WO2012029855A1 - Procédé de criblage permettant d'identifier une substance régulant la coagulation sanguine - Google Patents

Procédé de criblage permettant d'identifier une substance régulant la coagulation sanguine Download PDF

Info

Publication number
WO2012029855A1
WO2012029855A1 PCT/JP2011/069780 JP2011069780W WO2012029855A1 WO 2012029855 A1 WO2012029855 A1 WO 2012029855A1 JP 2011069780 W JP2011069780 W JP 2011069780W WO 2012029855 A1 WO2012029855 A1 WO 2012029855A1
Authority
WO
WIPO (PCT)
Prior art keywords
tmem16f
substance
blood coagulation
cells
cell
Prior art date
Application number
PCT/JP2011/069780
Other languages
English (en)
Japanese (ja)
Inventor
重一 長田
鈴木　淳
Original Assignee
国立大学法人京都大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人京都大学 filed Critical 国立大学法人京都大学
Priority to JP2012531917A priority Critical patent/JP5855003B2/ja
Publication of WO2012029855A1 publication Critical patent/WO2012029855A1/fr

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology
    • G01N2800/222—Platelet disorders

Definitions

  • the present invention relates to a method for screening a blood coagulation regulating substance, a composition for regulating blood coagulation, and a method for regulating blood coagulation targeting TMEM16F (transmembrane protein 16F). Furthermore, the present invention relates to a diagnostic method and diagnostic composition for Scott syndrome.
  • Non-patent Document 1 Choline-containing phospholipids (phosphatidylcholine [PC] and sphingomyelin [SM]) are basically located in the outer leaf, while anionic phospholipids and primary amine-containing phospholipids (phosphatidylserine [PS], phosphatidylethanol) Amine [PE], phosphoinositide [PIP], and phosphatidic acid ⁇ [PA]) are restricted to the cytoplasmic endoderm.
  • PC phosphatidylcholine
  • SM sphingomyelin
  • Non-patent Document 1 The asymmetry of phospholipid has been suggested in many cell functions (Non-patent Document 1).
  • the asymmetric distribution of phospholipids allows for tight packing of membrane lipids and appears to reduce membrane permeability to solutes.
  • Controlled disruption of asymmetric phospholipid distribution, particularly PS exposure at the cell surface is important in various systems.
  • platelets when activated by collagen or thrombin, they expose PS on their surface.
  • the exposed PS provides a catalytic surface for the complex of tenase and prothrombinase and induces clotting factor activation (Non-Patent Document 2).
  • Cells undergoing apoptotic cell death also expose PS, and the exposed PS functions as an “eat me” signal for phagocytes (Non-Patent Documents 3 and 4).
  • lipid transporters flippase, flippase, and scramble.
  • the flippase is also called an ATP-dependent aminophospholipid translocase, and transports aminophospholipid from the extracellular leaf to the inside of the cell (Non-patent Documents 1 and 3).
  • Type 4 P-type ATPase P4-ATPase
  • Floppase is a transporter that moves a wide range of lipids from the cytoplasm to the extracellular lobe in an ATP-dependent manner.
  • ABC ATP binding cassette
  • Non-Patent Documents 1 and 3 Two types of scrambling are envisaged. One functions constitutively in the endoplasmic reticulum and transports newly synthesized phospholipids from the cytoplasm to the inner lobe. The other is located in the cell membrane and is inactive when the cell is quiescent, but activated when intracellular Ca 2+ increases. Exposure of the activated PS on platelets from the need for Ca 2+, Ca 2+ dependent scrambling race is assumed. A family of proteins having this activity (phospholipid scrambled, PLSCR) has been isolated and characterized (Non-Patent Documents 9 and 10). However, there is controversy over whether the PLSCR protein actually provides endogenous cell membrane phospholipid scrambling activity, and recently there has been controversy (Non-Patent Documents 11-14).
  • TMEM16F Transmembrane protein 16F
  • SCR ⁇ scramble brace ⁇
  • the present invention provides the following: A method for screening a blood coagulation regulator, comprising the following steps: (1) contacting a TMEM16F-expressing cell with a candidate substance for a blood coagulation regulator, and (2) selecting a candidate substance that changes the exposure of phosphatidylserine on the surface of the cell,
  • a candidate substance that increases the exposure of phosphatidylserine is selected as a blood coagulation promoting substance
  • a candidate substance that decreases the exposure of phosphatidylserine is selected as a blood coagulation inhibitor.
  • the present invention also provides the following: A method for screening a blood coagulation regulator, comprising the following steps: (1) contacting a cell having a gene encoding TMEM16F with a candidate substance for a blood coagulation regulating substance, and (2) selecting a candidate substance that changes the expression of TMEM16F in the cell,
  • a candidate substance that increases the expression of TMEM16F is selected as a blood coagulation promoting substance
  • a candidate substance that decreases the expression of TMEM16F is selected as a blood coagulation inhibitor.
  • the present invention also provides a composition for promoting blood coagulation containing a substance that enhances the function of TMEM16F as an active ingredient, and a composition for inhibiting blood coagulation containing a substance that suppresses the function of TMEM16F as an active ingredient To do.
  • the present invention also inhibits blood coagulation, comprising administering to a subject a method that promotes blood coagulation, comprising administering a substance that enhances the function of TMEM16F to the subject, and a substance that inhibits the function of TMEM16F. Method.
  • the present invention also provides the following: A method for screening a substance that regulates the function of TMEM16F, which comprises the following steps: (1) contacting a TMEM16F-expressing cell with a candidate substance for a substance that regulates the function of TMEM16F, and (2) selecting a candidate substance that changes the exposure of phosphatidylserine on the surface of the cell,
  • a candidate substance that increases the exposure of phosphatidylserine is selected as a substance that enhances the function of TMEM16F
  • a candidate substance that decreases the exposure of phosphatidylserine is selected as a substance that suppresses the function of TMEM16F.
  • the present invention also provides the following: A method for screening a substance that regulates the function of TMEM16F, which comprises the following steps: (1) contacting a cell having a gene encoding TMEM16F with a candidate substance for a substance that regulates the function of TMEM16F; (2) selecting a candidate substance that changes the expression of TMEM16F in the cell;
  • a candidate substance that increases the expression of TMEM16F is selected as a substance that enhances the function of TMEM16F
  • a candidate substance that decreases the expression of TMEM16F is selected as a substance that suppresses the function of TMEM16F.
  • the present invention also provides a method for examining a subject's predisposition to Scott syndrome, comprising examining whether the subject has a mutation in a gene encoding TMEM16F.
  • the present invention also provides a diagnostic composition for Scott syndrome comprising an oligonucleotide that specifically hybridizes to the gene used as a probe or primer for detecting a mutation in the gene encoding TMEM16F.
  • the present invention also provides a diagnostic composition for Scott syndrome comprising an antibody that specifically recognizes TMEM16F.
  • the present invention makes it possible to screen for blood coagulation-regulating substances targeting TMEM16F.
  • the present invention makes it possible to provide a blood coagulation promoter and a blood coagulation inhibitor based on a novel mechanism.
  • Ba / F3 expressing Flag-tagged wild-type moth (WT) SCR ⁇ and mutant SCR ⁇ analyzed by Western blotting with anti-Flag. Arrows indicate SCR ⁇ monomers and multimers.
  • WT wild-type moth
  • c Human 293T cells expressing SCR ⁇ -mRFP observed with a fluorescent microscope. Scale bar: 10 ⁇ m.
  • d Ba / F3 transformed with blue vector, wild-type SCR ⁇ expression vector, or mutant SCR ⁇ expression vector and stained with annexin V (with or without pretreatment with BAPTA-AM).
  • e Ba / F3 transformed with vector or wild type SCR ⁇ pre-incubated with Annexin V and mixed with A23187. Fluorescence was monitored.
  • Ba / F3 expressing vector, wild-type SCR ⁇ , or D409G mutant SCR ⁇ (with or without pretreatment with BAPTA-AM), incubated with biotin-labeled Ro09-0198 and then stained with APC streptavidin b, Ba / F3 transformed with vector or SCR ⁇ preincubated with biotin-Ro09-0198 and APC streptavidin. A23187 was added and fluorescence was monitored.
  • Ba / F3 expressing vector or mutant SCR ⁇ incubated with 0.5 ⁇ M NBD-PC (c) or NBD-SM (e) in c and e, Ca 2+ containing HBSS at room temperature.
  • samples were diluted with chilled fatty acid-free BSA-containing buffer and analyzed by flow cytometry.
  • d or f vector or wild type SCR ⁇ expressing Ba / F3 preincubated with 0.1 ⁇ M NBD-PC (d) or NBD-SM (f) at 4 ° C.
  • A23187 was added and the incorporated NBD phospholipid was analyzed as described above at room temperature.
  • the y-axis is the fluorescence intensity observed by FACS analysis, and is expressed in arbitrary units. Experiments were performed at least three times independently.
  • cf the proportion of NBD phospholipids that could not be extracted by BSA at the indicated time was measured in triplicate and plotted with SD.
  • SCR ⁇ for Ca 2+ -dependent phospholipid scrambling.
  • a Ba / F3 transformants expressing shRNA for SCR ⁇ (shSCR) or scrambled shRNA (shCon) were established. SCR ⁇ mRNA is quantified by real-time PCR, normalized to ⁇ -actin mRNA and shown as relative expression. Ba / F3 expressing shSCR or scrambled shRNA, preincubated with b and c, Cy5-Annexin V (b) or biotin-Ro09-0198 and APC streptavidin (c) in Annexin V binding buffer. Cell fluorescence was monitored after addition of 0.5 ⁇ M A23187.
  • b RT-PCR for exons 1-12 and 11-20 of SCR ⁇ mRNA using RNA from cells of Scott patients and their parents.
  • the size of the marker DNA is shown on the left.
  • c Junction of exon 13 and intron 12 of SCR ⁇ gene sequenced from 3 'side.
  • the “CT” complementary to the “AG” at the consensus splice acceptor site is underlined. Arrows indicate mutations.
  • d RT-PCR using primers on exon 12 and exon 16 of SCR ⁇ . Arrows indicate wild type and exon 13 deficient fragments.
  • the cells are analyzed by flow cytometry using FACSAria and the staining profile is shown (colored portion). The staining operation is performed without MFG-E8, and the staining profile is shown in white.
  • NBD derivatives of phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM) were chromatographed in parallel, with their chromatogram positions to the right. Show. Effect of SCR ⁇ knockdown on Ca 2+ -dependent PS exposure.
  • a pRS shRNA vector for SCR ⁇ was introduced into Ba / F3 cells. Of the Ba / F3 stable transformed clones with reduced SCR ⁇ mRNA levels, 5 clones (shSCR # 1-5) were selected and examined. As a control, Ba / F3 transformed cells (shCon) established by a pRS shRNA vector having an invalid scrambled sequence were also analyzed.
  • SCR ⁇ mRNA levels in Ba / F3 transformed clones are measured by real-time PCR and shown as a ratio to levels in control Ba / F3 (shCon).
  • Ba / F3 transformed cells (shCon and shSCR # 1-5) were treated with 1.0 ⁇ M A23187 at 37 ° C. for 15 minutes, stained with Cy5-labeled annexin V, and analyzed by flow cytometry. In addition, the transformant was stained with annexin V without A23187 treatment, and the staining profile is shown in white. Sequence of exon 12-exon 16 of human TMEM16F chromosomal gene. CDNA sequence of human TMEM16F.
  • the start codon (ATG), stop codon (TAA), exon 6 (nucleotide numbers 649-762), 11 (nucleotide numbers 1001-1323), and 13 (nucleotide numbers 1402-1627) are bold and underlined.
  • Amino acid sequence of human TMEM16F Mutant TMEM16F cDNA sequence found in a patient with Scott syndrome. By exon 13 being skipped, the sequence of exon 14 is combined from nucleotide 1402. In this sequence, there is a TAG stop codon at 43 bases downstream (nucleotide numbers 1444-1446), and translation is completed.
  • Amino acid sequence of mutant TMEM16F found in a patient with Scott syndrome. The amino acid newly added in exon 14 because it was spliced from exon 12 to exon 14 is shown in bold and underlined.
  • TMEM16F transmembrane protein 16F
  • 8-transmembrane protein is a phospholipid scramblase involved in blood coagulation. Therefore, the present invention provides a screening method for a blood coagulation regulating substance targeting TMEM16F.
  • the “blood coagulation regulating substance” of the present invention includes a blood coagulation promoting substance and a blood coagulation inhibiting substance.
  • blood coagulation refers to a series of molecular actions in which platelets or platelets adhere to vascular endothelial cells, or blood cells adhere to a blood vessel wall to form a blood clot together with a clotting factor.
  • promoting blood coagulation means promoting the blood coagulation and promoting hemostasis
  • “inhibiting blood coagulation” means inhibiting the blood coagulation at any stage. .
  • Candidate substances for blood coagulation regulating substances may be either naturally occurring substances or synthesized substances such as microorganisms, plant or animal cell extracts or culture supernatants, synthetic low molecular weight compounds, nucleic acids, proteins. , Peptides, antibodies and the like.
  • the candidate substance may be in the form of a library such as a synthetic low-molecular compound library, a synthetic peptide library, or an antibody library.
  • TMEM16F-expressing cells and “cells having a gene encoding TMEM16F” in the present invention include platelets, megakaryocyte cells, and megakaryocyte-derived cell lines derived from various organisms such as humans, mice, rats, rabbits, and the like. Including various cell lines and cells into which a gene encoding TMEM16F has been introduced and expressed. B cell lines such as human Namalwa cells and mouse Ba / F3 cells can also be used.
  • the screening method of the present invention is performed by examining changes in the enzyme activity of TMEM16F. Since TMEM16F has a function as a scramble that exposes phosphatidylserine (PS) to the cell surface, changes in the enzyme activity of TMEM16F can be examined by changes in the exposure of PS on the surface of TMEM16F-expressing cells. Since PS exposure in platelets activates platelets and induces blood clotting, candidate substances that increase PS exposure are selected as procoagulants, and candidate substances that decrease PS exposure are as blood clotting inhibitors. Selected.
  • PS phosphatidylserine
  • PS exposure can be examined by binding of a substance having a property of binding to PS, for example, Annexin V or MFG-E8 (also called lactadherin) to PS on the cell surface.
  • a substance having a property of binding to PS for example, Annexin V or MFG-E8 (also called lactadherin)
  • a TMEM16F-expressing cell is treated with a candidate substance, incubated with annexin V labeled with fluorescence or the like, and the amount of annexin V bound to the cell surface may be examined.
  • the candidate substance that increases the binding of Annexin V compared to the control is selected as a blood coagulation promoting substance
  • the candidate substance that decreases the selected substance is selected as a blood coagulation inhibitor.
  • PS exposure can also be examined by the induction of a blood clotting reaction.
  • Methods for examining blood clotting reactions are well known. For example, cells treated with a calcium ionophore that treats TMEM16F-expressing cells with a candidate substance and further allows Ca 2+ to flow into the cells (after or simultaneously with the candidate substance treatment) (Factor Xa, Factor Va, prothrombin) and the presence of thrombin is examined. Alternatively, it may be further mixed with fibrinogen to examine the production of fibrin.
  • the screening method of the present invention is performed by examining whether a candidate substance changes the expression of TMEM16F.
  • a candidate substance that increases the expression of TMEM16F is selected as a blood coagulation promoting substance, and a candidate substance that decreases the expression of TMEM16F is selected as a blood coagulation inhibitor.
  • candidate substances for blood coagulation regulating substances in this embodiment include substances that act on regulatory sequences such as promoters and enhancers that regulate the expression of TMEM16F, and antisense oligonucleotides prepared based on the sequences of genes encoding TMEM16F ( DNA or RNA), siRNA, shRNA, miRNA, and ribozyme.
  • the method for examining the expression of TMEM16F is not particularly limited.
  • protein expression may be examined by Western blotting, ELISA, etc.
  • mRNA expression may be examined by Northern blotting, RT-PCR, real-time PCR, etc.
  • the present invention also provides a composition for regulating blood coagulation containing a substance that regulates the function of TMEM16F as an active ingredient, and a substance that regulates the function of TMEM16F.
  • a method of regulating blood coagulation comprising:
  • “substances that regulate the function of TMEM16F” include “substances that enhance the function of TMEM16F” and “substances that inhibit the function of TMEM16F”. “Enhance / suppress the function of TMEM16F” means to promote / suppress the biochemical function or biological function of TMEM16F as a phospholipid scramble in cells or organisms, and this includes the enzyme of TMEM16F. This includes promoting / suppressing activity and increasing / decreasing expression of TMEM16F. “Increased / decreased expression of TMEM16F” includes increased / decreased expression of mRNA from the gene encoding TMEM16F and increased / decreased expression of TMEM16F protein.
  • examples of the “substance that promotes the function of TMEM16F” include wild-type TMEM16F protein.
  • examples of the “substance that suppresses the function of TMEM16F” include anti-TMEM16F antibodies that suppress the functions of antisense oligonucleotides, siRNA, shRNA, miRNA, and TMEM16F that suppress the expression of TMEM16F.
  • composition for promoting blood coagulation can be used to stop bleeding due to vascular injury or the like, and also treats abnormal diseases for blood coagulation such as hemophilia, von Willebrand disease, disseminated intravascular coagulation syndrome, etc. Can also be used.
  • the “composition for inhibiting blood coagulation” of the present invention is a thromboembolism (for example, venous thrombosis, myocardium, etc.) that moves to a site other than the site where the thrombus is formed due to blood vessel damage or the like. Infarction, cerebral embolism, etc.).
  • composition for inhibiting blood coagulation of the present invention can be used for promoting blood circulation and preventing congestion by inhibiting abnormal blood coagulation. Furthermore, since excessive blood coagulation and thrombus formation induce arteriosclerosis and infarction, the “blood coagulation inhibiting composition” of the present invention can also be used for the prevention of arteriosclerosis and infarction.
  • the present invention also provides a screening method for substances that regulate the function of TMEM16F.
  • This screening method may be performed as described in “1.1. Screening Method for Blood Coagulation Regulator”.
  • TMEM16F is a causative gene for Scott syndrome, which is a hemorrhagic disease caused by impaired PS migration to the platelet surface. Therefore, the present invention provides a method for diagnosing Scott syndrome by examining mutations in the gene encoding TMEM16F.
  • the “gene encoding TMEM16F” in this specification includes not only the coding sequence of TMEM16F but also regulatory sequences such as a promoter that regulates the expression of TMEM16F.
  • the “gene encoding TMEM16F” includes both exons and introns.
  • a “mutation of a gene encoding TMEM16F” includes any mutation (for example, deletion, substitution, addition and / or insertion of one or several bases) that inactivates the enzymatic activity of TMEM16F in a cell or organism. included.
  • Such mutations include, for example, deletion or substitution, addition and / or insertion of one or several amino acid residues, deletion of exons due to abnormal splicing of mRNA, frameshift during translation of mRNA, etc. Cause mutation.
  • Examples of such mutations include the mutations described herein, Castoldi E et al., Compound heterozygosity for 2 novel TMEM16F mutations in a patient with Scott syndrome. Blood 117, 4399-4400 (2011) Non-patent document 47) can be mentioned. In the present specification, “several” means 2, 3, 4, 5, 6, 7, 8, 9, or about 10.
  • the diagnostic method of the present invention comprises examining a G to T mutation at position 11423 of SEQ ID NO: 1.
  • SEQ ID NO: 1 shows the sequence of exon 12 to exon 16 of the human TMEM16F chromosomal gene. Each sequence of exon 12 to exon 16 is shown in italics. The underline in the sequence indicates the positions of Ex12-FW and Ex16-RV used in PCR.
  • Mutation can be examined using genomic DNA, mRNA, and protein prepared by a known method from a sample collected from a subject.
  • the sample is not particularly limited, and for example, various cells, tissues, organs, blood, body fluids, and the like of a subject can be used.
  • Mutations can be detected by directly examining the sequence of the subject's genomic DNA or mRNA.
  • Various methods can be used to detect mutations, including the following: TaqMan PCR method (Genet. Anal., 14, 143-149 (1999), J. Clin. Microbiol., 34, 2933-2936 (1996)); Invader method (Science, 5109, 778-783 (1993), J. Biol. Chem., 30, 21387-21394 (1999), Nat. Biotechnol., 17, 292-296 (1999)); Molecular Beacons method (Nat.
  • the mutation in the patient with Scott syndrome identified by the present inventors is located at the splice acceptor site of intron 12 of the gene encoding TMEM16F, mRNA splicing is not normally performed in the subject having the mutation, and as a result Exon 13 is missing. Therefore, the presence or absence of the mutation can also be examined by examining the presence or absence of exon 13 deletion in the TMEM16F mRNA of the subject.
  • SEQ ID NOs: 2 and 3 The cDNA sequence and amino acid sequence of wild-type TMEM16F are shown in SEQ ID NOs: 2 and 3. Further, SEQ ID NOs: 4 and 5 show the cDNA sequence and amino acid sequence of mutant TMEM16F in a patient with Scott syndrome identified by the present inventors.
  • RT-PCR is performed using a primer that specifically hybridizes to a gene encoding TMEM16F to amplify a region containing exon 13, and control the length of DNA amplified from the subject's sample (healthy person) Compare with If the subject has the mutation, a short DNA fragment amplification is observed compared to the control.
  • RT-PCR is performed using a primer that specifically hybridizes to exon 13 of the gene encoding TMEM16F, a DNA fragment is not amplified from the sample of the subject.
  • the mutation of the gene encoding TMEM16F other than the mutation identified by the present inventors can be similarly detected by preparing a primer or probe corresponding to the mutation.
  • the presence of the mutation identified by the present inventors causes a frame shift in exon 14 in addition to the loss of exon 13, resulting in termination of protein translation in exon 14 encoding the third transmembrane domain of TMEM16F. To do. Therefore, the presence or absence of the mutation can also be examined by examining the expression of immature mutant TMEM16F in the subject.
  • mutant TMEM16F can be examined using an antibody that specifically recognizes TMEM16F.
  • an antibody that recognizes both wild-type TMEM16F and mutant-type TMEM16F is prepared using the amino acid sequence portion common to both wild-type TMEM16F and mutant-type TMEM16F as an antigen, and the size of the protein expressed in the subject by Western blotting is determined. What is necessary is just to investigate and compare this with the size of wild type TMEM16F.
  • an antibody that recognizes wild-type TMEM16F but does not recognize mutant-type TMEM16F is prepared using the amino acid sequence portion of the region that does not exist in mutant-type TMEM16F as an antigen, and whether or not the subject has a protein that is recognized by the antibody. You may investigate by a Western blot method, ELISA method, and dot blot method.
  • the mutation of the gene encoding TMEM16F other than the mutation identified by the present inventors can be similarly detected when the mutation results in a mutant TMEM16F having an amino acid sequence different from that of wild-type TMEM16F.
  • the present invention also provides a diagnostic composition for Scott syndrome comprising an oligonucleotide that specifically hybridizes to the gene used as a probe or primer for detecting a mutation in the gene encoding TMEM16F.
  • the diagnostic composition of the present invention is used in the diagnostic method.
  • the diagnostic composition of the invention comprises any of the following: (1) an oligonucleotide used as a probe that specifically hybridizes to a region containing position 11423 of SEQ ID NO: 1; (2) an oligonucleotide used as a probe that specifically hybridizes to the sequence of positions 11424 to 11649 of SEQ ID NO: 1; and (3) specifically hybridizes to the sequence of SEQ ID NO: 1 and SEQ ID NO: 1 Oligonucleotides used as primers for amplifying a region comprising all or part of positions 11424 to 11649.
  • the diagnostic composition of the present invention comprises an oligonucleotide that specifically hybridizes to the sequence of SEQ ID NO: 2 and is used as a primer for amplifying all or part of SEQ ID NO: 2. Including. Preferably, the oligonucleotide amplifies a region including all or part of positions 1402-1627 of SEQ ID NO: 2. In another embodiment, the diagnostic composition of the present invention comprises an oligonucleotide used as a probe that specifically hybridizes to the sequence at positions 1402-1627 of SEQ ID NO: 2.
  • sequence of the oligonucleotide need not be completely complementary to the sequence as long as it can specifically hybridize with the sequence of SEQ ID NO: 1 or 2, but is preferably complementary to part of the sequence of SEQ ID NO: 1 or 2 Is.
  • “specifically hybridizes” means stringent conditions determined according to the teaching of Molecular ⁇ ⁇ ⁇ Cloning: A Laboratory Manual, 3rd edition (2001) based on the Tm value of the oligonucleotide (for example, 6 ⁇ SSC, 0.5% SDS, 50% in formamide) means hybridization to the target sequence.
  • the oligonucleotide When used as a primer, the oligonucleotide is usually 15 to 100 bases, preferably 15 to 30 bases, more preferably 18 to 24 bases. When used as a probe, the oligonucleotide is usually 10 to 100 bases, preferably 10 to 50 bases, more preferably 15 to 25 bases. Oligonucleotides may be modified with radioisotopes, fluorescent materials, luminescent materials, enzymes, biotin, etc., depending on the method in which they are used. Further, when used as a probe, it may be fixed to a solid phase such as a glass plate, nylon membrane, microbead, capillary or the like.
  • the diagnostic composition of the present invention comprises an antibody that specifically recognizes TMEM16F.
  • the antibody of the present invention may be prepared using the whole or part of TMEM16F as an antigen, and may be a polyclonal antibody or a monoclonal antibody.
  • the antibodies of the present invention also include antibody fragments such as Fab, F (ab ′) 2 and Fv. Methods for producing antibodies are well known in the art (see, eg, “Antibodies: A Laboratory Manual”, Lane, HD, et al. Eds., Cold Spring Harbor Laboratory Press, New York, 1989).
  • Method (1) Cell line, recombinant protein, antibody, serum Mouse interleukin (IL-3) dependent Ba / F3 cells are RPMI (10% fetal calf serum (FCS, Gibco), 45 units / ml recombinant mouse). IL-3 and containing 50 ⁇ M ⁇ -mercaptoethanol). EBV transformed human cell lines from Scott syndrome patients and their parents were grown in RPMI 1640 (containing 10% FCS and 50 ⁇ M ⁇ -mercaptoethanol). Human 293T cells were cultured in DMEM containing 10% FCS. Plat-E packaging cells (Non-patent Document 42) were grown in DMEM containing 10% FCS.
  • mouse IL-3 was produced by mouse C127I cells transformed with a bovine papilloma virus expression vector having mouse IL-3 cDNA as previously reported (Non-patent Document 43).
  • Flag-labeled mouse MFG-E8 was produced in human 293T cells as previously reported (Non-patent Document 44), and secreted MFG-E8 was purified using anti-Flag M2 beads (Sigma-Aldrich).
  • Ca 2+ / Mg 2+ -free RPMI1640 medium was purchased from Cell Science & Technology Institute.
  • Ca 2+ -free RPMI medium contained 0.5 mM MgSO 4 .
  • Ca 2+ free FCS was prepared by dialyzing FCS against PBS for 4 days with 4 buffer changes.
  • the cells were diluted with Cy5-labeled annexin V (Biovision) diluted 15 to 2500 times on ice for 15 minutes in staining buffer (containing 10 mM Hepes-NaOH buffer [pH7.4], 140 mM NaCl and 2.5 mM CaCl 2 ). Stained in the presence of 5 ⁇ g / ml propidium iodide (PI). Flow cytometry was performed with FACSAria (BD Bioscience) or FACSCalibur (BD Bioscience) and data was analyzed with FlowJo software (True Star).
  • Sublines of Ba / F3 cells sensitive to Ca 2+ ionophore-induced PS exposure were selected by repeated FACS sorting. Briefly, 2 x 10 7 Ba / F3 cells (in HBSS) were treated with A23187 for 15 minutes at 37 ° C and suspended in 1 ml Annexin V staining buffer previously cooled to 4 ° C. . The cells were stained on ice with Cy5-Annexin V as described above, and sorted using FACSAria with the injection chamber maintained at 4 ° C.
  • Cells that provide the highest level of Cy5 fluorescence signal (top 0.5-5.0%) are collected and Ca2 + -free RPMI (5% dialyzed FCS, 45 units / ml) at a density higher than 1.0 x 10 5 cells / ml IL-3 and 50 ⁇ M ⁇ -mercaptoethanol). After 24 hours, cells were resuspended in normal Ca 2+ -containing RPMI medium and grown for subsequent sorting.
  • a cDNA synthesis kit SuperScript TM Choice System for cDNA Synthesis, Invitrogen
  • a DNA fragment longer than 2.5 kb was recovered from the gel using a DNA extraction kit (Wizard R SV Gel and PCR Clean-up System, Promega) and ligated to a Bst XI digested pMXs vector (Non-patent Document 45).
  • E. coli DH10B cells ElectroMax DH10B; Invitrogen
  • Gene Pulser Bio-Rad
  • About 9.3 ⁇ 10 5 clones were produced, and plasmid DNA was prepared by QIAfilter Plasmid Maxi Kit (Qiagen).
  • Plasmid DNA (108 ⁇ g) derived from cDNA library was grown in 18 10 cm dishes by lipofection using FuGENE6 (Roche Diagnostics) 7.2 x 10 7 PLAT-E It introduced into the packaging cell (nonpatent literature 42). Two days after transfection, the virus in the culture supernatant was centrifuged at 6,000 xg for 16 hours, centrifuged at 4 ° C, and resuspended in RPMI1640 medium (containing 10% FCS and 45 units / ml IL-3). Used to infect 7.2 ⁇ 10 6 Ba / F3 cells in the presence of 8 ⁇ g / ml polybrene (Sigma-Aldrich). After culturing for 24 hours, the medium was replaced with fresh medium and the cells were cultured for an additional 2 hours. Sorting of cells sensitive to ionophore-induced PS exposure was performed as described above.
  • PCR fragment was cloned into pGEM-T Easy vector (Promega) and DNA sequence analysis was performed using ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).
  • the primers used are as follows (Eco RI recognition sites are underlined in each primer): mTMEM16F, 5'-ATAT GAATTC GACATGCAGATGATGACTAGGAA-3 '(SEQ ID NO: 8) and 5'-ATAT GAATTC GACATGCAGATGATGACTAGGAA-3' (SEQ ID NO: 9); hTMEM16F, 5'-ATAT GAATTC GACATGAAAAAGATGAGCAGGAA-3 '(SEQ ID NO: 10) and 5'-ATAT GAATTC TTCTGATTTTGGCCGTAAAT-3' (SEQ ID NO: 11).
  • the PCR fragment was inserted into the Eco RI site of pMXs-puro c-Flag and the reliability of the cDNA was verified by DNA sequencing.
  • the coding sequence of mRFP in pcDNA-mRFP (Invitrogen) was linked to the C terminus of mouse TMEM16F in frame and introduced into the pMXs vector.
  • HRP-conjugated mouse anti-FLAG M2 (Sigma) was bound to the membrane, and peroxidase activity was detected by Western Lightning R- ECL system (PerkinElmer).
  • Ex1 -FW (5'- ATATGAATTC GACATGAAAAAGATGAGCAGGAA-3 ') (SEQ ID NO: 12) and Ex11 / 12-RV (5'-GCGTTCTTCTTCCTGAGTAA-3') (SEQ ID NO: 13); Ex11 / 12-FW (5'-TTACTCAGGAAGAAGAACGC -3 ') (SEQ ID NO: 14) and Ex20-RV (5'- ATATGAATTC TTCTGATTTTGGCCGTAAAAT-3 ') (SEQ ID NO: 15); Ex12-FW (5'-TCTGTGCCAGTGCTGTCTTT-3 ') (SEQ ID NO: 16) and Ex16-RV (5'- CTGCAGATGGTAGTCCTGTT- 3') (SEQ ID NO: 17).
  • Genomic DNA was prepared from a human cell line for sequence analysis of the human SCR ⁇ chromosomal gene, and 964-bp with 226-bp exon 13 and its 5'- and 3'-flanking regions (each about 370-bp)
  • the DNA fragments were amplified by PCR using the following primers: 5'-CCAGAGTATGCTACTAGTTG-3 '(SEQ ID NO: 18) and 5'-TCTCAGCAACCGAGGAACAT-3' (SEQ ID NO: 19).
  • the PCR product was purified by Wizard R SV PCR and Gel Clean-up System.
  • Cycle sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing kit with the primer 5'-ACATATGTGGATGCGCCTTC-3 '(SEQ ID NO: 20) and analyzed with the ABI PRIZM 3100 Genetic Analyzer.
  • MFG-E8 binding cells were suspended in RPMI 1640 containing 10% FCS and incubated with 0.4 ⁇ g / ml Flag-labeled MFG-E8 D89E mutant (44) on ice for 20 minutes. The cells were washed with the above medium and incubated on ice for 20 minutes with 1.0 ⁇ g / ml hamster monoclonal antibody (mAb) against mouse MFG-E8 (clone 2422) and then with PE-labeled mouse anti-hamster IgG (BD Bioscience). Analysis by flow cytometry using FACSAria.
  • mAb monoclonal antibody
  • cells 1.0 x 10 5 cells
  • 10 ⁇ M BAPTA-AM in RPMI1640 medium containing 10% FCS
  • 37 ° C for 5 minutes for PS exposure or for PE exposure
  • the cells were washed with Annexin V staining buffer and stained with Cy5-Annexin V or Biotin-Ro09-0198 as described above.
  • cells 1.0 x 10 6 cells were washed with PBS and added to 1.0 ml cold annexin V staining buffer with Cy5 labeled annexin V or biotin-Ro09- A mixture of 0198 and APC-labeled streptavidin and 5 ⁇ g / ml PI were suspended.
  • the cells were mixed with A23187 on ice at a final concentration of 0.25 or 0.5 ⁇ M and applied to a FACSAria injection chamber set at 20 ° C. (Ba / F3 cells) or 37 ° C. (human cell line) to induce A23187 reaction. Data were recorded for the period indicated and PI positive cells were excluded from the analysis.
  • NBD-PC and NBD-SM Uptake of NBD lipid analogues was basically analyzed by flow cytometry as described by Williamson et al. (Non-patent Document 17). Briefly, cells (10 6 cells) were washed with HBSS and resuspended in 0.5 ml HBSS (containing 2 mM CaCl 2 ).
  • HBSS 2 mM CaCl 2 and 1 ⁇ M 1-oleoyl-2- ⁇ 6-[(7-nitro-2-1,3-benzooxadiazol-4-yl) amino] hexanoyl ⁇ -sn-glycero -3-phosphocholine (NBD-PC), or 1-oleoyl-2- ⁇ 6-[(7-nitro-2-1,3-benzooxadiazol-4-yl) amino] hexanoyl ⁇ -sphingosine-1 -Phosphocholine (NBD-SM) (containing Avanti Polar Lipids) was added to the cell suspension and incubated at room temperature.
  • NBD-SM 1-oleoyl-2- ⁇ 6-[(7-nitro-2-1,3-benzooxadiazol-4-yl) amino] hexanoyl ⁇ -sphingosine-1 -Phosphocholine
  • Non-patent Document 46 Ca 2+ influx was measured as previously reported (Non-patent Document 46). Briefly, cells (1.0 ⁇ 10 6 ) were labeled with Fluo-4-AM (1 ⁇ M) (in RPMI containing 10% FCS) for 30 minutes at 37 ° C. After washing with annexin V staining buffer, the cells were suspended in annexin V staining buffer and maintained at 4 ° C. To this mixture was added Ca 2+ ionophore A23187 at a final concentration of 0.5 ⁇ M, and changes in mean fluorescence intensity (MFI) were recorded directly using a FACSCalibur system. Data was analyzed by FlowJo Software.
  • shRNA The shRNA expression plasmid for mouse SCR ⁇ in the pRS shRNA vector carrying the puromycin resistance gene was purchased from OriGene.
  • Ba / F3 cells were infected with retrovirus containing shRNA and cultured in the presence of 1.0 ⁇ g / ml puromycin. Puromycin resistant cells were subjected to cloning by limiting dilution.
  • SCR ⁇ mRNA was quantified by real-time PCR, and clones showing decreased expression of SCR ⁇ were used in subsequent experiments.
  • TMEM16A is another member of TMEM16 family Ca 2+ dependent Cl - was shown to be a channel (Non-Patent Document 19-21).
  • Cl ⁇ channel activity of TMEM16F was very low compared to TMEM16A (Non-patent Document 22).
  • Flag or mRFP was added to the C-terminus of the wild-type and mutant (D409G) forms of SCR ⁇ and expressed in Ba / F3 cells or human 293T cells.
  • Annexin V binds to Ba / F3 cells that express the D409G mutant, but does not bind to Ba / F3 cells that express wild-type SCR ⁇ (FIG. 2d), and cells that express the SCR ⁇ mutant are constitutive. It was suggested that PS was exposed to. This was confirmed by the fact that MFG-E8 (lactoadherin) (Non-patent Documents 23 and 24) that specifically binds to phosphatidylserine bound to the D409G mutant-expressing cells (FIG. 6). When intracellular Ca 2+ was chelated by BAPTA-AM, the level of exposed PS in SCR ⁇ mutant-expressing cells decreased (FIG. 2d).
  • phospholipid scrambles should mediate bidirectional transport of phospholipids.
  • Culture of 1-oleoyl-2- [6-[(7-nitro-2-1,3-benzooxadiazol-4-yl) amino] dodecanoyl] -sn-glycero-3-phosphocholine (NBD-PC) When added to the product, it was rapidly taken up by cells expressing the D409G mutant (FIG. 3c). That is, over 40% of NBD-PC bound to the cells became resistant to BSA extraction within 6 minutes. When cells overexpressing wild-type SCR ⁇ were treated with A23187, these cells took up NBD-PC faster than the parent cells, and about 25% of the NBD-PC bound to the cells was observed within the cell within 4 minutes.
  • Non-patent Document 27 Splice site mutation of SCR ⁇ in a patient with Scott syndrome Platelets and other blood cells obtained from a patient with Scott syndrome lack the ability to expose PS in response to Ca 2+ ionophore.
  • Non-patent Document 28 Kojima et al. (Non-Patent Document 28) established an EBV-transformed B cell line from Scott syndrome patients and their parents. Consistent with previous reports (Non-Patent Documents 17 and 28), patient-derived cells did not expose PS in response to Ca 2+ ionophore (FIG. 5a). In contrast, in a cell line derived from the patient's parents, A23187 induced PS exposure at the same level as a cell line from an unrelated healthy volunteer.
  • RNA was prepared from the patient and parents, and the SCR ⁇ mRNA was divided into two parts, the 5 ′ half (equivalent to exon 1-12) and the 3 ′ half (exon 11-20). Analyzed by PCR. The 5'-half 1320-bp DNA fragment was identical in the patient and parents, whereas the patient-derived 3'-half fragment was shorter than the parents' fragment ( Figure 5b). DNA sequence analysis suggested that the patient's cDNA lacks the 226-bp sequence corresponding to exon 13.
  • the patient's SCR ⁇ gene has a G-to-T homozygous mutation at the consensus splice acceptor site in intron 12, but parents found that the mutation at this position is heterozygous. Suggested ( Figure 5c). PCR analysis of SCR ⁇ mRNA using primers designed to bind to exons 12 and 16 showed a 608-bp band from the control cell line and a 382-bp band from the Scott syndrome patient cell line. As shown (FIG. 5d), suggesting that exon 13 was skipped by mutations in the splice acceptor site.
  • TMEM16F (SCR ⁇ ) is a phospholipid scramble that controls the distribution of phospholipids in the cell membrane, and TMEM16F is a cause of Scott syndrome, which is a hemorrhagic disease caused by impaired PS migration to the platelet surface. It became clear that it was a gene. Therefore, TMEM16F is useful as a novel target for blood coagulation regulating substances.
  • Non-patent literature [Non-patent literature] 1. Leventis, P. A. & Grinstein, S., The Distribution and Function of Phosphatidylserine in Cellular Membranes. Annu. Rev. Biophys. 39, 407-427 (2010). 2. Lentz, B., Exposure of platelet membrane phosphatidylserine regulates blood coagulation.Prog. Lipid Res. 42, 423-438 (2003). 3. Balasubramanian, K. & Schroit, A. J., Aminophospholipid asymmetry: A matter of life and death. Annu. Rev. Physiol. 65, 701-734 (2003). 4.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Hematology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Diabetes (AREA)
  • Food Science & Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Cette invention concerne un procédé de criblage permettant d'identifier une substance régulant la coagulation sanguine, une composition régulant la coagulation sanguine, et un procédé de régulation de la coagulation sanguine, chacun ciblant la TMEM6F (protéine transmembranaire 16 F). Cette invention concerne également un procédé et une composition pour diagnostiquer le syndrome de Scott.
PCT/JP2011/069780 2010-09-01 2011-08-31 Procédé de criblage permettant d'identifier une substance régulant la coagulation sanguine WO2012029855A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2012531917A JP5855003B2 (ja) 2010-09-01 2011-08-31 血液凝固調節物質のスクリーニング方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US37912410P 2010-09-01 2010-09-01
US61/379,124 2010-09-01

Publications (1)

Publication Number Publication Date
WO2012029855A1 true WO2012029855A1 (fr) 2012-03-08

Family

ID=45772933

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2011/069780 WO2012029855A1 (fr) 2010-09-01 2011-08-31 Procédé de criblage permettant d'identifier une substance régulant la coagulation sanguine

Country Status (2)

Country Link
JP (1) JP5855003B2 (fr)
WO (1) WO2012029855A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013157652A1 (fr) * 2012-04-16 2013-10-24 Kyoto University Procédé pour le criblage d'un modulateur d'un membre d'une famille tmem16
WO2014077279A1 (fr) 2012-11-14 2014-05-22 国立大学法人京都大学 Procédé de criblage pour identifier une substance qui régule la fonction de xkr8
WO2015156275A1 (fr) * 2014-04-08 2015-10-15 国立大学法人京都大学 Procédé de criblage pour un inhibiteur d'atp11c ou de cdc50a

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0873365A (ja) * 1994-09-02 1996-03-19 Agency Of Ind Science & Technol 血液凝固阻害物質及びその製造方法
JPH09509050A (ja) * 1994-01-24 1997-09-16 ネオルクス コーポレイション 放射標識したアネキシン
WO1998029453A1 (fr) * 1996-12-27 1998-07-09 Mochida Pharmaceutical Co., Ltd. Medicaments diriges vers la membrane cellulaire
JP2010512842A (ja) * 2006-12-13 2010-04-30 ハネウェル・インターナショナル・インコーポレーテッド 無機止血剤と他の止血剤との組み合わせ物
JP2010521421A (ja) * 2007-02-26 2010-06-24 インダストリー−アカデミック コーポレーション ファウンデーション，ヨンセイ ユニバーシティ アポトーシスを誘導するイミダゾール誘導体とその治療的用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09509050A (ja) * 1994-01-24 1997-09-16 ネオルクス コーポレイション 放射標識したアネキシン
JPH0873365A (ja) * 1994-09-02 1996-03-19 Agency Of Ind Science & Technol 血液凝固阻害物質及びその製造方法
WO1998029453A1 (fr) * 1996-12-27 1998-07-09 Mochida Pharmaceutical Co., Ltd. Medicaments diriges vers la membrane cellulaire
JP2010512842A (ja) * 2006-12-13 2010-04-30 ハネウェル・インターナショナル・インコーポレーテッド 無機止血剤と他の止血剤との組み合わせ物
JP2010521421A (ja) * 2007-02-26 2010-06-24 インダストリー−アカデミック コーポレーション ファウンデーション，ヨンセイ ユニバーシティ アポトーシスを誘導するイミダゾール誘導体とその治療的用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ROCK JR ET AL.: "Transmembrane protein 16A (TMEM16A) is a Ca2+-regulated Cl- secretory channel in mouse airways", J BIOL CHEM, vol. 284, no. 22, pages 14875 - 14880 *
SUZUKI J ET AL.: "Calcium-dependent phospholipid scrambling by TMEM16F", NATURE, vol. 468, no. 7325, 24 November 2010 (2010-11-24), pages 834 - 838 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013157652A1 (fr) * 2012-04-16 2013-10-24 Kyoto University Procédé pour le criblage d'un modulateur d'un membre d'une famille tmem16
JP2015514961A (ja) * 2012-04-16 2015-05-21 国立大学法人京都大学 Ｔｍｅｍ１６ファミリーメンバーの調節物質のスクリーニング方法
EP2839279A4 (fr) * 2012-04-16 2015-11-04 Univ Kyoto Procédé pour le criblage d'un modulateur d'un membre d'une famille tmem16
US9453835B2 (en) 2012-04-16 2016-09-27 Kyoto University Method for screening a modulator of a TMEM16 family member
WO2014077279A1 (fr) 2012-11-14 2014-05-22 国立大学法人京都大学 Procédé de criblage pour identifier une substance qui régule la fonction de xkr8
US9857357B2 (en) 2012-11-14 2018-01-02 Kyoto University Method of screening modulator of XKR8
WO2015156275A1 (fr) * 2014-04-08 2015-10-15 国立大学法人京都大学 Procédé de criblage pour un inhibiteur d'atp11c ou de cdc50a
JPWO2015156275A1 (ja) * 2014-04-08 2017-04-13 国立大学法人京都大学 Ａｔｐ１１ｃまたはｃｄｃ５０ａの阻害物質のスクリーニング方法
US10295528B2 (en) 2014-04-08 2019-05-21 Kyoto University Method of screening ATP11C or CDC50A inhibitor
JP2019164145A (ja) * 2014-04-08 2019-09-26 国立大学法人京都大学 Ａｔｐ１１ｃまたはｃｄｃ５０ａの阻害物質のスクリーニング方法
US11073510B2 (en) 2014-04-08 2021-07-27 Kyoto University Method of screening ATP11C or CDC50A inhibitor
US11692999B2 (en) 2014-04-08 2023-07-04 Kyoto University Method of screening ATP11C or CDC50A inhibitor

Also Published As

Publication number Publication date
JP5855003B2 (ja) 2016-02-09
JPWO2012029855A1 (ja) 2013-10-31

Similar Documents

Publication Publication Date Title
Devuyst et al. Uromodulin: from physiology to rare and complex kidney disorders
Carlosama et al. A homozygous donor splice-site mutation in the meiotic gene MSH4 causes primary ovarian insufficiency
Albrecht et al. A novel missense mutation in ABCA1 results in altered protein trafficking and reduced phosphatidylserine translocation in a patient with Scott syndrome
Suzuki et al. Calcium-dependent phospholipid scrambling by TMEM16F
US11035003B2 (en) TRPC6 involved in glomerulonephritis
Delmaghani et al. Defect in the gene encoding the EAR/EPTP domain-containing protein TSPEAR causes DFNB98 profound deafness
Cotterill et al. Multiple epiphyseal dysplasia mutations in MATN3 cause misfolding of the A‐domain and prevent secretion of mutant matrilin‐3
Kobiita et al. The diabetes gene JAZF1 is essential for the homeostatic control of ribosome biogenesis and function in metabolic stress
WO2017035017A1 (fr) Procédés de traitement de conditions auto-immunes chez des patients comprenant des variations génétiques dans dcr3 ou dans un gène de réseau de dcr3
WO2004092384A2 (fr) Moyens et methodes pour le diagnostic et le traitement de troubles affectifs
Sommers et al. A role for the Tec family tyrosine kinase Txk in T cell activation and thymocyte selection
JP2007512002A5 (fr)
Flister et al. SH2B3 is a genetic determinant of cardiac inflammation and fibrosis
Mehta et al. Changes in the expression of the type 2 diabetes-associated gene VPS13C in the β-cell are associated with glucose intolerance in humans and mice
Möller-Kerutt et al. Crumbs2 is an essential slit diaphragm protein of the renal filtration barrier
JP5855003B2 (ja) 血液凝固調節物質のスクリーニング方法
Yu et al. Identification and characterization of S2V, a novel putative siglec that contains two V set Ig-like domains and recruits protein-tyrosine phosphatases SHPs
EP1572084A2 (fr) Genes de mammifere impliques dans la suppression des infections virales et des tumeurs
JP2005505237A (ja) スルファターゼおよびそれらの使用の方法
US9857357B2 (en) Method of screening modulator of XKR8
CA2253433A1 (fr) Regulateur de la degradation de l'arn chez les mammiferes induite par des transcrits non-sens
JP2006524492A (ja) 血管形成に関連する核酸分子を同定するための方法
CN112996542A (zh) 用于淋巴系统病症的诊断和治疗的组合物和方法
Pontoglio et al. Hepatocyte nuclear factor 1α controls the expression of terminal complement genes
Huang et al. Identification of a novel splicing mutation in the fibrinogen gamma chain gene leading to dysfibrinogenaemia in a Chinese pedigree

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11821870

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2012531917

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11821870

Country of ref document: EP

Kind code of ref document: A1