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WO2010091581A1 - Détection de mutations du gène ptp1b et leurs utilisations dans le diagnostic des cancers - Google Patents

Détection de mutations du gène ptp1b et leurs utilisations dans le diagnostic des cancers Download PDF

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Publication number
WO2010091581A1
WO2010091581A1 PCT/CN2009/075005 CN2009075005W WO2010091581A1 WO 2010091581 A1 WO2010091581 A1 WO 2010091581A1 CN 2009075005 W CN2009075005 W CN 2009075005W WO 2010091581 A1 WO2010091581 A1 WO 2010091581A1
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Prior art keywords
gene
ptp1b
seq
ptpib
mutation
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Chinese (zh)
Inventor
郑新民
梅文翰
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Shanghai Jiao Tong University
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Shanghai Jiao Tong University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • Protein tyrosine phosphorylation is an important cellular signal regulation mechanism that affects important physiological processes such as cell growth and differentiation, cell cycle regulation, apoptosis and migration.
  • the tyrosine phosphorylation state in cells is a reversible dynamic balance that changes with cell function, and is maintained by protein tyrosine kinase (Protein Tyrosine Kinase, ⁇ ) and protein tyrosine phosphatase (Protein Tyrosine Phosphotase, ⁇ ). . ⁇ , ⁇ and their respective substrates together form an extensive signal transduction network.
  • PTP1B is a member of the ⁇ gene family.
  • Human PTP1B is a single-copy gene, which is encoded by PTPN1 gene and located at 20ql3.1-ql3.2. The gene is about 74 kb in length and contains 10 exons. Its cDNA is 3,318 bp in length and encodes 435 amino acids. It is 49,666 Da.
  • PTP1B is widely expressed in vivo, and its substrate proteins include insulin receptor (IR) epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGFR), insulin growth factor I receptor (IGF-IR), and p210Bcr-Abl.
  • IR insulin receptor
  • EGFR epidermal growth factor receptor
  • PDGFR platelet-derived growth factor
  • IGF-IR insulin growth factor I receptor
  • p210Bcr-Abl p210Bcr-Abl.
  • JAK2 Janus Kinase 2
  • TYK2 TYK2 and other receptor-type PTK (RTK)o
  • PTK receptor-type PTK
  • guanidine can negatively regulate a variety of PTK signaling pathways by affecting the phosphorylation of PTK tyrosine residues, thereby negatively regulating various PTK signaling pathways and affecting important physiological functions of cells.
  • the nucleotide sequence of the PTP1B gene is described in the PTP1B file (3318 bp in total), which contains 10 exons, namely: Exonl: l-237 bp; Exon 2: 238-328 bp; Exon 3: 329-429 bp; Exon 4 : 430-528 bp; Exon 5: 529-666 bp; Exon 6: 667-876 bp; Exon 7: 877-1038 bp; Exon 8: 1039-1262 bp; Exon 9: 1263-1458 bp; Exon 10: 1459-3318 bp, Exonl Exon2 Exon3 Exon4 Exon5 Exon6 Exon7 ⁇ Exon9 ExonlO
  • PTP1B mutant namely: Exonl: l-237 bp; Exon 2: 238-328 bp; Exon 3: 329-429 bp; Exon 4 : 430-528 bp; Ex
  • One of the objects of the present invention is to provide a PTP1B mutant gene related to cancer, which is characterized in that a mutation site exists in the PTP1B gene, and the mutation positions are 238-328 bp of the PTP1B gene, 309-328 bp of the PTPIB gene, and 529 of the PTPIB gene.
  • the sequences deleted at the above mutation positions are respectively SEQ ID: NoK SEQ ID : No. 2, SEQ ID: No3, SEQ ID: No4, SEQ ID: No5, SEQ ID: No. 6, SEQ ID: No. 7, SEQ ID: No. 8.
  • Another object of the present invention is to provide a method for detecting cancer, which is to detect whether a PTP1B gene in a sample from an individual to be tested has a mutation site, and the mutation positions are 238-328 bp of PTP1B gene and 309 bp of PTPIB gene, respectively.
  • the detection method includes the following steps:
  • the obtained PCR reaction product is subjected to direct sequencing analysis, and the obtained sequence is compared with the standard sequence of the PTP1B normal gene to determine whether or not the above-mentioned mutation site exists. 4) Based on the above results, it is judged whether the test subject has a cancer-associated PTP1B gene mutation.
  • the PCR primer used in the above detection method can be designed according to the known PTP1B gene sequence, usually 15-30 bases, and the GC content is about 45.50%, and specifically binds to the template at an appropriate temperature, which can be utilized. Specialized computer programming.
  • a set of PCR primers is designed using primer design software, the sequence of which is:
  • a further object of the present invention is to provide a kit for detecting a mutation in the PTP1B gene, which kit may include one or a combination of the following reagents:
  • a kit for detecting a mutation in the PTP1B gene comprises one or more containers containing one or more components for detecting a mutation in the PTP1B gene, which are simultaneously provided It is information about the manufacture, use and sale of pharmaceuticals or biological products that has been reviewed by government drug regulatory agencies.
  • a kit for directly detecting a PTP1B gene mutation site in a sample after PCR amplification may contain one or more of amplification primers, dNTPs, DNA polymerases for PCR reactions, and buffers thereof.
  • the primers described above may employ a pair of primers, and the DNA polymerase for PCR reaction can be used.
  • PCR amplified enzyme for the method of use, please refer to the embodiment in the specific embodiment.
  • the use of the PTP1B mutant gene for diagnosing a cancer disease is provided, and the cause and type of cancer of the individual are determined by detecting whether a PTP1B gene-specific mutation site is present in the test subject.
  • the present invention provides for the first time a novel mutation site of the PTP1B gene existing in the Chinese population, and proves that this type of mutant gene is specifically associated with the occurrence of cancer, which is of great significance for the diagnosis of cancer patients.
  • the present invention also proposes a method for detecting the cause and type of cancer occurrence by detecting the presence or absence of a mutation in the PTP1B gene in a patient. This will facilitate the clinical screening of PTP1B gene mutations in cancer patients and provide services for the diagnosis and treatment of cancer patients.
  • Figure 1A and Figure 1B are RT-PCR results of different types of tumor specimens
  • Figure 2 is a diagram showing the results of immunoblotting for detecting wild-type and mutant PTP1B fusion proteins
  • Figure 3 is a graph showing RT-PCR results of partial mutation sites of PTP1B gene. detailed description
  • the PTP1B gene was selected as a research object, and a new mutation site of a cancer-associated PTP1B gene was found by screening a sample of 105 cancer patients. These mutation sites are at home and abroad. None of them were reported. The specific results of the mutations are summarized in the table below.
  • RNAlug was taken, random primers lul and dNTP lul were added, and 10 ⁇ l was made up with DEPC-H 2 0, placed in a water bath at 65 ° C for 5 minutes, and immediately placed in an ice bath.
  • Add the cDNA synthesis mixture lOul leave it at 25 ° C for 10 minutes, then place it at 50 ° C for 50 minutes, then place it at 85 ° C for 5 minutes, then place it in an ice bath, add lul RNase H and place it in a 37 ° C water bath for 20 minutes.
  • the cDNA was stored at -20 °C.
  • the PTP1B gene was amplified by polymerase chain reaction (PC) of the sample DNA.
  • the amplified fragment was the 175-1482 reading frame of the PTP1B gene, which was 1308 bp long and encompassed 9 complete exons.
  • An upstream primer sequence designed to amplify the PTP1B gene 5,-ATGGAGATGGAAAAGGAGTTCGAGCAGATC-3,, downstream primer sequence: 5'-CTATGTGTTGCT GTTGAACAGGAACCTGTAG-3.
  • the amplification system is: 10x buffer 5ul 4NTP2ul, 10umol L upstream primer and downstream primer 0.5ul gCl 2 2ul, sample cDNA 3ul, supplemented with double distilled water volume 48ul 0 95.
  • the above PCR product was first separated by 1% agarose gel electrophoresis to determine whether the size of the amplified fragment was consistent with the expected 1212 bp, and then the correct PCR product was ligated to the 3.9 kb pCR2.1-TOPO vector and transformed into In E. coli cells (TOPO TA Cloning Kits, Invitrogen products, the specific steps are: take 4 ul of PCR product, add salt solution and TOPO carrier M, mix gently, let stand for 5 minutes at room temperature, then wait at 30 ° C for 10 minutes. Then connect 2ul to the One Shot R E.coli E.
  • FIG. 1A The results of RT-PCR for different types of tumor specimens are shown in Figure 1A and Figure 1B.
  • Figure 1A from left to right: l.lkb DNA Ladder; 2. normal thyroid tissue; 3. thyroid cancer 62; 4. thyroid cancer 186; 5. thyroid cancer 234; 6. thyroid cancer 330; 7. normal stomach Organization 227; 8. Gastric cancer 4.
  • Fig. 1B from the left, in order: l.lkb DNA Ladder; 2. normal colon tissue; 3. intestinal cancer 8, 4. intestinal cancer 36, 5. intestinal cancer 10, 6. intestinal cancer 13, 7. Cancer 93, 8. Intestinal cancer 69; 9. Intestinal cancer 45.
  • 3ug pTetsplice-PTP 1 B WT-HA and 3ug pTetsplice- ⁇ E6-HA were co-transfected with REF ( rat embryo fibroblast ) cells with 3ugpTet-tTAK and 0.3ugpSV2neo respectively. After 48 hours of transfection, G418 0.5mg/ml was screened for two. Week, pick up stable transfected cell clones. After stable cells were induced by doxycycline ( Dox ) for 16 hours, they were collected and made into whole cell suspensions. 30 ug of protein was taken from each whole cell suspension for PAGE electrophoresis, and then transferred to NC membrane.
  • Dox doxycycline
  • pTetsplice-PTPlBA E6-HA transfected REF cells do not add doxycycline 16 small days; 4.
  • the inventors designed a series of downstream primers to pair with the upstream and downstream primers for amplifying the PTP1B gene for detection of clinical pathological tissue samples, which were specific for the mutant gene. PCR amplification can obtain the corresponding mutant gene amplification product, thereby confirming that the sample has a mutation at the corresponding site.
  • This mutant gene-specific PCR method cannot amplify PTP1B in para-tumor tissues, ie, normal tissues, as shown in the table below.
  • PTP1B gene analysis was performed on various types of tumor tissues, and several PTP1B mutant genes found in the experiment have not been reported at home and abroad so far.
  • the invention provides a specific detection method and clinical application of PTP1B mutation gene in Chinese population, and the above detection method can judge and track important molecular indexes in the early occurrence, development and metastasis of malignant tumors, from molecular level to malignant tumors. Early diagnosis and observation of effective predictions after anti-tumor drug therapy are instructive.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
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  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Hospice & Palliative Care (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne de nouveaux sites mutants présents dans le gène PTP1B de la population chinoise, qui sont spécifiquement associés à l'apparition de cancers et utilisés de manière significative chez des individus souffrant de cancers. L'invention concerne également des méthodes de diagnostic des causes et des types de cancers par détection de la présence de sites mutants dans le gène PTP1B dans des échantillons d'individus à détecter. Ces méthodes penchent en faveur d'un criblage clinique des sites mutants présents dans le gène PTP1B d'individus, et offrent un service permettant de diagnostiquer et de traiter des individus souffrant de cancers.
PCT/CN2009/075005 2009-02-13 2009-11-18 Détection de mutations du gène ptp1b et leurs utilisations dans le diagnostic des cancers Ceased WO2010091581A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN200910046185.9 2009-02-13
CNA2009100461859A CN101487013A (zh) 2009-02-13 2009-02-13 Ptp1b基因突变的检测以及其在癌症诊断中的应用

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Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101487013A (zh) * 2009-02-13 2009-07-22 上海交通大学医学院 Ptp1b基因突变的检测以及其在癌症诊断中的应用
CN102108365A (zh) * 2009-12-29 2011-06-29 上海新号源生物科技有限公司 一组恶性肿瘤中突变PTPα基因及其应用
CN107699620B (zh) * 2017-11-20 2018-07-20 知几未来（成都）生物科技有限公司 一种用于诊断预示Luminal B型乳腺癌骨转移的基因诊断试剂盒
CN107904309B (zh) * 2017-11-20 2019-01-04 武汉迈特维尔生物科技有限公司 一种用于诊断预示三阴性型乳腺癌骨转移的基因诊断试剂盒

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1275722A1 (fr) * 2001-07-11 2003-01-15 Novo Nordisk A/S La variante P387L de la Protéine Tyrosine Phophatase 1B est associée avec le diabète de type 2 et démontre une phosphorylation réduite de la sérine de PTP-1B in vitro
CA2357559A1 (fr) * 2001-09-19 2003-03-19 Donald A. Fujita Diagnostic et pronostic d'une tumeur faisant appel a la proteine tyrosine phosphatase 1b (ptp 1b)
CN101487013A (zh) * 2009-02-13 2009-07-22 上海交通大学医学院 Ptp1b基因突变的检测以及其在癌症诊断中的应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1275722A1 (fr) * 2001-07-11 2003-01-15 Novo Nordisk A/S La variante P387L de la Protéine Tyrosine Phophatase 1B est associée avec le diabète de type 2 et démontre une phosphorylation réduite de la sérine de PTP-1B in vitro
CA2357559A1 (fr) * 2001-09-19 2003-03-19 Donald A. Fujita Diagnostic et pronostic d'une tumeur faisant appel a la proteine tyrosine phosphatase 1b (ptp 1b)
CN101487013A (zh) * 2009-02-13 2009-07-22 上海交通大学医学院 Ptp1b基因突变的检测以及其在癌症诊断中的应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SONG YIXUAN ET AL.: "Association of the Pro387Leu mutation of protein tyrosine phoshatase-lB gene with varying glucose tolerances subjects in Han Nationality population in Tianjin", TIANJIN MED J, vol. 36, no. 8, 31 August 2008 (2008-08-31), pages 584 - 587 *

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