CN107502667B - Gene functional genetic variation related to LDL-C level and related application - Google Patents
Gene functional genetic variation related to LDL-C level and related application Download PDFInfo
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Abstract
The invention provides functional genetic variation of genes related to LDL-C level and related applications; specifically, the invention provides an application of a reagent material and/or an instrument device for detecting the following protein amino acid sites and/or corresponding gene sites in a sample from an individual to be detected in preparing a detection system for evaluating the blood lipid level, dyslipidemia and/or coronary heart disease onset risk: the EV15 gene rs117711462, the APOB gene rs376825639 and/or the PKD1L3 gene rs17358402 cause the 354 th amino acid of the EVI5 protein, the 3768th amino acid of the APOB protein and/or the 1572 th amino acid of the PKD1L3 protein to code and change respectively. Individuals to be tested with variation of EVI5Arg354Cys and/or PKD1L3Arg1572His have higher total cholesterol and/or low density lipoprotein cholesterol levels and/or higher risk of dyslipidemia. The individuals to be detected with the APOBIle3768Thr variation have lower low-density lipoprotein cholesterol level, lower dyslipidemia risk and/or lower coronary heart disease risk.
Description
Technical Field
The invention relates to gene functional genetic variation related to LDL-C level and related application, in particular to application of reagent materials and/or instrument equipment for detecting variation conditions of EVI5Arg354Cys, APOBIle3768Thr and PKD1L3Arg1572His in a sample from an individual to be detected in preparation of a detection system for evaluating blood lipid level, blood lipid abnormality and/or coronary heart disease onset risk, and further relates to a detection system for evaluating blood lipid level, blood lipid abnormality and/or coronary heart disease onset risk.
Background
A large number of research data show that dyslipidemia is an independent risk factor for coronary heart disease, myocardial infarction, sudden cardiac death and ischemic stroke. It causes occult, gradual, progressive, systemic and organic damage to the body by accelerating systemic atherosclerosis. Research shows that the blood cholesterol level of people is increased by 2-3% when the blood cholesterol level is increased by 1% and the coronary heart disease (CAD) is increased by 2-3%. The increase of the total number of dyslipidemia patients in China is a remarkable trend, and the research results of diabetes and metabolic disorder in China in 2007-2008 indicate that the cholesterol level of people over 20 years old in China is increased by 23.9% compared with that in 2002, and dyslipidemia becomes an important public health problem for residents in China. The reported data of the sanitation and crowd health conditions of Beijing City in 2011 show that the prevalence rate of dyslipidemia of more than half of residents in Beijing City is 58.5% of the residents in the Beijing City, wherein the prevalence rate of dyslipidemia of 18-30-year-old men is already high.
Plasma Total Cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) and Triglyceride (TG) concentrations are the most important risk factors for cardiovascular disease and targets for therapeutic intervention. The latest data of the world health organization indicate that the contribution of the control of the risk factors of dyslipidemia is the largest in the benefit of the prevention and control of cardiovascular diseases. The death of coronary heart disease is reduced by half during 1980-2000 in the United states, which is completely attributed to the contribution of developing national cholesterol education.
Most dyslipidemia has a complex etiologic mechanism, which is the result of long-term interactions between multiple genes or between multiple genes and the environment. Genome-wide association studies successfully identified multiple genetic loci associated with blood lipids, however, almost all of these loci were initially identified in populations of european descent, lacking research data in asian populations. In addition, the functional sites of the known gene regions are frequently rare low-frequency sites, and have important functional significance and clinical significance, and research results show that most of the rare functional variations (80%) have ethnic specificity. In view of the huge difference of environmental exposure and genetic background between Asian population and European population, the need of identifying Asian population, especially Chinese population, for systematic screening of blood lipid related functional variation and rare variation is urgent, which will help to Chinese hyperlipidemia prevention, new drug development, diagnosis and individual treatment.
Disclosure of Invention
The invention mainly aims to determine the specific genetic functional genetic variation related to the blood lipid level of Asian people, particularly east Asian people, provide a method for evaluating the blood lipid level and/or the incidence risk of dyslipidemia by detecting related genetic loci, and provide a detection system for evaluating the blood lipid level and/or the incidence risk of dyslipidemia and related application.
The inventor takes east Asian population including Chinese, Philippines, American Huaren, Singapore Hispanic, Singapore Hispanic Malaysia and the like as research objects, analyzes and researches the correlation between a plurality of 11 ten thousand genetic variations and blood fat level, and determines the genetic functional genetic variation related to Total Cholesterol (TC) and/or low density lipoprotein cholesterol (LDL-C) level. The functional genetic variation of the gene comprises the following amino acid position variation of the protein and/or the corresponding gene position variation: the 354 th amino acid of the EVI5 protein, the 3768th amino acid of the APOB protein and/or the 1572 th amino acid of the PKD1L3 protein. Wherein, the gene locus corresponding to the protein amino acid locus refers to the nucleotide locus corresponding to the amino acid locus in the nucleotide sequence of the gene for coding the protein. Preferably, the gene site corresponding to amino acid 354 of the EVI5 protein comprises rs117711462 of the EVI5 gene; the gene site corresponding to 3768th amino acid of APOB protein comprises APOB gene rs 376825639; the gene site corresponding to the 1572 th amino acid of the PKD1L3 protein comprises the rs17358402 gene of PKD1L 3.
In the present invention, the term "EVI 5" includes EVI5 gene and/or EVI5 protein; the term "APOB" includes APOB genes and/or APOB proteins; the term "PKD 1L 3" includes the PKD1L3 gene and/or the PKD1L3 protein.
The research of the invention shows that individuals to be tested, wherein the 354 th amino acid of the EVI5 protein is Cys (for example, the rs117711462 of the EVI5 gene is A), have higher total cholesterol and/or low-density lipoprotein cholesterol level and/or higher dyslipidemia risk; the test individual of APOB protein with 3768th amino acid Thr (for example, the situation that APOB gene rs376825639 is G) has lower total cholesterol and/or low density lipoprotein cholesterol level, lower dyslipidemia risk and/or lower coronary heart disease risk; the individuals to be tested, wherein the amino acid at the 1572 th position of the protein PKD1L3 is His (for example, the situation that the rs17358402 of the PKD1L3 gene is T), have higher total cholesterol and/or low-density lipoprotein cholesterol level and/or higher dyslipidemia risk.
Therefore, by detecting whether variation conditions of the protein amino acid sites and/or the corresponding gene sites exist in a sample from the individual to be detected, the method can be used for evaluating the blood lipid level of the individual to be detected, particularly the Total Cholesterol (TC) and/or low-density lipoprotein cholesterol (LDL-C) level, and can also be used for evaluating the onset risk of dyslipidemia and/or coronary heart disease of the individual to be detected.
In one aspect, the invention provides the use of a reagent material and/or an apparatus for detecting the following protein amino acid sites and/or their corresponding genetic sites in a sample from an individual to be tested in the preparation of a detection system for assessing the risk of developing blood lipid levels, dyslipidemia and/or coronary heart disease:
the 354 th amino acid of the EVI5 protein, the 3768th amino acid of the APOB protein and/or the 1572 th amino acid of the PKD1L3 protein;
wherein, the gene locus corresponding to the protein amino acid locus refers to the nucleotide locus corresponding to the amino acid locus in the nucleotide sequence of the gene for coding the protein; preferably, the corresponding gene sites are rs117711462 of the EVI5 gene, APOB gene rs376825639 and/or PKD1L3 gene rs 17358402.
According to a particular embodiment of the invention, the blood lipid levels comprise Total Cholesterol (TC) and/or low density lipoprotein cholesterol (LDL-C).
According to a specific embodiment of the invention, the protein of EVI5 has Cys at the 354 th amino acid or the rs117711462 allele of the EVI5 gene has higher total cholesterol and/or low density lipoprotein cholesterol level and/or higher dyslipidemia risk because the carrier of A (the rs117711462 is A and the amino acid is changed into Cys) exists in the protein.
According to a specific embodiment of the present invention, the amino acid at position 3768 of the APOB protein is Thr or the carrier of the APOB gene rs376825639 allele as G (rs376825639 is G, the amino acid becomes Thr) has lower total cholesterol and/or low density lipoprotein cholesterol level, lower risk of dyslipidemia and/or lower risk of coronary heart disease onset.
According to a specific embodiment of the invention, the protein of PKD1L3 has His at amino acid position 1572 or the rs17358402 allele of the PKD1L3 gene has a higher total cholesterol and/or low density lipoprotein cholesterol level and/or a higher risk of dyslipidemia due to the carrier of T (rs17358402 is T and the amino acid is changed into His).
According to the specific implementation scheme of the invention, the individuals to be detected are east Asian people including Chinese, Philippines, American China, Singapore Hippocampus Hispanic, Singapore Hispanic Malaysia and the like. In particular, for detection, the sample may be from blood, urine, saliva, gastric juice, hair, biopsy, or the like of the subject, preferably blood.
The amino acid sites of the protein of the present invention can be detected at the protein level by any available technique in the art, and the gene sites corresponding to the amino acid sites of the protein of the present invention (such as the aforementioned single nucleotide polymorphism sites) can also be detected at the DNA level or RNA level to detect the functional genetic variation of the gene. Detection methods at the protein level are, for example: protein and peptide sequence analysis techniques, including chemical method of N-terminal sequence determination, Edman method, C-terminal enzymolysis method, C-terminal chemical degradation method, etc.; mass spectrometry-related protein detection techniques, such as matrix assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) and electrospray ionization-ionization mass spectrometry (ESI-MS); antibody-based detection methods, such as preparing antibodies that recognize different mutants, and detecting protein variations using immunoblotting (e.g., western blots) and/or enzyme-linked immunosorbent assay (ELISA). Detection methods at the DNA level and RNA level are, for example: the sequence difference between the control gene and the gene carrying the mutation can be directly revealed by direct DNA sequencing by a direct DNA sequencing method, specifically, the direct DNA sequencing can be directly carried out by using a traditional commercial sequencing kit or an automatic sequencer, or Pyrosequencing (Pyrosequencing), micro-sequencing (SNaPshot) and the like which are developed in recent years. Hybridization-based methods may also be employed, including specifically the Taqman probe method, DNA chip method, and the like. Primer extension-based methods such as matrix-assisted laser desorption ion time-of-flight mass spectrometry (MALDI-Tof-MS) can also be used. Conformation-based methods, such as Restriction Fragment Length Polymorphism (RFLP) analysis, single-strand conformation polymorphism (SSCP) analysis, Denaturing Gradient Gel Electrophoresis (DGGE) analysis, denaturing high performance liquid chromatography (hplc) analysis, and the like, may also be used. High resolution dissolution curve analysis techniques (HRM) may also be employed. In practice, one skilled in the art can select any one of the above techniques for in vitro detection of the amino acid sites and/or gene sites (including the single nucleotide polymorphism sites) of the protein of the present invention according to the actual situation, and can also use a combination of multiple techniques for in vitro detection.
According to a specific embodiment of the present invention, the reagent material and/or the apparatus for detecting the protein amino acid site and/or the gene site can be any reagent material and/or apparatus and the like used in any feasible technology for detecting the protein amino acid site and/or the gene site. Reagent materials for detecting 386 th amino acid of CD36 protein, 61 th amino acid of APOA1 protein and/or 74 th amino acid of CETP protein at protein level; and/or detecting the CD36 gene rs148910227, the APOA1 gene rs12718465 and/or the CETP gene rs201790757 at the DNA level and the RNA level. Reagent materials for detection at the protein level such as: reagent materials for use in techniques for sequence analysis of proteins and peptide fragments; or reagent materials for use in protein detection techniques associated with mass spectrometry; or reagent materials for antibody-based detection methods, and the like. Reagent materials for detection at DNA level, RNA level such as: reagents for direct sequencing; or reagents for polymerase chain reaction combined with restriction fragment length polymorphism analysis; or reagents for polymerase chain reaction coupled with direct sequencing; or reagents for polymerase chain reaction coupled with direct sequencing; or a reagent for use in any one of the following SNP typing methods: hybridization-based methods, primer extension-based methods, conformation-based methods, or high resolution melting curve analysis techniques, among others.
In another aspect, the present invention provides a detection system for evaluating the risk of developing blood lipid level, dyslipidemia and/or coronary heart disease, comprising: reagent materials and/or instrumentation to detect the following protein amino acid sites and/or their corresponding genetic loci in a sample from an individual to be tested:
the 354 th amino acid of the EVI5 protein, the 3768th amino acid of the APOB protein and/or the 1572 th amino acid of the PKD1L3 protein;
wherein, the gene locus corresponding to the protein amino acid locus refers to the nucleotide locus corresponding to the amino acid locus in the nucleotide sequence of the gene for coding the protein; preferably, the corresponding gene sites are rs117711462 of the EVI5 gene, APOB gene rs376825639 and/or PKD1L3 gene rs 17358402.
According to a specific embodiment of the present invention, the detection system for evaluating the onset risk of blood lipid level, dyslipidemia and/or coronary heart disease of the present invention comprises a detection unit and an evaluation unit, wherein:
the detection unit comprises a reagent material and/or instrument equipment for detecting the protein amino acid sites and/or the corresponding gene sites in a sample from an individual to be detected, and is used for obtaining the detection result of the protein and/or gene functional genetic variation condition of the individual to be detected;
the evaluation unit comprises a processing unit for carrying out evaluation processing according to the detection result of the detection unit; wherein, the EVI5 protein 354 amino acid is Cys or rs117711462 allele of the EVI5 gene has higher total cholesterol and/or low density lipoprotein cholesterol level and/or higher dyslipidemia incidence risk due to A carriers; the 3768th amino acid of APOB protein is Thr or the rs376825639 allele of APOB gene is G carrier with lower total cholesterol and/or low density lipoprotein cholesterol level and/or lower dyslipidemia risk; the protein of PKD1L3 has His as amino acid in 1572 position or rs17358402 allele of PKD1L3 gene, T carrier has higher total cholesterol and/or low density lipoprotein cholesterol level and/or higher dyslipidemia risk.
The detection system for evaluating the blood lipid level, the dyslipidemia and/or the risk of coronary heart disease can be a virtual device as long as the functions of the detection unit and the evaluation unit can be realized. The detection unit can comprise various detection reagent materials and/or detection instrument equipment and the like; the data analysis unit may be any operation instrument, module or virtual device capable of analyzing and processing the detection result of the detection unit to obtain the evaluation condition of the blood lipid level, the blood lipid abnormality and/or the coronary heart disease onset risk, for example, a data chart corresponding to various possible detection results and the corresponding blood lipid level (including the LDL-C level), the blood lipid abnormality and/or the coronary heart disease onset risk is prepared in advance, and the detection result of the detection unit is compared with the data chart to obtain the evaluation result of the blood lipid level, the blood lipid abnormality and/or the coronary heart disease onset risk.
By applying the technology provided by the invention, the blood lipid level and/or the blood lipid abnormality onset risk of east Asian population can be evaluated by detecting the functional genetic variation of the gene, an individual health action scheme aiming at a research object is made, and the technology is favorable for the prevention, new drug development, diagnosis and individual treatment of the hyperlipemia of Chinese people.
Detailed Description
In order that the invention may be more clearly understood, it will now be further described with reference to the following examples. The examples are for illustration only and do not limit the invention in any way. The experimental methods in the examples, in which specific conditions are not noted, are conventional methods and conventional conditions well known in the art, or conditions as recommended by the manufacturer.
Example one
The correlation between the genetic variation and the low-density lipoprotein cholesterol LDL-C is detected and analyzed respectively. First, an exon chip assay was performed on 47532 subjects of 23 studies in the east asian population. The study samples were from 23 studies such as Chinese ophthalmologic study (CHES), Chinese Health and Nutrition Survey (CHNS), FaMHES, Guizhou Bijie type 2 diabetes study (GBTDS), hong Kong university Special study program (HKU-TRS), Hubei coronary heart disease study (HuCAD), and Chinese elderly population nutrition and health status survey (NHAPC). The conditions of exon chip detection platforms, genotype-phenotype analysis software, etc. used in 23 independent blood lipid level studies are shown in table 1. All samples were pooled for association analysis, the final 110986 genetic variations at the single locus were excluded from association analysis, and the statistical significance of the study was set as P<4.5×10-7。
Exon chip detection technology platform and analysis software for 123 study samples in table
To further assess whether significant sites of blood lipids affect the risk of coronary heart disease CAD at the same time, we performed case-control studies using the east asian population CAD data, which included the study of myocardial infarction at the health science center of beijing university and michigan university college of medicine (PUUMA-MI), the project of research at hong kong university (HKU-TRS), the study of north hui coronary heart disease (HuCAD), the study of Beijing Atherosclerosis (BAS), and the study of Chinese Atherosclerosis (CAS), with 359661 CAD patients and 18558 controls, totaling in total to 28899.
Statistical analysis method
The LDL-C indices measured in each cohort were adjusted by age, age squared and study specific covariates and converted to a standard normal distribution with a mean of 0 and a standard deviation of 1 at meta analysis. The correlation between genetic variation and HDL-C in each research sample is analyzed by RARE ETALWORKER or RVTESST software, and then meta analysis is carried out by RAREMETALS software to combine the results of each research. And (3) adopting case contrast design with CAD (computer aided design) risk research, analyzing the relation between the blood fat significant site and CAD in each research, and combining the research results by applying fixed effect reciprocal weighted meta analysis of METAL.
Basic conditions of the study population
The meta analysis included 23 eastern asian population samples of studies, totaling 47532 subjects, from 23 studies such as the chinese ophthalmic study (CHES), the Chinese Health and Nutrition Survey (CHNS), the urban defense male health examination survey (FAMHES), the guizhou birch type 2 diabetes study (GBTDS), the hong kong university program of research (HKU-TRS), the north huichi coronary heart disease study (HuCAD), the chinese aged population nutrition and health survey (NHAPC), with the basic characteristics of the samples as given in table 2 below. The association study of coronary heart disease CAD included 9661 CAD patients and 18558 controls, study of myocardial infarction from the health sciences center of Beijing university and the medical college of Michigan university (PUUMA-MI), the Special research program of hong Kong university (HKU-TRS), the Hubei coronary heart disease study (HuCAD), the Beijing Atherosclerosis Study (BAS), and the Chinese Atherosclerosis Study (CAS). Each study was approved by its research institute and by the ethical committee of the local research institute. All participants signed informed consent in paper.
TABLE 2 exon chip detection of basic information of human population
Abbreviations: CHES, chinese ophthalmic study; CHNS, chinese health and nutrition survey; CLHNS, longitudinal health and nutrition survey of cebu; fames, urban harbor defense male health examination survey; GBTDS: type 2 diabetes study, Guizhou, graduation; HKU-TRS, hong Kong university Special research project; HuCAD, study of coronary heart disease in north of huh; NHAPC, a survey of nutritional and health status of the chinese elderly population; PUUMA, the health science center of beijing university and the study of myocardial infarction of michigan university medical school; SBCS, shanghai breast cancer study; SCES, singapore chinese ophthalmic study; SiMES, singapore mare ophthalmic study; SMHS, shanghai male health study; SP2, singapore prospective research project; SWHS, shanghai female health study; TUDR, the american taiwan diabetic retinopathy study; TC, total cholesterol; LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol; TG, triglycerides; BMI, body mass index.
Table 3 shows the results of meta analysis of the association of EVI5Arg354Cys (EV15 gene rs117711462 is A, EVI5 protein 354 amino acid is mutated from Arg to Cys), APOBIle3768Thr (APOB gene rs376825639 is G, APOB protein 3768 amino acid is mutated from Ile to Thr), PKD1L3Arg1572His (PKD1L3 gene rs 58173402 is T, PKD1L3 protein 1572 amino acid is mutated from Arg to His) with low density lipoprotein cholesterol. Table 4 shows the result of associating APOBIle3768Thr with CAD.
Table 3 correlation results of EVI5Arg354Cys, APOBIle3768Thr, PKD1L3Arg1572His with low density lipoprotein cholesterol
TABLE 4 correlation of APOB Gene Ile3768Thr with CAD
The analysis data of the invention show that EVI5Arg354Cys, APOBIle3768Thr and PKD1L3Arg1572His are all significantly related to LDL-C and reach the significance level of research (P values are respectively 3.23 × 10-7、3.35×10-9And 2.11X 10-8) The study of the invention also shows that the three sites of EVI5Arg354Cys, APOBIle3768Thr and PKD1L3Arg1572His and the TC level also reach the significance level of the study (the P values are respectively 1.41X 10-7、8.44×10-12And 1.96X 10-9). At the same time, APOBIle3768Thr and coronary heart disease are also obviousSignificantly, the G allele (amino acid mutation at position 3768 to Thr) carrier had a lower LDL-C level with a 80% reduction in the risk of coronary heart disease (OR 0.19, 95% CI-0.51-0.88, P-2.06 × 10)-6)。
Claims (5)
1. Use of a reagent material and/or an instrumental device for detecting the following genetic loci and/or their corresponding proteinogenic amino acid loci in a sample from an individual to be tested in the manufacture of a test system for assessing blood lipid levels:
EVI5rs117711462,APOBGene rs376825639 and/orPKD1L3Gene rs 17358402;
wherein the blood lipid level is total cholesterol and/or low density lipoprotein cholesterol level;
wherein the individual to be detected is east Asian population;
wherein,EVI5the rs117711462 allele has higher total cholesterol and/or low density lipoprotein cholesterol level because of the carrier of A,APOBthe rs376825639 allele has lower total cholesterol and/or low density lipoprotein cholesterol levels as a carrier of G,PKD1L3the rs17358402 allele has higher total cholesterol and/or low density lipoprotein cholesterol levels as a carrier of T.
2. Use according to claim 1, wherein the sample is from blood, urine, saliva, gastric juice, hair or a biopsy of the subject to be tested.
3. Use according to claim 2, wherein the sample is blood.
4. A test system for assessing blood lipid levels, wherein the blood lipid levels are total cholesterol and/or low density lipoprotein cholesterol levels, the test system comprising a test unit and an assessment unit, wherein:
the detection unit comprises reagent materials and/or instrument equipment for detecting the following gene loci and/or protein amino acid loci corresponding to the following gene loci in a sample from an individual to be detected, and is used for obtaining the detection result of the functional genetic variation condition of the gene of the individual to be detected:
EVI5rs117711462,APOBGene rs376825639 and/orPKD1L3Gene rs 17358402;
the evaluation unit comprises a processing unit for performing an evaluation process based on the detection result of the detection unit, wherein,EVI5the rs117711462 allele has higher total cholesterol and/or low density lipoprotein cholesterol level because of the carrier of A,APOBthe rs376825639 allele has lower total cholesterol and/or low density lipoprotein cholesterol levels as a carrier of G,PKD1L3the rs17358402 allele has higher total cholesterol and/or low density lipoprotein cholesterol levels as a carrier of T.
5. The detection system according to claim 4, wherein the reagent material and/or instrument device for detecting the gene locus and/or the corresponding protein amino acid locus in the sample from the individual to be detected comprises:
detection at protein levelEVI5Amino acid corresponding to gene rs117711462,APOBAmino acids corresponding to gene rs376825639 and/orPKD1L3Reagent material for amino acids corresponding to gene rs 17358402; and/or
Detection at DNA level, RNA levelEVI5Rs117711462,APOBGene rs376825639 and/orPKD1L3Reagent material for gene rs 17358402.
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