KR20190052890A - Composition, kit for predicting the risk of developing cardiovascular disease related to Cholesterol efflux capacity, and method using the same - Google Patents

Abstract

According to a microarray, kit and composition for predicting the risk of developing cardiovascular diseases, comprising polynucleotides capable of detecting single nucleotide polymorphic markers, and a method for providing information for predicting the risk of developing cardiovascular diseases in an individual, it is possible to predict or measure the risk for an individual to develop cardiovascular diseases. The predicted or measured risk level of cardiovascular diseases of an individual can be used as information to prevent the onset of coronary artery disease and the like early on and can be applied to personalized medical treatment to increase therapeutic effects.

Description

Technical Field [0001] The present invention relates to a composition, a kit, and a method for predicting the risk of developing cardiovascular diseases associated with cholesterol efflux,

The present invention relates to a composition and a kit for detecting a mutation of a gene to predict the risk of developing cardiovascular disease, and a method of providing information for predicting the risk of developing cardiovascular disease in an individual using the kit.

Cardiovascular disease (CVD) is the world's highest death rate. According to the World Health Organization report, 23.6 million people are expected to die from cardiovascular disease by 2030. Myocardial infarction is a fatal disease that is the number one cause of sudden death in adults. The causes of cardiovascular disease are smoking, eating, overweight, obesity, and metabolic problems. Studies are continuing to prevent and treat cardiovascular diseases through blood pressure lowering, serum cholesterol lowering, and psychological treatment.

Conventional methods of diagnosing cardiovascular disease include invasive methods such as intravascular ultrasound (IVUS), and biomarkers for the diagnosis of cardiovascular disease include glutamic oxaloacetic transaminase (GOT) Lactate dehydrogenase (LDH), creatine kinase-MB (MB), troponin I, troponin T, and C-reactive protein (CRP) And B-type natriuretic peptide (BNP). However, genetic indices that can predict the onset of cardiovascular disease using genetic characteristics of cardiovascular disease are still insufficient. Therefore, it is necessary to diagnose and prevent high risk of cardiovascular disease through genetic testing based on continuous research.

One aspect provides a composition for predicting the risk of developing cardiovascular disease.

Another aspect provides a kit for predicting the risk of developing cardiovascular disease.

Another aspect provides a method of providing information for predicting the risk of developing cardiovascular disease in an individual.

One aspect provides a composition for predicting the risk of developing cardiovascular disease.

Wherein the composition comprises a polynucleotide capable of detecting a single base polymorphism marker, wherein the single base polymorphism marker is selected from the group consisting of A or C of 112357897 base of human chromosome 2, 21041698 base of human 6th chromosome is G Or A, 21148021st base of human 6th G is A or G, 21196501th base of human 6th chromosome is C or G, 21226461 and 21226462th bases of human 6th chromosome are TG or deletion, or their It can be a combination.

Single nucleotide polymorphism (SNP) is used in the sense commonly known in the art. Single nucleotide polymorphism refers to a single nucleotide or nucleotide (A, T, C or G, A, T, C or G, nucleotide A adenine, nucleotide T thymine, nucleotide C cytosine, nucleotide G guanine) Quot; means a variant or polymorphism of the base or nucleotide sequence present in my genome. Single nucleotide polymorphisms are the most prevalent genetic polymorphisms on the human genome, and can cause large differences in each individual genetically based on single nucleotide polymorphisms. The single nucleotide polymorphism may be mixed with a single nucleotide variant (SNV). The single nucleotide mutation refers to a change in sequence that shows a difference in one base or nucleotide on the genome.

Wherein the polynucleotide is selected from the group consisting of A or C of 112357897 base of human chromosome 2, 21041698 base of human 6th chromosome is G or A, 21148021 base of human 6th chromosome is G or A , The 21196501 base of human 6 th chromosome is C or G, the 21226461 and 21226462 base of human 6 th chromosome include TG or deletion, or a combination thereof, or a complementary polynucleotide thereof .

The polynucleotide may be judged to be associated with the risk of developing cardiovascular disease if one single-stranded polynucleotide is associated with a risk of developing cardiovascular disease, as well as a polynucleotide complementary to the single-stranded polynucleotide. Thus, compositions according to certain embodiments comprise single-stranded polynucleotides and polynucleotides having a sequence complementary thereto that are associated with the risk of developing cardiovascular disease having one particular sequence.

For example, human 112357897 base (single base polymorphism marker) of chromosome 2 is "A" or "C". In this case, the composition contains not only the "A" or "C" nucleotide of human chromosome 2, the 112357897 base (single base polymorphism marker), but also the human chromosome number 112357897 base (single base polymorphism marker Quot; T " or " G " nucleotide at a position corresponding to the nucleotide sequence of SEQ ID NO: The composition can detect the 112357897 base (single base polymorphism marker) of human chromosome 2. Alternatively, the composition can detect A > C at the 112357897 base (single base polymorphism marker) of human chromosome 2.

Polynucleotides having complementary sequences are applied equally to other single nucleotide polymorphic markers.

The polynucleotide may be a polynucleotide for analyzing a single nucleotide polymorphism marker downstream of the LOC541471 gene. LOC541471 is known to be involved in glioblastoma microvesicles carrying RNA and proteins that promote tumor growth, but its function has not yet been clearly elucidated. In the case of human, LOC541471 may be a protein encoded by the LOC541471 gene. The LOC541471 gene may be located in GRCh37 / hg19, the second chromosome, and 2q13 in humans.

The polynucleotide may be an intron of the CDKAL1 gene And may be a polynucleotide for analyzing a single nucleotide polymorphism marker. CDKAL1 is known to be a member of the methylthiotransferase family, but its function has not yet been clearly established. In the case of human, CDKAL1 may be a protein encoded by the CDKAL1 gene. The CDKAL1 gene may be located in the case of human, GRCh37 / hg19, the sixth chromosome, and 6p22.3.

The term " gene " refers to a structural unit that determines genetic information, and includes a structural gene having information for determining the base sequence of the protein's amino acid sequence or functional RNA (tRNA, rRNA, etc.), and / For example, a promoter, a repressor, an operator, and the like. As used herein, the term " gene " is understood to mean a single stranded side comprising a nucleotide sequence that is transcribed to produce a product of the gene.

As a general practitioner, the single nucleotide polymorphism marker and sequence may be readily identified using the registration number. The specific sequence corresponding to the number registered in the UCSC genome browser or GenBank may change over time. It will be apparent to those of ordinary skill in the art that the scope of the present invention also affects the altered sequence.

The term " polynucleotide " refers to a nucleotide polymer of any length, which polynucleotide can be used interchangeably with nucleic acids, or oligonucleotides. The polynucleotide may specifically bind to the nucleotide sequence of the target region. Using the specific binding characteristics of such polynucleotides, it is possible to effectively isolate a target gene or a fragment thereof containing a target region from a mixture.

The polynucleotides may be singular or plural and may be in the form of a single strand or a double strand. The polynucleotide may also be modified so that the sugar, base or phosphate site of a natural nucleotide, an analogue of a natural nucleotide, or a natural nucleotide, as well as being composed of a natural nucleotide, may be modified so long as it has a property of being hybridizable with a complementary nucleotide by hydrogen bonding (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)), and nucleotides selected from the group consisting of . The polynucleotide may be DNA or RNA.

The polynucleotide may be a polynucleotide comprising a contiguous nucleotide sequence comprising the single nucleotide polymorphism marker or a complementary polynucleotide thereof.

The term " contiguous nucleotide sequence " refers to two or more contiguous nucleotide sequences.

The term " complementary " means having complementarity enough to selectively hybridize to the nucleotide sequence under any particular hybridization or annealing conditions.

The size of the polynucleotide may be 5 to 200 nt (nucleotide: nt). For example, the polynucleotide may be 10-150 nt in length, 10-100 nt in length, or 15-100 nt in length.

The polynucleotide may be a primer, a probe, or a combination thereof. The primer or the probe may be a nucleotide sequence complementary to the nucleotide sequence of the target region, but a nucleotide sequence substantially complementary to the nucleotide sequence that does not specifically interfere with hybridization may be used It is possible. In addition, the primer or the probe may have a modified nucleotide sequence within a range that does not interfere specifically with hybridization with respect to the nucleotide sequence of the target region.

The term " primer " means a single strand of oligonucleotides that can act as a starting point in the polymerization of a nucleotide by a polymerase. For example, the primer can serve as a starting point for template-directed DNA synthesis in the presence of suitable conditions, i.e., four different nucleoside triphosphates and polymerases, at the appropriate temperature and in the appropriate buffer Lt; RTI ID = 0.0 > oligonucleotides < / RTI > The appropriate length of the primer may vary depending on various factors, for example, temperature and use of the primer. The primer may have a length of 5 to 100 nt, 5 to 70 nt, 10 to 50 nt, or 15 to 30 nt. For example, the shorter the length of the primer, the more stable hybridization complex can be formed with the template at a lower annealing temperature. If the length of the primer is less than 5 nt, the accuracy for capturing the target region is low, and if the primer has a size of more than 100 nt, the synthesis cost is increased. Thus, the primers are economical and have a size optimized for gene polymorphism or mutation detection. The design of the primer can be easily carried out by a conventional technician with reference to the nucleotide sequence of the target region to be amplified. For example, it can be designed using a commercially available program for primer design. Examples of commercially available programs for primer design include the PRIMER 3 program.

The primer may further comprise a nucleotide analogue such as phosphorothioate, alkylphosphorothioate, a peptide nucleic acid or an intercalating agent. Further, it may further comprise a labeling substance that emits fluorescence, phosphorescence, or radioactivity. The fluorescent labeling substance may be VIC, NED, FAM, PET, or a combination thereof. The labeling substance may be labeled at the 5 ' end of the polynucleotide. In addition, the radioactive labeling substance may be incorporated into the amplification product through a PCR reaction using a polymerase chain reaction (PCR) in which a radioactive isotope such as ³² P or ³⁵ S is added.

The primers can be used for allele-specific PCR, PCR extension analysis, PCR-single strand conformation polymorphism (PCR-SSCP), TaqMan method and sequencing. have.

The term " probe " means a polynucleotide that is capable of sequence-specifically binding to a complementary polynucleotide strand. The probe may have a length of 5 to 100 nt, 10 to 90 nt, 15 to 80 nt, 20 to 70 nt, or 30 to 50 nt. If the length of the probe is less than 10 nt, the accuracy for capturing the target area is low, and if the length is more than 100 nt, the synthesis cost is increased. Therefore, the probe is economical and has a size optimized for gene polymorphism or mutation detection. The probe may comprise, for example, a perfect match probe consisting of a sequence complementary to the nucleotide sequence of the target region and a probe having a sequence completely complementary to all nucleotide sequences except for the single base polymorphism marker ≪ / RTI >

The probe may be used in hybridization methods such as microarray, Southern blotting, dynamic allele-specific hybridization, and DNA chip. The microarray is used in a manner known in the art, for example, a group of probes or probes immobilized on a plurality of divided regions on a substrate. The substrate can be any suitable rigid or semi-rigid support, such as a membrane, filter, chip, slide, wafer, fiber, magnetic bead or non-magnetic bead, gel, tubing, Microparticles, and capillaries. The probe or a complementary probe thereof may be used in a method capable of hybridizing with a nucleic acid obtained from an individual and measuring the hybridization degree obtained therefrom.

The cardiovascular disease may be coronary artery disease, peripheral vascular disease, atherosclerotic cardiovascular disease, cerebrovascular disease, or a combination thereof, and may be used in a manner known to those skilled in the art.

The "risk of developing cardiovascular disease" may be a relative risk of developing a cardiovascular risk. For example, the risk may be indicative of whether the probability of onset is increased or decreased relative to a healthy normal population. In addition, the risk may be that the individual having a particular allele or genotype has an increased or decreased probability of developing a cardiovascular disease as compared to an individual having another particular allele or genotype.

Cardiovascular disease may be caused by decreased cholesterol efflux compared to healthy or normal subjects.

The subject means a subject for predicting the risk of developing cardiovascular disease. The subject may include a vertebrate animal, a mammal, or a human ( Homo sapiens ). For example, the human being may be a Korean.

The single nucleotide polymorphism marker can be used as a marker for diagnosing or predicting the risk of developing cardiovascular disease. The single nucleotide polymorphism marker can be used as a marker to diagnose or predict cholesterol efflux inhibition. The single nucleotide polymorphism marker can be used as a marker for diagnosing or predicting the risk of developing cardiovascular disease caused by inhibition of cholesterol efflux.

Another aspect provides a kit for predicting the risk of developing cardiovascular disease.

The kit may be one comprising the composition. Wherein the kit comprises a polynucleotide capable of detecting a single nucleotide polymorphism marker, wherein the single nucleotide polymorphism marker is selected from the group consisting of A or C of 112357897 base of human chromosome 2, 21041698 base of human 6th G or A, 21148021 base of human 6th G is A or G, 21196501 base of human 6th chromosome is C or G, 21226461 and 21226462 base of human 6th chromosome are TG or deletion, or combinations thereof Lt; / RTI >

The kit may, for example, comprise the polynucleotide and the constructs necessary for its particular use. And the reagent necessary for the method of use thereof together with the polynucleotide. The kit may further comprise a known material required for hybridization of the polynucleotide with the nucleic acid. The kit may be a kit for specifically amplifying a nucleotide sequence of a target region and diagnosing an individual's cardiovascular disease through the presence or absence of an amplification product. Or a kit for hybridizing the polynucleotide or a probe derived therefrom with a nucleic acid in a sample and diagnosing the risk of developing an individual's cardiovascular disease from the hybridization result. For example, it may further comprise reagents, buffers, cofactors, and / or substrates necessary for hybridization of the nucleic acid. In addition, when the kit is applied to the PCR amplification process, it may optionally contain a reagent necessary for PCR amplification, for example, a buffer, a DNA polymerase, a DNA polymerase cofactor, and dNTPs. In addition, the kit may further include instructions for use to amplify the target region, and may be produced from a number of separate packaging or compartments containing the reagent components described above. For example, in the case where the target region is amplified in the amplification reaction using the specific primer and the target region is not amplified in the amplification reaction using the non-specific primer, Including an explanation of the determination of the outcome, including the determination of the presence of a disease, a region, a polymorphism or a variant, and a determination of the risk of developing an individual's cardiovascular disease from the result.

The polynucleotides and polymorphisms are as described above.

Another aspect provides a method of providing information for predicting the risk of developing cardiovascular disease in an individual. The method comprising: obtaining a nucleic acid sample from a biological sample of an individual; And analyzing the genotype of the single base polymorphism marker from the obtained nucleic acid sample, wherein the single base polymorphism marker is selected from the group consisting of human chromosome 2, chromosome 2, chromosome 112357897 of A or C, human 6th chromosome 21041698 base G or A of human 6 th chromosome is G or A, 21196501 base of human 6 th chromosome is C or G, 21226461 and 21226462 base of human 6 th chromosome are TG or deleted, or Or a combination thereof.

The method comprises obtaining a nucleic acid sample from a biological sample of an individual.

The subject means a subject for predicting the risk of developing cardiovascular disease. The subject may be a vertebrate animal, a mammal, a human ( Homo sapiens ), a mouse, a rat, a cow, a horse, a pig, a sheep, a goat, a dog, a cat and the like. For example, the human being may be Asian or Korean. &Quot; Subject " and " patient " are used interchangeably herein.

The biological sample refers to a sample obtained from an organism. The biological sample may be, for example, blood, tissue, urine, mucus, saliva, tears, plasma, serum, sputum, spinal fluid, pleural fluid, nipple aspirate, lymphatic fluid, airway fluid, intestinal fluid, , Cerebrospinal fluid, intracranial fluid, ascites, cystic tumor fluid, positive fluid, or a combination thereof. The biological sample may be one comprising a purely isolated nucleic acid, a crude nucleic acid, a cell lysate comprising a nucleic acid, or a cell free nucleic acid.

The method of separating the genomic nucleic acid from the biological sample can be performed by a conventional nucleic acid separation method. For example, the target nucleic acid can be amplified by polymerase chain reaction (LCR), ligase chain reaction (LCR), transcription amplification, or real-time nucleic acid sequence based amplification (NASBA) and purified.

The method comprises analyzing the genotype of a single base polymorphic marker from the resulting nucleic acid sample.

The step of analyzing the genotype of the single base polymorphism marker in the method means determining the nucleotide at each position of the target region or single base polymorphism marker in the chromosome. For example, the nucleotide sequencing method of a known nucleic acid can directly determine the nucleotide at the target region or at each position in the chromosome. The nucleotide sequence determination method may include a ginger (or dideoxy) sequencing method or a germ-gilbert (chemical cleavage) method. In addition, the nucleotide sequence of the target region can be determined by hybridizing the probe with the polynucleotide of interest and analyzing the result of hybridization. The degree of hybridization can be confirmed, for example, by marking a detectable label on the target nucleic acid, detecting the hybridized target nucleic acid, or confirming it by an electrical method or the like. In addition, a single base primer extension (SBE) method can be used. The nucleotide sequence determination method may also include the next generation base sequencing. The term " next generation sequencing " (NGS) involves fragmenting a full-length genome in a chip-based and PCR-based paired end format, Sequencing. By next-generation nucleotide sequencing, a large amount of nucleotide sequence data can be generated for a sample to be analyzed within a short time.

The step of analyzing the genotype may determine whether the subject or subject is a patient having a cardiovascular disease or a cardiovascular disease, based on the presence or absence of a specific allele in the single nucleotide polymorphism marker or the presence of a specific genotype .

Wherein the single nucleotide polymorphism marker comprises A or C of 112357897 base of human chromosome 2, G or A of 21041698 base of human 6 th chromosome, G or A of 21148021 base of human 6 th chromosome, The 21196501 base of the sixth chromosome may be C or G, the 21226461 and 21226462 base of the sixth human chromosome may be TG or deleted, or a combination thereof.

In this method, the 112357897 base of human chromosome 2 is C, 21041698 base of human 6th chromosome is A, 21148021 base of human 6th chromosome is A, 21196501 base of human 6th chromosome is G , And when the human 21226461 and 21226462 base of the sixth chromosome are deleted, or a combination thereof, it may be determined that the risk of developing a cardiovascular disease belongs to a high risk group.

In this method, the 112357897 base of human chromosome 2 is C, 21041698 base of human 6th chromosome is A, 21148021 base of human 6th chromosome is A, 21196501 base of human 6th chromosome is G , And determining that the cholesterol efflux ability of the human chromosome 21226461 and 21226462 base is deleted, or a combination thereof, belongs to a risk group in which cholesterol efflux is inhibited.

Providing information for predicting the risk of developing a cardiovascular disease of the subject may be for predicting or diagnosing the relative risk of developing a cardiovascular disease. For example, the risk may be for predicting or diagnosing whether the likelihood of developing a cardiovascular disease is increased relative to a group having a reference genomic sequence.

The term " reference genome sequence " may be a human genomic sequence that is referenced for polymorphism or mutation confirmation. The reference genomic sequence may be a nucleotide sequence of a reference genome, for example, a nucleotide sequence of a human gene, specifically NCBI37.1 or UCSC hg19 (GRCh37), published in the database of the Institute of Biotechnology Information of the National Institutes of Health . The comparison between the nucleotide sequence of the individual and the reference genomic sequence can be performed using various known sequence comparison analysis programs such as Maq, Bowtie, SOAP, GSNAP, and the like.

If the number of polymorphisms in the single nucleotide polymorphism marker is more than 1, more than 2, more than 3, more than 4, or more than 5, it can be judged that the risk of cardiovascular disease is high, have.

Analysis of the genotypes may include sequencing, hybridization by microarrays, allele specific PCR, dynamic allele-specific hybridization, PCR extension analysis, PCR-single strand conformation polymorphism, TaqMan method, or a combination thereof. The step of analyzing the genotype may utilize the composition or kit.

Sequencing analysis can be performed using conventional methods for sequencing and can be performed using an automated gene analyzer. Allele-specific PCR refers to a PCR method in which a DNA fragment in which a single nucleotide polymorphic marker is located is amplified with a primer set including a primer designed with a base at the 3 'end at which a single nucleotide polymorphic marker is located. The PCR extension assay first amplifies a DNA fragment containing a base in which a single nucleotide polymorphic marker is located into a pair of primers and then inactivates all the nucleotides added to the reaction by dephosphorylation and introduces a specific nucleotide polymorphic marker extension A primer, a dNTP mixture, a digoxinucleotide, a reaction buffer, and a DNA polymerase to perform a primer extension reaction. The TaqMan method is performed by designing and fabricating a primer and a TaqMan probe so as to amplify a desired DNA fragment, amplifying and analyzing the probe of different alleles with FAM and VIC (Applied Biosystems).

The composition, kit or method is specifically associated with Asian, Korean, or Korean people over 50 years of age with cardiovascular disease. Thus, the composition, kit or method may be useful for predicting or diagnosing the risk of developing cardiovascular disease in Asians or Koreans.

According to a composition, a kit and a microarray for predicting the risk of developing cardiovascular disease comprising a polynucleotide capable of detecting a single nucleotide polymorphism marker, and a method of providing information for predicting the risk of developing cardiovascular disease in an individual, The risk level of an individual's cardiovascular disease can be predicted or measured. The risk level of the predicted or measured cardiovascular disease of an individual can be used as information to prevent the onset of coronary artery disease early on and can be applied to personalized medical treatment to increase the therapeutic effect.

Figure 1 shows the genome-wide association of cholesterol efflux. Figure 1a shows a quantile-quantile (QQ) plot in the excavation group. The logarithm of the observed (y-axis) and expected (x-axis) p-numbers was constructed for each SNP. The red line represents the null hypothesis. This plot suggests that the biased signal is actually correlated.
Figure 1b shows the association of cholesterol efflux and genome-wide mutations in the excavation group. The single nucleotide polymorphism that passed the quality control criteria was plotted on the x-axis of the Manhattan plot according to the chromosomal location for -log ₁₀ P. P <5x10 ^-5 was considered significant. The four points marked with red letters indicate significant single nucleotide polymorphisms of CDKAL1 in the validation group.
Figure 2 shows a locuszoom area plot of the left side of the CDKAL1 gene and the surrounding region. The genome build and linkage disequilibrium (LD) is hg19 / 1000 Genomes Asian (Nov 2014) and the blue line represents the recombination rate using HapMap data. The dot represents an alternative single nucleotide polymorphism of CDKAL1.

The present invention will be described in more detail with reference to the following examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.

Example  1: cardiovascular disease Marker  Selection

1. Selection of research subjects

In this study, 607 patients with coronary artery disease were included in the cohort of the cardiovascular genome center of Yonsei University College of Medicine. The patients underwent coronary angiography according to chest symptoms. Coronary artery disease was diagnosed based on the presence of significant stenosis (≥50%) in at least one epicardial coronary artery. The replication group was 158 patients who were referred to Severance Hospital for coronary artery disease. The study was conducted with the approval of the Clinical Trials Committee of Severance Hospital (IRB no. 4-2001-0039) and all participants agreed in writing.

2. Coronary artery disease In patients  Plasma lipid levels and cholesterol Effluent  evaluation

Clinical data on history and demographic variables were obtained from the study subjects. Blood samples were taken from all subjects and placed in tubes and centrifuged. Plasma was then collected and stored at -80 [deg.] C.

Plasma lipid levels were measured by conventional methods, and cholesterol efflux analysis was measured by the following method. J774 cells were plated on plates and radiolabeled to the cells by adding 2 μCi of ³ H-cholesterol / mL and reacting for 24 h. To increase the adenosine triphosphate-binding cassette transporter subfamily member A1 (ABCA1), the cells were treated with 0.2% bovine serum albumin (BSA) and 0.3 mM 0.0 &gt; adenosine monophosphate &lt; / RTI &gt; for 2 hours. The medium was then replaced with medium containing 0.2% BSA and patient sample and incubated for 4 hours. Cells were supplemented with 2 [mu] g / ml acyl-coenzyme A: cholesterol acyltransferase inhibitor. The cholesterol efflux rate was calculated using the following equation.

Cholesterol efflux function (Cholesterol efflux capacity: CEC) ( %) = [3 in a culture medium containing a high-density lipoprotein cholesterol H- (μCi) / of the culture medium containing high-density lipoprotein cholesterol H- ³ μCi ^{3} + cells in H- ΜCi {μCi}] x 100 of cholesterol. The values were adjusted based on the efflux of pooled serum on each plate. All samples were repeatedly tested at least twice.

The mean age of the patients was 52 years and 61 years in the excavation group and the verification group, respectively. Men were dominant in both groups. About 25% of all patients had diabetes. The mean HDL-C levels were 42.1 and 42.6 mg / dL in the excavated and tested groups, respectively, and the mean LDL-C levels were approximately 99 mg / dL in both excavated and validated groups (Table 1).

Excavation group (n = 607) Verification group (n = 158) Age, years 52.3 ± 8.0 60.9 ± 8.0 male(%) 321 (52.9) 129 (81.6) diabetes(%) 150 (24.7) 39 (24.7) High blood pressure(%) 286 (47.1) 81 (51.3) Acute coronary syndrome (%) 349 (57.5) 90 (57.0) Current smoking status (%) 140 (23.1) 55 (34.8) Total cholesterol, mg / dL 168 ± 44 162 ± 45 Triglyceride
(Triglycerides: TG), mg / dL 139 ± 101 129 ± 71 HDL-C, mg / dL 42.1 + - 10.0 42.6 ± 11.8 LDL-C, mg / dL 99 ± 39 99 ± 40 Statin use (%) 121 (19.9) 126 (79.7)

3. Genetic analysis and imputation mutation analysis

The genotypes were analyzed using the Affymetrix genome-wide human SNP assay 6.0 (Affymetrix Inc., Santa Clara, CA, USA). For the validation group, genotypes were analyzed using the Axiom (TM) genome-total ASI-30 assay plate (Affymetrix Inc). In order to increase the level of statistics, the standardized method was substituted for the analysis.

The genotype of the autosomes was first screened using SHAPEIT2 (v.2.20) and the HapMap step II NCBI GRCh37 / hg19 genetic map, and the genome-total replacement analysis was performed using IMPUTE2 (v.2.3.2) (Delaneau, Nat Methods 2013 (Marchini, Nat Rev Genet 2010; Howie, Nat Genet 2012) and 1000 Genome Project Phase III as a cosmopolitan reference panel.

For quality control, 1) total mutation extraction rate <95% in both excavation and verification groups; 2) minor allele frequency <0.5%; Or 3) Hardy-Weinberg equilibrium P < 1.0 x 10 ^-6 , SNP was excluded.

Alternative variations with an imputation information score (r ² _info ) less than 0.3 were excluded using QCTOOL (v.1.4) and converted to PLINK using GTOOL (v.0.7.5). In the entire process of replacement and quality control, the GRCh36 / hg18 genotypic coordinates were converted to GRCh37 / hg19 using the UCSC genome browser and the UCSC liftOver algorithm, and the closest genes were annotated. As a result, about 5.8 million and about 7.1 million SNPs were identified in the excavation group and the verification group, respectively.

3. Statistical Analysis

Cholesterol efflux and genome-wide associations between genotyped or imputed mutations. To identify factors associated with cholesterol efflux, Spearman rank correlation coefficients between efflux and clinical parameters were calculated. Cholesterol efflux was adjusted by considering age, sex, and high-density lipoprotein-cholesterol (HDL-C). The association between adjusted cholesterol efflux and mutation was assessed by linear regression, assuming an additive genetic model. The dosage of each variation is equal to the number of minority alleles performed in the range 0 to 2. Correlation coefficient calculations and genome-wide statistical analysis were performed using the SciPy library in Python (V.3.5.2) and PLINK (v.1.07), respectively. Genomic inflation factor (λ) was calculated based on median chi-square statistics, and Manhattan plots and quantile-quantile (QQ) plots were calculated using the R software (V.3.3. 3) using the qqman library.

For the test group, loci were selected by examining mutations satisfying P < 5.0 x 10 &lt; ^{-5 &} gt ;. Linkage disequilibrium (LD) calculations were performed using the PLINK software. LocusZoom (v.0.4.8), which contains the mutations significantly associated with cholesterol efflux in both excavation and verification groups, .

The results of the analysis of the association of cholesterol efflux with the excavation group for about 5.8 million SNPs that passed through substitution and quality control are as follows. The genomic inflation factor (λ) was 0.99. This result implies that the bias of the group sub-structure is not affected. These results were also confirmed by a p-value deviation in the Q-Q plot. Figure 1 shows the genome-wide association of cholesterol efflux. Figure 1a shows a quantile-quantile (Q-Q) plot in the excavation group.

Table 2 shows the results of the correlation coefficient analysis between the Spearman rank and the P value between clinical parameters and cholesterol efflux. Unlike other parameters, HDL-C showed the highest coefficients and significant P values (ρ = 0.313, P = 2.89 × 10 ^-15 and ρ = 0.241, P = 0.002, respectively) in both excavation and validation groups. Age, sex, and HDL-C were adjusted prior to performing the association analysis.

Excavator
(Discovery group) Verification group
(Replication group) ρ P ρ P Age, years 0.073 0.07 0.020 0.81 male 0.097 0.02 0.100 0.21 diabetes 0.075 0.07 0.007 0.93 High blood pressure 0.047 0.25 0.185 0.02 Currently smoking 0.005 0.90 -0.138 0.08 Acute coronary syndrome 0.030 0.45 -0.013 0.08 Total cholesterol, mg / dL 0.062 0.13 -0.077 0.33 Triglyceride, mg / dL 0.015 0.71 -0.001 0.99 HDL-C, mg / dL 0.313 2.89 x 10 ^-15 0.241 0.002 LDL-C, mg / dL -0.033 0.42 -0.123 0.13 Using statins 0.024 0.55 -0.051 0.53

Figure 1b shows the association of cholesterol efflux and genome-wide mutations in the excavation group. The single nucleotide polymorphism that passed the quality control criteria was plotted on the x-axis of the Manhattan plot according to the chromosomal location for -log ₁₀ P. The four points marked with red letters indicate significant single nucleotide polymorphisms of CDKAL1 in the validation group.

In genome - wide association analysis of excavated group (n = 607), 631 varieties were statistically significant (P <5.0 × 10 ^-5 ) and the highest P value was 2.28 × 10 ^{- 10} . Also, in the genome-wide association analysis of the validation group (n = 158), it was found that five alternative mutations were significantly associated with cholesterol efflux. The five variations are shown in Table 3.

Located downstream (downstream) of LOC541471, variations of the left and 2q13 are P = 2.6 × 10 in the excavation group P = 2.5 × 10 ^-5 and verification group ^{in-indicated three.} Four other mutations (rs117835232, rs117252933, rs118065692, and rs150434350) were all located in the intron region of the CDKAL1 gene, identified at position 6p22.3. P value for each variation is 1.1 × 10 ^-7 to 3.8 × 10 in the excavation group ^- was ^5, it was 8.7 × 10 ^-6 to 3.0 × 10 ^-3 in the validation group. The left five species were the cholesterol efflux activity and significantly correlated, P value is in the comprehensive analysis of the two groups 3.8 x 10 ^-10 to 2.8 x 10 ^{- 7} was.

chromosome location rs number gene
(Closest gene) Opposition
gene Subject MAF beta P ^? 2q13 112357897 - LOC541471
(Downstream, 105kb) A> C Excavator
Verification group
Dugong synthesis 0.005
0.010
0.006 8.1
9.5
8.3 2.4 x 10 ^-5
2.6 x 10 ^-3
2.8x10 ^-7 6p22.3 21041698 rs117835232 CDKAL1
(Intron) G> A Excavator
Verification group
Dugong synthesis 0.007
0.013
0.008 6.9
11.8
8.6 3.8 x 10 ^-5
8.7 x 10 ^-6
1.1x10 ^-9 21148021 rs117252933 CDKAL1
(Intron) G> A Excavator
Verification group
Dugong synthesis 0.014
0.010
0.013 5.2
10.9
6.1 5.2 x 10 ^-6
3.1x10 ^-4
1.3x10 ^-8 21196501 rs118065692 CDKAL1
(Intron) C> G Excavator
Verification group
Dugong synthesis 0.010
0.010
0.010 7.0
10.9
7.8 1.1x10 ^-7
3.2 x 10 ^-4
1.5x10 ^-10 21226461 and 21226462 rs150434350 CDKAL1
(Intron) delTG- Excavator
Verification group
Dugong synthesis 0.014
0.020
0.015 4.9
6.5
5.3 1.8 x 10 ^-5
3.0 x 10 ^-3
1.3x10 ^-7

rs_id stands for rs-ID, an independent marker assigned to all single base polymorphisms for which NCBI was initially registered. The rs_id in the table means the SNP marker, which is a polymorphic marker.

Four variants in CDKAL1 were associated with high cholesterol efflux. Of these, rs117835232 did not appear to exhibit strong association criterion with rs117252933 (r ² = 0.50, D '= 0.75). However, between rs117252933 and ^{rs118065692 (r 2 = 0.82, D} '= 0.91), rs117252933 and ^{rs150434350 (r 2 = 0.75, D} ' = 0.93), and rs118065692 and ^{rs150434350 (r 2 = 1.0, D} '= 1.0) r ² and D ', the three variants showed strong association non-equilibrium. Specifically, rs117252933 and ^{rs150434350 (r 2 = 0.75, D} '= 0.93), and rs118065692 and ^{rs150434350 (r 2 = 1.0, D} ' = 1.0) was determined to be in the fully associative non-equilibrium. Figure 2 shows a plot of the region of CDKAL1 left.

To determine whether the effect of the mutation on cholesterol efflux was independent of other clinical variables regardless of the adjustment, age, sex, diabetes, hypertension, smoking, acute coronary syndrome, total cholesterol, triglyceride, HDL-C , Post-hoc linear regression analysis including low-density lipoprotein cholesterol and statin use.

The results of post - linear regression analysis are as follows. HDL-C (β = 0.155, p = 6.81 × 10-14) and CDKAL1 mutation (β = 4.945, p = 2.11 × 10-5) were independently associated with cholesterol efflux, while triglycerides , p = 0.01) and LDL-C levels (β = 4.945, p = 2.11 × 10-5) showed borderline association. Figure 2 shows a locuszoom area plot of the left side of the CDKAL1 gene and the surrounding region. The genome build and linkage disequilibrium (LD) is hg19 / 1000 Genomes Asian (Nov 2014) and the blue line represents the recombination rate using HapMap data. The dot represents an alternative single nucleotide polymorphism of CDKAL1.

variable beta SE P Age, years -0.029 0.036 0.42 male 0.756 0.600 0.21 diabetes 0.656 0.458 0.15 High blood pressure 0.493 0.399 0.22 Currently smoking 0.485 0.501 0.33 Acute coronary syndrome 0.643 0.394 0.10 Triglyceride, mg / dL 0.008 0.003 0.01 HDL-C, mg / dL 0.155 0.020 6.81 x 10 ^-14 LDL-C, mg / dL -0.010 0.005 0.051 Using statins 0.576 0.480 0.23 CDKAL1 Genetic mutation 4.945 1.153 2.11 x 10 ^-5

When assigning a genetic score to a CDKAL1 mutation, a score is given when at least one of the four mutations occurs and a zero score is assigned to the wild type.

Of the five mutations associated with cholesterol efflux identified in this study, one mutation was located in the vicinity of LOC541471 (downstream) of chromosome 2q13, and four species were located in CDKAL1 of chromosome 6p22.3. These mutations were associated with cholesterol efflux, independent of HDL-C levels and other variables.

Claims (9)

A composition for predicting the risk of developing cardiovascular disease comprising a polynucleotide capable of detecting a single nucleotide polymorphism marker,
The single nucleotide polymorphism marker is selected from the group consisting of A or C of 112357897 base of human chromosome 2, G or A of 21041698 base of human 6 th chromosome, G or A of 21148021 base of human 6 th chromosome, Wherein the 21196501 base of the sixth chromosome of the sixth chromosome is C or G, and the 21226461 and 21226462 base of the sixth chromosome of a human are TG or deletion, or a combination thereof, for predicting the risk of developing cardiovascular disease. The composition of claim 1, wherein the detectable polynucleotide is a primer, a probe, or a combination thereof. The composition of claim 1 wherein the size of the detectable polynucleotide is 5 to 200 nt. The composition according to any one of claims 1 to 3, wherein the cardiovascular disease is coronary artery disease, peripheral vascular disease, atherosclerotic cardiovascular disease, cerebrovascular disease or a combination thereof. The composition according to any one of claims 1 to 3, wherein the cardiovascular disease is caused by inhibition of cholesterol efflux. A kit for predicting the risk of developing cardiovascular disease comprising the composition of claim 1. CLAIMS 1. A method of providing information for predicting the risk of developing cardiovascular disease in an individual comprising the steps of:
Obtaining a nucleic acid sample from the biological sample of the individual; And
Analyzing the genotype of the single base polymorphic marker from the obtained nucleic acid sample,
The single nucleotide polymorphism marker is selected from the group consisting of A or C of 112357897 base of human chromosome 2, G or A of 21041698 base of human 6 th chromosome, G or A of 21148021 base of human 6 th chromosome, Wherein the 21196501 base of the sixth chromosome of the sixth chromosome is C or G, the 21226461 and 21226462 th bases of the human sixth chromosome are TG or deletion, or a combination thereof. [Claim 7] The method according to claim 7, wherein human chromosome 2 has chromosome 112357897 C, human 6th chromosome 21041698 base A, human 6th chromosome 21148021 base A, human 6th chromosome 21196501 base Is G, the 21226461 and 21226462 th bases of the human chromosome 6 are deleted, or a combination thereof, the risk of developing a cardiovascular disease belongs to a high risk group. The method according to claim 7, wherein the step of analyzing the genotypes comprises: sequencing, hybridization with a microarray, allele specific PCR, dynamic allele-specific hybridization, PCR extension analysis, PCR-SSCP (PCR-single strand conformation polymorphism), TaqMan method, or a combination thereof.

Images