CN104878080B - Kit for in vitro detection of c.602delTG mutation of Neurosporistosis 2 disease-causing gene NF2 - Google Patents

Abstract

The invention discloses a kit for detecting c.602delTG mutation of Neurosporibossis 2 disease-causing gene NF2 in vitro. The kit comprises the following primer pairs: SEQ ID NO: 1 and SEQ ID NO: 2. the invention discovers the c.602delTG mutation of NF2 disease-causing gene NF2 for the first time, and proves that the mutation site is related to NF2 diseases. Primers for detecting the mutation site are further designed and used for diagnosing NF2 diseases. Through prenatal screening and neonatal NF2 gene mutation screening, the birth rate of infants with NF2 diseases can be reduced, the living behaviors of patients carrying pathogenic genes are guided, the diseases are prevented, and the social pressure is greatly relieved. The kit for in vitro detection of c.602delTG mutation of NF2 disease-related gene NF2 is indispensable to research and development of the subjects.

Description

Kit for in vitro detection of c.602delTG mutation of Neurosporistosis 2 disease-causing gene NF2

Technical Field

The invention relates to a kit for detecting c.602delTG mutation of NF2 gene, which can be used for auxiliary diagnosis of the disease and development of new drugs and belongs to the technical field of biology.

Background

Neurofibromatosis type2 disease (NF2) is an autosomal genetic disease mainly characterized by bilateral auditory neuroma (FIG. 1), frequently accompanied by intracranial and intramedullary schwannoma, meningioma and ependymoma, most tumors are benign, but a series of serious symptoms mainly including hearing loss, visual deterioration, myasthenia of limbs, facial hypoesthesia, eye-face insufficiency, pharyngeal reflex disappearance, subcutaneous nodules and the like are caused by the compression of nerves or brain tissues due to the fact that the tumors are located in the intracranial or spinal canal, the incidence rate of NF2 is high, about 1/25000, and the spontaneous mutation rate is high, and the penetrance rate is 100%.

The NF2 gene is located in the two bands (22q12) of the long arm 1 region of chromosome 22, is about 110000 bp long, has a coding region cDNA length of 1785bp, contains 17 exons, and generates two main protein subtypes, namely schwannomin or Merlin, by alternative splicing. This protein belongs to the Ezrin, radiaxin, moesin (erm) family, and plays a major role in the connection between the cytoskeleton and the cell membrane. However, NF2 is also a tumor suppressor, and functional inactivation of NF2 is associated with not only familial but also sporadic nervous system tumors. The NF2 gene coding protein Merlin can inhibit cell proliferation, and simultaneously can interact with endoubiquitin ligase E3 to inhibit and regulate cell cycle gene expression and activate apoptosis gene expression. More importantly, Merlin is involved in tumor suppression through a variety of biological signaling pathways. Currently, Merlin is found to inhibit cell proliferation through tyrosine receptor kinase pathway, while Merlin protein of NF2 patient is inactivated, so that tyrosine receptor kinase (RTKs) pathway is activated to promote abnormal cell proliferation. Rac and Pakl proteins play an important role in RTKs-ERK/MAPK pathways. Paks (p212activated kinases) is a carcinogenic Rac/CDC42 dependent Ser/Thr kinase, and integrin signal peptides (cell adhesion related molecules) inactivate Merl in by Paks phosphorylation, causing normal cells to undergo mitosis; merlin negatively regulates Racl, inhibits its downstream effector Paks, and thereby inhibits cell growth, or Merlin directly inhibits Paks activity to block Rac-mediated signaling pathways. In addition, it has been found that the epidermal growth factor receptor family (ErbB family), including vascular growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFRb), the Receptor Tyrosine Kinase (RTKs) pathway is involved in NF2 tumor formation, and that drugs that inhibit the EGFR signaling pathway are found in cells with a deletion in the Merlin gene, which inhibits cell growth. Expression of many ErbB family proteins was found in tissue specimens of schwannomas. anti-ErbB family protein antibodies can inhibit the proliferation of schwannoma cells. In preclinical bilateral auditory neuroma model experiments, the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib is found to be capable of effectively inhibiting the proliferation of tumor cells. Recently, studies have reported that the angiogenic growth factor monoclonal antibody avastin improves hearing function in NF2 patients.

At present, no method or kit for screening NF2 type diseases at the gene level is reported.

Disclosure of Invention

An object of the present invention is to provide a kit for the auxiliary diagnosis of neurofibromatosis type2 disease.

The kit for auxiliary diagnosis of NF2 type diseases provided by the invention comprises the following primer pairs: SEQ ID NO: 1 and SEQ ID NO: 2.

it is another object of the present invention to provide the use of the following mutation sites: nucleotide TG at c.602-603 in exon 2 of the NF2 gene was deleted.

The application is the application in the preparation of a kit for the auxiliary diagnosis of NF2 type diseases. Specifically, a primer pair capable of detecting the mutation site is designed aiming at the mutation site, and the kit for auxiliary diagnosis of NF2 type diseases is obtained.

It is another object of the present invention to provide a mutation site associated with NF 2-type diseases.

The mutation sites related to NF2 type diseases provided by the invention are as follows: the C.602-603 bit TG in the 2 nd exon of the NF2 gene is deleted.

The invention discovers the c.602delTG mutation of a pathogenic gene NF2 of neurofibromatosis type2 (NF2) disease for the first time, and proves that the mutation site is related to NF2 disease. Primers for detecting the mutation site are further designed and used for diagnosing NF2 diseases. Experiments prove that the primer can specifically and accurately diagnose NF2 patients. The diagnosis method is simple, low in cost, direct and reliable in detection result, and suitable for large-scale screening and diagnosis of NF2 gene c.602delTG mutation of NF2 diseases. Through prenatal screening and neonatal NF2 gene mutation screening, the birth rate of infants with NF2 diseases can be reduced, the living behaviors of patients carrying pathogenic genes are guided, the diseases are prevented, and the social pressure is greatly relieved. The kit for in vitro detection of c.602delTG mutation of NF2 disease-related gene NF2 is indispensable to research and development of the subjects.

Drawings

Figure 1 is a graph of clinical presentation for NF2 patients: bilateral auditory neuroma (as indicated by arrows).

FIG. 2 shows the comparison of amino acids and nucleotide bases in the c.DNA coding region of NF2 gene: the mutation is at base 602, and the amino acid at position 54 becomes a translation termination signal (base inserted in grey scale, after which amino acid is expressed by deletion).

FIG. 3 is a schematic diagram showing the PCR reaction process in the heterozygous mutation of the NF2 gene according to the invention, and showing the reaction temperature and reaction time.

FIG. 4 shows the partial sequencing result of exon 2 of the NF2 gene according to the method of the present invention, wherein the upper part is a mutant sequence, the lower part is a normal control sequence, and the arrow indicates the mutation site.

Detailed Description

The reagent materials used in the present invention are commercially available products unless otherwise specified.

Example 1 discovery of NF2 Gene mutation site c.602delTG associated with neurofibromatosis type2 disease

NF2 patients are collected through an outpatient department of neurosurgery, and 5-10ml of blood samples are reserved after signing an informed consent on the premise that the patients and family members voluntarily, an in-hospital medical record database is established, and the disease conditions of the patients, the disease conditions in families and the contact ways are recorded in detail. Then extracting the genome DNA by a phenol chloroform extraction method, quantitatively warehousing the genome DNA at the temperature of minus 20 ℃, and storing each part of DNA accurately corresponding to the registered clinical data of the patient. Primers were designed according to Primer premier5.0, including the 602 th site and flanking sequence of NF2 gene, and PCR amplification was performed. And (3) directly sequencing the PCR product, wherein the sequencing primer is the same as the PCR amplification primer, and reverse sequencing is performed by using an ABI 3730DNA sequencer. The resulting sequence was aligned with standard sequences in Genbank to determine 602 the mutation site. Translation was performed in the normal reading frame to identify the site of NF2 gene mutation.

As a result: 36 cases of NF2 patients with diseases and 6 cases of normal control were screened for the exon of the coding region of the NF2 gene, and one patient was found to have a new mutation of the NF2 gene in exon 2. The mutation point is the deletion mutation of the dinucleotides. NF2 patients were heterozygous, i.e., one allele was deleted for the TG at nucleotides 602-603 in exon 2 of the NF2 gene, and this mutation was designated as the c.602delTG mutation. This mutation was not found in any of the 6 control groups. Normal humans do not have any mutation at this point and are homozygous genotypes, i.e. the TG is at nucleotide positions c.602-603 in exon 2 of the NF2 gene for both alleles (FIG. 2). Indicating that this mutation site is associated with NF2 disease. Patients with NF2 disease had the c.602deltg mutation.

Example 2 diagnostic kit for NF2 type diseases and use thereof

Kit composition

An upstream primer: NF 2602-F: 5'-CCTTCCCCATTGGTTTGTTATTG-3' (SEQ ID NO: 1);

a downstream primer: NF 2602-R: 5'-CCAGGGCCAGCAGCAGTCTAATC-3' (SEQ ID NO: 2);

50ul of 10 XPCR buffer (Pharmacia),

10ul of 10mM dNTP mix (Pharmacia),

5ul (5unit/ul) Taq DNA polymerase (Takara),

10ul (10pmol/ul) of each of F1(SEQ ID NO: 1) and R1(SEQ ID NO: 2) primers,

1ml of purified water (made by house).

Second, application of kit

(one) diagnostic material:

there were 36 NF2 patients with disease and 6 normal patients in total.

NF2 patients are collected through an outpatient department of neurosurgery, and 5-10ml of blood samples are reserved after signing an informed consent on the premise that the patients and family members voluntarily, an in-hospital medical record database is established, and the disease conditions of the patients, the disease conditions in families and the contact ways are recorded in detail. The study was approved by the ethical committee of the unit.

(II) diagnostic method

1. Extraction of genomic DNA:

the first day: centrifuging collected 5-10ml human peripheral blood at 2500rpm for 30 min in the presence of anticoagulant EDTA to remove serum; a0.2% NaCl solution was added to make the total volume 50 ml. Gently shake the solution 5-6 times and allow to stand on ice for 15 minutes; centrifuging at 2500rpm for 30 minutes, thereby collecting the precipitate; further washing with 0.2% NaCl solution in a similar manner to the above; to the thus-obtained precipitate, 10mM Tris-HCl (pH8.0) and 10mM EDTA (4ml) were added to suspend the precipitate; adding 10% SDS, 25mg/ml proteinase K and 10mg/ml RNaseA into the suspension in an amount of 4ml, 16ul and 20ul, respectively, and then slightly mixing the suspension by inverting the suspension upside down; the suspension was incubated at 37 ℃ overnight.

The next day: adding 4ml of phenol/Tris solution, and mixing the mixture by turning the mixture upside down; centrifuging at 3000rpm for 10 min to remove the water layer; the aqueous layer was mixed with 4ml of phenol/chloroform solution, followed by back mixing and centrifugation at 3000rpm for 10 minutes; the aqueous layer was removed, extracted twice with chloroform to obtain an aqueous phase, to which 1/10, 3M NaAC (pH5.2), twice the amount of cold absolute ethanol, was added to precipitate DNA; washing with 70% ethanol to obtain genomic DNA; the DNA thus obtained, genomic DNA, was dissolved in TE, and then the absorbance of the mixture at 260nm was quantitatively determined; the concentration of the DNA working solution is corrected to 50ng/ul and the DNA working solution is stored in a refrigerator at the temperature of 20 ℃ below zero.

2. PCR amplification

PCR reaction system

PCR reaction procedure: as shown in fig. 3.

3. Sequencing of PCR products

After the PCR was completed, the amount of the PCR product was quantified by electrophoresis on 1.5% agarose Gel, 3ul of each PCR reaction solution was electrophoresed on 8% polyacrylamide Gel to obtain a single band, which was observed on a BioRad imager using the Gel Doc2000 program. The purified PCR products were sequenced using the designed PCR primers.

4. Mutation analysis: sequence alignment analysis was performed using Seqmann software terminated by DNASTAT5.0(Lasergene Inc.). And comparing the sequence obtained by sequencing with a standard sequence retrieved by NCBI, finding out a mutant sequence and finding out a mutant site.

(III) results of diagnosis

The results are shown in FIG. 4.

The sequencing results for 6 normal persons were: no mutation exists, and the gene is homozygous genotype, namely the c.602-603 nucleotides in the 2 nd exon of the NF2 gene of both alleles are sequentially TG (figure 2).

The sequencing results of NF2 disease patients were: 1 NF2 disease patient has the following gene mutation: heterozygote, one allele is TG at c.602-603 th nucleotide in 2 nd exon of NF2 gene, and the other allele is TG at c.602-603 th nucleotide in 2 nd exon of NF2 gene corresponding to the above allele (FIG. 2).

In the above examples, the mutation site of NF2 gene can be detected by conventional methods for detecting point mutation in the art, such as high resolution melting curve analysis, high pressure liquid chromatography, or restriction fragment length polymorphism.

Claims (2)

1. A kit for assisting in diagnosing NF2 type diseases comprises the following primer pairs: SEQ ID NO: 1 and SEQ ID NO: 2, the kit assists in diagnosing NF2 type diseases by detecting the deletion of c.602-603 bit nucleotide TG in the 2 nd exon of the NF2 gene.

2. The following primer pairs: SEQ ID NO: 1 and SEQ ID NO: 2 in the preparation of a kit for the auxiliary diagnosis of NF 2-type diseases, wherein the kit is used for the auxiliary diagnosis of NF 2-type diseases by detecting the deletion of nucleotide TG at c.602-603 in the 2 nd exon of NF2 gene.

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