WO1999028459A1 - Examination method, examination reagent and remedy for diseases caused by variation in lkb1 gene - Google Patents
Examination method, examination reagent and remedy for diseases caused by variation in lkb1 gene Download PDFInfo
- Publication number
- WO1999028459A1 WO1999028459A1 PCT/JP1998/005357 JP9805357W WO9928459A1 WO 1999028459 A1 WO1999028459 A1 WO 1999028459A1 JP 9805357 W JP9805357 W JP 9805357W WO 9928459 A1 WO9928459 A1 WO 9928459A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- lkb1
- dna
- gene
- mutation
- lkb1 gene
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- 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
- the present invention relates to a method for testing a disease caused by a mutation in the LKB1 gene, and a test and therapeutic drug for the disease.
- MIM 175200 is an autosomal dominant syndrome characterized by lip, peri-oral, and similar dark brown pigmentation, and benign, hamartomatic, and adenomatous multiple gastrointestinal polyps . Patients with this syndrome are also known to frequently develop benign and malignant tumors in the gastrointestinal tract, kidney, ovary, testis, breast, and uterus. In particular, small benign tumors frequently occur in the ovaries, and these small tumors develop as multifocal bilateral sex cord structures having an annular tubule. It can then progress to granulosa carcinoma, leading to precocious maturity in girls.
- Multifocal sex cord cancer in boys has been found to cause gynecomastia and gynecology, albeit less frequently, as a result of excessive estrogen production. It is thought that deficiency of the causative gene of Koss syndrome (Boy-Jega gene) predisposes to a wide range of neoplastic diseases. It is known that 50% of carriers with one will develop cancer by age 60 (Giardiello, FM et al. Increasing risk oi cancer in the Peutz-Jeghers syndrome. N. Engl. J. Med. 316 , 1511-1514 (1987)., Spigelman, AD, Murday, V.
- the present invention elucidates the causative gene of Peutz-Jeghers syndrome, and provides a method for testing a disease caused by mutation of the gene, and a test and therapeutic agent for the disease.
- the present inventors In order to elucidate the causative gene of Peutz-Jeghers syndrome, the present inventors first constructed a continuous cosmid contig for 1.5 Mbp or more of the 19pl3.3 region of the chromosome where the Peutz-Jeger gene exists, and A map was made. Then Using the EST database, we searched for genes mapped in this region and determined the exact location of those genes. Next, through the evaluation of biological information and other factors, we identified some promising candidates for the Peutz-Jeger gene from among the many genes found.
- LKB1 gene, primers and probes based on its base sequence, LKB1 protein, and antibodies that bind to LKB1 protein can be used to test and treat diseases caused by mutations in LKB1 gene. Has been found to be possible.
- the present invention relates to a method for testing a disease caused by a mutation in the ⁇ LKB1 '' gene, which is a causative gene of Peutz-Jeghers syndrome, and a test and therapeutic agent for the disease, more specifically,
- a primer comprising a nucleotide sequence comprising at least a part of the nucleotide sequence of any one of SEQ ID NOs: 1 to 4, and used for testing a disease caused by a mutation in the LKB1 gene;
- a probe DNA comprising a nucleotide sequence containing at least a part of the nucleotide sequence of any of SEQ ID NOs: 1 to 4 and used for testing a disease caused by a mutation in LKB1 gene;
- a therapeutic drug for a disease caused by a mutation in the LKB1 gene comprising an LKB1 protein as an active ingredient
- a therapeutic agent for a disease caused by a mutation in the LKB1 gene comprising a compound that promotes the activity of the LKB1 protein as an active ingredient
- test agent for a disease caused by mutation in the LKB1 gene comprising an antibody that binds to the LKB1 protein as an active ingredient
- a method for examining a disease caused by a mutation in the LKB1 gene comprising detecting a mutation in the LKB1 gene.
- the present invention is based on the findings by the present inventors that Peutz-Jeghers syndrome develops due to a mutation in a gene named “LKB1”.
- the present invention relates to a nucleotide comprising at least a part of the nucleotide sequence of a genomic DNA region (including an intron region, a promoter region, and an enhancer region in addition to an exon region) encoding LKB1, and LKB1.
- the present invention relates to the use of the nucleotide for testing a disease caused by a gene mutation.
- the genomic DNA region of LKB1 is shown in SEQ ID NOs: 1 to 4.
- SEQ ID NO: 1 Is the gene sequence containing the 5 'upstream region of the LKB1 gene
- SEQ ID NO: 2 is exon 1 and intron 1 (part) of the LKB1 gene
- SEQ ID NO: 3 is exons 2 to 8 and Introns 1 (part) to 8 (part) and SEQ ID NO: 4 are intron 8 (part) and exon 9.
- Nucleotides containing a part of these regions can be used as primers or probes for testing diseases caused by mutations in the LKB1 gene.
- the nucleotide used as a primer is usually 15 bp to 100 bp, preferably 17 bp to 30 bp.
- the primer may be any primer as long as it can amplify at least a part of the LKB1 gene or a region that regulates its expression.
- Such regions include, for example, the exon region, intron region, promoter region, and enhancer region of the LKB1 gene.
- a nucleotide as a probe usually has a chain length of at least 15 bp or more if it is a synthetic oligonucleotide.
- the region used as a probe may be any region as long as it is at least a part of the LKB1 gene or a region that regulates its expression. Such regions include, for example, the exon region, intron region, promoter region, and enhancer region of the LKB1 gene.
- the oligonucleotide or double-stranded DNA is appropriately labeled and used.
- Labeling methods include, for example, labeling by phosphorylating the 5 'end of the oligonucleotide with ⁇ using T4 polynucleotide kinase, or random hexamer using DNA polymerase such as Klenow enzyme.
- a method of incorporating a substrate base labeled with an isotope such as 32 P, a fluorescent dye, or biotin using a primer such as a lignonucleotide as a primer random prime method, etc.
- PTEN and APC genes are hereditary cancers n disease (Cowden's disease) and familial adenomatous polyposis (FAP), but both genes are frequently found in non-hereditary general cancers. It is clear that the mutation has occurred.
- the PTEN gene or the APC gene works to control cell growth in normal tissues, but when they mutate and lose their function, the cells lose their control and one of the major steps toward canceration is initiated. It is thought to proceed.
- the LKB1 gene may be involved in the development of general cancer by mutating it.
- test for a disease caused by a mutation in the LKB1 gene is characterized by detecting the mutation in the LKB1 gene.
- the term "test for a disease caused by a mutation in the LKB1 gene” means not only a test for a patient who develops a specific symptom due to the mutation in the LKB1 gene, but also Testing for mutations in the LKB1 gene to determine whether a person is susceptible to a particular disease is also included. That is, a mutation in one of the LKB1 alleles greatly increases the risk of developing a specific disease caused by a mutation in the LKB1 gene even if the symptom has not yet appeared on the surface. it is conceivable that.
- Such a test for identifying a patient (carrier) having a mutation in one of the LKB1 alleles is also included in the present invention.
- the “detection of a mutation in the LKB1 gene” in the present invention includes detection in DNA, detection in RNA, and detection in protein.
- test method of the present invention is a method for directly determining the nucleotide sequence of the LKB1 gene of a patient.
- a part of the LKB1 gene of a patient is determined by PCR (Polymerase Chain Reaction) using DNA isolated from a patient suspected of having a disease caused by LKB1 mutation as a primer using the above nucleotide as a primer.
- the whole is amplified and its base sequence is determined. By comparing this with the nucleotide sequence of the LKB1 gene of a healthy subject, a disease caused by a mutation in the LKB1 gene can be examined.
- the direct determination of the nucleotide sequence of Various methods other than the method used can be used.
- One embodiment is (a) a step of preparing a DNA sample from a patient, (b) a step of amplifying DNA derived from a patient using the primer DNA of the present invention, and (c) a step of converting the amplified DNA to a single-stranded DNA. (D) separating the dissociated single-stranded DNA on a non-denaturing gel, (e) comparing the mobility of the separated single-stranded DNA on the gel with a healthy control, including.
- PCR-SSCP Single-strand conformation polymorphism, single-chain higher-order structural polymorphism
- PCR-SSCP Single-strand conformation polymorphism, single-chain higher-order structural polymorphism
- This method has advantages such as relatively simple operation and small sample size, and is particularly suitable for screening a large number of DNA samples.
- the principle is as follows. When a double-stranded DNA fragment is dissociated into single strands, each strand forms a unique higher-order structure depending on its base sequence. When the dissociated DNA strand is electrophoresed in a polyacrylamide gel containing no denaturant, the single-stranded DNA of the same complementary length moves to a different position according to the difference in its higher-order structure. I do.
- the single-stranded substitution also changes the higher-order structure of the single-stranded DNA, indicating different mobilities in polyacrylamide gel electrophoresis. Therefore, by detecting the change in mobility, it is possible to detect whether or not a mutation due to a point mutation, deletion, insertion or the like exists in the DNA fragment to be examined.
- a part or all of the LKB1 gene is amplified by a PCR method or the like.
- the amplification range is usually preferably about 200 to 400 bp.
- the region to be amplified includes the exon, intron, and LKB1 gene of the LKB1 gene. Includes child promoters and enhancers.
- PCR can be performed under normal conditions (for example, conditions for a reaction for amplifying each exon using the primer shown in Example 5).
- the DNA fragment synthesized by the PCR reaction is labeled by adding a base
- a Klenow enzyme is used to synthesize a substrate base labeled with an isotope such as 32 P, a fluorescent dye, or biotin.
- the DNA fragment thus obtained can be labeled by adding the DNA fragment to the DNA fragment ..
- the conditions for separation of DNA fragments can be improved by the addition of gel, and the electrophoresis conditions vary depending on the properties of each DNA fragment, but are usually performed at room temperature (20 to 25 ° C). If the desired separation cannot be obtained, it is preferable to study the temperature that gives the optimum mobility at a temperature of 4 to 30 ° C. After electrophoresis, the mobility of the DNA fragment is determined by autoradiography using an X-ray film.
- this band is directly excised from the gel, re-amplified by PCR, and directly sequenced.
- the gel after electrophoresis can be stained with ethidium gel or silver stain. By staining the like, it is possible to detect the band of the DNA fragment.
- test method of the present invention include (a) a step of preparing a DNA sample from a patient, (b) a step of amplifying a patient-derived DNA using the primer DNA of the present invention, and (c) amplified DNA. (D) separating the MA fragment according to its size, (e) hybridizing the probe DNA of the present invention to the separated DNA fragment. (F) comparing the size of the detected DNA fragment with a control of a healthy subject. Examples of such a method include a method using restriction fragment length polymorphism (RFLP) and a PCR-RFLP method.
- RFLP restriction fragment length polymorphism
- these mutations can be detected as a difference in the mobility of the band after electrophoresis.
- the presence or absence of the mutation can be detected by treating the patient-derived DNA with these restriction enzymes, electrophoresing, and performing Southern blotting using the probe DNA of the present invention.
- the restriction enzymes to be used are appropriately selected according to the mutations in addition to those described above. In this method, genomic DNA prepared from a patient is treated with a restriction enzyme to detect mutations, and cDNA prepared by treating RNA prepared from a patient with reverse transcriptase is directly treated with a restriction enzyme, followed by Southern enzyme treatment. It can also be detected by blotting.
- PCR is performed using this cDNA as type II, and the amplified product (part or all of the LKB1 gene) is cleaved with restriction enzymes, followed by electrophoresis to detect mutations as differences in the mobility of DNA fragments. You can also.
- detection can be performed in the same manner by using A instead of DNA prepared from patients.
- Such a method comprises: (a) a step of preparing an RNA sample from a patient; (b) a step of separating RNA prepared according to its size; and (c) a step of applying the probe DNA of the present invention to the separated RNA. And (d) comparing the size of the detected RNA with a control of a healthy subject.
- An example of a specific method is that the patient The prepared RNA is subjected to electrophoresis, and Northern blotting is performed using the probe of the present invention to detect a difference in mobility.
- test method of the present invention include (a) a step of preparing a DNA sample from a patient, (b) a step of amplifying patient-derived MA using the primer of the present invention, and (c) amplified DNA. (D) comparing the mobility of the separated DNA on the gel with a control of a healthy subject.
- a method include denaturant gradient gel electrophoresis (DGGE).
- DGGE denaturant gradient gel electrophoresis
- a part or all of the LKB1 gene is amplified by PCR or the like using the primer of the present invention or the like, and this is gradually increased as the concentration of a denaturant such as urea moves. Perform electrophoresis in the gel and compare with healthy subjects.
- the DNA fragment becomes single-stranded at a lower concentration of the denaturing agent, and the migration speed is significantly reduced.
- the presence or absence of the mutation can be determined by detecting the difference in mobility. Can be detected.
- the Allele Specific Oligonucleotide / ASO hybridization method can be used for the purpose of detecting only a mutation at a specific position.
- an oligonucleotide containing a base sequence that is considered to have a mutation is produced and hybridized with this and a sample MA. If mutations are present, the efficiency of hybridization is reduced.
- These methods are based on Southern blotting, or a method that utilizes the property of quenching by the special fluorescent reagent being quenched in the gap of the hybrid every night. Detect by such as.
- RNA mismatch cleavage method Detection by the ribonuclease A mismatch cleavage method is also possible.
- part or all of the LKB1 gene is amplified by PCR or the like, and this is hybridized with labeled RNA prepared from LKB1 cDNA or the like incorporated in a plasmid vector or the like.
- the hybrid has a single-stranded structure where the mutation exists This part is cleaved with ribonuclease A and autoradiography
- the present invention also relates to an agent for testing a disease caused by a mutation in the LKB1 gene, comprising an antibody that binds to the LKB1 protein as an active ingredient.
- Antibodies that bind to LKB1 protein can be prepared by methods known to those skilled in the art.
- LKB1 protein naturally protein, LKB1 protein expressed in Escherichia coli as a fusion protein with GST, and other suitable host cells (E. coli, yeast, and mammalian cells) can be used.
- the expressed recombinant LKB1 protein can be used, or a partial peptide thereof (eg, a peptide consisting of the amino acid sequence described in SEQ ID NO: 31 to SEQ ID NO: 34) is immunized to a small animal such as a egret to obtain serum.
- a partial peptide thereof eg, a peptide consisting of the amino acid sequence described in SEQ ID NO: 31 to SEQ ID NO: 34
- This can be prepared, for example, by purifying it with ammonium sulfate precipitation, protein A, protein G columns, DEAE ion exchange chromatography, affinity columns with LKB1 protein or synthetic peptides. is there.
- a monoclonal antibody In the case of a monoclonal antibody, a small animal such as a mouse is immunized with the LKB1 protein or a partial peptide thereof, the spleen is excised from the mouse, the cells are crushed to separate cells, and mouse myeloma cells and polyethylene are used. Fused with a reagent such as glycol, and a clone that produces an antibody that binds to the LKB1 protein is selected from the resulting fused cells (hybridomas).
- a reagent such as glycol
- the obtained hybridoma is transplanted into the peritoneal cavity of a mouse, ascites is collected from the mouse, and the obtained monoclonal antibody is subjected to, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, and LKB1 protein. It can be prepared by purifying a synthetic peptide with an affinity column to which a capping is applied.
- a tissue collected from a patient or The isolated cells are stained by an enzyme-labeled antibody method, a fluorescent-labeled antibody method, or the like, and examined for LKB1 protein deficiency, abnormal accumulation, or abnormal intracellular distribution.
- a cell extract is prepared from tissues or isolated cells collected from patients such as Koz-Jieger, separated by SDS-PAGE or other method, and then transferred to a nitrocellulose membrane or PVDF membrane, etc.
- detection can also be carried out by a method of staining proteins by the above-mentioned enzyme-antibody method (Western blotting, immnoblotting).
- the distance on the chromosomal DNA between the gene LKB1 causing the Peutz-Jiega syndrome and the microsatellite marker D19S886 was about 190 kbp. It was revealed that both exist in close proximity. Therefore, as a test for various diseases based on the LKB1 gene mutation, a method of testing for loss of heterozygosity (LOH) using the D19S886 marker may be effective.
- LH heterozygosity
- the present invention also relates to a therapeutic agent for a disease caused by a mutation in the LKB1 gene.
- the LKB1 gene is used as an active ingredient.
- a part or the entire length of LKB1 genomic DNA or LKB1 cDNA (SEQ ID NO: 5) can be converted into an appropriate vector, for example, an adenovirus vector, an adeno-associated virus vector, or a retrovirus vector.
- the drug is incorporated into plasmid DNA or the like, and administered to patients by, for example, oral administration, intravenous administration, local administration to the affected area, or the like.
- an ex vivo method can be used in addition to an in vivo method.
- tissue translocation and tissue absorption can also be enhanced by encapsulating the gene in ribosomes prepared by converting phospholipids into micelles.
- tissue transferability and tissue absorbability it is possible to enhance tissue transferability and tissue absorbability. This makes it possible to replace the mutated LKB1 gene in the patient with a normal gene, or to additionally administer a normal gene to the patient, thereby treating a disease caused by the LKB1 gene mutation. Can do You.
- the LKB1 protein is used as an active ingredient.
- LKB1 protein can be prepared as a natural protein or as a recombinant protein using gene recombination technology.
- the amino acid sequence of LKB1 protein is shown in SEQ ID NO: 6.
- the native protein can be expressed by a method known to those skilled in the art, for example, by performing affinity chromatography using an antibody against a partial peptide of the LKB1 protein described in Example 7 using an antibody against a partial peptide of the LKB1 protein. It can be isolated from testis, fetal liver, or cultured cells such as K562 cells.
- a recombinant protein can be prepared, for example, by culturing cells transformed with DNA encoding the LKB1 protein (SEQ ID NO: 5).
- cells used for producing a recombinant protein include mammalian cells such as COS cells, CH0 cells, and NIH3T3 cells, insect cells such as Sf9 cells, yeast cells, and E. coli.
- the vector for expressing the recombinant protein in the cell varies depending on the host cell.For example, as a vector for a mammalian cell, pcDNA3 (Invitrogen) or pEF-BOS (Nucleic Acids.
- Escherichia coli vectors include pGEX-5X-1 (Pharmacia) and QIAexpress systemj (Qiagen).
- Vectors can be introduced into host cells by the calcium phosphate method, DEAE dextran method, Nick liposome D0TAP (Boehringer Mannheim), a method using SuperFect (Quiagen), an electroporation method, a calcium chloride method, and other known methods can be used. Refining of the recombinant protein from the body, a conventional method, for example, can be performed using the document "The Qiaexpressionist handbook, Q iagen, Hilden, a method of Ge Rmanyj described.
- the LKB1 protein can be directly administered, or can be formulated and administered by a known pharmaceutical manufacturing method.
- a carrier or medium generally used as a drug for example, sterile water, physiological saline, vegetable oil, surfactant, lipid, solubilizing agent, stabilizer, preservative, and the like.
- the dose varies depending on various factors such as the patient's body weight, age, health degree, and administration method, and those skilled in the art can appropriately select an appropriate dose. Usually, it is in the range of O. Olmg / -1 000 mg / kg.
- Administration can be performed by, for example, oral administration, intravenous administration, intramuscular administration, subcutaneous administration and the like.
- a site-directed mutagenesis system using PCR (GIBCO-BRL, Gaithersburg, Maryland), a site-directed mutagenesis method using oligonucleotides (Kramer, W and Fritz, HJ (1987) Methods in Enzymol., 154: 350-367) and the Kunkel method (Methods Enzymol. 85, 763-Z766 (1988)).
- Amino acid substitutions, deletions, additions, and / or insertions in the amino acid sequence of the LKB1 protein can be easily performed to enhance stability and the like.
- Such a modified LKB1 protein can also be used for the drug of the present invention.
- a compound that promotes the activity of the LKB1 protein is used as an active ingredient.
- the LKB1 gene is a serine-threonine kinase with 82% high homology in amino acid sequence to the African megal serine-threonine kinase XEEK1. It is thought that the disruption of serine threonine kinase activity in the LKB1 protein is closely related to the onset of disease caused by mutations in the LKB1 gene. Therefore, by promoting the serine threonine kinase activity, it is conceivable to treat a disease caused by a mutation in the LKB1 gene.
- LKB1 protein expressed in E. coli as a fusion protein with GST or LKB1 protein expressed in mammalian cells, insect cells, etc.
- LKB1 protein expressed in mammalian cells, insect cells, etc. Using protein, the kinase activity of these proteins is measured in the presence of the test compound,
- a compound that promotes the activity of LKB1 protein is selected.
- the activity of phosphorylating the substrate protein of the LKB1 protein or the activity of autophosphorylation (autophosphorylation) by the LKB1 protein is determined using an appropriate reaction solution (for example, 50 mM Tris-HCl).
- the amount of transfer of 32 P from [[P] ATP to the substrate in [pH 7.2, ImM dithiothreitol (DTT), 10 mM MgCL, 10 mM MnCL, etc.) is measured using a liquid scintillation counter or the like.
- a compound that increases the amount a compound that promotes the activity of LKB1 protein can be isolated, and when the isolated compound is used as a therapeutic agent for a disease, the above LKB1 protein is treated.
- it can be formulated and administered by a known pharmaceutical manufacturing method, and the dosage is usually in the range of O. Olmg / to 1000 mg / kg.
- a method using a region that controls the expression of the LKB1 gene or a factor that binds to the region can be considered.
- the structures of the LKB1 gene and its 5 ′ upstream region (SEQ ID NO: 1) have been clarified. It is considered that these regions contain a region that regulates the expression of the LKB1 gene (such as promoter promoter), but for those skilled in the art, the LKB1 gene can be obtained by appropriately combining existing methods. It is easy to identify the gene region that regulates the expression of E. coli.
- a method of specifying a gene regulatory region for example,
- the upstream region of the LKB1 gene is cleaved with various restriction enzymes to obtain fragments of an appropriate size, which are then used as the luciferase gene, secreted alkaline phosphatase gene, or the like.
- Liposome such as chloramphenicol acetyltransferase (CAT) gene —Create an expression vector (such as PicaGeneTM Vector Wako Pure Chemical Industries, Ltd.) that is integrated upstream of the Yuichi gene.
- this is introduced into a suitable host cell such as COS cell, HEK293 cell, CH0 cell, etc., and after culturing for a certain period of time, the amount of the gene product inside and outside the cell is measured to determine the integrated gene.
- the activity of the fragment can be measured overnight. If the gene fragment having the activity is identified in this way, the same operation is repeated with a smaller fragment, whereby the active site can be specified in a narrower region.
- the activity can be measured after the nucleotide sequence of the specified region has been changed by site-directed mutagenesis or the like to confirm that it is an active site.
- the region that regulates the expression of the LKB1 gene can be, for example, linked to the upstream of the normal LKB1 gene and administered to a patient with a mutated LKB1 gene, under the natural regulation of in vivo expression. Since the LKB1 gene can be expressed, it is particularly useful in the above-described gene therapy.
- Such a screening method includes: (a) a step of constructing a vector in which a repo overnight gene is linked downstream of a portion of the promoter of the LKB1 gene; and (b) a step of: (C) contacting and / or introducing a test compound into the cells to detect the activity of the reporter gene.
- Test compounds include, but are not limited to, for example, proteins, peptides, synthetic compounds, natural compounds, genes, gene products, and the like.
- a compound that controls LKB1 gene expression can be screened by bringing a test sample into contact with a portion of the promoter and selecting a compound (such as a protein) that binds to the promoter site.
- a compound such as a protein
- a synthetic oligo DNA having a base sequence at a part of the promoter is prepared, and this is prepared using an appropriate primer such as Sepharose.
- a transcription regulatory factor that binds to this promoter site and controls LKB1 gene expression can be purified by affinity purification or the like.
- the present inventors have revealed that a mutation in the LKB1 gene causes a neoplasm such as a polyp in a Peutz'jega patient. For this reason, it can be said that by reducing the amount and activity of LKB1 protein, it is possible to impart temporary cell growth activity to normal cells. Therefore, by using antisense DNA against the LKB1 gene or cDNA, or a compound that inhibits the activity of the LKB1 protein, etc., the amount and activity of the LKB1 protein is artificially reduced, so that wound healing and tissue Treatment of diseases that require new cell proliferation, such as regeneration, may also be considered.
- Figure 1 a is LKB1 chromosomal gene, and the structure of the vicinity region, restriction enzyme c R is EcoRI showing a map, B is BamHI, S is Sacl, K is Kpnl, BssH indicates it it sites of BssH 11 c
- restriction enzyme c R is EcoRI showing a map
- B is BamHI
- S is Sacl
- K is Kpnl
- BssH indicates it it sites of BssH 11 c
- Figure lb schematically shows the gene rearrangement seen in Peutz-Jeger patient A. The upper part shows the normal gene structure, and the lower part shows the structure of the mutant LKB1 gene of P. jaeger patient A. Arrows indicate the orientation and positional relationship of the primers used for PCR analysis.
- Figure 2 shows a map of the Cosmidokun's contig and the EcoRI site in the vicinity of the microsatellite camera D19S886.
- the positional relationship between the microsatellite marker D19S886 in the human chromosome 19pl3.3 region and the LKB1 gene is schematically shown.
- tel indicates telomere
- cen indicates the direction in which the centromere exists.
- the aligned partially overlapping cosmid clones are shown.
- Fig. 3a shows a family tree of Poijega-Patient A over three generations. The right half is filled The shaded symbol indicates the affected member.
- b shows the results of long distance PCR analysis using the DJ666 primer and the DJ660 primer. Only the affected member has an abnormal amplification product of 2.5 kbp, indicated by a closed triangle.
- c shows the results of PCR analysis using DJ666 and DJ684. An amplification product was observed only in the affected member, indicating that an inversion was present.
- FIG. 4 shows an agarose electrophoresis image of each exon of LKB1 amplified by PCR. The number of the exon being amplified is shown above. MW represents a molecular weight marker.
- FIG. 5 is an electrophoretic image showing the results of analysis by PCR-RFLP. Ahdl, BsrBI, R sal, and Seal represent samples treated with the respective restriction enzymes. WT indicates a healthy person, B indicates a boy Jager patient B, FA indicates a boy Jager patient FA, MA indicates a boy Jager patient MA, D indicates a DNA sample obtained from a Peutz Jager patient D . MW indicates a molecular weight marker.
- FIG. 6 is a diagram showing the autophosphorylation ability of the immunoprecipitated LKB1 protein in the presence of various divalent cations. It can be seen that the autophosphorylation ability (kinase activity) of LKB1 is not activated very much by Mg 2+ but is strongly activated by Mn ".
- FIG. 7 is a diagram illustrating the autophosphorylation ability of the wild-type LKB1 protein and each mutant.
- the upper panel is an autoradiography image showing autophosphorylation ability
- the lower panel is the result of Western plotting stained with anti-c-Myc antibody. It can be seen that almost the same amount of each protein was produced.
- FIG. 8 is an electrophoresis image showing the results of Western blot analysis of the GST-fused LKB1-myc protein expressed in E. coli.
- M indicates a molecular weight marker
- 1 indicates Escherichia coli lysate before induction with IPTG
- 2 indicates Escherichia coli lysate after induction with IPTG
- 3 indicates GST-fused LKB myc protein purified by glutathione sepharose.
- the filter labeled “anti-Myc antibody” shows the results of staining with anti-myc antibody
- the filter labeled “anti-LKB1 antibody” shows the results stained with affinity-purified anti-LKB1 peptide antibody.
- FIG. 9 shows the results of Western blotting using each antibody.
- Lane 1 is a lysate of C0S7 cells transfected with the pcDNA3 vector alone
- lane 2 is a lysate of C0S7 cells transfected with pcDNA3 / LKBlmyc
- lane 3 is a lysate of HeLa S3 cells. . Stained with the antibody shown on the left and anti-C-Myc antibody. The peptide used for the pre-incubation is shown at the top.
- FIG. 10 is a diagram showing a tissue section of a human fetal colon stained with an anti-LKB1 P3 antibody.
- the cytoplasm of epithelial cells is stained. Also, some cells considered to be endocrine cells are stained very strongly.
- FIG. 11 is a diagram in which a tissue section of a human adult renal sulcus is stained with an anti-LKB1 P3 antibody. The islet cell group c is stained.
- FIG. 12 shows a tissue section of a human fetal testis stained with an anti-LKB1 P3 antibody. C Undeveloped germ cells are strongly stained. BEST MODE FOR CARRYING OUT THE INVENTION
- Cosmid clone contig in human chromosome 19pl3.3 region For the 19pl3.3 region of human chromosome, aiming at efficient identification of all genes in the region, a continuous clone consisting of cosmid clones etc. (Aligned clone. Contig) was constructed. First, from the group of cosmid clones mapped to this region by FISH mapping or the like, appropriate cosmids having short but different overlapping portions are selected, and the space between these clones is removed from the cosmid library, phosmi library. Screening BAC libraries and obtaining clones to fill them sequentially (cosmid walking, chromosome Ng).
- a continuous clone contig consisting of L 5 bp in the 19pter region of the chromosome was constructed, and a high-resolution physical map was prepared.
- the construction of the clone contig was performed as follows. Two chromosome 19-specific cosmid libraries cloned into the Lawrist5 vector and Lawristl6 vectors, a chromosome 19-specific fosmid library cloned into the pBeloBacll vector, a whole human genomic cosmid library (Stratagene), and all humans.
- GZMM locus Pierat, D. et al. The human Met-ase gene (GZMM): structure, sequence and close physical linkage to the e ).
- Cosmid walking was started from the ends of two contigs containing serine protease gene cluster on 19pl3.3. Genomics 24, 445-450 (1994). In other words, using the EcoRI fragment at the end of the contig as a probe, high-density cosmid filters and fosmid filters were screened with Cotl DNA (New England Biolabs) under reduced background conditions to obtain sequentially overlapping clones. Was.
- the ends of the cosmid insert were sequenced directly using the Llawrist vector-specific primers DJ180 (SEQ ID NO: 35 / CGACTCACTATAGGGAGACCCA) and DJ181 (SEQ ID NO: 36 / CCTCGAGMTTACCCTCA CTAA).
- the resulting sequence is used to (i) search a database to identify new genes that show significant homology to known genes, and (ii) to perform further chromosome tracking. It was used for preparing STS (Sequence Tagged Site).
- STS Sequence Tagged Site
- mapping was performed in this contig by the same method. Furthermore, by searching for sequences such as the LLNL database [http: ⁇ www-Mo.llnl.gov] using the sequence at the end of the cosmid, a newer gene can be added to this contig. Identified within.
- genes that could be the causative gene of Peutz-Jeghers syndrome were selected in consideration of various biological information such as information on analysis of counterpart genes in other species. In each case, the presence or absence of mutations in the chromosomal DNA of patients was searched using DNA samples prepared from Koz-Jeger patients.
- LKB1 gene was present in the R29114 clone and the R26552 clone adjacent to this clone, and it was confirmed that the LKB1 gene was present in this region.
- primers for PCR and sequencing were designed based on the LKBlcDNA sequence (1302 bp) to determine the entire exon-intron structure of the LKB1 gene (see Tables 1 and 2 below). 2) The analysis was performed. Individual gene segments were amplified by PCR from cosmid DNA and sequenced by directly sequencing the PCR products. By comparing the sequence thus obtained with the LKBlcDNA sequence, the positions of exons and splice sites of all introns were identified.
- the positions of all exons on the restriction map are determined by comparing restriction enzyme sites found in the genome sequence and by comparing the distance between each gene segment by long distance PCR (Long Distanse PCR). ( Figure la).
- DJ666 5 '-GGTGATGGAGTACTGCGTGTG-3' (SEQ ID NO: 26 / exon 3) DJ684 5 '-ACATCGGGAAGGGGAGCTACG-3' (SEQ ID NO: 27 / exon 6) DJ660 5 '-CCGGGCACCGTGAAGTCCTG-3' (SEQ ID NO: 28 / exon 8) DJ650 5 '-TCACTGCTGCTTGCAGGCC-3' (SEQ ID 29 / exon 9) DJ717 5 '-GCAGGCGGCCAGCCTCA-3' (SEQ ID 30 / exon 9)
- Mutation analysis was performed using DNA samples from five unrelated Peit's Jega patients (A, B, D, MA, FA,). First, gene deletion and rearrangement were screened by long-distance PCR using the primer sets DJ659 and DJ660 and DJ666 and DJ660.
- PCR using primers DJ666 and DJ660 resulted in a 3.9 kbp amplification product in MA samples from all patients and control healthy subjects.
- a single product of 2.5 gbp was simultaneously observed in a single patient, a patient of Poetry and Jega (Fig. 3b).
- Fig. 3b After subcloning this DNA fragment, determine the base sequence, Comparison with the corresponding normal chromosomal DNA sequence revealed that there was a complex chromosomal rearrangement in the LKB1 gene in this patient ( Figure lb). That is, the 2.5 kbp PCR product had two mutations, a 1286 bp deletion between introns 3 and 5, and an 81 bp deletion within intron 7, respectively. Larger deletions include exons 4 and 5.
- RNA isolated from peripheral blood cells of Patient A was isolated using the Blood RNA Isolation Kit (Qiagen) and reverse transcribed into cDNA using Superscript (GIBCO-BRL, Life Technologies). PCR using the primer set DJ660 and DJ666 yielded two products, 730 bp and 270 bp. The difference in length of about 460 bp between the two is well consistent with the transcript encoded by exons 4 to 7 being 456 bp, and the transcript in which exons 4 to 7 are spliced out due to this complex mutation. Was thought to have occurred.
- DJ674 5'-AAGGAGACGGGAAGAGGAGCAG-3 '(SEQ ID NO: 10 / exon 2 antisense) DJ690 5'-GAGGAGGGGCAAGGTGGGT-3' (SEQ ID NO: 11 / exon 3 sense)
- DJ693 5'-TCAGTCCTCTCAATGCCTGCTG-3 '(SEQ ID NO: 16 / exon 6 antisense) DJ696 5'-GCGGGGTCCCCCTTAGGAG-3' (SEQ ID NO: 17 / exon 7 sense)
- DJ702 5 '-TGCTCCCGTGGGACATCCTG-3' (SEQ ID NO: 20 / exon 8 antisense) DJ676 5 '-GTAAGTGCGTCCCCGTGGTG-3' (SEQ ID NO: 21 / exon 9 sense)
- exons 4 and 5 were simultaneously amplified in one PCR because of the short intron. Analysis revealed that mutations were present in one of the LKB1 alleles in all patients. Table 3 shows the results. The codon in the table is the last Indicates the number of the wild-type codon. Restriction enzyme sites were used to prove the presence or absence of mutations in PCR products from Peutz-Jeger patients and healthy subjects. Tens of restriction enzymes indicate the acquisition and elimination of restriction enzyme sites by mutation, respectively.
- the base number of LKB1 in B, MA, and FA patients was in accordance with the base sequence number of LKB1 cDNA registered in Genbank accession number U63333.
- the LKB1 base number of the D patient was in accordance with the base sequence number of the LKB1 gene registered in Genbank accession number AF032985.
- the LKB1 gene structure was also examined in a Japanese polyposis patient (SK1) suspected to be a Peutz-Jegher patient. Genomic DNA was extracted from the peripheral blood of this patient using the QIAamp Blood kit (Qiagen), and the LKB1 gene was amplified by PCR using the primers listed in Table 2 as type II. When this PCR product was directly sequenced and analyzed, there was a single base (C) deletion (the “GAC” “C” deletion corresponding to the 207th Asp) in the exon 5 coding region. I found that. It was thought that this mutation caused a frameshift after the Asp at position 207, resulting in a protein of incomplete length, probably without kinase activity.
- C single base
- the amplification of the LKB1 gene from the DNA sample by PCI was performed basically under the following conditions.
- As a reaction solution use a total of 50/1 reaction solution containing chromosomal DNA ( ⁇ DNA) lOOng, primer 50 pmol, PCR buffer I J (Invitrogen), Ampl iTaq (Perkin Elmer) 2.5 Unit, and DMSO 2.5 / 1.
- TaKaRa 0.4 ⁇ 1
- TaqStart TM Antibody (CLON TECH) 0.4 / 1
- a total of 50 1 reaction solution was used.
- the reaction was performed for 2 to 4 minutes at 94 ° C, followed by 35 cycles of ⁇ 30 to 45 seconds at 94 ° C, 30 seconds at 58 ° C or 62 ° C, and 45 seconds at 72 ° C '', Then, the reaction was performed at 72 ° C for 3 minutes.
- the PCR product is purified from the gel after agarose electrophoresis using the QI Aquick Gel Extraction kit (Qiagen), or from the PCR reaction solution using the QIAquick Nucleotide Removal Kit (Qiagen). It was sequenced directly from both directions.
- Sequencing was performed using a Drhoda mine terminator cycle sequencing kit (Applied Biosystems).
- Applied Biosystems After amplifying the region containing the mutation from the DNA sample prepared from each patient by PCR under the above conditions, cut it with restriction enzymes corresponding to each mutation, and examine the length of the cut fragment. As a result, it was confirmed that these mutations were not PCR errors or sequencing errors (PCR-RFLP analysis). That is, PCR amplification product restriction enzymes (Ahd I, BsrB I, Rsal, Seal) A total of 201 reaction solutions containing 4 Unit s restriction enzyme buffer 2 ⁇ 1 were incubated at 37 ° C for 1.5 hours, and agarose The length of each DNA fragment was examined by electrophoresis.
- LK E1 primer SEQ ID NO: 39 / 5'-gat g aa ttc ggg tec age atg gag gtg gtg gac-3 '
- E2 primer SEQ ID NO: 40 / 5'-
- a c-Myc epitope sequence at the C-terminus SEQ ID NO: 41 / Glu Gin
- pcDNA3 / LKBlmyc was type I, and an in vitro mutagenesis using GeneEditor (Promega) was used to produce an expression plasmid DNA into which mutations including amino acid substitution mutations found in Peutz-Jieger patients were introduced. did. That is, the expression plasmid KDNA (pcDNA3 / LKBl D176Nmyc) having the D176N mutation (mutation in which Asp at position 176 replaces Asn; the same applies hereinafter) was used for the LK D176N primer (SEQ ID NO: 45 / 5'-at).
- LK W308C primer SEQ ID NO: 46/5, -egg cag cac age tgc ttc egg aag aaa-3, for the W308C mutation (pcDNA3 / LKBl W308Cmyc)
- LK L67P primer SEQ ID NO: 47/5, -gtg aag gag gtg ccg gac tcg gag acg-3 '
- L67P mutation pcDNA3 / LKBl L67Pmyc
- K78I mutation pcDNA3 / LKBl K78Imyc
- Mutagenesis was carried out using KIl primer (SEQ ID NO: 48/5, -agg agg gcc gtc atcate etc aag aag-3,) as a primer for mutagenesis.
- Both the primer for mutation introduction and the primer for selection (for bottom strand) attached to the kit were annealed with the single-stranded type I plasmid DNA to synthesize a new DNA strand. This was introduced into Escherichia coli, and a clone retaining the plasmid having the mutation was selected as a GeneEditor TM antibiotic-resistant clone. The mutation was confirmed by sequencing and used for expression experiments.
- LK E4 primer (SEQ ID NO: 42 / 5'-gat ggg c cc tta cag gga ggc ata gtc agg cac ate ata tgg gta ctg ctg ctt gca ggc c ga-3 ') instead of LK E2 primer or LK Perform PCR using the E5 primer (SEQ ID NO: 43/5, -gat gaa ttc tta gtg atg gtg atg gtg atg ctg ctg gca ggc cga-3 '), and integrate the obtained fragments into various vectors.
- Epito Ichipu that is recognized by anti-HA antibody (SEQ ID NO: 4 4 / NH 2 - YPYDVPDYASL- C00H) and histidine (H) is Six consecutive sequences (histidine hexamer) will be added to the C-terminal.
- H histidine
- NP40 kinase lysis buffer (10 mM Tris-HCl pH 7.8, 13 ⁇ 4 NP40, 0.15 M sodium chloride, ImM EDTA, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 10 ⁇ g / ml aprotinin, ImM PMSF).
- the protein was turbid and solubilized by mixing at 4 ° C for 30 minutes. Protein A / G plus agarose (Santa Cruz) was added to the cell lysate thus obtained, and the mixture was mixed for 30 minutes to remove non-specifically adsorbed proteins to the beads.
- anti-c-Myc antibody A14 (Santa Cruz) was added, and the mixture was allowed to stand at 4 ° C for 1 hour. Then, protein A / G plus agarose was added, and the mixture was allowed to stand for 1 hour. The immunocomplex is precipitated by centrifugation, washed several times with NP40 Quina-Zelesis buffer, a buffer containing 1 M sodium chloride, and 50 mM Tris-HCl (pH 7.8). Was served.
- the kinase assay was performed in a reaction system containing 50 mM Tris-HCl (pH 7.8), ImM DTT, lOmM divalent cation (Mn, etc.) and 10 ° Ci [ ⁇ - 32 P] ATP in a total amount of 50 ⁇ 1.
- the immunoprecipitate was incubated at 37 ° C. for 30 minutes in this kinase assay solution to carry out the reaction, and then the reaction was stopped by adding an SDS-PAGE sample buffer and boiling, followed by SDS-PAGE.
- the gel was fixed with a methanol / acetic acid solution, dried, and the image was analyzed using a BAS200 image analyzer (Fuji Film). LKB1 kinase activity is autophosphorus It was determined as oxidizing ability.
- the expressed protein was detected after SDS-PAGE by Western plotting using anti-c-Myc antibody A14.
- pGEX / LKBlmyc was introduced into E. coli DH5 strain to obtain a single colony. After this was cultured at 37 ° C- ⁇ ⁇ in 10 ml of 2xY ⁇ medium, a part thereof was diluted 100-fold with a new 2xYT medium, and the culture was continued at 37 ° C until the 0D at 600 nm became 0.6. Thereafter, IPTG (isopropyl-3-D (-)-thiogalactovyranoside) was added to a final concentration of O. lmM, and the culture was further continued for several hours.
- IPTG isopropyl-3-D (-)-thiogalactovyranoside
- Escherichia coli was collected by centrifugation, suspended in 1% TritonX-100, 1% Tween 20, PBS, and subjected to sonication to disrupt cells and solubilize the protein.
- LKB1 protein expressed as a fusion protein with glutathione-S-transferase (GST) was purified from this solubilized sample by affinity purification using glutathione sepharose 4B (Pharmacia). did.
- LKB1 protein expressed in Escherichia coli was also detected by Western blotting using anti-C-Myc antibody A14 (FIG. 8).
- LKBl P6 (equivalent to 27-# 45) peptide NH 2 - CHMDSTEVIYQPRRK KL- C00H (SEQ ID NO: 3 4)
- LKB1 P3 (corresponding to 400- 417 No.) peptide NH 2 -CLSTKSRAEGRAPNPARKA- C00H (SEQ ID NO: 3 1)
- FIG. 9 shows the results of Western blotting using these antibodies (anti-LKB1 P6 antibody-recognizing the N-terminal side, anti-LKB1 P3 antibody-recognizing the C-terminal side). It was shown that both antibodies could detect a band (arrow) of about 55 kDa, which is considered to be LKBlmyc protein expressed in C0S7 cells. Since this reaction was blocked by pre-incubating the antibody with a large excess amount of the peptide used as the antigen in advance, it was considered that this reaction was specific to the epitope. In addition, no cross-reactivity was seen in HeLa S3 cell lysates, which are known not to express LKB1, indicating that these antibodies can detect LKB1 protein very specifically. Became clear. In addition, it was confirmed that LKB1 protein expressed as a GST fusion protein by E. coli was also detected by anti-LKB1 P3 antibody (Fig. 8).
- FIG. 10 is a diagram showing a tissue section of a human fetal colon stained. The cytoplasm of epithelial cells is stained. In addition, it can be seen that some cells considered to be endocrine cells are stained very strongly.
- FIG. 11 shows a tissue section of a human adult spleen stained. Islet cells are stained.
- FIG. 12 shows a tissue section of a human fetal testis stained. Immature germ cells are strongly stained. All of these stains were obtained with anti-LKB1 P3 antibody, but similar results were obtained with anti-LKB1 P6 antibody. In addition, these reactions disappeared by pre-incubation with each antigen peptide, and are considered to be specific staining.
- Industrial applicability was obtained with anti-LKB1 P3 antibody, but similar results were obtained with anti-LKB1 P6 antibody. In addition, these reactions disappeared by pre-incubation with each antigen peptide, and are considered to be specific staining.
- the Boyt-Jega syndrome is caused by a mutation in the LKB1 gene.
- This enables testing and treatment of diseases caused by mutations in the LKB1 gene, such as Koz-Jeghers syndrome, using the LKB1 gene, primers or probes based on its sequence, LKB1 protein, and its antibodies. It became possible.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002311414A CA2311414A1 (en) | 1997-11-27 | 1998-11-27 | Diagnostic method, diagnostic reagent and therapeutic preparation for diseases caused by variation in lkb1 gene |
US09/555,166 US6800436B1 (en) | 1997-11-27 | 1998-11-27 | Diagnostic method, diagnostic reagent and therapeutic preparation for diseases caused by variation in LKB1 gene |
AU12620/99A AU750564B2 (en) | 1997-11-27 | 1998-11-27 | Examination method, examination reagent and remedy for diseases caused by variation in LKB1 gene |
EP98955971A EP1036844A4 (en) | 1997-11-27 | 1998-11-27 | Test methods, test reagent and remedies for diseases caused by changes in the LKB1 gene |
NO20002697A NO20002697L (no) | 1997-11-27 | 2000-05-26 | Diagnostisk metode, diagnostisk reagens og terapeutisk preparat for sykdommer forÕrsaket av variasjon i LKB1-gen |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP9/344256 | 1997-11-27 | ||
JP34425697 | 1997-11-27 | ||
JP28035798 | 1998-10-01 | ||
JP10/280357 | 1998-10-01 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999028459A1 true WO1999028459A1 (en) | 1999-06-10 |
Family
ID=26553739
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP1998/005357 WO1999028459A1 (en) | 1997-11-27 | 1998-11-27 | Examination method, examination reagent and remedy for diseases caused by variation in lkb1 gene |
Country Status (6)
Country | Link |
---|---|
US (1) | US6800436B1 (ja) |
EP (1) | EP1036844A4 (ja) |
AU (1) | AU750564B2 (ja) |
CA (1) | CA2311414A1 (ja) |
NO (1) | NO20002697L (ja) |
WO (1) | WO1999028459A1 (ja) |
Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002006520A1 (fr) * | 2000-07-19 | 2002-01-24 | Chugai Seiyaku Kabushiki Kaisha | Methode de criblage de compose regulant la transduction de signaux mek/erk et utilisation medicale dudit compose |
US9155736B2 (en) | 2012-10-18 | 2015-10-13 | Signal Pharmaceuticals, Llc | Inhibition of phosphorylation of PRAS40, GSK3-beta or P70S6K1 as a marker for TOR kinase inhibitory activity |
US9358232B2 (en) | 2013-04-17 | 2016-06-07 | Signal Pharmaceuticals, Llc | Methods for treating cancer using TOR kinase inhibitor combination therapy |
US9359364B2 (en) | 2013-04-17 | 2016-06-07 | Signal Pharmaceuticals, Llc | Pharmaceutical formulations, processes, solid forms and methods of use relating to 1-ethyl-7-(2-methyl-6-(1H-1,2,4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3-b] pyrazin-2(1H)-one |
US9416134B2 (en) | 2014-04-16 | 2016-08-16 | Signal Pharmaceuticals, Llc | Solid forms of 1-ethyl-7-(2-methyl-6-(1H-1,2,4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(1H)-one, as TOR kinase inhibitors |
US9474757B2 (en) | 2013-04-17 | 2016-10-25 | Signal Pharmaceuticals, Llc | Methods for treating cancer using TOR kinase inhibitor combination therapy |
US9505764B2 (en) | 2013-04-17 | 2016-11-29 | Signal Pharmaceuticals, Llc | Treatment of cancer with dihydropyrazino-pyrazines |
US9512129B2 (en) | 2014-04-16 | 2016-12-06 | Signal Pharmaceuticals, Llc | Solid forms comprising 1-ethyl-7-(2-methyl-6-(1H-1,2,4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(1H)-one and a coformer |
US9555033B2 (en) | 2010-02-03 | 2017-01-31 | Signal Pharmaceuticals, Llc | Identification of LKB1 mutation as a predictive biomarker for sensitivity to TOR kinase inhibitors |
US9604939B2 (en) | 2013-05-29 | 2017-03-28 | Signal Pharmaceuticals, Llc | Pharmaceutical compositions of 7-(6-(2-hydroxypropan-2-YL)pyridin-3-YL)-1-((trans)-4-methoxycyclohexyl)-3,4-dihydropyrazino [2,3-B]pyrazin-2(1H)-one, a solid form thereof and methods of their use |
US9630966B2 (en) | 2013-04-17 | 2017-04-25 | Signal Pharmaceuticals, Llc | Treatment of cancer with dihydropyrazino-pyrazines |
US9782427B2 (en) | 2013-04-17 | 2017-10-10 | Signal Pharmaceuticals, Llc | Methods for treating cancer using TOR kinase inhibitor combination therapy |
US9937169B2 (en) | 2013-04-17 | 2018-04-10 | Signal Pharmaceuticals, Llc | Methods for treating cancer using dihydropyrazino-pyrazine compound combination therapy |
US11096940B2 (en) | 2017-06-22 | 2021-08-24 | Celgene Corporation | Treatment of hepatocellular carcinoma characterized by hepatitis B virus infection |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1186232A4 (en) | 1999-05-31 | 2004-11-10 | Chugai Pharmaceutical Co Ltd | 'KNOCKOUT' ANIMALS LACKING THE LKB1 GENE |
ES2313029T3 (es) * | 2003-06-17 | 2009-03-01 | Medical Research Council | Metodo para evaluar la actividad de fosforilacion de lkb1. |
SG156625A1 (en) * | 2004-12-08 | 2009-11-26 | Aventis Pharma Inc | Method for measuring resistance or sensitivity to docetaxel |
US20070048768A1 (en) * | 2005-06-30 | 2007-03-01 | Syngenta Participations Ag | Methods for screening for gene specific hybridization polymorphisms (GSHPs) and their use in genetic mapping and marker development |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH08308586A (ja) * | 1995-03-16 | 1996-11-26 | Chugai Pharmaceut Co Ltd | プロテインキナーゼをコードするdna |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5709999A (en) * | 1994-08-12 | 1998-01-20 | Myriad Genetics Inc. | Linked breast and ovarian cancer susceptibility gene |
US5827726A (en) | 1995-03-16 | 1998-10-27 | Chugai Seiyaku Kabushiki Kaisha | DNA coding protein kinase |
-
1998
- 1998-11-27 EP EP98955971A patent/EP1036844A4/en not_active Withdrawn
- 1998-11-27 AU AU12620/99A patent/AU750564B2/en not_active Ceased
- 1998-11-27 WO PCT/JP1998/005357 patent/WO1999028459A1/ja not_active Application Discontinuation
- 1998-11-27 CA CA002311414A patent/CA2311414A1/en not_active Abandoned
- 1998-11-27 US US09/555,166 patent/US6800436B1/en not_active Expired - Fee Related
-
2000
- 2000-05-26 NO NO20002697A patent/NO20002697L/no not_active Application Discontinuation
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH08308586A (ja) * | 1995-03-16 | 1996-11-26 | Chugai Pharmaceut Co Ltd | プロテインキナーゼをコードするdna |
Non-Patent Citations (9)
Title |
---|
AVIZIENYTE E., ET AL.: "SOMATIC MUTATIONS IN LKB1 ARE RARE IN SPORADIC COLORECTAL AND TESTICULAR TUMORS.", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 58., no. 10., 15 March 1998 (1998-03-15), US, pages 2087 - 2090., XP000938617, ISSN: 0008-5472 * |
BIGNELL G. R., ET AL.: "LOW FREQUENCY OF SOMATIC MUTATIONS IN THE LKB1/PEUTZ-JEGHERS SYNDROME GENE IN SPORADIC BREAST CANCER.", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 58., no. 07., 1 April 1998 (1998-04-01), US, pages 1384 - 1386., XP000938618, ISSN: 0008-5472 * |
DONG S. M., ET AL.: "FREQUENT SOMATIC MUTATIONS IN SERINE/THREONINE KINASE 11/PEUTZ-JEGHERS SYNDROME GENE IN LEFT-SIDED COLON CANCER.", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 58., no. 17., 1 September 1998 (1998-09-01), US, pages 3787 - 3790., XP000938616, ISSN: 0008-5472 * |
HEMMINKI A., ET AL.: "SERINE/THREONINE KINASE GENE DEFECTIVE IN PEUTZ-JEGHERS SYNDROME.", NATURE, NATURE PUBLISHING GROUP, UNITED KINGDOM, vol. 391., no. 6663., 1 January 1998 (1998-01-01), United Kingdom, pages 184 - 187., XP000861923, ISSN: 0028-0836, DOI: 10.1038/34432 * |
JENNE D. E., ET AL.: "PEUTZ-JEGHERS SYNDROME IS CAUSED BY MUTATIONS IN A NOVEL SERINE THREONINE KINASE.", NATURE GENETICS., NATURE PUBLISHING GROUP, NEW YORK, US, vol. 18., no. 01., 1 January 1998 (1998-01-01), NEW YORK, US, pages 38 - 43., XP000951587, ISSN: 1061-4036, DOI: 10.1038/ng0198-38 * |
NAKAGAWA H., ET AL.: "NINE NOVEL GERMLINE MUTATIONS OF STK11 IN TEN FAMILIES WITH PEUTZ-JEGHERS SYNDROME.", HUMAN GENETICS, SPRINGER, BERLIN, DE, vol. 103., no. 02., 1 August 1998 (1998-08-01), BERLIN, DE, pages 168 - 172., XP000861892, ISSN: 0340-6717, DOI: 10.1007/s004390050801 * |
STRATAKIS C. A., ET AL.: "CARNEY COMPLEX, PEUTZ-JEGHERS SYNDROME, COWDEN DISEASE, AND BANNAYAN-ZONANA SYNDROME SHARE CUTANEOUS AND ENDOCRINE MANIFESTATIONS, BUT NOT GENETIC LOCI.", JOURNAL OF CLINICAL ENDOCRINOLOGY AND METABOLISM, THE ENDOCRINE SOCIETY, US, vol. 83., no. 08., 1 August 1998 (1998-08-01), US, pages 2972 - 2977., XP000861842, ISSN: 0021-972X, DOI: 10.1210/jc.83.8.2972 * |
WALSH J H: "GASTROENTEROLOGY NEWS PEUTZ-JEGHERS CULPRIT IS REVEALED", GASTROENTEROLOGY, ELSEVIER, AMSTERDAM, NL, vol. 114, no. 03, 1 March 1998 (1998-03-01), AMSTERDAM, NL, pages 429, XP002918382, ISSN: 0016-5085 * |
WANG Z.-J., ET AL.: "GENETIC PATHWAYS OF COLORECTAL CARCINOGENESIS RARELY INVOLVE THE PTEN AND LKB1 GENES OUTSIDE THE INHERITED HAMARTOMA SYNDROMES.", JOURNAL OF PATHOLOGY, JOHN WILEY & SONS LTD, GB, vol. 153., no. 02., 1 August 1998 (1998-08-01), GB, pages 363 - 366., XP000911885, ISSN: 0022-3417 * |
Cited By (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002006520A1 (fr) * | 2000-07-19 | 2002-01-24 | Chugai Seiyaku Kabushiki Kaisha | Methode de criblage de compose regulant la transduction de signaux mek/erk et utilisation medicale dudit compose |
US9555033B2 (en) | 2010-02-03 | 2017-01-31 | Signal Pharmaceuticals, Llc | Identification of LKB1 mutation as a predictive biomarker for sensitivity to TOR kinase inhibitors |
US9557338B2 (en) | 2012-10-18 | 2017-01-31 | Signal Pharmaceuticals, Llc | Inhibition of phosphorylation of PRAS40, GSK3-beta or P70S6K1 as a marker for tor kinase inhibitory activity |
US9155736B2 (en) | 2012-10-18 | 2015-10-13 | Signal Pharmaceuticals, Llc | Inhibition of phosphorylation of PRAS40, GSK3-beta or P70S6K1 as a marker for TOR kinase inhibitory activity |
US9782427B2 (en) | 2013-04-17 | 2017-10-10 | Signal Pharmaceuticals, Llc | Methods for treating cancer using TOR kinase inhibitor combination therapy |
US9980963B2 (en) | 2013-04-17 | 2018-05-29 | Signal Pharmaceuticals, Llc | Treatment of cancer with dihydropyrazino-pyrazines |
US9505764B2 (en) | 2013-04-17 | 2016-11-29 | Signal Pharmaceuticals, Llc | Treatment of cancer with dihydropyrazino-pyrazines |
US10391092B2 (en) | 2013-04-17 | 2019-08-27 | Signal Pharmaceuticals, Llc | Methods for treating cancer using dihydropyrazino-pyrazine compound combination therapy |
US10183019B2 (en) | 2013-04-17 | 2019-01-22 | Signal Pharmaceuticals, Llc | Treatment of cancer with dihydropyrazino-pyrazines |
US9359364B2 (en) | 2013-04-17 | 2016-06-07 | Signal Pharmaceuticals, Llc | Pharmaceutical formulations, processes, solid forms and methods of use relating to 1-ethyl-7-(2-methyl-6-(1H-1,2,4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3-b] pyrazin-2(1H)-one |
US9474757B2 (en) | 2013-04-17 | 2016-10-25 | Signal Pharmaceuticals, Llc | Methods for treating cancer using TOR kinase inhibitor combination therapy |
US9630966B2 (en) | 2013-04-17 | 2017-04-25 | Signal Pharmaceuticals, Llc | Treatment of cancer with dihydropyrazino-pyrazines |
US9358232B2 (en) | 2013-04-17 | 2016-06-07 | Signal Pharmaceuticals, Llc | Methods for treating cancer using TOR kinase inhibitor combination therapy |
US10052322B2 (en) | 2013-04-17 | 2018-08-21 | Signal Pharmaceuticals, Llc | Pharmaceutical formulations, processes, solid forms and methods of use relating to 1-ethyl-7-(2-methyl-6-(1H-1,2,4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(1H)-one |
US9827243B2 (en) | 2013-04-17 | 2017-11-28 | Signal Pharmaceuticals, Llc | Pharmaceutical formulations, processes, solid forms and methods of use relating to 1-ethyl-7-(2-methyl-6-(1H-1,2,4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(1H)-one |
US9937169B2 (en) | 2013-04-17 | 2018-04-10 | Signal Pharmaceuticals, Llc | Methods for treating cancer using dihydropyrazino-pyrazine compound combination therapy |
US9604939B2 (en) | 2013-05-29 | 2017-03-28 | Signal Pharmaceuticals, Llc | Pharmaceutical compositions of 7-(6-(2-hydroxypropan-2-YL)pyridin-3-YL)-1-((trans)-4-methoxycyclohexyl)-3,4-dihydropyrazino [2,3-B]pyrazin-2(1H)-one, a solid form thereof and methods of their use |
US9974786B2 (en) | 2013-05-29 | 2018-05-22 | Signal Pharmaceuticals, Llc | Pharmaceutical compositions of 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-1-((trans)-4-methoxycyclohexyl)-3,4-dihydropyrazino[2,3- B]pyrazin-2(1H)-one, a solid form there of and methods of their use |
US10052323B2 (en) | 2013-05-29 | 2018-08-21 | Signal Pharmaceuticals, Llc | Pharmaceutical compositions of 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-1-(trans)-4-methoxycyclohexyl)-3,4-dihydropyrazino [2,3-b]pyrazin-2(1H)-one, a solid form thereof and methods of their use |
US9795603B2 (en) | 2013-05-29 | 2017-10-24 | Signal Pharmaceuticals, Llc | Pharmaceutical compositions of 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-1-((trans)-4-methoxycyclohexyl)-3,4-dihydropyrazino [2,3-B]pyrazin-2(1H)-one, a solid form thereof and methods of their use |
US9975898B2 (en) | 2014-04-16 | 2018-05-22 | Signal Pharmaceuticals, Llc | Solid forms of 1-ethyl-7-(2-methyl-6-(1H-1,2,4-triazol-3-yl)pyridin-3-YL)-3,4-dihydropyrazino [2,3-b]pyrazin-2(1H)-one as tor kinase inhibitors |
US9981971B2 (en) | 2014-04-16 | 2018-05-29 | Signal Pharmaceuticals, Llc | Solid forms of 1-ethyl-7-(2-methyl-6-(1H-1,2,4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(1H)-one as TOR kinase inhibitors |
US9416134B2 (en) | 2014-04-16 | 2016-08-16 | Signal Pharmaceuticals, Llc | Solid forms of 1-ethyl-7-(2-methyl-6-(1H-1,2,4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(1H)-one, as TOR kinase inhibitors |
US9512129B2 (en) | 2014-04-16 | 2016-12-06 | Signal Pharmaceuticals, Llc | Solid forms comprising 1-ethyl-7-(2-methyl-6-(1H-1,2,4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(1H)-one and a coformer |
US11096940B2 (en) | 2017-06-22 | 2021-08-24 | Celgene Corporation | Treatment of hepatocellular carcinoma characterized by hepatitis B virus infection |
Also Published As
Publication number | Publication date |
---|---|
AU1262099A (en) | 1999-06-16 |
EP1036844A4 (en) | 2002-12-04 |
US6800436B1 (en) | 2004-10-05 |
NO20002697L (no) | 2000-07-27 |
AU750564B2 (en) | 2002-07-25 |
NO20002697D0 (no) | 2000-05-26 |
EP1036844A1 (en) | 2000-09-20 |
CA2311414A1 (en) | 1999-06-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7510834B2 (en) | Gene mapping method using microsatellite genetic polymorphism markers | |
EP2300041B1 (en) | Method for determining risk of recurrence of prostate cancer | |
WO1999028459A1 (en) | Examination method, examination reagent and remedy for diseases caused by variation in lkb1 gene | |
US20020098530A1 (en) | Tumor suppressor gene | |
JP2010248210A (ja) | 疾患のイメージング、診断、及び治療 | |
JP2002501376A (ja) | 新規のldl−レセプター | |
US7335475B2 (en) | PR/SET-domain containing nucleic acids, polypeptides, antibodies and methods of use | |
US20090220967A1 (en) | Systemic Carnitine Deficiency Gene and Uses Thereof | |
Yang et al. | Genomic structure and mutational analysis of the human KIF1B gene which is homozygously deleted in neuroblastoma at chromosome 1p36. 2 | |
JPH11504803A (ja) | ヒト転移サプレッサー遺伝子kai1に由来する試薬を使用する診断方法及び遺伝子療法 | |
US20050130146A1 (en) | Histone deacetylase 9 | |
US6361948B1 (en) | Prognostic compositions for prostate cancer and methods of use thereof | |
JP2004533206A (ja) | 化学療法のための標的としてのガン関連遺伝子 | |
JP2002531066A (ja) | イカロス(ikaros)アイソフォームおよび突然変異体 | |
US20110052593A1 (en) | Breast cancer associated antigen | |
JP2001512969A (ja) | 緑内障の診断および治療 | |
CN114846156A (zh) | Hla-h、hla-j、hla-l、hla-v和hla-y作为治疗和诊断靶 | |
US7112419B2 (en) | Human hepatoma associated protein and the polynucleotide encoding said polypeptide | |
JP2004505637A (ja) | ガン関連sim2遺伝子 | |
JP4617257B2 (ja) | ヒト癌関連遺伝子、それがコードする産物および適用 | |
US20030049623A1 (en) | PR/SET-domain containing nucleic acids, polypeptides, antibodies and methods of use | |
US20030109027A1 (en) | TSLL2 gene | |
JP2002521062A (ja) | ヒトニューロキニン1レセプター遺伝子における遺伝子多形性および疾病の診断および処置におけるその使用 | |
US6994996B1 (en) | Polypeptide, human vacuolar H+ -ATPase C subunit 42 and polynucleotide encoding it | |
WO1999009164A9 (en) | Coding sequence haplotypes of the human brca2 gene |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IS JP KE KG KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) |
Free format text: (EXCEPT GD) |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
ENP | Entry into the national phase |
Ref document number: 2311414 Country of ref document: CA Ref country code: CA Ref document number: 2311414 Kind code of ref document: A Format of ref document f/p: F |
|
NENP | Non-entry into the national phase |
Ref country code: KR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 12620/99 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1998955971 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1998955971 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 09555166 Country of ref document: US |
|
WWG | Wipo information: grant in national office |
Ref document number: 12620/99 Country of ref document: AU |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1998955971 Country of ref document: EP |