WO2019129029A1 - Gène utilisé comme marqueur pour diagnostiquer et traiter la maladie de parkinson, et utilisation correspondante - Google Patents

Gène utilisé comme marqueur pour diagnostiquer et traiter la maladie de parkinson, et utilisation correspondante Download PDF

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WO2019129029A1
WO2019129029A1 PCT/CN2018/123794 CN2018123794W WO2019129029A1 WO 2019129029 A1 WO2019129029 A1 WO 2019129029A1 CN 2018123794 W CN2018123794 W CN 2018123794W WO 2019129029 A1 WO2019129029 A1 WO 2019129029A1
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gene
protein
detecting
product
disease
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PCT/CN2018/123794
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English (en)
Chinese (zh)
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肖枫
汪冰怡
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北京泱深生物信息技术有限公司
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Priority claimed from CN201711481574.5A external-priority patent/CN108103183B/zh
Priority claimed from CN201711477253.8A external-priority patent/CN107904304B/zh
Priority claimed from CN201711481575.XA external-priority patent/CN108220419B/zh
Priority claimed from CN201711483559.4A external-priority patent/CN108048554B/zh
Priority claimed from CN201810292179.0A external-priority patent/CN108611413B/zh
Application filed by 北京泱深生物信息技术有限公司 filed Critical 北京泱深生物信息技术有限公司
Publication of WO2019129029A1 publication Critical patent/WO2019129029A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention belongs to the field of molecular diagnosis, and relates to a gene as a marker for diagnosis and treatment of Parkinson's disease and a use thereof.
  • PD is the second most common, slow-onset, age-related neurodegenerative disease following Alzheimer's disease, also known as tremor.
  • Pathologically the selective loss of dopaminergic neurons (DNs) in the substantia nigra, the formation of Lewy body (LB) in the remaining neurons, and the reduction of striatum dopamine content, resulting in resting tremor, Four major clinical symptoms of muscle rigidity, bradykinesia, and abnormal posture gait (Braak H, Tedici K D, et al. Staging of brain pathology related to sporadic Parkinson, s disease. Neurobiology of Aging, 2003, 24(2) :197-211).
  • PD patients account for 3% of the total population, accounting for 1% of the population over 60 years old, and 8-18 PD patients per 100,000 people worldwide every year (de Lau L M, Breteler MM. Epidemiology of parkinson's Disease. The Lancet Neurology, 2006, 5(6): 525-535). Stephen (K S. Incidence of parkinson's Disease: Variation by age, Gender, and Race/Ethnicity, 2003: 157, 1015-1022) studied the relationship between PD incidence and age, gender, and ethnicity, and found incidence rates above 60 years of age. It will increase rapidly, and only 4% of cases under 50 years old; 1.9 times of male patients are female; the incidence of PD in different races is changing. In general, the incidence of white PD is higher than that of Asians, and blacks are black. It is the lowest.
  • the Unified Pakinson's Disease Rating Scale (UPDRS) and the modified Hoehn and Yahr Parkinson's Disease Staging Scale (HY staging) are clinical diagnostics for PD. And common methods of treatment evaluation.
  • exclusion of brain trauma, cerebrovascular disease, metal poisoning, etc. can cause the cause of secondary Parkinson's disease, and there are no signs of extraocular muscle paralysis, cerebellar sign, cone system damage and muscle atrophy, according to the British brain.
  • the library's diagnostic criteria for Parkinson's disease, patients with less movement, plus one of three symptoms of static tremor, myotonia, and abnormal posture gait can be diagnosed as PD.
  • decreased levels of dopamine and its metabolites in urine and cerebrospinal fluid, and a good response to the drug levodopa also help distinguish between PD and other neurodegenerative diseases.
  • Neurodegenerative diseases including Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis, which are characterized by the storage and specialization of abnormal proteins in different parts of the central nervous system.
  • Loss of sex neurons Ross C A, Poirier M A. Protein aggregation and neurodegenerative disease. Nat Med, 2004, 10 Suppl: S10-S17
  • Neurodegenerative diseases occur in the brain because of pathological changes, and brain tissue is obtained after necropsy after death, so the pathological mechanism is progressing slowly. In general, neurodegenerative diseases are the result of a combination of genes and the environment.
  • the gene expression regulatory network system has made remarkable progress in the field of cancer research.
  • tumor tissues such as lung cancer, breast cancer, gastric cancer, colorectal cancer, etc.
  • the expression levels of certain genes are stable and distinct from the normal tissues of the source.
  • Tumors of different tissue origins even tumors with different differentiation status of the same tissue source, have different gene expression profiles, indicating that gene expression has obvious tissue specificity and developmental stage specificity.
  • the pathological direct feature of neurodegenerative diseases is the overexpression of disease-specific proteins, it is speculated that abnormalities in specific genes alter the expression levels of functional proteins, thereby causing diseases.
  • a molecular marker that can be used for early diagnosis of Parkinson's disease.
  • the use of genetic markers to diagnose Parkinson's disease is timely, specific and sensitive, so that patients can know the disease risk in the early stage of the disease, and take appropriate measures for the risk level. Preventive and therapeutic measures.
  • the invention provides the use of a gene in the preparation of a tool for the diagnosis of Parkinson's disease, the gene being selected from the group consisting of THBD, KCTD20, DNASE2, NACC2, FGFBP2.
  • the present invention provides the use of a product for detecting a gene as described above for the preparation of a tool for the diagnosis of Parkinson's disease.
  • the product for detecting the gene described above includes a product for detecting expression of the gene described above.
  • the product for detecting expression of the gene described above includes a product for detecting the mRNA expression level of the aforementioned gene.
  • the product for detecting the expression level of the mRNA of the aforementioned gene comprises a product for detecting the mRNA expression level of the aforementioned gene by reverse transcription PCR, real-time quantitative PCR, in situ hybridization, gene chip or high-throughput sequencing platform.
  • the product for detecting the expression level of the mRNA of the aforementioned gene by the reverse transcription PCR comprises at least one pair of primers for specifically amplifying the gene described above; and the product for detecting the expression level of the mRNA of the aforementioned gene by the real-time quantitative PCR includes at least a pair of primers for specifically amplifying the aforementioned gene; the product for detecting the expression level of the mRNA of the aforementioned gene by the in situ hybridization comprises: a probe which hybridizes with the nucleic acid sequence of the gene described above; the gene chip detects the aforementioned Products of gene mRNA expression levels include probes that hybridize to the nucleic acid sequences of the genes described above.
  • the real-time quantitative PCR detects at least one pair of primers for specifically amplifying the aforementioned gene, and the primer is:
  • the primer sequence for specifically amplifying the THBD gene is shown in SEQ ID NOS. 1 to 2;
  • the primer sequences for specifically amplifying the KCTD20 gene are shown in SEQ ID NOS. 3 to 4;
  • the primer sequences for specifically amplifying the DNASE2 gene are shown in SEQ ID NOS. 5 to 6;
  • the primer sequences for specifically amplifying the NACC2 gene are shown in SEQ ID NOS. 7 to 8;
  • the primer sequence for specifically amplifying the FGFBP2 gene is shown in SEQ ID NOS. 9 to 10.
  • the product for detecting expression of the aforementioned gene comprises a product for detecting the expression level of a protein encoded by the aforementioned gene.
  • the product for detecting the expression level of the gene-encoded protein described above includes a product for detecting the expression level of the protein encoded by the aforementioned gene by ELISA, immunoblotting, or protein chip.
  • the ELISA detects a product of a protein-coding protein expression level of the aforementioned gene, comprising: an antibody or a ligand that specifically binds to a protein encoded by the aforementioned gene; and the immunoblot detects a product of a protein expression level of the aforementioned gene, including An antibody or a ligand which specifically binds to a protein encoded by a gene as described above; the protein chip detects a product of a protein expression level of the aforementioned gene, and comprises an antibody or a ligand which specifically binds to a protein encoded by the gene described above.
  • the present invention provides a tool for diagnosing Parkinson's disease that is capable of diagnosing Parkinson's disease by detecting a gene described above in a sample.
  • the tool is capable of diagnosing Parkinson's disease by detecting gene expression as described above in the sample.
  • the tool is capable of diagnosing Parkinson's disease by detecting mRNA expression of a gene as described above in the sample, and/or by encoding the protein encoded by the aforementioned gene.
  • the tool comprises a chip, a kit, a test strip or a high throughput sequencing platform.
  • the high-throughput sequencing platform is a special diagnostic tool, and the product detecting the gene expression described above can be applied to the platform to realize the detection of the expression of the aforementioned gene.
  • the chip comprises a gene chip, a protein chip;
  • the gene chip comprises a solid phase carrier and an oligonucleotide probe immobilized on a solid phase carrier, wherein the oligonucleotide probe comprises a gene for detecting the aforementioned gene Transcriptional level of an oligonucleotide probe directed against a gene as described above;
  • the protein chip comprising a solid phase carrier and a specific antibody or ligand immobilized on the solid phase vector of the gene encoding the protein;
  • the gene chip is available The expression level of a plurality of genes including the aforementioned genes (for example, a plurality of genes associated with Parkinson's disease) is detected.
  • the protein chip can be used to detect expression levels of a plurality of proteins (e.g., a plurality of proteins associated with Parkinson's disease) including the protein encoded by the aforementioned gene. By simultaneously detecting multiple markers of Parkinson's disease, the accuracy of Parkinson's disease diagnosis can be greatly improved.
  • a plurality of proteins e.g., a plurality of proteins associated with Parkinson's disease
  • the kit comprises a gene detection kit and a protein immunoassay kit;
  • the gene detection kit comprises an reagent for detecting a transcription level of the gene described above;
  • the protein immunoassay kit comprises the gene coding described above Protein specific antibody.
  • the reagent includes a reagent required for detecting a gene expression level of the aforementioned gene using RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or a microarray method.
  • the reagent comprises a primer and/or a probe for the gene described above. Primers and probes which can be used to detect the expression levels of the aforementioned genes are easily designed based on the nucleotide sequence information of the aforementioned genes.
  • the probe that hybridizes to the nucleic acid sequence of the aforementioned gene may be DNA, RNA, DNA-RNA chimera, PNA or other derivative.
  • the length of the probe is not limited, and any length can be used as long as the specific hybridization is completed and specifically bound to the nucleotide sequence of interest.
  • the length of the probe can be as short as 25, 20, 15, 13 or 10 bases in length. Also, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or longer, or even the entire gene.
  • the length of the probe is usually at least 14 base pairs, and the longest is generally no more than 30 base pairs, and the nucleotide sequence of interest Complementary lengths are best at 15-25 base pairs.
  • the probe self-complementary sequence is less than 4 base pairs so as not to affect the efficiency of hybridization.
  • the high throughput sequencing platform includes reagents for detecting the level of transcription of a gene as described above.
  • the test paper includes a test paper carrier and an oligonucleotide immobilized on a test paper carrier capable of detecting the transcription level of the aforementioned gene.
  • the specific antibody encoding the protein encoded by the aforementioned gene includes a monoclonal antibody and a polyclonal antibody.
  • Specific antibodies to the previously encoded genes include intact antibody molecules, any fragments or modifications of antibodies (eg, chimeric antibodies, scFv, Fab, F(ab')2, Fv, etc. as long as the fragments are capable of retaining The ability to bind to the gene-encoded protein described above is sufficient.
  • the preparation of antibodies for protein levels is well known to those skilled in the art, and the present invention can be used to prepare the antibodies using any method.
  • the primer sequence for the gene described above is:
  • the primer sequences for the THBD gene are shown in SEQ ID NOS. 1 to 2;
  • the primer sequences for the KCTD20 gene are shown in SEQ ID NOS. 3 to 4.
  • Primer sequences for the DNASE2 gene are set forth in SEQ ID NOS. 5-6;
  • Primer sequences for the NACC2 gene are set forth in SEQ ID NOS. 7-8;
  • Primer sequences directed against the FGFBP2 gene are set forth in SEQ ID NOS. 9-10.
  • Sources of the aforementioned genes and their expression products for diagnosing Parkinson's disease include, but are not limited to, blood, tissue fluid, urine, saliva, spinal fluid, and the like which can obtain genomic DNA.
  • the source of the aforementioned gene and its expression product for diagnosing Parkinson's disease is blood.
  • the present invention provides the use of the aforementioned gene or an expression product thereof for screening a drug for treating Parkinson's disease.
  • the test compound was added to the culture system, and the expression level of the above-described gene was determined; in the control group, the test compound was not added to the same culture system, and the aforementioned gene was determined.
  • the expression level wherein, if the expression level of the aforementioned gene in the experimental group is smaller than that of the control group, it indicates that the substance to be screened is a drug that inhibits the gene described above.
  • the method further comprises: further testing the drug obtained in the above step to inhibit the effect of Parkinson, and if the test compound has a significant inhibitory effect on Parkinson, the compound is for preventing or treating Parkinson's. drug.
  • the culture system includes, but is not limited to, a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system (such as an animal model, preferably an animal model of a non-human mammal such as a mouse, a rabbit, a sheep, or a monkey. and many more.
  • a compound isolated by the screening method of the present invention is administered as a medicament to a human or other mammal, including but not limited to a mouse, a rat, a guinea pig, a rabbit, a cat, a dog, a sheep, a pig, a cow, a monkey, a donkey,
  • the isolated compounds may be administered directly or may be formulated into various dosage forms using known pharmaceutical preparation methods.
  • the drug may be administered orally as a sugar-coated tablet, a capsule, an elixir, and a microcapsule, as needed; or a sterile solution or suspension with water or any other pharmaceutically acceptable liquid, orally in the form of an injection. Apply.
  • the compound may be mixed in a unit dosage form, such as, but not limited to, sterile water, physiological saline, in a unit dosage required for the generally accepted pharmaceutical administration.
  • a unit dosage form such as, but not limited to, sterile water, physiological saline, in a unit dosage required for the generally accepted pharmaceutical administration.
  • Vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, and the like According to the content of the active ingredient in these preparations, a suitable administration amount within a specified range can be obtained.
  • the present invention provides the use of the aforementioned gene and/or its expression product for the preparation of a medicament for treating Parkinson's disease.
  • the medicament includes an inhibitor of the aforementioned gene and/or an expression product thereof.
  • the inhibitor includes a substance which inhibits expression of the gene described above, a substance which affects the stability of the gene expression product described above, and/or a substance which inhibits the activity of the gene expression product described above.
  • the inhibitor comprises: a double-stranded ribonucleic acid which inhibits expression of a gene described above by interfering with RNA, or a tumor vaccine based on a protein encoded by the aforementioned gene, or for inhibiting a protein encoded by the aforementioned gene Active protein.
  • the inhibitor is an siRNA directed against the aforementioned gene.
  • RNA interference refers to endogenous or exogenous double-stranded RNA (dsRNA)-mediated degradation of intracellular mRNA, resulting in a target gene. Silence of expression results in a corresponding loss of functional phenotype.
  • RNAi technology is a typical negative regulation mechanism that can specifically knock out or turn off the expression of specific genes. This technology has been widely used to explore gene function, gene therapy and new drug development.
  • Cell-based RNAi screening has many advantages in functional genetics studies, mainly in the use of RNAi methods in most cell types, and relatively easy to down-regulate or silence expression of the gene of interest.
  • siRNA-specific fragments were designed based on the mRNA sequences of the genes described above.
  • the design of siRNA is based on published general design principles (Elbashir et. al 2001, Schwarz et. al 2003, Khvorova et. al 2003, Reynolds et. al 2004, Hsieh et. al 2004, Ui-Tei et.
  • the present invention provides a pharmaceutical composition for treating Parkinson, which comprises siRNA against the aforementioned gene and a pharmaceutically acceptable carrier.
  • sequence of the siRNA is shown in SEQ ID NO. 15 to 32.
  • the pharmaceutically acceptable carrier in the present invention includes, but is not limited to, a diluent, an excipient such as water, a filler such as starch, sucrose, etc.; a binder such as a cellulose derivative, an alginate, gelatin, and polyethylene. Pyrrolidone; wetting agent such as glycerin; disintegrating agent such as agar, calcium carbonate and sodium hydrogencarbonate; absorption enhancer quaternary ammonium compound; surfactant such as cetyl alcohol; adsorption carrier such as kaolin and soap clay; lubricant such as talc, Calcium stearate and magnesium, polyethylene glycol and the like.
  • a diluent an excipient such as water, a filler such as starch, sucrose, etc.
  • a binder such as a cellulose derivative, an alginate, gelatin, and polyethylene.
  • Pyrrolidone such as glycerin
  • disintegrating agent such as agar,
  • the medicament of the present invention can also be combined with other drugs for treating Parkinson's, and the combination of a plurality of drugs can greatly mention the success rate of the treatment.
  • a "DNASE2 gene” includes a polynucleotide of the DNASE2 gene and any functional equivalent of the DNASE2 gene.
  • the DNASE2 gene (Chromosome 19, NC_000019.10 (12875211..12881521, complement)) sequence can be found in the international public nucleic acid sequence database GeneBank.
  • THBD gene includes a BRAD gene as well as a polynucleotide of any functional equivalent of the THBD gene.
  • the THBD gene (Chromosome 20, NC_000020.11 (23045633.. 23049664, complement)) sequence can be found in the international public nucleic acid sequence database GeneBank.
  • the "KCTD20 gene” includes a polynucleotide of the KCTD20 gene and any functional equivalent of the KCTD20 gene.
  • the KCTD20 gene (Chromosome 6, NC_000006.12 (36442767.. 36491143)) sequence can be found in the international public nucleic acid sequence database GeneBank.
  • a "NACC2 gene” includes a polynucleotide of the NACC2 gene and any functional equivalent of the NACC2 gene.
  • the NACC2 gene (Chromosome 9, NC_000009.12 (136006537..136095285, complement)) sequence can be found in the international public nucleic acid sequence database GeneBank.
  • the "FGFBP2 gene” includes the human FGFBP2 gene and a polynucleotide equivalent to any function of the human FGFBP2 gene.
  • the sequence of a representative FGFBP2 gene has the same DNA sequence as the FGFBP2 gene (NM_031950.3) in the current international public nucleic acid sequence database GeneBank.
  • gene expression products include proteins as well as partial peptides of proteins.
  • a partial peptide of the protein contains a functional domain associated with Parkinson's disease.
  • DNASE2 protein includes the DNASE2 protein as well as any functional equivalent of the DNASE2 protein.
  • the functional equivalents include a DNASE2 protein conserved variant protein, or an active fragment thereof, or a reactive derivative thereof, an allelic variant, a natural mutant, an inducible mutant, DNA capable of binding to DNASE2 under high or low stringency conditions A protein encoded by a hybridized DNA.
  • THBD protein includes the THTHB protein as well as any functional equivalent of the THBD protein.
  • the functional equivalents include a conservative variant protein of the THBD protein, or an active fragment thereof, or a reactive derivative thereof, an allelic variant, a natural mutant, an inducible mutant, a DNA capable of interacting with THBD under high or low stringency conditions A protein encoded by a hybridized DNA.
  • KCTD20 protein includes the KCTD20 protein as well as any functional equivalent of the KCTD20 protein.
  • the functional equivalents include a KCTD20 protein conserved variant protein, or an active fragment thereof, or a reactive derivative thereof, an allelic variant, a natural mutant, an inducible mutant, and a DNA capable of interacting with KCTD20 under high or low stringency conditions.
  • a protein encoded by a hybridized DNA includes a KCTD20 protein conserved variant protein, or an active fragment thereof, or a reactive derivative thereof.
  • NACC2 protein includes the NACC2 protein as well as any functional equivalent of the NACC2 protein.
  • the functional equivalents include a conservative variant protein of the NACC2 protein, or an active fragment thereof, or a reactive derivative thereof, an allelic variant, a natural mutant, an inducible mutant, and a DNA capable of interacting with NACC2 under high or low stringency conditions.
  • a protein encoded by a hybridized DNA includes a conservative variant protein of the NACC2 protein, or an active fragment thereof, or a reactive derivative thereof, an allelic variant, a natural mutant, an inducible mutant, and a DNA capable of interacting with NACC2 under high or low stringency conditions.
  • FGFBP2 protein includes the FGFBP2 protein as well as any functional equivalent of the FGFBP2 protein.
  • the functional equivalents include a FGFBP2 protein conserved variant protein, or an active fragment thereof, or a reactive derivative thereof, an allelic variant, a natural mutant, an inducible mutant, and a DNA capable of interacting with FGFBP2 under high or low stringency conditions.
  • a protein encoded by a hybridized DNA is a protein that encoded by a hybridized DNA.
  • modification of one or more amino acids in a protein does not affect the function of the protein.
  • One skilled in the art will recognize that altering a single amino acid or a small percentage of amino acids or individual additions, deletions, insertions, substitutions to an amino acid sequence is a conservative modification in which a protein change produces a protein with similar function.
  • Conservative substitution tables that provide functionally similar amino acids are well known in the art.
  • a protein modified by the addition of one amino acid or a plurality of amino acid residues is a fusion protein of a protein encoded by the aforementioned gene.
  • a fusion protein of a protein encoded by the aforementioned gene There is no limitation on the peptide or protein fused to the gene-encoded protein described above, as long as the resulting fusion protein retains the biological activity of the protein.
  • diagnosing Parkinson's disease includes both determining whether a subject already has Parkinson's disease and also determining whether the subject is at risk of having Parkinson's disease.
  • the present invention finds for the first time that the expression of THBD, KCTD20, DNASE2, NACC2, and FGFBP2 is associated with Parkinson's disease.
  • detecting the expression of the former gene in the subject it is possible to determine whether the subject has Parkinson's disease or to judge the subject. Whether there is a risk of Parkinson's disease, instructing the clinician to provide a preventive or treatment plan for the subject.
  • the invention discovers a new molecular marker, which is more timely, more specific and more sensitive than the traditional detection means, and can realize the early diagnosis of Parkinson's disease, thereby reducing the mortality of Parkinson's disease.
  • Figure 1 shows the difference in expression of DNASE2 gene in Parkinson's disease patients and normal persons using a gene chip
  • Figure 2 shows the difference in expression of DNASE2 gene in Parkinson's disease patients and normal persons by QPCR
  • Figure 3 shows the difference in expression of the THBD gene in Parkinson's disease patients and normal persons using a gene chip
  • Figure 4 shows the difference in expression of the THBD gene in Parkinson's disease patients and normal persons by QPCR
  • Figure 5 shows the difference in expression of KCTD20 gene in Parkinson's disease patients and normal persons using a gene chip
  • Figure 6 shows the difference in expression of KCTD20 gene in Parkinson's disease patients and normal persons by QPCR
  • Figure 7 shows the difference in expression of the NACC2 gene in Parkinson's disease patients and normal persons using a gene chip
  • Figure 8 shows the difference in expression of the NACC2 gene in Parkinson's disease patients and normal persons by QPCR
  • FIG. 9 shows the expression of FGFBP2 gene in the blood of patients with Parkinson's disease by QPCR
  • Figure 10 shows the effect of detecting siRNA on gene expression by using QPCR
  • Figure 11 shows the effect of KCTD20 on the content of ⁇ -syn by ELISA
  • Figure 12 shows the effect of siRNA on the expression of THBD gene by QPCR
  • Figure 13 shows the effect of THBD on the content of ⁇ -syn by ELISA
  • Figure 14 shows the effect of siRNA on the expression of FGFBP2 gene by QPCR
  • Figure 15 shows the effect of detecting FGFBP2 gene expression on the growth of Parkinson's nerve cells using MTT.
  • PD inclusion criteria Diagnostic criteria are in line with PD clinical diagnostic criteria (refer to "Jiang Yuping, Wang Jian, Ding Zhengtong, et al., Diagnostic criteria for primary Parkinson's disease, 2005, Chinese Journal of Clinical Neuroscience, 2006, 14:40" ). Exclusion criteria: (1) idiopathic tremor; (2) secondary Parkinson's syndrome; (3) severe dementia, dysarthria; (4) patients with other mental disorders. The study was approved by the hospital ethics committee and all patients signed informed consent.
  • Normal group 10 healthy volunteers aged 32-80 years old, 5 males and 5 females.
  • Fresh blood (peripheral blood) was taken directly, and 3 volumes of red blood cell lysate was added, mixed, left at room temperature for 10 minutes, and centrifuged at 10,000 rpm for 1 minute. Thoroughly discard the supernatant and collect leukocyte pellets. The leukocyte pellet collected per 100-200 ⁇ l of blood was added to 1 ml of TRIzol.
  • RNA is mainly in the aqueous phase, and the aqueous phase (usually 550 ⁇ l can be absorbed) is transferred to a new tube.
  • RNA-free water 50-100 ⁇ l was added to the pellet, and the tube wall was lightly shaken to fully dissolve the RNA and stored at -70 °C.
  • RNA quality expressed by RNA integrity, integrity can be detected by ordinary agarose gel electrophoresis (electrophoresis conditions: 1.2% gel; 0.5 x TBE running buffer; 150v, 15 minutes).
  • RNA purity The OD260/OD280 ratio is an indicator of the extent of protein contamination in an RNA sample. High quality RNA samples with an OD260/OD280 value (10 mM Tris, pH 7.5) around 2.0.
  • Agilent DNA Microarray Scanner scanner was used to perform hybridization at 65 ° C for 17 h in a chip hybridization oven using a human whole gene expression profiling chip (4x 44K gene) from Agilent, USA.
  • the hybridized chip After the hybridized chip reads the data point by the chip scanner, the data is introduced into the analysis software, and the gene with the natural logarithm absolute value of the two ratios greater than 2.0 or less than 0.5 is used as the differentially expressed gene.
  • RNA in total RNA was removed using the Ribo-Zero kit.
  • the cDNA library was constructed using the Illumina TruseqTM RNA sample Prep Kit, and the specific procedures were carried out according to the instructions.
  • the cDNA library was sequenced using the Illumina X-Ten sequencing platform, and the specific procedures were performed according to the instructions.
  • RNA-seq read position was mapped by TopHat v1.3.1, the number of RNA-seq fragments was normalized by Cufflinks v1.0.3 to calculate the relative abundance of transcripts, and the differential expression was detected by cuffdiff.
  • the p value is ⁇ 0.05, the gene is considered to be significantly differentially expressed.
  • RNA-seq results showed that the expression level of FGFBP2 gene in the blood of patients with Parkinson's disease was significantly higher than that in normal human blood.
  • the screening criteria were the same as in Example 1, 50 patients with Parkinson's disease and 50 normal subjects.
  • the cDNA was reverse transcribed into 1 ⁇ g of total RNA using reverse transcription buffer.
  • 1 ⁇ g of total RNA was used as template RNA for each sample, and the following components were added to the PCR tube: DEPC water, 5 ⁇ reverse transcription buffer, 10 mmol/L dNTP, 0.1 mmol/l DTT, 30 ⁇ mmol/l Oligo dT, 200 U/ ⁇ l M-MLV, template RNA.
  • Incubate at 42 ° C for 1 h, 72 ° C for 10 min, and briefly centrifuge.
  • the QPCR amplification primers were designed according to the coding sequences of DNASE2 gene and GAPDH gene in Genbank, and synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.
  • the specific primer sequences are as follows:
  • the forward primer was 5'-CAGCATCACACTCACATC-3' (SEQ ID NO. 5);
  • the reverse primer is 5'-AGTACAGGTCATCTCCAA-3' (SEQ ID NO. 6),
  • the forward primer was 5'-CTCAATGCCAGTCAGATC-3' (SEQ ID NO. 1);
  • the reverse primer is 5'-GTTCAGTAGCAAGGAAATG-3' (SEQ ID NO. 2)
  • the forward primer was 5'-GCTGCTTAGTGGATGATAC-3' (SEQ ID NO. 3);
  • the reverse primer was 5'-AGATTATGAGGACCAGATGA-3' (SEQ ID NO. 4),
  • the forward primer was 5'-ATGGTGGAGGAGCAGTAC-3' (SEQ ID NO. 7);
  • the reverse primer was 5'-TGGCTCAGGCTTCTCTTG-3' (SEQ ID NO. 8),
  • the forward primer was 5'-TTTAACTCTGGTAAAGTGGATAT-3' (SEQ ID NO. 11);
  • the reverse primer was 5'-GGTGGAATCATATTGGAACA-3' (SEQ ID NO. 12).
  • the SYBR Green polymerase chain reaction system was purchased from Invitrogen.
  • Reagent volume Forward primer 1 ⁇ l Reverse primer 1 ⁇ l SYBR Green polymerase chain reaction system 12.5 ⁇ l template 2 ⁇ l Deionized water Make up 25 ⁇ l
  • PCR reaction conditions 95 ° C for 10 min, (95 ° C 10 s, 60 ° C 40 s) * 45 cycles.
  • the SYBR Green was used as a fluorescent marker, and the PCR reaction was carried out on a Light Cycler quantitative PCR machine.
  • the target band was determined by melting curve analysis and electrophoresis, and the ⁇ CT method was used for relative quantification.
  • the screening criteria were the same as in Example 1, 90 patients with Parkinson's disease and normal subjects.
  • RNA extraction step is the same as in Example 1.
  • a 25 ⁇ l reaction system was constructed, which included 5 ⁇ l of 5 ⁇ reverse transcription buffer, 5 ⁇ l of dNTP (2.5 mM), 40 U/ ⁇ l of RNasin, and 200 U/ ⁇ l of M-MLV, and the nuclease-free water was added to the expected volume.
  • the QPCR amplification primers were designed according to the coding sequences of FGFBP2 gene and GAPDH gene in Genbank, and synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.
  • the specific primer sequences are as follows:
  • the forward primer was 5'-ATGAGGAAGCAAAGAAGA-3' (SEQ ID NO. 9);
  • the reverse primer was 5'-GGAAGAAGCTGATGAGAA-3' (SEQ ID NO. 10).
  • the forward primer was 5'-AACTCTGGTAAAGTGGATATTG-3' (SEQ ID NO. 13);
  • the reverse primer was 5'-GGTGGAATCATATTGGAACA-3' (SEQ ID NO. 14).
  • the SYBR Green polymerase chain reaction system was purchased from Invitrogen.
  • PCR reaction conditions 95 ° C for 10 min, (95 ° C 15 s, 60 ° C 60 s) * 45 cycles.
  • the SYBR Green was used as a fluorescent marker, and the PCR reaction was carried out on a Light Cycler quantitative PCR machine.
  • the target band was determined by melting curve analysis and electrophoresis, and the ⁇ CT method was used for relative quantification.
  • the experiment used 3 repeated experiments, the results were expressed in the mean ⁇ standard deviation, using SPSS13.0 statistical software for statistical analysis, the difference between the two using t test, that when P ⁇ 0.05 has statistical significane.
  • Example 3 Western blotting experiments confirmed the expression products of differentially expressed genes in Parkinson's disease patients and normal subjects
  • HBSS Na 2 HPO 4 0.132g, KH 2 PO 4 0.06g, KCl 0.4g, phenol red 1ml, NaHCO 3 0.35g, D-glucose 1.0g, dissolved in 1000ml
  • Double distilled water Double distilled water
  • the cell suspension obtained in the above experiment (concentration: 1 ⁇ 10 6 /ml) was centrifuged at room temperature 1 000 r/min for 10 min, the supernatant was discarded, 100 ⁇ l of lysis buffer was added, and the mixture was shaken at 4 ° C for 1 h, and the cells were disrupted by a sonicator for 10 s each time. 10 times, centrifuged at 12000r/min for 1h at 4°C; the supernatant was used to quantify the protein by Brandford method, dispensed into 2.5 ⁇ g/ ⁇ l, and stored in a -80°C refrigerator for later use.
  • Total cellular protein was quantified by Brandford method, mixed with sample buffer for 5 min, cooled for 5 min; 30 pg of protein was applied to the prepared 15% polyacrylamide gel, electrophoresed, and set to 80V constant pressure, see Marker After the increase to 120V; the gel after electrophoresis was removed and transferred to 100V for 50 min using Bio.Rad semi-dry transfer system; after the membrane was finished, it was washed once with 1 ⁇ PBS, immersed in blocking solution, and overnight at 40 C; the blocking solution was drained and added to Western.
  • washing solution for 5-10min add to the anti-shaker bed for 2h at room temperature; dilute in the appropriate buffer with Western secondary antibody dilution solution in blocking buffer for 60min; wash the membrane solution 3 times for 10min each time; use ECL reagent to develop Fixation detects protein expression.
  • the gray value of the protein band was analyzed using Image J software, and the gray value of the target white band was normalized by using ⁇ -actin as an internal parameter. The results were expressed as mean ⁇ standard deviation. Statistical analysis was performed using SPSS 13.0 statistical software. The difference between the two was analyzed by t test, which was considered statistically significant when P ⁇ 0.05.
  • the diluted siRNA and the transfection reagent were mixed (the ratio of siRNA to transfection reagent was 1:2), allowed to stand at room temperature for 10-15 min, added to the cell culture medium, and gently mixed. After the cells were further cultured for 72 hours, they were centrifuged at 12,000 rpm for 10 min at 4 ° C, and the supernatant was collected for subsequent ELISA detection. RNA was extracted from the lysed cells, and QPCR was used to detect the effect of siRNA silencing KCTD20.
  • siRNA sequence of KCTD20 was designed according to the sequence of rat KCTD20 gene (Chromosome 20, NC_005119.4 (6205897..6222818)) as follows:
  • KCTD20-SIRNA1 (referred to as KCTD-1)
  • the sense strand is 5'-GGUCUGAAGUUAUCUACAATT-3' (SEQ ID NO. 15),
  • the antisense strand is 5'-UUGUAGAUAACUUCAGACCTT-3' (SEQ ID NO. 16);
  • KCTD20-SIRNA2 (referred to as KCTD-2)
  • the sense strand is 5'-CCAGGAGAUAAUGAUGCAUTT-3' (SEQ ID NO. 17),
  • the antisense strand is 5'-AUGCAUCAUUAUCUCCUGGTT-3' (SEQ ID NO. 18);
  • KCTD20-SIRNA3 (referred to as KCTD-3)
  • the sense strand is 5'-GCGACUAUCUCUGCAUUAATT-3' (SEQ ID NO. 19),
  • the antisense strand is 5'-UUAAUGCAGAGAUAGUCGCTT-3' (SEQ ID NO. 20).
  • RNA extraction Add 400 ⁇ L of Trizol to each well of a 12-well plate, gently pipette a few times to fully lyse the cells, then transfer the cell lysate to a 1.5 mL centrifuge tube, add 80 ⁇ L of chloroform to each tube, and vortex well. It was allowed to stand at room temperature for 3 min, centrifuged at 12,000 rpm for 15 min at 4 °C. Take the uppermost layer into a new centrifuge tube, add 200 ⁇ L of isopropanol to each tube, gently mix upside down, let stand for 10 min at room temperature, and centrifuge at 10 ° C for 10 min at 12000 rpm.
  • the ⁇ -syn-mAb (Abeam, ab138501) was coated into a 96-well plate (0.5 ⁇ g/well) at 4 ° C overnight. After washing three times with 0.1% PBST, 3% BSA was blocked. The PC12- ⁇ -syn cell supernatant was concentrated with a 3k ultrafiltration tube, added to a 96-well plate, 100 ⁇ L per well, and reacted at 37 ° C for 1 hour, and washed with 0.1% PBST six times.
  • Syn-1 (BD, 610787) was added as a detection antibody, and after reacting for 1 hour at 37 ° C, HRP-labeled goat anti-mouse secondary antibody was added, and TMB was developed, and the SpectraMax M5 microplate reader (Molecular Devices, LLC, Sunnyvale) was used. , CA, USA) Measurement of absorbance (450 nm).
  • the amount of ⁇ -syn in the cell supernatant was increased by 2.12 times after the siRNA inhibited the expression of KCTD20 as compared with the control group.
  • siRNA sequence of THBD was designed according to the sequence of rat THBD gene (Chromosome 3, NC_005102.4 (142748673..142752325, complement)) as follows:
  • THBD-siRNA1 (abbreviated as THBD-1)
  • the sense strand is 5'-GCUUCGAAUGCCGCUGCUATT-3' (SEQ ID NO. 21),
  • the antisense strand is 5'-UAGCAGCGGCAUUCGAAGCTT-3' (SEQ ID NO. 22);
  • THBD-siRNA2 (abbreviated as THBD-2)
  • the sense strand is 5'-GGUGCGAAAUGUUCUGCAATT-3' (SEQ ID NO. 23),
  • the antisense strand is 5'-UUGCAGAACAUUUCGCACCTT-3' (SEQ ID NO. 24);
  • THBD-siRNA3 (abbreviated as THBD-3)
  • the sense strand is 5'-GCGCAGAGCUGGAGUACAATT-3' (SEQ ID NO. 25),
  • the antisense strand is 5'-UUGUACUCCAGCUCUGCGCTT-3' (SEQ ID NO. 26).
  • RNA extraction Add 400 ⁇ L of Trizol to each well of a 12-well plate, gently pipette a few times to fully lyse the cells, then transfer the cell lysate to a 1.5 mL centrifuge tube, add 80 ⁇ L of chloroform to each tube, and vortex well. It was allowed to stand at room temperature for 3 min, centrifuged at 12,000 rpm for 15 min at 4 °C. Take the uppermost layer into a new centrifuge tube, add 200 ⁇ L of isopropanol to each tube, gently mix upside down, let stand for 10 min at room temperature, and centrifuge at 10 ° C for 10 min at 12000 rpm.
  • the ⁇ -syn-mAb (Abeam, ab138501) was coated into a 96-well plate (0.5 ⁇ g/well) at 4 ° C overnight. After washing three times with 0.1% PBST, 3% BSA was blocked. The PC12- ⁇ -syn cell supernatant was concentrated with a 3k ultrafiltration tube, added to a 96-well plate, 100 ⁇ L per well, and reacted at 37 ° C for 1 hour, and washed with 0.1% PBST six times.
  • Syn-1 (BD, 610787) was added as a detection antibody, and after reacting for 1 hour at 37 ° C, HRP-labeled goat anti-mouse secondary antibody was added, and TMB was developed, and the SpectraMax M5 microplate reader (Molecular Devices, LLC, Sunnyvale) was used. , CA, USA) Measurement of absorbance (450 nm).
  • the amount of ⁇ -syn in the cell supernatant was increased by 4.88 times after the siRNA inhibited the expression of THBD as compared with the control group.
  • Dopamine neuron cell SH-SY5Y in DMEM containing 10% fetal bovine serum, 1% penicillin/streptomycin (pH 7.2-7.4), at 37 ° C, 5% CO 2 , relative humidity of 90% Cultivate in an incubator. Change the liquid every 2 days, pass the cells until the cells grow to 90% contact, wash with PBS, add 0.25%-EDTA trypsin to separate the cells from the bottle wall, and terminate with DMEM medium containing fetal bovine serum.
  • the sense chain is 5'-UUAGAAACUCUCUUCUUCCAG-3' (SEQ ID NO. 27),
  • the antisense strand is 5'-GGAAGAAGAGAGUUUCUAAUC-3' (SEQ ID NO. 28);
  • the sense strand is 5'-AAGUAGUUGUGUAUGCUUGUC-3' (SEQ ID NO. 29),
  • the antisense strand is 5'-CAAGCAUACACAACUACUUAU-3' (SEQ ID NO. 30);
  • the sense strand is 5'-UACAGAUAAUAAGUAGUUGUG-3' (SEQ ID NO. 31),
  • the antisense strand is 5'-CAACUACUUAUUAUCUGUAGA-3' (SEQ ID NO. 32).
  • adenovirus-mediated siRNA the cells were divided into five groups, SH-SY5Y group: SH-SY5Y cells not transfected with any viral vector, as a blank control; Ad group: infected empty adenovirus plasmid cell group, siRNA1 Group: Adenovirus-mediated interference sequence 1 infected cell group: siRNA2 group: adenovirus-mediated interference sequence 2 infected cell group; siRNA3 group: adenovirus-mediated interference sequence 3 infected cell group.
  • the cells were inoculated into a 6-well cell culture plate at 1 ⁇ 10 5 /well, 2 ml per well, and cultured in a 37 ° C, 5% CO 2 incubator for 24 h, at which time the cell fusion density was about 50%-60%; The supernatant was aspirated, and washed twice with 1 ml of serum-free medium.
  • the adenoviruses of 50% MOI diluted in 1 ml of serum-free medium were shaken once every 20 minutes to increase the infection effect. After 48 hours of infection, Further, the concentration of 1000 ⁇ mol/L MPP + complete medium was added and incubated for 24 hours. Collecting cells for extracting RNA;
  • the specific method is:
  • the culture solution was aspirated, washed once with PBS, and an appropriate amount of TRIzol reagent was added. 1 mL was added to each well of a six-well plate, and the cells were lysed for 5 min at room temperature, uniformly blown, and dispensed into a 1.5 mL Eppendorf tube at 1 mL/tube. Chloroform was added to 400 ⁇ l of chloroform/ml Trizol, and the mixture was shaken up and down 30-50 times by hand and left at room temperature for 5 minutes.
  • RNA precipitate was transferred to a clean Eppendorf tube, 0.4 mL of isopropanol was added, and the mixture was gently mixed, left at room temperature for 10 min, and centrifuged at 7500 g for 10 min at 4 ° C. Discard the supernatant, wash the RNA precipitate with 75% ethanol, centrifuge at 7500g for 5min, dry the RNA precipitate at room temperature, and dissolve it in an appropriate amount of DEPC water after 5-10min. The integrity of the RNA samples was determined by agarose gel electrophoresis with a mass fraction of 1.0%, and the extracted RNA was quantitatively determined using a Bio-Photometer.
  • the 3.3QPCR amplification step is the same as in Example 2.
  • FGFBP2 gene The effect of FGFBP2 gene on cell proliferation of SH-SY5Y Parkinson's disease cell model was examined by MTT assay.
  • the SH-SY5Y cell density was adjusted to 5 ⁇ 10 4 /mL, and 100 ⁇ l of cells per well were seeded in a 96-well culture plate, and the cells were treated as in Example 3, and MTT assay was applied every 12 hours after the treatment until 72 h. MTT reduction assay was used to detect cell viability: the solution in the well was discarded, 100 ⁇ l of medium was added, and 10 ⁇ l of 5 mg/mL MTT solution was added to each well. After incubating at 37 ° C for 4 h, the medium was aspirated and 100 ⁇ l of DMSO was added to each well.
  • the experiment used 3 replicate experiments and statistical analysis using SPSS18.0 statistical software. The difference between the two was analyzed by t test, which was considered statistically significant when P ⁇ 0.05.

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Abstract

L'invention concerne l'utilisation d'un gène THBD, d'un gène KCTD20, d'un gène DNASE2, d'un gène NACC2, d'un gène FGFBP2 et de leurs produits d'expression dans la préparation d'un produit destiné à diagnostiquer la maladie de Parkinson. La détection des taux du gène THBD, du gène KCTD20, du gène DNASE2, du gène NACC2, du gène FGFBP2 et de leurs produits d'expression permet de déterminer qu'un sujet présente un risque de contracter la maladie de Parkinson dans le futur ou de confirmer qu'un sujet souffre de la maladie de Parkinson. L'invention concerne également l'utilisation des gènes ci-dessus dans la préparation d'un médicament destiné au traitement de la maladie de Parkinson, procurant ainsi des cibles candidates pour le développement clinique de médicaments destinés au traitement de la maladie de Parkinson.
PCT/CN2018/123794 2017-12-29 2018-12-26 Gène utilisé comme marqueur pour diagnostiquer et traiter la maladie de parkinson, et utilisation correspondante WO2019129029A1 (fr)

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
CN201711481574.5A CN108103183B (zh) 2017-12-29 2017-12-29 Kctd20基因作为早期诊断帕金森症的新型标志物
CN201711477253.8A CN107904304B (zh) 2017-12-29 2017-12-29 Dnase2作为帕金森症诊断标志物的用途
CN201711483559.4 2017-12-29
CN201711481575.XA CN108220419B (zh) 2017-12-29 2017-12-29 血液中nacc2基因的诊断用途
CN201711483559.4A CN108048554B (zh) 2017-12-29 2017-12-29 Thbd基因作为帕金森症诊断的分子标志物
CN201711477253.8 2017-12-29
CN201711481574.5 2017-12-29
CN201711481575.X 2017-12-29
CN201810292179.0 2018-03-30
CN201810292179.0A CN108611413B (zh) 2018-03-30 2018-03-30 一种帕金森相关生物标志物及其应用

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012074933A1 (fr) * 2010-11-29 2012-06-07 Genentech, Inc. Procédés pour la détection de maladies ou de troubles neurodégénératifs
CN107012238A (zh) * 2017-05-04 2017-08-04 上海交通大学医学院附属瑞金医院 易栓相关基因突变检测试剂盒
CN108048554A (zh) * 2017-12-29 2018-05-18 北京泱深生物信息技术有限公司 Thbd基因作为帕金森症诊断的分子标志物

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012074933A1 (fr) * 2010-11-29 2012-06-07 Genentech, Inc. Procédés pour la détection de maladies ou de troubles neurodégénératifs
CN107012238A (zh) * 2017-05-04 2017-08-04 上海交通大学医学院附属瑞金医院 易栓相关基因突变检测试剂盒
CN108048554A (zh) * 2017-12-29 2018-05-18 北京泱深生物信息技术有限公司 Thbd基因作为帕金森症诊断的分子标志物

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