CN116240226A - LDL-C related gene and in vitro detection reagent, preparation or kit and application thereof - Google Patents
LDL-C related gene and in vitro detection reagent, preparation or kit and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of genes related to dyslipidemia in bioengineering, in particular to a gene related to LDL-C, a reagent, a preparation or a kit for in-vitro detection and application thereof, and the gene related to LDL-C, namely an rs35605 locus mutated ABCC1 gene. The invention discloses a low-density lipoprotein cholesterol related gene, an in-vitro detection system and application thereof, wherein the in-vitro detection system can be used for detecting, preventing, diagnosing or treating hypercholesterolemia besides the polymorphism of the low-density lipoprotein cholesterol related gene in vitro, thereby reducing the incidence rate of the hypercholesterolemia and having great significance for preventing and treating dyslipidemia related diseases.
Description
Technical Field
The invention relates to the technical field of genes related to dyslipidemia in bioengineering, in particular to genes related to LDL-C, and a reagent, a preparation or a kit for in-vitro detection and application thereof.
Background
Hypercholesterolemia is one of the major risk factors for cardiovascular disease, and particularly elevated low-density lipoprotein cholesterol (LDL-C for short) is one of the most risk factors for the development and progression of atherosclerotic cardiovascular disease, and both the FOURIER and ODYSSEY studies have shown that maximal lowering of LDL-C levels can bring about maximal cardiovascular benefit. Although the diagnosis and treatment of hypercholesterolemia have been greatly developed in recent years, new chemical and biological agents for lipid-lowering therapy are emerging, and lipid-lowering therapy greatly improves the prognosis of hypercholesterolemia patients, the etiology and pathogenesis thereof are still unclear. Since the discovery of Low Density Lipoprotein Receptor (LDLR) gene mutation by scientists in the last 70 th century, familial Hypercholesterolemia (FH) and early cardiovascular diseases have been greatly improved, and it has been recognized that hypercholesterolemia is not a single factor disease but a complex disease determined by various endogenous and exogenous factors, and besides conventional risk factors such as hyperlipidemia, hypertension, diabetes, smoking and other environmental factors, genetic factors play an important role in the development and progress of hypercholesterolemia. Currently, 157 genes associated with lipid levels have been established by whole genome association studies (GWAS for short). In the early stage, a novel gene LIMA1 affecting intestinal cholesterol uptake is screened out through a biological analysis by adopting a whole exon sequencing (WES for short) combined with family research; the induced degradation sub IDOL gene of LDLR is discovered and verified.
Cholesterol metabolism is mainly divided into four ways of synthesis, ingestion, excretion and esterification. Wherein the ATP-binding cassette transporter superfamily plays a key role in the cholesterol outer row. GWAS, WES, basic research, etc. have found and confirmed that ATP-binding cassette transporter superfamily members such as ABCA1, ABCG5, ABCG8, etc. are associated with cholesterol efflux and that the extent of atherosclerosis in knockout animals is significantly increased. ABCA1 and ABCG1 are widely expressed in systemic tissues, wherein ABCA1 and ABCG1, which are highly expressed in macrophages, affect cholesterol metabolism by coordinating the mechanisms of actions such as efflux and reverse transport degradation of cholesterol, and these two gene deletions lead to massive deposition of lipids in macrophages, thus exacerbating atherosclerosis. ABCG5 and ABCG8 are expressed almost exclusively on the apical surface of hepatocytes and intestinal cells where they function as heterodimers, mediating excretion of neutral sterols (including plant sterols and cholesterol) into bile and intestinal lumen, respectively. Single or combined mutations in ABCG5 or ABCG8 can lead to sitosterolemia.
Meanwhile, the ABCC1 gene is positioned at 16p13.11, has 35 exons, is a protein coding gene, and the coded protein belongs to ATP binding cassette transport protein superfamily members, is widely expressed in human body whole body tissues, and has high expression in mononuclear macrophages as the ABCA1 which is proved to be involved in cholesterol efflux. ABCC1 is also involved in transmembrane material transport across cell membranes, such as the transport of steroid hormones and bile salts, and has also been reported to be involved in fatty acid metabolism. Thus, research analysis of the ABCC1 gene is also more helpful in the discovery of new pathogenic genes for hypercholesterolemia.
Disclosure of Invention
The invention provides a gene related to LDL-C, and a reagent, a preparation or a kit for in vitro detection and application thereof, overcomes the defects of the prior art, is more beneficial to finding a new pathogenic related gene of hypercholesterolemia, and has great significance for preventing and treating diseases related to dyslipidemia.
One of the technical schemes of the invention is realized by the following measures: the nucleotide sequence of the gene related to LDL-C, namely the ABCC1 gene with rs35605 site mutation and the ABCC1 gene with rs35605 site mutation is shown in a sequence table SEQ ID NO. 1.
The second technical scheme of the invention is realized by the following measures: a reagent for detecting in vitro a gene related to LDL-C for detecting mutation at the rs35605 locus of the ABCC1 gene, comprising the following primers:
an upstream primer: 5 'ACTGCTCTACAGGATGACTCACCT3'
A downstream primer: 5 'CCTCCTCGGAAGTCGCCACAGC 3'.
The following is a further optimization and/or improvement of the second technical scheme of the invention:
the reagent is a reagent for combining polymerase chain reaction and restriction fragment length polymorphism analysis.
The third technical scheme of the invention is realized by the following measures: a preparation or kit comprising reagents for in vitro detection of genes associated with LDL-C, the preparation or kit comprising reagents, PCR amplification enzymes and corresponding buffers, pspPI restriction enzymes and corresponding cleavage maps.
The above reagents include the following primers:
an upstream primer: 5 'ACTGCTCTACAGGATGACTCACCT3'
A downstream primer: 5 'CCTCCTCGGAAGTCGCCACAGC 3'.
The fourth technical scheme of the invention is realized by the following measures: use of a reagent for in vitro detection of a gene associated with LDL-C in the preparation of a formulation or kit for in vitro detection of a gene associated with LDL-C.
The preparation or the kit comprises reagents, PCR (polymerase chain reaction) amplification enzyme, corresponding buffer solution, pspPI restriction enzyme and corresponding enzyme digestion patterns.
The above reagents include the following primers:
an upstream primer: 5 'ACTGCTCTACAGGATGACTCACCT3'
A downstream primer: 5 'CCTCCTCGGAAGTCGCCACAGC 3'.
The invention discloses a low-density lipoprotein cholesterol related gene, an in-vitro detection system and application thereof, wherein the in-vitro detection system can be used for detecting, preventing, diagnosing or treating hypercholesterolemia besides the polymorphism of the low-density lipoprotein cholesterol related gene in vitro, thereby reducing the incidence rate of the hypercholesterolemia and having great significance for preventing and treating dyslipidemia related diseases.
Drawings
FIG. 1 is a cleavage map of the ABCC1 rs35605 locus of the invention.
Detailed Description
The present invention is not limited by the following examples, and specific embodiments can be determined according to the technical scheme and practical situations of the present invention. The various chemical reagents and chemicals mentioned in the present invention are all commonly known in the art unless specifically stated otherwise.
The invention is further described below with reference to examples:
example 1: the nucleotide sequence of the gene related to LDL-C, namely the ABCC1 gene with rs35605 site mutation and the ABCC1 gene with rs35605 site mutation is shown in a sequence table SEQ ID NO. 1.
The sample to be tested containing the ABCC1 gene according to the present invention may be obtained from cells from a tester, such as cells from blood, urine, saliva, gastric juice, hair, biopsy and autopsy material, preferably from blood.
Example 2: the reagent for detecting the gene related to LDL-C in vitro is used for detecting the mutation of the rs35605 locus of the ABCC1 gene, and comprises the following primers:
an upstream primer: 5 'ACTGCTCTACAGGATGACTCACCT3'
A downstream primer: 5 'CCTCCTCGGAAGTCGCCACAGC 3'.
Example 3: as an optimization of example 2 above, the reagent is a reagent combining polymerase chain reaction with restriction fragment length polymorphism analysis.
In the present invention, the term "gene polymorphism" refers to a difference in nucleotide sequence of each individual gene in a population. As known to those skilled in the art, the polymorphic site according to the present invention is a Single Nucleotide Polymorphism (SNP) site, i.e., a single nucleotide in the genomic sequence is changed; the nucleotide sequence differences may be present at the DNA level or at the RNA level, so that the hypercholesterolemia-related gene of the present invention may be present at the DNA level, at the RNA level, preferably at the DNA level, more preferably at the genomic DNA.
Example 4: the preparation or the kit containing the reagent for detecting the gene related to LDL-C in vitro comprises the reagent, PCR amplification enzyme, corresponding buffer solution, pspPI restriction enzyme and corresponding enzyme digestion map.
Example 5: the application of the reagent for in vitro detection of genes related to LDL-C in preparation of a preparation or a kit for in vitro detection of genes related to LDL-C.
Example 6: as an optimization of example 5 above, the preparation or kit comprises PCR amplification enzymes and corresponding buffers.
There are two strategies for the current study of complex trait diseases, linkage analysis methods and case control studies. Because hypercholesterolemia is a multifactorial disease, which is a result of both genetic and environmental factors, linkage analysis is difficult. From a genetic perspective, hypercholesterolemia belongs to a complex genetic trait disease. Among the factors contributing to the occurrence of the disease, in addition to environmental factors, the total number of genes contributing is estimated to be as many as hundreds, and there are interactions between the individual genes and between the genes and the environment. The occurrence of hypercholesterolemia has a certain family aggregation, but more so some sporadic cases. The present invention uses case control studies. The case control study was to compare the frequency of an allele in two groups of people (high and low LDL-C levels) with random selection.
Example 7:
(1) Selection of study objects
Inclusion and exclusion criteria: the study subjects were selected from 18-24-year-old universities who participated in routine physical examination at Xinjiang medical university at 2018, 9-2018, 12, and signed informed consent prior to inclusion in the study. The medical history of heart insufficiency, liver and kidney insufficiency and other diseases can be eliminated, and researchers cannot cooperate with the medical history. With reference to the latest European and American guidelines for blood lipid management, it was suggested that 2824 subjects who were finally studied were divided into 1405 cases of the high LDL-C group (LDL-C value. Gtoreq.2.5 mmol/L) and 1419 cases of the low LDL-C group (LDL-C value < 2.5 mmol/L), both groups being Han population, with the LDL-C value of 100mg/dL, i.e., 2.5mmol/L, as a critical value.
(2) The polymorphism of the rs35605 locus of the hypercholesterolemia related gene is detected by combining polymerase chain reaction and restriction fragment length polymorphism analysis
The method comprises the following steps: PCR reaction system (50 μl): 50ng of genomic template DNA (from a blood sample, DNA in white blood cells is extracted by phenol-atmosphere imitation or salting-out method according to a conventional method), 2*powder Taq PCR master mix 25ul, 21ul of deionized water, 1ul of each of upper and lower primers, and PCR reaction on a 96-well PCR automatic cycler, and PCR cycle parameters: pre-denaturation at 95 ℃ for 2 min; 94℃for 20 seconds, 65.0℃for 30 seconds and 72℃for 1.5 minutes; after 35 weeks of circulation, the extension was carried out at 72℃for 2 minutes; finally, storing at 4 ℃ or observing on a machine. The primers were as follows:
an upstream primer: 5 'ACTGCTCTACAGGATGACTCACCT3'.
A downstream primer: 5 'CCTCCTCGGAAGTCGCCACAGC 3'.
The length of the amplified product is 245 base, and PspPI restriction enzyme is selected for enzyme digestion. The enzyme cutting conditions are referred to the manufacturer's manual. The length was then observed by UV-transilluminator after electrophoresis on a 1% agarose gel.
Results: as shown in fig. 1. As can be seen from FIG. 1, when only one fragment with a length of 245 bases is provided, it is indicated that the allele at the rs35605 locus of the individual to be tested is T; when a PCR product with the length of 245 bases is cut to generate fragments with the lengths of 177 and 68 bases, the allele of the rs35605 locus of the individual to be detected is indicated as C; when there are three fragments of 245, 177 and 68 bases in length, it is indicated that the alleles at the rs35605 locus of the individual to be tested are T and C. Therefore, lane 1 shows that the genotype of the rs35605 locus of the tested individual is T/T homozygote, lane 3 shows that the genotype of the rs35605 locus of the tested individual is C/C homozygote, and lane 5 shows that the genotype of the rs35605 locus of the tested individual is T/C heterozygote.
(3) Association study of polymorphic sites with hypercholesterolemia
To further investigate the relationship between ABCC1 gene and high LDL-C from the genetic point of view and provide evidence of genetic epidemiology for the same kind of research existing at home and abroad, the distribution difference of rs35605 locus in the population of high LDL-C and low LDL-C was analyzed by detecting polymorphism at rs35605 locus in vitro in the collected 1405 cases of high LDL-C and 1419 cases of low LDL-C controls in the sample from the individual to be tested by using the internationally prevailing genotyping method combining polymerase chain reaction with restriction fragment length polymorphism analysis.
Hardy-Weinberg equilibrium test was first used. Hardy-Weinberg equilibrium is a concept of population genetics: the genetic information of the group is called as the gene library of the group, and the genetic characteristics of the local area and the ethnic group are directly reflected. The expression form gene frequency and genotype frequency of the genetic information are kept unchanged under the condition of no mutation, migration and genetic drift, namely Hardy-Weinberg equilibrium. Thus, the genetic polymorphism and the genetic stability are provided.
Analysis of the genotyping results revealed that the genotyping results met Hardy-Weinberg equilibrium, so experimental errors could be eliminated, the genotyping results were more reliable, and the analysis results are shown in Table 2.
Conclusion: the single factor analysis shows that the distribution of three genotypes of the polymorphic locus rs35605 in the high LDL-C group and the low LDL-C group is obviously different (P is less than 0.05), and the T/C obvious recessive genetic pattern is further analyzed, so that the distribution frequency of C alleles and C/C, T/C genotypes is obviously higher than that of the low LDL-C group in the high LDL-C group (the distribution frequency of the alleles C is 0.731 in the high LDL-C group, 0.640 in the low LDL-C group and 51.3 in the high LDL-C group and 45.9 in the low LDL-C group); distribution frequency of T/C genotypes: the high LDL-C group was 43.5 and the low LDL-C group was 36.2), and was statistically significant (P < 0.001). The above results indicate that high LDL-C risk is higher for C allele carrying persons than for allele T carrying persons.
Factors which may influence the research results of case control include genetic confounding of the crowd, disease confounding caused by non-strict standards during case enrollment, and the like, although the inventor selects objects from young and young people with fewer basic diseases, on the basis of excluding main disease confounding factors such as heart, liver and kidney dysfunction, the inventor selects 1405 research samples and corresponding controls, and in order to further analyze the contribution of the rs35605 locus to high LDL-C risk degree, multivariate logistic regression model analysis is performed after other possible influencing factors such as age, sex, smoking, body mass index, uric acid, and the like are adjusted. The logistic regression model analysis is a common statistical analysis method, which is mainly used for analysis of two classified variables and can be used for adjusting the factors causing mixing of the whole analysis. The results are shown in Table 3.
Results: as shown in table 3, multivariate logistics regression model analysis shows that after adjusting age, sex, smoking, body mass index, uric acid and other possible influencing factors, rs35605 locus is found to be still a risk factor of high LDL-C [ T/C genotype and C/C genotype are significantly increased in risk of hypercholesterolemia compared with T/T genotype, respectively OR value=1.611, 95% ci is (1.104-2.253); OR value = 1.526, 95% ci (1.056-2.205); and has statistical significance (P < 0.001).
Conclusion: the ABCC1 gene with the rs35605 mutation of the polymorphic site is an LDL-C related gene, and the risk of carrying the hypercholesterolemia of a C allele individual is obviously increased compared with that of a T allele individual, so that the preparation or the kit for detecting the rs35605 mutation of the ABCC1 gene in vitro can be clinically used for detecting, preventing, diagnosing or treating the hypercholesterolemia.
In summary, the invention discloses a low-density lipoprotein cholesterol related gene, an in vitro detection system and application thereof, wherein the in vitro detection system can be used for detecting, preventing, diagnosing or treating hypercholesterolemia besides the polymorphism of the low-density lipoprotein cholesterol related gene in vitro, thereby reducing the occurrence rate of the hypercholesterolemia and having great significance for preventing and treating dyslipidemia related diseases.
The technical characteristics form the embodiment of the invention, have stronger adaptability and implementation effect, and can increase or decrease unnecessary technical characteristics according to actual needs so as to meet the requirements of different situations.
Claims (8)
1. A gene related to LDL-C is characterized in that the gene related to LDL-C, namely an rs35605 site mutated ABCC1 gene, and the nucleotide sequence of the rs35605 site mutated ABCC1 gene is shown in a sequence table SEQ ID NO. 1.
2. A reagent for in vitro detection of LDL-C related gene according to claim 1, characterized in that the reagent is used for detecting mutation at rs35605 site of ABCC1 gene, the reagent comprising the following primers:
an upstream primer: 5 'ACTGCTCTACAGGATGACTCACCT3'
A downstream primer: 5 'CCTCCTCGGAAGTCGCCACAGC 3'.
3. The reagent for in vitro detection of LDL-C related genes according to claim 2, wherein the reagent is a reagent for combination of polymerase chain reaction and restriction fragment length polymorphism analysis.
4. A preparation or kit comprising a reagent for detecting an LDL-C associated gene according to claim 2 or 3 in vitro, characterized in that the preparation or kit comprises a reagent, a PCR amplification enzyme and a corresponding buffer, a PspPI restriction enzyme and a corresponding cleavage map.
5. The preparation or kit of claim 4, wherein the reagents comprise the following primers:
an upstream primer: 5 'ACTGCTCTACAGGATGACTCACCT3'
A downstream primer: 5 'CCTCCTCGGAAGTCGCCACAGC 3'.
6. Use of the reagent for in vitro detection of LDL-C associated gene according to claim 2 or 3 for preparing a preparation or kit for in vitro detection of LDL-C associated gene.
7. The method according to claim 6, wherein the preparation or kit comprises reagents, PCR amplification enzymes and corresponding buffers, pspPI restriction enzymes and corresponding cleavage maps.
8. Use according to claim 6 or 7, characterized in that the reagents comprise the following primers:
an upstream primer: 5 'ACTGCTCTACAGGATGACTCACCT3'
A downstream primer: 5 'CCTCCTCGGAAGTCGCCACAGC 3'.
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| CN101565752A (en) * | 2009-05-27 | 2009-10-28 | 张鹏辉 | Fluorescent quantitative PCR detection kit of tumor multidrug resistance gene and detection method thereof |
| US20170166977A1 (en) * | 2015-11-04 | 2017-06-15 | Duke University | Biomarkers for the Identification of Prostate Cancer and Methods of Use |
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| CN101565752A (en) * | 2009-05-27 | 2009-10-28 | 张鹏辉 | Fluorescent quantitative PCR detection kit of tumor multidrug resistance gene and detection method thereof |
| US20170166977A1 (en) * | 2015-11-04 | 2017-06-15 | Duke University | Biomarkers for the Identification of Prostate Cancer and Methods of Use |
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