CN109652532A - A kind of marker detecting drug for cardiovascular disease - Google Patents
A kind of marker detecting drug for cardiovascular disease Download PDFInfo
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Abstract
The present invention is a kind of marker for detecting drug for cardiovascular disease, provides a kind of application of the reagent of drug metabolism related gene for detecting drug for cardiovascular disease in the product of preparation research drug metabolism, can at most detect 66 related genes.Reagent of the present invention, the product containing reagent are determined for the drug metabolism study of drug for cardiovascular disease, drug screening, medicine curative effect evaluation or dosage, the mass survey for carrying out the detection of medication individual molecular science of heredity, influencing curative effect of medication polymorphic site, to reduce the incidence of medication failure, adverse drug reaction event.Meanwhile probe groups of the present invention, chip and kit have efficiently, more, accurate, easy to operate, the specific good, high sensitivities of detection site, quick, practical and low-cost advantage.
Description
Technical field
The present invention relates to genetic engineering and molecular genetic technique fields, and in particular to one kind is used for drug for cardiovascular disease
Drug metabolism gene personalized capture, sequencing probe groups and kit.
Background technique
The end of the fifties in last century, the pharmacogenetics that research individual inheritance variation influences drug response individual difference as
A pharmacological subdiscipline is formally proposed.The concept of pharmacogenomics also occurs therewith.Pharmacogenomics are logical
Cross associated gene expression or the suction of single nucleotide polymorphism (single nucleotide polymorphism, SNP) and drug
It receives, distribution, metabolism, drain (ADME) process and drug receptor target, to study congenital heredity or the day after tomorrow of patient's carrying
The hereditary variation of acquisition is on the subject of pharmaceutically-active influence.Patent CN107273710A, which is disclosed, a kind of establishes drug metabolic enzyme
The method of gene and the relational model of drug metabolism, this method include determining single polymorphic site and phannacokinetic profiles relationship
And the relationship of multiple polymorphic sites combination and phannacokinetic profiles, it is final to obtain drug metabolic enzyme gene and drug metabolism
Relational model.Pharmacogenomics are the important components of accurate medical treatment, selection of clinical optimal treatment drug are being instructed, with most
In terms of suitable dosage improves curative effect of medication, reduces or avoid adverse reaction, improves prognosis and Economy type medicine cost, have important
Meaning.From nineteen ninety, cardiovascular disease is continuously China resident first place cause of death, and is on the rise.It is cardiovascular
The use of drug is similarly subjected to the guidance of pharmacogenomics.Patent CN101760528B discloses a kind of drug metabolism relevant bits
Point detecting method, including detection drug metabolism related gene CYP2C9 gene, CYP2C19 gene, CYP2D6 gene and CYP3A4
Gene, the invention are whether to need consulting profession doctor when taking some drugs to determine whether to take and taking dose etc. provides
Reference frame.Patent CN107022611A discloses one kind and precisely uses for detecting 4 kinds of common clinical cardiovascular and cerebrovascular disease drugs
The method and primer special of medicine.Efficiently disposably experiment is completed and aspirin resistance, nitroglycerin resistance, warfarin for the invention
Aspirin, nitroglycerin, China are completed in the relevant 8 SNP sites detection of individual difference, clopidogrel Resistant, disposable experiment
The accurate medication genetic test of method woods, clopidogrel.More, clinically used cardiovascular drugs such as Statins is studied at present to drop
Rouge medicine, antiplatelet drug take anticoagulation, beta-blockers and angiotensin converting enzyme inhibitors (angiotensin
Converting enzyme inhibitors, ACEI) etc., curative effect of medication caused by all existing due to individual inheritance difference
Individual difference.
The curative effect of lipid-lowering statins is related to individual inheritance difference.Statins are most widely used in the world at present
A kind of blood lipid-lowering medicine, can pass through reduce blood lipid reduce cardiovascular and cerebrovascular disease risk.But individual difference results in some trouble
Person's Bloodlipid-lowering is bad and the side effects such as myalgia and myasthenia.Have multiple candidate genes and Bloodlipid-lowering at present, reduce the heart
Vascular death is related to Risk of myocardial infarction.Patent CN107099602A is disclosed a kind of while being detected statins metabolism
The kit of gene multisite mutation includes that APOE, SLCO1B1, CETP, ABCB1, mthfr gene site are drawn including detecting
Object to and probe pair, be mainly used for the personalized medicine auxiliary diagnosis of statins: Simvastatin, general is cut down Atorvastatin
Statin etc..Some researches show that the protein of the genes such as SLCO1B1, ABCB1 coding is statins transhipment and metabolic pathway
Major protein, these gene polynorphisms will affect the function of its protein, and then influence the curative effect of statins.Organic yin
Ion transport polypeptide (Organic Anion-Transporting Polypeptide, OATP) is one in intake type transporter
Major class is under the jurisdiction of Solute Transport body superfamily (super family of solute carriers, SLC), for inside and outside source
Property the absorption of substance, especially drug, distribution, eliminate there is great influence, encoding gene is referred to as SLCO gene.
There are 20 or more function genetic polymorphisms, the variations of single base nucleotide polymorphic site to cause for SLCO1B1 gene
The change of statins drug effect.1 transport protein (ATP-binding of atp binding cassette B subfamily member
Cassette subfamiliy B member 1 transporter, ABCB1) gene, the product of gene coding is P- sugar
Albumen (P-gp).In the case that the major function of P-gp is hydrolysis consumption ATP, inverse concentration gradient transhipment drug or endogenous object
Matter, so that intracellular concentration declines.ABCB1 is mainly distributed on the cell membrane of epithelial cell and endothelial cell.Physiological status
Under, the toxin of body and exogenous material can be transported out cell by ABCB1 transporter, play the role of protection.The base of ABCB1
Because polymorphism can also cause the change of some drugs drug effect, the study found that the haplotype of ABCB1 potentially affects statins
The curative effect of object.
Coumarin anticoagulant class (such as warfarin), anti-platelet aggregation medicinal (such as aspirin, chlorine in anticoagulant
Pyrrole Gray etc.), also all with relevant drug metabolism gene-correlation.Aspirin antiplatelet effects mechanism is directly and irreversibly
Inhibit Cycloxygenase 1 and 2 (COX-1 and COX-2), subtract the synthesis of prostaglandin, inhibits blood platelet synthesis thromboxane A2 (TXA2),
To inhibit platelet aggregation.That studies at present has PTGS1, GP1BA, LTC4S with aspirin metabolism related gene.Patent
CN107142307A also discloses primer sets, kit and the side for instructing aspirin personalized medicine related gene to detect
Method, the primer sets include being directed to three GPIa (807C > T), GPIa (873G > A) and COX1 (- 1676A > G) gene locis
The specific primer and probe of polymorphic detection.Patent CN106434943A discloses related for aspirin personalized medicine
The kit and its detection method of gene SNP detection, the kit include detection PTGS1 gene, CYP2C9 gene, CYP2D6
Two forward primers and a reverse primer of gene and CYP2C19 gene corresponding site.Patent CN104975016B is disclosed
The relevant P2Y1 gene polymorphism sites of aspirin resistance and application.Patent CN106947815A discloses a kind of for detecting
The method and primer special of aspirin and the accurate medication of nitroglycerin.If these site mutations are to lose function allele to make
The curative effect of aspirin reduces, and increases the incidence of patient's cardiovascular event.
Warfarin keeps liver synthesis factor and the reduction of the factor VII, Ⅸ and Ⅹ anticoagulant by antagonism vitamin K, is to face
Common anticoagulant on bed.The warfarin of normal dose can be anticoagulant, underdosage, and the treatment effect of anti-tampon is not achieved
Fruit；It is excessive then be easy to cause various bleedings again.Since medicament metabolism ability has differences between individual patients, Different Individual is due to base
Because of factors such as polymorphism, BMI, ages, it is different using the effective dose of warfarin.The active constituent of warfarin is by drug
Metablic enzyme of cytochrome P4502C9 subtribe (CYP2C9) metabolism, the mutation in the certain sites of CYP2C9 gene or missing will lead to
The reduction of CYP2C9 enzymatic activity, so that the accretion rate of warfarin in vivo slows down.VKORC1 be vitamin K oxide also
Protoenzyme complex subunit 1, it can promote the formation of vitamin K dependent form coagulation factor by the oxidation of inhibition vitamin K.
The variation of VKORC1 gene will lead to increasing or decreasing for such coagulation factor, cause being raised and lowered for patient's clotting ability.
Correlative study discovery CYP2C9 and VKORC1 gene pleiomorphism explains 50% or so warfarin individual dose.Such as: patent
CN101182573A discloses the determination method of warfarin Chinese population individual dosage, including extracts patient gene's group DNA,
Then the genotype of patient age, weight and CYP2C9 and VKORC1 are easily derived to the dosage of individual patients.Specially
Sharp CN102719524A is disclosed for the detection of gene associated with individualized medication of warfarin (CYP2C9 and VKORC1) SNP site
Kit and its PCR amplification method.Patent TWI334886B discloses the sequence of VKORC1 gene promoter or activity and can be used to
The warfarin of prediction study subject answers dosage.In August, 2007, U.S. FDA make clear stipulaties, it is desirable that in the quotient of warfarin
The variation that CYP2C9 and VKORC1 gene is indicated on product label can make one to generate drug the information such as different reactions, this abundant body
The necessity that associated genotype detection is carried out to the patient for taking warfarin is showed.
Clopidogrel belongs to platelet aggregation inhibitor as widely applied anticoagulant, and there is also individual weak curative effects
Different obvious phenomenon.Clopidogrel is by selectively inhibiting the combination of ADP and platelet receptor and inhibiting the sugared egg of ADP mediation
The activation of white II b/ of GP, III a compound, and inhibit platelet aggregation.A kind of pro-drug of clopidogrel, i.e., must be thin through internal liver
Born of the same parents' cytochrome p 450 (CYP450) metabolic conversion can play drug effect for active metabolite.Studies have shown that CYP2C19 gene and chlorine pyrrole
The metabolism of Gray is related.The reaction of clopidogrel significantly drops in the blood platelet for carrying CYP2C19*2 missing function allele person
Low, active metabolite and the blood platelet inhibition that can be detected in blood plasma are decreased obviously, and cardiovascular event incidence obviously increases.
Patent CN101978073B discloses the method and composition for assessing drug response, and the method includes using gene
(CYP2C19) polymorphism analysis determines subject to the therapeutic scheme of platelet aggregation related disease or the adaptability of intervention.Specially
Sharp CN106048076A is disclosed to be examined for clopidogrel personalized medicine related gene (CYP2C19*2, CYP2C19*3) SNP
The kit and its detection method of survey.
With the development of gene sequencing technology, the expense of genome sequence determination is lower and lower, collects individual specimen heredity
The speed of information is also getting faster.Second generation high throughput sequencing technologies (next-generation sequencing
Technology) have the advantages that quick, accurate, low cost, the various types of multiple genes can be mutated simultaneously and detected,
Be widely used in the cause of disease detection and molecular genetics diagnosis of genetic defect, however up to the present, always there has been no
Specifically for high-throughput probe, chip or the kit of the drug metabolism genetic test of drug for cardiovascular disease, so that cardiovascular
The progress in the drug metabolism gene screening field of medication seriously lags, because curative effect of medication failure and adverse drug reactions event are frequent
Occur, the screening of the relevant drug metabolism gene of medication patient can not be implemented, and instinct assesses suitable dose early, predicts whether to deposit
In adverse drug reactions, avoid causing because of medication problem aggravation, deteriorate, due to do not have the screening of drug metabolism gene without
It can be paid close attention to early, not only seriously affect the clinical treatment of patient, also to patient and its huge pain of family's bring
With heavy financial burden.At present commercially the mankind's full-length genome exon sequencing price between 6 thousand to 8 thousand RMB, and
The gene sequencing cost of probe groups of the invention only has 2 thousand to 3 thousand RMB, can substantially reduce the financial burden of patient, be
A kind of screening instruments of the drug metabolism gene of very useful drug for cardiovascular disease.
In conclusion develop a kind of drug metabolism gene of efficient, accurate, quick, low price drug for cardiovascular disease
Kit assists individuation to take drugs, meaning is very great for the drug metabolism gene screening of drug for cardiovascular disease.
Summary of the invention
The present inventor filters out about 66 kinds of bases relevant to the drug metabolism of drug for cardiovascular disease by creative work
Cause, preferably 39 kinds provide probe groups, chip or the kit of the corresponding combination, Ke Yiyong according to any combination of these genes
In drug metabolism study, drug screening, medicine curative effect evaluation or determine the dosage taken drugs, it is preferred that research patient carries
Congenital heredity or acquired hereditary variation on pharmaceutically-active influence.By detecting the drug for cardiovascular disease
The relevant gene of drug metabolism, the detection of progress medication individual molecular science of heredity, the crowd for influencing curative effect of medication polymorphic site are general
It looks into, to reduce the incidence of medication failure, adverse drug reaction event.
Meanwhile probe groups of the present invention, chip and kit are with efficient, detection site is more, accurate, quick, real
The advantage strong and at low cost with property.
Preferably, the method for the relevant gene of the screening drug metabolism is in conjunction with mass data library searching and/or to trouble
Person's gene sequencing result is screened.It is further preferred that the database is omim database, by inquiring OMIM data
Library and standard literature determine gene relevant to the drug metabolism of drug for cardiovascular disease.
The first aspect of the present invention, the reagent for being related to a kind of drug metabolism related gene for detecting drug for cardiovascular disease exist
Application in the product of preparation research drug metabolism, the drug metabolism related gene be ABCB1, ACE, ACE2, ACY3,
ADD1、ADRA1A、ADRA2A、ADRB1、ADRB2、AGT、AGTR1、AOX1、APOB、APOE、BDKRB2、C18orf21、
CACNA1C、CAMK1D、COQ2、CYP11B2、CYP2C19、CYP2C9、CYP2D6、CYP3A4、CYP3A5、DPYS、GALNT2、
GNB3、GP1BA、HMGCR、KCNE1、KCNH2、KCNJ1、KIF6、LDLR、LTC4S、MMP3、MTHFR、MTR、MTRR、NAT2、
NEDD4L、NOS1AP、PRKCA、NOS3、NR1H3、NR3C2、PEAR1、PLCD3、PPARA、PRCP、YEATS4、PROX1、
PTGS1, PTGS2, PTPRD, SCN1A, SLC14A2, SLCO1B1, STK39, TCF7L2, TLR4, TRIB3, VKORC1, WNK1 or
Two or more combination in CYP4F2.
Preferably, the drug metabolism related gene be CYP2C9, CYP2C19, CYP2D6, SLCO1B1, APOE,
NAT2、CYP3A5、SLC14A2、KCNH2、CACNA1C、NOS1AP、PLCD3、GNB3、ABCB1、AGTR1、ACE、BDKRB2、
PTPRD、CYP11B2、ADRB2、ADRB1、ACY3、ADRA2A、STK39、YEATS4、KCNJ1、ADD1、PTGS1、LTC4S、
Two kinds or two kinds in CYP3A4, PEAR1, COQ2, KCNE1, SCN1A, AOX1, VKORC1, CYP4F2, TLR4 or MTHFR with
On combination.
In the specific embodiment of the present invention, the reagent can 66/39 angiocarpy of specificity capture simultaneously
The drug metabolism gene of Systemic administration, the drug metabolism related gene be ABCB1, ACE, ACE2, ACY3, ADD1,
ADRA1A、ADRA2A、ADRB1、ADRB2、AGT、AGTR1、AOX1、APOB、APOE、BDKRB2、C18orf21、CACNA1C、
CAMK1D、COQ2、CYP11B2、CYP2C19、CYP2C9、CYP2D6、CYP3A4、CYP3A5、DPYS、GALNT2、GNB3、
GP1BA、HMGCR、KCNE1、KCNH2、KCNJ1、KIF6、LDLR、LTC4S、MMP3、MTHFR、MTR、MTRR、NAT2、
NEDD4L、NOS1AP、PRKCA、NOS3、NR1H3、NR3C2、PEAR1、PLCD3、PPARA、PRCP、YEATS4、PROX1、
PTGS1, PTGS2, PTPRD, SCN1A, SLC14A2, SLCO1B1, STK39, TCF7L2, TLR4, TRIB3, VKORC1, WNK1 and
The combination of CYP4F2.
In the specific embodiment of the present invention, the drug metabolism related gene be CYP2C9, CYP2C19,
CYP2D6、SLCO1B1、APOE、NAT2、CYP3A5、SLC14A2、KCNH2、CACNA1C、NOS1AP、PLCD3、GNB3、
ABCB1、AGTR1、ACE、BDKRB2、PTPRD、CYP11B2、ADRB2、ADRB1、ACY3、ADRA2A、STK39、YEATS4、
KCNJ1、ADD1、PTGS1、LTC4S、CYP3A4、PEAR1、COQ2、KCNE1、SCN1A、AOX1、VKORC1、CYP4F2、TLR4
With the combination of MTHFR.
Preferably, the drug metabolism related gene is the drug metabolism gene of drug for cardiovascular disease.
Preferably, probe sequence is designed and synthesized by known, general method according to above-mentioned each gene, to target fragment
Specific capture, amplification, sequencing are carried out, to achieve the purpose that detect sample.
Studying medicament metabolism of the present invention includes drug metabolism study, drug screening, the medicine of drug for cardiovascular disease
Object curative effect evaluation or dosage determine.
Preferably, the studying medicament metabolism is to study the congenital heredity or acquired heredity change that patient carries
It is different on pharmaceutically-active influence.
Preferably, the drug metabolism be drug absorption, distribution, metabolism, excretion (ADME) process and drug by
Body target.
Drug metabolism related gene of the present invention individually or combinations thereof drug metabolism as drug for cardiovascular disease
The target spot of research.
Preferably, each drug metabolism related gene of the present invention, which mutates, can indicate the metabolic condition of the drug
And individual tolerance, anti-medicine situation.
Preferably, each gene or the abrupt climatic change of its assortment of genes are carried out by the method for gene sequencing.
Preferably, the reagent is selected from the probe groups of the drug metabolism related gene of detection drug for cardiovascular disease, draws
Object group or genetic chip；The product includes the reagent, alternatively, the product is kit.
The second aspect of the present invention is related to a kind of method for carrying out genetic test using reagent of the present invention, including
DNA to be checked is mixed with the reagent.
Preferably, the reagent is probe groups, primer sets or genetic chip.
Preferably, the genetic test include detect the gene with or without, alternatively, mutation with or without.Into one
Step is preferred, and the mutation is selected from shearing site mutation, nonsense mutation or frameshift mutation.
Preferably, the genetic test includes detecting the level of the gene.
The method of genetic test of the present invention can be non-diagnostic purpose or diagnostic purpose.
Non-diagnostic purpose of the present invention is the prominent of each gene in combining using the probe groups detection said gene
Become with or without and detect the level of each gene in said gene combination.
Diagnostic purpose of the present invention is to diagnose or predict that patient is using after the probe groups detection said gene
It is no to be suitble to take the drug, the side effect taken drugs or the dosage taken drugs determination.
The third aspect of the present invention is related to a kind of drug metabolism containing detection drug for cardiovascular disease of the present invention
The genetic chip of the probe groups of related gene.
Preferably, the genetic chip further includes solid phase carrier.It is further preferred that the solid phase carrier is selected from nitre
The combination of one or more of acid cellulose film, nylon membrane, polystyrene, sheet glass, silicon wafer or polypropylene screen.
Preferably, the genetic chip further includes corresponding primer sets.
Preferably, the probe be fixed on the method on solid phase carrier be selected from in-situ synthesis, point sample method or other fix
Method.
Wherein, the probe in genetic chip is fixed on the solid phase carrier.Preferably, probe sequence is intensively had
It arranges to sequence and is fixed in the pre-set region of solid phase carrier, form micro-detector part.
The fourth aspect of the present invention is related to a kind of drug metabolism containing detection drug for cardiovascular disease of the present invention
The kit of the probe groups of related gene or genetic chip of the present invention.
Preferably, the kit further includes hybridization solution and/or buffer and/or cleaning solution.
It is further preferred that the kit further include harvesting buffer, hybridization buffer, combination buffer, rinsing liquid,
NaOH solution, Tris-HCl buffer, PCR reaction solution, TE buffer.
The fifth aspect of the present invention provides the preparation method of the kit described in one kind containing probe groups, comprising:
1) corresponding probe sequence is prepared according to the drug metabolism gene of above-mentioned drug for cardiovascular disease；
2) biotin labeling is added for the probe sequence that step 1) obtains；
3) hybridization solution needed for preparing and/or buffer and/or cleaning solution.
The sixth aspect of the present invention is related to a kind of reagent of drug metabolism related gene for detecting drug for cardiovascular disease,
The drug metabolism related gene be ABCB1, ACE, ACE2, ACY3, ADD1, ADRA1A, ADRA2A, ADRB1, ADRB2,
AGT、AGTR1、AOX1、APOB、APOE、BDKRB2、C18orf21、CACNA1C、CAMK1D、COQ2、CYP11B2、CYP2C19、
CYP2C9、CYP2D6、CYP3A4、CYP3A5、DPYS、GALNT2、GNB3、GP1BA、HMGCR、KCNE1、KCNH2、KCNJ1、
KIF6、LDLR、LTC4S、MMP3、MTHFR、MTR、MTRR、NAT2、NEDD4L、NOS1AP、PRKCA、NOS3、NR1H3、
NR3C2、PEAR1、PLCD3、PPARA、PRCP、YEATS4、PROX1、PTGS1、PTGS2、PTPRD、SCN1A、SLC14A2、
Two or more combination in SLCO1B1, STK39, TCF7L2, TLR4, TRIB3, VKORC1, WNK1 or CYP4F2.
Preferably, the drug metabolism related gene be CYP2C9, CYP2C19, CYP2D6, SLCO1B1, APOE,
NAT2、CYP3A5、SLC14A2、KCNH2、CACNA1C、NOS1AP、PLCD3、GNB3、ABCB1、AGTR1、ACE、BDKRB2、
PTPRD、CYP11B2、ADRB2、ADRB1、ACY3、ADRA2A、STK39、YEATS4、KCNJ1、ADD1、PTGS1、LTC4S、
Two kinds or two kinds in CYP3A4, PEAR1, COQ2, KCNE1, SCN1A, AOX1, VKORC1, CYP4F2, TLR4 or MTHFR with
On combination.
Wherein, the reagent is selected from probe groups, primer sets or genetic chip.
The seventh aspect of the present invention is related to a kind of detection method of the drug metabolism related gene of drug for cardiovascular disease,
Include:
(1) DNA to be detected is extracted, full-length genome library is prepared；
(2) the full-length genome library obtained step (1) is related to the detection drug metabolism of drug for cardiovascular disease
The reagent of gene mixes, and carries out PCR；
(3) PCR product described in purification step (2)；
(4) PCR product of step (3) after purification is sequenced；
(5) sequencing result of step (4) is analyzed；
Wherein, the reagent is selected from probe groups, primer sets or gene core.
Preferably, the analysis includes snp analysis and/or InDel analysis.
The eighth aspect of the present invention, is related to a kind of marker of studying medicament metabolism, and the marker is selected from gene
ABCB1、ACE、ACE2、ACY3、ADD1、ADRA1A、ADRA2A、ADRB1、ADRB2、AGT、AGTR1、AOX1、APOB、APOE、
BDKRB2、C18orf21、CACNA1C、CAMK1D、COQ2、CYP11B2、CYP2C19、CYP2C9、CYP2D6、CYP3A4、
CYP3A5、DPYS、GALNT2、GNB3、GP1BA、HMGCR、KCNE1、KCNH2、KCNJ1、KIF6、LDLR、LTC4S、MMP3、
MTHFR、MTR、MTRR、NAT2、NEDD4L、NOS1AP、PRKCA、NOS3、NR1H3、NR3C2、PEAR1、PLCD3、PPARA、
PRCP、YEATS4、PROX1、PTGS1、PTGS2、PTPRD、SCN1A、SLC14A2、SLCO1B1、STK39、TCF7L2、TLR4、
Two or more combination in TRIB3, VKORC1, WNK1 or CYP4F2.
Preferably, the marker be selected from gene be CYP2C9, CYP2C19, CYP2D6, SLCO1B1, APOE, NAT2,
CYP3A5、SLC14A2、KCNH2、CACNA1C、NOS1AP、PLCD3、GNB3、ABCB1、AGTR1、ACE、BDKRB2、PTPRD、
CYP11B2、ADRB2、ADRB1、ACY3、ADRA2A、STK39、YEATS4、KCNJ1、ADD1、PTGS1、LTC4S、CYP3A4、
Two or more group in PEAR1, COQ2, KCNE1, SCN1A, AOX1, VKORC1, CYP4F2, TLR4 or MTHFR
It closes.
The ninth aspect of the present invention is related to the drug metabolism related gene of drug for cardiovascular disease in preparation research drug generation
The application in product thanked, the drug metabolism related gene be ABCB1, ACE, ACE2, ACY3, ADD1, ADRA1A,
ADRA2A、ADRB1、ADRB2、AGT、AGTR1、AOX1、APOB、APOE、BDKRB2、C18orf21、CACNA1C、CAMK1D、
COQ2、CYP11B2、CYP2C19、CYP2C9、CYP2D6、CYP3A4、CYP3A5、DPYS、GALNT2、GNB3、GP1BA、
HMGCR、KCNE1、KCNH2、KCNJ1、KIF6、LDLR、LTC4S、MMP3、MTHFR、MTR、MTRR、NAT2、NEDD4L、
NOS1AP、PRKCA、NOS3、NR1H3、NR3C2、PEAR1、PLCD3、PPARA、PRCP、YEATS4、PROX1、PTGS1、
PTGS2, PTPRD, SCN1A, SLC14A2, SLCO1B1, STK39, TCF7L2, TLR4, TRIB3, VKORC1, WNK1 or CYP4F2
In two or more combination.
Preferably, the drug metabolism related gene be CYP2C9, CYP2C19, CYP2D6, SLCO1B1, APOE,
NAT2、CYP3A5、SLC14A2、KCNH2、CACNA1C、NOS1AP、PLCD3、GNB3、ABCB1、AGTR1、ACE、BDKRB2、
PTPRD、CYP11B2、ADRB2、ADRB1、ACY3、ADRA2A、STK39、YEATS4、KCNJ1、ADD1、PTGS1、LTC4S、
Two kinds or two kinds in CYP3A4, PEAR1, COQ2, KCNE1, SCN1A, AOX1, VKORC1, CYP4F2, TLR4 or MTHFR with
On combination.
The tenth aspect of the present invention, the reagent for being related to a kind of drug metabolism related gene for detecting drug for cardiovascular disease exist
The application in product is prepared, the product is that drug metabolism study, drug screening, the curative effect of medication of drug for cardiovascular disease are commented
Estimate or dosage determine product, alternatively, research patient carry congenital heredity or acquired hereditary variation to cardiovascular drug
The influence of object effect, alternatively, cardiovascular system Prognostic, pregnant preceding early warning indicate offspring to the medicine of drug for cardiovascular disease
The product of the evaluation of hazard grade of object metabolism.Preferably, the product is selected from probe groups, kit, genetic chip or drug.
The eleventh aspect of the present invention is related to a kind of dosing method of drug for cardiovascular disease, the method packet
The drug metabolism for including the reagent detection drug for cardiovascular disease of the drug metabolism related gene of detection drug for cardiovascular disease is related
The mutation or level of gene.
Drug for cardiovascular disease of the present invention is selected from lipid-lowering statins, antiplatelet drug, antianginal drug, the anti-heart
It restrains not normal medicine, antihypertensive, anti-cardiac insufficiency medicine, peripheral vasodilators, calcium antagonist, take anticoagulation, beta receptor blocking
Agent or angiotensin converting enzyme inhibitors (angiotensin converting enzyme inhibitors, ACEI)；Institute
The antianginal drug stated is selected from nitrate esters, nitroglycerin class, nifedipine class or diltiazem class.Preferably, the heart
Vascular system medication is selected from aspirin, nitroglycerin, warfarin, clopidogrel, Simvastatin, Atorvastatin, general cuts down him
Spit of fland, nitroglycerin.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only section Example of the invention, rather than all.Based on the embodiments of the present invention, those of ordinary skill in the art exist
Every other embodiment obtained under the premise of creative work is not made, shall fall within the protection scope of the present invention.
The probe groups of the present invention of embodiment 1 design and prepare
One, the screening of the drug metabolism gene of drug for cardiovascular disease
In the present embodiment detection drug for cardiovascular disease drug metabolism related gene are as follows: ABCB1, ACE, ACE2,
ACY3、ADD1、ADRA1A、ADRA2A、ADRB1、ADRB2、AGT、AGTR1、AOX1、APOB、APOE、BDKRB2、C18orf21、
CACNA1C、CAMK1D、COQ2、CYP11B2、CYP2C19、CYP2C9、CYP2D6、CYP3A4、CYP3A5、DPYS、GALNT2、
GNB3、GP1BA、HMGCR、KCNE1、KCNH2、KCNJ1、KIF6、LDLR、LTC4S、MMP3、MTHFR、MTR、MTRR、NAT2、
NEDD4L、NOS1AP、PRKCA、NOS3、NR1H3、NR3C2、PEAR1、PLCD3、PPARA、PRCP、YEATS4、PROX1、
PTGS1, PTGS2, PTPRD, SCN1A, SLC14A2, SLCO1B1, STK39, TCF7L2, TLR4, TRIB3, VKORC1, WNK1 and
CYP4F2。
Being diagnosed as disease of cardiovascular system to 35, (5 are diagnosed as pheochromocytoma, and 4 are diagnosed as primary pulmonary arterial
High pressure, 3 are diagnosed as familial hypercholesterolemia, and 2 are diagnosed as familial hypertriglyceridemia, and 8 are diagnosed as heredity
Property arotic disease, 13 are diagnosed as arrhythmia cordis) patient (disease of cardiovascular system made a definite diagnosis of patient's position hospital clinical, and
Each patient signs informed and voluntary compliance agreement, and ratifies through hospital's Medical Ethics Committee), and take cardiovascular drugs (ammonia chlorine
Horizon, Losartan, Losartan, captopril, clopidogrel, warfarin, Simvastatin, Atorvastatin, phenytoinum naticum) 3 months
Above patient carries out full sequencing of extron group, and covering, detection range are whole more than 30,000 genes known to human genome
All exons, wherein remaining gene (accounting for the overwhelming majority) of mutated gene high concentration, genome involves although also having,
The frequency of mutation is very low, many to be even only found in single patient, is difficult to determine with the correlation of disease.Therefore it is designing
When probe groups of the present invention, highest above-mentioned 66 genes of this patient mutation probability are incorporated in, it is equal that frequency is involved in mutation
Greater than 5, testing cost can be rationally controlled while increasing detection drug metabolism related gene to the maximum extent in this way.
Preferably 39 kinds of genes from above-mentioned 66 kinds of drug metabolism related genes are as follows: CYP2C9, CYP2C19, CYP2D6,
SLCO1B1、APOE、NAT2、CYP3A5、SLC14A2、KCNH2、CACNA1C、NOS1AP、PLCD3、GNB3、ABCB1、AGTR1、
ACE、BDKRB2、PTPRD、CYP11B2、ADRB2、ADRB1、ACY3、ADRA2A、STK39、YEATS4、KCNJ1、ADD1、
PTGS1, LTC4S, CYP3A4, PEAR1, COQ2, KCNE1, SCN1A, AOX1, VKORC1, CYP4F2, TLR4 and MTHFR.
Two, the preparation of probe
According to the genome sequence of said medicine metabolic gene (66 kinds/39 kinds), those skilled in the art can pass through public affairs
Know, general method designs and synthesizes probe sequence (such as patent WO2013/003585), to the probe of synthesis in the present embodiment
Sequence carries out biotin labeling, concrete operations are as follows: uses approach well known synthesising probing needle sequence, is evenly mixed in total volume
The dH of 1.2mL2In O, taking wherein 15 μ L using general PCR primer, (5 ' terminal sequences are GACTACATGGGACAT (SEQ ID
NO:1), the sequence at 3 ' ends is GGAACCTACGACGTA (SEQ ID NO:2)), point three pipes carry out PCR amplification, wherein primer
GACTACATGGGACAT (SEQ ID NO:1) is the primer with biotin labeling.
PCR amplification system: above-mentioned probe solution, 5 μ L；Forward primer (25 μM), 2 μ L；Reverse primer (25 μM), 2 μ L；
MgCl2(50mM), 4 μ L；10x Platinum Taq polymerase buffer (is purchased from Life Technologies), 5 μ L；dNTPs
(every kind of 10mM), 4 μ L；Platinum Taq polymerase (5U/ μ L is purchased from Life Technologies), 1 μ L；H2O, 27 μ L；
50 μ L of total volume.
Amplification condition: 98 DEG C, 30s；(98 DEG C, 30s, 60 DEG C, 25s, 72 DEG C, 45s) 35 circulations；72 DEG C, 5min.
After purification with MinElute PCR purification kit (being purchased from Life Technologies) by PCR product, it takes
500ng is combined PCR product with MyOne streptavidin magnetic bead (being purchased from Invitrogen).Then alkalinity NaOH is added
By the complementary strand denaturation of not biotin, elute；Then by 100 DEG C of the formamide liquid scrubbing of entire magnetic bead, make to visit
Needle is separated from magnetic bead.With the probe groups of the present embodiment for obtaining biotin labeling after ethanol precipitation.
Embodiment 2: kit forms, preparation and use of the present invention
It is to pass through detection for detecting the kit of the drug metabolism gene of drug for cardiovascular disease described in the present embodiment
The personalized medicine or curative effect of the mutation of above-mentioned 66/39 drug metabolism gene comes adjuvant drug person is insufficient, bad kickback of using medicine
The kit of risk profile.
One, the composition of kit
The ingredient that the kit includes are as follows: the probe groups obtained of embodiment 1 (160 μ L, 150ng/ μ L), enrichment buffering
Liquid (208 μ L), hybridization buffer (800 μ L), combination buffer (3.2mL), rinsing liquid 1 (9mL), rinsing liquid 2 (45mL), NaOH
Solution (0.1M, 1mL), Tris-HCl buffer (1M, pH 7.5,1.2mL), PCR reaction solution (580 μ L), TE buffer (800 μ
L, 10mM Tris-HCl, 1mM EDTA adjust pH to 8.0, and water is settled to 500mL).Wherein each buffer composition is as follows:
(1) harvesting buffer (every 20 μ L):
People cot-1DNA (is purchased from Invitrogen), 7 μ L；Salmon sperm dna (is purchased from Invitrogen), 3 μ L；
Specificity closing primer, totally 10 μ L, every kind of primer concentration are 1nmol/ μ L:
Primer 1:
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCT (SEQ ID NO:3)；
Primer 2:
CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGC (SEQ ID NO:4)；
(2) hybridization buffer: the SSPE (being purchased from AMRESCO company) of 5 times of concentration, the Denhardt solution (purchase of 5 times of concentration
From USB company), 5mM EDTA (0.5M, pH8.0 are purchased from Mediatech company), 0.1%SDS；
(3) combination buffer: 1M NaCl, 10mM Tris-HCl (pH 7.5), 1mM EDTA
(4) (NaCl 175g, trisodium citrate 88g adjust pH to 7.4, dH to the SSC solution of 1:1 times of concentration of rinsing liquid2O is fixed
Hold to 1 liter), 0.1%SDS
(5) (NaCl 175g, trisodium citrate 88g adjust pH to 7.4, dH to the SSC solution of 2:0.1 times of concentration of rinsing liquid2O
It is settled to 1 liter), 0.1%SDS
(6) PCR reaction solution: 2 μ L dNTPs (every kind of 10mM),
0.5 μ L primer 1 (AATGATACGGCGACCACCGA*G (SEQ ID NO:5), 50pmol),
0.5 μ L primer 2 (CAAGCAGAAGACGGCATACG*A (SEQ ID NO:6), 50pmol), 20 5 times of μ L concentration
Phusion buffer (New England Biolab company), 1 μ L Hotstart Phusion enzyme (are purchased from New England
Biolabs), 5 μ L DMSO, 51 μ L dH2O；* indicating intermediate has thio-modification；
Two, the application of kit
1, DNA is extracted and prepared by full-length genome library
The DNA in peripheral blood is extracted using Qiagen DNA mini kit (250) (being purchased from Qiagen) kit.With point
Light photometric quantification, agarose gel electrophoresis detect sample quality, and complete genome dna electrophoresis band should usually be not less than
20kb.Concentration is adjusted to 75ng/ μ L by the DNA of quality inspection qualification, and total amount 3-5 μ g DNA is interrupted at random, expanded, to establish full base
Because of a group library.
2, the specificity capture and sequencing of drug target metabolic gene segment
1) it prepares following mixed system: taking the full-length genome library of 1 μ g step 1 built, 13 μ L harvesting buffers, 5 μ
The probe groups of the present invention of L embodiment 1；It is placed in PCR instrument: 95 DEG C, 7min, 65 DEG C later, 2min；
2) the 23 μ L of hybridization buffer of 65 DEG C of preheatings is taken to be added to above-mentioned steps 1) in obtained mixed liquor, then in PCR
Hybridize 22 hours for 65 DEG C on instrument, obtains enrichment system mixture.
3) magnetic bead is made sufficiently to suspend MyOne C1 Streptavidin MagneSphere (being purchased from Invitrogen) whirlpool concussion, it is of short duration
Centrifugation, is centrifuged magnetic bead to bottom of the tube, takes 50 μ L MyOne C1 Streptavidin MagneSpheres to the centrifuge tube of new 1.5mL.
4) the 1.5mL centrifuge tube whirlpool equipped with 50 μ L MyOne C1 Streptavidin MagneSpheres is shaken at least 5s, makes magnetic bead
It sufficiently suspends, is put on magnetic frame and remain stationary one minute (should not rotating centrifugal pipe) after of short duration centrifugation, careful inhale abandons supernatant.
5) centrifuge tube is removed, the combination buffer of 1 times of concentration of 50 μ L is added, rotation nest shakes at least 5s, after of short duration centrifugation
It is put on magnetic frame static one minute, careful inhale abandons supernatant, in triplicate.
6) centrifuge tube is removed, the combination buffer of 2 times of concentration of 100 μ L is added, rotation nest shakes at least 5s, after of short duration centrifugation
It is put on magnetic frame static one minute.
7) the enrichment system mixture that step 2) obtains is added in rapid centrifuge tube 6), rotation nest concussion at least 5s is (no
With centrifugation), it is placed in room temperature on gyroscope and rotates 1 hour (60 revs/min).
8) then, utilize 1 room temperature cleaning step 7 of rinsing liquid) magnetic bead it is primary, 15 minutes, then again with rinsing liquid 2 clean
3 times, 65 DEG C, every time 15 minutes.
9) magnetic bead of step 8) is eluted 10 minutes at room temperature with 0.1M NaOH, then shakes eluent whirlpool at least
5s is put on magnetic frame static one minute after of short duration centrifugation, then supernatant is transferred to containing 70 μ L Tris-HCl buffers
In the clean centrifuge tube of (1M, pH 7.5).
10) DNA solution that step 9) obtains is carried out using Qiagen MinElute Column (being purchased from Qiagen) pure
Change, Laboratory Manual of the purification step referring to Qiagen MinElute Kit.
11) DNA purified finally expands 15 circulations by PCR:
PCR reaction solution: 2 μ L dNTPs (every kind of 10mM),
0.5 μ L primer 1 (AATGATACGGCGACCACCGA*G (SEQ ID NO:5), 50pmol),
0.5 μ L primer 2 (CAAGCAGAAGACGGCATACG*A (SEQ ID NO:6), 50pmol), 20 5 times of μ L concentration
Phusion buffer (is purchased from New England Biolabs), and 1 μ L Hotstart Phusion enzyme (is purchased from New England
Biolabs), 5 μ L DMSO, 51 μ L dH2O；* indicating intermediate has thio-modification.PCR program: 98 DEG C, 30s (1cycle)；98
DEG C, 25s；65 DEG C, 30s；72 DEG C, 30s (15cycles)；72 DEG C, 5min (1cycle).
12) using Agencourt AMPure XP Nucleic acid purification kits (be purchased from Beckman Coulter), according to making
With handbook purification step 11) PCR product.
13) DNA product that step 12) obtains is sequenced on 500 sequenator of Illumina Nexseq.
3, bioinformatic analysis process and result output:
(1) snp analysis process
1. obtaining original short sequence；
2. removing connector and the low quality data etc. in sequencing data；
3. short sequence is navigated on the corresponding position of people's MHC3.6M genomic data with SOAPaligner software, it is used
To parameter: soap2.20-a-b-t-v 3-l 42-s 63-m100-x 400, wherein sequence mismatch number is 3, design parameter meaning
With reference to: http://soap.genomics.org.cn/soapaligner.html；
4. counting sequencing result information, short sequence quantity, target area covering size, average sequencing depth etc.；
5. SOAPsnp is used to find out the genotype in site, used parameter: soapsnp-i-d-o-r in target area
0.00005-e 0.0001-M-t-u-L-s-2-T, design parameter meaning reference: http://soap.genomics.org.cn/
soapsnp.html；
6. filtering the SNP of low quality value (mass value >=20) and low cover degree (depth >=10)；
7. being annotated using CCDS, human genome database (NCBI 37.2), dbSNP (v138) information to SNP, really
Determine gene, coordinate, the site mRNA, the amino acid change, (missense mutation/nonsense mutation/variable of SNP function of mutational site generation
Shearing site), SIFT prediction SNP influence protein function prediction etc.；
8. selecting and being not present common to disease sample and in normal group according to disease sample and normal specimens information
SNP as candidate SNPs, got rid of in candidate SNPs dbSNP, HAPMAP, 1000 people MHC3.6M genomes, other
The SNP occurred in exon sequencing project.Meanwhile SNPs of the SIFT prediction on protein function without influence is filtered out as last disease
The relevant candidate SNP s of disease；
(2) InDel analysis process
1. removal joint sequence and low-quality sequencing data are compared with Burrows-Wheeler Aligner (BWA)
Onto people's MHC3.6M genome, used parameter: bwa aln-L-l 31-i 10-k2-t 7-e 40, design parameter meaning ginseng
It examines: http://bio-bwa.sourceforge.net/bwa.shtml；
2. with GATK software find out sequence contained in insertion/deletion (InDel) information；
3. InDel is annotated using CCDS, human genome database (NCBI 37.2), dbSNP (v138) information,
Determine gene, coordinate, the site mRNA, the change of Coding region sequence, the influence to amino acid, InDel that mutational site occurs
Function (amino acid insertion/amino acid deletions/frameshift mutation).
Embodiment 3: the using effect verifying of kit of the present invention
Utilize kit of the present invention detection (detection probe comprising 66 kinds of genes that embodiment 2 prepares) 50
As a result sample confirms that the capture rate of purpose drug metabolism gene is satisfied, the average effective sequencing data amount of target area reaches
221.97Mb, the average sequencing depth of target area are 512.3X (being shown in Table 1), significantly larger than general diagnosis of genetic disorders requirement
(generally 20X).
The drug metabolism gene sequencing quality of data of 1 mesh of table
In conclusion provided by the present invention for the probe groups and examination of the drug metabolism gene for detecting drug for cardiovascular disease
Agent box has the characteristics that easy to operate, low in cost, specific good, high sensitivity, can at most detect simultaneously, define 66/39
The various types of the drug metabolism gene of a drug for cardiovascular disease are mutated, therefore probe groups and kit of the invention can be applied
It is diagnosed in the molecular genetics of medication individual, while the adverse drug reaction people at highest risk in screening drug user family members also can be used,
And carry out corresponding genetic counselling or antenatal intervention offer reference.Since Drugs for Cardiovascular Diseases is generally taken for a long time, therefore its
The consequence of the medication failure and adverse drug reaction that may occur be it is very serious, acute cardiocerebrovasculaevents events, tight such as occur
Weight organic impairment etc., detects the drug metabolism gene of medication patient, can accurately instruct the personalized medicine of patient, guarantees the heart
The use curative effect of blood vessel drug eluting guarantees the health of medication patient.Use of the invention can for patient take it is correct, reasonable,
The medication guide of individuation provides foundation, meets the development trend of accurate medical treatment.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
Sequence table
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Claims (10)
1. a kind of reagent for the drug metabolism related gene for detecting drug for cardiovascular disease is in the product of preparation research drug metabolism
In application, which is characterized in that the drug metabolism related gene be ABCB1, ACE, ACE2, ACY3, ADD1, ADRA1A,
ADRA2A、ADRB1、ADRB2、AGT、AGTR1、AOX1、APOB、APOE、BDKRB2、C18orf21、CACNA1C、CAMK1D、
COQ2、CYP11B2、CYP2C19、CYP2C9、CYP2D6、CYP3A4、CYP3A5、DPYS、GALNT2、GNB3、GP1BA、
HMGCR、KCNE1、KCNH2、KCNJ1、KIF6、LDLR、LTC4S、MMP3、MTHFR、MTR、MTRR、NAT2、NEDD4L、
NOS1AP、PRKCA、NOS3、NR1H3、NR3C2、PEAR1、PLCD3、PPARA、PRCP、YEATS4、PROX1、PTGS1、
PTGS2, PTPRD, SCN1A, SLC14A2, SLCO1B1, STK39, TCF7L2, TLR4, TRIB3, VKORC1, WNK1 or CYP4F2
In two or more combination.
2. application according to claim 1, which is characterized in that the drug metabolism related gene be CYP2C9,
CYP2C19、CYP2D6、SLCO1B1、APOE、NAT2、CYP3A5、SLC14A2、KCNH2、CACNA1C、NOS1AP、PLCD3、
GNB3、ABCB1、AGTR1、ACE、BDKRB2、PTPRD、CYP11B2、ADRB2、ADRB1、ACY3、ADRA2A、STK39、
YEATS4、KCNJ1、ADD1、PTGS1、LTC4S、CYP3A4、PEAR1、COQ2、KCNE1、SCN1A、AOX1、VKORC1、
Two or more combination in CYP4F2, TLR4 or MTHFR.
3. application according to claim 1 or 2, which is characterized in that the drug metabolism related gene be ABCB1,
ACE、ACE2、ACY3、ADD1、ADRA1A、ADRA2A、ADRB1、ADRB2、AGT、AGTR1、AOX1、APOB、APOE、BDKRB2、
C18orf21、CACNA1C、CAMK1D、COQ2、CYP11B2、CYP2C19、CYP2C9、CYP2D6、CYP3A4、CYP3A5、
DPYS、GALNT2、GNB3、GP1BA、HMGCR、KCNE1、KCNH2、KCNJ1、KIF6、LDLR、LTC4S、MMP3、MTHFR、
MTR、MTRR、NAT2、NEDD4L、NOS1AP、PRKCA、NOS3、NR1H3、NR3C2、PEAR1、PLCD3、PPARA、PRCP、
YEATS4、PROX1、PTGS1、PTGS2、PTPRD、SCN1A、SLC14A2、SLCO1B1、STK39、TCF7L2、TLR4、TRIB3、
The combination of VKORC1, WNK1 and CYP4F2.
4. application according to claim 1 to 3, which is characterized in that the studying medicament metabolism includes cardiovascular system
Drug metabolism study, drug screening, medicine curative effect evaluation or the dosage of system medication determine.
5. a kind of marker of studying medicament metabolism, which is characterized in that the marker be selected from Gene A BCB1, ACE, ACE2,
ACY3、ADD1、ADRA1A、ADRA2A、ADRB1、ADRB2、AGT、AGTR1、AOX1、APOB、APOE、BDKRB2、C18orf21、
CACNA1C、CAMK1D、COQ2、CYP11B2、CYP2C19、CYP2C9、CYP2D6、CYP3A4、CYP3A5、DPYS、GALNT2、
GNB3、GP1BA、HMGCR、KCNE1、KCNH2、KCNJ1、KIF6、LDLR、LTC4S、MMP3、MTHFR、MTR、MTRR、NAT2、
NEDD4L、NOS1AP、PRKCA、NOS3、NR1H3、NR3C2、PEAR1、PLCD3、PPARA、PRCP、YEATS4、PROX1、
PTGS1, PTGS2, PTPRD, SCN1A, SLC14A2, SLCO1B1, STK39, TCF7L2, TLR4, TRIB3, VKORC1, WNK1 or
Two or more combination in CYP4F2.
6. application of the drug metabolism related gene of drug for cardiovascular disease in the product of preparation research drug metabolism, feature
Be, the drug metabolism related gene be ABCB1, ACE, ACE2, ACY3, ADD1, ADRA1A, ADRA2A, ADRB1,
ADRB2、AGT、AGTR1、AOX1、APOB、APOE、BDKRB2、C18orf21、CACNA1C、CAMK1D、COQ2、CYP11B2、
CYP2C19、CYP2C9、CYP2D6、CYP3A4、CYP3A5、DPYS、GALNT2、GNB3、GP1BA、HMGCR、KCNE1、KCNH2、
KCNJ1、KIF6、LDLR、LTC4S、MMP3、MTHFR、MTR、MTRR、NAT2、NEDD4L、NOS1AP、PRKCA、NOS3、
NR1H3、NR3C2、PEAR1、PLCD3、PPARA、PRCP、YEATS4、PROX1、PTGS1、PTGS2、PTPRD、SCN1A、
Two or more in SLC14A2, SLCO1B1, STK39, TCF7L2, TLR4, TRIB3, VKORC1, WNK1 or CYP4F2
Combination.
7. a kind of reagent for the drug metabolism related gene for detecting drug for cardiovascular disease, which is characterized in that the drug generation
Thank related gene be ABCB1, ACE, ACE2, ACY3, ADD1, ADRA1A, ADRA2A, ADRB1, ADRB2, AGT, AGTR1,
AOX1、APOB、APOE、BDKRB2、C18orf21、CACNA1C、CAMK1D、COQ2、CYP11B2、CYP2C19、CYP2C9、
CYP2D6、CYP3A4、CYP3A5、DPYS、GALNT2、GNB3、GP1BA、HMGCR、KCNE1、KCNH2、KCNJ1、KIF6、
LDLR、LTC4S、MMP3、MTHFR、MTR、MTRR、NAT2、NEDD4L、NOS1AP、PRKCA、NOS3、NR1H3、NR3C2、
PEAR1、PLCD3、PPARA、PRCP、YEATS4、PROX1、PTGS1、PTGS2、PTPRD、SCN1A、SLC14A2、SLCO1B1、
Two or more combination in STK39, TCF7L2, TLR4, TRIB3, VKORC1, WNK1 or CYP4F2；Wherein, described
Reagent be selected from probe groups, primer sets or genetic chip.
8. a kind of detection method of the drug metabolism related gene of drug for cardiovascular disease, which is characterized in that the detection side
Method includes:
(1) DNA to be detected is extracted, full-length genome library is prepared；
(2) examination of the drug metabolism related gene in the full-length genome library and detection drug for cardiovascular disease that obtain step (1)
Agent mixing, carries out PCR；
(3) PCR product described in purification step (2)；
(4) PCR product of step (3) after purification is sequenced；
(5) sequencing result of step (4) is analyzed；
Wherein, the reagent is selected from probe groups, primer sets or genetic chip.
9. a kind of reagent for the drug metabolism related gene for detecting drug for cardiovascular disease is preparing the application in product, feature
It is, the product is that drug metabolism study, drug screening, medicine curative effect evaluation or the dosage of drug for cardiovascular disease determine
Product, alternatively, congenital heredity that research patient carries or the influence that acts on cardiovascular drugs of acquired hereditary variation
Product, alternatively, the drug metabolism of cardiovascular system Prognostic, pregnant preceding early warning instruction offspring to drug for cardiovascular disease
Evaluation of hazard grade product.
10. a kind of dosing method of drug for cardiovascular disease, which is characterized in that the method includes application detection painstaking effort
The mutation of the drug metabolism related gene of the reagent detection drug for cardiovascular disease of the drug metabolism related gene of guard system medication
Or it is horizontal.
Priority Applications (1)
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