CN101831501A - Han children asthma susceptibility gene detection model and kit thereof - Google Patents

Abstract

The invention relates to a Han children asthma susceptibility gene detection model which consists of nine SNP loci including FcER1 E237G, FcER1 C-109T, ADRB2 R16G, IL4RA Q551R, IL4RA I75V, IL4 C-590T, IL13 A2044G, IL13 C1923T and IL13 C-1112T. The invention also provides a Han children asthma susceptibility gene detection kit. The invention has the advantages that the Han children asthma susceptibility gene detection model is established for the first time in China, thus having innovation; and compared with foreign similar research, the Han children asthma susceptibility gene detection model has species specificity. The Han children asthma susceptibility gene detection kit which detects the nine SNP loci has good asthma detection efficiency (OR=345.3478, P value is less than 0.0001, the accuracy is 92.97%, the sensitivity is 97.92%, and the specificity is 88.02%).

Description

Han children asthma susceptibility gene detection model and test kit thereof

[technical field]

The present invention relates to a kind of detection model, specifically, is about a kind of Han children asthma susceptibility gene detection model and test kit thereof.

[background technology]

Bronchial asthma is one of modal chronic inflammatory airway disease in the world today, also is children's respiratory system common disease, frequently-occurring disease, and its M ﹠ M is ascendant trend year by year, causes serious harm for children's health and society.Along with molecular biology and genetic continuous development, the research of asthma gene day by day being goed deep in recent years, generally acknowledged that at present asthma has obvious family and assembles tendency, is a kind of multiplefactor, disease of multifactorial inheritance of complexity.

The tumor susceptibility gene of seeking asthma morbidity on the human genome is a big focus of genome times afterwards comprehensively disease-susceptible humans Journal of Sex Research.2006 is the 10th year that the mankind finish asthma and allergic disease gene susceptibility loci screening plan, also is to untie the 10th days that these have complicated phenotype disease genetic basis in the worldwide.At the beginning of 2006, processes such as Ober are browsed lot of documents, draw as drawing a conclusion: up to now, had 492 pieces of bibliographical informations 118 genes and asthma or allergic disease phenotypic correlation, wherein 54 genes are able to repetition in 2～5 independent samples, 15 gene (GSTM1, IL10, CTLA4, SPINK5, LTC4S, LTA, GRPA, NOD1, CC16, GSTP1, STAT6, NOS1, CCL5, TBXA2R, TGFB1) in 6～10 samples, be able to repetition, 10 gene (IL4 (interleukin 4), IL13 (interleukin-11 3), CD14, ADRB2 (alpha 1 beta-adrenergic 2 acceptors), HLA-DRB1, HLA-DQB1, TNF, FcER1 (IgE high-affinity receptor), IL4RA (interleukin 4 acceptor α), ADAM33) in＞10 samples, obtain repetition, thereby think and in 6 or more independent sample, be able to 25 genes of multiple and asthma or allergic disease phenotypic correlation, be the real asthma or the tumor susceptibility gene of allergic disease.

Along with the human genome genetic map draw perfect day by day, third generation polymorphism sign is single nucleotide polymorphism (single nucleotide polymorphism, SNP, single nucleotide polymorphism) meaning has exceeded the genetic mapping category, become a kind of new tool of research genome diversity, identification and location disease related gene, be subjected to molecular biology worker's common concern and research.For example, people such as Munthe-Kaas MC confirms first that in 2008 TBX21 gene SNP and allergic asthma are closely related; Taiwan there are some researches show that Eotaxin 1 gene Thr23Thr homozygote has the asthma protective effect, can significantly reduce blood plasma eotaxin concentration, or the like.These researchs focus mostly in finding and verify the distributional difference of single SNP site between case and normal population.Since asthma is a kind of polygenic inheritance disease, pathogenesis relates to many biochemical pathway, and is inevitable interrelated between these SNP sites so.Bibliographical information Korea S scholar in 2007 carries out gene-gene interaction research by 12 SNP sites to 7 asthma candidate genes of national children, finds that two SNP sites of KDR and tnf gene have synergy to asthma.Up to the present various countries are all rarely found about the research of interdependence between the different SNP of the asthma site.We think that seeking out these related SNP has important scientific meaning to understanding pathogenesis of asthma mechanism with from the susceptible physique that the genetics angle detects asthma.

Yet, traditional statistical method can only carry out joint study to two SNP sites at most simultaneously, if can find a kind of novel statistical method that can carry out the high-order interactive analysis simultaneously to a plurality of sites, then may obtain comparatively ideal asthma susceptibility gene detection model.A kind of main flow statistical method of studying the interrelated relation in a plurality of SNP site is arranged at present---and the multiplefactor method of descent (multifactor dimensionality reduction, MDR).This statistical method equals the calendar year 2001 proposition by Ritchie, " factor " is the variable (genotype) in the interaction research, " dimension " is meant the number of the factor in the multiplefactor combination of research, mode modeling with disease susceptibility classification (susceptible type, non-susceptible type), regard a plurality of factors in the research as a multiplefactor combination (genotype combination), so just can be reduced to one dimension two levels (being susceptible type and non-susceptible type) to the data structure of higher-dimension, this process is " dimensionality reduction ".This is a kind of distribution free, need not the statistical analysis method of hereditary pattern, is applicable to case control study, only need possess the genetic data (SNP genotype) in each site, can carry out the interactive analysis of gene-gene, and need not other special conditions.Compare with the traditional statistics modeling method, the advantage of " multiplefactor method of descent " is to reduce the required degree of freedom of modeling greatly, its principal feature is: 1. do not need to specify hereditary pattern (dominance or recessive inheritance) and interaction model (linearity or nonlinear model, addition or multiplied model); 2. in conjunction with the MDR software package, can discern the high-order interaction between a plurality of SNP site.

Owing to there is racial diversify, for understanding the hereditary basis of China Han childhood asthma morbidity, tentatively set up the asthma susceptibility gene detection model, this experiment is based on the above-mentioned theory background, choose 10 SNP sites that in＞10 samples, are able to 5 asthma susceptibility genes of multiple and (be respectively IL13 gene C-1112T, C1923T and A2044G site, IL4 gene C-590T site, IL4RA gene I75V and Q551R site, ADRB2 gene R16G and Q27E site and FcER1 gene C-109T and E237G site) and 5 SNP sites that in 6～10 samples, are able to 3 asthma susceptibility genes of multiple (be respectively TGFB1 (transforminggrowthfactor-) gene C-509T, the R25P site, NOS1 (nitricoxide synthase 1) gene C 5266T site and RANTES, i.e. CCL5 (chemokine ligand 5) gene A-403G and G-28C site) in the crowd of Han nationality, carry out case control study.Each 192 example of asthma cases group and normal control group; Employing polymerase chain reaction-restriction fragment length polymorphism (polymerase chain reaction-restrictionfragment length polymorphism, PCR-RFLP) method is carried out above-mentioned candidate locus gene type to each sample; Integrate all experimental datas, carry out traditional statistics analysis and MDR research, tentatively set up Han children asthma susceptibility gene detection model.Establishing of this model helps differentiate the asthma high risk child, intervenes early, prevents trouble before it happens.This still belongs to the first time at home, has novelty.

[summary of the invention]

The objective of the invention is provides a kind of Han children asthma susceptibility gene detection model at deficiency of the prior art.

One purpose more of the present invention is that a kind of Han nationality children asthma susceptibility gene detection kit is provided.

For achieving the above object, the technical scheme that the present invention takes is: a kind of Han children asthma susceptibility gene detection model, described detection model is made up of FcER1 E237G, FcER1 C-109T, ADRB2R16G, IL4RA Q551R, IL4RA I75V, IL4 C-590T, IL13 A2044G, IL13 C1923T and nine SNP sites of IL13 C-1112T.

For realizing above-mentioned second purpose, the technical scheme that the present invention takes is:

A kind of Han nationality children asthma susceptibility gene detection kit, described test kit is made up of nine little test kits, described nine little test kits are respectively: detect the little test kit in SNP site of FcER1 E237G, FcER1C-109T, ADRB2 R16G, IL4RA Q551R, IL4RA I75V, IL4 C-590T, IL13 A2044G, IL13 C1923T and IL13 C-1112T, the related reagent of dress DNA extracting, pcr amplification, digestion with restriction enzyme and agarose gel electrophoresis step in every little test kit.

A kind of Han nationality children asthma susceptibility gene detection kit, described test kit is made up of two little test kits, described two little test kits are respectively: detect the little test kit in SNP site of ADRB2 R16G and FcER1C-109T, the related reagent of dress DNA extracting, pcr amplification, digestion with restriction enzyme and agarose gel electrophoresis step in every little test kit.

A kind of Han nationality children asthma susceptibility gene detection kit, described test kit is made up of two little test kits, described two little test kits are respectively: detect the little test kit in SNP site of ADRB2 R16G and FcER1 E237G, the related reagent of dress DNA extracting, pcr amplification, digestion with restriction enzyme and agarose gel electrophoresis step in every little test kit.

The invention has the advantages that:

1, Han children asthma susceptibility gene detection model be based upon domestic still belonging to the first time, have novelty; Compare with external similar research, have kind of a group specificity.

2, detect the Han nationality children asthma susceptibility gene detection kit in nine SNP sites among the present invention, it has good asthma and detects usefulness (OR=345.3478, P value＜0.0001, accuracy 92.97%, sensitivity 97.92%, specific degree 88.02%).

3, detect the Han nationality children asthma susceptibility gene detection kit in two SNP sites among the present invention, better than detecting single SNP site (R16G AA or E237G G) and detecting other two SNP sites (IL13 A2044G and ADRB2 R16G) Han nationality children asthma susceptibility gene detection kit detection usefulness.

[description of drawings]

Fig. 1: each SNP (IL13 C-1112T, IL13 C1923T, IL13 A2044G, IL4C-590T) (annotate: numerical markings is the respective segments size behind the Marker electrophoresis to the PCR product in site, and all the other each hole electrophoretic bands are the individual PCR product fragment of part.)

Fig. 2: each SNP (ADRB2 R16G FcER1 C-109T, TGFB1 C-509T, FcER1E237G, ADRB2 Q27E, TGFB1 R25P) (annotate: numerical markings is the respective segments size behind the Marker electrophoresis to the PCR product in site, and all the other each hole electrophoretic bands are the individual PCR product fragment of part.)

Fig. 3: each SNP (IL4RA I75V, NOS1 C5266T, IL4RA Q551R, RANTESA-403G, RANTES G-28C) (annotate: numerical markings is the respective segments size behind the Marker electrophoresis to the PCR product in site, and all the other each hole electrophoretic bands are the individual PCR product fragment of part.)

Fig. 4: each SNP (IL13 C-1112T, IL13 C1923T, IL13 A2044G, IL4C-590T, IL4RA I75V) enzyme in site is cut product (annotate: numerical markings is the respective segments size behind the Marker electrophoresis, and all the other each hole electrophoretic bands are cut the product fragment for the individual enzyme of part.)

Fig. 5: each SNP (IL4RA Q551R, ADRB2 R16G, ADRB2 Q27E, FcER1 C-109T, FcER1 E237G) enzyme in site is cut product (annotate: numerical markings is the respective segments size behind the Marker electrophoresis, and all the other each hole electrophoretic bands are cut the product fragment for the individual enzyme of part.)

Fig. 6: each SNP (TGFB1 C-590T, TGFB1 R25P, NOS1 C5266T, RANTESA-403G, RANTES G-28C) enzyme in site is cut product (annotate: numerical markings is the respective segments size behind the Marker electrophoresis, and all the other each hole electrophoretic bands are cut the product fragment for the individual enzyme of part.)

[embodiment]

Below in conjunction with accompanying drawing embodiment provided by the invention is elaborated.

Embodiment 1

1 materials and methods

1.1 material

1.1.1 research object

(1) asthma cases group: from 192 routine asthma infants of hospital of Xinhua asthma special outpatient clinic, the men and women half and half during 2007 to 2008, and the age is 3～12 years old.All meet global asthma in 2006 and propose (Global Initiative for Asthma, GINA) the childhood asthma Case definition of council's revision: the frequent outbreak of panting (maybe can review relevant) with certain allergen or stimulus; Two lungs news and expiratory phase are main wheezing sound during outbreak, and expiratory phase prolongs; Bronchodilators has obvious curative effects, has the reversibility flow limitation; Except other cause pant, uncomfortable in chest and the cough disease.Because the rsv infection infant often has the generation of panting, and is decided to be 3 years old so will go into to organize lower age limit.75% have an IgE data go into to organize infant, its total IgE level is all more than 400IU/ml.

(2) normal control group: the same period, the men and women half and half from 192 healthy students of Shanghai Normal University and East China University of Science, and the age is 18～22 years old.Inclusion criteria: I and three generations be not to have asthma history, other history of disease of no lung, ametaboly reaction history among the interior lineal relative.Why selecting 18～22 years old this age group in contrast, is for sufficiently long medical diagnosis on disease such as asthma except the time being arranged, anaphylactic disease history such as no asthma before this age at least being described.

More than two groups be the crowd of Han nationality, mutual affinity-less relation.

1.1.2 main agents

1.1.2.1 buccal swab extracting genome DNA reagent

Buccal swab genome DNA extracting reagent kit: available from TIANGEN Biotech (Beijing) Co., Ltd., include damping fluid GA, damping fluid GB, damping fluid GD, damping fluid PW, elution buffer TB, Proteinase K, 4 ℃ of preservations.

12.1.2.2PCR reaction reagent

(1) TaKaRa Ex Taq

Hot Start Version: available from precious biotechnology (Dalian) company limited, include TaKaRa Ex Taq HS (5U/ μ l), 10 * Ex Taq damping fluid (contains Mg ²⁺), dNTP mixed solution (each 2.5mM of dATP, dTTP, dGTP and dCTP) ,-20 ℃ of preservations.

(2) gold medal Taq enzyme: available from u.s.a. applied biosystem company, include gold medal Taq enzyme, 10 * gold medal Taq enzyme HS damping fluid, Mg ²⁺(50mmol/l).

(3) Q-solution: available from the safe bio tech ltd of Ji ,-20 ℃ of preservations.

1.1.2.3 agarose gel electrophoresis reagent

(1) 10 * tbe buffer liquid: contain Tris alkali 108 grams, boric acid 55 grams, 0.5 * MDTA 40ml (PH8.0) in the 1L damping fluid, room temperature preservation.

(2) electrophoresis sample-loading buffer: 6 * Loading Buffer and 10 * Loading Buffer are all available from precious biotechnology (Dalian) company limited, room temperature preservation.

(3) 10mg/ml ethidium bromide: final concentration is 0.5 μ g/ml, room temperature preservation.

(4) Marker: available from TIANGEN Biotech (Beijing) Co., Ltd., 4 ℃ of preservations.

1.1.2.4 restriction enzyme

(1) BstU I and NEBuffer 2 damping fluids: available from New England BioLabs Inc..

(2) BsaA I and NEBuffer 3 damping fluids: available from New England BioLabs Inc..

(3) Alu I and NEBuffer 4 damping fluids: available from New England BioLabs Inc..

(4) BsmF I and NEBuffer 4+BSA buffer solution system: available from New England BioLabsInc..

(5) Bfu I and NEBuffer 4 damping fluids: available from New England BioLabs Inc..

(6) Alwn I and NEBuffer 4 damping fluids: available from New England BioLabs Inc..

(7) Nco I and 10 * Buffer Tango ^TMDamping fluid: available from MBI Fermentas company.

(8) Bse XI and 10 * Buffer BseXI damping fluid: available from MBI Fermentas company.

(9) Tru9 I and NEBuffer 4+BSA buffer solution system: available from New England BioLabsInc..

(10) Xmn I and NEBuffer 2+BSA buffer solution system: available from New England BioLabsInc..

(11) Eco81 I and 10 * Buffer Tango ^TMDamping fluid: available from MBI Fermentas company.

(12) Sau96 I and NEBuffer 4 damping fluids: available from New England BioLabs Inc..

(13) Rsa I and NEBuffer 1 damping fluid: available from New England BioLabs Inc..

(14) Mnl I and 10 * Buffer G+10 * Buffer Tango ^TMBuffer solution system: available from MBIFermentas company.

1.1.3 key instrument

(1) TP600 type grads PCR instrument (Japanese TaKaRa company)

(2) 4-15 type large vol whizzer (German SIGMA company)

(3) TGL-16G type desk centrifuge (Anting Scientific Instrument Factory, Shanghai)

(4) the biological electrophoresis image analysis system (Shanhai Furi Science and Technology Co., Ltd.) of FR-980 type

(5) UV-254 type camera bellows formula ultraviolet transilluminator (Beijing ancient cooking vessel state biotechnology limited liability company)

(6) PowerBC 3002SI type numerical control electrophoresis apparatus (Shanghai Shenergy Biocolor BioScience ﹠ Technology Company)

(7) DYCP-32A type agarose horizontal strip electrophoresis groove (Beijing Liuyi Instrument Factory)

(8) HE-90 level trough glue device (sky, Shanghai energy Science and Technology Ltd.)

(9) DK-8D type electric heating constant temperature tank (going up the grand testing installation of Nereid company limited)

(10) P7021TP-6 type Glanz microwave oven (Shunde District, Foshan City Glanz microwave oven Electrical Appliances Co., Ltd)

(11) JT10001 type electronic balance (Shanghai Jingtian Electronic Instrument Co., Ltd.)

(12) QL-901 type vortice (its woods Bel instrument Manufacturing Co., Ltd of Haimen City)

(13) manual adjustable pipettor (big dragon medical facilities company limited)

1.2 method

1.2.1 collection of specimens

According to the informed consent principle, gather oral cavity buccal mucosa swab.Concrete grammar is as follows:

(1) goes into to organize children's fasting half an hour before gathering.

(2) use cotton swab to scrape the inboard mucous membrane of cheek 10 times, during rotate cotton swab to obtain the maximum cell collecting amount.

(3) cut cotton swab head, put into 2ml sterilization centrifuge tube, lid upper tube cap ,-20 ℃ of preservations.

1.2.2 buccal swab extracting genome DNA

(1) in the 2ml of built-in cotton swab head centrifuge tube, adds 600 μ l damping fluid GA.

(2) add 30 μ l Proteinase K (20mg/ml) solution, the lid upper tube cap, 60 seconds mixings of vortex were placed 60 minutes for 56 ℃, during per 10 minutes vortex mixings once.

(3) add 600 μ l damping fluid GB, 30 seconds mixings of vortex were placed 10 minutes for 70 ℃, and this moment, the solution strain was limpid, and are brief centrifugal to remove the drop of cap wall.

(4) add 300 μ l dehydrated alcohols, 30 seconds mixings of vortex, brief centrifugal to remove the drop of cap wall.

(5) 700 μ l previous step gained solution are added among the adsorption column CR (adsorption column CR puts into collection tube), and 12000rpm (～13400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube, CR puts back in the collection tube with adsorption column.

(6) remaining (4) step gained solution all is transferred among the adsorption column CR repeating step (5).

(7) in adsorption column CR, add 500 μ l damping fluid GD (checking whether added dehydrated alcohol earlier before using), and 12000rpm (～13400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube, CR puts back in the collection tube with adsorption column.

(8) in adsorption column CR, add 700 μ l rinsing liquid PW (checking whether added dehydrated alcohol earlier before using), and 12000rpm (～13400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube, CR puts back in the collection tube with adsorption column.

(9) in adsorption column CR, add 500 μ l rinsing liquid PW, and 12000rpm (～13400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube.

(10) adsorption column CR is put back in the collection tube, and 12000rpm (～13400 * g) centrifugal 2 minutes, outwell waste liquid.Adsorption column CR room temperature was placed several minutes, thoroughly to dry rinsing liquid remaining in the sorbing material.

(11) adsorption column is put into the 1.5ml sterilization centrifuge tube that cuts off pipe lid, to the unsettled dropping 100 μ l elutriant TB in adsorption film mid-way, room temperature was placed 5 minutes, 12000rpm (～13400 * g) centrifugal 30 seconds.

(12) the centrifugal solution that obtains is added in the adsorption column again, room temperature was placed 5 minutes, and 12000rpm (～13400 * g) centrifugal 2 minutes, elutriant is transferred in the new 1.5ml sterilization centrifuge tube.

(13) genomic dna of purifying is put-20 ℃ of preservations.

1.2.3SNP the selection in site and primer design and synthetic

(1) with reference to the report of Ober etc., choose 10 SNP sites that in＞10 samples, are able to 5 asthma susceptibility genes of multiple and (be respectively IL13 gene C-1112T, C1923T and A2044G site, IL4 gene C-590T site, IL4RA gene I75V and Q551R site, ADRB2 gene R16G and Q27E site and FcER1 gene C-109T and E237G site) and 5 SNP sites that in 6～10 samples, are able to 3 asthma susceptibility genes of multiple (be respectively TGFB1 gene C-509T, the R25P site, NOS1 gene C 5266T site and RANTES, i.e. CCL5 gene A-403G and G-28C site).

(2) adopt HotStart PCR, TouchDown PCR and nested PCR method to be increased in above-mentioned 15 SNP sites respectively, use 2.2 version muPlex multiplex PCR design system (online softwares, network address: http://genomics14.bu.edu:8080/MuPlex/MuPlex.html) design of primers is carried out in each site, the RS in these sites number and primer sequence see Table 1～3.Primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.

1.2.4PCR amplification

1.2.4.1HotStart?PCR

(1) HotStart PCR reaction system: the reaction cumulative volume is 10 μ l, includes

TaKaRa?Ex?Taq?HS(5U/μl)????????0.05μl

10 * Ex Taq damping fluid (contains Mg ²⁺) 1 μ l

DNTP mixed solution (each 2.5mM) 1 μ l

Forward primer (50pmol/ μ l) 0.1 μ l

Reverse primer (50pmol/ μ l) 0.1 μ l

Genomic dna 0.4 μ l

ddH ₂O???????????????????????????7.35μl

(2) HotStart pcr amplification reaction condition:

95℃????5min

72℃????10min

4℃?????∞

1.2.4.2TouchDown PCR (claiming touchdown PCR again)

1.2.4.2.1IL13 the TouchDown PCR in C-1112T site

(1) reaction system: the reaction cumulative volume is 10 μ l, includes

Gold medal Taq enzyme 0.05 μ l

10 * gold medal Taq enzyme HS damping fluid, 1.0 μ l

Mg ²⁺(50mmol/l)???????????????0.3μl

dNTP(2.5mM?each)?????????????0.8μl

Forward primer (50pmol/ μ l) 0.1 μ l

Reverse primer (50pmol/ μ l) 0.1 μ l

Genomic dna 0.4 μ l

ddH ₂O????????????????????????7.25μl

(2) amplification reaction condition:

95℃????5min

72℃????10min

4℃?????∞

1.2.4.2.2IL13 the TouchDown PCR in A2044G site

(1) reaction system: the reaction cumulative volume is 10 μ l, includes

TaKaRa?Ex?Taq?HS(5U/μl)????????0.05μl

10 * Ex Taq damping fluid (contains Mg ²⁺) 1 μ l

DNTP mixed solution (each 2.5mM) 1 μ l

Forward primer (50pmol/ μ l) 0.1 μ l

Reverse primer (50pmol/ μ l) 0.1 μ l

Genomic dna 0.4 μ l

ddH ₂O???????????????????????????7.35μl

(2) amplification reaction condition:

95℃????5min

72℃????10min

4℃?????∞

1.2.4.2.3 the TouchDown PCR in IL4RA Q551R site

(1) reaction system: the reaction cumulative volume is 10 μ l, includes

Gold medal Taq enzyme 0.05 μ l

10 * gold medal Taq enzyme HS damping fluid, 1.0 μ l

Mg ²⁺(50mmol/l)???????????????0.3μl

dNTP(2.5mM?each)?????????????0.8μl

Forward primer (50pmol/ μ l) 0.1 μ l

Reverse primer (50pmol/ μ l) 0.1 μ l

Genomic dna 0.4 μ l

ddH ₂O????????????????????????7.25μl

(2) amplification reaction condition:

95℃????5min

72℃????10min

4℃?????∞

1.2.4.3 nest-type PRC (claiming sleeve type PCR again)

1.2.4.3.1 first round PCR

(1) reaction system: the reaction cumulative volume is 10 μ l, includes

Gold medal Taq enzyme 0.05 μ l

10 * gold medal Taq enzyme HS damping fluid, 1.0 μ l

Mg ²⁺(50mmol/l)????????????????0.3μl

5×Q-solution?????????????????2.0μl

dNTP(2.5mM?each)??????????????0.8μl

Outside forward primer (50pmol/ μ l) 0.1 μ l

Outside reverse primer (50pmol/ μ l) 0.1 μ l

Genomic dna 0.4 μ l

ddH ₂O?????????????????????????5.25μl

(2) amplification reaction condition:

95℃????5min

72℃????10min

4℃?????∞

1.2.4.3.2 second takes turns PCR

IL13C-1112T and IL4RA Q551R two sites adopt nest-type PRC+TouchDown PCR method to increase, and promptly after above-mentioned first round PCR reaction finishes, the corresponding TouchDown PCR reaction that 2.4.2 partly introduces are carried out in 1000 times of PCR product dilutions.Following reaction then after above-mentioned first round PCR reaction finishes, is proceeded for 1000 times with the dilution of PCR product in IL4RA I75V and RANTESG-28C two sites:

(1) reaction system: the reaction cumulative volume is 10 μ l, includes

Gold medal Taq enzyme 0.05 μ l

10 * gold medal Taq enzyme HS damping fluid, 1.0 μ l

Mg ²⁺(50mmol/l)?????????????????0.3μl

dNTP(2.5mM?each)???????????????0.8μl

Inboard forward primer (50pmol/ μ l) 0.1 μ l

Inboard reverse primer (50pmol/ μ l) 0.1 μ l

Genomic dna 0.4 μ l

ddH ₂O??????????????????????????7.25μl

(2) amplification reaction condition:

95℃??????5min

72℃??????10min

4℃???????∞

1.2.5PCR product detects

After pcr amplification reaction finishes, (sepharose has added ethidium bromide in preparation process to get behind PCR product 5 μ l and the 1 μ l electrophoresis sample-loading buffer mixing sepharose at 2.0% (w/v), final concentration is 0.5 μ g/ml) go up electrophoresis, press voltage electrophoresis in 0.5 * tbe buffer liquid of 5V/cm; On the biological electrophoresis image analysis system of FR-980 type, carry out gel after electrophoresis finishes and take pictures, analyze the pcr amplification situation.

1.2.6 restriction fragment length polymorphism analysis

(1) restriction enzyme of each SNP site correspondence sees Table 4.

(2) endonuclease reaction system: the reaction system volume is 10 μ l, includes

PCR product 5 μ l

10 * Buffer buffer system, 1 μ l

100 * BSA, 0.1 μ l (only BsmF I, Tru9 I and Xmn I)

Restriction enzyme 0.5U

DdH ₂O complements to cumulative volume and reaches 10 μ l

(3) endonuclease reaction temperature and time: the endonuclease reaction temperature in each SNP site sees Table 4; Reaction times is 16 hours.

The restriction enzyme in each SNP site of table 4, endonuclease reaction temperature and enzyme are cut the product sheet segment length

				??GC：164bp；125bp；39bp
					??NOS1	??C5266T	??NcoⅠ	??37	??CC：146bp??TT：125bp，21bp??CT：146bp，125bp，21bp

Table 4 (continuing)

1.2.7 enzyme is cut product and is detected

Getting every pipe enzyme cuts behind product 5 μ l and the 1 μ l electrophoresis sample-loading buffer mixing sepharose at 4.0% (w/v) (sepharose has added ethidium bromide in preparation process, final concentration is 0.5 μ g/ml) go up electrophoresis, press voltage electrophoresis in 0.5 * tbe buffer liquid of 2V/cm; On the biological electrophoresis image analysis system of FR-980 type, carry out gel after electrophoresis finishes and take pictures, the record result.

1.2.8 statistical analysis

1.2.8.1 genotype frequency and gene frequency calculate

(1) calculating of genotype frequency: A, a represent two kinds of allelotrope respectively, N typical example number

A A frequency=N _{A A}/ (N _{A A}+ N _Aa+ N _Aa)

Aa frequency=N _Aa/ (N _{A A}+ N _Aa+ N _Aa)

Aa frequency=N _Aa/ (N _{A A}+ N _Aa+ N _Aa)

(2) calculating of gene frequency: A, a represent two kinds of allelotrope respectively, N typical example number

A frequency=(N _{A A}+ 1/2N _Aa)/(N _{A A}+ N _Aa+ N _Aa)

A frequency=(N _Aa+ 1/2N _Aa)/(N _{A A}+ N _Aa+ N _Aa)

1.2.8.2Hardy-Weinberg balance goodness of fit check

Calculate the expection genotype of each SNP site in asthma group and control group according to the Hardy-Weinberg equilibrium law

Frequency is used the SPSS11.0 statistical software and is carried out difference between genotype frequency that the chi square test comparative observation arrives and expection genotype frequency, represents that with P＜0.05 difference has statistical significance.Determine that asthma group and the control group chosen all are in the genetic equilibrium state in each SNP site of being studied, have colony's representativeness.

2.2.8.3 using the SPSS11.0 statistical software, genotype, allelotrope and genotype combination distribution difference statistical analysis carry out chi square test to analyze each SNP site base between asthma group and control group

Because of type, allelotrope and genotype combination frequency difference, has statistical significance with P＜0.05 expression difference.If the genotype in certain SNP site and gene frequency have significant difference between group, illustrate that this polymorphic site and asthma have dependency, otherwise then do not have dependency with asthma.

1.2.8.4 the high-order interactive analysis in many SNP sites

Use MDR 2.0beta software and carry out the high-order interactive analysis in a plurality of SNP site, set up the asthma detection model.The freeware (can in http://www.epistasis.org/mdr.html free download) that the MDR software program package is based on the open source that java applet writes all can carry out MDR and analyze under several operation systems.Java 2 Runtime Environment (JRE, can in http://www.java.com/ free download) are installed earlier, direct click mdr.jar running paper program under the Windows operating system.

2 results

2.1 the PCR product and the enzyme in each SNP site are cut the product detected result

Adopt the PCR-RFLP method that gene type is carried out in 15 SNP sites, the PCR product in each SNP site and enzyme are cut the product gel electrophorogram and are seen Fig. 1 and Fig. 2 respectively.

2.2Hardy-Weinberg balance goodness of fit assay

The check of the Hardy-Weinberg balance goodness of fit is carried out in 15 SNP sites respectively in asthma cases group and normal control group, the P value shows that all greater than 0.05 these 15 SNP sites all are in the genetic equilibrium state in case group and control group, tool colony representativeness.

2.3 genotype and the gene frequency distribution situation of each SNP site between asthma cases group and normal control group

15 genotype and the gene frequency distribution situations of SNP site between asthma cases group and normal control group see Table 5.In these 15 sites, IL13 A2044G, ADRB2R16G and FcER1C-109T three sites genotype and the gene frequency distributional difference between two groups has statistical significance.Case group A2044G site GG genotype and G gene frequency, R16G site AA genotype and A gene frequency, C-109T site TT genotype and T gene frequency all are higher than control group.Genotype and the gene frequency distributional difference not statistically significant of all the other 12 sites between two groups.

2.4 the high-order interactive analysis in ten SNP sites

Adopting the MDR method (is IL13C-1112T, C1923T, A2044G to 10 SNP sites that are able to 5 asthma susceptibility genes of multiple in＞10 samples, IL4C-590T, IL4RA I75V, Q551R, ADRB2 R16G, Q27E, FcER1 C-109T, E237G) carry out the high-order interactive analysis, analytical results sees Table 6.The nine site models that FcER1 E237G, FcER1 C-109T, ADRB2 R16G, IL4RAQ551R, IL4RA I75V, IL4 C-590T, IL13 A2044G, IL13 C1923T and IL13C-1112T form have best cross validation consistence.The asthma of this model detects index: accuracy 92.97%, sensitivity 97.92%, specific degree 88.02%, OR (95%CI)=345.3478 (117.0514,1018.9122), χ ²Value=286.3984, P value＜0.0001." the If-Then Rules " of this model is as follows (because length is long, only to take passages wherein few part; Class=1 represents the patient, and Class=0 represents non-patient):

IF?FcER1_E237G＝AA?AND?FcER1_C-109T＝CT?AND?ADRB2_R16G＝GGAND?IL4RA_Q551R＝GG?AND?IL4RA_I75V＝AA?AND?IL4_C-590T＝TT?ANDIL13_A2044G＝GG?AND?IL13_C1923T＝CT?AND?IL13_C-1112T＝CC?THENCLASSIFY?AS?1.

IF?FcER1_E237G＝AA?AND?FcER1_C-109T＝CT?AND?ADRB2_R16G＝AG?ANDIL4RA_Q551R＝AA?AND?IL4RA_I75V＝GG?AND?IL4_C-590T＝TT?ANDIL13_A2044G＝AG

AND?IL13_C1923T＝CT?AND?IL13_C-1112T＝CT?THEN?CLASSIFY?AS?0.

IF?FcER1_E237G＝AA?AND?FcER1_C-109T＝TT?AND?ADRB2_R16G＝AAAND

IL4RA_Q551R＝AG?AND?IL4RA_I75V＝AA?AND?IL4_C-590T＝TT?ANDIL13_A2044G＝GG?AND?IL13_C1923T＝TT?AND?IL13_C-1112T＝CC?THENCLASSIFY?AS?1.

IF?FcER1_E237G＝AA?AND?FcER1_C-109T＝CT?AND?ADRB2_R16G＝AGAND?IL4RA_Q551R＝AA?AND?IL4RA_I75V＝AG?AND?IL4_C-590T＝TT?ANDIL13_A2044G＝AG?AND?IL13_C1923T＝CC?AND?IL13_C-1112T＝CC?THENCLASSIFY?AS?0.

IF?FcER1_E237G＝AG?AND?FcER1_C-109T＝CC?AND?ADRB2_R16G＝AGAND?IL4RA_Q551R＝AG?AND?IL4RA_I75V＝AG?AND?IL4_C-590T＝TT?ANDIL13_A2044G＝AA?AND?IL13_C1923T＝CT?AND?IL13_C-1112T＝CT?THENCLASSIFY?AS?1.

IF?FcER1_E237G＝AG?AND?FcER1_C-109T＝CT?AND?ADRB2_R16G＝AGAND?IL4RA_Q551R＝AA?AND?IL4RA_I75V＝GG?AND?IL4_C-590T＝CT?ANDIL13_A2044G＝AG?AND?IL13_C1923T＝CT?AND?IL13_C-1112T＝CC?THENCLASSIFY?AS?1.

2.5SNP site synergy analysis between any two

Above-mentioned nine site models are carried out the synergy analysis between the site in twos.The joint study in ADRB2 R16G and FcER1 C-109T two sites sees Table 7, is divided into different genotype combinations by whether carrying R16G AA homozygote or C-109T TT homozygote.Carrying AA+TT combination person more only carries single AA (being the non-TT of AA+) or TT (being non-AA+TT) person and suffers from the dangerous of asthma and significantly raise.

The joint study in IL13 A2044G and ADRB2 R16G two sites sees Table 8, is divided into different genotype combinations by whether carrying A2044G GG homozygote or R16G AA homozygote.Carry the danger rising that non-AA of GG+AA, GG+ or the non-AA person of the more non-GG+ of non-GG+AA person suffer from asthma, and GG+AA rising degree is higher than non-AA of GG+ or non-GG+AA.

The joint study of ADRB2 R16G and FcER1 E237G sees Table 9, is divided into different genotype combinations by whether carrying R16G AA homozygote or E237G GG homozygote.Carry non-AA+GG person and compare, suffer from the dangerous no difference of science of statistics of asthma, raise but carry the danger that AA+GG or the non-GG person of the more non-AA+ of the non-GG person of AA+ suffer from asthma, and AA+GG rising degree is higher than the non-GG of AA+ with the non-GG person of non-AA+.

The synergy analysis [routine number (%)] of table 7ADRB2 R16G and FcER1C-109T

	The example number	??AA+TT	The non-TT of AA+	Non-AA+TT	The non-TT of non-AA+
						The case group	??192	??64(33.3)	??22(11.5)	??46(24.0)	??60(31.1)
Control group	??192	??14(7.3)	??32(16.7)	??64(33.3)	??82(42.7)
						??χ 2Value			??23.984	??30.432	??32.421
The P value			??0.001	??0.000	??0.000
						??OR??(95％CI)			??6.65??(3.01～14.70)	??6.36??(3.19～12.70)	??6.25??(3.21～12.18)

Annotate: the AA+TT group is compared for three groups with the non-TT of AA+, non-AA+TT, the non-TT of non-AA+ respectively

The synergy analysis [routine number (%)] of table 8IL13 A2044G and ADRB2 R16G

	The example number	??GG+AA	The non-AA of GG+	Non-GG+AA	The non-AA of non-GG+
						The case group	??192	??38(19.8)	??52(27.1)	??48(25.0)	??54(28.1)
Control group	??192	??13(6.8)	??41(21.4)	??33(17.2)	??105(54.7)
						??χ 2Value		??25.790	??11.603	??14.052
The P value		??0.000	??0.001	??0.000
						??OR??(95％CI)		??5.68??(2.79～11.56)	??2.47??(1.46～4.17)	??2.83??(1.63～4.91)

Annotate: non-AA of GG+AA, GG+ and non-GG+AA are compared with the non-AA group of non-GG+ respectively for three groups

The synergy analysis [routine number (%)] of table 9ADRB2 R16G and FcER1 E237G

	The example number	??AA+GG	The non-GG of AA+	Non-AA+GG	The non-GG of non-AA+
						The case group	??192	??6(3.1)	??80(41.7)	??12(6.3)	??94(49.0)
Control group	??192	??1(0.5)	??45(23.4)	??7(3.6)	??139(72.4)
						??χ 2Value		??4.040	??18.226	??3.752
The P value		??0.044	??0.000	??0.053
						??OR??(95％CI)		??8.87??(1.05～74.89)	??2.63??(1.68～4.12)	??2.54??(0.96～6.68)

Annotate: non-GG of AA+GG, AA+ and non-AA+GG are compared with the non-GG group of non-AA+ respectively for three groups

3 discuss

Asthma is a kind of multiplefactor, disease of multifactorial inheritance of complexity.Several genes may be distinguished or the control and the expression of asthma features relevant phenotype jointly, synthetic and the secretion etc. as serum total Ig E level, serum antigen specificity Ig E level, airway hyperreactivity, cytokine, the last or common pathway of its regulation and control then is the air flue chronic inflammatory diseases.Seek out these gene pairss understanding pathogenesis of asthma mechanism and have important scientific meaning of being mutually related from the susceptible physique that the genetics angle detects asthma.

Ten years in the past, the asthma gene studies has obtained remarkable progress.At the beginning of 2006, processes such as Ober are browsed lot of documents, reach a conclusion, think and in 6 or more independent sample, be able to 25 genes of multiple and asthma or allergic disease phenotypic correlation, be real asthma or allergic disease tumor susceptibility gene, wherein 10 genes obtain repetition in＞10 samples, and 15 genes are able to repetition in 6～10 samples.

Owing to there is racial diversify, for understanding the hereditary basis of China Han childhood asthma morbidity, seek out the gene locus that is mutually related, tentatively set up asthma gene test model, above-mentioned 5 SNP sites that are able to 10 SNP sites of 5 asthma susceptibility genes of multiple and are able to 3 asthma susceptibility genes of multiple in＞10 samples in 6～10 samples have been chosen in this experiment, have carried out single site, high-order interaction and synergy analysis in twos.

One, single site is analyzed

This experiment is to comprising each 192 example of allergic asthma infant and Health College Students, and the crowd of Han nationality of totally 384 examples has carried out case control study.Why selecting Health College Students and do not select healthy children in contrast, is not have the medical history of being correlated with at least for sufficiently long medical diagnosis on disease such as asthma except the time being arranged, illustrating before growing up.

Above-mentioned 15 candidate SNP locus are carried out the single-point analysis, find that IL13 A2044G, ADRB2R16G and FcER1 C-109T three sites genotype and the gene frequency distributional difference between case group and control group have statistical significance; Case group A2044G site GG genotype and G gene frequency, R16G site AA genotype and A gene frequency, C-109T site TT genotype and T gene frequency all are higher than control group, this shows that IL13 A2044G, ADRB2 R16G are all relevant with the generation of Han nationality children asthma with FcER1 C-109T three sites, is the main effect site; A2044G GG homozygote, R16G AA homozygote and C-109T TT homozygote are the asthma susceptibility gene type.

Documents and materials show that IL-13 A2044G is obviously relevant with the Total IgE in Serum rising, and the latter is a big high risk factor of asthma progress; This polymorphism reduces the avidity of IL13 and receptors bind, causes local I L13 concentration to raise, and strengthens with signal transduction after the receptors bind, causes serious bronchial asthma; A2044G SNP also reduces relevant with the severity and the pulmonary function of atopy, exercise induced airway hyperreactivity.ADRB2 R16G can increase airway reactivity, and influences the curative effect of bronchodilator by remarkable downward modulation acceptor quantity.It is relevant that FcER1-109T allelotrope and the MS4A2 gene promoter activity of coding FcER1 β chain increase, and C-109T SNP increases mastocyte MS4A2 genetic expression, causes air flue proinflammatory medium to discharge and increases; C-109T TT homozygote genotype raises relevant with allergic asthma patient total plasma IgE level.

This result of study also shows, in 15 candidate SNP locus, except that above-mentioned three main effect sites, genotype and the gene frequency distributional difference not statistically significant of all the other 12 sites between case group and control group, the no obvious dependency of these polymorphisms and Han nationality children asthma morbidity is described, is non-main effect site.This conforms to some similar research conclusion of Chinese population, but it is not consistent with some external bibliographical informations, its reason may be the genetic background difference between the different ethnic populations, also may how much relevant with this experiment sample amount, intend strengthening sample content further research is done in above-mentioned SNP site.

Two, high-order interactive analysis

The multiplefactor method of descent of propositions such as calendar year 2001 Ritchie is a main flow statistical method of studying the interrelated relation in a plurality of SNP site at present.Adopt the MDR method that the high-order interactive analysis is carried out in a plurality of SNP site and can obtain comparatively ideal asthma susceptibility gene detection model.Because this experiment sample amount is limited, the SNP site sum that participates in statistical study can not be too much, otherwise influence the usefulness that MDR analyzes.

This experiment is carrying out finding when single-point is analyzed that these three genes of IL13, ADRB2 and FcER1 take place relevant with Han nationality children asthma to the SNP site, the three is in＞10 samples and is able to the multiple asthma susceptibility gene, therefore adopting the MDR method to carry out the interactive analysis of multidigit point high-order when tentatively setting up asthma gene test model, 10 candidate SNP locus that only are chosen at＞are able in 10 samples 5 asthma susceptibility genes of multiple are studied.

The result shows, the nine site models that the two site models that ADRB2 R16G and IL13 A2044G form and FcER1E237G, FcER1 C-109T, ADRB2 R16G, IL4RA Q551R, IL4RA I75V, IL4 C-590T, IL13 A2044G, IL13 C1923T and IL13 C-1112T form all have good cross validation consistence (the former is 9/10, and the latter is 10/10).The asthma of two site models detects index: accuracy 64.06%, sensitivity 73.96%, specific degree 54.17%, OR (95%CI)=3.3564 (2.1844,5.1571), χ ²Value=31.6133, P value＜0.0001.Nine site models are: accuracy 92.97%, sensitivity 97.92%, specific degree 88.02%, OR (95%CI)=345.3478 (117.0514,1018.9122), χ ²Value=286.3984, P value＜0.0001.During the best model interpretation, in the model of all grades, should select the model of cross validation consistence maximum.Every asthma of above-mentioned nine site models detects index and all is better than two site models, therefore selects this nine sites model as the optimum detection model, and it has comprised aforesaid three main effect sites and other six non-main effect sites.What this model " If-Then Rules " showed is the asthma susceptibility classification (susceptible type, non-susceptible type) of all genotype combinations of nine SNP sites formation.

The MDR method is fit to the interactive analysis of a plurality of gene locuss is carried out in case control study, has been successfully applied to the research of multiple diseases such as asthma at present.Korea S scholar report, the two site models that KDR V297I and TNF G-308A form are 64.1% to the accuracy that detects national children and suffer from allergic disease, the cross validation consistence is 10/10.Studies show that of relevant Eotaxin gene and asthma, ((the five site model gook groups' of Eot2+304C＞A, Eot3+716A＞G and Eot3+1579G＞A) form asthma accuracy in detection is 64.3% for Eot2+1272A＞G and Eot3+77C＞T) and three non-main effect sites by two main effect sites, the cross validation consistence is 10/10, P value＜0.001.The Leung TF in Hong Kong analyzes by MDR, reaches a conclusion: there are significant interactions in IL13 R130Q, ADRB2 R16G and STAT6 C1570T three sites, determine the change of Chinese asthmatic children FVC jointly.Up to now, still do not have the relevant report of Han nationality children asthma detection model, this research belongs to first, has novelty.

Three, synergy is analyzed in twos

The OR value in IL13 A2044G, ADRB2 R16G and three main effect sites of FcER1 C-109T is respectively 2.255,2.575 and 1.961, and the nine site model OR values of being made up of these three main effect sites and other six non-main effect sites are up to 345.3478.The risk level that nine site models are suffered from asthma is significantly higher than single SNP site, has synergy mutually between the prompting different loci.

This experiment is further carried out the synergy analysis between the site in twos to nine site models, finds to carry ADRB2 R16G AA and FcER1 C-109T TT combination person and more only carries single R16GAA or C-109T TT person and suffer from the dangerous of asthma and significantly raise; Carrying IL13 A2044G GG and ADRB2 R16G AA combination person more only carries the danger that single A2044G GG or R16G AA person suffer from asthma and raises; Carrying ADRB2 R16G AA and FcER1 E237G GG combination person more only carries the danger that single R16G AA or E237G GG person suffer from asthma and raises, show ADRB2 R16G and FcER1C-109T, all there are synergy between any two in IL13 A2044G and ADRB2 R16G, ADRB2 R16G and FcER1 E237G.This has just explained why the dangerous of asthma suffered from the more single SNP of nine site models site and has significantly raise, and the OR value of also having explained nine site models why is apparently higher than aforesaid two site models.

At present, synergistic research report is much between relevant two SNP sites.For example, Llanes E discovers that single SNP site IL4RA I50V or Q551R are all irrelevant with the asthma phenotype, but The combined is then closely related with the asthma phenotype, shows to have synergy between these two non-main effect sites.One piece of treatise that this seminar delivered in 2008 has been reported the synergy that IL13 A2044G and ADRB2 R16G polymorphism take place childhood asthma.Hong Kong scholar studies show that IL13 R130Q, ADRB2 R16G and STAT6 C1570T three interaction, determines the change of Chinese asthmatic children FVC jointly.Up to now, do not see documents and materials report ADRB2 R16G and FcER1 C-109T as yet, ADRB2 R16G and FcER1 E237G collaborative research between any two.

In sum, in 15 candidate SNP locus, IL13 A2044G, ADRB2 R16G are relevant with the morbidity of Han nationality children asthma with FcER1 C-109T, are the main effect site, all the other 12 sites and the no obvious dependency of Han nationality children asthma morbidity are non-main effect site; Between the main effect site, there is synergy in twos between main effect site and the non-main effect site; The nine site models of being made up of three main effect sites and six non-main effect sites have good detection usefulness to Han nationality children asthma, become the optimum detection model that this research is established.

Han children asthma susceptibility gene detection model be based upon domestic still belonging to the first time, have novelty; Compare with external similar research, have kind of a group specificity.Yet this is the preliminary foundation of detection model, and it also need finally to determine constantly perfect.Ritchie points out, when case, each 200 example of contrast number, detects two site interactions, and the usefulness of MDR reaches more than 80%.Even under the situation of wrong branch of the genotype 5% and 5% missing data, this result is still true to most models.But, this sample content equally, when carrying out 10 site interactions, the usefulness of MDR drops to about 60%.Therefore, the perfect work of primarily carrying out of model is exactly further enlarged sample content.In addition, the asthma susceptibility gene that Ober etc. determine has 25 more than, and this experiment is only studied 8 genes wherein, for obtaining detection model more accurately, needs to increase the gene locus number of intending research.The expansion of experiment sample amount and the increase of waiting to study the gene locus number cause the requirement of experimental technique is improved.The PCR-RFLP technology that this experiment is adopted is classical methods of genotyping, and cheap, but its consumed time and manpower are more, are only applicable to the small sample quantity research.Follow-up study need improve experimental technique, adopts real-time fluorescence quantitative PCR technology or high-throughput SNP genotyping technique to carry out the large sample quantity research.In addition, in these years, statistical method constantly develops, and should pay close attention to, and in time receives, and strives for obtaining on existing method basis better detection model.

At present, still be in the experimental exploring stage, after obtaining comparatively perfect model, also need the clinical experiment checking through above-mentioned various improvement about the foundation of asthma susceptibility gene detection model.What MDR created is to distinguish the individual ideal identification and classification model of high-risk, low danger.The final establishment of detection model will help to differentiate the asthma high risk child, intervene early, prevent trouble before it happens, and effective reduction asthma morbidity is had positive effect.In addition, following gene-interactive result of study of gene statistics, also can carry out the biology interaction research on the corresponding function level, this perhaps will open another pathogenetic gate of fan understanding asthma.

In the genome times afterwards comprehensively, the major objective of childhood asthma gene studies is to understand the function of each genes involved, comprising complicated interaction between gene-gene, the gene-environment.Though also can't accomplish all genes of full confirmation-gene interaction at present, can be inquired into some important relatively in this multigenic disease interactions at least, this will help from now on asthma more fully to be familiar with.

4 conclusions

Han children asthma susceptibility gene detection model is tentatively set up, form by FcER1 E237G, FcER1C-109T, ADRB2 R16G, IL4RA Q551R, IL4RA I75V, IL4C-590T, IL13 A2044G, IL13 C1923T and nine SNP sites of IL13 C-1112T, have good asthma and detect usefulness, but this model still need be further perfect.

Embodiment 2

The clinical application of Han children asthma susceptibility gene detection model

The youngster of hospital of Xinhua internal medicine will be from each 192 example of the Hans group's case-control as research object, 15 single nucleotide polymorphism (single nucleotide polymorphisms with regard to 8 genes, SNPs) gene type is carried out in the site, adopt main flow statistical method---multiplefactor method of descent (the multifactor dimensionality reduction of the interrelated relation in research a plurality of SNP site, MDR) carry out statistical study, tentatively set up E237G by FcER1, FcER1 C-109T, ADRB2R16G, IL4RA Q551R, IL4RA I75V, IL4C-590T, IL13 A2044G, the Han children asthma susceptibility gene detection model that IL13 C1923T and IL13 C-1112T form in nine SNP sites, it has good asthma and detects usefulness (OR=345.3478, P value＜0.0001, accuracy 92.97%, sensitivity 97.92%, specific degree 88.02%).Han children asthma susceptibility gene detection model be based upon domestic still belonging to the first time, have novelty; Compare with external similar research, have kind of a group specificity.What MDR created is to distinguish the individual ideal identification and classification model of high-risk, low danger, the asthma susceptibility classification (susceptible type, non-susceptible type) of all genotype combinations that nine SNP sites of this models show form.The establishment of detection model will help to differentiate the asthma high risk child, intervene early, prevent trouble before it happens, and effective reduction asthma morbidity is had positive effect.Now that the clinical application brief introduction of Han children asthma susceptibility gene detection model is as follows:

1, this model is applied for a patent, and it is developed to Han nationality children asthma susceptibility gene detection kit.This test kit is made up of nine capsules, and gene type is carried out in a SNP site in the corresponding nine site models of every capsule difference.The related reagent of dress DNA extracting, pcr amplification, digestion with restriction enzyme and agarose gel electrophoresis four steps in every capsule.

2, the applicable object of this detection kit is the potential asthma infant that is dispersed in community, and promptly those have individual or family's allergies, but does not still have asthma person, and these potential infants just may be sent out under certain condition and be asthma.The external notion that proposes the asthma tertiary prevention, wherein primary prevention is exactly just to take actively promising measure when disease does not take place as yet, as the living environment that changes the high risk population, food habits etc., prevents the generation of asthma.We can adopt Han nationality children asthma susceptibility gene detection kit that those potential infants are carried out early screening, determine the asthma high risk child, it is taked to prevent trouble before it happens such as avoiding measures such as anaphylactogen targetedly, effectively reduce the asthma morbidity, save ample resources.

3, detect step: (1) gathers examinee's oral cavity buccal mucosa swab; (2) extract the buccal swab genomic dna; (3) DNA fragment specific is carried out pcr amplification; (4) adopt agarose gel electrophoresis method for detecting to detect the PCR product; (5) the PCR product is carried out the digestion with restriction enzyme reaction; (6) adopt agarose gel electrophoresis method for detecting to detect the endonuclease reaction product, determine genotype; (7) methods of genotyping in each SNP site is same as above, repeats nine times, obtains the genotype in nine SNP sites; (8) according to the genotype result in nine sites, differentiate the classification of asthma susceptibility, determine that promptly this examinee is the high-risk or low danger children of asthma; (9) asthma high risk child and family thereof are carried out asthma education and management.

The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the inventive method; can also make some improvement and replenish, these improvement and replenish and also should be considered as protection scope of the present invention.

SEQUENCE?LISTING

＜110〉Xinhua Hospital Attached to Medical School, Shanghai Jiaotong Univ.

 

＜120〉Han children asthma susceptibility gene detection model and test kit thereof

 

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<210>20

<211>27

<212>DNA

＜213〉artificial sequence

 

<400>20

gttcctgctt?attcattaca?gatcgta????????????????????????????????????27

 

<210>21

<211>43

<212>DNA

＜213〉artificial sequence

 

<400>21

gcggtcccaa?aagggtcagt?ggaatccagc?atgccttgtg?agg??????????????????43

<210>22

<211>44

<212>DNA

＜213〉artificial sequence

 

<400>22

gcggtcccaa?aagggtcagt?gtcgcctttt?cctgctcttc?ccgc?????????????????44

 

<210>23

<211>42

<212>DNA

＜213〉artificial sequence

 

<400>23

gcggtcccaa?aagggtcagt?gctttcgaag?tttcagttga?gc???????????????????42

 

<210>24

<211>41

<212>DNA

＜213〉artificial sequence

 

<400>24

gcggtcccaa?aagggtcagt?tcatgtgctg?acctctttgt?c????????????????????41

 

<210>25

<211>42

<212>DNA

＜213〉artificial sequence

 

<400>25

gcggtcccaa?aagggtcagt?ctctctgagc?caaccactgt?gc???????????????????42

 

<210>26

<211>40

<212>DNA

＜213〉artificial sequence

 

<400>26

gcggtcccaa?aagggtcagt?ccaccgcatg?taccagctcc??????????????????????40

 

<210>27

<211>20

<212>DNA

＜213〉artificial sequence

 

<400>27

aagcttcgag?tgtggacaga????????????????????????????????????????????20

 

<210>28

<211>20

<212>DNA

＜213〉artificial sequence

 

<400>28

agtgttccta?gtgccactgg????????????????????????????????????????????20

 

<210>29

<211>43

<212>DNA

＜213〉artificial sequence

 

<400>29

gcggtcccaa?aagggtcagt?ggaatccagc?atgccttgtg?agg??????????????????43

 

<210>30

<211>44

<212>DNA

＜213〉artificial sequence

 

<400>30

gcggtcccaa?aagggtcagt?gtcgcctttt?cctgctcttc?ccgc?????????????????44

 

<210>31

<211>20

<212>DNA

＜213〉artificial sequence

 

<400>31

gtcctcacat?ccgtgatcgg????????????????????????????????????????????20

 

<210>32

<211>20

<212>DNA

＜213〉artificial sequence

 

<400>32

cttgaaggag?cccttccaca????????????????????????????????????????????20

 

<210>33

<211>22

<212>DNA

＜213〉artificial sequence

 

<400>33

ggaagctgga?agagtctgat?gc?????????????????????????????????????????22

 

<210>34

<211>22

<212>DNA

＜213〉artificial sequence

 

<400>34

cactgaccac?gtcatccatg?ag?????????????????????????????????????????22

 

<210>35

<211>20

<212>DNA

＜213〉artificial sequence

 

<400>35

gtgtcccaga?gagctgggtc????????????????????????????????????????????20

 

<210>36

<211>20

<212>DNA

＜213〉artificial sequence

<400>36

gaaggccttg?taaccagcct????????????????????????????????????????????20

 

<210>37

<211>42

<212>DNA

＜213〉artificial sequence

 

<400>37

gcggtcccaa?aagggtcagt?ctctctgagc?caaccactgt?gc???????????????????42

 

<210>38

<211>40

<212>DNA

＜213〉artificial sequence

 

<400>38

gcggtcccaa?aagggtcagt?ccaccgcatg?taccagctcc??????????????????????40

 

<210>39

<211>20

<212>DNA

＜213〉artificial sequence

 

<400>39

accattggtg?cttggtcaaa????????????????????????????????????????????20

 

<210>40

<211>20

<212>DNA

＜213〉artificial sequence

 

<400>40

tggtggtcaa?gaccaggact????????????????????????????????????????????20

 

<210>41

<211>20

<212>DNA

＜213〉artificial sequence

 

<400>41

aatttccgga?ggctatttca????????????????????????????????????????????20

 

<210>42

<211>20

<212>DNA

＜213〉artificial sequence

 

<400>42

gtacctgtgg?gagaggctgt????????????????????????????????????????????20

Claims (4)

1. Han children asthma susceptibility gene detection model, it is characterized in that described detection model is made up of FcER1 E237G, FcER1 C-109T, ADRB2 R16G, IL4RA Q551R, IL4RA I75V, IL4 C-590T, IL13 A2044G, IL13 C1923T and nine SNP sites of IL13 C-1112T.

2. Han nationality children asthma susceptibility gene detection kit, it is characterized in that, described test kit is made up of nine little test kits, described nine little test kits are respectively: detect the little test kit in SNP site of FcER1 E237G, FcER1C-109T, ADRB2 R16G, IL4RA Q551R, IL4RA I75V, IL4 C-590T, IL13 A2044G, IL13 C1923T and IL13 C-1112T, the related reagent of dress DNA extracting, pcr amplification, digestion with restriction enzyme and agarose gel electrophoresis step in every little test kit.

3. Han nationality children asthma susceptibility gene detection kit, it is characterized in that, described test kit is made up of two little test kits, described two little test kits are respectively: detect the little test kit in SNP site of ADRB2 R16G and FcER1C-109T, the related reagent of dress DNA extracting, pcr amplification, digestion with restriction enzyme and agarose gel electrophoresis step in every little test kit.

4. Han nationality children asthma susceptibility gene detection kit, it is characterized in that, described test kit is made up of two little test kits, described two little test kits are respectively: detect the little test kit in SNP site of ADRB2 R16G and FcER1E237G, the related reagent of dress DNA extracting, pcr amplification, digestion with restriction enzyme and agarose gel electrophoresis step in every little test kit.

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