and carry out hot start PCR amplification in the presence of biotin marked extension primer SEQ ID No.4 to obtain PCR amplification products;
s3, detecting chr21 in the sample by adopting a pyrophosphoric acid sequencing methylation detection method: 43642366 methylation level at the site.
As an embodiment of the present invention, step S3 includes the steps of: methylation level detection was performed on the site cg27243685 of the samples taken using a pyrophosphorymeter.
As one embodiment of the present invention, in step S2, the length of the amplification product is 50-200bp.
As one embodiment of the present invention, in step S2, the primer pair has a length of 15-25bp.
As one embodiment of the present invention, in step S2, the total volume of the amplification reaction system for the amplification is 25. Mu.l, wherein,
10×CoralLoad Concentrate 2.5μl，
2×PyroMark PCR Master Mix 12.5μl，
bisulfite treated genomic DNA 1. Mu.g,
5×Q solution 5μl，
10uM forward primer 0.5. Mu.l,
extension primer of 10uM 0.5. Mu.l,
the double distilled water is filled up to 25 mu l;
amplification reaction conditions: pre-denaturation at 95℃for 15min; 45X (94 ℃ C. 30s;56 ℃ C. 30s,72 ℃ C. 30 s); and at 72℃for 10mins.
As one embodiment of the present invention, biotin-labeled PCR products were mixed with avidin-labeled microbeads in a system of 80. Mu.L:
PCR product 5. Mu.L
PyroMark Binding 34μL
Streptavidin Sepharose High Performance 1μL
Separating and purifying the biotin-marked single-stranded DNA by using a PyroMark Q24 Advanced vacuum workstation;
binding the single stranded DNA template to the reverse primer in a system of 20. Mu.L comprising:
reverse primer 0.75. Mu. Mol/L
PyroMark Annealing Buffer 19.25μL
Annealing at 80 ℃ for 5min, and finally sequencing on a pyroMark Q24 Advanced pyrosequencer; the data were analyzed using PyroMark Q24 Advanced software.
In a sixth aspect, the invention also provides a special primer pair for detecting SNP loci containing new variation loci, wherein the sequence of the nucleotides is shown as SEQ ID NO. 1;
the locus is positioned at 4100 th site, cg27243685 is C, and the primer pair is:
forward primer 5'-CCTATCAATCTAAAATAAACCAACTCTCT-3' SEQ ID No.2,
reverse primer 5'-CTAAAAACACATCCTCC-3' SEQ ID No.3;
the primer 5'-GTGGGGGAAGGTTAAAGATAAT-3' SEQ ID No.4 was extended.
Compared with the prior art, the invention has the following beneficial effects:
the invention lays a foundation for researching the relation between the cg27243685 methylation level on the ABCG1 gene and the drug-induced metabolic syndrome, and provides a molecular target for predicting the occurrence risk of the drug-induced metabolic syndrome.
Drawings
Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:
FIG. 1 shows the methylation level difference at cg27243685 site between the case group (Mets) and the control group (Non-Mets);
fig. 2 is a graph showing changes in Triglyceride (TG) and Total Cholesterol (TC) levels of HerpG2 cells treated with olanzapine or empty vector 24 hours after CRISPR-based methylation modification.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address specific conditions in the following examples, is generally followed by conventional conditions, such as molecular cloning by Sambrook et al: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.
Example 1
The invention relates to a method for detecting the methylation level of cg27243685 on the ABCG1 gene, and also relates to related isolated nucleotides and primers. The invention discovers that methylation site cg27243685 on the ABCG1 gene can be used as a molecular marker of drug-induced metabolic syndrome through genome methylation association analysis, the specific information of the gene is the ABCG1 gene, and the position is chr21:43,639,267-43,717,354, and the full length is 78,088bp.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available products unless otherwise indicated. Specifically, the experiment uses Infinium MethylationEPIC Kit for whole genome methylation detection. The quality evaluation and processing are carried out on the off-machine data, and then the high-quality methylation data are analyzed through a minfi analysis flow. The method comprises the following specific steps:
a) Extraction of blood genomic DNA: subjects meeting the group entry conditions were fasted to collect 5mL whole blood in EDTA anticoagulation blood collection tubes in the morning. The extraction of blood genome DNA adopts QIAamp DNA Blood Mini Kit kit.
B) Detection of methylation sites: the methylation chip Infinium MethylationEPIC BeadChip (850 k chip for short) of Illumina company is selected for detecting methylation sites in the whole genome range. The experiment first uses EZ DNA Methylation Kit kit to bisulphite convert the DNA. The experimental procedure was as follows:
1) 750 μl of ddH2O and 210 μl of M-division Buffer were added to CT Conversion Reagent to dissolve the reagent;
2) Shaking the reagent at room temperature for 10min, taking care that the reagent needs to be protected from light;
3) 200-500ng of DNA is used for bisulphite conversion, 5 mu L of M-partition Buffer is added into a DNA sample, the total volume is supplemented to 50 mu L by ddH2O, and a pipetting gun is used for gentle blowing and mixing;
4) Placing the sample in a water bath at 37 ℃ for 15min;
5) After the incubation, 100 μl of the CT Conversion Reagent prepared above was added to each DNA sample and mixed well, and the liquid on the tube cap was centrifuged off briefly;
6) Placing the treated DNA into a dark environment and incubating for 12-16 hours at 50 ℃;
7) After the incubation is finished, the sample is placed on ice (0-4 ℃) for 10min;
8) The kit is provided with a filter Column Tube (Zymo-Spin IC Column) and a Collection Tube (Collection Tube), wherein the filter Column Tube is placed in the Collection Tube for purifying DNA, and 400 mu L M-Binding Buffer is sucked into the filter Column Tube;
9) Adding the DNA sample incubated in the front into the treated filtering column tube, covering the tube cover and uniformly mixing;
10 Maximum centrifugation speed (> 10,000 g) for 30 seconds;
11 100 mu L M-Wash Buffer into the filter column tube, maximum centrifugation speed (> 10,000 g) for 30 seconds;
12 200. Mu.L of M-Des mu. Lphonation Buffer was added to each tube, left at room temperature for 20 minutes, and after incubation, centrifuged at 10000 rpm for 30 seconds;
13 Adding 200 mu L M-Wash Buffer into each tube, centrifuging for 30 seconds in a centrifuge at 10,000 rpm, adding 200 mu L M-Wash Buffer, and centrifuging for 30 seconds;
14 The filter cartridge was placed in a new 1.5mL tube, 10. Mu.l ddH2O was added to each tube to dissolve DNA, and then the transformed DNA was obtained by centrifugation at maximum speed for 30 seconds, to be used in the next step.
The bisulfite converted DNA can be analyzed using a pyrophosphate sequencer.
C) By performing correlation analysis on methylation data, and re-screening out sites related to the significance of the drug-induced metabolic syndrome, the cg27243685 of the ABCG1 gene is found to have higher methylation level in the case group than the control group (see figure 1).
The invention adopts the pyrophosphoric acid sequencing technology to detect cg27243685 locus (chr 21: 43642366) of the ABCG1 gene, and finds that the methylation level of the locus is higher in an experimental group. cg27243685 site at chromosomal position: chr21:43642366 (hg 19). The specific nucleotide fragment sequence of the ABCG1 gene is shown as SEQ ID NO.1, wherein the cg27243685 locus is positioned at 4100 th site of the sequence.
The invention designs a related primer pair aiming at the methylation site.
Specifically, the invention is characterized in that the gene chr21 of ABCG 1: primers are designed at the upstream and downstream of 43642366, and the length of amplified products is 50-200bp. The DNA samples were then analyzed using a pyrosequencer. Analysis revealed that the cg27243685 site had a higher methylation level in the experimental group than in the control group.
The present invention has a number of analytical techniques available for detecting the methylation level of said site in the ABCG1 gene. These techniques include (but are not limited to): sulfite whole genome sequencing technology and hybridization sequencing; enzymatic mismatch cleavage, heteroduplex analysis, dot hybridization, oligonucleotide arrays (DNA chips), mini-Sequencing, molecular beacons, etc., ELISA, methylation-specific PCR, methyl light, bisulfite cloning & Sequencing, guide Positioning Sequencing, third generation Sequencing, denaturing High Performance Liquid Chromatography (DHPLC). In another aspect, the detection method of the invention is used to evaluate the relationship between the methylation level of the cg27243685 locus of the ABCG1 gene of the drug induced metabolic syndrome and the risk of the drug induced metabolic syndrome. The test sample used in the present invention is a sample of DNA extracted from blood. For the ABCG1 gene, any sample containing the region of the ABCG1 gene, such as blood, etc., may be used.
By "isolated or purified" is meant that at the genomic DNA level, the gene sequence of each new mutation site is amplified using a primer pair and the amplified product is purified using a purification kit.
The following is a specific implementation procedure of the scheme of the invention:
samples used in this example were taken from 260 metabolic syndrome samples (age 18-60 years, table 1) from a multicenter collection network established in this laboratory, all participants agreed to be signed by family members on formal informed consent. The ABCG1 genomic sequence to which this example relates is obtained from the UCSC database and is set forth in SEQ ID NO:1.
primer design: primer-BLAST software is adopted, and the UCSC database ABCG1 gene sequence is used as a template to design a Primer, and the Primer is synthesized by Invitrogen company.
Primer information:
forward primer 5'-CCTATCAATCTAAAATAAACCAACTCTCT-3' SEQ ID No.2,
reverse primer 5'-CTAAAAACACATCCTCC-3' SEQ ID No.3;
extension primer 5'-GTGGGGGAAGGTTAAAGATAAT-3' seq ID No.4;
PCR amplification conditions: (reagents for the system are supplied by ABI, also available from published sources), the total volume of the reaction system is 25. Mu.l, wherein:
10×CoralLoad Concentrate 2.5μl，
2×PyroMark PCR Master Mix 12.5μl，
5×Q solution 5μl，
bisulfite treated genomic DNA 1. Mu.g,
10uM forward primer 0.5. Mu.l,
extension primer of 10uM 0.5. Mu.l,
the double distilled water is filled up to 25 mu l;
amplification reaction conditions: pre-denaturation at 95℃for 15min; 45X (94 ℃ C. 30s;56 ℃ C. 30s,72 ℃ C. 30 s); and at 72℃for 10mins.
The PCR reaction was performed in 96-well plates, and the samples were read out in a pyrosequencer to determine the methylation level of the cg27243685 locus of the ABCG1 gene (Table 2, FIG. 1).
TABLE 1
TABLE 2
D) Illustrating how application of the formula evaluates a patient's risk of drug induced metabolic syndrome
Patient a was selected here and known to be diagnosed with schizophrenia for 2 years, without family history, with olanzapine treatment for one year, blood glucose 6.19, blood pressure 150/90, triglycerides 2.2, hdl-C1.4, and to be diagnosed with metabolic syndrome. Cg27243685 methylation level was 0.92 as detected by pyrosequencing.
The cg27243685 methylation level and other information for patient a were substituted into the formula:
y= -21.62+24.34 cg27243685 (methylation level) +2.84 family history (1 no, 2 is, 0 is unknown) + -0.035 course (month);
log [ p/(1-p) ] = -21.62+24.34 x 0.92+2.84 x 1+ -0.035 x 24 = 2.77
The risk of metabolic syndrome p=exp (Y)/(1+exp (Y))=0.941
I.e. patient a has a 94.1% risk of developing the primary metabolic syndrome.
In conclusion, the method lays a foundation for researching the relation between the methylation level of the ABCG1 gene and the drug induced metabolic syndrome, and provides a molecular target for predicting the risk of the drug induced metabolic syndrome.
The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.

Claims (10)

1. An isolated nucleotide comprising a methylation region having the sequence set forth in SEQ ID No.1, said methylation region being at chr21:43642366.
2. use of an isolated nucleotide containing a methylation region according to claim 1 as a molecular marker for drug-induced metabolic syndrome, or for the preparation of a reagent or kit for the assisted control of drug-induced metabolic syndrome or for the assisted detection of the risk of occurrence of drug-induced metabolic syndrome.
3. The use according to claim 2, wherein the reagent comprises a primer, a probe or a chip.
4. The use according to claim 2, wherein the kit contains one or more reagents selected from the group consisting of:
(a) Specific primers for detecting the methylation level of said region;
(b) A specific probe for detecting the methylation level of said region;
(c) A chip for detecting the methylation level of said region.
5. The use according to claim 3 or 4, wherein methylation site cg27243685 is located at chr21:43642366, the primer is:
forward primer 5'-CCTATCAATCTAAAATAAACCAACTCTCT-3' SEQ ID No.2,
reverse primer 5'-CTAAAAACACATCCTCC-3' SEQ ID No.3;
the primer 5'-GTGGGGGAAGGTTAAAGATAAT-3' SEQ ID No.4 was extended.
6. A kit for aiding in screening or detecting a risk of developing a drug-induced metabolic syndrome, the kit comprising: according to chr21 of the sequence shown in SEQ ID No. 1:43642366 region.
7. The kit of claim 6, wherein the primer pair is used to detect the methylation level thereof; substituting the site methylation level into the formula yields the risk of the patient developing the primary metabolic syndrome:
the calculation formula is as follows:
y= -21.62+24.34×cg27243685 (methylation level) +2.84×family history (1 no, 2 is, 0 is unknown) + -0.035×course (month);
metabolic syndrome risk p=exp (Y)/(1+exp (Y)).
8. A method of using the kit of claim 6 for non-diagnostic purposes, comprising the steps of:
s1, extracting DNA of a blood sample to be detected and carrying out bisulfite conversion treatment;
s2, using the genomic DNA treated by S1 as a template, using chr21 according to the sequence shown in SEQ ID No. 1:43642366 region designed primer SEQ ID No.2, and performing hot start PCR amplification in the presence of biotin-labeled extension primer SEQ ID No.4 to obtain PCR amplification product;
s3, detecting chr21 in the sample by adopting a pyrophosphoric acid sequencing methylation detection method: 43642366 methylation level at the site.
9. The method according to claim 8, wherein the total volume of the amplified amplification reaction system is 25. Mu.l, wherein,
amplification reaction conditions: pre-denaturation at 95℃for 15min; 45X (94 ℃ C. 30s;56 ℃ C. 30s,72 ℃ C. 30 s); and at 72℃for 10mins.
10. The method of claim 8, wherein the biotin-labeled PCR product is mixed with avidin-labeled microbeads in an amount of 80. Mu.L:
the PCR product was used in an amount of 5. Mu.L,
PyroMark Binding 34μL，
Streptavidin Sepharose High Performance 1μL，
separating and purifying the biotin-marked single-stranded DNA by using a PyroMark Q24 Advanced vacuum workstation;
the single stranded DNA template was combined with the reverse primer SEQ ID No.3 in a system of 20. Mu.L, comprising:
reverse primer 0.75. Mu. Mol/L
PyroMark Annealing Buffer 19.25μL；
Annealing at 80 ℃ for 5min, and finally sequencing on a pyroMark Q24 Advanced pyrosequencer; the data were analyzed using PyroMark Q24 Advanced software.
CN202211000092.4A 2022-08-19 2022-08-19 Methylation marker and kit for predicting occurrence risk of drug-induced metabolic syndrome Pending CN117587022A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202211000092.4A CN117587022A (en) 2022-08-19 2022-08-19 Methylation marker and kit for predicting occurrence risk of drug-induced metabolic syndrome

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202211000092.4A CN117587022A (en) 2022-08-19 2022-08-19 Methylation marker and kit for predicting occurrence risk of drug-induced metabolic syndrome

Publications (1)

Publication Number Publication Date
CN117587022A true CN117587022A (en) 2024-02-23

Family

ID=89920703

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202211000092.4A Pending CN117587022A (en) 2022-08-19 2022-08-19 Methylation marker and kit for predicting occurrence risk of drug-induced metabolic syndrome

Country Status (1)

Country Link
CN (1) CN117587022A (en)

Similar Documents

Publication Publication Date Title
AU2009246199C1 (en) Genetic markers for weight management and methods of use thereof
JP5864431B2 (en) Genetic markers for weight management and uses thereof
US20150159220A1 (en) Methods for predicting and detecting cancer risk
CN107058538B (en) Primer composition, kit composed of primer composition and application of kit
WO2013043554A1 (en) Methods for creating recommended dietary regime
KR20150067151A (en) Method for screening cancer
CN102758010A (en) Combination of multiple genetic single nucleotide polymorphisms and environmental factors related to coronary heart disease and application of combination
CN110699440A (en) Primer and method for detecting SNP (single nucleotide polymorphism) locus of gene related to metformin personalized medicine
KR101979633B1 (en) SNP markers for metabolic syndrome and use thereof
CN105431552B (en) Use of multiomic markers for predicting diabetes
CN106337081B (en) Correlation of SNP locus rs1054135 of FABP4 gene and triple negative breast cancer prognosis
CN117587022A (en) Methylation marker and kit for predicting occurrence risk of drug-induced metabolic syndrome
KR101992952B1 (en) Composition, kit for predicting the risk of developing cardiovascular disease related to Cholesterol efflux capacity, and method using the same
WO2009055596A2 (en) Methods of using genetic variants to diagnose and predict metabolic syndrome and associated traits
CN112553326B (en) Primer, probe and fluorescent PCR kit for detecting neonatal jaundice UGT1A1 genotype and GST gene deletion type
CN115820845B (en) Polyadenylation functional site marker related to colorectal cancer diagnosis and application thereof
CN111235255A (en) Method for distinguishing individual medication of nitrendipine by using primer composition through mass spectrometry
CN117802223B (en) Based on 2-ΔΔCTKit for detecting copy numbers of SMN1 gene and SMN2 gene by using method and application method of kit
CN113943791B (en) Application of UC002yug.2-rs2246640 as female obesity biomarker
CN110656165B (en) Gene SNP detection kit for determining related therapeutic effect of clopidogrel medication
KR20230101467A (en) Single-nucleotide polymorphic biomarkers for predicting obesity risk according to eating habits and use thereof
CN113943797A (en) Application of hypertensive inflammation response gene as molecular marker in preparation of Xinjiang Kazakh family essential hypertension diagnostic reagent
CN118028454A (en) Primer composition for detecting endocrine-type personalized medicine genes and detection method thereof
CN116814778A (en) DNA methylation markers, methods and kits for colorectal and/or adenoma diagnosis
CN113943814A (en) Primer probe composition, kit and method for early screening of liver cancer

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination