KR101700945B1 - DDX58 as A Causative Gene Responsible for Congenital Glaucoma, Hereditary Vascular Calcification or Skeletal Abnormalities and Diagnosis Method and Composition for the Disease - Google Patents

Abstract

The present invention discloses that the novel mismatch mutation identified in the DDX58 gene is a genetic mutation that causes congenital glaucoma, hereditary calcification or skeletal dystrophy, and that the DDX58 mutant gene and the protein encoded thereby are congenital glaucoma, hereditary vascular calcification As a diagnostic marker for skeletal dyskinesia. The present invention provides compositions, kits, and methods for diagnosis of congenital glaucoma, hereditary vascular calcification, or skeletal dystrophy using the DDX58 mutant gene and protein. According to the present invention, it is possible to perform accurate early diagnosis through simple genetic test for congenital glaucoma, hereditary calcification or skeletal dystrophy, and customized treatment according to precise diagnosis according to accurate diagnosis.

Description

TECHNICAL FIELD [0001] The present invention relates to a DDX58 mutant gene as a causative gene for congenital glaucoma, hereditary vascular calcification, or skeletal dystrophy, and a method for diagnosing and diagnosing the above diseases using the DDX58 mutant gene. for the Disease}

The present invention relates to a DDX58 mutant gene as a causative gene for congenital glaucoma, hereditary vascular calcification, or skeletal dystrophy, and a diagnostic method and diagnostic composition for the disease using the same.

Glaucoma, vascular calcification, and skeletal abnormalities can be caused by a variety of causes. Congenital glaucoma is a rare cause of glaucoma. It is known that genetic causes are important, and CYP11B1, MYOC and LTBP2 genes are the causative genes. However, the rate of detection of three gene abnormalities among congenital glaucoma patients is approximately 20-50% depending on race and ethnicity, suggesting that there is another causative gene. Congenital glaucoma is a fatal disease that causes vision loss if it is not diagnosed early after birth. However, it is very important to diagnose glaucoma early by using genetic tests.

Vascular calcification is observed in various diseases and is frequently associated with inflammatory diseases such as vasculitis. Early diagnosis is very important because congenital vascular calcification occurs without any obvious preceding disease and it can be diagnosed after abnormalities such as blood vessels and heart valves due to severe symptoms without special symptoms.

Skeletal abnormalities can be caused by genetic or acquired causes. To date, a number of causative genes have been identified, and the types of skeletons and types of skeletal involvements vary widely. Therefore, genetic testing for diagnosis is easy if the causative gene according to the above form is revealed.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made efforts to develop diagnostic markers for the definitive diagnosis of congenital glaucoma, hereditary calcification or skeletal abnormalities, diagnosis of symptoms and prenatal diagnosis. As a result, we found that the new mismatch mutation identified in the DDX58 gene is a genetic mutation that causes congenital glaucoma, hereditary calcification, or skeletal dystrophy, and that the DDX58 mutant gene and the protein encoded thereby are congenital glaucoma, hereditary vascular calcification The present inventors have completed the present invention by confirming that they can be diagnostic markers of skeletal dystrophy.

Accordingly, an object of the present invention is to provide a DDX58 mutant gene.

Another object of the present invention is to provide a DDX58 mutant protein.

It is another object of the present invention to provide a diagnostic composition for congenital glaucoma, hereditary calcification or skeletal dystrophy.

Another object of the present invention is to provide a diagnostic kit for congenital glaucoma, hereditary vascular calcification or skeletal dystrophy.

It is another object of the present invention to provide information necessary for diagnosing the possibility of developing congenital glaucoma, hereditary calcification or skeletal dystrophy.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention and claims.

According to one aspect of the present invention, there is provided a diagnostic marker for congenital glaucoma, hereditary calcification or skeletal dystrophy, wherein the adenine at position 1118 in the nucleotide sequence of SEQ ID No. 1 is substituted with cytosine or at position 803 A DDX58 mutant gene in which guanine is substituted with thymine.

According to another aspect of the present invention, there is provided a DDX58 mutant protein encoded by a DDX58 mutant gene as a diagnostic marker for congenital glaucoma, hereditary calcification or skeletal dystrophy.

The present inventors have made efforts to develop diagnostic markers for the definitive diagnosis of congenital glaucoma, hereditary calcification, or skeletal dystrophy, diagnosis of symptoms and prenatal diagnosis. As a result, it has been found that a new mismatch mutation identified in DDX58 gene is a congenital glaucoma, The present inventors have discovered that the DDX58 mutant gene and the protein encoded by the DDX58 mutant gene can be diagnostic markers of congenital glaucoma, hereditary calcification, or skeletal dystrophy.

According to one embodiment of the present invention, in a patient showing all or part of the symptoms of congenital glaucoma, hereditary calcification or skeletal dystrophy, adenine, which is the 1118th base in the nucleotide sequence of Sequence Listing First Sequence, is substituted with cytosine and / The nucleotide guanine is replaced with thymine, and glutamate, which is the 373rd amino acid in the amino acid sequence of the sequence listing of SEQ ID NO: 2, which is encoded from the mutant gene, is substituted with alanine and / or cysteine which is the 268th amino acid is substituted with phenylalanine.

The present invention demonstrates that the DDX58 mutant gene and the DDX58 mutant protein encoded thereby can be used as diagnostic markers for congenital glaucoma, hereditary calcification or skeletal dysfunction.

In the present invention, congenital glaucoma is a type of glaucoma in which a symptom occurs due to an increase in intraocular pressure at birth. In most cases, the glaucoma is caused by the developmental disruption of the anterior chamber, Glaucoma, etc., but usually refers to primary congenital glaucoma. Symptoms include tearing, glare, and palpitation of the eyelids, blurred black pupils (corneas), high IOPs, and consequently, black pupils continue to grow and progressive myopia without symptoms such as enlargement of the eye after 3 years of age .

In the present invention, calcification of blood vessels refers to a phenomenon in which blood vessels are hardened by accumulation of calcium in blood vessels. In the present invention, particularly, they are caused by genetic causes.

In the present invention, skeletal dystrophy refers to skeletal growth disorder, which means that the bone is abnormally grown and developed and the skeleton exhibits an abnormal size and shape.

 In accordance with one aspect of the present invention, the present invention provides a method for detecting the expression of a DDX58 mutant gene encoded by the mRNA of the DDX58 mutant gene or an expression of the DDX58 mutant protein encoded by the gene, A diagnostic composition is provided.

According to another aspect of the present invention, there is provided a diagnostic kit for congenital glaucoma, hereditary vascular calcification, or skeletal dystrophy, which comprises the diagnostic composition described above.

As used herein, the expression " diagnostic kit for congenital glaucoma, hereditary calcification or skeletal dystrophy " refers to a kit containing a diagnostic composition for congenital glaucoma, hereditary calcification or skeletal dystrophy. Therefore, the above expression " Diagnostic kit for congenital glaucoma, hereditary calcification or skeletal dystrophy " can be used interchangeably or in combination with " a diagnostic composition for congenital glaucoma, hereditary calcification or skeletal dystrophy ".

The term " diagnosing " herein is used to determine the susceptibility of an object to a particular disease or disorder, to determine whether an object currently has a particular disease or disorder, Determining the prognosis of the object, or therametrics (e.g., monitoring the status of the object to provide information about the therapeutic efficacy).

The agent capable of detecting mRNA expression in a composition for diagnosis of congenital glaucoma, hereditary calcification or skeletal dystrophy according to the present invention is a primer pair or a probe that specifically binds to the DDX58 mutation gene.

The expression " mRNA expression detection " used for diagnosing congenital glaucoma, hereditary calcification or skeletal dystrophy in the present invention is a process for confirming the presence and the degree of expression of the polynucleotide encoding the DDX58 mutant gene of the present invention in a biological sample, Means measuring the amount of nucleotides. RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection (RPA), and reverse transcriptase-polymerase chain reaction assay, Northern blotting, DNA chip, and the like.

According to one embodiment of the present invention, the mRNA detection of the present invention is carried out according to a polymerase chain reaction (PCR). According to another embodiment of the present invention, the primers of the present invention are used for amplification reactions.

The term " amplification reaction " as used herein refers to a reaction to amplify a nucleic acid molecule. A variety of amplification reactions have been reported in the art, including polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159), reverse-transcription polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. (McPherson and Moller, 2000), the method of Davey, C. et al (EP 329,822), the method of Miller, HI (WO 89/06700) Ligase chain reaction (LCR), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA; WO 88/10315) , Self sustained sequence replication (WO 90/06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276), consensus sequence priming polymerase chain reaction sequence primed polymerase chain reaction, CP-PCR; Patent No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Patent Nos. 5,413,909 and 5,861,245), nucleic acid sequence based amplification (NASBA 5,309,238, 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification and loopmediated isothermal amplification. LAMP), but is not limited thereto. Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617, and U.S. Patent No. 09 / 854,317.

PCR is the most well-known nucleic acid amplification method, and many variations 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. In addition, 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 TAIL-PCR (thermal asymmetric interlaced PCR) have been developed for specific applications. For more information on PCR see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference.

When the method of the present invention is carried out using a primer, a gene amplification reaction can be performed to detect a target gene from an analysis target (for example, a sample derived from an object). Therefore, the present invention uses a primer that binds to DNA extracted from a sample to perform a gene amplification reaction. Extraction of DNA from the 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); (1987) and Chomczynski, P. et < RTI ID = 0.0 > et al., & al., Anal. Biochem. 162: 156 (1987)).

The primer used in the present invention is hybridized or annealed at one site of the template to form a double-stranded structure. Nucleic acid hybridization conditions suitable for forming such a double-stranded structure can be found in Nucleic Acid Hybridization < RTI ID = 0.0 > (" , A Practical Approach, IRL Press, Washington, DC (1985).

When performing the polymerization reaction, it is preferable to provide the reaction vessel with an excessive amount of the components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. It is desirable to provide the reaction mixture with such joins as Mg ²⁺ , dATP, dCTP, dGTP and dTTP to such an extent that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, buffers make all enzymes close to optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reaction without changing conditions such as the addition of reactants.

In the present invention, annealing is carried out under stringent conditions that allow specific binding between the target nucleotide sequence and the primer. The stringent conditions for annealing are sequence-dependent and vary with environmental variables. The amplified target gene (specifically, DDX58 gene) is analyzed by a suitable method to specifically detect the presence or absence of DDX58 mutation in the sample. For example, a target gene can be selectively detected by observing and analyzing patterns of bands formed by scanning the result of the amplification reaction through fluorescence measurement.

Therefore, 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 pair of primers and a probe annealed to a DDX58 mutant nucleotide sequence; And (ii) analyzing the product of the amplification reaction through fluorescence, whereby the occurrence or nonexistence of DDX58 mutation in the DNA extracted from the sample can be comprehensively detected or quantified.

The term " hybridization " as used herein means that two single-stranded nucleic acids form a duplex structure by pairing complementary base sequences. Hybridization can occur either in perfect match between single stranded nucleic acid sequences or in the presence of some mismatching nucleotides. The degree of complementarity required for hybridization can vary depending on hybridization reaction conditions, and can be controlled by temperature. The terms " annealing " and " hybridization " are no different and are used interchangeably herein. The target nucleic acid used in the present invention is not particularly limited and includes all of DNA (gDNA or cDNA) or RNA molecules, more preferably gDNA. When the target nucleic acid is an RNA molecule, reverse transcription is used with the cDNA.

 Methods for annealing or hybridizing a target nucleic acid to an extension primer and a probe can be carried out by a hybridization method known in the art. In the present invention, suitable hybridization conditions can be determined by a series of procedures by an optimization procedure. This procedure is performed by a person skilled in the art in a series of procedures to establish a protocol for use in the laboratory. Conditions such as, for example, 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 are described 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 'to 3' nuclease activity. The template-dependent nucleic acid polymerase used in the present invention is preferably a DNA polymerase. Typically, DNA polymerases have 5 '→ 3' nuclease activity. The template-dependent nucleic acid polymerase used in the present invention includes E. coli DNA polymerase I, thermostable DNA polymerase, and bacteriophage T7 DNA polymerase. Specifically, the template-dependent nucleic acid polymerase is a thermostable DNA polymerase obtainable from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, Pyrococcus furiosus Pfu), Thermus antranikianii, Thermus caldophilus, Thermus chliarophilus, Thermus flavus, Thermus species, Thermus lactis, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus species Z05 , Thermotoga neapolitana and Thermosipho africanus . A "template-dependent extension reaction" catalyzed by a template-dependent nucleic acid polymerase refers to a reaction that synthesizes a nucleotide sequence complementary to the sequence of a template.

The probe or primer used in the diagnostic composition of the present invention has a polynucleotide sequence encoding the DDX58 mutant gene and a sequence complementary to the polynucleotide sequence.

The term " primer " of the present invention means a short nucleic acid sequence capable of forming a base pair with a complementary template with a short free 3-terminal hydroxyl group and functioning as a starting point for template strand replication. The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. The primer of the present invention is a sense and antisense nucleic acid having 7 to 50 nucleotide sequences for each marker gene-specific primer. Primers can incorporate additional features that do not alter the primer's basic properties that serve as a starting point for DNA synthesis.

The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with one or more homologues, and modification between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, (E.g., dodecanedioic acid, dodecanedioic acid, dodecanedioic acid, tartaric acid, tartaric acid, tartaric acid, The nucleic acid can be in the form of one or more additional covalently linked residues such as a protein such as a nuclease, a toxin, an antibody, a signal peptide, a poly-L-lysine, an intercalator such as acridine, ), Chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. The nucleic acid sequences of the present invention may also be modified using a label capable of directly or indirectly providing a detectable signal. Examples of labels include radioactive isotopes, fluorescent molecules, biotin, and the like.

As used herein, the term " probe " refers to a linear oligomer of natural or modified monomer or linkages and includes deoxyribonucleotides and ribonucleotides and can specifically hybridize to a target nucleotide sequence, Present or artificially synthesized. The probe of the present invention is preferably a single strand, and is an oligodioxyribonucleotide.

When a probe is used, the probe is hybridized with the cDNA molecule. In the present invention, suitable hybridization conditions can be determined by a series of procedures by an optimization procedure. This procedure is performed by a person skilled in the art in a series of procedures to establish a protocol for use in the laboratory. Conditions such as, for example, temperature, concentration of components, hybridization and washing time, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. The 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). For example, high stringency conditions were hybridized at 65 ° C in 0.5 M NaHPO ₄ , 7% SDS (sodium dodecyl sulfate) and 1 mM EDTA, followed by addition of 0.1 x SSC / 0.1% SDS Lt; RTI ID = 0.0 > 68 C < / RTI > Alternatively, high stringency conditions means washing at < RTI ID = 0.0 > 48 C < / RTI > in 6 x SSC / 0.05% sodium pyrophosphate. Low stringency conditions mean, for example, washing in 0.2 x SSC / 0.1% SDS at 42 ° C.

The agent capable of detecting the expression of a protein in a diagnostic composition for congenital glaucoma, hereditary calcification or skeletal dystrophy according to the present invention is an antibody specific to a protein encoded by the DDX58 mutation gene.

In the present invention, the expression " detection of protein expression " used for diagnosing congenital glaucoma, hereditary calcification or skeletal dystrophy is a process for confirming the presence and the degree of expression of the protein expressed from the mutant gene in a biological sample, Means that the amount of protein is confirmed using an antibody that specifically binds to the protein. Methods for this analysis include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), and Protein Chip are examples of immunoprecipitation, protein immunoassay, immunohistochemical staining, The method of analysis of the invention is not limited.

In the present invention, an antibody that specifically binds to a marker protein includes, for example, an oligopeptide, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA) )to be.

&Quot; Antibody " in the present invention means a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein, and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

Since the novel marker proteins have been identified as described above, the production of antibodies using the novel marker proteins can be easily carried out using techniques well known in the art.

Polyclonal antibodies can be produced by methods well known in the art for obtaining serum containing antibodies by injecting the marker protein antigen described above into an animal and collecting it from the animal. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like.

Monoclonal antibodies can be prepared using hybridoma methods well known in the art (see Kohler and Milstein (1976) European Journal of Immunology 6: 511-519), or phage antibody libraries (Clackson et al, Nature , 352: 1992, Marks et al . , J. Mol. Biol. , 222: 58, 1-597, 1991). The antibody prepared by the above method can be isolated and purified by gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

In addition, the antibodies of the present invention include functional fragments of antibody molecules as well as complete forms with 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 an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

In the present invention, the aptamer binding to the marker protein is an oligonucleotide or a peptide molecule, and 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).

According to a preferred embodiment of the present invention, the present invention provides a method for diagnosing congenital glaucoma, hereditary calcification or skeletal dystrophy, comprising the steps of: (a) preparing an oligopeptide, monoclonal antibody, polyclonal antibody, chimeric chimeric antibody, a ligand, a peptide nucleic acid (PNA) or an aptamer, more preferably an oligopeptide, a monoclonal antibody, a polyclonal antibody or a chimeric antibody, and still more preferably a monoclonal antibody Or a polyclonal antibody, most preferably a monoclonal antibody.

In the present invention, the antibody is preferably a conjugated antibody conjugated with microparticles. The fine particles are preferably colored latex or colloidal gold particles.

According to one embodiment of the present invention, 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, protein microarray kit and ELISA kit comprise a polyclonal antibody and a monoclonal antibody to the protein, and a polyclonal antibody to which the labeling substance is bound and a secondary antibody to the monoclonal antibody .

Examples of the kit in the present invention include an immunochromatography strip kit, a lumenex assay kit, a protein microarray kit, an ELISA kit, or an immunological dot kit. The kind is not limited.

The kit may additionally include essential elements necessary for performing ELISA. ELISA kits contain antibodies specific for the marker protein. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for the marker protein and little cross-reactivity to other proteins. The ELISA kit may also include antibodies specific for the control protein. Other ELISA kits can be used to detect antibodies that can bind a reagent capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, an enzyme (e. G., Conjugated to an antibody) Other materials, and the like.

In addition, the kit may additionally include essential elements necessary for performing a protein microarray to simultaneously analyze complex markers. Microarray kits contain antibodies specific for the marker protein bound to the solid phase. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for the marker protein and little cross-reactivity to other proteins. The protein microarray kit may also include antibodies specific for the control protein. Other protein microarray kits can be used to detect a bound antibody, such as a labeled secondary antibody, a chromophore, an enzyme (e.g., fused with an antibody), and a substrate or other substance capable of binding to the antibody And the like. A method of analyzing a sample using a protein microarray is a method of separating a protein from a sample, hybridizing the separated protein with a protein chip to form an antigen-antibody complex, reading the protein, , Congenital glaucoma, hereditary vascular calcification, or skeletal dystrophy.

Luminex Assay is a high-throughput quantitative assay that can simultaneously measure up to 100 different analyte without pretreatment small (10-20 μl) patient samples Is an analytical method capable of replacing existing ELISA or ELISPOT with high sensitivity (in pg) and rapid quantification (3-4 hours). The Luminex Assay is a multiplexed fluorescent microplate assay capable of analyzing more than 100 biological samples simultaneously in each well in a 96-well plate. And more than 100 different color groups of polystyrene beads are discriminated and quantified by carrying out signal transmission in real time using a laser detector of the present invention. The 100 beads are configured to be distinguished in the following manner. On one side, the red fluorescence bead is divided into more than ten stages. On the other side, the orange fluorescence bead is divided into ten stages to show the intensity difference and the bead between them. Are mixed in different ratios of red and orange to form a total of 100 color-coded bead sets. In addition, each bead has an antibody of the protein to be analyzed, so that it is possible to quantify the protein by the immunoassay using the antibody. The sample is analyzed using two lasers, one laser detects the bead's unique number and the other laser reacts with the antibody attached to the bead The protein in the sample is detected. Thus, 100 in vivo protein analysis is possible simultaneously in one well. This assay has the advantage of being detectable with as little as 15 μl of sample.

Luminex kits capable of carrying out the Luminex assays of the present invention include antibodies specific for marker proteins. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for the marker protein and little cross-reactivity to other proteins. The lumenex kit may also include antibodies specific for the control protein. Other luminex kits may also include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated to antibodies) and other substrates capable of binding to the substrate or antibody . ≪ / RTI > The antibody may be a conjugated antibody conjugated with microparticles, and the microparticles may be colored latex or colloidal gold particles.

In the kit for the diagnosis of congenital glaucoma, hereditary calcification or skeletal dystrophy according to the present invention, the kit comprising the immunochromatographic strip for diagnosis of congenital glaucoma, hereditary calcification or skeletal dystrophy, And may include an essential element necessary for performing a rapid test. The immunochromatographic strip comprises (a) a sample pad on 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 containing a monoclonal antibody against the protein of the gene are treated; (d) an absorption pad on which a residual amount of the sample is absorbed; And (e) a support. Also, a rapid test kit containing an immunochromatographic strip contains antibodies specific for the marker protein. The antibody is a monoclonal antibody, polyclonal antibody or recombinant antibody, which has high specificity and affinity for the marker protein and little cross-reactivity to other proteins. The rapid test kit may also include an antibody specific for the control protein. Other rapid test kits include reagents capable of detecting conjugated antibodies, for example, a nitrocellulose membrane with a specific antibody and a secondary antibody immobilized thereon, a membrane bound to an antibody-bound bead, an absorbent pad and a sample pad Other substances necessary for diagnosis, and the like.

The measurement of the level of protein expression by the immunodet assay in the present invention includes (a) dotting a biological sample on the membrane; (b) reacting an antibody specific for the protein of the gene to the dormant membrane; And (c) a step of adding a secondary antibody conjugated with a label to the membrane to be subjected to the reaction, followed by color development, wherein the ELISA assay comprises the steps of: (a) contacting the protein of the gene having the nucleotide sequence with the marker Adsorbing a specific antibody 1 on a solid body; (b) contacting the biological sample of the suspect patient with the antibody 1 adsorbed on the solid body to form an antigen-antibody complex; (c) treating antibody 2 specific for the protein encoded by the gene having the nucleotide sequence for the marker to which the marker substance is bound, to bind to the complex; And (d) measuring the concentration of the protein by detecting the labeling substance, wherein the protein microarray assay comprises (a) a polyclonal antibody specific to 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 the monoclonal antibody specific to the protein encoded by the gene having the nucleotide sequence for the marker to which the marker substance is bound, to bind to the complex; And (d) measuring the concentration of the protein by detecting the labeled substance.

Through the above analysis methods, it is possible to compare the amount of the antigen-antibody complex formed in the normal control group with the amount of the antigen-antibody complex formed in the suspected patient, and whether the expression level of the marker gene is significantly increased or not The patient can be diagnosed as having a congenital glaucoma, hereditary calcification, or skeletal abnormalities in a suspected patient.

The term " antigen-antibody complex " in the present invention means a combination of a marker protein and an antibody specific thereto, and the amount of the antigen-antibody complex formed can be quantitatively measured through the size of a signal of a detection label .

Such detection labels may be selected from the group consisting of enzymes, chromophores, ligands, emitters, microparticles, redox molecules, and radioisotopes, but are not necessarily limited thereto. When an enzyme is used as the detection label, available enzymes include? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholine Glucoamylase, terazo, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokutase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decar ≪ / RTI > beta-lactamase, and the like. The minerals include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, fluororescamine and the like. Ligands include, but are not limited to, biotin derivatives. Emitters include, but are not limited to, acridinium esters, luciferin, luciferase, and the like. Fine particles include, but are not limited to, colloidal gold, colored latex, and the like. Examples of the redox molecules include ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4 W (CN) 8, [Os (bpy) [RU (bpy) 3] 2+, [MO (CN) 8] 4-, and the like. Radiation isotopes include, but are not limited to, 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, 186Re.

Measurement of protein expression levels is preferably performed using an ELISA method. ELISAs include direct ELISA using labeled antibodies that recognize the antigen attached to the solid support, indirect ELISA using labeled antibodies that recognize the capture antibody in a complex of antibodies recognizing the antigen attached to the solid support, A direct sandwich ELISA using another labeled antibody that recognizes an antigen in the complex of antibody and antigen, a method of reacting with another antibody recognizing an antigen in a complex of an antibody and an antigen attached to a solid support, Indirect sandwich ELISA using a secondary antibody, and various ELISA methods. More preferably, the antibody is attached to a solid support, the sample is reacted, and the labeled antibody recognizing the antigen of the antigen-antibody complex is adhered to produce an enzyme, or an antibody that recognizes the antigen of the antigen-antibody complex Is detected by a sandwich ELISA method in which a labeled secondary antibody is attached and the enzyme is developed. The presence and extent of complex formation of the marker protein and antibody can be confirmed to confirm the onset of congenital glaucoma, hereditary calcification or skeletal dysfunction.

In addition, preferably, Western blotting using one or more antibodies against the marker is used. The whole protein is separated from the sample, and the protein is separated according to size by electrophoresis, and then transferred to the nitrocellulose membrane to react with the antibody. The amount of the produced antigen-antibody complex is confirmed by using a labeled antibody to confirm whether or not the protein is produced by the expression of the gene or the amount of the produced protein to determine whether or not the congenital glaucoma, hereditary calcification, Can be confirmed. The detection method is performed by examining the amount of expression of the marker gene in the control group and the expression level or expression level of the marker gene in the cell in which congenital glaucoma, hereditary calcification or skeletal dystrophy has developed. The level of mRNA or protein may be expressed as the absolute (e.g., [mu] g / ml) or relative (e.g., the relative intensity of the signal) difference of the marker protein.

In addition, preferably, immunostaining is performed using one or more antibodies against the marker. Normal tissues and congenital glaucoma, hereditary vascular calcifications or suspected suspected skeletal abnormalities are collected and fixed, and then paraffin-embedded blocks are prepared by methods well known in the art. These are cut into pieces of several μm thick and attached to a glass slide, and then reacted with a selected one of the above antibodies by a known method. Thereafter, the unreacted antibody is washed and labeled with one of the above-mentioned detection labels, and the labeling of the antibody is read on a microscope.

Preferably, one or more antibodies against the marker is arranged at a predetermined position on the substrate and is immobilized at a high density. A method for analyzing a sample using a protein chip is a method of separating a protein from a sample, hybridizing the separated protein with a protein chip to form an antigen-antibody complex, reading the resultant protein, Glaucoma, hereditary vascular calcification, or skeletal dystrophy.

The biological sample in the present invention means a tissue, a cell, a blood, a serum, a plasma, a saliva, a cerebrospinal fluid or urine, and preferably means blood or serum.

According to one aspect of the present invention, the present invention provides a method of providing information necessary for diagnosing the likelihood of developing congenital glaucoma, hereditary calcification or skeletal dystrophy, comprising the steps of:

(a) detecting the expression of the mRNA of the DDX58 mutant gene or the DDX58 mutant protein encoded from the gene from the individual sample; And

(b) determining the probability of developing congenital glaucoma, hereditary calcification, or skeletal dystrophy in the subject when the expression of the mRNA of the DDX58 mutant gene or the DDX58 mutant protein encoded by the gene is detected in the individual sample .

According to one embodiment of the present invention, the mRNA expression in step (a) is measured using a primer pair or a probe that specifically binds to the DDX58 mutation gene.

The method using the primer and the probe of the present invention can detect DDX58 mutation very efficiently and easily in a sample.

According to one embodiment of the present invention, the protein expression in step (a) is measured using an antibody specific for the protein encoded by the DDX58 mutant gene.

The features and advantages of the present invention are summarized as follows:

(I) The present invention discloses that a mutation in the DDX58 gene is a mutation that causes congenital glaucoma, hereditary calcification, or skeletal dystrophy, and that the DDX58 mutant gene and the protein encoded thereby are congenital glaucoma, hereditary As a diagnostic marker for vascular calcification or skeletal dystrophy.

(Ii) In the present invention, a composition, a kit and a method for diagnosis of congenital glaucoma, hereditary vascular calcification or skeletal dystrophy using the DDX58 mutant gene and protein are provided.

(Iii) According to the present invention, it is possible to perform accurate early diagnosis through simple genetic test for congenital glaucoma, hereditary calcification or skeletal dystrophy, and it is possible to make customized treatment according to accurate diagnosis according to accurate diagnosis.

FIG. 1 is a diagram showing family lineage and body characteristics of a mutant for DDX58. FIG.
(a) Family line of family A with DDX58 p.E373 mutation. Square, male; Circle, female; Colored squares or circles, jamming bottles; Arrow, foot terminal; Diagonal, death; Question mark, unclear disease state.
(b) DDX58 family line of the family B with p.C268F mutation.
(c) Computed tomographic angiography (CTA) results, aortic calcification (black arrowheads) and calcification of the aortic valve (white arrowheads) were found in Family A II: 6 (left) and III: 7 (right). (d) Panoramic radiographs of Family A II: 6 and III: 7. No tooth abnormalities except mild tooth decay were found. (e) A picture of the family A Ⅲ: 7. Severe optic disc depression of both eyes was diagnosed as primary congenital glaucoma at the age of 3 years (left and upper right). Patients who underwent glaucomatous filtering surgery in both eyes were further treated with bilateral glaucoma implants.
(f) The result of photographing the skeleton of the hands and feet. Radiographs of Family A (FA) 33-year-old III and 2-year-olds (IV: 3) confirmed acro-osteolysis of the distal phalanges, Represents a triangle shape (arrow). Hand radiographs of son (Ⅳ: 3) and daughter (Ⅳ: 4, 5 years old) showed changes in erosion of terminal tufts resulting in dysplasia at all distal ends of the phalanges. At 3-4 years of age, limb calcification was delayed in Ⅳ: 3. The medullary cavities of the metacarpals and metatarsals were not found in the diseased family A all. In the family B (FB), radiographs of 61-year-old II: 4 revealed sprains, erosion of the big toe, and fusional melting of the tip of the phalanx, resulting in short toes (top). Family B Ⅲ: 2 is similar to the lower degree of erosion at the distal end of the phalanx. The thumb toe is triangular (upper, arrow). In the hand radiographs of family B Ⅱ: 4 and Ⅲ: 2, Ⅱ: 4 showed severe fusional melting and flexion contractures at the distal end of the phalanx, and Ⅲ: 2 showed a slight erosion change at the distal end of the phalanx. The palm and metatarsals do not spread in Family B.
Figure 2 is a diagram showing the ortholog preservation of the novel DDX58 mutant and the molecular structure of the RIG-I helicase Protein Data Bank ID 3ZD7).
A schematic representation of the DDX58 mutation associated with the protein domain. Alignment with the RIG-I protein sequence of vertebrates. Shows the alignment part corresponding to a missense mutation. Cys (C) at codon 268 and Glu (E) at codon 373 are well conserved across all species (open box). The RefSeq numbers for the RIG-I amino acid sequences aligned in the present invention are as follows: human, HIT000390839; Chimpanzee, ENSPTRT00000038562; Orangutans, ENSPPYT00000022324; Mouse, AK128929; Rats, ENSRNOT00000008465; Dogs, ENSCAFT00000002841; Horses, ENSECAT00000023725; Small, XM_580928; Zebrafish, ENSDART00000058276. Hel-2i is the two conserved core helicase domains, Hel-2i is the insertion domain conserved in the RIG-I-like helicase family, And P is a bridge region pincer that connects Hel-2 to the CTD (C-terminal domain) associated with the binding dsRNA.
FIG. 3 shows the results of measuring cytotoxicity by DDX58 mutation in HTM (human trabecular meshwork) cells.
(a) A phase contrast microscope photograph of HTM cells with DDX58 mutation. Scale bar = 100 μm. (b) FITC fluorescence (green) of RIG-I overlapped with DAPI (blue) is displayed in the left column, and Vimentin stained Cy3 image (red) superimposed with DAPI is displayed in the right column. Scale bar = 100 μm. (c) Cell viability is expressed as the ratio of living cells to wild type. Data are expressed as mean percentage ± standard deviation. * Indicates p < 0.01 compared to wild type.
Figure 4 shows the sequencing results of the exon of the DDX58 gene.
(a) showing the IGV browser view of the DDX58 gene region from the exome sequencing data. All of the diseased individuals in Family A have the c.1118A> C (p.E373A) mutation (arrow). (b) Identification of DDX58 mutation by sequencing of fish (arrow). (c) As a result of the seaweed sequencing, all the diseased persons in the family B have the c.803G> T (p.C268F) mutation (arrow).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

Individuals and families affected

In Family A, feet (Ⅱ: 6) and their sons (Ⅲ: 7) are patients with congenital glaucoma, vascular calcification and skeletal dysfunction. Other family members were evaluated with aortic and ophthalmic tests and / or skeletal examinations. Three patients in family B (II: 4, III: 2 and IV: 3) are patients with congenital glaucoma and skeletal dysfunction. GDNA (genomic DNA) was extracted from peripheral blood leukocytes under the permission of each individual. This study was approved by the Institutional Ethics Review Committee of Samsung Seoul Hospital (IRB # 2013-09-063).

Exome Sequencing

The gDNA samples from 5 patients (II: 6, III: 2, III: 7, IV: 3 and IV: 4) of family A and 2 normal individuals (II: 4 and II: 5) were analyzed by Agilent SureSelect Human All Exon Total exomes were obtained using v3 (Agilent Technologies, Santa Clara, Calif.) and sequenced using HiSeq2000 (Illumina, Inc., San Diego, Calif.) sequencing platform. Sequence reads were aligned to the hg19 human reference sequence using Burrows-Wheeler Aligner (BWA, version 0.6.2). The duplicated leads were removed with Picard and the local alignment was optimized with the Genome Analysis Tool kit (GATK). Single nucleotide variation and insertion-deletion were called with the GATK Unified Genotyper program and the decoy parameter was set at 5,000. GATK was used to filter variations according to the following conditions: allele balance> 0.8, quality <30, lead depth <25x, quality by depth <5, and window for clustered variation.

Sanger sequencing

A primer was designed to amplify the coding exon of the DDX58 gene (primers available upon request). The purified PCR amplification products were sequenced using the BigDye Terminator cycle sequencing Ready Reaction kit (Applied Biosystems, Foster City, Calif.) And the ABI 3730xl Genetic Analyzer (Applied Biosystems). The description of the mutation is based on the reference cDNA sequence NM_014314.3, which includes the nucleotide numbering starting from the first A in the ATG start codon.

Effect of RIG-I Mutation on HTM Cells

HTM cells derived from the normal eyes of 27-year-old female donors were obtained from Paul L. Kaufman (University of Wisconsin, Madison, WI). HTM cells were cultured in DMEM medium containing 10% FBS and antibiotics at 37 ° C, 5% CO ₂ -95% air wetting conditions. Replication-deficient recombinant adenoviruses carrying human wild-type and mutant genes of DDX58 were prepared using the AdEasy ™ XL adenoviral vector system (Agilent). To facilitate detection and visualization, all DDX58 genes were designed to be expressed as a fusion protein in which the FLAG epitope was tagged at its N-terminus. Viral amplification was performed in AD-293 cells (Agilent) and virus titer was determined using QuickTiter ™ adenovirus titer ELISA kits (Cell Biolabs, San Diego, Calif.). PCR confirmed the absence of the E1a gene in the virus construct. Viral infections were carried out for 2 hours with a multiplicity of infection (MOI) of 10-100 pfu (plaque-forming units) per cell, and the infected cells were further cultured for 72 hours.

To perform the immunochemical method, infected HTM cells were washed with PBS, fixed with 4% paraformaldehyde for 20 minutes, and then permeabilized with 0.5% Triton X-100 for 5 minutes. Cells were blocked with 2% bovine serum albumin for 1 hour and then incubated with 200-fold diluted anti-FLAG antibody (Santa Cruz Biotechnology) or 500-fold diluted Cy3-conjugated anti-vimentin antibody (Sigma-Aldrich) Lt; / RTI &gt; Cells were washed and reacted with 500-fold diluted FITC-conjugated secondary antibody (Life Technologies) for 2 hours. The nuclei were counterstained with DAPI (4 ', 6'-diamidino-2-phenylindole) for 20 minutes and then observed with a suitable filter set and 200X magnification under a fluorescence microscope.

For viability assay, infected HTM cells were treated with trypsin, and then the distant cells were mixed with 0.4% trypan blue. After 5 minutes, only the cells were counted using a disposable hemocytometer. Data were analyzed by one-way analysis of variance (ANOVA) followed by Newman-Keuls multiple comparison test, and p <0.05 was considered significant.

Experiment result

Recently, the present inventors have found a family (family A) exhibiting aortic calcification, glaucoma, and skeletal system abnormalities (Fig. 1A). To identify the causative genes, exome sequencing was performed on 5 patients and 2 normal individuals. The present inventors have found that a novel mutation in the DDX58 gene coding for a cytoplasmic helicase mediating induction of an IFN response to viral RNA (c (SEQ ID NO: 1)), except for the common polymorphism described in the dbSNP (Nucleotide Polymorphism database) . 1118A> C; p.E373A) (Fig. 2 and Fig. 4A, b) (5 1).

Because glaucoma is one of the major manifestations of the disease in Family A, we sequenced the entire coding exon for the DDX58 gene in 100 patients with primary congenital glaucoma (PCG) through a biofilm sequencing method. We have found another novel mutation (c.803G> T; p.C268F) in a female patient, her mother and maternal grandmother (family B) (FIG. 1B and FIG. 4C). All three showed acro-osteolysis, and the mother and grandmother were blinded because of congenital glaucoma. No dental abnormalities or cardiac calcification were observed in the family B. Detailed clinical characteristics of Families A and B are set forth in Table 1 and Figures 1c-f.

Clinical phenotypes of patients with DDX58-related disease phenotype - Family A Family B Case 1 (II: 6) 2 (III: 7) 3 (III: 1) 4 (III: 2) 5 (IV: 1) 6 (IV: 2) 7 (IV: 3) 8 (IV: 4) 9 (II: 4) 10 (III: 2) 11 (IV: 3) gender F M F M F F M F F F F Current age 56 22 35 33 15 13 8 6 61 38 20 Age at onset (years) 6 3 - - 6 4 4 2 10 3 2 First symptoms glaucoma glaucoma glaucoma none glaucoma glaucoma glaucoma glaucoma glaucoma glaucoma glaucoma Aortic & Valvular Calcification + + + + N / A N / A N / A N / A - - - Mediastinal calcification + + + + N / A N / A N / A N / A - - - Osteomalacia + - N / A - N / A N / A - - + - - Corrosion of horseshoe fissure or endometrial bones + + N / A + N / A N / A + + + + + Tooth problems ^a - ^b - ^b - - - - - - - - - glaucoma + + + - + + + + + + +

N / A, not available.

^a Tooth problem was assessed by a dentist with panoramic radiographs from two patients ^b of family A. Other problems are confirmed by physical examination.

The affected residues of p.C268F and p.E373A are strictly conserved across zebrafish-to-human organisms (Fig. 2), and the p.C268F and p.E373A variants are named Sorting Intolerant From Tolerant (SIFT 1.9 c) and Polymorphism phenotyping v2 (PolyPhen-2), the mutations are considered to be pathogenic. Also, the mutation was not found in 500 Koreans and was not found on 11,906 chromosomes of the NHLBI (National Heart, Lung, and Blood Institute) exome sequencing project database.

RIG-I, also known as DDX58 (Asp-Glu-Ala-Asp box polypeptide 58), is an 825-residue cytoplasmic viral RNA receptor and is associated with melanoma differentiation associated gene 5 (MDA5) and LGP2 (laboratory of genetics and physiology 2) RIG-I-like receptor family (2). RIG-I is an important intracellular sensor for several viruses, recognizing viral dsRNAs and causing an antiviral IFN response (1). RIG-I includes N-terminal CARDs (caspase activation recruitment domains) involved in activating mitochondrial antiviral signaling proteins (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).

We also investigated the effect of RIG-I mutations on primary HTM (human trabecular meshwork) cells. Previous studies have shown that mutations in the MYOC gene encoding myosylin cause glaucoma and that myosylin mutants can cause cytotoxicity in HTM cells (4-6). Since HTM cells excrete aqueous humor from the eye, the death of HTM cells may be a fundamental mechanism of glaucoma-induced elevation of IOP [7]. We transduced HTT cells with WT, two benign SNPs (p.I406T and p.S183I) and two pathogenic predictors (p.C268F and p.E373A) and a phase contrast microscope with p.C268F And the cytopathic effect of p.E373A mutation (Fig. 3A). Immunohistochemical staining revealed that green fluorescence was widely distributed throughout the cytoplasm, whereas HTM cells transfected with the p.C268F or p.E373A mutant showed a higher rate of vimentin compared to HTT cells transfected with WT and two SNPs Expression shrinkage (red fluorescence) was prominent (Fig. 3B). Vimentin is one of the five major groups of intermediate filament proteins, and vimentin contraction indicates that the stressed cells contract morphologically (8). In addition, the present inventors observed a remarkable decrease in cell number in cells transfected with p.C268F and p.E373A (Fig. 3C). This finding implies that DDX58 mutations such as the MYOC mutation show cytotoxic effects in HTM cells.

Type 1 IFN systems are very important for human antiviral immunity. However, excessive production or defective 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 a mutation in IFIH1 encoding MDA5 in patients with Aicardi-Gouti syndrome (AGS; MIM 225750) and found that these mutations lead to inadequate stimulation of type 1 IFN (9). AGS is an immune disease that affects the brain and skin, and the high induction of type I IFN-induced genes such as IFI27, IFI44L, IFIT1, ISG15, RSAD2, and SIGLEC1 is maintained constant (9). Funabiki et al. Reported that MDA5 dysregulation disorders induce autoimmune diseases without viral infection (10). Mutant mice with a single mismatch mutation (p.G821S) in IFIHl generated by the N-ethyl-N-nitroso urea mutation in the above study spontaneously developed lupus-like nephritis and systemic autoimmune symptoms (10). This finding implies that the IFIHl mutation activates downstream signaling for which no ligand is present, and paradoxically indicates that the mutation induces a morphological change in MDA5 without ligand- and virus-induced signaling.

We hypothesized that the clinical phenotype of patients with the DDX58 mutation is associated with the acquisition of RIG-I function, such as acquisition of MDA5 by IFIH1 mutation in AGS (9). Congenital glaucoma was an important sign in all patients except one, and three of these patients (family A to II: 6, family B to II: 4) And III: 2) were completely blinded. Aortic and vascular calcifications were observed only in Family A, and skeletal system abnormalities such as acro-osteolysis were more prominent in Family B.

The pathogenesis of aortic and vascular calcification found in family A with DDX58 p.E373A mutation is unknown. Cardiovascular calcification, once considered a passive degenerative disease, is now recognized as an active process (11). The chronic inflammatory stage, represented by increased production of pro-inflammatory cytokines induced by the acquisition of RIG-I function, appears to induce mineralization of the aorta and valves. 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 large vessel vasculitis, such as Takayasu's arteritis or giant cell arteritis. Indeed, in the diabetic LDLR - / - and murine models of calcified vascular disease, osteogenic transcription factors (Sox9, Runx2, Msx2, Osx) are activated when immune cytokines such as IL-6 and TNF are increased in the aortic wall 12, 15).

All patients of the present invention with DDX58 mutations did not show dental anomalies. Although the reason why the DDX58 mutation does not cause systemic inflammation or autoimmune disease is not well understood, it is believed that the negative regulatory mechanism plays a role in regulating activated RLR-IFN signaling. Further studies are needed to elucidate the precise mechanisms for the correlation between these genotypic-phenotypes.

In conclusion, the inventors have identified novel mutations in the DDX58 gene with various phenotypes including glaucoma, aortic calcification, and dysplasia except skeletal abnormalities.

references

1. Yoneyama, M. et al., Nat Immunol 5, 730-7 (2004).

2. Liu, F. & Gu, J. Protein Cell 2, 351-7 (2011).

3. Kowalinski, E. et al., Cell 147, 423-35 (2011).

4. Joe, MK et al., Biochem Biophys Res Commun 312, 592-600 (2003).

5. Alward, WL et al., N Engl J Med 338, 1022-7 (1998).

6. Stone, EM et al., Science 275, 668-70 (1997).

7. Alvarado, J., Murphy, C. & Juster, R. Ophthalmology 91, 564-79 (1984).

Minin, AA & Moldaver, MV Biochemistry (Mosc) 73, 1453-66 (2008).

9. Rice, GI et al., Nat Genet 46, 503-9 (2014).

10. Funabiki, M. et al., Immunity 40, 199-212 (2014).

11. New, SE & Aikawa, E. Circ Res 108, 1381-91 (2011).

12. Shao, JS, Cheng, SL, Sadhu, J. & Towler, DA Hypertension 55, 579-92 (2010).

13. Al-Aly, Z. et al., Arterioscler Thromb Vasc Biol 27, 2589-96 (2007).

14. Kohlway, A., Luo, D., Rawling, DC, Ding, SC & Pyle, AM EMBO Rep 14, 772-9 (2013).

15. Brooks, BR et al., J Comput Chem 30, 1545-614 (2009).

16. Im, W., Lee, MS & Brooks, CL, J. Comput. Chem. 24, 1691-702 (2003).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> SAMSUNG LIFE PUBLIC WELFARE FOUNDATION <120> DDX58 as A Causative Gene Responsible for Congenital Glaucoma,          Hereditary Vascular Calcification or Skeletal Abnormalities and          Diagnosis Method and Composition for the Disease <130> PN140394 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 4759 <212> RNA <213> Homo sapiens DDX58 mRNA <400> 1 cctcatttcc cctgctttcc ccgctcgcca cgccctcctc ctacccggct ttaaagctag tgaggcacag 120 cctgcgggga acgtagctag ctgcaagcag aggccggcat gaccaccgag cagcgacgca 180 gcctgcaagc cttccaggat tatatccgga agaccctgga ccctacctac atcctgagct 240 acatggcccc ctggtttagg gaggaagagg tgcagtatat tcaggctgag aaaaacaaca 300 agggcccaat ggaggctgcc acactttttc tcaagttcct gttggagctc caggaggaag 360 gctggttccg tggctttttg gatgccctag accatgcagg ttattctgga ctttatgaag 420 ccattgaaag ttgggatttc aaaaaaattg aaaagttgga ggagtataga ttacttttaa 480 aacgtttaca accagaattt aaaaccagaa ttatcccaac cgatatcatt tctgatctgt 540 ctgaatgttt aattaatcag gaatgtgaag aaattctaca gatttgctct actaagggga 600 tgatggcagg tgcagagaaa ttggtggaat gccttctcag atcagacaag gaaaactggc 660 ccaaaacttt gaaacttgct ttggagaaag aaaggaacaa gttcagtgaa ctgtggattg 720 tagagaaagg tataaaagat gttgaaacag aagatcttga ggataagatg gaaacttctg 780 acatacagat tttctaccaa gaagatccag aatgccagaa tcttagtgag aattcatgtc 840 caccttcaga agtgtctgat acaaacttgt acagcccatt taaaccaaga aattaccaat 900 tagagcttgc tttgcctgct atgaaaggaa aaaacacaat aatatgtgct cctacaggtt 960 gtggaaaaac ctttgtttca ctgcttatat gtgaacatca tcttaaaaaa ttcccacaag 1020 gacaaaaggg gaaagttgtc ttttttgcga atcagatccc agtgtatgaa cagcagaaat 1080 ctgtattctc aaaatacttt gaaagacatg ggtatagagt tacaggcatt tctggagcaa 1140 cagctgagaa tgtcccagtg gaacagattg ttgagaacaa tgacatcatc attttaactc 1200 cacagattct tgtgaacaac cttaaaaagg gaacgattcc atcactatcc atctttactt 1260 tgatgatatt tgatgaatgc cacaacacta gtaaacaaca cccgtacaat atgatcatgt 1320 ttaattatct agatcagaaa cttggaggat cttcaggccc actgccccag gtcattgggc 1380 tgactgcctc ggttggtgtt ggggatgcca aaaacacaga tgaagccttg gattatatct 1440 gcaagctgtg tgcttctctt gatgcgtcag tgatagcaac agtcaaacac aatctggagg 1500 aactggagca agttgtttat aagccccaga agtttttcag gaaagtggaa tcacggatta 1560 gcgacaaatt taaatacatc atagctcagc tgatgaggga cacagagagt ctggcaaaga 1620 gaatctgcaa agacctcgaa aacttatctc aaattcaaaa tagggaattt ggaacacaga 1680 aatatgaaca atggattgtt acagttcaga aagcatgcat ggtgttccag atgccagaca 1740 aagatgaaga gagcaggatt tgtaaagccc tgtttttata cacttcacat ttgcggaaat 1800 ataatgatgc cctcattatc agtgagcatg cacgaatgaa agatgctctg gattacttga 1860 aagacttctt cagcaatgtc cgagcagcag gattcgatga gattgagcaa gatcttactc 1920 agagatttga agaaaagctg caggaactag aaagtgtttc cagggatccc agcaatgaga 1980 atcctaaact tgaagacctc tgcttcatct tacaagaaga gtaccactta aacccagaga 2040 caataacaat tctctttgtg aaaaccagag cacttgtgga cgctttaaaa aattggattg 2100 aaggaaatcc taaactcagt tttctaaaac ctggcatatt gactggacgt ggcaaaacaa 2160 atcagaacac aggaatgacc ctcccggcac agaagtgtat attggatgca ttcaaagcca 2220 gtggagatca caatattctg attgccacct cagttgctga tgaaggcatt gacattgcac 2280 agtgcaatct tgtcatcctt tatgagtatg tgggcaatgt catcaaaatg atccaaacca 2340 gaggcagagg aagagcaaga ggtagcaagt gcttccttct gactagtaat gctggtgtaa 2400 ttgaaaaaga acaaataaac atgtacaaag aaaaaatgat gaatgactct attttacgcc 2460 ttcagacatg ggacgaagca gtatttaggg aaaagattct gcatatacag actcatgaaa 2520 aattcatcag agatagtcaa gaaaaaccaa aacctgtacc tgataaggaa aataaaaaac 2580 tgctctgcag aaagtgcaaa gccttggcat gttacacagc tgacgtaaga gtgatagagg 2640 aatgccatta cactgtgctt ggagatgctt ttaaggaatg ctttgtgagt agaccacatc 2700 ccaagccaaa gcagttttca agttttgaaa aaagagcaaa gatattctgt gcccgacaga 2760 actgcagcca tgactgggga atccatgtga agtacaagac atttgagatt ccagttataa 2820 aaattgaaag ttttgtggtg gaggatattg caactggagt tcagacactg tactcgaagt 2880 ggaaggactt tcattttgag aagataccat ttgatccagc agaaatgtcc aaatgatatc 2940 aggtcctcaa tcttcagcta cagggaatga gtaactttga gtggagaaga aacaaacata 3000 gtgggtataa tcatggatcg cttgtacccc tgtgaaaata tattttttaa aaatatcttt 3060 agcagtttgt actatattat atatgcaaag cacaaatgag tgaatcacag cactgagtat 3120 tttgtaggcc aacagagctc atagtacttg ggaaaaatta aaaagcctca tttctagcct 3180 tctttttaga gtcaactgcc aacaaacaca cagtaatcac tctgtacaca ctgggataga 3240 tgaatgaatg gaatgttggg aatttttatc tccctttgtc tccttaacct actgtaaact 3300 ggcttttgcc cttaacaatc tactgaaatt gttcttttga aggttaccag tgactctggt 3360 tgccaaatcc actgggcact tcttaacctt ctatttgacc tctgcgcatt tggccctgtt 3420 gagcactctt cttgaagctc tccctgggct tctctctctt ctagttctat tctagtcttt 3480 ttttattgag tcctcctctt tgctgatccc ttccaagggt tcaatatata tacatgtata 3540 tactgtacat atgtatatgt aactaatata catacataca ggtatgtata tgtaatggtt 3600 atatgtactc atgttcctgg tgtagcaacg tgtggtatgg ctacacagag aacatgagaa 3660 cataaagcca tttttatgct tactactaaa agctgtccac tgtagagttg ctgtatgtag 3720 caatgtgtat ccactctaca gtggtcagct tttagtagag agcataaaaa tgataaaata 3780 cttcttgaaa acttagttta ctatacatct tgccctatta atatgttctc ttaacgtgtg 3840 ccattgttct ctttgaccat tttcctataa tgatgttgat gttcaacacc tggactgaat 3900 gtctgttctc agatcccttg gatgttacag atgaggcagt ctgactgtcc tttctacttg 3960 aaagattaga atatgtatcc aaatggcatt cacgtgtcac ttagcaaggt ttgctgatgc 4020 ttcaaagagc ttagtttgcg gtttcctgga cgtggaaaca agtatctgag ttccctggag 4080 atcaacggga tgaggtgtta cagctgcctc cctcttcatg caatctggtg agcagtggtg 4140 caggcgggga gccagagaaa cttgccagtt atataacttc tctttggctt ttcttcatct 4200 gtaaaacaag gataatactg aactgtaagg gttagtggag agtttttaat taaaagaatg 4260 tgtgaaaagt acatgacaca gtagttgctt gataatagtt actagtagta gtattcttac 4320 taagacccaa tacaaatgga ttatttaaac caagtttatg agttggtttt ttttcatttt 4380 ctatttgtat tttattaaga gtgtcttttc ttatgtgatt ttttttaatt gctatttgat 4440 atggtttggc tatatgtccc cacccaaatc tcatcttgaa ttataatccc catgtgtcaa 4500 gggagggacc tgacgggagg tgattggatc acgggggcag ttgtccccat gctgttcttg 4560 ggatagtgag ttagttctca tgagatctga tggttttata agtgtttgac aattcctcct 4620 ttacacacac tctctctctc atctgctgcc atgtaagact tgcctgcttc cccttctgcc 4680 atgattgtaa gtttcctgag gcctcctcag ccatgtggaa ctgtgaatct attaagcctc 4740 ttttctttat aaatgaaaa 4759 <210> 2 <211> 925 <212> PRT <213> Homo sapiens DDX58 protein <400> 2 Met Thr Thr Glu Gln Arg Arg Ser Leu Gln Ala Phe Gln Asp Tyr Ile   1 5 10 15 Arg Lys Thr Leu Asp Pro Thr Tyr Ile Leu Ser Tyr Met Ala Pro Trp              20 25 30 Phe Arg Glu Glu Glu Val Gln Tyr Ile Gln Ala Glu Lys Asn Asn Lys          35 40 45 Gly Pro Met Glu Ala Ala Thr Leu Phe Leu Lys Phe Leu Leu Glu Leu      50 55 60 Glu Glu Glu Gly Trp Phe Arg Gly Phe Leu Asp Ala Leu Asp His Ala  65 70 75 80 Gly Tyr Ser Gly Leu Tyr Glu Ala Ile Glu Ser Trp Asp Phe Lys Lys                  85 90 95 Ile Glu Lys Leu Glu Glu Tyr Arg Leu Leu Leu Lys Arg Leu Gln Pro             100 105 110 Glu Phe Lys Thr Arg Ile Ile Pro Thr Asp Ile Ile Ser Asp Leu Ser         115 120 125 Glu Cys Leu Ile Asn Gln Glu Cys Glu Glu Ile Leu Gln Ile Cys Ser     130 135 140 Thr Lys Gly Met Met Ala Gly Ala Glu Lys Leu Val Glu Cys Leu Leu 145 150 155 160 Arg Ser Asp Lys Glu Asn Trp Pro Lys Thr Leu Lys Leu Ala Leu Glu                 165 170 175 Lys Glu Arg Asn Lys Phe Ser Glu Leu Trp Ile Val Glu Lys Gly Ile             180 185 190 Lys Asp Val Glu Thr Glu Asp Leu Glu Asp Lys Met Glu Thr Ser Asp         195 200 205 Ile Gln Ile Phe Tyr Gln Glu Asp Pro Glu Cys Gln Asn Leu Ser Glu     210 215 220 Asn Ser Cys Pro Pro Ser Glu Val Ser Asp Thr Asn Leu Tyr Ser Pro 225 230 235 240 Phe Lys Pro Arg Asn Tyr Gln Leu Glu Leu Ala Leu Pro Ala Met Lys                 245 250 255 Gly Lys Asn Thr Ile Ile Cys Ala Pro Thr Gly Cys Gly Lys Thr Phe             260 265 270 Val Ser Leu Leu Ile Cys Glu His His Leu Lys Lys Phe Pro Gln Gly         275 280 285 Gln Lys Gly Lys Val Val Phe Phe Ala Asn Gln Ile Pro Val Tyr Glu     290 295 300 Gln Gln Lys Ser Val Phe Ser Lys Tyr Phe Glu Arg His Gly Tyr Arg 305 310 315 320 Val Thr Gly Ile Ser Gly Ala Thr Gly Asn Val Val Glu Gln                 325 330 335 Ile Val Glu Asn Asn Asp Ile Ile Leu Thr Pro Gln Ile Leu Val             340 345 350 Asn Asn Leu Lys Lys Gly Thr Ile Pro Ser Leu Ser Ile Phe Thr Leu         355 360 365 Met Ile Phe Asp Glu Cys His Asn Thr Ser Lys Gln His Pro Tyr Asn     370 375 380 Met Ile Met Phe Asn Tyr Leu Asp Gln Lys Leu Gly Gly Ser Ser Gly 385 390 395 400 Pro Leu Pro Gln Val Ile Gly Leu Thr Ala Ser Val Gly Val Gly Asp                 405 410 415 Ala Lys Asn Thr Asp Glu Ala Leu Asp Tyr Ile Cys Lys Leu Cys Ala             420 425 430 Ser Leu Asp Ala Ser Val Ile Ala Thr Val Lys His Asn Leu Glu Glu         435 440 445 Leu Glu Gln Val Val Tyr Lys Pro Gln Lys Phe Phe Arg Lys Val Glu     450 455 460 Ser Arg Ile Ser Asp Lys Phe Lys Tyr Ile Ile Ala Gln Leu Met Arg 465 470 475 480 Asp Thr Glu Ser Leu Ala Lys Arg Ile Cys Lys Asp Leu Glu Asn Leu                 485 490 495 Ser Gln Ile Gln Asn Arg Glu Phe Gly Thr Gln Lys Tyr Glu Gln Trp             500 505 510 Ile Val Thr Val Gln Lys Ala Cys Met Val Phe Gln Met Pro Asp Lys         515 520 525 Asp Glu Glu Ser Arg Ile Cys Lys Ala Leu Phe Leu Tyr Thr Ser His     530 535 540 Leu Arg Lys Tyr Asn Asp Ala Leu Ile Ile Ser Glu His Ala Arg Met 545 550 555 560 Lys Asp Ala Leu Asp Tyr Leu Lys Asp Phe Phe Ser Asn Val Arg Ala                 565 570 575 Ala Gly Phe Asp Glu Ile Glu Gln Asp Leu Thr Gln Arg Phe Glu Glu             580 585 590 Lys Leu Gln Glu Leu Glu Ser Val Ser Arg Asp Pro Ser Asn Glu Asn         595 600 605 Pro Lys Leu Glu Asp Leu Cys Phe Ile Leu Gln Glu Glu Tyr His Leu     610 615 620 Asn Pro Glu Thr Ile Thr Ile Leu Phe Val Lys Thr Arg Ala Leu Val 625 630 635 640 Asp Ala Leu Lys Asn Trp Ile Glu Gly Asn Pro Lys Leu Ser Phe Leu                 645 650 655 Lys Pro Gly Ile Leu Thr Gly Arg Gly Lys Thr Asn Gln Asn Thr Gly             660 665 670 Met Thr Leu Pro Ala Gln Lys Cys Ile Leu Asp Ala Phe Lys Ala Ser         675 680 685 Gly Asp His Asn Ile Leu Ile Ala Thr Ser Val Ala Asp Glu Gly Ile     690 695 700 Asp Ile Ala Gln Cys Asn Leu Val Ile Leu Tyr Glu Tyr Val Gly Asn 705 710 715 720 Val Ile Lys Met Ile Gln Thr Arg Gly Arg Gly Arg Ala Arg Gly Ser                 725 730 735 Lys Cys Phe Leu Leu Thr Ser Asn Ala Gly Val Ile Glu Lys Glu Gln             740 745 750 Ile Asn Met Tyr Lys Glu Lys Met Met Asn Asp Ser Ile Leu Arg Leu         755 760 765 Gln Thr Trp Asp Glu Ala Val Phe Arg Glu Lys Ile Leu His Ile Gln     770 775 780 Thr His Glu Lys Phe Ile Arg Asp Ser Gln Glu Lys Pro Lys Pro Val 785 790 795 800 Pro Asp Lys Glu Asn Lys Lys Leu Leu Cys Arg Lys Cys Lys Ala Leu                 805 810 815 Ala Cys Tyr Thr Ala Asp Val Arg Ile Glu Glu Cys His Tyr Thr             820 825 830 Val Leu Gly Asp Ala Phe Lys Glu Cys Phe Val Ser Arg Pro His Pro         835 840 845 Lys Pro Lys Gln Phe Ser Ser Phe Glu Lys Arg Ala Lys Ile Phe Cys     850 855 860 Ala Arg Gln Asn Cys Ser His Asp Trp Gly Ile His Val Lys Tyr Lys 865 870 875 880 Thr Phe Glu Ile Pro Val Ile Lys Ile Glu Ser Phe Val Val Glu Asp                 885 890 895 Ile Ala Thr Gly Val Gln Thr Leu Tyr Ser Lys Trp Lys Asp Phe His             900 905 910 Phe Glu Lys Ile Pro Phe Asp Pro Ala Glu Met Ser Lys         915 920 925

Claims (11)

A DDX58 mutant gene in which the 1118th base adenine substituted with cytosine or the 803th base guanine is substituted with thymine is a diagnostic marker for glaucoma, horsesolubus, or endometrial erosion.
A DDX58 mutant protein encoded from the DDX58 mutation gene according to claim 1 as a diagnostic marker for glaucoma, horseradish or horseshoe erosion.
3. The method according to claim 2, wherein glutamic acid, which is the 373rd amino acid in the amino acid sequence of the second sequence of the sequence listing, is substituted with alanine, or cysteine, which is the 268th amino acid in the amino acid sequence of the second sequence of the sequence listing, is substituted with phenylalanine The DDX58 mutant protein.
1. A diagnostic composition for glaucoma, endoluminal or endoleal corrosion, comprising an agent capable of detecting the expression of the gDNA, mRNA or DDX58 mutant protein encoded by the DDX58 mutant gene according to claim 1 from the individual sample.
5. The diagnostic composition according to claim 4, wherein the agent capable of detecting the gDNA or mRNA expression is a primer pair or a probe that specifically binds to the DDX58 mutation gene.
The diagnostic composition according to claim 4, wherein the agent capable of detecting the expression of the protein is an antibody specific to a protein encoded by the DDX58 mutation gene.
A kit for the diagnosis of glaucoma, horsesolubus or equine erosion comprising the composition of any one of claims 4 to 6.
A method of providing information necessary to diagnose the potential for development of glaucoma, horsesolubage, or horseshoe erosion comprising the steps of:
(a) detecting the expression of the gDNA, mRNA of the DDX58 mutant gene or the DDX58 mutant protein encoded from the gene from the individual sample; And
(b) when the expression of the gDNA, mRNA of the DDX58 mutant gene, or the DDX58 mutant protein encoded by the gene is detected in the individual sample, it is determined that the individual has a high possibility of developing glaucoma, .
9. The method according to claim 8, wherein in step (a), the gDNA, mRNA expression is measured using a primer pair or a probe that specifically binds to the DDX58 mutation gene.
9. The method of claim 8, wherein the protein expression in step (a) is measured using an antibody specific for the protein encoded from the DDX58 mutant gene. The DDX58 mutant gene according to claim 1, wherein the DDX58 mutant gene in which the adenine at position 1118 base in the nucleotide sequence shown in the Sequence Listing Sequence 1 is substituted with cytosine can be used as a diagnostic marker for vascular calcification.

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