CN100342032C - Schizophrenia susceptible gene detection method, susceptible gene and application - Google Patents
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Abstract
The invention provides a method for detecting a polymorphic site of a schizophrenia susceptible gene FZD3 gene, a method for detecting the schizophrenia susceptibility of a tester in vitro, a schizophrenia susceptible gene FZD3 gene and application. The method for detecting the schizophrenia susceptible gene provided by the invention is to detect the FZD3 gene sequence in vitro by a polymerase chain reaction direct sequencing method and/or a polymerase chain reaction-restriction fragment length polymorphism analysis method, wherein rs2241802 polymorphic site, rs2323019 polymorphic site and/or rs352203 polymorphic site. The invention meets the requirement of correctly identifying the susceptibility gene of schizophrenia in the field, and provides a new idea for the deep research and prevention, even diagnosis and treatment of schizophrenia.
Description
Technical field
The present invention relates to schizophrenia tumor susceptibility gene detection method, the method of vitro detection trier's schizophrenia susceptibility and schizophrenia tumor susceptibility gene and purposes, specifically, the present invention relates to the detection method of schizophrenia tumor susceptibility gene FZD3 gene polymorphism sites, the method for vitro detection trier's schizophrenia susceptibility and schizophrenia tumor susceptibility gene FZD3 gene and purposes.
Background technology
According to the end of the year 1999, the statistical information of China Ministry of Health shows: the life time limit index of adjusting by permanent disability, estimate all kinds of diseases shared ratio in China's disease burden on society, mental disease accounts for 1/5 of the total burden of disease, surpassed illness such as cardiovascular, respiratory system and malignant tumour, rank ranks first.The schizophrenia sickness rate is only second to dysthymia disorders, accounts for second of whole mental disease.Schizoid symptom comprises: disturbance in thinking, chimae and mood and action change etc.
Since oneth century, people have made various hypothesis and deduction to the observation of aspects such as physiology, biochemistry, image, pharmacological agent and the social family of mental disorder, environment to the pathogeny of mental disorder.Progress along with science and technology, people recognize that more and more genetic flaw is the major reason that many serious mental disorderes produce, the people is when running into psychology and social environment pressure, and those people that carry diseases predisposing gene more may suffer from the mental health illness than the people who does not carry diseases predisposing gene.Inherited genetic factors also obtains family, twins basically and entrusts one's child to the care of sb. the epidemiology survey result's of son support, the later stage eighties, and to the large-scale inquiry of mental disorder genetic epidemiology, the more abundant hereditary basis that shows mental disorder.Science and technology growing today how to mental disease, especially schizophrenia, how to detect tumor susceptibility gene and individual disease susceptibility,, become the severe problem that numerous scientific and technical personnel and health care personnel face to such an extent as to carry out further risk profile and diagnosis from hereditary angle.Although diseases predisposing gene research is both at home and abroad carried out for many years, do not make a breakthrough as yet, rarely have the Study on Value result.For the genetic predisposition of how identifying inheritance susceptible gene and qualification test person, this area lacks comprehensively always, system, effective recognition method, for the achievement in research of schizophrenia susceptibility still less.
Nearest genome scanning results suggest, 8p22-21 is one of schizoid susceptible zone.The FZD3 assignment of genes gene mapping is in 8p21, genbank registration number: GI:22047956, and total length 70187bp is the important acceptor in the signal transducting system.
Summary of the invention
At the problems referred to above, the invention provides a kind of method that detects the schizophrenia tumor susceptibility gene, thereby, satisfied the demand of this area for correct identification schizophrenia tumor susceptibility gene, be schizoid further investigation and control, even diagnoses and treatment provides new thinking.
The present invention provides a kind of method of vitro detection trier's schizophrenia susceptibility simultaneously.
The present invention also provides the test kit that detects the schizophrenia tumor susceptibility gene.
On the other hand, the present invention further provides a kind of schizophrenia tumor susceptibility gene.
The present invention further also provides the application of method in schizophrenia prevention, diagnosis and treatment of vitro detection schizophrenia tumor susceptibility gene.
The present invention also provides the application of method in the ill risk profile of schizophrenia, diagnosis and treatment of vitro detection trier's schizophrenia susceptibility.
The present invention also provides the application of the test kit that detects the schizophrenia susceptibility in schizophrenia prevention, diagnosis and treatment.
At last, the present invention also provides the application of schizophrenia tumor susceptibility gene of the present invention in schizophrenia prevention, diagnosis and treatment.
The present invention is by the statistical study of large sample, studied the rs2241802 of FZD3 gene order, rs2323019 and rs352203 pleomorphism site are at the gene frequency of schizophrenia core families, and, carry out transmission disequilibrium check (TDT) and haplotyping according to the transmission situation of parent gene type in ill children.Found that all distribution of the genotype of sample and gene frequency all meet the Hardy-Weinburg balance; After the rectification of Bonferroni method, the TDT of three pleomorphism sites analyzes still significant significance,statistical; It is too much that the analysis of single haplotype is also shown among the patient transmission of GAT haplotype, and difference has significance; Thereby with evidence the rs2241802 of FZD3 gene order, rs2323019 and/or rs352203 pleomorphism site influence schizoid susceptibility, wherein the rs2241802 pleomorphism site is G in the FZD3 gene order, and the rs2323019 pleomorphism site is that A and/or rs352203 pleomorphism site are that the trier of T is the high person of schizophrenia susceptibility.
Therefore, the invention provides a kind of method that detects the schizophrenia tumor susceptibility gene, this method comprises by polymerase chain reaction-direct sequencing and/or polymerase chain reaction-restriction fragment length polymorphism analytical procedure vitro detection FZD3 gene order, the rs2241802 pleomorphism site is wherein arranged, rs2323019 pleomorphism site and/or rs352203 pleomorphism site.
So-called " gene pleiomorphism " refers in the crowd, the difference that the nucleotide sequence of each genes of individuals exists.Those of ordinary skills are known, and pleomorphism site of the present invention is single nucleotide polymorphism (SNP) site, and promptly single Nucleotide changes in the genome sequence; The difference of nucleotide sequence can be embodied on the dna level or on the rna level, so, can be by detecting DNA, RNA detects polymorphism, preferred DNA, more preferably genomic dna.
It is known to those skilled in the art that the pleomorphism site that to use multiple technologies external detection FZD3 gene order on dna level.Can through with the dna sequence dna after radiolabeled sense-rna or dna probe and amplification hybridization, with the differential point polymorphism.Also can be based on the change of known nucleotide sequence, the PCR primer that synthesizes normal and polymorphism, in the substrate of polymerase chain reaction (PCR reaction), add fluorescently-labeled Nucleotide, according to having or not fluorescence to occur in the reaction product, determine in the used primer of amplification, to have or not base to change, thereby detect polymorphism.Can directly disclose crt gene and carry sequence difference between the polymorphism gene by dna direct order-checking.When being used in combination with PCR, the susceptibility of this method improves greatly.For example, the single-stranded template molecule with sequencing primer and double-stranded PCR product or the generation of asymmetric TRAP uses together.Also available ordinary method of the mensuration of the nucleotide sequence of various DNA and dna fragmentation such as dideoxy chain termination (people such as Sanger, PNAS, 1977,74:5463-5467).In addition, also available commercial sequencing kit of nucleotide sequencing or automatic sequencer etc.Conventional automatic sequencing method is come the definite kernel acid sequence with radio-labeling or fluorescent mark.
Because gene pleiomorphism; cause the restriction enzyme digestion sites change, disappear or produce new site; if with certain digestion with restriction enzyme genomic dna; then enzyme is cut the dna fragmentation of back generation and normal gene group different lengths; through detecting, just can detect the position and the size of these bands with suitable probe hybridization.The principle of polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) method is: when the experiment of design pcr amplification, primer is positioned at the both sides at gene pleiomorphism position, now goal gene is increased, make it be easy to detect, because polymorphism causes existing restriction endonuclease sites and changes, then can be earlier with corresponding digestion with restriction enzyme amplified production, carry out agarose gel electrophoresis again and observe, judge according to product clip size or quantity and normal control.The present invention preferably adopts polymerase chain reaction-direct sequencing, polymerase chain reaction-restriction fragment length polymorphism analytical procedure.More preferably polymerase chain reaction-restriction fragment length polymorphism analytical procedure.
In an embodiment of the invention, utilize polymerase chain reaction-restriction fragment length polymorphism analytical procedure to detect the method for schizophrenia tumor susceptibility gene, described polymorphism analyzing method comprises:
A: extract DNA, at rs2241802, design PCR primer carries out the PCR reaction near rs2323019 and/or the rs352203 pleomorphism site;
B:, utilize restriction enzyme to carry out enzyme and cut at above-mentioned pleomorphism site;
C: gel electrophoresis separates with identifying enzyme cuts the result;
Wherein, rs2241802 pleomorphism site, rs2323019 pleomorphism site and rs352203 pleomorphism site are schizophrenia susceptibility allelotrope.
The method of detection schizophrenia tumor susceptibility gene of the present invention, may further include after polymorphism analysis, utilize allelic transmission frequency of transmission disequilibrium check analysis FZD3 and haplotype to transmit frequency, having significant difference is the schizophrenia tumor susceptibility gene.As described in embodiment 1, the present invention has carried out the haplotype frequency analysis to 3 mononucleotide polymorphism sites in 246 familys (every example all contains father and mother parents and 1 the ill son/woman who has relationship by blood).The result draws, and after correcting through the Bonferroni method, the TDT of 3 pleomorphism sites analyzes still significance,statistical (Rs2241802:X
2=18.028, P=0.00002; Rs2323019:X
2=13.018, P=0.0003; Rs352203:X
2=20.260, P=0.000007).
The present invention provides a kind of method of vitro detection trier's schizophrenia genetic predisposition simultaneously, this method is for detecting trier FZD3 gene order rs2241802, rs2323019 and/or rs352203 pleomorphism site, wherein the rs2241802 pleomorphism site is that G and/or rs2323019 pleomorphism site are that A and/or rs352203 pleomorphism site are the trier of T, is the high person of schizophrenia susceptibility.
Here said " genetic predisposition " is meant the proneness (susceptibility) that is easy to suffer from certain (certain class) disease by the heredity decision, promptly passes by normal " quality " called of people (diathesis).The existence of inheritance susceptible gene is the basis of genetic predisposition.The people is when running into psychology and social environment pressure, and those people that carry the schizophrenia tumor susceptibility gene more may suffer from the schizophrenia illness than the people who does not carry tumor susceptibility gene.
The sample that contains FZD3 gene order to be measured can obtain Tathagata autoblood, urine, saliva, gastric juice, hair, the cell of examination of living tissue and necrotomy material from the cell from the trier.Preferably come autoblood.
Statistical study by large sample of the present invention, can use method of the present invention separately, promptly detect trier FZD3 nucleotide sequence variation and detect relevant schizophrenia genetic predisposition, simultaneously, it be known to those skilled in the art that schizoid generation, development are multifactor coefficient results, genetic predisposition also has the complicacy of himself, so the present invention also can unite use with other method, to reach the purpose that detects the schizophrenia susceptibility.
The method of vitro detection trier's schizophrenia genetic predisposition provided by the invention, detect FZD3 gene order rs2241802, the method of rs2323019 and rs352203 polymorphism can adopt the method for the pleomorphism site of above-mentioned detection gene order, direct sequencing for example, the restriction fragment length polymorphism analytical procedure.Preferred polymeric polymerase chain reaction-direct sequencing, polymerase chain reaction-restriction fragment length polymorphism analytical procedure, more preferably polymerase chain reaction-restriction fragment length polymorphism analytical procedure.
In an embodiment of the invention, utilize polymerase chain reaction-restriction fragment length polymorphism analytical procedure to detect trier's schizophrenia susceptibility, wherein said polymorphism analyzing method comprises:
A: extract trier DNA, at rs2241802, design PCR primer carries out the PCR reaction near rs2323019 and/or the rs352203 pleomorphism site;
B:, utilize restriction enzyme to carry out enzyme and cut at above-mentioned pleomorphism site;
C: gel electrophoresis separates with identifying enzyme cuts the result.
The present invention also provides a kind of test kit that is used to detect the schizophrenia susceptibility.One or more containers are housed in the described test kit, are equipped with in the container in order to detect FZD3 gene order rs2241802, one or more components of rs2323019 and/or rs352203 pleomorphism site.According to the difference of concrete detection method and detection pleomorphism site, test kit can contain different components.What provide simultaneously with it can be through medication management mechanism of government audit, relevant medicine or biological products manufacturing, the information using and sell.Preferably contain and utilize polymerase chain reaction-restriction fragment length polymorphism analytical procedure, detect FZD3 gene order rs2241802, the component of rs2323019 and/or rs352203 pleomorphism site:
1) amplification rs2241802, the primer of rs2323019 and/or rs352203 pleomorphism site;
2) pcr amplification enzyme, enzyme are cut the corresponding restriction enzyme of pleomorphism site, and corresponding damping fluid;
3)dNTP；
1) 4) described pleomorphism site restriction enzyme mapping.
Those of ordinary skills are known, the primer in above-mentioned amplification polymorphism site, can be according to known nucleotide sequence design, be generally 15-30 base, GC content is about 45%-50%, combine with template specificity under suitable temperature, it can utilize special computer programming, for example (OLIGO 4.06 primer analysis software).Shown in the embodiment of the invention 1, provide a kind of amplimer:
| Pleomorphism site | Primer sequence (5 ' → 3 ') |
| rs2241802 | CTATGAAATAGCGAGCAAATGACA(SEQ ID No 4) GGAAATCCAAACTGTTAGATCGTG(SEQ ID No 5) |
| rs2323019 | AGCCACTGCTCCCACCAAAG(SEQ ID No 6) CAAAAACCCAGGGATACCCAAAC(SEQ ID No 7) |
| rs352203 | ATGACTTCCTAGGGCCAAACCTC(SEQ ID No 8) GCAAAAACTAATGGCCAGCAATGT(SEQ ID No 9) |
Described Tag archaeal dna polymerase can be the Klenow fragment, the Tth archaeal dna polymerase, and VENT archaeal dna polymerase etc. can be used in the enzyme of pcr amplification.Enzyme is cut the corresponding restriction enzyme those of ordinary skills of pleomorphism site and also can be designed according to known technique, for example, according to being respectively restriction enzyme A luI, SspI, NlaIII described in the embodiment 1, after restriction enzyme was determined, inscribe collection of illustrative plates on the other side is also corresponding to be determined.
The present invention proposes a kind of schizophrenia tumor susceptibility gene simultaneously, and it is the FZD3 gene order, and rs2241802 pleomorphism site and/or rs2323019 pleomorphism site and/or rs352203 pleomorphism site are arranged.The polymorphism of FZD3 gene of the present invention can show dna level or rna level.Preferred DNA, more preferably genomic dna.
The present invention also provides the application of method in schizophrenia prevention, diagnosis and treatment of vitro detection schizophrenia susceptibility.
The present invention also provides the application of method in the ill risk profile of schizophrenia, diagnosis and treatment of vitro detection trier's schizophrenia tumor susceptibility gene.
The present invention also provides the application of the test kit that detects the schizophrenia susceptibility in schizophrenia prevention, diagnosis and treatment.
At last, the present invention also provides the application of schizophrenia tumor susceptibility gene of the present invention in schizophrenia prevention, diagnosis and treatment.
Description of drawings
Fig. 1 shows the gene synoptic diagram of FZD3 gene, and wherein black bar shaped frame is represented exon, and white bar shaped frame is represented intron, and the arrow indication is mononucleotide polymorphism site (SNP) position.
Fig. 2 shows the schema that utilizes polymerase chain reaction-restriction fragment length polymorphism analytical procedure to detect trier's schizophrenia susceptibility.
Fig. 3 shows the cleavage map of pleomorphism site rs2241802, and wherein, 1 is 100bp molecular weight standard (marker), and 2 is 307 homozygotes, and 3 is 124/183 homozygote, and 4 is 124/183/307 heterozygote.
Fig. 4 shows the cleavage map of pleomorphism site rs2323019, and wherein, 1 is 321 homozygotes, and 2 is 100bp molecular weight standard (marker), and 3 is 131/190/321 heterozygote, and 4 is 131/190 homozygote.
Fig. 5 shows the cleavage map of pleomorphism site rs352203, and wherein, 1 is 100bp molecular weight standard (marker), and 2 is 120/294/414 heterozygote, and 3 is 120/294 homozygote, and 4 is 414 homozygotes.
Embodiment
1. research object
The research object in this stage is the patient that diagnosis and treatment are carried out in Peking University's mental health institute outpatient service and inpatient department in calendar year 2001-2002 year, merge total schizophrenia core families 246 examples (every example all contains father and mother parents and 1 the ill son/woman who has relationship by blood) in back with the sample of previous stage, be Han nationality.All patients meet schizoid Case definition in the International Classification of Diseases handbook the 10th edition (ICD-10), and have accepted the clinical interview of structural formula.In the patient, the male sex is 138 examples (56%), women's 108 examples (44%), and the mean age is 29 years old.Average course of disease is 5 years.
All research objects have all been signed Informed Consent Form.This research obtains the approval of Department Of Medicine, Peking University Ethics Committee.
2. method
Detect all research object rs2241802 with polymerase chain reaction-restriction fragment length polymorphism (PCR-based RFLP) analytical procedure, rs2323019 and/or rs352203 pleomorphism site genotype.Flow process is referring to accompanying drawing 2.
2.1 the collection of blood preparation and processing
All patients have extracted peripheric venous blood 5-10ml, place anticoagulant tube, preserve in 4 ℃ of refrigerators, extract genomic dna in 1 week.
2.2 the extraction of genomic dna and evaluation
2.2.1 the extraction of genomic dna
In the extraction of genomic dna previous stage, need 5ml blood, it is frozen to fail to stay part blood.In order to reduce the blood consumption, the extraction in this stage is finished with genome DNA extraction purification kit (Shanghai China Shun biotechnology company limited, poba gene group DNA extracting and purifying test kit).Method is as follows: add the fresh whole blood that 1ml contains antithrombotics in the 5ml centrifuge tube, add 1*BP (erythrocyte cracked liquid) liquid of 3ml precooling again, put upside down centrifuge tube back and forth with thorough mixing.Behind the ice bath 10 minutes, centrifugal 2 minutes of 4500g with the thorough sucking-off of supernatant liquid, adds 1*BP (erythrocyte cracked liquid) liquid of 1ml precooling in centrifuge tube.Thoroughly behind the mixing, centrifugal 2 minutes of 4500g is with the thorough sucking-off of supernatant liquid.Add 200 μ l DT (suspension) liquid in precipitation, thoroughly vibration suspends.Add 400 μ l DL (lysate) liquid and 25 μ l Proteinase Ks, the mixing that vibrates is rapidly put 65 ℃ of temperature and was bathed 15-30 minute, during put upside down centrifuge tube back and forth repeatedly.Add 400 μ l Virahols, after acutely putting upside down centrifuge tube and making the solution mixing, pipette 600 μ l to adsorption column, centrifugal 30 seconds, discard the liquid in the collection tube, adsorption column is put into same collection tube, all move in the adsorption column centrifugal 30 seconds with remaining.Discard the liquid in the collection tube, adsorption column is put into same collection tube.Add 500 μ l W1 (washings) liquid, leave standstill 1 minute after, centrifugal 30 seconds.Adsorption column is moved in another clean collection tube, add 500 μ l W1 liquid, centrifugal 15 seconds.Discard the liquid in the collection tube, again adsorption column is put into same collection tube, centrifugal 1 minute.Adsorption column is moved in the clean 1.5ml centrifuge tube, and central authorities add 100 μ l T1 (elutriant) liquid at adsorption film, 65 ℃ leave standstill 5 minutes after, centrifugal 1 minute.Add 60 μ l T1 liquid, centrifugal 1 minute.1.5ml centrifuge tube (DNA) is put in-20 ℃ of preservations.
2.3 the segmental amplification of purpose
Polymerase chain reaction 2.3.1 (PCR)
The pcr amplification reaction system of 25-μ l is as follows: 10mM Tris-HCl (pH 8.3), 50mM KCl, 1.5mM magnesium chloride, 200 μ M dNTP, 0.4 μ M primer, 1.0U Taq archaeal dna polymerase, 30-50ng genomic dna.The pcr amplification reaction condition is: 94 ℃ of sex change 5 minutes, and 94 ℃ of sex change 30 seconds, 57 ℃-62 ℃ annealing 30 seconds, 72 ℃ were extended 40 seconds, and 32 circulations were extended 7 minutes after last 72 ℃.
2.3.2 primer
By the retrieval of information biology, chosen 3 mononucleotide polymorphism site: rs2241802 on the FZD3 gene respectively, rs2323019 and/or rs352203, physical resource sees Table 1.
The sequence and the relevant information of 3 SNPs primers of table 1
| SNP | Primer sequence (5 ' → 3 ') | Product (bp) | Annealing temperature (℃) | Restriction endonuclease | Allelotrope (bp) | |
| rs2241802 | CTATGAAATAGCGAGCAAATGACA (SEQ ID No 4) GGAAATCCAAACTGTTAGATCGTG (SEQ ID No 5) | 307 | AluI | A 124/183 | G 307 | |
| rs2323019 | AGCCACTGCTCCCACCAAAG (SEQ ID No 6) CAAAAACCCAGGGATACCCAAAC (SEQ ID No 7) | 321 | SspI | A 131/190 | G 321 | |
| rs352203 | ATGACTTCCTAGGGCCAAACCTC (SEQ ID No 8) GCAAAAACTAATGGCCAGCAATGT (SEQ ID No 9) | 414 | NlaIII | C 120/294 | T 414 | |
2.3.3 the complete sequence of PCR product
rs2241802：(SEQ ID No 1)
rs2323019：(SEQ ID No 2)
rs352203：(SEQ ID No 3)
2.4 the position of pleomorphism site on gene
Particular location is seen Fig. 1.
2.5 restriction fragment length polymorphism analysis
2.5.1 digestion with restriction enzyme reaction
Get 15 μ l PCR products and place 5 μ l restriction endonucleases and enzyme cutting buffering liquid system, in 37 ℃ of incubator reaction overnight.
2.5.2 agarose gel electrophoresis separates, identifies
Get 6-8 μ l enzyme and cut product, separate the purpose fragment, after gel becomes phase system scanning, read genotype with 3% agarose gel electrophoresis.
Conclusion:
As shown in Figure 3, be 3% agarose gel electrophoresis after, the cleavage map of pleomorphism site rs2241802, wherein, 1 is 100bp molecular weight standard (marker), 2 is 307 homozygotes, 3 is 124/183 homozygote, 4 is 124/183/307 heterozygote.
As shown in Figure 4, be 3% agarose gel electrophoresis after, the cleavage map of pleomorphism site rs2323019, wherein, 1 is 321 homozygotes, 2 is 100bp molecular weight standard (marker), 3 is 131/190/321 heterozygote, 4 is 131/190 homozygote.
As shown in Figure 5, be 3% agarose gel electrophoresis after, the cleavage map of pleomorphism site rs352203, wherein, 1 is 100bp molecular weight standard (marker), 2 is 120/294/414 heterozygote, 3 is 120/294 homozygote, 4 is 414 homozygotes.
2.6 statistical analysis
The genetic statistics mathematical analysis
(the transmission disequilibrium test TDT) analyzes the relation of allelotrope and disease in all schizophrenia core families, and analytic process is studied identical with the fs with transmission disequilibrium check.In association study, because haplotype is more accurate than one SNP site, and have higher statistics effectiveness, so the contriver has carried out the analysis of haplotype frequency with 3 mononucleotide polymorphism sites based on linkage disequilibrium.The haplotype frequency adopts TRANSMIT software (2.5.2) to analyze.After statistical study repeatedly, correct with the Bonferroni method.
The result:
1. three SNPs genotype distribute and gene frequency
Genotype distributes and gene frequency sees Table 2.
The genotype of 3 pleomorphism sites of table 2 FZD3 gene distributes and gene frequency
| Genotype distribution (%) | Gene frequency (%) | ||||
| Rs2241802 patient father and mother | GG 118 196 | GA 116 246 | AA 12 50 | G 352(0.72) 638(0.65) | A 140(0.28) 346(0.35) |
| Rs2323019 patient father and mother | GG 57 144 | GA 115 224 | AA 74 124 | G 229(0.47) 512(0.52) | A 263(0.53) 472(0.48) |
| Rs352203 patient father and mother | TT 110 181 | TC 113 235 | CC 23 76 | T 333(0.68) 597(0.61) | C 159(0.32) 387(0.39) |
2. the TDT of three SNPs check
tAccording to the transmission situation of parent gene type in ill children, in 246 familys, carry out transmission disequilibrium check (TDT).TDT analysis in table 3.As shown in table 3, after the rectification of Bonferroni method, the TDT of three pleomorphism sites analyzes still significance,statistical (Rs2241802:X
2=18.028, P=0.00002; Rs2323019:X
2=13.018, P=0.0003; Rs352203:X
2=20.260, P=0.000007).
The allelic transmission disequilibrium check of table 3 FZD3 gene
| SNPs | Allelotrope | Transmit | Do not transmit | X 2 | The P value |
| Rs2241802 | G A | 158 91 | 91 158 | 18.028 | 0.00002 |
| Rs2323019 | G A | 85 139 | 139 85 | 13.018 | 0.0003 |
| Rs352203 | T C | 152 83 | 83 152 | 20.260 | 0.000007 |
3.FZD3 the haplotyping of gene
The overall check of haplotype transmission shows that the FZD3 gene has stronger related (X with schizophrenia
2=48.84, degree of freedom=7, p＜0.000001).It is too much that the analysis of single haplotype is also shown among the patient transmission of GAT haplotype, and difference has significance (X
2=34.21, degree of freedom=1, p＜0.000001) (seeing Table 4).
The haplotype of table 4 FZD3 gene transmits frequency analysis
| Haplotype | Observed value | Expected value | X 2 | The p value |
| Total X 2Check | 48.84 | p＜0.000001 | ||
| GGT | 69.644 | 68.755 | 0.026 | 0.871 |
| AGT | 17.326 | 25.257 | 5.503 | 0.019 |
| GAT | 210.98 | 170.42 | 34.207 | ＜0.000001 |
| AAT | 35.051 | 34.072 | 0.066 | 0.797 |
| GGC | 58.364 | 60.158 | 0.132 | 0.716 |
| AGC | 83.667 | 101.83 | 8.797 | 0.003 |
| GAC | 12.013 | 18.67 | 5.884 | 0.015 |
| AAC | 4.9566 | 12.841 | 11.478 | 0.0007 |
Annotate: GGT is Rs2241802G+Rs2323019G+Rs352203T; AGT is Rs2241802A+Rs2323019G+Rs352203T; GAT is Rs2241802G+Rs2323019A+Rs352203T; AAT is Rs2241802A+Rs2323019A+Rs352203T; GGC is Rs2241802G+Rs2323019G+Rs352203C; AGC is Rs2241802A+Rs2323019G+Rs352203C; GAC is Rs2241802G+Rs2323019A+Rs352203C; AAC is Rs2241802A+Rs2323019A+Rs352203C.
Conclusion:
Can draw from above-mentioned test: the rs2241802 of FZD3 gene order, rs2323019 and/or rs352203 pleomorphism site influence schizoid susceptibility, wherein the rs2241802 pleomorphism site is G in the FZD3 gene order, and the rs2323019 pleomorphism site is that A and/or rs352203 pleomorphism site are that T is the high person of schizophrenia susceptibility.
Method:
Detect trier rs2241802, the genotype of rs2323019 and/or rs352203 pleomorphism site with polymerase chain reaction-restriction fragment length polymorphism (PCR-based RFLP) analytical procedure.
Main method is as follows:
The collection of 1 blood preparation and processing
The extraction of 2 genomic dnas and evaluation
The segmental amplification of 3 purposes
4 restriction fragment length polymorphism analyses
Concrete grammar is referring to the 2.1-2.5 of embodiment 1.
Wherein the rs2241802 pleomorphism site is G, and the rs2323019 pleomorphism site is that A and/or rs352203 pleomorphism site are the trier of T, is the high person of schizophrenia susceptibility.
The test kit of embodiment 3 vitro detection schizophrenia tumor susceptibility genes
1) primer in amplification polymorphism site
| SNP | Primer sequence (5 ' → 3 ') |
| rs2241802 | CTATGAAATAGCGAGCAAATGACA(SEQ ID No 4) GGAAATCCAAACTGTTAGATCGTG(SEQ ID No 5) |
| rs2323019 | AGCCACTGCTCCCACCAAAG(SEQ ID No 6) CAAAAACCCAGGGATACCCAAAC(SEQ ID No 7) |
| rs352203 | ATGACTTCCTAGGGCCAAACCTC(SEQ ID No 8) GCAAAAACTAATGGCCAGCAATGT(SEQ ID No 9) |
2) pcr amplification enzyme and restriction enzyme A luI, SspI, NlaIII and corresponding damping fluid;
3)dNTP；
4) described pleomorphism site restriction enzyme mapping:
| SNP | Restriction endonuclease | Allelotrope (bp) | |
| rs2241802 | AluI | A 124/183 | G 307 |
| rs2323019 | SspI | A 131/190 | G 321 |
| rs352203 | NlaIII | C 120/294 | T 414 |
And explanation using method is following comprises:
The collection of 1 blood preparation and processing
The extraction of 2 genomic dnas and evaluation
The segmental amplification of 3 purposes
4 restriction fragment length polymorphism analyses
Concrete grammar is referring to embodiment 1 method 2.1-2.5.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read foregoing description content of the present invention, but the equivalent form of value of changing or revising drops on equally in the application's claims institute restricted portion.
Sequence table
＜110〉Zhang Dai
Mental health institute of Peking University
＜120〉schizophrenia tumor susceptibility gene detection method and tumor susceptibility gene and purposes
<130>PD0305023
<160>9
<170>PatentIn version 3.1
<210>1
<211>307
<212>DNA
＜213〉Homo sapiens (homo sapiens)
<400>1
ctatgaaata gcgagcaaat gacagttctt ctcttctgaa gtacatattt ggacaaggag 60
gtgaacaatc acgcacatgc agaaaagaat aacccagatc aggatcaatt tttaactctc 120
ggggacacca aaaaccatag tctctctgca ctgccactgg ggctccttca gttggttctc 180
cagctaaatt cagatccaca agtcgaggat atggctcatc acaatctggg aacctaaaaa 240
acaaaacaag atgagaacta tttttactta tggggaaatc tatcacgatc taacagtttg 300
gatttcc 307
<210>2
<211>321
<212>DNA
＜213〉Homo sapiens (homo sapiens)
<400>2
agccactgct cccaccaaag attatctttt taattcaatt atttcttgtc ttatttcatc 60
cctactgctt tctttagaat tactttgttt ttctagattc ttgaattgaa agcttaactc 120
atttattgtc aatgttttaa aatgtatctt aaaaatacat ttaatagtta ctttaaattt 180
aaccaaaaga ccattattaa ccaggttggt cattcacact cattctgtct tctgaactta 240
actttaagta actttatgtt tgttttgttt tgttttgttt tgttttgttt tgttttgttt 300
gagaaccttt atttattgaa g 321
<210>3
<211>415
<212>DNA
＜213〉Homo sapiens (homo sapiens)
<400>3
atgacttcct agggccaaac ctcaaaatta tttagtttct gtaatagagg aaaagaatga 60
gaattccctg aaaaaatatt ctatatctct tgttttgcca gaaaatatta cagaatacga 120
catgaaggaa acatagacta ttatagaaat ttatagaaac tgtaatactg ttatgtcctg 180
gttgtaaaat actgtcaaat gacacttttg gataaatatg ataaaatata caagttatta 240
ttctggcctc tgttaaacag aataacctaa gttgtgtgtt cagtttaagt ctccactgca 300
gaaatacctg atatgcaaat agtttataca tttttaggag aaaattcaat gtacttgcca 360
ttatctacag tagggcatgg caaactgtaa aagatcagat aataaatatt ttaga 415
<210>4
<211>24
<212>DNA
＜213〉artificial (artificial sequence)
<220>
＜223〉primer (primer)
<400>4
ctatgaaata gcgagcaaat gaca 24
<210>5
<211>24
<212>DNA
＜213〉artificial (artificial sequence)
<220>
＜223〉primer (primer)
<400>5
ggaaatccaa actgttagat cgtg 24
<210>6
<211>20
<212>DNA
＜213〉artificial (artificial sequence)
<220>
＜223〉primer (primer)
<400>6
agccactgct cccaccaaag 20
<210>7
<211>23
<212>DNA
＜213〉artificial (artificial sequence)
<220>
＜223〉primer (primer)
<400>7
caaaaaccca gggataccca aac 23
<210>8
<211>23
<212>DNA
＜213〉artificial (artificial sequence)
<220>
＜223〉primer (primer)
<400>8
atgacttcct agggccaaac ctc 23
<210>9
<211>24
<212>DNA
＜213〉artificial (artificial sequence)
<220>
＜223〉primer (primer)
<400>9
gcaaaaacta atggccagca atgt 24
Claims (2)
1, a kind of test kit that is used to detect the schizophrenia susceptibility, it comprises:
1) primer of amplification FZD3 gene order rs2241802, rs2323019 and/or rs352203 pleomorphism site;
2) pcr amplification enzyme, enzyme are cut the corresponding restriction enzyme of pleomorphism site, and corresponding damping fluid;
3)dNTP；
4) described pleomorphism site restriction enzyme mapping.
2, the test kit of detection schizophrenia susceptibility as claimed in claim 1, it comprises:
1) primer:
The primer of amplification rs2241802 pleomorphism site:
5’-CTATGAAATAGCGAGCAAATGACA-3’
5’-GGAAATCCAAACTGTTAGATCGTG-3’；
The primer of amplification rs2323019 pleomorphism site:
5’-AGCCACTGCTCCCACCAAAG-3’
5’-CAAAAACCCAGGGATACCCAAAC-3’；
The primer of amplification rs352203 pleomorphism site:
5’-ATGACTTCCTAGGGCCAAACCTC-3’
5’-GCAAAAACTAATGGCCAGCAATGT-3’；
2) pcr amplification enzyme, restriction enzyme A luI, SspI and NlaIII and corresponding damping fluid;
3)dNTP；
4) described pleomorphism site restriction enzyme mapping.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2003/000367 WO2004101818A1 (en) | 2003-05-19 | 2003-05-19 | Detecting method of the schizophrenia susceptible gene and use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1771332A CN1771332A (en) | 2006-05-10 |
| CN100342032C true CN100342032C (en) | 2007-10-10 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB038264838A Expired - Fee Related CN100342032C (en) | 2003-05-19 | 2003-05-19 | Schizophrenia susceptible gene detection method, susceptible gene and application |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN100342032C (en) |
| AU (1) | AU2003231562A1 (en) |
| WO (1) | WO2004101818A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5943301B2 (en) * | 2012-09-26 | 2016-07-05 | 大日本印刷株式会社 | Negative resist composition, method for producing resist pattern using the resist composition, and electronic component |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1183122A (en) * | 1995-05-03 | 1998-05-27 | 罗纳-布朗克·罗莱尔股份有限公司 | Method for diagnosing schizophrenia |
| CN1423696A (en) * | 2000-02-28 | 2003-06-11 | 解码遗传Ehf公司 | Human schizophrenia gene |
-
2003
- 2003-05-19 CN CNB038264838A patent/CN100342032C/en not_active Expired - Fee Related
- 2003-05-19 AU AU2003231562A patent/AU2003231562A1/en not_active Abandoned
- 2003-05-19 WO PCT/CN2003/000367 patent/WO2004101818A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1183122A (en) * | 1995-05-03 | 1998-05-27 | 罗纳-布朗克·罗莱尔股份有限公司 | Method for diagnosing schizophrenia |
| CN1423696A (en) * | 2000-02-28 | 2003-06-11 | 解码遗传Ehf公司 | Human schizophrenia gene |
Non-Patent Citations (1)
| Title |
|---|
| genebank登录号GI:22047956 genebank数据库 2003 * |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2003231562A1 (en) | 2004-12-03 |
| WO2004101818A1 (en) | 2004-11-25 |
| CN1771332A (en) | 2006-05-10 |
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