CN104878080A - Kit for external detection of Neurofibromastosis 2 disease causative gene NF2 c.602delTG mutation - Google Patents

Abstract

The invention discloses a kit for external detection of Neurofibromastosis 2 disease causative gene NF2 c.602delTG mutation. The kit comprises a primer pair shown in the formulas of SEQ ID NO: 1 and SEQ ID NO: 2. The invention firstly discloses Neurofibromastosis 2 disease causative gene NF2 c.602delTG mutation and proves a relationship of the mutation site and the NF2 disease. The invention further designs the primers for detecting the mutation site and discloses a use of the primers in diagnosis of the NF2 disease. Through prenatal screening and neonatal NF2 gene mutation screening, a birth rate of the children suffering from the NF2 disease is reduced, behavior of the patients with the causative gene is guide, disease is prevented and social pressure is greatly reduced. The kit for external detection of Neurofibromastosis 2 disease causative gene NF2 c.602delTG mutation is necessary for disciplinary research and development.

Description

A kind of test kit c.602delTG suddenlyd change for vitro detection Neurofibromastosis2 disease Disease-causing gene NF2

Technical field

The present invention relates to the test kit that c.602delTG a kind of NF2 of detection gene suddenlys change, this test kit can be used for auxiliary diagnosis and the new drug development of this disease, belongs to biological technical field.

Background technology

Neurofibromatosis 2 type disease (neurofibromatosis type2, NF2) be a kind of autosomal inheritance disease, its principal character is Bilateral Acoustic Neroumas (Fig. 1), the kinds of tumors such as schwannoma, meningioma and ependymoma in often occur together encephalic and marrow.Most tumors is optimum, but is positioned at the compressing that encephalic or intraspinal tube cause nerve or cerebral tissue due to these tumours, causes a series of serious symptoms, mainly comprise hearing disability, visual deterioration, four limbs muscular strength goes down, facial perception goes down, eye face dysraphism, pharyngeal reflex disappears, subcutaneous nodule etc., the sickness rate of NF2 is high, about 1,/25 000, and spontaneous mutation rate is high, penetrance 100%.Because NF2 phenotype is various, NIH determines the Case definition of NF2: 1. CT or MRI shows Bilateral Acoustic Neroumas; 2. lineal first degree relative is suffered from NF2 and Study of unilateral auditory neuropathy or is had 2 in following pathology at least: muddy under coating after neurofibroma, meningioma, glioma, schwannoma and teenager's lens.

The NF2 assignment of genes gene mapping is with (22q12) in No. 22 chromosome long arm 1 districts two, is about 110 000 bp, and coding region cDNA is long is 1785bp, containing 17 exons, produces two major protein hypotype schwannomin or Merlin by alternative splicing.This albumen belongs to Ezrin, and Radixin, Moesin (ERM) family, mainly plays ligation between cytoskeleton and cytolemma.But NF2 is also a tumor suppressor gene, NF2 functionally inactive not only with familial nervous system neoplasm about and also relevant with the nervous system neoplasm distributed.NF2 gene coded protein Merlin can antiproliferative effect, and it can interact with ubiquitin ligase E3 in core simultaneously, suppression cell cycle regulation genetic expression and activating cells expression of apoptotic gene.More importantly, Merlin take part in tumor inhibition effect by multiple biological signaling pathway.At present, find that Merlin is by tyrosine receptor kinase path, antiproliferative effect, and NF2 patient Merlin protein inactivation, make tyrosine receptor kinase (RTKs) Pathway Activation, promotes abnormal cell proliferation.In RTKs-ERK/MAPK path, Rac, Pakl albumen plays important effect.Paks (p212activated kinases) is the Ser/Thr kinases that the Rac/CDC42 of tool carinogenicity relies on, Merl in phosphorylation is made its inactivation by Paks by integrin signal peptide (cell adhesion associated molecule), impels normal cell to carry out mitotic division; Merlin plays negativity to Racl and regulates, and suppresses its downstream effect thing Paks, thus inhibits Growth of Cells, or the signal path that Merlin directly suppresses Paks activity blocks Rac to mediate.In addition, find Epidermal Growth Factor Receptor Family (ErbB family), comprise the growth factor receptors (PDGFRb) in Angiogenesis factor receptors (EGFR) and thrombocyte source, dredging collateral tyrosine receptor kinase (RTKs) path take part in the formation of NF2 tumour, finds to suppress the medicine of EGFR signal path can the growth of T suppression cell in Merlin genetically deficient cell.The expression of many ErbB family proteins is found in the tissue sample of schwannoma.Anti-ErbB family protein antibody can suppress the propagation of schwannoma cell.In the model experiment of preclinical phase Bilateral Acoustic Neroumas, find that epidermal growth factor recipient tyrosine kinase inhibitor erlotinib can the propagation of effective inhibition tumor cell.Recently, research report angiogenesis factor monoclonal antibody Arastin improves the hearing function of NF2 patient.

At present the method for examination is carried out to NF2 type disease or test kit was not also reported at gene level.

Summary of the invention

An object of the present invention is to provide a kind of test kit for auxiliary diagnosis neurofibromatosis 2 type disease.

Test kit for auxiliary diagnosis NF2 type disease provided by the present invention, comprises following primer pair: SEQ ID NO:1 and SEQ ID NO:2.

Another object of the present invention is to provide the purposes in following mutational site: in the 2nd exon of NF2 gene, c.602-603 the Nucleotide TG at position place lacks.

This purposes is the application in the test kit preparing auxiliary diagnosis NF2 type disease.Specifically can detect the primer pair in this mutational site for the design of this mutational site, namely obtain the test kit of auxiliary diagnosis NF2 type disease.

Another object of the present invention is to provide mutational site that is a kind of and NF2 type disease-related.

Provided by the present invention as follows with the mutational site of NF2 type disease-related: in the 2nd exon of NF2 gene, c.602-603 Nucleotide TG in position lacks.

The Late Cambrian of the present invention c.602delTG sudden change of neurofibromatosis 2 type (NF2) disease Disease-causing gene NF2, and prove this mutational site and NF2 disease-related.Devise the primer detecting this mutational site further, and use it for diagnosis NF2 disease.Experiment proves, this primer can specifically, accurately must diagnose out NF2 Disease.This diagnostic method is simple, cost is low, detected result directly, reliable, be applicable to large-scale examination that c.602delTG NF2 disease NF2 gene is suddenlyd change and diagnosis.By Prenatal Screening and newborn infant NF2 gene mutation for screening, can reduce the natality of NF2 disease infant, instruct minimal invasive treatment's behavior of carrying Disease-causing gene, preventing disease occurs, and greatly alleviates social pressures.C.602delTG the test kit suddenlyd change of vitro detection NF2 disease related gene NF2 is all indispensable for these disciplinary study and development.

Accompanying drawing explanation

Fig. 1 is about NF2 patients with clinical manifestations: Bilateral Acoustic Neroumas (as shown by arrows).

Fig. 2 is NF2 gene c.DNA coding region amino acid and the contrasting of nucleotide base: suddenly change and be positioned at the 602nd base, the amino acid of the 54th becomes translation termination signal (base that grey mark inserts, aminoacid deletion is expressed thereafter).

Fig. 3 is PCR reaction process schematic diagram in NF2 genetic heterozygosis of the present invention sudden change, and temperature of reaction and reaction times are shown.

Fig. 4 is the part sequencing result of the inventive method NF2 gene extron 2, and top is mutant nucleotide sequence, lower orientation normal control sequences, and arrow indication is mutational site.

Embodiment

Agents useful for same material of the present invention, if no special instructions, is commercially available purchase product.

Embodiment 1, with the NF2 gene mutation site discovery c.602delTG of neurofibromatosis 2 type disease-related

Collect NF2 patient by Neurosurgery Clinic, under patient and the voluntary prerequisite of family members, signature informed consent postscript, leaves and takes 5-10ml blood sample, sets up inpatient cases database, incidence and contact method in the detailed record patient state of an illness, family.Then apply phenol chloroform extraction methods and extract genomic dna, warehouse-in after quantitative ,-20 DEG C of preservations, every part of DNA all accurately corresponding patient clinical data of registering.Carry out design primer according to Primer Premier5.0, comprise NF2 gene 602 site and both sides sequence, carry out pcr amplification.Carry out direct Sequencing to PCR primer, sequencing primer is identical with pcr amplification primer, and application ABI company 3730DNA sequenator carries out backward sequencing.By the standard sequence comparison in the sequence that obtains and Genbank, determine 602 mutational sites.Undertaken translating to determine NF2 gene mutation site by normal reading frame.

Result: find that a patient has the new sudden change of NF2 gene on exon 2 by the examination of the NF2 gene coding region exon to 36 routine NF2 Diseases and 6 routine normal controls.This catastrophe point is dinucleotide deletion mutantion.NF2 Disease is heterozygote, and namely an allelotrope is that in the 2nd exon of NF2 gene, c.602-603 Nucleotide TG in position lacks, and this sudden change is denoted as and c.602delTG suddenlys change.This sudden change is all undiscovered in 6 routine control groups.There is not any sudden change in normal people, is homozygous genotype herein, and namely two allelotrope to be in the 2nd exon of NF2 gene c.602-603 position Nucleotide is TG (Fig. 2).This mutational site and NF2 disease-related are described.What c.602delTG existence suddenlyd change is NF2 Disease.

Embodiment 2, for the diagnostic kit of NF2 type disease and application thereof

One, test kit composition

Upstream primer: NF2602-F:5 '-CCTTCCCCATTGGTTTGTTATTG-3 ' (SEQ ID NO:1);

Downstream primer: NF2602-R:5 '-CCAGGGCCAGCAGCAGTCTAATC-3 ' (SEQ ID NO:2);

50ul 10X PCR damping fluid (Pharmacia),

10ul 10mM dNTP mixed solution (Pharmacia),

5ul (5unit/ul) Taq archaeal dna polymerase (Takara),

Each 10ul (10pmol/ul) F1 (SEQ ID NO:1) and R1 (SEQ ID NO:2) primer,

1ml pure water (self-control).

Two, test kit application

(1) diagnostic materials:

The peripheral blood blood sample of totally 36 routine NF2 Diseases and 6 routine normal peoples.

Collect NF2 patient by Neurosurgery Clinic, under patient and the voluntary prerequisite of family members, signature informed consent postscript, leaves and takes 5-10ml blood sample, sets up inpatient cases database, incidence and contact method in the detailed record patient state of an illness, family.This research has obtained the approval of Ethics Committee of our unit.

(2) diagnostic method

1, the extraction of genomic dna:

First day: under antithrombotics EDTA exists, by the 5-10ml human peripheral of collection at 2500rpm, centrifugation removes serum deprivation in 30 minutes; Add 0.2%NaCl solution, make cumulative volume be 50ml.Vibration solution 5-6 time gently, and make it be positioned over 15 minutes on ice; At 2500rpm centrifugation 30 minutes, whereby collecting precipitation thing; By the NaCl solution of 0.2%, wash again in a manner similar to before; In the throw out so obtained, add 10mMTris-HCl (pH8.0) and 10mM EDTA (4ml), with this throw out that suspends; Add in suspension by the Proteinase K of 10%SDS, 25mg/ml and the RNaseA of 10mg/ml, its add-on is respectively 4ml, 16ul and 20ul, and the suspension that then turns upside down mixes gently; At 37 DEG C of Overnight incubation suspensions.

Second day: add 4ml phenol/Tris solution, the mixture of the mixture mixing gained that turns upside down; Centrifugation 10 minutes removing water layers are carried out with 3000rpm; By water layer and 4ml phenol/chloroformic solution mixing, then against mixing and with 3000rpm centrifugation 10 minutes; Removing water layer, with chloroform extraction twice, to obtain aqueous phase, toward wherein adding 1/10,3M NaAC (pH5.2), the cold dehydrated alcohol of doubling dose, makes DNA precipitate; Washing with alcohol with 70% is to obtain genomic dna; Make the DNA obtained like this, genomic dna is dissolved in TE, and then quantitative assay mixture is in the specific absorption of 260nm; DNA working fluid concentration correction, to 50ng/ul, puts-20 DEG C of Refrigerator stores.

2, pcr amplification

PCR reaction system

PCR response procedures: as shown in Figure 3.

3, PCR primer order-checking

After PCR terminates, use the sepharose of 1.5% to carry out electrophoresis quantitatively, 3ul is often planted PCR reaction solution electrophoresis on the polyacrylamide gel of 8% and obtain single band, observe at BioRad imager, application Gel Doc2000 program.The PCR primer of the PCR primer pair purifying of design is used to check order.

4, mutation analysis: the SeqmanTM software at application DNAStat5.0 (Lasergene Inc.) software package end carries out sequence comparative analysis.The standard sequence that the sequence obtained checking order and NCBI retrieve is compared, and finds mutant nucleotide sequence, finds mutational site.

(3) diagnostic result

Result as shown in Figure 4.

The sequencing result of 6 routine normal peoples is: all there is not any sudden change, is homozygous genotype, and namely two allelotrope to be in the 2nd exon of NF2 gene c.602-603 position Nucleotide and to be followed successively by TG (Fig. 2).

The sequencing result of NF2 Disease is: 1 routine NF2 Disease exists following transgenation: heterozygote, namely an allelotrope is that in the 2nd exon of NF2 gene, c.602-603 position Nucleotide is followed successively by TG, and another allelotrope is that in the 2nd exon of the NF2 gene corresponding with above-mentioned allelotrope, c.602-603 Nucleotide TG in position lacks (Fig. 2).

In above-described embodiment, the ordinary method that the check point of this area also can be adopted to suddenly change to detect the mutational site of NF2 gene, as high resolution solubility curve analyze, high pressure liquid chromatography or by restriction fragment length polymorphism method etc.

Claims (3)

1. for a test kit for auxiliary diagnosis NF2 type disease, comprise following primer pair: SEQ ID NO:1 and SEQ ID NO:2.

2. the following application of mutational site in the test kit preparing auxiliary diagnosis NF2 type disease: in the 2nd exon of NF2 gene, c.602-603 Nucleotide TG in position lacks.

3. one kind with the mutational site of NF2 type disease-related: in the 2nd exon of NF2 gene, c.602-603 Nucleotide TG in position lacks.