CN101768630A - SNP locus of human renin-binding protein gene, detection method and diagnostic application thereof - Google Patents
SNP locus of human renin-binding protein gene, detection method and diagnostic application thereof Download PDFInfo
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Abstract
The invention discloses an SNP locus existing in a promoter region of a human renin binding protein gene, which is an A/G polymorphism of-1590 site of the promoter region of the human renin binding protein gene and has a nucleotide sequence shown in SEQ ID NO. 1. In addition, the invention also discloses a method for detecting the SNP locus of the human renin binding protein gene and a detection primer thereof. In addition, the invention also discloses the application of the SNP locus of the human renin binding protein gene in diagnosing coronary heart disease.
Description
Technical field
The present invention relates to the biological medicine technology field.More specifically, the SNP site and detection method and the diagnostic uses that relate to a kind of human renin binding-protein gene.
Background technology
Renin-angiotensin system (Renin-Angiotensin System, RAS) be important endocrine regulation system in the body, participate in vasoconstriction, metabolism and orthosympathetic adjusting directly, be to keep water-electrolyte balance, adjusting antiotasis, Q volume of blood and blood pressure, participate in the important step of cardiovascular systems growth and reconstruct.Previously think that RAS mainly comprises proangiotensin (Angiotensinogen, AGT), feritin (Renin), angiotensin I (Angiotensin I, Ang I), Zinc metallopeptidase Zace1 (Angiotensin-converting enzyme, ACE), I type angiotensin-ii receptor (Angiotensin II type 1 receptor, AGTR1) and II type angiotensin-ii receptor (Angiotensin II type 2 receptor, AGTR2).In recent years, some new researchs are again successively for this system has increased new member, for example: and RBP (Renin-bindingprotein, RENBP) etc.In view of RAS and cardiovascular system diseases take place closely relatedly, this systematic research is received publicity day by day.
The activation of RAS, initiating is in the release of feritin.Feritin is mainly derived from the kidney juxtaglomerular cell, and it is former that at first feritin mRNA produces prorenin in the kidney juxtaglomerular cell, and removing a single peptide and glycosylation then, to change feritin into former.Some feritins are former to be converted into feritin in the kidney juxtaglomerular cell, other feritin principles directly enter blood circulation.Vascular tissue is more to the former picked-up of feritin, so blood vessel may be the former main position that is converted into feritin of feritin.Local organization such as suprarenal gland, brain, heart etc. also can synthesize feritin.But the feritin that local organization produces is to be mainly derived from the blood plasma picked-up, still is mainly derived from self the synthetic further research that awaits.It has been generally acknowledged that blood pressure drops or cardiac output reduce the blood perfusion quantity not sufficient that can cause kidney, renal perfusion lowers or uriniferous tubules na concn minimizing can stimulation juxtaglomerular cell release feritin is gone into blood.Human renin conjugated protein (RENBP) is a kind of albumen lyase of discovered in recent years, is made up of 417 amino acid, belongs to N-acyl group-D-osamine 2-epimerase (NAGE), by participating in the acid of N-acetylneuraminic amine (sialic acid, NeuAc) synthetic and involved in sugar metabolism.In vitro study is found, RBP can combine with 1: 1 Idiotype of feritin, in conjunction with the time need leucine zipper structure, feritin 232-253 amino acid may be the site with the RBP effect, both form high molecular feritin mixture (high molecular mass rennin) jointly, suppress renin activity, infer that it may participate in the regulating effect of RAS system.RBP mainly may be synthetic by mesangial cell, belong to desmo enzyme, in uriniferous tubules, collecting tubule, fatty tissue and cardiac endothelial cells etc. expression is arranged, in blood plasma, can not be recorded, how RENBP interacts with feritin in vivo actually, what its physiological function is, still do not know at present.
Both at home and abroad to studies show that of RAS, the generation of numerous cardiovascular disordeies such as RAS genovariation and hypertension, coronary heart disease, atherosclerosis and develop closely related.Up to now, it is polymorphic and AGTR1 gene 11 66A/C is polymorphic in AGT gene M 235T, ACE gene I/D that the research of RAS genovariation and cardiovascular disorder is focused mostly on, similar conclusion is arranged, the report that fails to agree is also arranged, research is carried out in the crowd of Caucasia mostly, and dabbles limited to the variation of these other genes of system.People RENBP gene is positioned at Xq28, and 10 exons and 9 introns are arranged, and is at the early-stage to the research of people RENBP genovariation.Knoll etc. have found that in the crowd of Caucasia the T61C of No. 6 intron-No. 7 exon junctions is polymorphic, and the C gene frequency is 0.18.The male sex carries T allelotrope person, and the former level of plasma renin increases, but does not find the dependency between T61C polymorphic and plasma renin and blood pressure.Laan etc. find that among Saami people and the Finn, the C gene frequency is respectively 0.21 and 0.19 in genetic evolution research.Sunder-Plassmann etc. do not find the dependency between the morbidity of the polymorphic and hypertensive crisis of T61C in Austrian crowd.The present invention attempts in the Chinese East China crowd of Han nationality the promoter region of people RENBP gene and the SNP of coding region to be detected, the main dependency of inquiring into people's RENBP genovariation and incidence of coronary heart disease, whether clear and definite people RENBP genovariation increases the danger of coronary heart disease and/or various hypotypes.
Summary of the invention
One of the technical problem to be solved in the present invention provides a kind of SNP site of human renin binding-protein gene.
Two of the technical problem to be solved in the present invention provides a kind of primer that detects the SNP site of human renin binding-protein gene.
Three of the technical problem to be solved in the present invention provides a kind of method that detects the SNP site of human renin binding-protein gene.
Four of the technical problem to be solved in the present invention provides a kind of purposes of SNP site in diagnosis of coronary heart disease of human renin binding-protein gene.
In one aspect of the invention, provide a kind of SNP site of human renin binding-protein gene, this site is that the A/G of human renin binding-protein gene promoter region-1590 is polymorphic, has the nucleotide sequence shown in the SEQ ID NO.1.The A/G of described human renin binding-protein gene promoter region-1590 is polymorphic to be that the GA genotype is polymorphic.
In another aspect of this invention, provide one group of primer that detects the SNP site of human renin binding-protein gene, be the base sequence shown in SEQ ID NO.2～SEQ ID NO.4, SEQ ID NO.2 and SEQ ID NO.3 are that primer is right.
In another aspect of this invention, provide a kind of method that detects the SNP site of human renin binding-protein gene, comprise the steps:
1) seeks SNP by the PCR-direct sequencing in the control region and the coding region of human renin binding-protein gene;
2) carry out pcr amplification reaction in SNP to be measured place fragment;
3) carry out the SNaPshot reaction;
4) carry out capillary electrophoresis.
In the step 1), the primer that the PCR-direct sequencing adopts is a sequence shown in SEQ ID NO.2 and the SEQ ID NO.3; Step 2) in, the primer of pcr amplification reaction is a sequence shown in SEQ ID NO.2 and the SEQ ID NO.3; In the step 3), the primer of SNaPshot reaction is a sequence shown in the SEQ ID NO.4.
A kind of purposes of SNP site (this site is that the A/G of human renin binding-protein gene promoter region-1590 is polymorphic) in diagnosis of coronary heart disease of human renin binding-protein gene is provided in another aspect of this invention.
Among the present invention, " SNP " refers to the single nucleotide polymorphism molecule marker, belongs to third generation genetic marker, and after human genome DNA's sequence order-checking and analytical work were finished, it can provide strong application guarantee for systematically studying gene function and correlated inheritance disease.Because it is distributed widely in human genome and relatively stable existence, thereby in using, the hereditary mechanism research of disease related gene location and disease has prospect.By the SNP mark of finding to exist in the special group, and the design different schemes carries out the crowd size with the SNP that finds and divides the type examination, provides technical foundation and means with the direct mechanism research for heredopathia; Simultaneously, etiological diagnosis, treatment and the control to the human genetic disease produces great effect.
The present invention has carried out research to the SNP of human renin binding-protein gene in the Chinese East China crowd of Han nationality.Promoter region SNP of new discovery (A/G that is human renin binding-protein gene promoter region-1590 is polymorphic).Because the human renin binding-protein gene is positioned at Xq28, when carrying out the case-control association analysis, according to the different separately statistics of sex.The present invention finds, in women crowd, the A/G of human renin binding-protein gene promoter region-1590 is polymorphic to be the Hazard Factor of women's incidence of coronary heart disease.Especially the GA heterozygote with the coronary heart disease significant correlation, carries GA genotype person, and overall incidence of coronary heart disease danger is 3.9 times of GG genotype person.Find through the subgroup analysis, the A/G of human renin binding-protein gene promoter region-1590 polymorphic with coronary heart disease with hypertension morbidity, with anginal generation, and related with the pathology severity of coronary atherosclerosis, the initiation potential that carries GA genotype person is 4-5 a times of GG genotype person.Although not in the accompanied with hypertension group, the significant difference that genotype distributes is not found in the present invention's research in myocardial infarction and coronary heart disease, because this two subgroups number (22 people and 38 people) very little needs more sample just can obtain believable conclusion.And male sex crowd, the A/-genotype of CHD group is higher than control group (25.2% vs 20.9%), but does not reach significant difference.
The function of RBP gene, it be not immediately clear, the RBP genetically deficient mouse that the gene knockout technology of utilizing Schmitz etc. causes, appearance is normal, the kidney structure is no abnormal, plasma renin activity and expression are all unaffected, and the supposition RBP may be regulated renin activity in cell.For the polymorphic function of human renin binding-protein gene promoter region-1590 A/G, also there is not relevant report so far.The present invention may be some transcription factor bonded sites by this site of Matinspector software prediction, and A is polymorphic may to be the binding site of V$OCT1_06, and G polymorphic may be the binding site of V$ARNT_01.Imagine thus, it is polymorphic to be positioned at this A/G of promoter region, may produce and damage the binding site of some transcription factors, influence the RBP expression of gene, if RBP expression amount deficiency, the function reduction of its antagonism feritin, local RAS increased activity makes cause of coronary heart disease danger increase.
According to the data of Framingham, the result that 35～84 years old crowd was followed up a case by regular visits to 26 years shows: the sickness rate of males with coronary disease is 2 times of women, and 60% coronary heart disease betides the male sex, the clinical symptom that coronary heart disease appears in the women generally than the male sex late 10 years.The modal symptom of women is a stenocardia, and has only 60～70% to have narrowly through coronary angiography, and the male sex then more shows as myocardial infarction, and coronary angiography has narrow more than 90%.The geographic epidemiologic data in Shanghai shows that also the ratio of men and women's incidence of coronary heart disease sex is 2.74: 1.All the time; research to incidence of coronary heart disease mechanism exists a kind of hypothesis; the incidence of coronary heart disease of inferring masculinity and femininity exists different mechanism and path; as: sex hormone levels such as female estrogen to provide protection of cardiovascular systems etc.; women's coronary heart disease after climacteric develops reason faster; may be owing to lost the provide protection of female sex hormone, can change the function of distribution, thrombin and the blood vessel endothelium of blood fat climacteric.The present invention from another side-gene epidemiology angle illustrates, men and women's incidence of coronary heart disease may exist different mechanism and path really, the gene susceptibility also may be an important factor.
The present invention first openly human renin binding-protein gene promoter region have a SNP, be that the A/G of human renin binding-protein gene promoter region-1590 is polymorphic.This A/G is polymorphic may to be the Hazard Factor of women's incidence of coronary heart disease.Carry GA genotype person, overall incidence of coronary heart disease danger is 3.9 times of GG genotype person.The morbidity of this polymorphic and coronary heart disease with hypertension, with anginal generation, and and the pathology severity of coronary atherosclerosis related.Therefore, the A/G of human renin binding-protein gene promoter region-1590 is polymorphic, can be used for the early stage auxiliary diagnosis and the examination of coronary heart disease.
Description of drawings
Fig. 1 is the electrophoretic image of SNaPshot in the embodiment of the invention; Primer extension length numerical value among Fig. 1 adds 1 for each SNP site SNaPshot primer length; The different fluorescent dyes of ddNTP: redness-T blueness-G black-C green-A; NO.6 is people RENBP gene promoter area-1590 a G/A heterozygote among Fig. 1, all the other numberings are SNPs of other genes, NO.1 is the C/T heterozygote, NO.2 is the G/A heterozygote, NO.3 is the C/T heterozygote, and NO.4 is the G/G homozygote, and NO.5 is the C/T heterozygote, NO.7 is the C/C homozygote, and NO.8 is the G/T heterozygote.
Fig. 2 is the sequence synoptic diagram of the new SNP of inventor's RBP Gene Partial promoter region sequence and discovery.
Fig. 3 be the embodiment of the invention SNP detection and the checking in sequencing reaction program synoptic diagram.
Fig. 4 is the PCR response procedures synoptic diagram in the SNP gene type of the embodiment of the invention.
Fig. 5 is the PCR purification reaction program synoptic diagram in the SNP gene type of the embodiment of the invention.
Fig. 6 is the SNaPshot response procedures synoptic diagram in the SNP gene type of the embodiment of the invention.
Fig. 7 is the SNaPshot purification reaction program synoptic diagram in the SNP gene type of the embodiment of the invention.
Embodiment
The invention will be further elaborated by the following examples:
Embodiment
One, experiment sample
1. CHD group (CAD): derive from the intracardiac section of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine ward, according to the ischemic heart disease Case definition of WHO promulgation in 1979, all the underwent coronary radiography is made a definite diagnosis and/or clear and definite acute myocardial infarction medical history person is arranged.Coronarography shows coronary artery luminal stenosis＞70%, is diagnosed as coronary artery pathological changes; Left side trunk luminal stenosis＞50%, be diagnosed as two coronary artery pathological changes.Get rid of diabetes, cerebral infarction, immunological disease, thyroid disease, neural system disease, malignant tumour and liver kidney disease patient.According to analyzing needs, CHD group is divided into 3 subgroup groups again, totally 6 subgroups are other: 1. coronary heart disease with hypertension group (CAD+HBP) and coronary heart disease accompanied with hypertension group (CAD-HBP) not; 2. myocardial infarction group (MI) and angina pectoris group (AP); 3. single pathology group and many pathology groups.
2. essential hypertension group (EH): derive from Ruijin Hospital, Shanghai Jiao Tong University School of Medicine intracardiac section ward and outpatient service.Case definition is: systolic pressure is equal to or higher than 140mmHg and/or diastolic pressure is equal to or higher than 90mmHg, or accepts the antihypertensive drug treatment more than at least 1 year.Get rid of secondary hypertension, coronary heart disease, cerebral infarction, serious liver kidney and the Tiroidina person of taking a disease.
3. control group (control): derive from Ruijin Hospital, Shanghai Jiao Tong University School of Medicine health check-up outpatient service, check performances such as no coronary heart disease, hypertension, diabetes and cerebral infarction through medical history, health check-up, electrocardiogram(ECG etc., do not have the persons of taking a disease such as serious liver kidney, Tiroidina, hyperlipidemia.
The experimenter is all by family histories such as inquiry detailed medical history, past medical history, cardiovascular, the cerebrovascular, diabetes, smoking, drink, food habits.Case group perquisition drug use situation comprises depressor, lipid lowerers, anti-freezing medicine and antiplatelet drug etc.Experimenter's physical examination comprises: somatometry, blood pressure, heart rate and electrocardiogram(ECG.The biochemistry detection index comprises blood fat and fasting blood glucose level.Blood sugar and blood fat are measured by the biochemical chamber of Rui Jin hospital: wherein total cholesterol (TC) and triglyceride level (TG) adopt enzymatic assays; High-density lipoprotein (HDL) ester (HDL-C) and low-density lipoprotein ester (LDL-C) adopt phospho-wolframic acid magnesium precipitate method to measure.
4. sample is prepared: blood sample adopts classical phenol-chloroform method extracting human gene group DNA after Sodium Citrate anti-freezing in 1: 9 is handled, and according to the OD value of DNA sample, adds TE DNA is diluted to final concentration 20 μ g/ml.
Two, the detection of SNP and checking
Select 12 coronary disease cases and 12 normal control samples, all affinity-less relation.Seek SNP by the PCR-direct sequencing in the control region and the coding region of gene.Detected SNP in small sample, is verified through the PCR-direct sequencing again.Purpose fragment PCR amplification step comprises:
1.PCR reaction system (25 μ l): 10 * Buffer (damping fluid), 2.5 μ l, dNTPmix (deoxynucleoside triphosphate mixed solution 10mM) 0.75 μ l, MgCl
2(25mM) 2 μ l, Taq archaeal dna polymerase (concentration 5U/ul) 0.2-0.3 μ l, primer (20 μ M) 0.75 μ l * 2, dna profiling (20ng/ μ l) 1 μ l, all the other are distilled water.
2.PCR response procedures: 95 ℃ of sex change 2min; 94 ℃ of sex change 30sec, 62 ℃ of annealing 1min, 72 ℃ are extended 40sec, totally 35 circulations; Last 72 ℃ are extended 5min; 4 ℃.
3.PCR product purification (SAP and Exon I purifying)
Purification system (7 μ l): PCR product 5 μ l, shrimp alkali enzyme (SAP, concentration 7.633U/ μ l, ABI Biosystem) 1 μ l, excision enzyme (Exon I, concentration 20U/ μ l, ABI Biosystem) 1 μ l.
Purification reaction: hatch 15min for 37 ℃, 80 ℃ of 15min.
4. sequencing reaction
1) reaction system: sequencing primer (primer is F:gggaggcatcctctgtgtg (SEQ ID NO.2), R:gagcagagcggtaggagtcat (SEQ ID NO.3)) (0.8 μ M), PCR product and fluorescence order-checking MIX each 2 μ l, totally 6 μ l.
2) the sequencing reaction program is seen Fig. 3.
3) ethanol sedimentation.
4) at the up capillary electrophoresis of ABI3700 sequenator, about 2 hours/plate of order-checking time.
5) determine the SNP site.
Three, the gene type of SNP
1. the present invention uses the SNaPshot method and carries out gene type
1) principle of work
Clearly need to carry out the SNPs of gene type, 6-10 SNPs is combined into a SNaPshot reaction system, utilize single base extension (Single Base Extension, SBE) principle, at each SNP to be measured, the unidirectional oligonucleotide primer that design length is different, under the situation that the different fluorescently-labeled ddNTP with 4 kinds of AmpliTaqDNA polysaccharase exist, each bar primer combines with complementary dna profiling separately, many primer extends in a reaction system, has constructed multiple SBE reaction.Polysaccharase only promptly comes to an end in the single base reaction of 3 ' terminal extension of primer, and the length of product is primer length+1, and the base of extension is exactly the genotype of this sample in this site, and homozygote shows as unimodal, and heterozygote shows as bimodal.At ABI
On the 3700 dna sequencing instrument in the sample system, the GeneScan-120 LIZ Size Standard that adds custom-designed fluorescent orange mark is as marking in the length, per 5 base length are done a mark at interval, reach the purpose of distinguishing different SNP site in order to the primer of demarcating different lengths.This method is similar with order-checking, also is micrometering preface technology (minisequencing).
Can control owing to participate in the unidirectional oligonucleotide primer length of reaction, therefore designing the unidirectional oligonucleotide primer of different lengths and making it to detect in same reaction system is the experiment key of success.Unidirectional oligonucleotide primer also has some special requirements except the requirement of following general design of primers:
1. for avoiding the overlapping of final SnaPshot product, each bar primer is distinguished with length, need to add different lengths poly (dT) (or poly (dA) at 5 ' end of primer, poly (dC), poly (dGACT)), so neither influence the Tm value and the primer specificity extension of primer, again can be when ABI PRISM 3700 capillary electrophoresis separately with each segment.
2. the shortest primer generally is set at 20bp, and too short mobility influence to capillary electrophoresis is bigger, the long experimental cost that then increases.
3. the Tm value of each primer best near and be higher than 50 ℃, cross and lowly then can not form pairing.
Length generally differs 4-6 nucleic acid between the primer in 4. adjacent two SNP sites, if the SNP type in adjacent two sites (for example A/G and C/T) inequality, primer length differs 4 bases and gets final product; If the SNP type in adjacent two sites has coincidence (for example A/G and A/C), primer length preferably differs 6 bases, in order to interpretation;
5. design of primers can be from positive and negative two to carrying out.For example, are identical SNP types (for example A/G and A/G) for adjacent two sites, can avoid overlapping from positive and negative two to design primer (for example A/G and T/C) respectively.
Therefore, multiple SNaPshot reaction promptly utilizes 4 kinds of different fluorescently-labeled ddNTP of difference and single-basic extension of length between primer and finally reaches the effect of distinguishing different SNPs, primary first-order equation can be carried out gene type to 6～10 SNPs simultaneously, is the higher methods of genotyping of a kind of flux.The SNaPshot test kit is provided by U.S. biologic applications system house, and reaction system is optimized stable by the present invention.
2) experimental procedure
1. the segmental pcr amplification reaction in SNPs to be measured place:
The a.PCR reaction
Reaction system (10 μ l):
10×Buffer 1.0μl
Primer(20μM) 0.16μl×2
dNTPmix(10mM) 0.2μl
Mgcl
2(25mM) 1.2μl
TaqDNA polysaccharase (concentration 5U/ul) 0.08 μ l
Dna profiling (20ng/ μ l) 1.0 μ l
DdH
2O mends to 10 μ l
The PCR response procedures: adopt Touch-down PCR, see Fig. 4, wherein * represents every through circulating temperature decline 0.5 degree.
B. after Touchdown PCR reaction, get 1.5 μ l PCR products and carry out 1.5% agarose gel electrophoresis evaluation product, balanced mix after each segment demarcation concentration that increases is formed the PCR product mixtures.
C. get 5 μ l PCR product mixtures, the template of behind enzyme (SAP and Exo I) purifying, reacting as next step SNaPshot.
The purification reaction system:
SAP (concentration 7.633U/ μ l) 0.065 μ l
Exo I (concentration 20U/ μ l) 0.05 μ l
The purification reaction program is seen Fig. 5.
2. SNaPshot reaction:
Reaction system:
Purified PCR product mixtures 1 μ l
(each unidirectional primer balanced mix, final concentration 0.5 μ M)
(containing AmpliTaqDNA polysaccharase and 4 kinds of fluorescently-labeled ddNTP)
Response procedures is seen Fig. 6.
The SNaPshot reaction product is stand-by behind the SAP purifying again:
Purified SNaPshot reaction product 5 μ l
SAP (concentration 7.633U/ μ l) 0.065 μ l
The purification reaction program is seen Fig. 7.
3. the capillary electrophoresis of 3700DNA sequenator
A. go up the sample system
Methane amide 9 μ l
GeneScan-120LIZ?Size?Standard 0.2μl
The SNaPshot reaction product 2 μ l of purifying
B.95 ℃ sex change is 5 minutes, and the cooling back is at ABI
Carry out capillary electrophoresis on the 3700 dna sequencing instrument, operation
3.7 analysis software experimental result.The polymorphic amplimer data of human renin binding-protein gene promoter region-1590 A/G sees Table 1.The SNaPshot electrophoretic image is seen Fig. 1.
Table 1 human renin binding-protein gene part amplimer data
Four, statistical study
1. use SPSS 10.0 for Windows softwares and carry out data analysis and statistics.
The diversity ratio of each clinical variable mean adopts the t check between group;
Calculate the genotype frequency and the gene frequency of each group, frequency ratio adopts χ between group
2Check;
The allelotrope of relative disease and genotype relative risk adopt than number than (odds ratio OR) represents, and carries out the risk level correlation analysis.
2. use the linkage disequilibrium analysis that ARLEQUIN software carries out each SNP in the candidate gene, confirm whether each SNP meets the Hardy-Weinberg balance (with the control group sample calculation, the statistics genotype is the expecterd frequency of homozygote and heterozygote, and will expect that number and observed number carry out χ
2Check when P 〉=0.05, thinks that the genotype frequency that observation post gets meets the Hardy-Winberg balance);
3. control region binding site prediction: utilize TRANSFAC4.0 matrices and matrics searching procedure MatInspector (version 2 .2,
Http:// transfac.gbf.de/programs.html) carry out the inquiry of possible transcription factor binding site point.
Five, result
1, clinical and lab index comparison (table 2) between CHD group, hypertension group and control group
Table 2 control group, essential hypertension group and CHD group is clinical and biochemical indicator relatively
* compare P＜0.01 with control group
2, the detection of human renin binding-protein gene SNP
Human renin binding-protein gene (GenBank Accession #U52112) is positioned at Xq28,10 exons and 9 introns are arranged, the present invention detects human renin binding-protein gene 5607bp altogether, comprising promoter region 1942bp, 5 ' non-translational region 191bp, coding region 1254bp, exon-intron junction region 1867bp, 3 ' non-translational region 16bp, 3 ' terminal 337bp.The result finds a new SNP at promoter region, be the A/G polymorphic (Position in reference Seq.99132) of human renin binding-protein gene promoter region-1590, the T61C polymorphic (including the subarea) that once reported is found in research in the crowd of Caucasia.
3, the case of human renin binding-protein gene SNP-contrast association analysis
Among the present invention, carried out case-contrast association analysis to the A/G of human renin binding-protein gene promoter region-1590 is polymorphic.
1) through Hardy-Weinberg balance goodness of fit check, the polymorphic Hardy-Weinberg balance that meets of A/G of human renin binding-protein gene promoter region-1590.
2), when statistics genotype and gene frequency, calculate respectively by the sex difference because the human renin binding-protein gene is positioned at Xq28:
In male sex's sample, polymorphic G/-and two kinds of genotype of A/-of existing of A/G of human renin binding-protein gene promoter region-1590, genotype (isoallele type frequency) distribute (table 4) at the comparative result of each group be:
(1) total CHD group is compared with control group: the A/-genotype accounts for 25.2%, is higher than control group (20.9%), but does not reach significant difference.
(2) comparison between each subgroup of coronary heart disease
The three groups of subgroups (1. coronary heart disease with hypertension group and coronary heart disease not the accompanied with hypertension group 2. myocardial infarction group and angina pectoris group 3. singly prop up pathology group and many pathology groups respectively with control group) in, 6 groups are compared with control group respectively, the no significant difference of genotype distribution; Compare between every group of subgroups, genotype distributes does not also have significant difference.
(3) hypertension group is compared with control group: genotype distribution no difference of science of statistics.
The polymorphic genotype of people RENBP gene promoter area-1590 A/G (allelotrope) distributes among table 4 male sex crowd
P=NS represents P in the test of significance＞0.1
In women's sample, polymorphic GG, GA and three kinds of genotype of AA of existing of A/G of human renin binding-protein gene promoter region-1590, genotype and gene frequency distribute (table 5) at the comparative result of each group be:
(1) total CHD group is compared with control group: three kinds of genotype of GG, GA and AA are respectively 56,31,3 and 63,9,6 in two groups, genotype be distributed in that there were significant differences in two groups (Fisher exact test, P=0.001).A allelotrope accounts for 20.6% in total CHD group, be higher than 13.5% of control group, but do not reach statistical significance (χ as yet
2=2.95, P=0.08).
(2) comparison between each subgroup of coronary heart disease
1. coronary heart disease with hypertension group and coronary heart disease accompanied with hypertension group not: in the coronary heart disease with hypertension group, significant difference (Fisher exact test has been compared in three kinds of genotype distributions of GG, GA and AA with control group, P=0.0002), the accompanied with hypertension group is (not fewer in number for coronary heart disease, 22 people only) compare genotype and the gene frequency no significant difference that distributes with control group; Two subgroups are compared, and genotype and gene frequency distribute does not then have significant difference.
2. myocardial infarction group and angina pectoris group: in the angina pectoris group, significant difference (Fisher exact test has been compared in three kinds of genotype distributions of GG, GA and AA with control group, P=0.0001), the myocardial infarction group is (fewer in number, 38 people only) compare genotype and the gene frequency no significant difference that distributes with control group; Two subgroups are compared, and genotype and gene frequency distribute does not then have significant difference.
3. single pathology group and many pathology groups: compare with control group respectively, show that all genotype is distributed with significant difference, the former is through the definite probability inspection of Fisher, and the P value reaches 0.0002; The latter is through chi square test (χ
2=10.493) the P value is 0.005.Two subgroups are compared, and genotype and gene frequency distribute does not then have significant difference.
(3) hypertension group is compared with control group: genotype and gene frequency distribution not statistically significant.
Polymorphic genotype of people RENBP gene promoter area-1590 A/G and allele distributions among the table 5 women crowd
P=NS represents P in the test of significance＞0.1
3) the polymorphic initiation potential degree to women's coronary heart disease (and subgroup) of human renin binding-protein gene promoter region-1590 A/G is analyzed
Through risk level analysis (table 6), the A/G of human renin binding-protein gene promoter region-1590 is polymorphic to be the Hazard Factor of women's incidence of coronary heart disease.Especially the GA heterozygote with the coronary heart disease significant correlation, carries GA genotype person, and overall incidence of coronary heart disease danger is 3.9 times of GG genotype person.Wherein, in the coronary heart disease with hypertension group, the danger of GA genotype person incidence of coronary heart disease is 4.4 times of GG genotype person; In the myocardial infarction group, the danger of GA genotype person incidence of coronary heart disease is 2.8 times of GG genotype person, and in the angina pectoris group, the danger of GA genotype person incidence of coronary heart disease is 4.7 times of GG genotype person; In single the pathology group, the danger of GA genotype person incidence of coronary heart disease is 3.4 times of GG genotype person; In many pathology groups, the danger of GA genotype person incidence of coronary heart disease is 4.9 times of GG genotype person.
The polymorphic risk level analysis of table 6 people RENBP gene promoter area-1590 A/G to women's coronary heart disease (and subgroup)
* OR checks P＜0.05
#OR checks P＜0.01
4) adopt the polymorphic binding site that whether contains transcription factor of Matinspector software analysis human renin binding-protein gene promoter region-1590 A/G, analytical results sees Table 7.
The polymorphic binding site of transcribing of people RENBP gene promoter area-1590 A/G that table 7 is inferred
4, conclusion
There is a SNP in reported first human renin binding-protein gene promoter region of the present invention, is that the A/G of human renin binding-protein gene promoter region-1590 is polymorphic, and this A/G is polymorphic may to be the Hazard Factor of women's incidence of coronary heart disease.Carry GA genotype person, overall incidence of coronary heart disease danger is 3.9 times of GG genotype person.The morbidity of this polymorphic and coronary heart disease with hypertension, with anginal generation, and and the pathology severity of coronary atherosclerosis related.Therefore, the A/G of human renin binding-protein gene promoter region-1590 is polymorphic, can be used for the early stage auxiliary diagnosis and the examination of coronary heart disease.
＜110〉Ruijin Hospital, Shanghai Jiao Tong University School of Medicine
＜120〉the SNP site of human renin binding-protein gene and detection method and diagnostic uses
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gacggaaacg?ccgtattttc?agataaacac?cagaggcatg?actgatgtgg?cccttgcctt 240
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ccccacaggt?gccagctaca?gaagtggggt?aggaagaagc?cttgtaccac?ccccaccagg 360
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Claims (6)
1. the SNP site of a human renin binding-protein gene is characterized in that, this site is that the A/G of human renin binding-protein gene promoter region-1590 is polymorphic, has the nucleotide sequence shown in the SEQ ID NO.1.
2. the SNP site of human renin binding-protein gene as claimed in claim 1 is characterized in that, the A/G of described human renin binding-protein gene promoter region-1590 is polymorphic to be that the GA genotype is polymorphic.
3. one group of primer that detects the SNP site of human renin binding-protein gene, it is characterized in that: be the base sequence shown in SEQ IDNO.2～SEQ ID NO.4, SEQ ID NO.2 and SEQ ID NO.3 are that primer is right.
4. a method that detects the SNP site of human renin binding-protein gene is characterized in that, comprises the steps:
1) seeks SNP by the PCR-direct sequencing in the control region and the coding region of human renin binding-protein gene;
2) carry out pcr amplification reaction in SNP to be measured place fragment;
3) carry out the SNaPshot reaction;
4) carry out capillary electrophoresis.
5. the method in the SNP site of detection human renin binding-protein gene as claimed in claim 4 is characterized in that, in the step 1), the primer that the PCR-direct sequencing adopts is a sequence shown in SEQ ID NO.2 and the SEQ ID NO.3; Step 2) in, the primer of pcr amplification reaction is a sequence shown in SEQ ID NO.2 and the SEQ ID NO.3; In the step 3), the primer of SNaPshot reaction is a sequence shown in the SEQ ID NO.4.
6. the purposes of SNP site in diagnosis of coronary heart disease of a human renin binding-protein gene as claimed in claim 1.
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| CN110433282A (en) * | 2018-05-04 | 2019-11-12 | 上海交通大学医学院附属瑞金医院 | Application of the glucagon-like-peptide-1 in terms of calcific aortic disease medicament is treated in preparation |
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| CN110433282B (en) * | 2018-05-04 | 2023-03-14 | 上海交通大学医学院附属瑞金医院 | Application of glucagon-like peptide-1 in preparation of medicine for treating calcified aortic valve diseases |
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