CN100436595C - Method for diagnosis and treatment of hair eipidermal tumor using human's CYLD gene and its coded products - Google Patents

Abstract

The invention discloses a method for diagnosing trichoepithelial tumors, which includes detecting whether there is variation in the CYLD gene, transcript and/or protein of an individual compared with normal ones, and the presence of variation indicates that the possibility of the individual suffering from trichoepithelial tumors is greater than normal crowd. The invention also discloses a corresponding detection kit, a method for treating piloma and a pharmaceutical composition.

Description

Use of human CYLD gene and its coding product in the preparation of diagnostic reagents

technical field

The invention relates to the fields of bioengineering and medicine. More specifically, the present invention relates to a method for diagnosing and treating piloma by using human CYLD gene and its coded product, as well as a pharmaceutical composition containing CYLD gene and/or protein.

Background technique

Trichoepithelialoma is a skin tumor. It usually occurs before puberty, and is more likely to occur on the face, especially the nasolabial folds and upper lip. The back, scalp, neck, trunk, and extremities can also be affected. The lesions are hemispherical or conical in shape, firm in texture, translucent, yellow or reddish in color, and sometimes larger lesions indicate visible telangiectasia. Trichoepithelial tumor not only affects beauty, because it affects the back, scalp, neck, trunk, and limbs, so severe cases will also encounter difficulties in life. The disease is an autosomal dominant genetic disease, and its pathogenesis is still unclear. There is currently no satisfactory treatment for this disease.

However, until now, the exact cause of piloma has not been fully revealed, nor has anyone revealed a direct correlation between piloma and a certain protein. In addition, the field also lacks an effective method for early diagnosis of piloma and an effective means for non-surgical treatment of piloma.

CYLD is a known protein, and its basic information is as follows:

English: Gene from: Homo sapiens chromosome 16working draft sequence segment.

NCBI: Contig NT_030834

GENE: Gene from: Homo sapiens chromosome 16 working draft sequencesegment.gi|17488444：243550-308161

The DNA sequence of CYLD is shown in SEQ ID NO: 1, and the ORF is located at positions 392-3262, encoding a protein with a full length of 956 amino acids (SEQ ID NO: 2). Additional information on CYLD is available at Http://www.ncbi.nlm.nih.gov.

It has been reported that mutations in the exons of CYLD gene lead to the occurrence of cylindrical tumors, but there is no international article reporting the relationship between CYLD gene and piloma. Information on the genetic mechanism of piloma is available from OMIM at NCBI.

Therefore, there is an urgent need in this field to develop new effective methods for diagnosing and treating piloma, as well as related therapeutic drugs, diagnostic techniques and reagents.

Contents of the invention

One object of the present invention is to provide a new method and detection kit for diagnosing (especially early diagnosis) piloma.

Another object of the present invention is to provide a new method for treating piloma.

Another object of the present invention is to provide a pharmaceutical composition for treating piloma.

In a first aspect of the present invention there is provided a method of diagnosing an individual's susceptibility to piloma, comprising the steps of:

detecting the individual's CYLD gene, transcript and/or protein, and comparing it with normal CYLD gene, transcript and/or protein,

A difference indicates that the individual is more likely to have piloma than the normal population.

Preferably, the gene or transcript of CYLD is detected, and the difference is compared with the normal CYLD nucleotide sequence. More preferably, the difference is selected from: A deletion at position 1853 in SEQ ID NO:1.

In the second aspect of the present invention, there is provided a method for treating piloma, which comprises the step of administering a safe and effective amount of normal CYLD protein to a patient in need of said treatment. Preferably, the CYLD protein is locally applied to the lesion.

In the third aspect of the present invention, a use of CYLD protein in preparing a pharmaceutical composition and a corresponding pharmaceutical composition are provided, which contain a safe and effective amount of human CYLD protein and a pharmaceutically acceptable carrier. Preferably, it is an ointment.

In the fourth aspect of the present invention, a kit for detecting piloma is provided, which includes primers for specifically amplifying CYLD gene or transcript. Preferably, it also contains a probe that binds to the mutation site and/or an enzyme that recognizes the mutation site.

Other aspects of the invention will be apparent to those skilled in the art from the technical disclosure herein.

Description of drawings

Figure 1 shows the pedigree of an autosomal dominant pilochepithelial tumor family.

Figure 2 is a photograph of a typical patient's face.

Figure 3 is a biopsy pathological photo of the patient.

Figure 4 shows the sequence changes in the CYLD gene, in which a base deletion occurred at position 1853 of SEQ ID NO: 1, resulting in a frameshift mutation and shortening of the encoded protein.

Detailed ways

After extensive and in-depth research, the inventors first discovered and proved that CYLD is closely related to piloma, and discovered its new function: the change of CYLD will directly lead to piloma. The present invention has been accomplished on this basis.

Through genetic linkage analysis, candidate gene screening and large-scale sequencing of an autosomal dominant piloma family, it has been confirmed that all patients in the family have a base at position 1853 of exon 10 of the CYLD gene The deletion of the protein leads to a frameshift mutation, which shortens the amino acid code and abnormally translates the protein, eventually leading to the occurrence of piloma. Normal CYLD encodes a protein with a full length of 956 amino acids (SEQ ID NO: 2), and if the deletion of base A occurs at position 1853, it only encodes a protein with a full length of 496 amino acids (SEQ ID NO: 5) and Some abnormal small peptides.

Mutations in CYLD lead to human individuals exhibiting skin neoplastic lesions, especially pilomas. Our study demonstrates that the presence of normal CYLD is critical for the normal physiological state of the skin.

Given that the variation of human CYLD is one of the direct causes of piloma. Therefore, the medicine and diagnosis and treatment technology that can be designed according to the gene and its expression product can be used for diagnosis and treatment of human hair epithelioma.

The full-length human CYLD nucleotide sequence or its fragments of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequence of CYLD, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art can be used as a template , to amplify the related sequence. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

The CYLD protein can be isolated by inserting the CYLD coding sequence into a suitable expression vector and then transferring it into a host cell.

Based on the new risk of the present invention, the CYLD protein or polypeptide has many new uses. These uses include (but are not limited to): direct drug treatment of diseases caused by CYLD protein dysfunction or loss (such as piloma), and screening for substances that promote the function of CYLD protein, such as antibodies, peptides or other ligands . Screening the polypeptide library with the expressed recombinant human CYLD protein can be used to find therapeutically valuable polypeptide molecules that can stimulate the function of the human CYLD protein.

On the other hand, the present invention also includes polyclonal antibodies and monoclonal antibodies specific to human CYLD DNA or polypeptides encoded by its fragments, especially monoclonal antibodies. Here, "specificity" means that the antibody can bind to human CYLD gene product or fragment. Preferably, it refers to those antibodies that can bind to human CYLD gene products or fragments but do not recognize and bind to other irrelevant antigen molecules. Antibodies in the present invention include those molecules capable of binding and inhibiting human CYLD protein, as well as those antibodies that do not affect the function of human CYLD protein.

The present invention includes not only complete monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) ₂ fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules; or chimeric antibodies.

Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, purified human CYLD gene products, or antigenic fragments thereof, can be administered to animals to induce polyclonal antibody production. Similarly, cells expressing human CYLD protein or antigenic fragments thereof can be used to immunize animals to produce antibodies. Various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant and the like.

Antibodies of the invention may also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology. The antibodies of the present invention include antibodies capable of blocking the function of human CYLD protein and antibodies that do not affect the function of human CYLD protein. Various antibodies of the present invention can be obtained by conventional immunization techniques using fragments or functional regions of human CYLD gene products. These fragments or functional regions can be prepared using recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to unmodified forms of human CYLD gene products can be produced by immunizing animals with gene products produced in prokaryotic cells (e.g., E. coli); antibodies that bind to post-translationally modified forms (such as glycosylated or phosphorylated Proteins or polypeptides), which can be obtained by immunizing animals with gene products produced in eukaryotic cells (such as yeast or insect cells).

Antibodies against human CYLD protein can be used in immunohistochemical techniques to detect human CYLD protein in biopsy specimens. A preferred anti-CYLD antibody is an antibody that does not recognize normal CYLD but recognizes mutant CYLD (since the protein encoded by the mutant CYLD is much smaller than the protein encoded by normal CYLD, an antibody that recognizes only the abnormally small protein can be designed) , or an antibody that recognizes normal CYLD but not mutant CYLD. Utilizing the specific difference of the antibody to normal and abnormal CYLD proteins, the susceptibility detection of piloma at the protein level can be conveniently performed.

Using the CYLD protein of the present invention, substances that interact with the CYLD protein, such as inhibitors, agonists or antagonists, can be screened out through various conventional screening methods.

When the CYLD protein of the present invention and its antibody, inhibitor, agonist, antagonist, etc. are administered (administered) therapeutically, various effects can be provided. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated. The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intravenous, subcutaneous, or local administration (including lesion sites). Topical administration is preferred.

Normal CYLD polypeptides can be directly used in the treatment of diseases, for example, in the treatment of piloma. When using the CYLD protein of the present invention, other medicaments for treating piloma can also be used at the same time.

The present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the CYLD protein of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as ointments, tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as ointments, injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 0.1 microgram/kg body weight to about 10 mg/kg body weight per day. In addition, the polypeptides of the invention can also be used with other therapeutic agents.

When using the pharmaceutical composition, a safe and effective amount of CYLD protein or its antagonist, agonist is administered to the mammal, wherein the safe and effective amount is usually at least about 0.1 microgram/kg body weight, and in most cases no more than about 10 mg/kg body weight, preferably the dosage is about 0.1 μg/kg body weight to about 100 μg/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

Polynucleotides of human CYLD protein can also be used for various therapeutic purposes. Gene therapy technology can be used to treat cell proliferation, development or metabolic abnormalities caused by non-expression of CYLD protein or expression of abnormal/inactive CYLD protein. The method for constructing a recombinant viral vector carrying the CYLD gene can be found in existing literature (Sambrook, et al.). In addition, the recombinant human CYLD gene can be packaged into liposomes and then transferred into cells.

The methods for introducing polynucleotides into tissues or cells include: directly injecting polynucleotides into tissues in the body; or first introducing polynucleotides into cells in vitro through vectors (such as viruses, phages, or plasmids, etc.) , and then transplant the cells into the body, etc.

The invention also relates to a diagnostic test method for quantitative and localized detection of human CYLD protein level. These assays are well known in the art and include FISH assays and radioimmunoassays.

A method for detecting whether there is CYLD protein in a sample is to use a specific antibody for CYLD protein to detect, which includes: contacting the sample with a specific antibody for CYLD protein; observing whether an antibody complex is formed, which means that the antibody complex is formed CYLD protein is present in the sample.

The polynucleotide of CYLD protein can be used for the diagnosis and treatment of diseases related to CYLD protein. In terms of diagnosis, the polynucleotide of CYLD protein can be used to detect the expression of CYLD protein or the abnormal expression of CYLD protein in a disease state. For example, the CYLD DNA sequence can be used for hybridization of biopsy specimens to determine the abnormal expression of CYLD protein. Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These technical methods are all open and mature technologies, and relevant kits are available from commercial sources. Part or all of the polynucleotides of the present invention can be immobilized as probes on microarrays or DNA chips (also known as "gene chips") for analysis of differential expression of genes in tissues and gene diagnosis. RNA-polymerase chain reaction (RT-PCR) in vitro amplification with CYLD protein-specific primers can also detect the transcripts of CYLD protein.

The present invention also provides a method for detecting abnormal expression of human CYLD gene, which comprises the steps of: (a) determining the 1853rd nucleotide in the sequence shown in SEQ ID NO: 1 of human CYLD gene; and (b ) to detect whether there is a base A deletion at the position.

Testing for mutations in the CYLD gene can also be used to diagnose piloma. Detection can be against cDNA or genomic DNA. The form of CYLD protein mutation includes point mutation, translocation, deletion, recombination and any other abnormality compared with the normal wild-type CYLD DNA sequence. Mutations can be detected using established techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene has a mutation.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions suggested conditions.

Example 1

Determination of an autosomal dominant pilochepithelial tumor family

1.1 Object

A large pedigree with piloma, a total of 4 generations, 53 people. Among them, there were 20 patients with piloma (Figure 1).

Example 2

CYLD mutation identified as direct cause of piloma

2.1 Genetic linkage analysis

Linkage analysis of this family was performed using microsatellite markers. A total of 384 pairs of microsatellite primers (RESEARCH GENETICS) were used to scan the entire genome, and the piloma gene of this family was located on human chromosome 16. The primers were further synthesized to locate the pathogenic gene within the genetic distance of 14 (cM) centimorgans between the microsatellite markers d16s753 and d16s3253, as shown in FIG. 1 . This physical distance is about 14 million base pairs and more than 100 genes.

2.2 Candidate genes

Genomic DNA was extracted from family blood samples with a DNA extraction kit (QIAGEN) as a template.

For the candidate genes, PCR primers are designed, the candidate genes are amplified by PCR, and then sequenced. After sequencing the candidate genes, it was found that CYLD was closely related to the piloma in this family.

34 pairs of primers were designed for CYLD for sequencing. Among them, when the PCR product amplified by the following pair of primers was sequenced, it was found that the CYLD of each patient in the piloma family was compared with the normal person in the family and found that the gene had a base at position 1853 of exon 10 The deletion, that is, the deletion of base A at position 1853 of SEQ ID NO: 1, resulting in a frameshift mutation, shortening the encoded protein to 496 amino acids. This mutation ultimately leads to the development of piloma (see Figure 4).

name Sequence (5'→3') serial number CYLD_15 positive tctgcagtga tagcttttct gaca SEQ ID NO: 3 CYLD_15 Reverse cagtctcacc aagatgccca atac SEQ ID NO: 4

In addition, in order to verify the specificity of this site for the occurrence of piloma, 100 people with no parental relationship at this site were randomly checked, and no mutation was found. This shows that the mutation of this site is not caused by polymorphism in the population, but a specific mutation site of piloma. Therefore, it is shown that CYLD is closely related to the occurrence of human hair epithelioma, and this gene and its encoded product may play an important role in human hair epithelioma.

Example 3

Preparation of Trichoepithelialoma Detection Kit

A kit is prepared which contains:

Name Sequence (5’→3’) Number Concentration

Forward primer tctgcagtga tagcttttct gaca SEQ ID NO: 3 dry powder 20D

Reverse primer cagtctcacc aagatgccca atac SEQ ID NO: 4 dry powder 20D

PCR reaction solution contains Taq enzyme dNTP magnesium ion PCR reaction buffer

PCR product purification kit Contains a small amount of PCR product purification solution, DNA adsorption column

Sequencing reaction solution contains Big Dye

Take 3ml of blood from the patient to be tested, and use a conventional method (or use a specific kit) to extract DNA from the blood. Dilute the PCR primers in the piloma detection kit to 1 μmol/μl, and use the extracted DNA as a template to carry out PCR reaction with the provided primers. Use the PCR product purification kit provided by the detection kit to purify the PCR product. The purified product is directly sequenced after the sequencing reaction. Observe whether there is a frameshift mutation in the sequence obtained by sequencing.

Example 4

Preparation of pharmaceutical composition

The normal protein encoded by CYLD is mixed with common ointment materials in a specific ratio to make an ointment containing the normal protein encoded by CYLD. When in use, the ointment is applied to the affected area, and the normal protein encoded by CYLD in the affected area is supplemented, so that the skin lesions caused by the lack of normal protein encoded by CYLD can be alleviated until the final disappearance. Alternatively, the normal protein encoded by CYLD can be made into an injection, and injected subcutaneously into the affected area to directly supplement the protein encoded by CYLD at the lesion, so that the physiological condition of the patient's skin can be improved, thereby achieving the purpose of treatment.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

<110>Shanghai Bioengineering Research Center, Chinese Academy of Sciences

<120> Method for Diagnosing and Treating Trichoepithelial Tumor Using Human CYLD Gene and Its Coding Product

<130>020613

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<170>PatentIn version 3.0

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Gly Lys Asp Phe Lys Leu Phe Lys Lys Ile Phe Pro Ser Leu Glu Leu

760 765 770 775

aat ata aca gat tta ctt gaa gac act ccc aga cag tgc cgg ata tgt 2764

Asn Ile Thr Asp Leu Leu Glu Asp Thr Pro Arg Gln Cys Arg Ile Cys

780 785 790

gga ggg ctt gca atg tat gag tgt aga gaa tgc tac gac gat ccg gac 2812

Gly Gly Leu Ala Met Tyr Glu Cys Arg Glu Cys Tyr Asp Asp Pro Asp

795 800 805

atc tca gct gga aaa atc aag cag ttt tgt aaa acc tgc aac act caa 2860

Ile Ser Ala Gly Lys Ile Lys Gln Phe Cys Lys Thr Cys Asn Thr Gln

810 815 820

gtc cac ctt cat ccg aag agg ctg aat cat aaa tat aac cca gtg tca 2908

Val His Leu His Pro Lys Arg Leu Asn His Lys Tyr Asn Pro Val Ser

825 830 835

ctt ccc aaa gac tta ccc gac tgg gac tgg aga cac ggc tgc atc cct 2956

Leu Pro Lys Asp Leu Pro Asp Trp Asp Trp Arg His Gly Cys Ile Pro

840 845 850 855

tgc cag aat atg gag tta ttt gct gtt ctc tgc ata gaa aca agc cac 3004

Cys Gln Asn Met Glu Leu Phe Ala Val Leu Cys Ile Glu Thr Ser His

860 865 870

tat gtt gct ttt gtg aag tat ggg aag gac gat tct gcc tgg ctc ttc 3052

Tyr Val Ala Phe Val Lys Tyr Gly Lys Asp Asp Ser Ala Trp Leu Phe

875 880 885

ttt gac agc atg gcc gat cgg gat ggt ggt cag aat ggc ttc aac att 3100

Phe Asp Ser Met Ala Asp Arg Asp Gly Gly Gln Asn Gly Phe Asn Ile

890 895 900

cct caa gtc acc cca tgc cca gaa gta gga gag tac ttg aag atg tct 3148

Pro Gln Val Thr Pro Cys Pro Glu Val Gly Glu Tyr Leu Lys Met Ser

905 910 915

ctg gaa gac ctg cat tcc ttg gac tcc agg aga atc caa ggc tgt gca 3196

Leu Glu Asp Leu His Ser Leu Asp Ser Arg Arg Ile Gln Gly Cys Ala

920 925 930 935

cga aga ctg ctt tgt gat gca tat atg tgc atg tac cag agt cca aca 3244

Arg Arg Leu Leu Cys Asp Ala Tyr Met Cys Met Tyr Gln Ser Pro Thr

940 945 950

atg agt ttg tac aaa taa ctggggtcat cgggaaaggc aaagaaactg 3292

Met Ser Leu Tyr Lys

955

aaggcagagt cctaacgttg catcttattc gagctggcag ttctgttcac gtccattgcc 3352

ggcaatggat gtctttgtgg tgatgatcct tcagaaaagg atgcctctgt ttaaaaacaa 3412

attgcttttg tgtccctgaa gtatttaata agaagcattt tgcactctag aaagtatgtt 3472

tgtgttggtt ttttaagaag tctaaatgaa gttattaata cctgaagctt taagttaagt 3532

gcattgatca tatgatattt ttggaagcat acaattttaa ttgtggaagt ttaaagcctc 3592

ttttagtcca ttgagaatgt aaataaatgt gtcttcttta tggaccaagg atatgaaatc 3652

atttttcttt tgtagctaac ggttgccttg aggaagaaat aatttggttt tattaagagt 3712

ctactctcaa tccagttatt agagatgtac tgagtttgat ttgttaatcc tttctatata 3772

ctgctgatct tgcatgtcta caatctgctc agtttttctg tgtttctgca atagtggtca 3832

gaaaaatact taaattccct taatggtgtt gttttctatt tgttctggtt ttgagataaa 3892

tgagtgattc tgtccccaaa tgtccatttt tgaagtgatt ttcctggagg attagggtat 3952

ttagcagttg aagctcttca ttcatagtag ttactgtcag ctaacaggtt ttttaaggct 4012

tttaactatt aatattttat ggaatggggc aaagtaaatt gatgaaagaa ttggagtgat 4072

aatagtcctt tacaaacata cagtccataa gaaaatgaat ttggcatata gaattattac 4132

aatttcctgg gagagatgga tattaaacc tctattattt tagacaagac tgtctagaac 4192

ttaagtttga tctgtcagcc agtactccca ttaaattcag tgtagtttca cttgatagaa 4252

tcagatatgt tatcgaaatg ttagcagcag cttcatcctc cttctgatta aagtaagtag 4312

aaatgggatg ttttgtttaa taacagccat agtgtgtgtt tagaccacag cggatgttgt 4372

agaccaggac catagatgat acatgtcagt gctgtggaat gtgcattctc tgagtgttgt 4432

tttgtggtat cattgtcttt cctgaatgac tttctaactg tgcagaaagg cagaaaagtc 4492

atcatatgta tatgtcatat gactttataa aatattaat gtgacaaaaa gtggaaagaa 4552

tctttacaaa ccctgcaatt acttttttaa aggcactttt actctttggt tttatcattc 4612

cattttgcta atatttacta gctttataaa ttacagtaag gtacaaaaac tcatcttgta 4672

atattttcat ttttgaagtg aaaaagtaca tatattttgc acaaggtttt atactgctaa 4732

gtgcttggtt gggtggtga gatgatgatt agatcagggg tgaggctgag agactctggg 4792

tttagggcta gccctgcctc catctccctt gggtaaaatg aagggtgtgg ggtaaaagat 4852

gcataaggcc ttttctagct ctgacagcct agaagtccaa tcaccctgta ataaatatgt 4912

gttgaatgaa gaaatgggtg aatgagcttg tcaatgtgat tttaaaaaat tgactacctg 4972

gaggaatgat taggaatcta aatgaagcca gccctcggta tctgcaggtt tctcatccat 5032

ggattcaacc aactgcaaat ggaaaatacg atttttttta aaaaaaggat ggttacatcc 5092

gtattgaaca tgtacagact tttttcttgt cattattctc tgaacaatac aagaactctt 5152

tatgtagcat ttacatttat taggtattat aagtaatcta gagattattt aattaaaata 5212

tacaggagga tgtgtgttta tatgccagaa attctgtacc attttgtatc agggaattga 5272

gcatcttcag atgttggtat ctgcagggat cctggaacca aacccctgca gatactaagg 5332

gctgacgatc taggtaagac tggatttaac agttggaaa 5371

<210>2

<211>956

<212>PRT

<213> Homo sapiens

<400>2

Met Ser Ser Gly Leu Trp Ser Gln Glu Lys Val Thr Ser Pro Tyr Trp

1 5 10 15

Glu Glu Arg Ile Phe Tyr Leu Leu Leu Gln Glu Cys Ser Val Thr Asp

20 25 30

Lys Gln Thr Gln Lys Leu Leu Lys Val Pro Lys Gly Ser Ile Gly Gln

35 40 45

Tyr Ile Gln Asp Arg Ser Val Gly His Ser Arg Ile Pro Ser Ala Lys

50 55 60

Gly Lys Lys Asn Gln Ile Gly Leu Lys Ile Leu Glu Gln Pro His Ala

65 70 75 80

Val Leu Phe Val Asp Glu Lys Asp Val Val Glu Ile Asn Glu Lys Phe

85 90 95

Thr Glu Leu Leu Leu Ala Ile Thr Asn Cys Glu Glu Arg Phe Ser Leu

100 105 110

Phe Lys Asn Arg Asn Arg Leu Ser Lys Gly Leu Gln Ile Asp Val Gly

115 120 125

Cys Pro Val Lys Val Gln Leu Arg Ser Gly Glu Glu Lys Phe Pro Gly

130 135 140

Val Val Arg Phe Arg Gly Pro Leu Leu Ala Glu Arg Thr Val Ser Gly

145 150 155 160

Ile Phe Phe Gly Val Glu Leu Leu Glu Glu Gly Arg Gly Gln Gly Phe

165 170 175

Thr Asp Gly Val Tyr Gln Gly Lys Gln Leu Phe Gln Cys Asp Glu Asp

180 185 190

Cys Gly Val Phe Val Ala Leu Asp Lys Leu Glu Leu Ile Glu Asp Asp

195 200 205

Asp Thr Ala Leu Glu Ser Asp Tyr Ala Gly Pro Gly Asp Thr Met Gln

210 215 220

Val Glu Leu Pro Pro Leu Glu Ile Asn Ser Arg Val Ser Leu Lys Val

225 230 235 240

Gly Glu Thr Ile Glu Ser Gly Thr Val Ile Phe Cys Asp Val Leu Pro

245 250 255

Gly Lys Glu Ser Leu Gly Tyr Phe Val Gly Val Asp Met Asp Asn Pro

260 265 270

Ile Gly Asn Trp Asp Gly Arg Phe Asp Gly Val Gln Leu Cys Ser Phe

275 280 285

Ala Cys Val Glu Ser Thr Ile Leu Leu His Ile Asn Asp Ile Ile Pro

290 295 300

Ala Leu Ser Glu Ser Val Thr Gln Glu Arg Arg Pro Pro Lys Leu Ala

305 310 315 320

Phe Met Ser Arg Gly Val Gly Asp Lys Gly Ser Ser Ser His Asn Lys

325 330 335

Pro Lys Ala Thr Gly Ser Thr Ser Asp Pro Gly Asn Arg Asn Arg Ser

340 345 350

Glu Leu Phe Tyr Thr Leu Asn Gly Ser Ser Val Asp Ser Gln Pro Gln

355 360 365

Ser Lys Ser Lys Asn Thr Trp Tyr Ile Asp Glu Val Ala Glu Asp Pro

370 375 380

Ala Lys Ser Leu Thr Glu Ile Ser Thr Asp Phe Asp Arg Ser Ser Pro

385 390 395 400

Pro Leu Gln Pro Pro Pro Val Asn Ser Leu Thr Thr Glu Asn Arg Phe

405 410 415

His Ser Leu Pro Phe Ser Leu Thr Lys Met Pro Asn Thr Asn Gly Ser

420 425 430

Ile Gly His Ser Pro Leu Ser Leu Ser Ala Gln Ser Val Met Glu Glu

435 440 445

Leu Asn Thr Ala Pro Val Gln Glu Ser Pro Pro Leu Ala Met Pro Pro

450 455 460

Gly Asn Ser His Gly Leu Glu Val Gly Ser Leu Ala Glu Val Lys Glu

465 470 475 480

Asn Pro Pro Phe Tyr Gly Val Ile Arg Trp Ile Gly Gln Pro Pro Gly

485 490 495

Leu Asn Glu Val Leu Ala Gly Leu Glu Leu Glu Asp Glu Cys Ala Gly

500 505 510

Cys Thr Asp Gly Thr Phe Arg Gly Thr Arg Tyr Phe Thr Cys Ala Leu

515 520 525

Lys Lys Ala Leu Phe Val Lys Leu Lys Ser Cys Arg Pro Asp Ser Arg

530 535 540

Phe Ala Ser Leu Gln Pro Val Ser Asn Gln Ile Glu Arg Cys Asn Ser

545 550 555 560

Leu Ala Phe Gly Gly Tyr Leu Ser Glu Val Val Glu Glu Asn Thr Pro

565 570 575

Pro Lys Met Glu Lys Glu Gly Leu Glu Ile Met Ile Gly Lys Lys Lys

580 585 590

Gly Ile Gln Gly His Tyr Asn Ser Cys Tyr Leu Asp Ser Thr Leu Phe

595 600 605

Cys Leu Phe Ala Phe Ser Ser Val Leu Asp Thr Val Leu Leu Arg Pro

610 615 620

Lys Glu Lys Asn Asp Val Glu Tyr Tyr Ser Glu Thr Gln Glu Leu Leu

625 630 635 640

Arg Thr Glu Ile Val Asn Pro Leu Arg Ile Tyr Gly Tyr Val Cys Ala

645 650 655

Thr Lys Ile Met Lys Leu Arg Lys Ile Leu Glu Lys Val Glu Ala Ala

660 665 670

Ser Gly Phe Thr Ser Glu Glu Lys Asp Pro Glu Glu Phe Leu Asn Ile

675 680 685

Leu Phe His His Ile Leu Arg Val Glu Pro Leu Leu Lys Ile Arg Ser

690 695 700

Ala Gly Gln Lys Val Gln Asp Cys Tyr Phe Tyr Gln Ile Phe Met Glu

705 710 715 720

Lys Asn Glu Lys Val Gly Val Pro Thr Ile Gln Gln Leu Leu Glu Trp

725 730 735

Ser Phe Ile Asn Ser Asn Leu Lys Phe Ala Glu Ala Pro Ser Cys Leu

740 745 750

Ile Ile Gln Met Pro Arg Phe Gly Lys Asp Phe Lys Leu Phe Lys Lys

755 760 765

Ile Phe Pro Ser Leu Glu Leu Asn Ile Thr Asp Leu Leu Glu Asp Thr

770 775 780

Pro Arg Gln Cys Arg Ile Cys Gly Gly Leu Ala Met Tyr Glu Cys Arg

785 790 795 800

Glu Cys Tyr Asp Asp Pro Asp Ile Ser Ala Gly Lys Ile Lys Gln Phe

805 810 815

Cys Lys Thr Cys Asn Thr Gln Val His Leu His Pro Lys Arg Leu Asn

820 825 830

His Lys Tyr Asn Pro Val Ser Leu Pro Lys Asp Leu Pro Asp Trp Asp

835 840 845

Trp Arg His Gly Cys Ile Pro Cys Gln Asn Met Glu Leu Phe Ala Val

850 855 860

Leu Cys Ile Glu Thr Ser His Tyr Val Ala Phe Val Lys Tyr Gly Lys

865 870 875 880

Asp Asp Ser Ala Trp Leu Phe Phe Asp Ser Met Ala Asp Arg Asp Gly

885 890 895

Gly Gln Asn Gly Phe Asn Ile Pro Gln Val Thr Pro Cys Pro Glu Val

900 905 910

Gly Glu Tyr Leu Lys Met Ser Leu Glu Asp Leu His Ser Leu Asp Ser

915 920 925

Arg Arg Ile Gln Gly Cys Ala Arg Arg Leu Leu Cys Asp Ala Tyr Met

930 935 940

Cys Met Tyr Gln Ser Pro Thr Met Ser Leu Tyr Lys

945 950 955

<210>3

<211>24

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>3

tctgcagtga tagcttttct gaca 24

<210>4

<211>24

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>4

cagtctcacc aagatgccca atac 24

<210>5

<211>496

<212>PRT

<213> Homo sapiens

<400>5

Met Ser Ser Gly Leu Trp Ser Gln Glu Lys Val Thr Ser Pro Tyr Trp

1 5 10 15

Glu Glu Arg Ile Phe Tyr Leu Leu Leu Gln Glu Cys Ser Val Thr Asp

20 25 30

Lys Gln Thr Gln Lys Leu Leu Lys Val Pro Lys Gly Ser Ile Gly Gln

35 40 45

Tyr Ile Gln Asp Arg Ser Val Gly His Ser Arg Ile Pro Ser Ala Lys

50 55 60

Gly Lys Lys Asn Gln Ile Gly Leu Lys Ile Leu Glu Gln Pro His Ala

65 70 75 80

Val Leu Phe Val Asp Glu Lys Asp Val Val Glu Ile Asn Glu Lys Phe

85 90 95

Thr Glu Leu Leu Leu Ala Ile Thr Asn Cys Glu Glu Arg Phe Ser Leu

100 105 110

Phe Lys Asn Arg Asn Arg Leu Ser Lys Gly Leu Gln Ile Asp Val Gly

115 120 125

Cys Pro Val Lys Val Gln Leu Arg Ser Gly Glu Glu Lys Phe Pro Gly

130 135 140

Val Val Arg Phe Arg Gly Pro Leu Leu Ala Glu Arg Thr Val Ser Gly

145 150 155 160

Ile Phe Phe Gly Val Glu Leu Leu Glu Glu Gly Arg Gly Gln Gly Phe

165 170 175

Thr Asp Gly Val Tyr Gln Gly Lys Gln Leu Phe Gln Cys Asp Glu Asp

180 185 190

Cys Gly Val Phe Val Ala Leu Asp Lys Leu Glu Leu Ile Glu Asp Asp

195 200 205

Asp Thr Ala Leu Glu Ser Asp Tyr Ala Gly Pro Gly Asp Thr Met Gln

210 215 220

Val Glu Leu Pro Pro Leu Glu Ile Asn Ser Arg Val Ser Leu Lys Val

225 230 235 240

Gly Glu Thr Ile Glu Ser Gly Thr Val Ile Phe Cys Asp Val Leu Pro

245 250 255

Gly Lys Glu Ser Leu Gly Tyr Phe Val Gly Val Asp Met Asp Asn Pro

260 265 270

Ile Gly Asn Trp Asp Gly Arg Phe Asp Gly Val Gln Leu Cys Ser Phe

275 280 285

Ala Cys Val Glu Ser Thr Ile Leu Leu His Ile Asn Asp Ile Ile Pro

290 295 300

Ala Leu Ser Glu Ser Val Thr Gln Glu Arg Arg Pro Pro Lys Leu Ala

305 310 315 320

Phe Met Ser Arg Gly Val Gly Asp Lys Gly Ser Ser Ser His Asn Lys

325 330 335

Pro Lys Ala Thr Gly Ser Thr Ser Asp Pro Gly Asn Arg Asn Arg Ser

340 345 350

Glu Leu Phe Tyr Thr Leu Asn Gly Ser Ser Val Asp Ser Gln Pro Gln

355 360 365

Ser Lys Ser Lys Asn Thr Trp Tyr Ile Asp Glu Val Ala Glu Asp Pro

370 375 380

Ala Lys Ser Leu Thr Glu Ile Ser Thr Asp Phe Asp Arg Ser Ser Pro

385 390 395 400

Pro Leu Gln Pro Pro Pro Val Asn Ser Leu Thr Thr Glu Asn Arg Phe

405 410 415

His Ser Leu Pro Phe Ser Leu Thr Lys Met Pro Asn Thr Asn Gly Ser

420 425 430

Ile Gly His Ser Pro Leu Ser Leu Ser Ala Gln Ser Val Met Glu Glu

435 440 445

Leu Asn Thr Ala Pro Val Gln Glu Ser Pro Pro Leu Ala Met Pro Pro

450 455 460

Gly Asn Ser His Gly Leu Glu Val Gly Ser Leu Ala Glu Val Lys Glu

465 470 475 480

Asn Pro Pro Phe Tyr Gly Val Ser Val Gly Ser Val Ser His Gln Asp

485 490 495

Claims (4)

1. the CYLD gene purposes in the reagent of preparation diagnosis trichoepithelioma susceptibility, the nucleotide sequence of wherein said CYLD gene is the 1853rd A disappearance among sequence shown in the SEQ ID NO:1 and the SEQ ID NO:1.

2. the CYLD albumen purposes in the reagent of preparation diagnosis trichoepithelioma, the proteic aminoacid sequence of wherein said CYLD is the aminoacid sequence shown in the SEQ ID NO:5.

3. test kit that detects trichoepithelioma, it is characterized in that, it comprises the primer of specific amplification CYLD gene or transcript, the PCR reaction solution, PCR product purification box, and sequencing reaction liquid, wherein said PCR product purification box contains solution and the DNA adsorption column that is useful on the PCR product purification, and described test kit also contains the reagent that is selected from down group:

(a) with mutable site bonded probe;

(b) restriction enzyme in identification mutational site;

Wherein said mutable site or mutational site are the 1853rd A disappearances among the SEQ ID NO:1.

4. test kit as claimed in claim 3 is characterized in that, described primer is that sequence is the primer of SEQ ID NO:3 and 4.

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