CN104263724A

CN104263724A - Low frequency SNV marker associated with sporadic non-syndromic CHD (congenital heart disease) auxiliary diagnosis and application of low frequency SNV (single nucleotide variant) marker

Info

Publication number: CN104263724A
Application number: CN201410470273.2A
Authority: CN
Inventors: 胡志斌; 沈洪兵; 沙家豪; 陈亦江; 林苑; 许晶
Original assignee: Nanjing Medical University
Current assignee: Nanjing University; Nanjing Medical University
Priority date: 2014-09-15
Filing date: 2014-09-15
Publication date: 2015-01-07
Anticipated expiration: 2034-09-15
Also published as: CN104263724B

Abstract

The invention belongs to the field of genetic engineering and reproductive medicine and discloses a low frequency SNV marker associated with sporadic non-syndromic CHD (congenital heart disease) auxiliary diagnosis and an application of the low frequency SNV marker. The marker is a combination of 41 low frequency SNVs such as rs75423398, rs7527798, rs200531164, rs6756629, rs139026854, rs79043251, rs1801212, rs4956145, rs7689099, rs145248317, rs34525648, rs16891235, rs13194053, rs41269281, rs41270593, rs3130250 and rs3130379. The marker can be used for preparing a sporadic non-syndromic CHD auxiliary diagnosis kit.

Description

A kind of low frequency SNV mark relevant to distributing type nonsyndromic CHD auxiliary diagnosis and application thereof

Invention field

The invention belongs to genetically engineered and reproductive medicine field, relating to a kind of low frequency SNV mark relevant to distributing type nonsyndromic CHD auxiliary diagnosis and application thereof.

Background technology

Congenital heart disease (congenital heart disease, 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 (syndromic CHD) of syndrome and type accounts for patient populations's about 25%, all the other are nonsyndromic CHD (non-syndromic CHD), namely show as cardiovascular malformation and do 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 distributing type 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 distributing type 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 distributes the Important cause of disease of type nonsyndromic CHD, and wherein single nucleotide variations (single nucleotide variant, SNV) plays an important role.SNV 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 SNV is considered to impart individual different phenotypic character, and for the differential responses of the factor such as environmental exposure, pharmacological agent.SNV comprises common genetic variation (common variants) and low frequency heritable variation (rare variants), the former minimum gene frequency >=5%, for low penetrance is suddenlyd change, and the minimum gene frequency < 5% of the latter, for high penetrance is suddenlyd change.Research finds, the number of human genome medium and low frequency heritable variation far exceedes common genetic variation, and low frequency heritable variation can explain the Genetic Contributions of a large portion in common complex disease.Therefore the heritable variation of low frequency height penetrance may be cause the individual important hereditary basis to common disease morbidity and prognosis susceptibility difference.The low frequency height penetrance genetic marker of disease-susceptible humans is utilized to compose the generation diagnosed the illness, sensitive, accurate and quick, have broad application prospects.In recent years, the generation development utilizing low frequency height penetrance genetic marker to diagnose the illness has become clinical and study hotspot that is researcher, in the using value first meeting clue of the common major disease such as tumour and cardiovascular disorder.

But, also low frequency height penetrance genetic marker is not applied to the report distributing the diagnosis of type nonsyndromic congenital heart disease at present, the low frequency height penetrance genetic marker of type nonsyndromic congenital heart disease susceptible is distributed as biomarker if can filter out, and develop corresponding diagnostic kit, distributing type nonsyndromic congenital heart disease diagnosis present situation to China will be once strong promotion, 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, proposing a kind of low frequency height penetrance genetic marker relevant to distributing type nonsyndromic congenital heart disease auxiliary diagnosis and application thereof.

Second object of the present invention is to provide the specificity amplification primer of above-mentioned low frequency height penetrance genetic marker.

The specificity that 3rd object of the present invention is to provide above-mentioned low frequency height penetrance genetic marker extends primer.

4th object of the present invention is to provide above-mentioned low frequency height penetrance genetic marker and specificity amplification primer and specificity thereof and extends the application of primer in the sporadic nonsyndromic congenital heart disease auxiliary diagnostic box of preparation.

The present invention's the 5th object is to provide distributes type nonsyndromic congenital heart disease auxiliary diagnostic box.

Contriver by be separated and research distribute type nonsyndromic patients with congenital heart and with its age, single nucleotide polymorphism in the normal healthy controls peripheral blood DNA of gender matched, find one group with the low frequency SNV of the high specific and susceptibility that distribute type nonsyndromic congenital heart disease height correlation, and develop the type that the distributes nonsyndromic congenital heart disease auxiliary diagnostic box can being convenient to clinical application, for the examination and diagnosis of distributing type nonsyndromic congenital heart disease 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 kind of low frequency height penetrance genetic marker relevant to distributing type nonsyndromic congenital heart disease auxiliary diagnosis, this mark is rs75423398, rs7527798, rs200531164, rs6756629, rs139026854, rs79043251, rs1801212, rs4956145, rs7689099, rs145248317, rs34525648, rs16891235, rs13194053, rs41269281, rs41270593, rs3130250, rs3130379, rs3130785, rs2523480, rs2524276, rs743400, rs140770834, rs213212, rs498114, rs45504893, rs142301665, rs142508708, rs145591245, rs199643484, rs1107947, rs1326331, rs3781411, rs4353250, rs10830430, rs200700939, rs10139931, rs187319098, rs1624825, rs149543099, the combination of rs79298157 and rs9978445.

The specificity amplification primer of described low frequency height penetrance genetic marker, these primers are:

The amplimer sequence of rs75423398 is SEQ ID No:1 and SEQ ID No:2; The amplimer sequence of rs7527798 is SEQ ID No:4 and SEQ ID No:5; The amplimer sequence of rs200531164 is SEQ ID No:7 and SEQ ID No:8; The amplimer sequence of rs6756629 is SEQ ID No:10 and SEQ ID No:11; The amplimer sequence of rs139026854 is SEQ ID No:13 and SEQ ID No:14; The amplimer sequence of rs79043251 is SEQ ID No:16 and SEQ ID No:17; The amplimer sequence of rs1801212 is SEQ ID No:19 and SEQ ID No:20; The amplimer sequence of rs4956145 is SEQ ID No:22 and SEQ ID No:23; The amplimer sequence of rs7689099 is SEQ ID No:25 and SEQ ID No:26; The amplimer sequence of rs145248317 is SEQ ID No:28 and SEQ ID No:29; The amplimer sequence of rs34525648 is SEQ ID No:31 and SEQ ID No:32; The amplimer sequence of rs16891235 is SEQ ID No:34 and SEQ ID No:35; The amplimer sequence of rs13194053 is SEQ ID No:37 and SEQ ID No:38; The amplimer sequence of rs41269281 is SEQ ID No:40 and SEQ ID No:41; The amplimer sequence of rs41270593 is SEQ ID No:43 and SEQ ID No:44; The amplimer sequence of rs3130250 is SEQ ID No:46 and SEQ ID No:47; The amplimer sequence of rs3130379 is SEQ ID No:49 and SEQ ID No:50; The amplimer sequence of rs3130785 is SEQ ID No:52 and SEQ ID No:53; The amplimer sequence of rs2523480 is SEQ ID No:55 and SEQ ID No:56; The amplimer sequence of rs2524276 is SEQ ID No:58 and SEQ ID No:59; The amplimer sequence of rs743400 is SEQ ID No:61 and SEQ ID No:62; The amplimer sequence of rs140770834 is SEQ ID No:64 and SEQ ID No:65; The amplimer sequence of rs213212 is SEQ ID No:67 and SEQ ID No:68; The amplimer sequence of rs498114 is SEQ ID No:70 and SEQ ID No:71; The amplimer sequence of rs45504893 is SEQ ID No:73 and SEQ ID No:74; The amplimer sequence of rs142301665 is SEQ ID No:76 and SEQ ID No:77; The amplimer sequence of rs142508708 is SEQ ID No:79 and SEQ ID No:80; The amplimer sequence of rs145591245 is SEQ ID No:82 and SEQ ID No:83; The amplimer sequence of rs199643484 is SEQ ID No:85 and SEQ ID No:86; The amplimer sequence of rs1107947 is SEQ ID No:88 and SEQ ID No:89; The amplimer sequence of rs1326331 is SEQ ID No:91 and SEQ ID No:92; The amplimer sequence of rs3781411 is SEQ ID No:94 and SEQ ID No:95; The amplimer sequence of rs4353250 is SEQ ID No:97 and SEQ ID No:98; The amplimer sequence of rs10830430 is SEQ ID No:100 and SEQ ID No:101; The amplimer sequence of rs200700939 is SEQ ID No:103 and SEQ ID No:104; The amplimer sequence of rs10139931 is SEQ ID No:106 and SEQ ID No:107; The amplimer sequence of rs187319098 is SEQ ID No:109 and SEQ ID No:110; The amplimer sequence of rs1624825 is SEQ ID No:112 and SEQ ID No:113; The amplimer sequence of rs149543099 is SEQ ID No:115 and SEQ ID No:116; The amplimer sequence of rs79298157 is SEQ ID No:118 and SEQ ID No:119; The amplimer sequence of rs9978445 is SEQ ID No:121 and SEQ ID No:122.

The specificity of described low frequency height penetrance genetic marker extends primer, and these primers are:

The extension primer sequence of rs75423398 is SEQ ID No:3; The extension primer sequence of rs7527798 is SEQ ID No:6; The extension primer sequence of rs200531164 is SEQ ID No:9; The extension primer sequence of rs6756629 is SEQ ID No:12; The extension primer sequence of rs139026854 is SEQ ID No:15; The extension primer sequence of rs79043251 is SEQ ID No:18; The extension primer sequence of rs1801212 is SEQ ID No:21; The extension primer sequence of rs4956145 is SEQ ID No:24; The extension primer sequence of rs7689099 is SEQ ID No:27; The extension primer sequence of rs145248317 is SEQ ID No:30; The extension primer sequence of rs34525648 is SEQ ID No:33; The extension primer sequence of rs16891235 is SEQ ID No:36; The extension primer sequence of rs13194053 is SEQ ID No:39; The extension primer sequence of rs41269281 is SEQ ID No:42; The extension primer sequence of rs41270593 is SEQ ID No:45; The extension primer sequence of rs3130250 is SEQ ID No:48; The extension primer sequence of rs3130379 is SEQ ID No:51; The extension primer sequence of rs3130785 is SEQ ID No:54; The extension primer sequence of rs2523480 is SEQ ID No:57; The extension primer sequence of rs2524276 is SEQ ID No:60; The extension primer sequence of rs743400 is SEQ ID No:63; The extension primer sequence of rs140770834 is SEQ ID No:66; The extension primer sequence of rs213212 is SEQ ID No:69; The extension primer sequence of rs498114 is SEQ ID No:72; The extension primer sequence of rs45504893 is SEQ ID No:75; The extension primer sequence of rs142301665 is SEQ ID No:78; The extension primer sequence of rs142508708 is SEQ ID No:81; The extension primer sequence of rs145591245 is SEQ ID No:84; The extension primer sequence of rs199643484 is SEQ ID No:87; The extension primer sequence of rs1107947 is SEQ ID No:90; The extension primer sequence of rs1326331 is SEQ ID No:93; The extension primer sequence of rs3781411 is SEQ ID No:96; The extension primer sequence of rs4353250 is SEQ ID No:99; The extension primer sequence of rs10830430 is SEQ ID No:102; The extension primer sequence of rs200700939 is SEQ ID No:105; The extension primer sequence of rs10139931 is SEQ ID No:108; The extension primer sequence of rs187319098 is SEQ ID No:111; The extension primer sequence of rs1624825 is SEQ ID No:114; The extension primer sequence of rs149543099 is SEQ ID No:117; The extension primer sequence of rs79298157 is SEQ ID No:120; The extension primer sequence of rs9978445 is SEQ ID No:123.

Described low frequency height penetrance genetic marker is preparing the application of distributing in type nonsyndromic congenital heart disease auxiliary diagnostic box.

The specificity amplification primer of described low frequency height penetrance genetic marker is preparing the application of distributing in type nonsyndromic congenital heart disease auxiliary diagnostic box.

The specificity of described low frequency height penetrance genetic marker extends primer pair and is preparing the application of distributing in type nonsyndromic congenital heart disease auxiliary diagnostic box.

One distributes type nonsyndromic congenital heart disease auxiliary diagnostic box, this test kit is for detecting rs75423398 in peripheral blood DNA, rs7527798, rs200531164, rs6756629, rs139026854, rs79043251, rs1801212, rs4956145, rs7689099, rs145248317, rs34525648, rs16891235, rs13194053, rs41269281, rs41270593, rs3130250, rs3130379, rs3130785, rs2523480, rs2524276, rs743400, rs140770834, rs213212, rs498114, rs45504893, rs142301665, rs142508708, rs145591245, rs199643484, rs1107947, rs1326331, rs3781411, rs4353250, rs10830430, rs200700939, rs10139931, rs187319098, rs1624825, rs149543099, rs79298157 and rs9978445.

Described diagnostic kit, this test kit contains above-mentioned specificity amplification primer and/or specificity extends primer.

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 to distribute type nonsyndromic congenital heart disease case, with distribute type nonsyndromic congenital heart disease case age, the contrasting of gender matched, utilize high-density low frequency height penetrance genetic marker chip, within the scope of full exon, find out the low frequency height penetrance genetic marker relevant to distributing type nonsyndromic congenital heart disease.(3) to the positive association low frequency genetic marker filtered out, Sequenom MassARRAY gene type platform is adopted to detect further, to verify the repeatability being applied to diagnose.(4) development of type nonsyndromic congenital heart disease auxiliary diagnostic box is distributed: according to distributing the low frequency height penetrance genetic marker that there were significant differences of genotype distribution frequency in type nonsyndromic congenital heart disease case and normal healthy controls exploitation auxiliary diagnostic box.

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 Illumina human Exome Beadchip (Illumina Inc., San Diego, CA) chip carries out the scanning of full exon 1, and the positive site of primary dcreening operation adopts Sequenom MassARRAY 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 7937 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 l DNA, purity (ultraviolet 260OD and 280OD ratio) is at 1.6-2.0.

3.Illumina human Exome Beadchip (Illumina Inc., San Diego, CA) chip detection

(1) experimenter's complete genome DNA sample is got;

(2) at Illumina human Exome Beadchip (Illumina Inc., San Diego, CA) chip carries out the scanning of full exon 1;

(3) detect and more each genotype is distributing the difference difference in type nonsyndromic congenital heart disease case and normal healthy controls.

4.Sequenom MassARRAY genotype tests

(1) experimenter's DNA sample is got;

(2) specificity amplification primer and the specificity that design genetic marker 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 distributing different genotype in type nonsyndromic congenital heart disease case and normal healthy controls.

5. diagnostic reagent box preparation method

Illumina Infinium zero RHuman Exome BeadChip (Illumina Inc., San Diego, CA) chip determines to distribute the genotype distribution frequency low frequency genetic marker that there were significant differences in type nonsyndromic congenital heart disease case and normal healthy controls, as the index of distributing the diagnosis of type nonsyndromic congenital heart disease after carrying out the scanning of full exon group and the detection of single genetic marker.The low frequency genetic marker relevant with distributing type nonsyndromic congenital heart disease finally filtered out forms auxiliary diagnostic box (rs75423398, rs7527798, rs200531164, rs6756629, rs139026854, rs79043251, rs1801212, rs4956145, rs7689099, rs145248317, rs34525648, rs16891235, rs13194053, rs41269281, rs41270593, rs3130250, rs3130379, rs3130785, rs2523480, rs2524276, rs743400, rs140770834, rs213212, rs498114, rs45504893, rs142301665, rs142508708, rs145591245, rs199643484, rs1107947, rs1326331, rs3781411, rs4353250, rs10830430, rs200700939, rs10139931, rs187319098, rs1624825, rs149543099, rs79298157 and rs9978445).Diagnostic reagent can comprise specificity amplification primer and/or the specificity extension primer of these genetic markers, and the reagent such as Taq enzyme, dNTP.

6. statistical analysis technique

Utilization χ2-test,chi-square test (for classified variable) or student t 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.

In order to study the effect of comprehensive indication for early diagnosis of these 41 low frequency height penetrance genetic markers formations further, we construct a mathematical formula, consider each low frequency SNV and distribute the positive and negative of type nonsyndromic congenital heart disease and associate situation and relation intensity.Specifically, we mark to three kinds of genotype of each low frequency genetic marker, wild homozygous=" 0 ", heterozygous=" 1 ", make a variation homozygous=" 2 ", regression coefficient under additive model when analyzing with single genetic marker is for weight, and the situation considering each low frequency genetic marker determines a dangerous score value to each research research contrast.The following calculation method of risk: risk score = score (0.973 x rs75423398 score) + (0.446 x rs7527798 score) + (1.027 x rs200531164 score) + (0.848 * rs6756629 + score) (0.834 x rs139026854 score) + (0.842 x rs79043251 score) + (2.150 x rs1801212 score) + (0.443 * rs4956145 + score) (1.094 x rs7689099 score) + (2.207 x rs145248317 score) + (1.334 x rs34525648 score) + (0.738 * rs16891235 + score) (0.499 x rs13194053 score) + (1.334 x rs41269281 score) + (0.521 x rs41270593 score) + (0.730 * rs3130250 + score) (0.501 x rs3130379 score) + (0.534 x rs3130785 score) + (0.742 x rs2523480 score) + (1.024 * rs2524276 + score) (1.596 x rs743400 score) + (0.691 x rs140770834 score) + (0.494 x rs213212 score) + (0.558 * rs498114 + score) (0.940 x rs45504893 score) + (0.488 x rs142301665 score) + (0.798 x rs142508708 score) + (2.208 * rs145591245 + score) (1.254 x rs199643484 score) + (1.016 x rs1107947 score) + (2.026 x rs1326331 score) + (0.406 * rs3781411 + score) (0.613 x rs4353250 score) + (1.113 x rs10830430 score) + (1.173 x rs200700939 score) + (0.418 * rs10139931 + score) (1.671 x rs187319098 score) + (1.662 x rs1624825 score) + (0.997 x rs149543099 score) + (0.619 * rs79298157 + score) (0.395 x rs9978445 score), risk score coefficients obtained and the limit values of 3937 cases of sample is directly applied to the use of exon microarray scanning all exons in.(be the regression coefficient (see table 1) of rs75423398 for rs75423398: 0.973; The scoring of rs75423398, wild homozygous=" 0 ", heterozygous=" 1 ", make a variation homozygous=" 2 ", and the genotype of certain SNV is determined by Instrumental results; The overall score of certain sample is the summation that this few SNV marks respectively, and the genotype of single SNV 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:

Qualifiedly distribute in type nonsyndromic congenital heart disease case and 2953 routine normal healthy controls in above-mentioned 984 examples, two groups of ages, sex equilibrium are comparable.We by these two groups of crowds through Illumina human Exome Beadchip (Illumina Inc, San Diego, CA) chip carries out the scanning of full exon 1 and obtains correlated results.

According to Illumina human Exome Beadchip (Illumina Inc, San Diego, CA) chip detection, the present inventor detects that the genetic marker that genotype distribution frequency there are differences in " distributing type nonsyndromic congenital heart disease case " group and " normal healthy controls " group comprises: rs75423398, rs7527798, rs200531164, rs6756629, rs139026854, rs79043251, rs1801212, rs4956145, rs7689099, rs145248317, rs34525648, rs16891235, rs13194053, rs41269281, rs41270593, rs3130250, rs3130379, rs3130785, rs2523480, rs2524276, rs743400, rs140770834, rs213212, rs498114, rs45504893, rs142301665, rs142508708, rs145591245, rs199643484, rs1107947, rs1326331, rs3781411, rs4353250, rs10830430, rs200700939, rs10139931, rs187319098, rs1624825, rs149543099, rs79298157 and rs9978445.

According to above-mentioned detected result, we by these 41 low frequency genetic markers relevant to distributing type nonsyndromic congenital heart disease other 2000 examples distribute type nonsyndromic congenital heart disease case and with 2000 routine normal healthy controls of its age, gender matched in verify at Sequenom MassARRAY gene type platform, result and Illumina human Exome Beadchip (Illumina Inc, San Diego, CA) chip detection is consistent.

Single factor test and logistic Regression Analysis result all show, these 41 low frequency genetic markers also exist remarkable association with the morbidity of distributing type nonsyndromic congenital heart disease.

The combination of these 41 low frequency genetic markers of further analysis, for distributing the effect of type 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 distributing type nonsyndromic congenital heart disease auxiliary diagnosis, comprises the specificity amplification primer, specificity extension primer and other detection reagent that measure above-mentioned low frequency genetic marker in experimenter's blood specimen DNA.

Specifically, the combination of these 41 low frequency genetic markers, or the dependent diagnostic test kit that the combination that the specificity amplification primer of these 41 low frequency genetic markers and specificity extend primer is formed contributes to the auxiliary diagnosis distributing type 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:

Low frequency height penetrance genetic marker genetic marker provided by the invention is as the superiority of the mark distributing type nonsyndromic congenital heart disease auxiliary judgment:

(1) low frequency height penetrance genetic marker 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 distributing type 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) genetic marker test kit is a kind of system, comprehensively diagnostic kit, can be used for the auxiliary diagnosis distributing type 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 low frequency height penetrance genetic marker spectrum of disease-related, and verifies on Sequenom MassARRAY gene type platform; The application acceleration of above methods and strategies and ensure that low frequency height penetrance genetic marker biomarker and diagnostic kit application clinically 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, research low frequency height penetrance genetic marker is in the application prospect of distributing type nonsyndromic congenital heart disease auxiliary diagnosis, set forth low frequency height penetrance genetic marker for the impact of distributing type nonsyndromic Congenital Heart disease progression, disclose its diagnostic value.Therefore, present invention obtains distribute type nonsyndromic congenital heart disease be correlated with low frequency height penetrance genetic marker spectrum and Specific marker; By the development and application of genetic marker and diagnostic kit, the diagnosis of distributing type 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 showing full exon group association study case group and control group.

The ROC curve that type nonsyndromic congenital heart disease case group is reference to normal healthy controls group is distributed in display.

Embodiment

The invention will be further elaborated by the following examples.

The collection of embodiment 1 sample and the arrangement of sample data

Contriver started to have collected a large amount of type that distributes 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 7937 examples and meets the sample full exon group chip scanning of following standard and the laboratory sample of Sequenom MassARRAY 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 full exon group scanning of embodiment 2 peripheral blood DNA medium and low frequency height penetrance genetic marker

Distribute in type nonsyndromic patients with congenital heart and 2953 routine normal healthy controls in above-mentioned qualified 984 examples, two groups of ages, gender matched.By these two groups of crowds through Illumina human Exome Beadchip (Illumina Inc., San Diego, CA) chip detection obtains correlated results.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 (amresco 0694) 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 122.5ml 0.2M sodium-chlor in every 300ml in precipitation, 14.4ml 0.5M ethylenediamine tetraacetic acid (EDTA), 15ml 10% sodium lauryl sulphate, 148.1 ml distilled waters, 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, centrifugal 10 min 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 l DNA, purity (ultraviolet 260OD and 280OD ratio) is at 1.6-2.0.

8, the scanning of full exon group is carried out: at Illumina human Exome Beadchip (Illumina Inc., San Diego, CA) chip carries out the scanning of full exon 1.

9, data analysis and process: the genotype distribution frequency low frequency height penetrance genetic marker that there were significant differences organized " distributing type nonsyndromic congenital heart disease case " and find in " normal healthy controls " group is enumerated out hereinbefore, the results are shown in Table 1.

Table 1. case group and control group full exon group association analysis result

The Sequenom MassARRAY gene type of embodiment 3 low frequency height penetrance genetic marker

Distributed in type nonsyndromic congenital heart disease case and 2000 routine normal healthy controls in other 2000 examples by low frequency height penetrance genetic marker relevant with congenital heart disease for above-mentioned full exon group scanning discovery and detect, 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.

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).

8, Sequenom MassARRAY gene type is carried out:

1) Sequenom company Genotyping Tools and MassARRAY Assay Design software is used to design pcr amplification primer and single-basic extension primer (table 2) to the SNP of 41 positive association that genome-wide screening finds.Reaction system comprises 4 μ l Sequenom MassARRAY gene type PCR master mix (Hotstar Taq0.5 U, the 25mM dNTPs of every bar amplimer 0.5pmol, 0.1 μ l, 1.9 μ l distilled waters), adds 1 μ l DNA.Instrument uses Sequenom MassARRAY Nanodispenser, 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 (shrimp alkaline phosphatase, 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 l EXTEND Mix (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 l SAP 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, IV 4 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) Clean Resin 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 MassARRAY Nanodispenser RS1000 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-assisted laser desorption/ionization – time of fligh, matrix solid-dispersion flight time mass spectrum) to analyze, detected result uses TYPER 4.0 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 genetic marker of additive model in logistic regression model, result and full exon scan similarly no longer to be listed.

Embodiment 4 utilizes MELD method further analysing low frequency height penetrance genetic marker and distributes type nonsyndromic congenital heart disease

According to the above results, the present inventor is by the comparison to 2 groups of samples (" distributing type nonsyndromic congenital heart disease case group " and " normal healthy controls group ") genotype distribution frequency, select the genetic marker of positive association, with low frequency genetic marker regression coefficient single in full exon group scanned samples for weight, try to achieve dangerous score value further, draw susceptibility and specificity that ROC assesses prediction, and then assess these low frequency height penetrance genetic markers to the judgement of distributing type nonsyndromic congenital heart disease.The Conjoint Analysis of 41 low frequency genetic markers is found, normal healthy controls group and congenital heart disease case group are separated with the AUC of 69.5% by these 41 low frequency genetic markers, the sensitivity of best cut point is 50.6%, specific degree: 79.1% (Fig. 1).

Therefore, the present inventor demonstrates and adopts rs75423398, rs7527798, rs200531164, rs6756629, rs139026854, rs79043251, rs1801212, rs4956145, rs7689099, rs145248317, rs34525648, rs16891235, rs13194053, rs41269281, rs41270593, rs3130250, rs3130379, rs3130785, rs2523480, rs2524276, rs743400, rs140770834, rs213212, rs498114, rs45504893, rs142301665, rs142508708, rs145591245, rs199643484, rs1107947, rs1326331, rs3781411, rs4353250, rs10830430, rs200700939, rs10139931, rs187319098, rs1624825, rs149543099, the combination of rs79298157 and rs9978445 can well by normal healthy controls with distribute type nonsyndromic patients with congenital heart and distinguish.

Embodiment 5 is for distributing the making of type nonsyndromic congenital heart disease auxiliary diagnosis low frequency height penetrance genetic marker test kit

Making and the operating process of low frequency height penetrance genetic marker test kit are based on Illumina human Exome Beadchip (Illumina Inc., San Diego, CA) chip detection and Sequenom MassARRAY gene type detection of platform technology.Test kit contains a collection of low frequency height penetrance genetic marker specificity amplification primer and (comprises following primer: the amplimer sequence of rs75423398 is SEQ ID No:1 and SEQ ID No:2; The amplimer sequence of rs7527798 is SEQ ID No:4 and SEQ ID No:5; The amplimer sequence of rs200531164 is SEQ ID No:7 and SEQ ID No:8; The amplimer sequence of rs6756629 is SEQ ID No:10 and SEQ ID No:11; The amplimer sequence of rs139026854 is SEQ ID No:13 and SEQ ID No:14;

The amplimer sequence of rs79043251 is SEQ ID No:16 and SEQ ID No:17; The amplimer sequence of rs1801212 is SEQ ID No:19 and SEQ ID No:20; The amplimer sequence of rs4956145 is SEQ ID No:22 and SEQ ID No:23; The amplimer sequence of rs7689099 is SEQ ID No:25 and SEQ ID No:26; The amplimer sequence of rs145248317 is SEQ ID No:28 and SEQ ID No:29; The amplimer sequence of rs34525648 is SEQ ID No:31 and SEQ ID No:32; The amplimer sequence of rs16891235 is SEQ ID No:34 and SEQ ID No:35; The amplimer sequence of rs13194053 is SEQ ID No:37 and SEQ ID No:38; The amplimer sequence of rs41269281 is SEQ ID No:40 and SEQ ID No:41; The amplimer sequence of rs41270593 is SEQ ID No:43 and SEQ ID No:44; The amplimer sequence of rs3130250 is SEQ ID No:46 and SEQ ID No:47;

The amplimer sequence of rs3130379 is SEQ ID No:49 and SEQ ID No:50; The amplimer sequence of rs3130785 is SEQ ID No:52 and SEQ ID No:53; The amplimer sequence of rs2523480 is SEQ ID No:55 and SEQ ID No:56; The amplimer sequence of rs2524276 is SEQ ID No:58 and SEQ ID No:59; The amplimer sequence of rs743400 is SEQ ID No:61 and SEQ ID No:62; The amplimer sequence of rs140770834 is SEQ ID No:64 and SEQ ID No:65; The amplimer sequence of rs213212 is SEQ ID No:67 and SEQ ID No:68;

The amplimer sequence of rs498114 is SEQ ID No:70 and SEQ ID No:71; The amplimer sequence of rs45504893 is SEQ ID No:73 and SEQ ID No:74; The amplimer sequence of rs142301665 is SEQ ID No:76 and SEQ ID No:77; The amplimer sequence of rs142508708 is SEQ ID No:79 and SEQ ID No:80;

The amplimer sequence of rs145591245 is SEQ ID No:82 and SEQ ID No:83; The amplimer sequence of rs199643484 is SEQ ID No:85 and SEQ ID No:86; The amplimer sequence of rs1107947 is SEQ ID No:88 and SEQ ID No:89; The amplimer sequence of rs1326331 is SEQ ID No:91 and SEQ ID No:92;

The amplimer sequence of rs3781411 is SEQ ID No:94 and SEQ ID No:95; The amplimer sequence of rs4353250 is SEQ ID No:97 and SEQ ID No:98; The amplimer sequence of rs10830430 is SEQ ID No:100 and SEQ ID No:101; The amplimer sequence of rs200700939 is SEQ ID No:103 and SEQ ID No:104;

The amplimer sequence of rs10139931 is SEQ ID No:106 and SEQ ID No:107; The amplimer sequence of rs187319098 is SEQ ID No:109 and SEQ ID No:110; The amplimer sequence of rs1624825 is SEQ ID No:112 and SEQ ID No:113; The amplimer sequence of rs149543099 is SEQ ID No:115 and SEQ ID No:116;

The amplimer sequence of rs79298157 is SEQ ID No:118 and SEQ ID No:119; The amplimer sequence of rs9978445 is SEQ ID No:121 and SEQ ID No:122) and/or specificity extend primer (comprise following extension primer: the extension primer sequence of rs75423398 is SEQ ID No:3; The extension primer sequence of rs7527798 is SEQ ID No:6;

The extension primer sequence of rs200531164 is SEQ ID No:9; The extension primer sequence of rs6756629 is SEQ ID No:12; The extension primer sequence of rs139026854 is SEQ ID No:15; The extension primer sequence of rs79043251 is SEQ ID No:18; The extension primer sequence of rs1801212 is SEQ ID No:21; The extension primer sequence of rs4956145 is SEQ ID No:24; The extension primer sequence of rs7689099 is SEQ ID No:27; The extension primer sequence of rs145248317 is SEQ ID No:30; The extension primer sequence of rs34525648 is SEQ ID No:33; The extension primer sequence of rs16891235 is SEQ ID No:36; The extension primer sequence of rs13194053 is SEQ ID No:39; The extension primer sequence of rs41269281 is SEQ ID No:42; The extension primer sequence of rs41270593 is SEQ ID No:45;

The extension primer sequence of rs3130250 is SEQ ID No:48; The extension primer sequence of rs3130379 is SEQ ID No:51; The extension primer sequence of rs3130785 is SEQ ID No:54; The extension primer sequence of rs2523480 is SEQ ID No:57; The extension primer sequence of rs2524276 is SEQ ID No:60; The extension primer sequence of rs743400 is SEQ ID No:63; The extension primer sequence of rs140770834 is SEQ ID No:66; The extension primer sequence of rs213212 is SEQ ID No:69; The extension primer sequence of rs498114 is SEQ ID No:72; The extension primer sequence of rs45504893 is SEQ ID No:75; The extension primer sequence of rs142301665 is SEQ ID No:78; The extension primer sequence of rs142508708 is SEQ ID No:81; The extension primer sequence of rs145591245 is SEQ ID No:84; The extension primer sequence of rs199643484 is SEQ ID No:87; The extension primer sequence of rs1107947 is SEQ ID No:90; The extension primer sequence of rs1326331 is SEQ ID No:93; The extension primer sequence of rs3781411 is SEQ ID No:96; The extension primer sequence of rs4353250 is SEQ ID No:99; The extension primer sequence of rs10830430 is SEQ ID No:102;

The extension primer sequence of rs200700939 is SEQ ID No:105; The extension primer sequence of rs10139931 is SEQ ID No:108; The extension primer sequence of rs187319098 is SEQ ID No:111; The extension primer sequence of rs1624825 is SEQ ID No:114; The extension primer sequence of rs149543099 is SEQ ID No:117; The extension primer sequence of rs79298157 is SEQ ID No:120; The extension primer sequence of rs9978445 is SEQ ID No:123), 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 low frequency height penetrance genetic marker, type nonsyndromic congenital heart disease is distributed again by low frequency height penetrance genetic marker spectrum auxiliary judgment, 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 2. is correlated with low frequency height penetrance genetic marker primer information

F:Forward Primer, upstream primer; R:Reverse Primer, downstream primer; E:Extended Primer, extends primer.

Claims

1. a low frequency height penetrance genetic marker relevant to distributing type nonsyndromic congenital heart disease auxiliary diagnosis, it is characterized in that this mark is rs75423398, rs7527798, rs200531164, rs6756629, rs139026854, rs79043251, rs1801212, rs4956145, rs7689099, rs145248317, rs34525648, rs16891235, rs13194053, rs41269281, rs41270593, rs3130250, rs3130379, rs3130785, rs2523480, rs2524276, rs743400, rs140770834, rs213212, rs498114, rs45504893, rs142301665, rs142508708, rs145591245, rs199643484, rs1107947, rs1326331, rs3781411, rs4353250, rs10830430, rs200700939, rs10139931, rs187319098, rs1624825, rs149543099, the combination of rs79298157 and rs9978445.

2. the specificity amplification primer of low frequency height penetrance genetic marker according to claim 1, is characterized in that this primer is:

3. the specificity of low frequency height penetrance genetic marker according to claim 1 extends primer sequence, it is characterized in that this primer is:

4. low frequency height penetrance genetic marker according to claim 1 is preparing the application of distributing in type nonsyndromic congenital heart disease auxiliary diagnostic box.

5. the specificity amplification primer of low frequency height penetrance genetic marker according to claim 2 is preparing the application of distributing in type nonsyndromic congenital heart disease auxiliary diagnostic box.

6. the specificity of low frequency height penetrance genetic marker according to claim 3 extends the application that primer distributes in type nonsyndromic congenital heart disease auxiliary diagnostic box in preparation.

7. a low frequency height penetrance genetic marker detection kit relevant to distributing type nonsyndromic congenital heart disease auxiliary diagnosis, is characterized in that this test kit is for detecting rs75423398 in peripheral blood DNA, rs7527798, rs200531164, rs6756629, rs139026854, rs79043251, rs1801212, rs4956145, rs7689099, rs145248317, rs34525648, rs16891235, rs13194053, rs41269281, rs41270593, rs3130250, rs3130379, rs3130785, rs2523480, rs2524276, rs743400, rs140770834, rs213212, rs498114, rs45504893, rs142301665, rs142508708, rs145591245, rs199643484, rs1107947, rs1326331, rs3781411, rs4353250, rs10830430, rs200700939, rs10139931, rs187319098, rs1624825, rs149543099, rs79298157 and rs9978445.

8. diagnostic kit according to claim 7, is characterized in that this test kit contains specificity amplification primer according to claim 2 and/or specificity according to claim 3 extends primer.

9. diagnostic kit according to claim 7 or 8, is characterized in that this test kit also comprises the conventional reagent of round pcr.