CN104195228B - A kind of SNP mark relevant to nonsyndromic congenital heart disease auxiliary diagnosis and application thereof - Google Patents
A kind of SNP mark relevant to nonsyndromic congenital heart disease auxiliary diagnosis and application thereof Download PDFInfo
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Abstract
The invention belongs to genetically engineered and reproductive medicine field, disclose a kind of SNP mark relevant to nonsyndromic congenital heart disease auxiliary diagnosis and application thereof.This mark is the combination of rs3789612, rs4140836, rs13391263, rs13101737, rs1400558, rs6872396, rs13189951, rs9266596, rs7863990, rs2433752, rs7259736, rs490514, rs2474937, rs1531070, rs9533839, rs17019472, rs10182004, rs9461938, rs72759270 and rs1886441.This mark can be used for preparing nonsyndromic congenital heart disease auxiliary diagnostic box.
Description
Invention field
The invention belongs to genetically engineered and reproductive medicine field, relate to the relevant SNP mark of nonsyndromic congenital heart disease auxiliary diagnosis and application thereof.
Background technology
Congenital heart disease (congenitalheartdisease, CHD) is the exception in heart caused by obstacle in embryo development procedure of heart, blood vessel, vascular morphology, structure, function, metabolism.In numerous inborn defect, CHD accounts for about 28%, is a kind of congenital disorders the most common.According to the data of the World Health Organization, generally believe that the sickness rate of CHD is 0.60% ~ 0.80% newborn infant that is mature, life birth, in the case of premature labor, stillbirth or miscarriage, sickness rate is then higher.According to measuring and calculating, newly-increased CHD case is more than 130,000 examples every year in China, and CHD has become China's peri-natal infant first deformity occurred frequently.From 2000, perinatal period, CHD incidence was quick ascendant trend, and national CHD incidence in 2011 is 3.56 times of 2000.Meanwhile, CHD is also the one of the main reasons causing infant's cause of the death or deformity, and it is serious harm children living and quality of life not only, affects happy family life harmonious, also can cause huge potential years o f life lost and social economical burden.
Epidemiological study proves, the CHD (syndromicCHD) of syndrome and type accounts for patient populations's about 25%, and all the other are nonsyndromic CHD (non-syndromicCHD), namely shows as cardiovascular malformation and does not accompany other system deformity.Nonsyndromic CHD is most in distributing in crowd, according between arranged on left and right sides heart and great vessels with or without shunting, 3 large classes can be divided into: left to right shunt type (livid purple type of hiding), right-left shunt type (livid purple type), without bypass type (without livid purple type).Left to right shunt type mainly comprises: VSD, PDA, ASD etc.Right-left shunt type mainly comprises: tetralogy of Fallot and transposition of conducting arteries etc.Mainly comprise without bypass type: pulmonary stenosis and aortic coaractation etc.Cause the reason of nonsyndromic CHD a lot, comprise that pregnant mother infects rubella virus, contact environment teratogen, pregnancy period medication, maternal disease, Maternal Smoking Smoking, is addicted to drink, takes drugs, chromosome abnormalty, single gene mutation and polygene defect etc.It should be noted that 71% infant can be survived after the child-bearing age, therefore, CHD, especially nonsyndromic CHD has become the great public health problem affecting Chinese children physical and mental health and population life quality.
At present, the diagnostic method of congenital heart disease is mainly disease history inquire, physical examination, imaging examination (using color Doppler echocardiography, x-ray, multi-layer spiral CT, nuclear magnetic resonance), cardiac catheterization and angiocardiography, Radionuclide ventriculography video picture and electrocardiogram(ECG etc.But still there are some drawbacks in present clinical conventional inspection: multi-layer spiral CT and x-ray exist certain radiation hazradial bundle to human body, can not as a line image check approach of CHD; The nuclear magnetic resonance time long (at least needing 30 minutes), check that noise is large, and the movement of patient is very large on the impact of picture quality, therefore is not suitable for the children that can not cooperate; Radionuclide ventriculography injection technique requires high; The gold standard that cardiac catheterization and angiocardiography are diagnosed as CHD, occupy critical role in clinical congenital heart disease is made a definite diagnosis, but its belong to have wound check, complicated operation, there is certain surgical risk, there is the possibility causing pulmonary edema, be therefore not suitable as routine screening means.
Heart development is an extremely complicated process, and its critical period is after becoming pregnant 2 ~ 8 weeks, experienced by the processes such as core barrel formation, cyclisation, interior separation, blood vessel connection, and each stage is subject to the regulation and control of many important gene and signal path.Research shows, inherited genetic factors is the Important cause of disease of nonsyndromic CHD, and wherein single nucleotide polymorphism (singlenucleotidepolymorphism, SNP) plays an important role.SNP refers to the change of the DNA sequence dna caused by the change of the variation of single core thuja acid-A, T, C or G in genomic level, cause comprise the mankind species between the diversity of chromogene group, be that the mankind can modal one in heritable variation.The existence of SNP is considered to impart individual different phenotypic character, and for the differential responses of the factor such as environmental exposure, pharmacological agent.Therefore SNP causes the individual important hereditary basis to common disease morbidity and prognosis susceptibility difference.In recent years, the SNP research for the common major disease such as tumour and cardiovascular disorder has become clinical and study hotspot that is researcher.
But, also SNP is not applied to the report of nonsyndromic congenital heart disease diagnosis at present, if the SNP of nonsyndromic congenital heart disease susceptible can be filtered out as biomarker, and develop corresponding diagnostic kit, diagnose present situation will be once strong promotion to China's nonsyndromic congenital heart disease, also for its drug screening, evaluating drug effect and targeted therapy open new approach.
Summary of the invention
The object of the invention is for above-mentioned technical problem, propose a kind of SNP mark relevant to nonsyndromic congenital heart disease auxiliary diagnosis.
Second object of the present invention is to provide the specificity amplification primer of above-mentioned SNP mark.
The specificity that 3rd object of the present invention is to provide above-mentioned SNP mark extends primer.
4th object of the present invention is to provide above-mentioned SNP mark and specificity amplification primer and/or specificity thereof and extends the application of primer in preparation congenital heart disease auxiliary diagnostic box.
The present invention's the 5th object is to provide nonsyndromic congenital heart disease auxiliary diagnostic box.
Contriver by be separated and study nonsyndromic patients with congenital heart and with the single nucleotide polymorphism in the normal healthy controls peripheral blood DNA of its age, gender matched, find one group with the high specific of nonsyndromic congenital heart disease height correlation and the SNP of susceptibility, and develop the nonsyndromic congenital heart disease auxiliary diagnostic box can being convenient to clinical application, for the examination of nonsyndromic congenital heart disease and diagnosis provide Data support, for finding that the new small molecule medicine with potential therapeutic value provides Data support.
The object of the invention is to be realized by following technical proposal:
A SNP mark relevant to nonsyndromic congenital heart disease auxiliary diagnosis, this mark is the combination of rs3789612, rs4140836, rs13391263, rs13101737, rs1400558, rs6872396, rs13189951, rs9266596, rs2433752, rs7259736, rs490514, rs2474937, rs1531070, rs9533839, rs17019472, rs10182004, rs9461938, rs72759270 and rs1886441.
The specificity amplification primer of described SNP mark, this primer is:
The amplimer sequence of rs3789612 is SEQIDNo:1 and SEQIDNo:2;
The amplimer sequence of rs4140836 is SEQIDNo:4 and SEQIDNo:5;
The amplimer sequence of rs13391263 is SEQIDNo:7 and SEQIDNo:8;
The amplimer sequence of rs13101737 is SEQIDNo:10 and SEQIDNo:11;
The amplimer sequence of rs1400558 is SEQIDNo:13 and SEQIDNo:14;
The amplimer sequence of rs6872396 is SEQIDNo:16 and SEQIDNo:17;
The amplimer sequence of rs13189951 is SEQIDNo:19 and SEQIDNo:20;
The amplimer sequence of rs9266596 is SEQIDNo:22 and SEQIDNo:23;
The amplimer sequence of rs2433752 is SEQIDNo:25 and SEQIDNo:26;
The amplimer sequence of rs7259736 is SEQIDNo:28 and SEQIDNo:29;
The amplimer sequence of rs490514 is SEQIDNo:31 and SEQIDNo:32;
The amplimer sequence of rs2474937 is SEQIDNo:34 and SEQIDNo:35;
The amplimer sequence of rs1531070 is SEQIDNo:37 and SEQIDNo:38;
The amplimer sequence of rs9533839 is SEQIDNo:40 and SEQIDNo:41;
The amplimer sequence of rs17019472 is SEQIDNo:43 and SEQIDNo:44;
The amplimer sequence of rs10182004 is SEQIDNo:46 and SEQIDNo:47;
The amplimer sequence of rs9461938 is SEQIDNo:49 and SEQIDNo:50;
The amplimer sequence of rs72759270 is SEQIDNo:52 and SEQIDNo:53;
The amplimer sequence of rs1886441 is SEQIDNo:55 and SEQIDNo:56.
The specificity of described SNP mark extends primer, and this primer is:
The extension primer sequence of rs3789612 is SEQIDNo:3;
The extension primer sequence of rs4140836 is SEQIDNo:6;
The extension primer sequence of rs13391263 is SEQIDNo:9;
The extension primer sequence of rs13101737 is SEQIDNo:12;
The extension primer sequence of rs1400558 is SEQIDNo:15;
The extension primer sequence of rs6872396 is SEQIDNo:18;
The extension primer sequence of rs13189951 is SEQIDNo:21;
The extension primer sequence of rs9266596 is SEQIDNo:24;
The extension primer sequence of rs2433752 is SEQIDNo:27;
The extension primer sequence of rs7259736 is SEQIDNo:30;
The extension primer sequence of rs490514 is SEQIDNo:33;
The extension primer sequence of rs2474937 is SEQIDNo:36;
The extension primer sequence of rs1531070 is SEQIDNo:39;
The extension primer sequence of rs9533839 is SEQIDNo:42;
The extension primer sequence of rs17019472 is SEQIDNo:45;
The extension primer sequence of rs10182004 is SEQIDNo:48;
The extension primer sequence of rs9461938 is SEQIDNo:51;
The extension primer sequence of rs72759270 is SEQIDNo:54;
The extension primer sequence of rs1886441 is SEQIDNo:57.
Described SNP mark is preparing the application in nonsyndromic congenital heart disease auxiliary diagnostic box.
The specificity amplification primer of described SNP mark is preparing the application in nonsyndromic congenital heart disease auxiliary diagnostic box.
The specificity of described SNP mark extends primer and is preparing the application in nonsyndromic congenital heart disease auxiliary diagnostic box.
A kind of nonsyndromic congenital heart disease auxiliary diagnostic box, this test kit is for detecting rs3789612, rs4140836, rs13391263, rs13101737, rs1400558, rs6872396, rs13189951, rs9266596, rs2433752, rs7259736, rs490514, rs2474937, rs1531070, rs9533839, rs17019472, rs10182004, rs9461938, rs72759270 and rs1886441 in peripheral blood DNA.
Described diagnostic kit, this test kit contains specificity amplification primer and/or the specificity extension primer of above-mentioned SNP mark.
Wherein, specificity amplification primer is:
The amplimer sequence of rs3789612 is SEQIDNo:1 and SEQIDNo:2;
The amplimer sequence of rs4140836 is SEQIDNo:4 and SEQIDNo:5;
The amplimer sequence of rs13391263 is SEQIDNo:7 and SEQIDNo:8;
The amplimer sequence of rs13101737 is SEQIDNo:10 and SEQIDNo:11;
The amplimer sequence of rs1400558 is SEQIDNo:13 and SEQIDNo:14;
The amplimer sequence of rs6872396 is SEQIDNo:16 and SEQIDNo:17;
The amplimer sequence of rs13189951 is SEQIDNo:19 and SEQIDNo:20;
The amplimer sequence of rs9266596 is SEQIDNo:22 and SEQIDNo:23;
The amplimer sequence of rs2433752 is SEQIDNo:25 and SEQIDNo:26;
The amplimer sequence of rs7259736 is SEQIDNo:28 and SEQIDNo:29;
The amplimer sequence of rs490514 is SEQIDNo:31 and SEQIDNo:32;
The amplimer sequence of rs2474937 is SEQIDNo:34 and SEQIDNo:35;
The amplimer sequence of rs1531070 is SEQIDNo:37 and SEQIDNo:38;
The amplimer sequence of rs9533839 is SEQIDNo:40 and SEQIDNo:41;
The amplimer sequence of rs17019472 is SEQIDNo:43 and SEQIDNo:44;
The amplimer sequence of rs10182004 is SEQIDNo:46 and SEQIDNo:47;
The amplimer sequence of rs9461938 is SEQIDNo:49 and SEQIDNo:50;
The amplimer sequence of rs72759270 is SEQIDNo:52 and SEQIDNo:53;
The amplimer sequence of rs1886441 is SEQIDNo:55 and SEQIDNo:56.
Specificity extends primer:
The extension primer sequence of rs3789612 is SEQIDNo:3;
The extension primer sequence of rs4140836 is SEQIDNo:6;
The extension primer sequence of rs13391263 is SEQIDNo:9;
The extension primer sequence of rs13101737 is SEQIDNo:12;
The extension primer sequence of rs1400558 is SEQIDNo:15;
The extension primer sequence of rs6872396 is SEQIDNo:18;
The extension primer sequence of rs13189951 is SEQIDNo:21;
The extension primer sequence of rs9266596 is SEQIDNo:24;
The extension primer sequence of rs2433752 is SEQIDNo:27;
The extension primer sequence of rs7259736 is SEQIDNo:30;
The extension primer sequence of rs490514 is SEQIDNo:33;
The extension primer sequence of rs2474937 is SEQIDNo:36;
The extension primer sequence of rs1531070 is SEQIDNo:39;
The extension primer sequence of rs9533839 is SEQIDNo:42;
The extension primer sequence of rs17019472 is SEQIDNo:45;
The extension primer sequence of rs10182004 is SEQIDNo:48;
The extension primer sequence of rs9461938 is SEQIDNo:51;
The extension primer sequence of rs72759270 is SEQIDNo:54;
The extension primer sequence of rs1886441 is SEQIDNo:57.
Described diagnostic kit, this test kit can also comprise the conventional enzyme of PCR reaction and reagent, as Taq enzyme, dNTP mixed solution, Mgcl
2solution, deionized water etc.; Standard substance and/or reference substance can also be contained.
Specifically, the technical scheme that the present invention deals with problems comprises: (1) sets up sample storehouse and the database of unified standard: gather standard compliant blood sample with Standard operation procedure SOP (SOP), the demographic data that systematic collection is complete and clinical data.(2) genotype detection: select nonsyndromic congenital heart disease case, with nonsyndromic congenital heart disease case age, the contrasting of gender matched, utilize high-density SNP chip, within the scope of full-length genome, find out the SNP relevant to nonsyndromic congenital heart disease.(3) for the positive association SNP screened, SequenomMassARRAY gene type platform is adopted to detect further, to verify the repeatability being applied to diagnose.(4) development of nonsyndromic congenital heart disease auxiliary diagnostic box: develop SNP auxiliary diagnostic box according to the genotype distribution frequency SNP that there were significant differences in nonsyndromic congenital heart disease case and normal healthy controls.
The present inventor gathers standard compliant blood sample with Standard operation procedure SOP (SOP), the demographic data that systematic collection is complete, clinical data etc., and have employed Affymetrix6.0 chip and carry out genome-wide screening, the positive site of primary dcreening operation adopts SequenomMassARRAY time-of-fight mass spectrometry biochip system to verify.
The experimental technique studied specifically mainly comprises following components:
1. study the selection of sample
(1) through nonsyndromic patients with congenital heart that ultrasonic cardiogram, heart catheterization or operation are clarified a diagnosis;
(2) merging other organ congenital anomaly, abnormal chromosome patients are got rid of;
(3) get rid of first degree relative and suffer from the patient that congenital heart disease, mother suffer from diabetes, pku, mother's pregnancy period contact teratogen (as agricultural chemicals or organic solvent) and drug administration;
(4) with case age, the contrasting of gender matched;
This research adopts 8217 routine standard compliant samples to study altogether.
2. phenol-chloroform method extracts peripheral blood genomic dna, operates according to a conventional method.Usually, can obtain 20-50ng/ μ lDNA, purity (ultraviolet 260OD and 280OD ratio) is at 1.6-2.0.
3.Affymetrix6.0 chip detection
(1) experimenter's complete genome DNA sample is got;
(2) on Affymetrix6.0 chip (being purchased from Ang Fei company of the U.S., lower same), genome-wide screening is carried out;
(3) detect and the difference difference of more each genotype in nonsyndromic congenital heart disease case and normal healthy controls.
4.SequenomMassARRAY genotype tests
(1) experimenter's DNA sample is got;
(2) specificity amplification primer and the specificity that design SNP extend primer;
(3) pcr amplification reaction and product purification is carried out;
(4) single base extension and product purification is carried out;
(5) detect and compare the distributional difference of different genotype in nonsyndromic congenital heart disease case and normal healthy controls.
5. diagnostic reagent box preparation method
Affymetrix6.0 chip determines the genotype distribution frequency SNP that there were significant differences in nonsyndromic congenital heart disease case and normal healthy controls after carrying out genome-wide screening and SequenomMassARRAY gene type platform validation, as the index of nonsyndromic congenital heart disease auxiliary diagnosis.The SNP relevant with nonsyndromic congenital heart disease finally filtered out forms auxiliary diagnostic box (rs3789612, rs4140836, rs13391263, rs13101737, rs1400558, rs6872396, rs13189951, rs9266596, rs2433752, rs7259736, rs490514, rs2474937, rs1531070, rs9533839, rs17019472, rs10182004, rs9461938, rs72759270 and rs1886441).Diagnostic reagent can comprise specificity amplification primer and the specificity extension primer of these SNP, and the reagent such as Taq enzyme, dNTP.
6. statistical analysis technique
Utilization chi square test (for classified variable) or studentt inspection (for continuous variable) compare the difference that demographic characteristics etc. distributes between research object group.Association analysis is carried out with the additive model in logistic regression analysis.Ratio calculated ratio (OddsRatio, OR) and their 95% credibility interval.Defining the SNP that those OR are less than 1 is protectiveness SNP; Defining the SNP that those OR are greater than 1 is dangerous SNP.
In order to study the effect of comprehensive indication for early diagnosis of these 19 SNP formations further, we construct a mathematical formula, consider each SNP and associate situation and relation intensity with the positive and negative of nonsyndromic congenital heart disease.Specifically, we mark to three of each SNP kinds of genotype, wild homozygous=" 0 ", heterozygous=" 1 ", make a variation homozygous=" 2 ", with the regression coefficient under additive model during single snp analysis for weight, the situation considering each SNP determines a dangerous score value to each research research contrast.Risk score calculation method is as follows: risk score = (0.447 x rs3789612 score) + (0.490 x rs4140836 score) + (0.338 x rs13391263 score) + (0.325 x rs13101737 score) + (0.266 x rs1400558 score) + (0.570 x rs6872396 score) + (0.397 x rs13189951 score) + (0.477 x rs9266596 score) + (0.329 x rs2433752 ratings) + (0.332 x rs7259736 score) + (0.255 x rs490514 score) + (0.499 x rs2474937 score) + (0.479 x rs1531070 score) + (0.392 x rs9533839 score) + (0.284 x rs17019472 ratings) + (0.270 x rs10182004 score) + (0.570 x rs9461938 score) + (0.341 x rs72759270 score) + (0.311 x rs1886441 score), the risk score coefficient and threshold value is directly used in the whole genome association studies of 2191 cases of sample.(be the regression coefficient (see table 1) of rs3789612 for rs3789612: 0.447; The scoring of rs3789612, wild homozygous=" 0 ", heterozygous=" 1 ", make a variation homozygous=" 2 ", and the genotype of certain SNP is determined by Instrumental results; The overall score of certain sample is the summation that this few SNP marks respectively, and the genotype of single SNP just calculates a pilot process of scoring, does not need to know concrete genotype.)
Statistical analysis is all completed (PLINK1.07) by special statistical analysis software.The horizontal P value of significance,statistical is set to 0.05, and all statistical test are two-tailed test.
Below further instruction of the present invention:
In above-mentioned 945 routine qualified nonsyndromic congenital heart disease cases and 1246 routine normal healthy controls, two groups of ages, sex equilibrium are comparable.These two groups of crowds are carried out genome-wide screening through Affymetrix6.0 chip and obtain correlated results by us.
According to Affymetrix6.0 chip detection, the present inventor detects that the SNP that genotype distribution frequency there are differences in " nonsyndromic congenital heart disease case " group and " normal healthy controls " group comprises: rs3789612, rs4140836, rs13391263, rs13101737, rs1400558, rs6872396, rs13189951, rs9266596, rs2433752, rs7259736, rs490514, rs2474937, rs1531070, rs9533839, rs17019472, rs10182004, rs9461938, rs72759270 and rs1886441.
According to above-mentioned detected result, we by these 20 SNPs relevant to nonsyndromic congenital heart disease other 2160 routine nonsyndromic congenital heart disease cases and with 3866 routine normal healthy controls of its age, gender matched in verify at SequenomMassARRAY gene type platform, result is consistent with Affymetrix6.0 chip detection.
Single factor test and logistic Regression Analysis result all show, these 19 SNP also exist remarkable association with the morbidity of nonsyndromic congenital heart disease.
The combination of these 19 SNP of further analysis is used for the effect of nonsyndromic congenital heart disease diagnosis, finds that its combination can be good at distinguishing case and contrasting.
According to above-mentioned experimental result, the present inventor has prepared a kind of test kit that can be used for nonsyndromic congenital heart disease auxiliary diagnosis, comprises the specificity amplification primer, specificity extension primer and other detection reagent that measure above-mentioned SNP in experimenter's blood specimen DNA.
Specifically, the combination of these 19 SNP, or the dependent diagnostic test kit that the combination that the specificity amplification primer of these 19 SNP, specificity extend primer is formed contributes to the auxiliary diagnosis of nonsyndromic congenital heart disease, for clinician quick and precisely grasps morbid state and the coincident with severity degree of condition of patient, the control prece of more personalized is taked to provide support in time.
Beneficial effect of the present invention:
SNP mark provided by the invention is as the superiority of the mark of nonsyndromic congenital heart disease auxiliary judgment:
(1) SNP is a kind of novel gene biomarker, be different from traditional biological mark, stable, Wicresoft, be easy to detect, to greatly improve the Sensitivity and Specificity of medical diagnosis on disease, Diagnosis and Treat for nonsyndromic congenital heart disease is started brand-new situation by the successful exploitation of such biomarker, for the development of other diseases biomarker is offered reference.
(2) SNP test kit is a kind of system, comprehensively diagnostic kit, can be used for the auxiliary diagnosis of nonsyndromic congenital heart disease, contribute to the morbid state reflecting patient, for clinician quick and precisely grasps conditions of patients, takes the control prece of more personalized to provide support in time.
(3) adopt tight checking and appraisement system, the present inventor's initial stage adopts full-length genome chip scanning to obtain the SNP spectrum of disease-related, and verifies on SequenomMassARRAY gene type platform; The application acceleration of above methods and strategies and ensure that the application clinically of SNP biomarker and diagnostic kit is also the reference on the development supplying method of other diseases biomarker and strategy.
The present invention is by controlling age, sex etc. to the influence factor of disease progression, and research SNP, in the application prospect of nonsyndromic congenital heart disease auxiliary diagnosis, sets forth SNP for the impact of nonsyndromic Congenital Heart disease progression, discloses its diagnostic value.Therefore, present invention obtains nonsyndromic congenital heart disease related SNP spectrum and Specific marker; By the development and application of SNP biomarker and diagnostic kit, the diagnosis of nonsyndromic congenital heart disease can be made more convenient and easy, for clinician quick and precisely grasps conditions of patients, for clinical therapeutic efficacy evaluation lays the foundation, and for finding that the new small molecule drug targets with potential therapeutic value is offered help.
Accompanying drawing explanation
Fig. 1: the ROC curve of display genome-wide association study case group and control group.
Display nonsyndromic congenital heart disease case group is the ROC curve of reference to normal healthy controls group.
Embodiment
The collection of embodiment 1 sample and the arrangement of sample data
Contriver started to have collected a large amount of nonsyndromic patients with congenital heart blood specimens to 2012 from No.1 Attached Hospital, Nanjing Medical Univ, Nanjing children's hospital, Xijing, Xi'an hospital in 2006, by the arrangement to sample data, contriver therefrom have selected 8217 examples and meets the sample full-length genome chip scanning of following standard and the laboratory sample of single SNPSequenomMassARRAY gene type:
1, through nonsyndromic patients with congenital heart that ultrasonic cardiogram, heart catheterization or operation are clarified a diagnosis;
2, merging other organ congenital anomaly, abnormal chromosome patients are got rid of;
3, get rid of first degree relative and suffer from the patient that congenital heart disease, mother suffer from diabetes, pku, mother's pregnancy period contact teratogen (as agricultural chemicals or organic solvent) and drug administration;
4, with the case age, gender matched normal healthy controls;
And the system acquisition situation such as demographic data and clinical data of these samples.
The genome-wide screening of SNP in embodiment 2 peripheral blood DNA
In above-mentioned qualified 945 routine nonsyndromic patients with congenital heart and 1246 routine normal healthy controls, two groups of ages, gender matched.These two groups of crowds are obtained correlated results through Affymetrix6.0 chip detection.Concrete steps are:
1, hemolyzing reagent (i.e. lysate is added to the peripheral blood be stored in 2ml cryopreservation tube, 40 deal collocation methods are as follows: after sucrose 219.72g, magnesium chloride 2.02g and triton x-100 (amresco0694) 20ml mix, 2000ml is settled to TrisHcl solution, lower same), proceed to completely after putting upside down mixing.
2, red corpuscle is removed: mend 5ml centrifuge tube to 4ml with hemolyzing reagent, put upside down mixing, centrifugal 10 minutes of 4000rpm, abandons supernatant.In precipitation, add 4ml hemolyzing reagent, again put upside down mixing cleaning once, centrifugal 10 minutes of 4000rpm, abandons supernatant.
3, extracting DNA: add 1ml extract (containing 122.5ml0.2M sodium-chlor in every 300ml in precipitation, 14.4ml0.5M ethylenediamine tetraacetic acid (EDTA), 15ml10% sodium lauryl sulphate, 148.1ml distilled water, lower same) and 8 μ l Proteinase Ks, oscillator fully shakes mixing, and 37 DEG C of water-baths are spent the night.
4, protein is removed: add the saturated phenol of 1ml and fully mix (have gentle hands shakes 15 minutes), centrifugal 10 minutes of 4000rpm, get supernatant and proceed in new 5ml centrifuge tube.Equal-volume chloroform and primary isoamyl alcohol mixed solution (chloroform: primary isoamyl alcohol=24:1 is added in supernatant liquor, v/v, lower same), fully after mixing (hand 15 minutes), centrifugal 10 minutes of 4000rpm, gets supernatant (being divided into the centrifuge tube of two 1.5ml).
5, DNA precipitation: the sodium-acetate 60 μ l adding 3M in supernatant liquor, then add isopyknic ice dehydrated alcohol with supernatant liquor, upper and lower jog, visible white flocculent precipitate, then with the centrifugal 10min of 12000rpm.
6, DNA washing: add ice dehydrated alcohol 1ml in precipitation, the centrifugal 10min of 12000rpm, abandon supernatant final vacuum and drain or be placed in clean dry environment evaporate to dryness.
7, concentration is measured: usually can obtain 20-50ng/ μ lDNA, purity (ultraviolet 260OD and 280OD ratio) is at 1.6-2.0.
8, on Affymetrix6.0 chip (being purchased from Ang Fei company of the U.S., lower same), genome-wide screening is carried out;
9, data analysis and process: the genotype distribution frequency SNP that there were significant differences organizing in " nonsyndromic congenital heart disease case " and find in " normal healthy controls " group is enumerated out hereinbefore, the results are shown in Table 1.
The SequeomMassARRAY gene type of the single SNP of embodiment 3
Above-mentioned genome-wide screening is found that the SNP relevant with nonsyndromic congenital heart disease detects in 2160 routine nonsyndromic congenital heart disease cases and 3866 routine normal healthy controls, and concrete steps are:
1, add hemolyzing reagent to the peripheral blood be stored in 2ml cryopreservation tube, proceed to completely after putting upside down mixing.
2, red corpuscle is removed: mend 5ml centrifuge tube to 4ml with hemolyzing reagent, put upside down mixing, centrifugal 10 minutes of 4000rpm, abandons supernatant.In precipitation, add 4ml hemolyzing reagent, again put upside down mixing cleaning once, centrifugal 10 minutes of 4000rpm, abandons supernatant.
3, extracting DNA: add 1ml extract and 8 μ l Proteinase Ks in precipitation, oscillator fully shakes mixing, and 37 DEG C of water-baths are spent the night.
4, protein is removed: add the saturated phenol of 1ml and fully mix (have gentle hands shakes 15 minutes), centrifugal 10 minutes of 4000rpm, get supernatant and proceed in new 5ml centrifuge tube.In supernatant liquor, add equal-volume chloroform and primary isoamyl alcohol mixed solution (chloroform: primary isoamyl alcohol=24:1), fully after mixing (hand 15 minutes), centrifugal 10 minutes of 4000rpm, gets supernatant (being divided into the centrifuge tube of two 1.5ml).
5, DNA precipitation: the sodium-acetate 60 μ l adding 3M in supernatant liquor, then add isopyknic ice dehydrated alcohol with supernatant liquor, upper and lower jog, visible white flocculent precipitate, then with the centrifugal 10min of 12000rpm.
6, DNA washing: add ice dehydrated alcohol 1ml in precipitation, the centrifugal 10min of 12000rpm, abandon supernatant final vacuum and drain or be placed in clean dry environment evaporate to dryness.
7, concentration is measured: usually can obtain 20-50ng/ μ lDNA, purity (ultraviolet 260OD and 280OD ratio) is at 1.6-2.0.
8, SequenomMassARRAY gene type is carried out:
1) Sequenom company GenotypingTools and MassARRAYAssayDesign software is used to design pcr amplification primer and single-basic extension primer (table 2) to the SNP of 19 positive association that genome-wide screening finds.Reaction system comprises 4 μ lSequenomMassARRAY gene type PCRmastermix (HotstarTaq0.5U, the 25mMdNTPs of every bar amplimer 0.5pmol, 0.1 μ l, 1.9 μ l distilled waters), adds 1 μ lDNA.Instrument uses SequenomMassARRAYNanodispenser, and PCR reaction conditions is: 94 DEG C 4 minutes; 94 DEG C 20 seconds, 56 DEG C 30 seconds, 72 DEG C 1 minute, 45 circulations; 72 DEG C 3 minutes; 4 DEG C of maintenances.
2) PCR reaction terminate after, by PCR primer with SAP (shrimpalkalinephosphatase, shrimp alkaline phosphotase) process, with remove system middle reaches from dNTPs.
3) after alkaline phosphatase treatment terminates, carry out single base extension, reaction system comprises: 2 μ lEXTENDMix (single base extension liquid, comprise wherein each extension primer mixture 0.94 μ l, iPLEX enzyme 0.041 μ l, extend mixture 0.2 μ l), PCR primer after 7 μ lSAP process.PCR reaction conditions: I.94 DEG C 30 seconds; II.94 DEG C 5 seconds; III.52 DEG C 5 seconds; IV.80 DEG C 5 seconds; V. III, IV4 circulation is repeated; VI. II, III, IV, V39 circulation is repeated; VII.72 DEG C 3 minutes; VII.4 DEG C of maintenance
4) resin purification:
(1) CleanResin resin is tiled in the resin board of 6mg;
(2) 16 μ l water are added in the corresponding aperture of extension products;
(3) pour in extension products plate by dried resin, sealer, slow speed vertical rotates 30 minutes, and resin is fully contacted with reactant;
(4) the centrifugal resin that makes sinks to bottom hole.
5) chip point sample: start MassARRAYNanodispenserRS1000 point sample instrument, the extension products after resin purification is moved on 384 hole SpectroCHIP (Sequenom) chips
6) mass spectrometric detection: the SpectroCHIP chip after point sample is used MALDI-TOF (matrix-assistedlaserdesorption/ionization – timeoffligh, matrix solid-dispersion flight time mass spectrum) to analyze, detected result uses TYPER4.0 software (sequenom) somatotype and Output rusults.
9, data processing and analysis: the difference utilizing three kinds of genotype distribution frequency in case group and control group of the more each SNP of additive model in logistic regression model, result and full exon scan similarly no longer to be listed.
Embodiment 4 utilizes MELD method to analyze SNP and nonsyndromic congenital heart disease further
According to the above results, the present inventor is by the comparison to 2 groups of samples (" nonsyndromic congenital heart disease case group " and " normal healthy controls group ") genotype distribution frequency, select the SNP of positive association, with SNP regression coefficient single in genome-wide screening sample for weight, try to achieve dangerous score value further, draw susceptibility and specificity that ROC evaluates detection, and then diagnose these SNP to the judgement of nonsyndromic congenital heart disease.Find the Conjoint Analysis of 19 SNP marks, normal healthy controls group and congenital heart disease case group are separated with the AUC of 71.3% by these 19 SNP, and the sensitivity of best cut point is 68.0%, specific degree: 63.8% (Fig. 1).
Therefore, the present inventor demonstrates the combination adopting rs3789612, rs4140836, rs13391263, rs13101737, rs1400558, rs6872396, rs13189951, rs9266596, rs2433752, rs7259736, rs490514, rs2474937, rs1531070, rs9533839, rs17019472, rs10182004, rs9461938, rs72759270 and rs1886441 and can normal healthy controls and nonsyndromic patients with congenital heart be distinguished well.
Embodiment 5 is for the making of nonsyndromic congenital heart disease auxiliary diagnosis SNP test kit
The making of SNP test kit and operating process are based on Affymetrix6.0 chip detection and SequenomMassARRAY genotyping technique.Test kit contains a collection of SNP specificity amplification primer and (comprises following primer: the amplimer sequence of rs3789612 is SEQIDNo:1 and SEQIDNo:2; The amplimer sequence of rs4140836 is SEQIDNo:4 and SEQIDNo:5; The amplimer sequence of rs13391263 is SEQIDNo:7 and SEQIDNo:8; The amplimer sequence of rs13101737 is SEQIDNo:10 and SEQIDNo:11; The amplimer sequence of rs1400558 is SEQIDNo:13 and SEQIDNo:14; The amplimer sequence of rs6872396 is SEQIDNo:16 and SEQIDNo:17; The amplimer sequence of rs13189951 is SEQIDNo:19 and SEQIDNo:20; The amplimer sequence of rs9266596 is SEQIDNo:22 and SEQIDNo:23; The amplimer sequence of rs2433752 is SEQIDNo:25 and SEQIDNo:26; The amplimer sequence of rs7259736 is SEQIDNo:28 and SEQIDNo:29; The amplimer sequence of rs490514 is SEQIDNo:31 and SEQIDNo:32; The amplimer sequence of rs2474937 is SEQIDNo:34 and SEQIDNo:35; The amplimer sequence of rs1531070 is SEQIDNo:37 and SEQIDNo:38; The amplimer sequence of rs9533839 is SEQIDNo:40 and SEQIDNo:41; The amplimer sequence of rs17019472 is SEQIDNo:43 and SEQIDNo:44; The amplimer sequence of rs10182004 is SEQIDNo:46 and SEQIDNo:47; The amplimer sequence of rs9461938 is SEQIDNo:49 and SEQIDNo:50; The amplimer sequence of rs72759270 is SEQIDNo:52 and SEQIDNo:53; The amplimer sequence of rs1886441 is SEQIDNo:55 and SEQIDNo:56.) specificity extend primer (comprise following extension primer: the extension primer sequence of rs3789612 is SEQIDNo:3; The extension primer sequence of rs4140836 is SEQIDNo:6; The extension primer sequence of rs13391263 is SEQIDNo:9; The extension primer sequence of rs13101737 is SEQIDNo:12; The extension primer sequence of rs1400558 is SEQIDNo:15; The extension primer sequence of rs6872396 is SEQIDNo:18; The extension primer sequence of rs13189951 is SEQIDNo:21; The extension primer sequence of rs9266596 is SEQIDNo:24; The extension primer sequence of rs2433752 is SEQIDNo:27; The extension primer sequence of rs7259736 is SEQIDNo:30; The extension primer sequence of rs490514 is SEQIDNo:33; The extension primer sequence of rs2474937 is SEQIDNo:36; The extension primer sequence of rs1531070 is SEQIDNo:39; The extension primer sequence of rs9533839 is SEQIDNo:42; The extension primer sequence of rs17019472 is SEQIDNo:45; The extension primer sequence of rs10182004 is SEQIDNo:48; The extension primer sequence of rs9461938 is SEQIDNo:51; The extension primer sequence of rs72759270 is SEQIDNo:54; The extension primer sequence of rs1886441 is SEQIDNo:57.), the common agents needed for corresponding round pcr can also be had, as: dNTPs, MgCl
2, distilled water, Taq enzyme etc., these common agents are all well known to those skilled in the art, can also have standard substance and contrast (as determined genotypic standard substance and blank etc.) in addition.The value of this test kit is only to need peripheral blood and does not need other tissue sample, by simplifying most with special amplimer and extending primer detection SNP, auxiliary judgment nonsyndromic congenital heart disease is composed again by SNP, not only stablize, easy to detect, and accurately, greatly improve the Sensitivity and Specificity of medical diagnosis on disease, therefore this test kit is dropped into practice, can help to instruct diagnosis and more effective individualized treatment.
Table 1. case group and control group whole-genome association result
Table 2. related SNP primer information
F:ForwardPrimer, upstream primer; R:ReversePrimer, downstream primer; E:ExtendedPrimer, extends primer.
Claims (5)
1. a SNP mark relevant to nonsyndromic congenital heart disease auxiliary diagnosis, is characterized in that this mark is the combination of rs3789612, rs4140836, rs13391263, rs13101737, rs1400558, rs6872396, rs13189951, rs9266596, rs2433752, rs7259736, rs490514, rs2474937, rs1531070, rs9533839, rs17019472, rs10182004, rs9461938, rs72759270 and rs1886441.
2. test right requires the Auele Specific Primer group of the SNP mark described in 1, and it is characterized in that this Auele Specific Primer group comprises specificity amplification primer group and specificity extends primer sets, this specificity amplification primer group is:
The amplimer sequence of rs3789612 is SEQIDNo:1 and SEQIDNo:2;
The amplimer sequence of rs4140836 is SEQIDNo:4 and SEQIDNo:5;
The amplimer sequence of rs13391263 is SEQIDNo:7 and SEQIDNo:8;
The amplimer sequence of rs13101737 is SEQIDNo:10 and SEQIDNo:11;
The amplimer sequence of rs1400558 is SEQIDNo:13 and SEQIDNo:14;
The amplimer sequence of rs6872396 is SEQIDNo:16 and SEQIDNo:17;
The amplimer sequence of rs13189951 is SEQIDNo:19 and SEQIDNo:20;
The amplimer sequence of rs9266596 is SEQIDNo:22 and SEQIDNo:23;
The amplimer sequence of rs2433752 is SEQIDNo:25 and SEQIDNo:26;
The amplimer sequence of rs7259736 is SEQIDNo:28 and SEQIDNo:29;
The amplimer sequence of rs490514 is SEQIDNo:31 and SEQIDNo:32;
The amplimer sequence of rs2474937 is SEQIDNo:34 and SEQIDNo:35;
The amplimer sequence of rs1531070 is SEQIDNo:37 and SEQIDNo:38;
The amplimer sequence of rs9533839 is SEQIDNo:40 and SEQIDNo:41;
The amplimer sequence of rs17019472 is SEQIDNo:43 and SEQIDNo:44;
The amplimer sequence of rs10182004 is SEQIDNo:46 and SEQIDNo:47;
The amplimer sequence of rs9461938 is SEQIDNo:49 and SEQIDNo:50;
The amplimer sequence of rs72759270 is SEQIDNo:52 and SEQIDNo:53;
The amplimer sequence of rs1886441 is SEQIDNo:55 and SEQIDNo:56;
This specificity extends primer sets:
The extension primer sequence of rs3789612 is SEQIDNo:3;
The extension primer sequence of rs4140836 is SEQIDNo:6;
The extension primer sequence of rs13391263 is SEQIDNo:9;
The extension primer sequence of rs13101737 is SEQIDNo:12;
The extension primer sequence of rs1400558 is SEQIDNo:15;
The extension primer sequence of rs6872396 is SEQIDNo:18;
The extension primer sequence of rs13189951 is SEQIDNo:21;
The extension primer sequence of rs9266596 is SEQIDNo:24;
The extension primer sequence of rs2433752 is SEQIDNo:27;
The extension primer sequence of rs7259736 is SEQIDNo:30;
The extension primer sequence of rs490514 is SEQIDNo:33;
The extension primer sequence of rs2474937 is SEQIDNo:36;
The extension primer sequence of rs1531070 is SEQIDNo:39;
The extension primer sequence of rs9533839 is SEQIDNo:42;
The extension primer sequence of rs17019472 is SEQIDNo:45;
The extension primer sequence of rs10182004 is SEQIDNo:48;
The extension primer sequence of rs9461938 is SEQIDNo:51;
The extension primer sequence of rs72759270 is SEQIDNo:54;
The extension primer sequence of rs1886441 is SEQIDNo:57.
3. the Auele Specific Primer group of SNP mark according to claim 2 is preparing the application in nonsyndromic congenital heart disease auxiliary diagnostic box.
4. a nonsyndromic congenital heart disease auxiliary diagnostic box, it is characterized in that this test kit contains specificity amplification primer group and the specificity extension primer sets of SNP mark described in claim 2, for detecting in peripheral blood DNA whether there is rs3789612, rs4140836, rs13391263, rs13101737, rs1400558, rs6872396, rs13189951, rs9266596, rs2433752, rs7259736, rs490514, rs2474937, rs1531070, rs9533839, rs17019472, rs10182004, rs9461938, rs72759270 and rs1886441.
5. diagnostic kit according to claim 4, is characterized in that this test kit also comprises the conventional reagent of round pcr.
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| Identification and functional analysis of CITED2 mutations in patients with congenital heart defects.;Sperling S等;《Hum Mutat.》;20051231;第26卷(第6期);575-582 * |
| 胎儿先天性心脏病多个相关基因ＳＮＰ位点突变的初步研究;解珺淑等;《中国妇产科临床杂志》;20140531;第15卷(第3期);209-212 * |
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