CN103555837A - Human ROR2 gene mutation and application thereof - Google Patents

Human ROR2 gene mutation and application thereof Download PDF

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CN103555837A
CN103555837A CN201310533358.6A CN201310533358A CN103555837A CN 103555837 A CN103555837 A CN 103555837A CN 201310533358 A CN201310533358 A CN 201310533358A CN 103555837 A CN103555837 A CN 103555837A
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郑芳
董素芳
韩明君
宋贵波
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Wuhan University WHU
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Abstract

The invention relates to human ROR2 gene mutation and application thereof. The invention provides a method for detecting human ROR2 gene mutation, and a related detection kit. The method aims to determine the 2472rd nucleotide (887th nucleotide of 9th exon of ROR2 gene) in the sequence disclosed as SEQ ID NO:1 by sequencing, thereby detecting whether C->A mutation exists. The research detects that when the 2472rd nucleotide of the human ROR2 gene (of which the sequence is disclosed as SEQ ID NO:1) has C->A mutation, S758X in the ROR2 protein amino acid sequence SEQ ID NO:2 is changed to form a truncated ROR2 protein, which is related to occurrence of Brachydactyly Type B. The method can be used in auxiliary diagnosis of Brachydactyly Type B, is used for finding out the potential carrier of hereditary cataract, and provides reference for preventing hereditary cataract.

Description

People ROR2 transgenation and uses thereof
Technical field
The present invention relates to the method for the transgenation of people ROR2 and the sudden change of analyst ROR2 gene, and this sudden change is in the purposes of brachydactyly Type B auxiliary diagnosis.
Background technology
Brachydactyly (Brachydactyly), has another name called brachydactylia disease, is a kind of autosome dominant disease.Refer to brothers' congenital malformation that finger (toe) bone that (toe) bone, the palm (sole of the foot) osteodysplasty cause shortens, lacks as or merges.Nineteen fifty-one, Bell is according to the difference of lopsided happening part and patient's involved, heredity brachydactylia is divided into five types of A, B, C, D, E, and A type is further subdivided into A1, A2, tri-kinds of hypotypes of A3.Wherein brachydactyly Type B (Brachydactyly type B, BDB) is the most serious a kind of of phenotype in many somatotypes, and phenotype is unique.BDB patient 2-5 refers to that distal phalanx and hypoplastic nails even lack, and middle phalanxes dysplasia, with the partially or completely disappearance of nail; Thumb can be acted normally also can present flat wide or phalangette bifurcated deformity; Foot phenotype is similar to hand, but involved is lighter.
Receptor tyrosine kinase sample orphan receptor 2(Tyrosine-protein kinase transmembrane receptor2, ROR2) the main Disease-causing gene [Oldridge that gene is BDB1, M., Fortuna, A.M., Maringa, M., Propping, P., Mansour, S., Pollitt, C., DeChiara, T.M., Kimble, R.B., Valenzuela, D.M., Yancopoulos, G.D., Wilkie, A.O.M.Dominant mutations in ROR2, encoding an orphan receptor tyrosine kinase, cause brachydactyly type B.Nature Genet.24:275-278, 2000.], by Oldridge, equal to be positioned for 1999 9q22.The about 235kb of this full length gene, totally 9 exons, code length is 943 amino acid.The chondrocyte that it has participated in getting up early forms growth, [DeChiara, T.M. relevant to cartilage and bone growth, Kimble, R.B., Poueymirou, W.T., Rojas, J., Masiakowski, P., Valenzuela, D.M., Yancopoulos, G.D.Ror2, encoding a receptor-like tyrosine kinase, is required for cartilage and growth plate development.Nature Genet.2000.24:271-274].ROR2 gene is combined with its part Wnt5a and is suppressed typical Wnt signal pathway, affect fetal development, cell proliferation and differentiation regulation and control [Mikels, A.J., Nusse, R.Purified Wnt5a protein activates or inhibits beta-catenin-TCF signaling depending on receptor context.PLoS Biol.4:e115,2006.Note:Electronic Article.].
Cause the transgenation of BDB very rare, up to the present, find altogether in the world approximately 10 kinds of pathogenic mutations, domesticly only find 2 kinds, all be positioned at the 8th, 9 exons of ROR2 gene, with the form of nonsense mutation or phase shift mutation, cause the generation of truncated protein or abnormal C-end.
The present invention be take BDB family as research object, in conjunction with gene sequencing technology and single-strand conformation polymorphism, has identified a new pathogenic mutation of ROR2.Also about ROR2 gene S758X, do not suddenly change and cause the report of brachydactyly Type B disease in the world at present.
Summary of the invention
First object of the present invention is to provide a kind of people of detection ROR2 method of transgenation;
Second object of the present invention is to be provided for detecting the test kit of people ROR2 transgenation.
The present invention studies and finds that the 887th Nucleotide C → A of ROR2 gene 9 exons nonsense mutation is associated with BDB.This sudden change causes the 758th coded amino acids and just by Serine (TCG), becomes terminator codon (TAG), and this sudden change causes ROR2 albumen by 185 amino acid of brachymemma, thereby its physico-chemical property and function are changed.Find after deliberation, closely related with mankind BDB disease.
For this reason, first aspect present invention, provides a kind of method of sudden change of the people of detection ROR2 gene, and it comprises the following steps:
(a) determine the 758th amino acids of people ROR2 genes encoding in sample, or the 2471st to 2473 Nucleotide of SEQ ID NO:1;
(b) detect in described position, whether there is amino acid S → X sudden change.
Particularly, described sudden change is the sudden change at the 2273rd Nucleotide C → A of SEQ ID NO:1, makes it form herein termination codon.
A second aspect of the present invention, provides a kind of nucleic acid of separation, and it has the sequence shown in SEQ ID NO:1, and the 2472nd be A, or comprises the specific fragment of sequence shown in the SEQ ID NO.1 of the 2472nd Nucleotide.
And then the invention provides a kind of molecule marker, it is the nucleotide sequence described in above-mentioned SEQ ID NO.1, or described specific fragment, by this molecule marker, can judge whether to exist the ill risk of BDB.
In a third aspect of the present invention, be provided for the Auele Specific Primer that amplification comprises said mutation site.For example, its length of this primer is 15-50bp, and hybridizes specifically and amplify the amplified production containing C → A sudden change at the 887th place of 9 exons of the 2472nd Nucleotide C in sequence shown in the SEQ ID NO:1 of people ROR2 gene → A sudden change or genome ROR2 gene.
In a fourth aspect of the present invention, provide a kind of and can and detect the oligonucleotide probe in described mutational site with above-mentioned SEQ ID NO.1 specific hybrid.For example, its length is 15-50bp, and hybridizes specifically and detect the C → A base mutation containing the 887th place of 9 exons of the 2472nd Nucleotide C → A sudden change or genome ROR2 gene in sequence shown in the SEQ ID NO:1 of people ROR2 gene.
In a fifth aspect of the present invention, provide the test kit of BDB disease molecules diagnosis.This test kit comprises the reagent that the 2472nd Nucleotide for people ROR2 gene detects, or the reagent detecting for the mRNA sequence corresponding positions point of this genetic transcription, or for the reagent of this gene expression product corresponding positions point detection.It can be: 1) detect genomic PCR test kit; Or 2) detect the fluorescent quantificationally PCR detecting kit of mRNA, or Northern detection kit.For example, can comprise the oligonucleotide probe that above-mentioned primer and/or the present invention are above-mentioned; Or 3) detect the immunity detection reagent of this mutain.
The present invention studies shown in the SEQ ID NO:1 of finder ROR2 gene C → A sudden change at the 887th place of 9 exons of the 2472nd Nucleotide C → A sudden change in sequence or genome ROR2 gene, this sudden change causes the change of S758X in ROR2 Argine Monohydrochloride sequence SEQ ID NO:2, formed the ROR2 albumen of brachymemma, relevant to mankind BDB disease incidence.The present invention can be for the auxiliary diagnosis of BDB disease, for finding the potential carrier of BDB disease, for the prevention of this disease provides foundation.
Accompanying drawing explanation
Fig. 1 shows is the sequencer map (forward) contrasting in contrast and crowd in family, and in figure, arrow indication is the 2472nd of SEQ ID No.1, is wild-type (C/C);
What Fig. 2 showed is the sequencer map (forward) of patient in family, and in figure, arrow indication is the 2472nd of SEQ ID No.1, is saltant type (C/A);
Embodiment
The inventor is by extensive and deep research, had been found that sudden change (the ﹥ A c.2273G of people ROR2 gene, S758X, NM_004560.3) in a BDB family, present heterozygous mutant, have typical clinical manifestation, middle phalanges shortens or merges with distal phalanx, with nail heteroplasia and thumb distal phalanx bifurcated, show that this sudden change may have influence on the function of gene, make skeleton development abnormal, caused the generation of BDB.The mouse model that has had ROR2 gene specific to knock out, has caused serious dyschondroplasia and has died young with other heteroplasia.
The genome sequence of people ROR2 gene is classified SEQ ID NO:1 as, and the aminoacid sequence of coding is SEQ ID NO:2.The genome sequence of people ROR2 can obtain from GenBank.
Those skilled in the art person all knows, has many analytical technologies can be for detection of the sudden change of people ROR2 gene.These technology include but is not limited to: the high performance liquid chromatography (DHPLC) of DNA sequencing, sequencing by hybridization, enzymatic mispairing cutting, heteroduple analysis, dot blot, oligonucleotide arrays (DNA chip), Mini-sequencing, Taqman technology, high resolving power melting curve (HRM), sex change and molecular beacon etc.
On the other hand, detection method of the present invention is used to the individual susceptibility to BDB of assessment.
Detection sample of the present invention can be from body fluid, organize genomic dna or the mRNA of extracting the sample such as hair even.
Primer and probe for the inventive method or detection kit, design according to the genome sequence of ROR2 gene (sequence of SEQ ID NO:1) or cDNA sequence, and synthesize by conventional synthetic technology.
Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in embodiment, conventionally carries out routinely or completes according to the condition of test kit suggestion below.
The genomic extracting of embodiment 1 and order-checking
By conventional phenol chloroform method, from people's blood, extract genomic dna, for conventional pcr amplification.Primer sequence is:
Sense primer: 5'CCGATGACTGTCCCGCCTG 3'
Antisense primer: 5'GCGTTGCTCACATTGCTCACTG 3'
Amplification system: (cumulative volume 50 μ L)
Figure BDA0000407096790000051
Response procedures:
Figure BDA0000407096790000062
Amplify the product of 211bp, carry out direct Sequencing.
The detection of embodiment 2 sudden changes
Take the peripheral blood of research object, and extract its genomic dna.In BDB family, carry out complete genomic linkage analysis, candidate region, location.Adopt sequencing technologies to obtain the base sequence of ROR2 gene SNP site.In conjunction with the analysis of sequencing result (sequence measurement is undertaken by embodiment 1) and information biology, through the SNP examination of the case-control analysis in family and large sample contrast, determine that the sudden change of ROR2 gene and BDB are chained together.
The equal informed consent of family member in following form participates in this research, is checked in the Central-South Hospital Physical Examination of all selected member Jun Wuhan Universitys center, makes a definite diagnosis or gets rid of BDB.
Individual numbering The 9th the 887th of exon Brachydactyly Type B
1 C/C Nothing
2 C/C Nothing
3 C/A Have
4 C/C Nothing
5 C/C Nothing
6 C/C Nothing
7 C/A Have
8 C/C Nothing
9 C/C Nothing
10 C/C Nothing
11 C/C Nothing
12 C/A Have
13 C/C Nothing
14 C/C Nothing
15 C/C Nothing
16 C/C Nothing
17 C/C Nothing
18 C/C Nothing
19 C/C Nothing
20 C/C Nothing
21 C/C Nothing
22 C/C Nothing
23 C/C Nothing
24 C/C Nothing
Embodiment 3 detection kit
The molecule diagnosis kit of preparation BDB disease, wherein contains the following primer that amplifies the 2472nd sudden change of nucleotide sequence (SEQ ID NO:1):
Forward primer: 5'CCGATGACTGTCCCGCCTG 3'
Reverse primer: 5'GCGTTGCTCACATTGCTCACTG 3'
The amplified production of patient and contrast is carried out to high resolving power melting curve analysis, can easier detect the sudden change of the 2472nd.
In addition, the present invention also provides a kind of molecule diagnosis kit that uses probe in detecting, comprises the probe for detection of described specificity site, and its sequence is as shown in SEQ ID No.5.Due to the sequence of not undergoing mutation can not with this probe hybridization, thereby can by hybridization can draw whether there is transgenation.
Should understand, after having read foregoing of the present invention, those skilled in the art can carry out various modifications or polishing to the present invention, yet be all to detect SNP of the present invention site to be correlated with, or the various forms of application in this site, these modifications or polishing all belong to scope of the present invention.
Sequence table explanation:
SEQ ID No.1 is the nucleotide sequence of ROR2 gene, comprises the sudden change of the 2472nd, and SEQ ID No.2 is the aminoacid sequence of its coding; SEQ ID No.3 & 4 is amplimers; SEQ ID No.5 is detection probes.
Figure IDA0000407096870000011
Figure IDA0000407096870000021
Figure IDA0000407096870000031
Figure IDA0000407096870000041
Figure IDA0000407096870000051
Figure IDA0000407096870000071
Figure IDA0000407096870000091
Figure IDA0000407096870000111
Figure IDA0000407096870000121
Figure IDA0000407096870000141

Claims (9)

1. one kind is detected people rOR2the method of transgenation, it comprises step:
(a) determine people in sample rOR2the 758th amino acids of the aminoacid sequence of genes encoding, or the 2471st to 2473 bit bases of this gene;
(b) detect and in described position, whether have the sudden change of S → X.
2. the method for claim 1, is characterized in that, described sudden change is the sudden change at the 2472nd Nucleotide C → A of SEQ ID NO:1, corresponding to the 758th amino acids of SEQ ID NO:2, has Ser → Ter sudden change.
3. a separated nucleic acid, is characterized in that, the nucleotide sequence of described separation is the 2472nd sequence shown in the SEQ ID NO:1 for A.
4. a molecule marker, it is separated nucleic acid described in claim 3.
5. for a test kit for auxiliary diagnosis brachydactyly Type B disease, it comprises for people rOR2the reagent of the 2472nd Nucleotide specific detection of gene, or the reagent detecting for the mRNA sequence corresponding positions point of this genetic transcription, or for detection of the detection reagent in this corresponding site of gene expression product.
6. test kit according to claim 1, is characterized in that, described test kit is: 1) detect genomic PCR test kit; Or 2) detect the PCR kit for fluorescence quantitative of mRNA, or Northern detection kit; Or 3) detect the immunoassay kit of this mutain.
7. test kit according to claim 6, is characterized in that, described PCR test kit comprises that sequence is the primer pair of SEQ ID NO:3 and 4.
8. one kind is detected people rOR2the primer pair of transgenation, its nucleotide sequence is as shown in SEQ ID No.3 and 4.
9. an oligonucleotide probe, it is hybridized with SEQ ID NO:1 specifically and detects its 2472nd Nucleotide.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108660210A (en) * 2018-05-24 2018-10-16 上海浦东解码生命科学研究院 Detect the primer and kit and method that apc gene is mutated
CN116286895A (en) * 2023-02-27 2023-06-23 青岛市妇女儿童医院（青岛市妇幼保健院、青岛市残疾儿童医疗康复中心、青岛市新生儿疾病筛查中心） ROR2 mutant and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
吕丹: "三种先天性肢端畸形的分子遗传学研究", 《中国博士学位论文全文数据库》 *
李聪敏 等: "一个中国B1型短指家系致病基因的突变分析", 《遗传》 *
杨威 等: "中国人遗传性B型短指(趾)家系中ROR2基因突变的鉴定", 《中华医学遗传学杂志》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108660210A (en) * 2018-05-24 2018-10-16 上海浦东解码生命科学研究院 Detect the primer and kit and method that apc gene is mutated
CN116286895A (en) * 2023-02-27 2023-06-23 青岛市妇女儿童医院（青岛市妇幼保健院、青岛市残疾儿童医疗康复中心、青岛市新生儿疾病筛查中心） ROR2 mutant and application thereof
CN116286895B (en) * 2023-02-27 2023-09-05 青岛市妇女儿童医院（青岛市妇幼保健院、青岛市残疾儿童医疗康复中心、青岛市新生儿疾病筛查中心） ROR2 mutant and application thereof

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