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WO2017039028A1 - Gène mutant ddx58 comme gène causal du glaucome congénital, de la calcification vasculaire héréditaire ou des anomalies du squelette, et procédé et composition de diagnostic de maladies l'utilisant - Google Patents

Gène mutant ddx58 comme gène causal du glaucome congénital, de la calcification vasculaire héréditaire ou des anomalies du squelette, et procédé et composition de diagnostic de maladies l'utilisant Download PDF

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WO2017039028A1
WO2017039028A1 PCT/KR2015/009168 KR2015009168W WO2017039028A1 WO 2017039028 A1 WO2017039028 A1 WO 2017039028A1 KR 2015009168 W KR2015009168 W KR 2015009168W WO 2017039028 A1 WO2017039028 A1 WO 2017039028A1
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ddx58
gene
vascular calcification
congenital glaucoma
mutant
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PCT/KR2015/009168
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English (en)
Korean (ko)
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기창석
김덕경
기창원
장미애
김은경
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사회복지법인 삼성생명공익재단
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Priority to PCT/KR2015/009168 priority Critical patent/WO2017039028A1/fr
Publication of WO2017039028A1 publication Critical patent/WO2017039028A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a DDX58 mutant gene as a causative gene for congenital glaucoma, hereditary vascular calcification or skeletal dysfunction, and a method for diagnosing and diagnosing the disease using the same.
  • Glaucoma which occurs after birth without a specific cause, is called congenital glaucoma. Genetic causes are known to be important, and CYP11B1, MYOC, and LTBP2 genes have been identified as causative genes. However, the rate at which abnormalities of three genes are found among congenital glaucoma patients is only about 20-50%, depending on ethnicity and race, suggesting that another causal gene exists. Congenital glaucoma is a fatal disease that causes vision loss if it is not diagnosed early after birth, but early diagnosis using genetic tests is very important because it often takes a long time to detect and diagnose symptoms.
  • Vascular calcification is observed in various diseases and is frequently associated with inflammatory diseases such as vasculitis.
  • Congenital vascular calcification occurs without any obvious preceding disease, and since there is no particular symptom, it becomes very severe and can be diagnosed after abnormalities in the function of blood vessels and heart valves, so early diagnosis is a very important disease.
  • Skeletal dysfunction can be caused by genetic or acquired causes. To date, a number of causal genes have been identified, and each genotype has a wide variety of types and abnormalities. Therefore, it is easy to test the gene for diagnosis only by identifying the cause genes according to the above forms.
  • the present inventors have tried to develop diagnostic markers for conclusive diagnosis of congenital glaucoma, hereditary vascular calcification or skeletal dysfunction, symptomatic diagnosis and prenatal diagnosis.
  • the novel missense mutations identified in the DDX58 gene are found to be genetic mutations that cause congenital glaucoma, hereditary vascular calcification or skeletal dysfunction, and the DDX58 mutant gene and the proteins encoded therefrom are congenital glaucoma, hereditary vascular calcification or
  • the present invention has been completed by identifying that it can be a diagnostic marker for skeletal abnormalities.
  • Another object of the present invention is to provide a DDX58 mutant protein.
  • Another object of the present invention to provide a diagnostic composition for congenital glaucoma, hereditary vascular calcification or skeletal abnormality.
  • Another object of the present invention is to provide a diagnostic kit for congenital glaucoma, hereditary vascular calcification or skeletal abnormality.
  • Another object of the present invention is to provide information necessary for diagnosing the possibility of developing congenital glaucoma, hereditary vascular calcification or skeletal abnormality.
  • the present invention provides a diagnostic marker for congenital glaucoma, hereditary vascular calcification, or skeletal dysfunction, wherein adenine, which is the 1118th base in the base sequence described in SEQ ID NO: 1, is substituted with cytosine or is the 803th base.
  • adenine which is the 1118th base in the base sequence described in SEQ ID NO: 1
  • cytosine or is the 803th base.
  • the invention provides a DDX58 mutant protein encoded from the DDX58 mutant gene as a diagnostic marker for congenital glaucoma, hereditary vascular calcification or skeletal dysfunction.
  • the present inventors have tried to develop diagnostic markers for definitive diagnosis, symptomatic diagnosis, and prenatal diagnosis of congenital glaucoma, hereditary vascular calcification or skeletal dysfunction.
  • the new missense mutations identified in the DDX58 gene are congenital glaucoma, hereditary. It was found that the gene mutation is the cause of vascular calcification or skeletal dysfunction, and it was found that this DDX58 mutant gene and the protein encoded therefrom could be a diagnostic marker of congenital glaucoma, hereditary vascular calcification or skeletal dysfunction.
  • adenine which is the 1118th base in the nucleotide sequence of SEQ ID NO: 1
  • cytosine and / or the 803th A mutation is shown in which the base guanine is substituted with thymine, and glutamic acid, which is the 373th amino acid, is substituted with alanine and / or cysteine, which is the 268th amino acid, is substituted with phenylalanine in the amino acid sequence of SEQ ID NO: 2 of the sequence coded from the mutant gene.
  • the present invention has revealed that the DDX58 mutant gene and the DDX58 mutant protein encoded therefrom can be used as diagnostic markers for congenital glaucoma, hereditary vascular calcification or skeletal dysfunction.
  • congenital glaucoma is a form of glaucoma in which symptoms develop due to an increase in intraocular pressure at birth, which occurs in most cases because of the developmental stoppage of the anterior angle, in which the waterproofing drains, and primary congenital glaucoma, primary combustion glaucoma, and abnormal developmental development with congenital abnormalities Glaucoma, but usually includes primary congenital glaucoma.
  • Symptoms include tearing, glare, and eyelid tremors, black eyes (corneas), clouding, and high intraocular pressure, which leads to an increase in black eyes, and even after 3 years of age, progressive myopia occurs without symptoms such as eye expansion. .
  • Vascular calcification in the present invention refers to a phenomenon in which calcium is accumulated in blood vessels and the blood vessels are hardened, and in the present invention, it is caused by a genetic cause.
  • skeletal dysfunction refers to skeletal growth disease, which means that the bone grows abnormally and develops, thereby showing an abnormal size and shape of the skeleton.
  • the invention provides for congenital glaucoma, hereditary vascular calcification or skeletal dysfunction comprising an agent capable of detecting the expression of the mRNA of the DDX58 mutant gene or the DDX58 mutant protein encoded from the gene from an individual sample. It provides a diagnostic composition.
  • the present invention provides a diagnostic kit for congenital glaucoma, hereditary vascular calcification or skeletal abnormality comprising the diagnostic composition described above.
  • the expression “kit for diagnosis of congenital glaucoma, hereditary vascular calcification or skeletal abnormality” means a kit containing a diagnostic composition for congenital glaucoma, hereditary vascular calcification or skeletal abnormality. Therefore, the expression "diagnosis kit for congenital glaucoma, hereditary vascular calcification or skeletal abnormality” can be used in combination with each other or "diagnostic composition for congenital glaucoma, hereditary vascular calcification or skeletal abnormality”.
  • diagnosis refers to determining the susceptibility of an object to a particular disease or condition, determining whether an object currently has a particular disease or condition, or as long as a person has a particular disease or condition. Determining the prognosis of the object, or therametrics (eg, monitoring the condition of the object to provide information about treatment efficacy).
  • Agents capable of detecting mRNA expression in a diagnostic composition for congenital glaucoma, hereditary vascular calcification or skeletal dysfunction of the present invention are primer pairs or probes that specifically bind to the DDX58 mutant gene.
  • mRNA expression detection used to diagnose congenital glaucoma, hereditary vascular calcification or skeletal dysfunction in the present invention is a process of confirming the presence and degree of expression of a polynucleotide encoding the DDX58 mutant gene of the present invention in a biological sample. It is meant to measure the amount of nucleotides. Analytical methods for this purpose include reverse transcriptase (RT-PCR), competitive reverse transcriptase (RT) PCR, real-time reverse transcriptase (Real-time RT-PCR), RNase protection assay (RPA). assays, Northern blotting, DNA chips, and the like.
  • RT-PCR reverse transcriptase
  • RT competitive reverse transcriptase
  • Real-time RT-PCR real-time reverse transcriptase
  • RNase protection assay RNase protection assay
  • mRNA detection of the present invention is carried out according to a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the primers of the present invention are used for gene amplification reactions.
  • amplification reaction means a reaction that amplifies a nucleic acid molecule.
  • Various amplification reactions have been reported in the art, which include polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcriptase-polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • PCR is the best known nucleic acid amplification method, and many modifications and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR, and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR.
  • multiplex PCR, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), inverse polymerase chain reaction ( Inverse polymerase chain reaction (IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications.
  • DD-PCR differential display PCR
  • RACE rapid amplification of cDNA ends
  • IPCR Inverse polymerase chain reaction
  • TAIL-PCR thermal asymmetric interlaced PCR
  • a gene amplification reaction can be performed to detect a target gene in the analyte (eg, an object derived sample). Therefore, the present invention performs the gene amplification reaction using a primer that binds to the DNA extracted from the sample. Extracting DNA from a sample can be carried out according to conventional methods known in the art (see Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001); Tesniere, C. et al., Plant Mol. Biol.
  • Primers used in the present invention are hybridized or annealed to one site of the template to form a double chain structure.
  • Suitable nucleic acid hybridization conditions for forming such a double-chain structure include Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization , A Practical Approach, IRL Press, Washington, DC (1985).
  • the amplification reaction When carrying out the polymerization reaction, it is preferable to provide an excess amount of components necessary for the reaction to the reaction vessel.
  • components required for the amplification reaction means an amount such that the amplification reaction is not substantially limited to the concentration of the components. So that the degree of amplification to a desired joinja, dATP, dCTP, dGTP and dTTP, such as Mg 2 + can be achieved to provide the reaction mixture is desired.
  • All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer ensures that all enzymes are close to optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of reactants.
  • Annealing in the present invention is carried out under stringent conditions allowing specific binding between the target nucleotide sequence and the primer. Stringent conditions for annealing are sequence-dependent and vary depending on the surrounding environmental variables.
  • This amplified target gene (specifically, DDX58 gene) is analyzed by a suitable method to specifically detect the presence or absence of a DDX58 mutation in a sample.
  • the target gene may be selectively detected by observing and analyzing a pattern of a band formed by scanning the amplification reaction product described above through fluorescence measurement.
  • the method of the present invention when the method of the present invention is carried out based on an amplification reaction using DNA, specifically, (i) performing an amplification reaction using a primer pair and probe annealed to a DDX58 mutant nucleotide sequence; And (ii) analyzing the product of the amplification reaction through fluorescence, thereby comprehensively detecting or quantifying the occurrence and presence of a DDX58 mutation in DNA extracted from a sample.
  • hybridization means that two single stranded nucleic acids form a duplex structure by pairing complementary base sequences. Hybridization may occur when the complementarity between single stranded nucleic acid sequences is perfect or even when some mismatch base is present. The degree of complementarity required for hybridization may vary depending on the hybridization reaction conditions, and in particular, may be controlled by temperature. The terms “annealing” and “hybridization” do not differ and are used interchangeably herein.
  • the target nucleic acid used in the present invention is not particularly limited, and includes all DNA (gDNA or cDNA) or RNA molecules, more preferably gDNA. If the target nucleic acid is an RNA molecule, reverse transcription to cDNA is used.
  • the method of annealing or hybridizing the target nucleic acid to the extension primers and probes may be carried out by hybridization methods known in the art.
  • suitable hybridization conditions can be determined in a series of procedures by an optimization procedure. This procedure is carried out by a person skilled in the art in order to establish a protocol for use in the laboratory.
  • conditions such as temperature, concentration of components, hybridization and reaction time, buffer components and their pH and ionic strength depend on various factors such as the length and GC amount of the oligonucleotide and the target nucleotide sequence.
  • Detailed conditions for hybridization can be found in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); And M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y. (1999).
  • the template-dependent nucleic acid polymerase used in the present invention is an enzyme having 5 ' ⁇ 3' nuclease activity.
  • the template-dependent nucleic acid polymerase used in the present invention is preferably a DNA polymerase.
  • DNA polymerases typically have 5 ′ ⁇ 3′nuclease activity.
  • Template-dependent nucleic acid polymerases used in the present invention include E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase.
  • the template-dependent nucleic acid polymerase is a thermostable DNA polymerase obtained from various bacterial species, which is Thermus aquaticus ( Taq ) , Thermus thermophilus ( Tth ) , Thermus filiformis , Thermis flavus , Thermococcus literalis, Pyrococcus furiosus ( Pfu ) , Thermus antranikianii , Thermus caldophilus, Thermus chliarophilus , Thermus flavus , Thermus igniterrae , Thermus lacteus , Thermus oshimai , Thermus ruber , Thermus rubens, Thermus scotoductus , Thermus silvanus , Thermus species Z05, Thermus species sps 17, Thermus thermophilus , Thermotoga maritima , Thermotoga DNA polymerases of neapolitana and Thermosi
  • the probe or primer used in the diagnostic composition of the present invention has a sequence complementary to the polynucleotide sequence and the polynucleotide sequence encoding the DDX58 mutant gene.
  • primer of the present invention is meant a nucleic acid sequence having a short free 3-terminal hydroxyl group which can form complementary templates and base pairs and which serves as a starting point for template strand copying.
  • Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures.
  • Primers of the invention are sense and antisense nucleic acids having 7 to 50 nucleotide sequences as primers specific for each marker gene. Primers can incorporate additional features that do not change the basic properties of the primers that serve as a starting point for DNA synthesis.
  • Primers of the invention can be chemically synthesized using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonate, phosphoester, phosphoroami Date, carbamate, etc.) or charged linkages such as phosphorothioate, phosphorodithioate and the like.
  • Nucleic acids may be selected from one or more additional covalently linked residues, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), inserts (eg, acridine, psoralene, etc.). ), Chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. Nucleic acid sequences of the invention can also be modified using a label that can provide a detectable signal directly or indirectly. Examples of labels include radioisotopes, fluorescent molecules, biotin, and the like.
  • probe refers to a linear oligomer of natural or modified monomers or linkages, includes deoxyribonucleotides and ribonucleotides, and can specifically hybridize to a target nucleotide sequence, naturally Present or artificially synthesized. Probes of the invention are preferably single chain and oligodioxyribonucleotides.
  • suitable hybridization conditions can be determined in a series of procedures by an optimization procedure. This procedure is carried out by a person skilled in the art in order to establish a protocol for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and wash times, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. Detailed conditions for hybridization are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); And MLM Anderson, Nucleic Acid Hybridization , Springer-Verlag New York Inc. NY (1999).
  • the higher stringency conditions were hybridized to 65 ° C. in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA, and at 0.1 ⁇ standard saline citrate / 0.1% SDS. It means to wash at 68 °C conditions.
  • high stringency conditions mean washing at 48 ° C. in 6 ⁇ SSC / 0.05% sodium pyrophosphate.
  • Low stringency means washing at 42 ° C. conditions, for example, at 0.2 ⁇ SSC / 0.1% SDS.
  • the agent capable of detecting the expression of a protein in a diagnostic composition for congenital glaucoma, hereditary vascular calcification or skeletal abnormality of the present invention is an antibody specific for a protein encoded from a DDX58 mutant gene.
  • the expression “protein expression detection” used in diagnosing congenital glaucoma, hereditary vascular calcification or skeletal abnormality is a process of confirming the presence and degree of expression of a protein expressed from the mutant gene in a biological sample.
  • an antibody that specifically binds to the protein it means to confirm the amount of the protein.
  • Analytical methods for this purpose include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, and rocket. Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, Fluorescence Activated Cell Sorter (FACS), protein chip, etc.
  • the method of analysis of the invention is not limited.
  • Antibodies that specifically bind to marker proteins in the present invention are, for example, oligopeptides, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, ligands, PNA (Peptide nucleic acid) or aptamers (aptamers). )to be.
  • antibody refers to a specific protein molecule directed against an antigenic site.
  • an antibody refers to an antibody that specifically binds to a marker protein and includes both polyclonal antibodies, monoclonal antibodies and recombinant antibodies.
  • Polyclonal antibodies can be produced by methods well known in the art for injecting the marker protein antigens described above into an animal and collecting blood from the animal to obtain serum comprising the antibody.
  • Such polyclonal antibodies can be prepared from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, bovine dog.
  • Monoclonal antibodies are well known in the art by the hybridoma method (see Kohler and Milstein (1976) European Journal of Immunology 6: 511-519), or phage antibody libraries (Clackson et al, Nature , 352: 624-628, 1991; Marks et al, J. Mol . Biol . , 222: 58, 1-597, 1991).
  • Antibodies prepared by the above method can be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.
  • antibodies of the invention include functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains.
  • a functional fragment of an antibody molecule refers to a fragment having at least antigen binding function, and includes Fab, F (ab '), F (ab') 2 and Fv.
  • the aptamer binding to the marker protein in the present invention is an oligonucleic acid or peptide molecule, the general contents of the aptamer are described in Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine”. J Mol Med . 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library”. Proc Natl Acad Sci USA. 95 (24): 142727 (1998).
  • the present invention provides oligopeptides, monoclonal antibodies, polyclonal antibodies, chimeric (specifically binding to each of the proteins for diagnosing congenital glaucoma, hereditary vascular calcification or skeletal dysfunction).
  • chimeric) antibody, ligand, Peptide nucleic acid (PNA) or aptamer more preferably oligopeptide, monoclonal antibody, polyclonal antibody or chimeric antibody, even more preferably monoclonal Local antibody or polyclonal antibody, most preferably monoclonal antibody.
  • the antibody is preferably a conjugated antibody (micro particle) (conjugated antibody).
  • the microparticles are preferably colored latex or colloidal gold particles.
  • the diagnostic kit of the present invention is an RT-PCR kit, a microarray chip kit or an immunoassay kit.
  • the immunoassay kit is a luminex assay kit, a protein microarray kit or an ELISA kit.
  • the Luminex Assay Kit, Protein Microarray Kit, and Eliza Kit include polyclonal and monoclonal antibodies directed against the protein, and secondary antibodies against the polyclonal and monoclonal antibodies bound to a label. .
  • kits in the present invention examples include immunochromatography strip kits, luminex assay kits, protein microarray kits, eliza kits, or immunological dot kits. kind is not limited.
  • the kit may further include the necessary elements necessary to perform the ELISA.
  • ELISA kits include antibodies specific for the marker protein. Antibodies are antibodies that have high specificity and affinity for marker proteins and have little cross-reactivity to other proteins and are monoclonal, polyclonal, or recombinant antibodies.
  • the ELISA kit can also include antibodies specific for the control protein.
  • Other ELISA kits can bind reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (eg conjugated with the antibody) and substrates or antibodies thereof. Other materials and the like.
  • the kit may additionally include the necessary elements necessary for performing protein microarrays to simultaneously analyze the complex markers.
  • the microarray kit includes antibodies specific for the marker protein bound to the solid phase. Antibodies are antibodies that have high specificity and affinity for marker proteins and have little cross-reactivity to other proteins and are monoclonal, polyclonal, or recombinant antibodies.
  • the protein microarray kit can also include antibodies specific for the control protein.
  • Other protein microarray kits include reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (such as fused with antibodies), and substrates thereof or other materials that can bind to antibodies. And the like.
  • the protein is separated from the sample, and the separated protein is hybridized with a protein chip to form an antigen-antibody complex, which is read to confirm the presence or expression level of the protein.
  • Luminex Assay is a high-throughput quantitative method that can simultaneously measure up to 100 different analytes without pretreatment of small (10-20 ⁇ l) patient samples As a result, it has good sensitivity (pg unit) and can be quantified in a short time (3-4 hours), and it can replace ELISA or ELISPOT.
  • the Luminex Assay is a multiplexed fluorescent microplate assay that can simultaneously analyze more than 100 biological samples from each well in a 96-well plate. By using the laser detector of the real-time signal transmission to distinguish the polystyrene bead (polystyrene bead) of more than 100 different color groups.
  • the 100 beads are configured to be distinguished in the following manner.
  • the red fluorescence bead is divided into ten or more steps, and on the other, the orange fluorescence bead is divided into ten steps, showing the difference in intensity, and the beads therebetween.
  • the red and orange ratios are mixed in different proportions, making up a total of 100 color-coded bead sets.
  • each bead is attached to the antibody of the protein to be analyzed, it is possible to quantify the protein by an immune antibody reaction using the same.
  • the sample is analyzed using two lasers, one of which detects the beads to determine the bead identification number, and the other laser reacts with the antibody attached to the beads.
  • the protein in the sample is detected.
  • 100 in vivo proteins can be analyzed simultaneously in one well. This analysis has the advantage of being able to detect samples as small as 15 ⁇ l.
  • Luminex kits capable of performing the Luminex assay of the present invention include antibodies specific for the marker protein.
  • Antibodies are antibodies that have high specificity and affinity for marker proteins and have little cross-reactivity to other proteins and are monoclonal, polyclonal, or recombinant antibodies.
  • Luminex kits can also include antibodies specific for control proteins.
  • Other Luminex kits include reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated with antibodies) and their substrates or other substances that can bind to antibodies, and the like. It may include.
  • the antibody may be a conjugated antibody to microparticles, and the microparticles may be colored latex or colloidal gold particles.
  • the kit containing the diagnostic immunochromatography strip for congenital glaucoma, hereditary vascular calcification or skeletal dysfunction the rapid analysis of the analysis results within 5 minutes It may be a diagnostic kit characterized by including the necessary elements necessary to perform a rapid test (Rapid test).
  • the immunochromatography strip may include (a) a sample pad into which a sample is absorbed; (b) a conjugate pad that binds to the protein of the gene in the sample; (c) a test membrane in which a test line and a control line including a monoclonal antibody against the protein of the gene are treated; (d) an absorption pad on which the remaining sample is absorbed; And (e) a support.
  • Rapid test kits which also include immunochromatographic strips, include antibodies specific for the marker protein.
  • the antibody is an antibody having high specificity and affinity for a marker protein and having little cross-reactivity to other proteins.
  • the antibody is a monoclonal antibody, a polyclonal antibody, or a recombinant antibody.
  • Rapid test kits may also include antibodies specific for the control protein.
  • Other rapid test kits include reagents that can detect bound antibodies, such as nitrocellulose membranes to which specific and secondary antibodies are immobilized, membranes bound to beads to which antibodies are bound, absorbent pads and sample pads, and the like. Other substances necessary for diagnosis, and the like.
  • Determination of protein expression levels by immunological dot assay in the present invention comprises the steps of (a) dotting a biological sample on the membrane; (b) reacting the antibody specific for the protein of the gene to the dipped membrane; And (c) adding and developing a secondary antibody conjugated with a marker to the reacted membrane, wherein the ELISA assay comprises (a) a protein of a gene having a nucleotide sequence for the marker.
  • Adsorbing specific antibody 1 to solid phase (b) contacting the antibody 1 adsorbed to the solid body with a biological sample of a suspected patient to form an antigen-antibody complex; (c) treating the antibody 2 specific for the protein encoded by the gene having a nucleotide sequence for the marker to which the labeling substance is bound and binding to the complex; And (d) is preferably a sandwich ELISA assay comprising the step of detecting the concentration of the protein by detecting the label, the protein microarray assay is (a) polyclonal specific for the protein of the marker gene Immobilizing the antibody on the chip; (b) contacting the immobilized polyclonal antibody 1 with a biological sample of a suspected patient to form an antigen-antibody complex; (c) treating a monoclonal antibody specific for a protein encoded by a gene having a nucleotide sequence for the marker to which the labeling agent is bound and binding to the complex; And (d) detecting the label and measuring the concentration of the protein.
  • the suspected patient can be diagnosed as to whether the actual congenital glaucoma, hereditary vascular calcification or skeletal abnormality has developed.
  • antigen-antibody complex refers to a combination of a marker protein and an antibody specific thereto, and the amount of antigen-antibody complex formed can be quantitatively measured through the size of a signal of a detection label. .
  • Such a detection label may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but is not necessarily limited thereto.
  • enzymes include ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholinese Therapies, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphphenolpyruvate deca Carboxylase, ⁇ -latamase, and the like, but are not limited thereto.
  • Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, and the like.
  • Ligands include, but are not limited to, biotin derivatives.
  • Luminescent materials include, but are not limited to, acridinium ester, luciferin, luciferase, and the like.
  • Microparticles include, but are not limited to, colloidal gold, colored latex, and the like.
  • Redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4 W (CN) 8, [Os (bpy) 3] 2+, [RU (bpy) 3] 2+, [MO (CN) 8] 4- and the like.
  • Radioisotopes include, but are not limited to, 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, 186Re, and the like.
  • Protein expression level measurement is preferably by using an ELISA method.
  • ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, attached to a solid support
  • Direct sandwich ELISA using another labeled antibody that recognizes the antigen in the antibody-antigen complex, a labeled antibody that recognizes the antibody after reacting with another antibody that recognizes the antigen in the complex of the antigen with the antibody attached to the solid support
  • Various ELISA methods include indirect sandwich ELISA using secondary antibodies.
  • the antibody is enzymatically developed by attaching the antibody to the solid support, reacting the sample, and then attaching a labeled antibody that recognizes the antigen of the antigen-antibody complex, or to an antibody that recognizes the antigen of the antigen-antibody complex. It is detected by the sandwich ELISA method which attaches a labeled secondary antibody and enzymatically develops. The formation and extent of complexes between the marker protein and the antibody can be confirmed to determine whether congenital glaucoma, hereditary vascular calcification, or skeletal dystrophy develop.
  • Western blot using at least one antibody against the marker.
  • the whole protein is isolated from the sample, electrophoresed to separate the protein according to size, and then transferred to the nitrocellulose membrane to react with the antibody.
  • the amount of the generated antigen-antibody complex is confirmed using a labeled antibody to determine whether the protein is produced by the expression of the gene or the amount of the produced protein, thereby determining whether the incidence of congenital glaucoma, hereditary vascular calcification, or skeletal dystrophy. You can check it.
  • the detection method consists of examining the expression level of the marker gene in the control group and whether or not the expression level of the marker gene in cells with congenital glaucoma, hereditary vascular calcification or skeletal dysfunction.
  • mRNA or protein levels can be expressed as absolute (eg ⁇ g / ml) or relative (eg relative intensity of signals) differences of the marker proteins described above.
  • immunohistostaining using at least one antibody against the marker is performed.
  • Normal tissues and tissues suspected of congenital glaucoma, hereditary vascular calcification or skeletal abnormalities are collected and fixed, and paraffin embedding blocks are prepared by methods well known in the art. These are sliced to a thickness of several micrometers and attached to glass slides, and then reacted with one of the above antibodies by a known method. The unreacted antibody is then washed and labeled with one of the above-mentioned detection labels to read whether or not the antibody is labeled on a microscope.
  • one or more antibodies against the marker are arranged at a predetermined position on the substrate to use a protein chip immobilized at a high density.
  • the protein is separated from the sample, and the separated protein is hybridized with the protein chip to form an antigen-antibody complex, which is read to confirm the presence or expression level of the protein and thereby innate.
  • the presence of glaucoma, hereditary vascular calcification or skeletal abnormalities can be identified.
  • Biological sample in the present invention means tissue, cells, blood, serum, plasma, saliva, cerebrospinal fluid or urine, preferably blood or serum.
  • the invention provides a method of providing information necessary for diagnosing the likelihood of developing congenital glaucoma, hereditary vascular calcification or skeletal dysfunction comprising the following steps:
  • mRNA expression in step (a) is measured using a primer pair or probe specifically binding to the DDX58 mutant gene.
  • the method using the primers and probes of the present invention can very effectively and conveniently isolate and detect DDX58 mutations in a sample.
  • the protein expression in step (a) is measured using an antibody specific for the protein encoded from the DDX58 mutant gene.
  • the present invention identifies that the new missense mutation identified in the DDX58 gene is a gene mutation that causes congenital glaucoma, hereditary vascular calcification or skeletal dysfunction, and the DDX58 mutant gene and the protein encoded therefrom are congenital glaucoma, hereditary. It serves as a diagnostic marker for vascular calcification or skeletal dysfunction.
  • compositions, kits and methods for the diagnosis of congenital glaucoma, hereditary vascular calcification or skeletal abnormality using the DDX58 mutant gene and protein are provided.
  • 1 is a diagram showing the family tree and body features of the family of mutations for DDX58.
  • Computed tomographic angiography revealed aortic calcification (black arrowheads) and aortic valve calcification (white arrowheads) found in family A: 6 (left) and III: 7 (right).
  • Family BIII: 2 is similar, except for a lower degree of erosive change at the phalanx end, and the big toe is triangular in shape (top, arrow).
  • radiographs of family B II: 4 and III: 2 II: 4 showed severe osteolysis and flexion contractures at the phalanx end, and III: 2 showed mild erosive change at the phalanx end. Carpal and metatarsal courses do not widen in family B.
  • RefSeq numbers for aligned RIG-I amino acid sequences in the present invention are as follows: human, HIT000390839; Chimpanzee, ENSPTRT00000038562; Orangutan, ENSPPYT00000022324; Mouse, AK128929; Rats, ENSRNOT00000008465; Dog, ENSCAFT00000002841; Horses, ENSECAT00000023725; Cow, XM_580928; Zebrafish, ENSDART00000058276.
  • Casase activation recruitment domain (CARD); helicase domain-1 and Hel-2 are two conserved core helicase domains, and Hel-2i is a conserved insertion domain in the RIG-I-like helicase family P is the pincer of the bridge region that connects Hel-2 to the C-terminal domain (CTD) associated with the binding dsRNA.
  • CCD C-terminal domain
  • Figure 3 is the result of measuring the cytotoxicity by DDX58 mutation in human trabecular meshwork (HTM) cells.
  • family A the initiator (II: 6) and his son (III: 7) are patients with congenital glaucoma, vascular calcification and skeletal dysfunction. Other family members were evaluated by aortic and ophthalmic tests and / or skeletal examination. Three patients in family B (II: 4, III: 2 and IV: 3) are patients with congenital glaucoma and skeletal abnormalities. With the permission of each individual, genomic DNA (gDNA) was extracted from peripheral blood leukocytes. This study was approved by Samsung Medical Center Institutional Review Board (IRB # 2013-09-063).
  • GATK Unified Genotyper program and the decoy parameter was set to 5,000.
  • GATK was used to filter mutations according to the following conditions: allele balance> 0.8, quality ⁇ 30, lead depth ⁇ 25x, quality by depth ⁇ 5, and window 10 for clustered mutations.
  • Primers were designed to amplify the coding exons of the DDX58 gene (primers available upon request). Purified PCR amplification products were sequenced using BigDye Terminator cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, Calif.) And ABI 3730xl Genetic Analyzer (Applied Biosystems). The description of the mutation is based on the reference cDNA sequence NM_014314.3 which includes nucleotide numbering starting from the first A in the ATG start codon.
  • HTM cells from normal eyes of a 27 year old female donor were provided by Paul L. Kaufman (University of Wisconsin, Madison, Wis.). HTM cells were cultured at 37 ° C., 5% CO 2 -95% air wet conditions in DMEM medium containing 10% FBS and antibiotics.
  • a replication-deficient recombinant adenovirus carrying human wild-type and mutant genes of DDX58 was prepared using the AdEasyTM XL adenoviral vector system (Agilent). To facilitate detection and visualization, all DDX58 genes were designed to be expressed as fusion proteins tagged with FLAG epitopes at their N-terminus.
  • Virus amplification was carried out in AD-293 cells (Agilent) and virus titers were determined using QuickTiterTM adenovirus titer ELISA kits (Cell Biolabs, San Diego, Calif.). PCR confirmed that the E1a gene does not exist in the viral construct. Viral infection was carried out for 2 hours with multiplicity of infection (MOI) of 10-100 pfu (plaque-forming units) per cell, and the infected cells were further cultured for 72 hours.
  • MOI multiplicity of infection
  • infected HTM cells were washed with PBS, fixed with 4% paraformaldehyde for 20 minutes, and then permeated with 0.5% Triton X-100 for 5 minutes.
  • Block cells for 1 hour with 2% bovine serum albumin and then 2 hours with anti-FLAG antibody (Santa Cruz Biotechnology) diluted 200-fold or Cy3-linked anti-mententin antibody (Sigma-Aldrich) diluted 500-fold. Reacted for a while.
  • the cells were washed and then reacted with a 500-fold diluted FITC-binding secondary antibody (Life Technologies) for 2 hours. Nuclei were counterstained with DAPI (4 ', 6'-diamidino-2-phenylindole) for 20 minutes, and then cells were observed with an appropriate filter set and 200 ⁇ magnification under fluorescence microscopy.
  • Infected HTM cells were trypsinized for viability assay and then the fallen cells were mixed with 0.4% trypan blue. After 5 minutes, only cells were counted using a disposable hemocytometer. Data were analyzed statistically using ANOVA (One-way analysis of variance) followed by Newman-Keuls multiple comparison test, p ⁇ 0.05 was determined to be significant.
  • a Dental problem is assessed by the dentist on a panoramic radiograph of two patients b of family A. Other problems are checked by physical examination.
  • Affected residues of p.C268F and p.E373A are strictly conserved across the biomass from zebrafish to humans (FIG. 2), and p.C268F and p.E373A variants are sorted Intolerant From Tolerant (SIFT 1.9). c) and the variants are considered to be pathogenic because they were predicted to be harmful by bioinformatics analysis using Polymorphism Phenotyping v2 (PolyPhen-2). In addition, the mutation was not found in 500 Koreans, nor in 11,906 chromosomes from the National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project Database.
  • RIG-I also known as Asp-Glu-Ala-Asp box polypeptide 58 (DDX58), is a 825-residue cytoplasmic viral RNA receptor and is associated with melanoma differentiation associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). Part of the RIG-I-like receptor family (2).
  • RIG-I is an important intracellular sensor for many viruses and recognizes viral dsRNAs to induce antiviral IFN responses (1).
  • RIG-I includes N-terminal activation activation domains (CARDs) involved in activating mitochondrial antiviral signaling protein (MAVS), helicase and C-terminal domains (1). Cooperative binding of ATP and RNA substrates to the helicase domain leads to morphological changes and MAVS downstream signaling by CARD (3).
  • HTM cells primary human trabecular meshwork (HTM) cells.
  • HTM cells have shown that mutations in the MYOC gene encoding myocillin cause glaucoma, and that myocillin mutations can cause cytotoxicity in HTM cells (4-6). Since HTM cells play a role in draining aqueous humor from the eye, the death of HTM cells may be a basic mechanism of intraocular pressure elevation leading to glaucoma (7).
  • Non-mentin is one of the five major groups of intermediate filament proteins, and non-mentin contractions indicate morphological contraction of stressed cells (8).
  • Type 1 IFN systems are very important for human antiviral immunity. However, excessive production or incomplete negative regulation of type 1 IFN may be associated with the development of immune disease (9) or autoimmune disease (10).
  • Gillian et al. Recently discovered that there is a mutation in IFIH1 encoding MDA5 in patients with Aicardi-Goutie syndrome (AGS; MIM 225750), and found that this mutation leads to inadequate stimulation of type 1 IFN (9).
  • AGS is an immune disease that affects the brain and skin and maintains high expression of genes induced by type I IFNs such as IFI27, IFI44L, IFIT1, ISG15, RSAD2, and SIGLEC1 (9).
  • Funabiki et al. Reported that MDA5 dysregulation induces autoimmune diseases without viral infection (10).
  • mutant mice with a single missense mutation (p.G821S) in IFIH1 caused by the generation of N-ethyl-N-nitrosouurea mutations developed spontaneously developed lupus-like nephritis and systemic autoimmune symptoms without viral infection. (10).
  • This finding implies that an IFIH1 mutation activates downstream signaling without a ligand for it, paradoxically that the mutation induces a morphological change in MDA5 without ligand- and virus-induced signaling.
  • Cardiovascular calcification once considered a passive degenerative disease, is now recognized as an active process (11).
  • Chronic inflammatory stages represented by increased production of pro-inflammatory cytokines induced by the acquisition of RIG-I, appear to cause aortic and valve mineralization.
  • Inflammation is a major cause of vascular calcification, and some vascular inflammation is found in most human arterial calcifications (12).
  • Severe calcification of the aorta is characteristic of late stages of large vasculitis, such as Takayasu's arteritis or giant cell arteritis.
  • immune cytokines such as IL-6 and TNF are increased in the aortic wall to activate bone formation transcription factors (Sox9, Runx2, Msx2, Osx). 12, 15).

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Abstract

La présente invention établit une nouvelle mutation faux-sens identifiée à partir du gène DDX58 comme mutation génique responsable du glaucome congénital, de la calcification vasculaire héréditaire ou des anomalies du squelette, et décrit le gène mutant DDX58 et les protéines codées de celui-ci comme marqueurs de diagnostic du glaucome congénital, de la calcification vasculaire héréditaire ou des anomalies du squelette. L'invention concerne également une composition, un nécessaire et un procédé de diagnostic du glaucome congénital, de la calcification vasculaire héréditaire ou des anomalies du squelette utilisant le gène mutant DDX58 et les protéines. Selon la présente invention, un diagnostic précis et précoce peut être effectué par un simple test génique concernant le glaucome congénital, la calcification vasculaire héréditaire ou les anomalies du squelette, ce qui permet des thérapies ciblées sur la base des causes précises de la maladie selon le diagnostic précis.
PCT/KR2015/009168 2015-09-01 2015-09-01 Gène mutant ddx58 comme gène causal du glaucome congénital, de la calcification vasculaire héréditaire ou des anomalies du squelette, et procédé et composition de diagnostic de maladies l'utilisant WO2017039028A1 (fr)

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CN110938684A (zh) * 2019-11-25 2020-03-31 福州福瑞医学检验实验室有限公司 一种编码ltbp2基因突变体的核酸及其应用

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* Cited by examiner, † Cited by third party
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CN110938684A (zh) * 2019-11-25 2020-03-31 福州福瑞医学检验实验室有限公司 一种编码ltbp2基因突变体的核酸及其应用

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