CN101748196A - SNP rs762551 of CYP1A2 gene and application thereof in relevant drug metabolism activity detection - Google Patents
SNP rs762551 of CYP1A2 gene and application thereof in relevant drug metabolism activity detection Download PDFInfo
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Abstract
The invention discloses SNP rs762551 of a CYP1A2 gene and application thereof in relevant drug metabolism activity detection, and also provides a method for prejudging drug metabolism activity. The method comprises the steps of detecting whether the cytochrome oxidase P4501A2 gene (CYP1A2) and a transcript of a human individual have variation or not compared with a normal cytochrome oxidase P4501A2 gene and a normal transcript, and if so, showing that the drug metabolism activity of the individual is different from common or normal people, for the individual. The invention also discloses a corresponding detection kit used for prejudging the drug metabolism activity of the individual.
Description
Technical field
The present invention relates to molecular biology and medical field.Relate more specifically to Terminal oxidase P4501A2 gene (Cytochrome P4501A2, single nucleotide polymorphism CYP1A2) (single nucleotidepolymorphism, SNP) and and drug metabolism activity detect between dependency.The invention still further relates to the method and the test kit that detect described SNP.
Background technology
The individual difference of drug effect effect has been a kind of ubiquitous phenomenon, and because the invalid or serious medicine toxic side effects of medicine that this species diversity causes more and more causes the concern of clinician and pharmacogenetics man.The difference of medicament metabolism ability and drug effect is the one of the main reasons of drug effect individual difference, some drug metabolisms and effect genes involved have been found in the variation that not only has dna sequence dna among the crowd, and also there is very big individual difference in its rna expression level.
The enzyme of being responsible for drug metabolism mainly contains two big classes: a class is called I phase drug metabolism enzyme, and the main oxidation of their effect activates drug molecule, for next step reaction provides substrate; Another kind of is II phase drug metabolism enzyme, and their effect is to add the high polarity group on I phase metabolic enzyme reaction product, glycosyl for example, and sulfonic group, ethanoyl or amino, thus increase the solvability of drug molecule, be beneficial to excrete.
I phase drug metabolism enzyme mainly comprises Terminal oxidase P450 (CYP450) and riboflavin mono-oxygenase (FMO), also comprises the part hydroxylase, peroxidase and monoamine oxidase etc.Terminal oxidase is topmost drug metabolism enzyme, and is widely studied.Having found has 55 these genoids approximately in the human genome, wherein 4 genes (CYP3A4, CYP2C9, CYP2C19 and CYP2D6) are main.
II phase drug metabolism enzyme mainly comprises UDP glycosyltransferase (UGTs), Thiadiazolidine isomerase (GSTs), sulfotransferase (SULTs) and N-acetyl-transferase (NATs) etc.
(cytochromes P4501A2 CYP1A2) is a kind of important oxydo-reductase in people's liver to Terminal oxidase P4501A2.In recent years, big quantity research finds that there is very big individual difference in CYP1A2 in the intravital expression of people.The differential expression of transcriptional level can be up to 40 times between Different Individual.Therefore, the multifarious hereditary basis of understanding human medicine metabolism and effect genes involved is one of main purpose of biomedical research Chinese traditional medicine genomics research.
CYP1A2 is important I phase drug metabolism enzyme, is one of P450 main metabolic enzyme, in human liver, accounts for 13% of P450 enzyme total amount.The difference of mRNA expression amount between individuality of CYP1A2 can be up to 40 times, cause between the Different Individual significant difference to medicament metabolism ability thus.
Known at present, be mainly to carry out the drug metabolism of I phase as medicines such as theophylline, tacrine, clozapine, olanzapine clinically by CYP1A2.For example, for caffeine (Caffeine), the 90%th, undertaken metabolic by CYP1A2; For clozapine (leoponex), about 30% is to be undertaken metabolic by CYP1A2.
The drug metabolism enzyme detection chip of the clinical application of FDA approval at present is the common exploitation of Roche Holding Ag and Affymetrix company, integrated some functional site information of having reported of CYP2D6 and CYP2C19 above it, be used to judge individual to these two enzymes as the main metabolic capacity of the medicine of drug metabolism enzyme.CYP1A2 clinically face to participate in metabolic medicament categories a lot of.Its differential expression between individuality can be the main source of its medicine for the ability individual difference up to 40 times.Therefore want the activity of individual CYP1A2 is made prediction, just be necessary to carry out somatotype influencing SNP site its expression amount or that can predict its expression amount.Can instruct clinical rational drug use with it, improve the security and the validity of medicine, realize the target of individualized treatment.
Present studies show that some polymorphisms of CYP1A2 gene are relevant with the metabolic capacity of medicine, and wherein most polymorphisms do not cause the proteic amino acid of CYP1A2 to change.This prompting, under the immovable situation of CYP1A2 enzymic activity (because the aminoacid sequence of enzyme does not change), these polymorphisms (comprising SNP) of CYP1A2 are to realize by the expression amount that influences CYP1A2.Because the quantity of proteic expression amount and its mRNA is proportional basically, therefore identifying the SNP relevant with the expression amount of CYP1A2 just becomes research emphasis.
Disclose genotypic SNP and the corresponding gene chip of measuring the several genes that comprises CYP1A2 among the WO 2008032921, and carried out the application of gene type with these SNP and gene chip.
Primer collection, primer and the probe of the polymorphism that is used to measure drug metabolism enzyme CYP1A2 gene are disclosed among the JP 2007006713, and the detection reagent that is used to detect the CYP1A2 enzymic activity.In the document, using by CYP1A2 before the metabolic medicine, detect earlier the gene pleiomorphism that experimenter's the metabolic capacity relevant with CYP1A2 is correlated with, thereby avoid or reduce untoward reaction relevant or side effect with this medicine.
Disclose a kind of genotypic method that detects gene CYP 1 A 2 among the WO 03014387, pointed out in the document, the CYP1A2 gene is relevant with medicament metabolism ability.
Disclose a kind of genotype of gene CYP 1 A 2 method that is associated with medicament metabolism ability in the Chinese patent application 200510088646.0, wherein G-3860A site, G-3113A and the T5347C site of CYP1A2 gene have been detected.
In addition, the existing correlationship that has of reporting CYP1A2 and part cancer for example mentions all in WO 03014387 and Chinese patent application 200510088646.0 that also there are certain dependency in CYP1A2 gene and liver cancer susceptibility.
In sum, though the polymorphism of known CYP1A2 enzyme in this area or CYP1A2 gene is relevant with the metabolic capacity of some drugs, but people do not influence as yet and have those concrete polymorphisms (comprising SNP) in CYP1A2, do not know which polymorphism (comprising SNP) is relevant with the size of drug metabolism activity or ability yet.
Therefore, in order to reduce the side effect of medicine to Different Individual, this area presses for the SNP of the searching CYP1A2 relevant with the size of drug metabolism activity or ability, and the relevant method and the test kit that can be used for the drug metabolism activity detection.
Summary of the invention
Purpose of the present invention just provides single nucleotide polymorphism (SNP) relevant with the size of drug metabolism activity or ability in the CYP1A2 gene.
Another object of the present invention provides method and the test kit that can be used for assessing in advance individual drug metabolism activity.
In a first aspect of the present invention, provide a kind of vitro detection (especially being non-diagnostic assays) sample whether to have the method for the single nucleotide polymorphism of Terminal oxidase P4501A2 gene CYP 1 A 2, comprise step:
(a) with the CYP1A2 gene of CYP1A2 gene-specific primer amplification sample, obtain amplified production; With
(b) detect whether there is following single nucleotide polymorphism in the amplified production:
830 A → C;
Wherein, the nucleotide position numbering is based on SEQ ID NO:1.
In another preference, described gene-specific primer has the sequence of SEQ ID NO:2 or 3.
In another preference, the length of described amplified production is 50-3000bp, and contains among the SEQ ID NO:1 the 830th.
In a second aspect of the present invention, a kind of test kit that is used to predict individual drug metabolism activity is provided, it comprises the primer of specific amplification CYP1A2 gene or transcript, and described primer amplification goes out length to be 50-3000bp and to contain among the SEQ ID NO:1 the 830th amplified production.
In another preference, described sudden change is following single nucleotide polymorphism:
830 A → C;
Wherein, the nucleotide position numbering is based on SEQ ID NO:1.
In another preference, described test kit also contains the reagent that is selected from down group:
(i) with SEQ ID NO:1 in the 830th sudden change bonded probe; Or
(ii) discern among the SEQ ID NO:1 the 830th sudden change restriction enzyme.
In another preference, described primer has the sequence of SEQ ID NO:2 or 3.
In a third aspect of the present invention, a kind of isolating polynucleotide are provided, the sequence of described polynucleotide is the genome nucleotide sequence of Terminal oxidase P4501A2 gene CYP 1 A 2 wild-type, and has the 830th A → C sudden change among the SEQ ID NO:1.
In another preference, described polynucleotide are the 830th and are the sequence shown in the SEQ ID NO:1 of C.
In a fourth aspect of the present invention, the purposes of the polynucleotide described in the third aspect present invention is provided, it is used to prepare reagent or the test kit that detects individual drug metabolism activity.
In another preference, described reagent is primer, primer collection, probe or nucleic acid chip.
In another preference, described primer or primer collection can amplify specifically and contain among the SEQ ID NO:1 the 830th amplified production; Described probe can with contain SEQ ID NO:1 in the 830th nucleic acid generation specificity combine; The probe that contains on the described nucleic acid chip, described probe can with contain SEQ IDNO:1 in the 830th nucleic acid generation specificity combine.
In a fifth aspect of the present invention, a kind of nucleic acid chip that can be used for the detection of drugs metabolic activity is provided, described chip comprises substrate and is fixed on oligonucleotide probe on the substrate, described probe can with contain SEQ ID NO:1 in the 830th nucleic acid generation specificity combine, and to identify among the SEQ ID NO:1 the 830th Nucleotide be A or C.
In another preference, described medicine is selected from down group:
Theophylline, tacrine, leoponex, olanzapine, Drogenil, frovatriptan, lignocaine, melatonin, mexiletine, ropivacaine, tizanidine, triamterene, Zomitriptan; Or
Paracetamol, almotriptan, amitriptyline, chlorimipramine, cyclobenzaprine, Myroxim, imipramine, maprotiline, Naproxen Base, estradiol, ondansetron, trilafon, Propafenone, Proprasylyte, Riluzole, thioridazine, verapamil, dextrorotation warfarin, Zileuton or zotepine.
In a sixth aspect of the present invention, provide a kind of drug metabolism activity has been carried out forecast method, it comprises step:
Detect the CYP1A2 gene and/or the transcript of individuality to be checked, and compare whether there is following single nucleotide polymorphism with normal CYP1A2 gene and/or transcript:
830 A → C;
Wherein, nucleotide position is numbered based on SEQ ID NO:1,
If exist described single nucleotide polymorphism its drug metabolism activity with regard to this individuality to be higher than the general population with regard to showing.
Should be understood that two or more technical characterictics of appointing above-mentioned and that this paper is hereinafter described in detail all can make up mutually, to constitute new technical scheme.In this application,, list no longer one by one in order to save space.
Embodiment
The inventor is through deeply and extensive studies, and the SNP of CYP1A2 gene is measured and analyzes.But find and proved the mRNA copy number of the part SNP remarkably influenced CYP1A2 of CYP1A2 gene first, and then influence drug metabolism activity.The association study result shows, (830 A → C) can significantly cause the mRNA copy number (P＜0.01) of CYP1A2, therefore can be used as the specificity SNP of prediction drug metabolism activity at the 830th SNP of sequence shown in the SEQ of the CYP1A2 ID NO:1.Finished the present invention on this basis.
The CYP1A2 gene
As used herein, " CYP1A2 gene " refers to the polynucleotide molecule of Codocyte chromo-oxidase P4501A2, comprises DNA and RNA.This term also comprises the genome sequence of CYP1A2, especially contains the polynucleotide of promoter region.
As used herein, " CYP1A2 albumen " or " CYP1A2 enzyme " is used interchangeably, phalangeal cell chromo-oxidase P4501A2.
The detailed sequence of people's Terminal oxidase P4501A2 gene (CYP1A2) can be referring to network address
Http:// www.ncbi.nlm.nih.gov/).In SEQ ID NO:1, provided the partial sequence in people CYP1A2 gene promoter district.
Of the present invention studies show that when 830 A → C take place Terminal oxidase P4501A2 in SEQ ID NO:1 the 830th, cause the quantity of the transcript of CYP1A2 obviously to rise, and then the CYP1A2 albumen quantity that causes translating generation increases.Because this SNP can not cause the proteic amino acid of CYP1A2 to change, so this SNP can not cause the proteic enzymic activity of CYP1A2 to change, therefore have cell or the individuality of SNP shown in the 830th A → C, its medicament metabolism ability is the cell or the individuality of wild-type greater than this site.
Main or part is by the metabolic medicine of CYP1A2
The at present known medicine that relates to CYP1A2 in drug metabolism is a lot, mainly is divided into following two classes:
One, main or part is by the CYP1A2 metabolism, and pointed out the various phenotypes of CYP1A2 to produce the medicine of different clinical meanings, comprising (but being not limited to): theophylline (theophylline) (Sarkar et al.1992), tacrine (tacrine) (Spaldin et al.1994), clozapine (leoponex) (Bertilsson et al.1994), olanzapine (olanzapine) (Callaghan et al.1999), Flutamide (Drogenil), Frovatriptan (frovatriptan), Lidocaine (lignocaine), Melatonin (melatonin), Mexiletine (mexiletine), Ropivacaine (ropivacaine), Tizanidine (tizanidine), Triamterene (triamterene), Zolmitriptan (Zomitriptan).
Two, part is carried out metabolism by CYP1A2, and the medicine that each phenotype clinical meaning of CYP1A2 is still uncertain or influence is more weak, comprising (but being not limited to): Acetaminophen (paracetamol), Almotriptan (almotriptan), Amitriptyline (amitriptyline), Clomipramine (chlorimipramine), Cyclobenzaprine (cyclobenzaprine), Fluvoxamine (Myroxim), Imipramine (imipramine), Maprotiline (maprotiline), Naproxen (Naproxen Base), Oestrogen (estradiol), Ondansetron (ondansetron), Perphenazine (trilafon), Propafenone (Propafenone), Propranolol (Proprasylyte), Riluzole (Riluzole), Thioridazine (thioridazine), Verapamil (verapamil), (R)-and Warfarin (dextrorotation warfarin), Zileuton (Zileuton), Zotepine (zotepine).
The inventor measures and analyzes the SNP of CYP1A2 gene, is wherein checked order in the almost whole zone in the CYP1A2 gene, has found many SNP, and wherein most of SNP and drug metabolism activity are also uncorrelated.Yet association study shows that the 830th A → C but is the SNP very high with the drug metabolism activity cognation among the SEQ ID NO:1.
The proteic polynucleotide of CYP1A2 can be used for prejudging drug metabolism activity.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of CYP1A2 as the CYP1A2DNA sequence.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene test of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of CYP1A2 albumen and also can detect the proteic transcription product of CYP1A2.
The sudden change that detects the CYP1A2 gene also can be used for prejudging drug metabolism activity and detects.Detection can be at cDNA, also can be at genomic dna.The form of CYP1A2 protein mutation comprises that the point mutation compared with normal wild type CYP1A2DNA sequence, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.
The method of the detection SNP of the present invention of most convenient is by the CYP1A2 gene with CYP1A2 gene-specific primer amplification sample, obtains amplified production; Detect then and whether have following single nucleotide polymorphism in the amplified production: the 830th A → C, wherein, nucleotide position is numbered based on SEQ ID NO:1.
Should understand, after the present invention has disclosed the dependency that the SNP of CYP1A2 gene and drug metabolism activity detect first, but those skilled in the art can design the amplified production that specific amplification goes out to contain this SNP position easily, determine whether to exist 830 A → C by methods such as order-checkings then.Usually, the length of primer is 15-50bp, preferably is 20-30bp.Though the complete complementation of primer and template sequence is preferred, one skilled in the art will appreciate that at primer and template to have under the situation of certain not complementary (especially 5 of primer ' end) also can increase specifically (promptly only amplifying required fragment).The method that contains the test kit of these primers and use these primers is all within the scope of the invention, as long as the amplified production that this primer amplification goes out contains the correspondence position of SNP of the present invention.A kind of preferred primer is to having the sequence of SEQ ID NO:2 or 3.
Though the length of amplified production is not particularly limited, the length of amplified production is 50-3000bp usually, preferably is 150-2000bp, more preferably is 200-1000bp.These amplified productions all should contain among the SEQ ID NO:1 the 830th.
Because detecting, SNP of the present invention and drug metabolism activity have very high cognation, therefore can be used for before administration, just prejudging individual drug metabolism activity, and can reduce against a rainy day with by all or part of metabolic Side effects of pharmaceutical drugs of Terminal oxidase P4501A2, thereby therefore the security of drug use has earth shaking using value and social benefit.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
Experiment material and method
Collect 96 routine normal Chinese's liver samples clinically.Sample collection is in normal pathological biopsy part for liver, and the every example of about 50～200mg immerses after collection among the 1mL RNALater at once, and 4 ℃ are spent the night, treat that sample is fully soaked into by RNALater after, be transferred to-70 ℃ of preservations.This research work sample collection and use all obtain the ethics approval.
The sample of-70 ℃ of preservations after room temperature is redissolved, is taken out the ddH that handles at DEPC-from RNALater
2Rinse RNALater among the O, be transferred among the 1mL Trizol (Invitrogen Inc.), carry out homogenate with Pro200homogenizer (Pro scientific Inc.).Scheme (protocol) according to Trizol obtains DNA and RNA then.Carry out quantitatively preliminary with Biophotometer (Eppendorf Inc.).The RNA that obtains is handled with DNase I (Takara Inc.), and with phenol/chloroform extracting and purifying, ethanol sedimentation.With DEPC-treated ddH
2The O redissolution also is diluted to the about 1ug/ul of final concentration.-70 ℃ of preservations.
With 20 μ l systems: the 1RT damping fluid, 0.5mM dNTP, 0.5 μ M poly24dT, 5 μ M N9,20URnase inhibitor (BIO BASIC INC.), 100U M-MLV (H-) reversed transcriptive enzyme (Promega Inc.) carries out reverse transcription.Reaction conditions is 25 ℃ of 10min, 42 ℃ of 60min, 70 ℃ of 15min then.
As standard substance, GAPDH, ACTB and 18srRNA are as reference gene with human gene group DNA (available from Promega, Catalog No.G152A).(AppliedBiosystems, Foster City carry out absolute quantitation PCR above CA), determine the expression amount of cyp1a2 in each sample at ABI7900HT real-time quantitative PCR instrument.With commercially available Primer3 software design PCR primer, select the fragment of the 80-180bp size within the single exon to increase.Reaction system is 20uL:1X QuantiTect SYBRGreen PCR Master Mix (Qiagen Inc.), 0.3 each primer of μ M, the standard substance genomic dna G152A of 2 μ l RT dilution after product (with the 40000 times dilutions of 18s rRNA as reference gene, other 20 times of dilutions) or doubling dilution.Reaction conditions is 95 ℃ of 15min; 94 ℃ of 15s, 57 ℃ of 30s, 40 circulations of 72 ℃ of 30s.
According to document and HapMap data, utilize LD, the cyp1a2 gene region and with the regulation and control zone of sharing of cyp1a1 in, selected 16 tagSNP altogether.R between any two tagSNP
2Less than 0.9.TagSNP12 and 16 is used to detect two expression ratios between the allelotrope as marker SNP.All the other 12 tagSNP carry out somatotype with the order-checking means.
10 microlitre PCR systems: 1x HotStarTaq damping fluid, 2.0mM Mg
2+, 0.2mM dNTP, 0.2M primer, 0.3U HotStarTaq polysaccharase (Qiagen Inc.) and 1 microlitre template DNA (about 10ng/l), reaction conditions: 95 ℃ of 15min; 94 ℃ of 20s, (62 ℃-0.5 ℃/circulation) 40s, 72 ℃ of 80s, 11 circulations; 94 ℃ of 20s, 56 ℃ of 30s, 72 ℃ of 80s, 25 circulations; 72 ℃ of 5min.The PCR product carries out sequencing reaction with BigDye3.0 after carrying out purifying with SAP and Exo I, and (AppliedBiosystems, Foster City check order on CA), read sequence with Polyphred at ABI3730XL.
With SNaPshot Multiplex kit (Applied Biosystems) the mark SNP (marker SNP) of all cDNA and 5 dna samples is carried out somatotype, as reference, determine the situation of two allele expression amounts with two allelotrope of dna sample (allele) peak height ratios by the ratio that detects two allele of marker SNP in the cDNA sample.With this phenotype as subsequent association research.This step repeats 4 times, averages.All 16 tagSNP's isozygotys and the genotype of heterozygous state as research.
Carry out rank test with SPSS VERSION15.0 (SPSS Inc., Chicago is IL) to 6) described genotype and phenotype, express the tagSNP site that ratio has correlationship to detect whether to have with allelotrope.With Kruska-Wallis test and ANOVA the expression amount of tagSNPs and CYP1A2 is carried out correlation analysis.
Obtain in rank test on the basis in part site, further picking one express with remarkable allelotrope that uneven (allele expression imbalance AEI) has the site tag12 of significant correlation, has carried out the in-vitro transcription test.Designed a fragment that comprises the 618bp of tag12, the position is from the 5 ' non-translational region of cyp1a2.The heterozygous individual of choosing a tag12 carries out pcr amplification.This PCR upstream and downstream primer has designed KpnI and XhoI restriction enzyme site respectively.The PCR product carries out enzyme with KpnI and XhoI and cuts, be cloned into pGL-3 promoter vector (available from Promega company) then. then it is converted into conventional E.coli DH10B bacterial strain, cultivation back picking comprises the bacterium of the sequence of two allele respectively and cultivates, sequence does not have the New Development sudden change with sequence verification, and the bacterium colony that the conclusive evidence picking goes out only comprises two different sequences of tag12 site.Carry out plasmid extraction with QIAGEN plasmid purification kit (Qiagen) after the microbial culture.
Human HepG2 cell is cultivated in 24 hole culture plates, and density is 1x10
5It is that 50-80% is as transfectional cell that cells/well, overnight incubation make fraction of coverage.Form transfection DNA mix with plasmid and pRL-SV40.(Polyplus Transfect ion Inc.) carries out cell transfecting with the jetPEI transfection reagent.37 ℃ are spent the night behind the cell transfecting, handle with 0.1%DMSO or 10nM TCDD then and spend the night.(TMO46 Promega) carries out after cell handles, and (Berthold Technologies, Bad Wildbach Germany) carry out fluoroscopic examination with Microlumat Plus LB 96Vluminometer with commercially available luciferase assay kit.
Test-results
The result shows, has significant correlation between the copy number of the CYP1A2 in Tag SNP12 (rs762551) and the sample.(the C allelotrope of the 830th A among the SEQ ID NO:1 → C) is than voluminous about 22% the copy number of giving birth to of A allelotrope for Tag12.This prompting, SNP rs762551 regulated and control the function of CYP1A2 gene by influencing genetic expression, and influenced this gene the metabolism situation of metabolic medicine.
Embodiment 2
The checking of SNP rs762551
2.1DNA extract
Extract DNA with conventional phenol chloroform method from people's blood, concentration correction is used for conventional pcr amplification to 20ng/ul.
2.2PCR and the design of sequencing primer
According to the sequence of the CYP1A2 gene shown in the SEQ ID NO:1, design and synthetic following primer.Concrete primer is as shown in table 1 below.
Table 1 primer sequence table
| The primer title | Sequence (5 '-3 ') | ?SEQ?ID?NO： |
| Adopted primer is arranged | ??gtttgaggcc?tcgggtcccc?aaaggc | ?2 |
| Antisense primer | ??cagtctccac?gaactcatga?gtgtt | ?3 |
1.2.3CYP1A2 the pcr amplification of gene
DNA with extraction is a template, uses the Taq enzyme, carries out pcr amplification with the Touchdown program on GeneAmp 9700PCR instrument.Reaction conditions is: 94 ℃ of pre-sex change 2 minutes, and 94 ℃ of sex change 30 seconds, 63 ℃ of annealing 40 seconds, 72 ℃ were extended totally 10 circulations, 0.5 ℃ of each cycle annealing lapse of temperature 40 seconds; Later 94 ℃ of sex change 30 seconds were annealed 40 seconds for 58 ℃, and 72 ℃ were extended totally 30 circulations 40 seconds; Last 72 ℃ were extended 7 minutes.Pcr amplification product is verified through agarose gel electrophoresis.
As a result, obtain the amplified production of 1680bp.
1.2.4SNP discovery and detection
The PCR product is used ABI-PRISM behind the Resin resin purification
TM377DNA sequenator (appliedbiosystems of u.s.a. applied biosystem company (ABI)) carries out the two-way order-checking of the terminal cessation method of fluorescent mark, and the interpretation and the SNP that carry out sequence with Polyphred software (the http://droog.mbt.washington.edu/Polyphred.html of Washington, DC university) confirm.
As a result, find to exist following SNP:830 position A → C.
Embodiment 3
Individual drug metabolism activity is measured
Because known in human body caffeine about 90% so have dependency between the metabolism speed of the active height of CYP1A2 and caffeine by the CYP1A2 metabolism.
In the present embodiment, the method for Application Example 2, in the healthy Chinese crowd to the CYP1A2 gene based among the SEQ ID NO:1: the SNP site of 830 A → C carried out gene type.And the relation between observation different genotype and the blood plasma caffeine metabolite ratio.
The health volunteer who chooses 20 (masculinity and femininity each 10) ages and be 18-30 year (21 ± 2 years old mean age) participates in this test, and wherein among the SEQ ID NO:1: 830 is A and C respectively 10.Whole experiment is carried out according to the national human genome criterion that studies ethics, and has obtained all experimenters' informed consent.
All experimenters are non-smoking individuality, detect and not to have taken in coffee, tea, Coca-Cola, chocolate within preceding 1 week and other contains the beverage of caffeine carrying out the caffeine metabolite rate.Simultaneously, all experimenter tests within preceding 1 week and had not taken any medicine.
, because of testing drug the activity in vivo of all experimenter CYP1A2 is measured with coffee.The experimenter plays that (7:00～7:30AM) is oral coffee internal cause capsule 100mg on an empty stomach morning.(0 hour) and back (5 little H) the extracting vein blood 5ml that takes medicine before taking medicine use the EDTA anti-freezing respectively.
Centrifugal separation plasma and hemocyte, and be stored in-20 ℃, be respectively applied for the mensuration of carrying out the caffeine metabolite phenotype.Use high performance liquid chromatography.Uv detection method is to caffeine (C in the blood plasma
Caffeine) concentration and meta-bolites 1 thereof, 7-dimethyl xanthine (C
Meta-bolites) concentration measure by the following method:
0.5ml add p-hydroxyethyltheophylline (interior mark) 100 microlitres and the 300mg sulfuric acid amine of 80 μ M in the blood plasma to be measured, add the 5ml chloroform then: Virahol (volume ratio is 9: 1), vibrated after 2 minutes in 2000g centrifugal 8 minutes, take off layer organic phase volatile dry under nitrogen, 50 microlitre moving phases are dissolved again, get chromatographic column on 20 microlitres.
Highly effective liquid phase chromatographic system comprises Agilent 1100 series, C18 chromatographic column (250mm * 4mm, particle diameter are 5 microns).The composition of moving phase comprises methyl alcohol (A), 0.05% acetic acid (B) and acetonitrile (C).Adopt the mode of gradient elution to carry out wash-out, the condition of washing out is: the volume ratio of 0-8 minute A: B: C is 10: 82: 8, and flow velocity is 0.75ml/min; The volume ratio of 8-12 minute A: B: C is 10: 78: 12, and flow velocity is 1ml/min, and column temperature is constant in 45 ℃.The wavelength of ultraviolet detection is 282nm.
The result shows, as follows caffeine (C in the 5th hour blood plasma behind the oral caffeine 100mg of Ji Suaning
Caffeine) concentration and meta-bolites 1 thereof, 7-dimethyl xanthine (C
Meta-bolites) the ratio 1 and 2 of concentration.
The result: ratio 1 is pointed out among the SEQ ID NO:1 greater than 1 (about 160%): behind the SNP site of 830 A → C, individual CYP1A2 activity and drug metabolism activity increase (being higher than the general population).
Ratio 2 is also pointed out among the SEQ ID NO:1 less than 1: behind the SNP site of 830 A → C, individual CYP1A2 activity and drug metabolism activity increase (being higher than the general population).
Embodiment 4
The drug metabolism activity detection kit
As described in embodiment 1, the sudden change of 830 A → C and drug metabolism activity detect closely related among the SEQ ID NO:1.Therefore, can be that template increases and detects with DNA of individual to be measured based on this sudden change design CYP1A2 gene-specific primer.
Prepare a test kit (100 person-times), it contains:
Extract male sex's patients'blood 3ml to be measured, use ordinary method (or using specific test kit) from blood, to extract DNA.The PCR primer that drug metabolism activity is detected in the prediction test kit is diluted to 1 ì mol/ ì l, is that template is carried out the PCR reaction with the primer that is provided with the DNA that is extracted.Behind the PCR product purification, use ABI-PRISM
TMThe 377DNA sequenator carries out the two-way order-checking of the terminal cessation method of fluorescent mark, and the interpretation and the SNP that carry out sequence with Polyphred software confirm.
Perhaps, amplified production and normal control are carried out stratographic analysis with sex change high performance liquid chromatograph (DHPLC), also can detect 830 A → C.
The result shows, the copy number that contains the mRNA of CYP1A2 in the tested object of 830 A → C is high more about 20% than normal population, and this site be A individuality caffeine is converted into 1, the ability of 7-dimethyl xanthine is also than normal population height (high approximately 25%).
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Reference:
1)Aklillu?E，Carrillo?JA，Makonnen?E，Hellman?K，Pitarque?M，Bertilsson?L，and?Ingelman-Sundberg?M(2003)Geneticpolymorphism?of?CYP1A2?in?Ethiopians?affecting?induction?andexpression：characterization?of?novel?haplotypes?withsingle-nucleotide?polymorphisms?in?intron?1.Mol?Pharmacol64：659-69
2)Bae?SY，Choi?SK，Kim?KR，Park?CS，Lee?SK，Roh?HK，Shin?DW，PieJE，Woo?ZH，and?Kang?JH(2006)Effects?of?genetic?polymorphismsof?MDR1，FMO3?and?CYP1A2?on?susceptibility?to?colorectal?cancerin?Koreans.Cancer?Sci.97：774-9
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4)Chung?I，and?Bresnick?E(1997)Identification?of?Positive?andNegative?Regulatory?Elements?of?the?Human?Cytochrome?P4501A2(CYP1A2)Gene.Arch?Biochem?Biophys?338：220-226
5)Cirulli?ET，and?Goldstein?DB(2007)In?vitro?assays?fail?topredict?in?vivo?effects?of?regulatory?polymorphisms.Hum?MolGenet?16：1931-9
6)Ghotbi?R，Christensen?M，Roh?HK，Ingelman-Sundberg?M，AklilluE，and?Bertilsson?L(2007)Compari?sons?of?CYP1A2?geneticpolymorphisms，enzyme?activity?and?the?genotype-phenotyperelationship?in?Swedes?and?Koreans.Eur?J?Clin?Pharmacol63：537-46
7)Gonzalez?FJ，and?Gelboin?HV(1994)Role?of?human?cytochromes?P450in?the?metabolic?activation?of?chemical?carcinogens?and?toxins.Drug?Metab?Rev?26：165-83
8)Jiang?Z，Dalton?TP，Jin?L，Wang?B，Tsuneoka?Y，Shertzer?H.G，DekaR，and?Nebert?DW(2005)Toward?the?evaluation?of?function?ingenetic?variability：characterizing?human?SNP?frequencies?andestablishing?BAC-transgenic?mice?carrying?the?humanCYP1A1_CYP1A2?locus.Hum.Mutat?25：196-206.
9)Kress?S，Reichert?J，and?Schwarz?M(1998)Functional?analysisof?the?human?cytochrome?P4501A1(CYP1A1)gene?enhancer.Eur?JBiochem?258：803-812
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11)Marlowe?JL，and?Puga?A(2005)Aryl?hydrocarbon?receptor，cellcycle?regulation，toxicity，and?tumorigenesis.J?Cell?Biochem96：1174-84
12)Martin?J，Cleak?J，Willis-Owen?SA，Flint?J，and?Shifman?S(2007)Mapping?regulatory?variants?for?the?serotonin?transporter?genebased?on?allelic?expression?imbalance.Mol?Psychiatry?12：421-2
13)Mimura?J，and?Fujii-Kuriyama?Y(2003)Functional?role?of?AhR?inthe?expression?of?toxic?effects?by?TCDD.Biochim?Biophys?Acta1619：263-268
14)Nebert?DW，and?Gonzalez?FJ(1987)P450?genes：structure，evolution?and?regulation.Annu?Rev?Biochem?56：945-993
15)Obase?Y，Shimoda?T，Kawano?T，Saeki?S，Tomari?SY，Mitsuta-IzakiK，Matsuse?H，Kinoshita?M，and?Kohno?S(2003)
16)Polymorphisms?in?the?CYP1A2?gene?and?theophylline?metabolism?inpatients?with?asthma.Clin?Pharmacol?Ther?73：468-74
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18)Rodrguez-Antona?C，Donato?MT，Pareja?E，Gmez-Lechn?MJ，andCastell?JV(2001)Cytochrome?P-450mRNA?Expression?in?HumanLiver?and?Its?Relationship?with?Enzyme.Arch?Biochem?Biophys393：308-15
19)Sachse?C，Brockmller?J，Bauer?S，and?Roots?I(1999)unctionalsignificance?of?a?C--＞A?polymorphism?in?intron?1?of?thecytochrome?P450?CYP1A2?gene?tested?with?caffeine.Br?J?ClinPharmacol?47：445-9
20)Ueda?R，Iketaki?H，Nagata?K，Kimura?S，Gonzalez?FJ，Kusano?K，Yoshimura?T，and?Yamazoe?Y(2006)A?common?regulatory?regionfunctions?bidirectionally?in?transcriptional?activation?of?thehuman?CYP1A1?and?CYP1A2?genes.Mol?Pharmacol?69：1924-30
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Sequence table
＜110〉Research Center of Shanghai Human Genome
＜120〉the SNP rs762551 of CYP1A2 gene and the application in relevant drug metabolism activity detects thereof
<130>085802
<160>3
<170>PatentIn?version?3.4
<210>1
<211>1680
<212>DNA
＜213〉people (Homo sapiens)
<220>
<221>misc_feature
<222>(830)..(830)
<223>SNP?rs762551?A→C
<400>1
ttgttcagtg?atccagcttt?catatcaggt?gatcaggaca?accaggccaa?tctgataggg???60
ggcggtgttt?ataaaaaggc?cactcaccta?gagccagaag?ctccacacca?gccattacaa??120
ccctgccaat?ctcaagcacc?tgcctctaca?ggtacctttc?ttgggaccaa?tttacaatct??180
ctgggatccc?caactataga?acctggaagc?tagtggggac?agaaagacgg?ggagcctggg??240
ctaggtgtag?gggtcctgag?ttccgggctt?tgctacccag?ctcttgactt?ctgtttcccg??300
attttaaatg?agcagtttgg?actaagccat?ttttaaggag?agcgatgggg?agggcttccc??360
ccttagcaca?agggcagccc?tggccctggc?tgaagcccaa?ccccaacctc?caagactgtg??420
agaggatggg?gactcatccc?tggaggaggt?gcccctcctg?gtattgataa?agaatgccct??480
ggggaggggg?catcacaggc?tatttgaacc?agccctggga?ccttggccac?ctcagtgtca??540
ctgggtaggg?ggaactcctg?gtcccttggg?tatatggaag?gtatcagcag?aaagccagca??600
ctggcaggga?ctctttggta?caatacccag?catgcatgct?gtgccagggg?ctgacaaggg??660
tgctgtcctt?ggcttcccca?ttttggagtg?gtcacttgcc?tctactccag?ccccagaagt??720
ggaaactgag?atgatgtgtg?gaggagagag?ccagcgttca?tgttgggaat?cttgaggctc??780
ctttccagct?ctcagattct?gtgatgctca?aagggtgagc?tctgtgggcm?caggacgcat??840
ggtagatgga?gcttagtctt?tctggtatcc?agctgggagc?caagcacaga?acacgcatca??900
gtgtttatca?aatgactgag?gaaatgaatg?aatgaatgtc?tccatctcaa?ccctcagcct??960
ggtccctcct?tttttccctg?cagttggtac?agatggcatt?gtcccagtct?gttcccttct??1020
cggccacaga?gcttctcctg?gcctctgcca?tcttctgcct?ggtattctgg?gtgctcaagg??1080
gtttgaggcc?tcgggtcccc?aaaggcctga?aaagtccacc?agagccatgg?ggctggccct??1140
tgctcgggca?tgtgctgacc?ctggggaaga?acccgcacct?ggcactgtca?aggatgagcc??1200
agcgctacgg?ggacgtcctg?cagatccgca?ttggctccac?gcccgtgctg?gtgctgagcc??1260
gcctggacac?catccggcag?gccctggtgc?ggcagggcga?cgatttcaag?ggccggcctg??1320
acctctacac?ctccaccctc?atcactgatg?gccagagctt?gaccttcagc?acagactctg??1380
gaccggtgtg?ggctgcccgc?cggcgcctgg?cccagaatgc?cctcaacacc?ttctccatcg??1440
cctctgaccc?agcttcctca?tcctcctgct?acctggagga?gcatgtgagc?aaggaggcta??1500
aggccctgat?cagcaggttg?caggagctga?tggcagggcc?tgggcacttc?gacccttaca??1560
atcaggtggt?ggtgtcagtg?gccaacgtca?ttggtgccat?gtgcttcgga?cagcacttcc??1620
ctgagagtag?cgatgagatg?ctcagcctcg?tgaagaacac?tcatgagttc?gtggagactg??1680
<210>2
<211>26
<212>DNA
＜213〉people (Homo sapiens)
<400>2
gtttgaggcc?tcgggtcccc?aaaggc?????????????????????????????????????????26
<210>3
<211>25
<212>DNA
＜213〉people (Homo sapiens)
<400>3
cagtctccac?gaactcatga?gtgtt??????????????????????????????????????????25
Claims (10)
1. whether a vitro detection sample exists the method for the single nucleotide polymorphism of Terminal oxidase P450 1A2 gene CYP 1 A 2, it is characterized in that, comprises step:
(a) with the CYP1A2 gene of CYP1A2 gene-specific primer amplification sample, obtain amplified production; With
(b) detect whether there is following single nucleotide polymorphism in the amplified production:
830 A → C;
Wherein, the nucleotide position numbering is based on SEQ ID NO:1.
2. the method for claim 1 is characterized in that, described gene-specific primer has the sequence of SEQID NO:2 or 3.
3. test kit that is used to predict individual drug metabolism activity, it is characterized in that, it comprises the primer of specific amplification CYP1A2 gene or transcript, and it is 50-3000bp that described primer amplification goes out length, and contains among the SEQ ID NO:1 the 830th amplified production.
4. test kit as claimed in claim 3 is characterized in that, it also contains the reagent that is selected from down group:
(i) with SEQ ID NO:1 in the 830th sudden change bonded probe; Or
(ii) discern among the SEQ ID NO:1 the 830th sudden change restriction enzyme.
5. test kit as claimed in claim 3 is characterized in that described primer has the sequence of SEQ ID NO:2 or 3.
6. isolating polynucleotide is characterized in that, the sequence of described polynucleotide is the genome nucleotide sequence of Terminal oxidase P450 1A2 gene CYP 1 A 2 wild-type, and have the 830th A → C sudden change among the SEQ ID NO:1.
7. the purposes of polynucleotide as claimed in claim 6 is characterized in that, is used to prepare reagent or the test kit that detects individual drug metabolism activity.
8. purposes as claimed in claim 7 is characterized in that, described reagent is primer, primer collection, probe or nucleic acid chip;
More preferably, described primer or primer collection can amplify specifically and contain among the SEQ ID NO:1 the 830th amplified production; Described probe can with contain SEQ ID NO:1 in the 830th nucleic acid generation specificity combine; With the probe that contains on the described nucleic acid chip, described probe can with contain SEQ ID NO:1 in the 830th nucleic acid generation specificity combine.
9. nucleic acid chip that can be used for the detection of drugs metabolic activity, it is characterized in that, described chip comprises substrate and is fixed on oligonucleotide probe on the substrate, described probe can with contain SEQ ID NO:1 in the 830th nucleic acid generation specificity combine, and to identify among the SEQ ID NO:1 the 830th Nucleotide be A or C.
10. one kind is carried out forecast method to drug metabolism activity, it is characterized in that it comprises step:
Detect the CYP1A2 gene and/or the transcript of individuality to be checked, and compare whether there is following single nucleotide polymorphism with normal CYP1A2 gene and/or transcript:
830 A → C;
Wherein, nucleotide position is numbered based on SEQ ID NO:1,
If exist described single nucleotide polymorphism its drug metabolism activity with regard to this individuality to be higher than the general population with regard to showing.
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Cited By (6)
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| CN102399899A (en) * | 2011-12-09 | 2012-04-04 | 邵棠 | Liquid phase chip method and kit for detecting polymorphism of cytochrome P450 1A2 (CYP1A2) genes |
| CN102912004A (en) * | 2011-08-02 | 2013-02-06 | 广州益善生物技术有限公司 | CYP1A2 gene polymorphism detection specific primers and liquid chip |
| CN106222281A (en) * | 2016-08-10 | 2016-12-14 | 中南大学湘雅三医院 | Test kit, application and method of based on the gene pleiomorphism accurate medication of guiding children patient |
| CN106319073A (en) * | 2016-10-09 | 2017-01-11 | 深圳联合医学科技有限公司 | Primer, probe, kit and method for detecting subtypes of human gene CYP1A2 |
| CN108300779A (en) * | 2018-02-05 | 2018-07-20 | 广州和康医疗技术有限公司 | A kind of kit and method for predicting the SNP site of leflunomide curative effect of medication |
| CN108949947A (en) * | 2017-05-25 | 2018-12-07 | 上海市预防医学研究院 | Cytochrome P450 gene polymorphic site relevant to anti-tubercular drug physical property hepatic injury generation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102912004A (en) * | 2011-08-02 | 2013-02-06 | 广州益善生物技术有限公司 | CYP1A2 gene polymorphism detection specific primers and liquid chip |
| CN102912004B (en) * | 2011-08-02 | 2014-06-18 | 益善生物技术股份有限公司 | CYP1A2 gene polymorphism detection specific primers and liquid chip |
| CN102399899A (en) * | 2011-12-09 | 2012-04-04 | 邵棠 | Liquid phase chip method and kit for detecting polymorphism of cytochrome P450 1A2 (CYP1A2) genes |
| CN106222281A (en) * | 2016-08-10 | 2016-12-14 | 中南大学湘雅三医院 | Test kit, application and method of based on the gene pleiomorphism accurate medication of guiding children patient |
| CN106319073A (en) * | 2016-10-09 | 2017-01-11 | 深圳联合医学科技有限公司 | Primer, probe, kit and method for detecting subtypes of human gene CYP1A2 |
| CN108949947A (en) * | 2017-05-25 | 2018-12-07 | 上海市预防医学研究院 | Cytochrome P450 gene polymorphic site relevant to anti-tubercular drug physical property hepatic injury generation |
| CN108300779A (en) * | 2018-02-05 | 2018-07-20 | 广州和康医疗技术有限公司 | A kind of kit and method for predicting the SNP site of leflunomide curative effect of medication |
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