WO2014065323A1 - 精神関連疾患の検査方法および検査キット - Google Patents
精神関連疾患の検査方法および検査キット Download PDFInfo
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- WO2014065323A1 WO2014065323A1 PCT/JP2013/078703 JP2013078703W WO2014065323A1 WO 2014065323 A1 WO2014065323 A1 WO 2014065323A1 JP 2013078703 W JP2013078703 W JP 2013078703W WO 2014065323 A1 WO2014065323 A1 WO 2014065323A1
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- 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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
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- 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
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- 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
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/38—Pediatrics
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/50—Determining the risk of developing a disease
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7057—(Intracellular) signaling and trafficking pathways
- G01N2800/7066—Metabolic pathways
- G01N2800/7085—Lipogenesis or lipolysis, e.g. fatty acid metabolism
Definitions
- the present invention relates to a test method and test kit for a mentally related disease, particularly an autism spectrum disorder.
- Autism spectrum disorder a psychological disorder, may be difficult to test and detect early because the etiology is unknown.
- recuperation early education intervention
- Non-Patent Document 1 lipid metabolism abnormality may be involved.
- Smith-Lemli-Opitz syndrome known as an autosomal recessive hereditary disease, results in low cholesterol levels and elevated 7-dehydrocholesterol levels due to deficiency of 7-dehydrocholesterol reductase. About half of them are known to merge autistic disorders (Non-Patent Document 1).
- Non-patent Document 2 reports such as changes in the amount of fatty acids in plasma or erythrocyte membranes (Non-patent Document 3) suggest that lipids may be involved in the pathology of autism spectrum disorders.
- lipids and lipid metabolites themselves are expected to have low disease specificity, it is difficult to use them as molecular markers for autism spectrum disorders.
- the present invention has been made in view of the above circumstances, and an object thereof is to provide a test method and test kit for an autism spectrum disorder using a new molecular marker.
- the present inventors have found that among the fatty acid binding protein FABP family, the amount of fatty acid binding protein FABP3, FABP4, FABP5, and FABP7 in a sample prepared from a human living body, or FABP3 in the sample. It has been found that the expression level of the gene, FABP4 gene, FABP5 gene, and FABP7 gene can be used as a molecular marker for autism spectrum disorder. That is, the present invention includes any of the following inventions.
- a method for examining the presence or absence of a predisposition to or development of an autism spectrum disorder wherein the amount of at least one of fatty acid binding proteins FABP3, FABP4, FABP5 and FABP7 in a sample prepared from a human body is examined.
- a test method comprising a test step of examining the expression level of at least one of the FABP3 gene, the FABP4 gene, the FABP5 gene and the FABP7 gene in the sample.
- a test kit for examining the presence or absence of a predisposition or onset of autism spectrum disorder wherein at least one of fatty acid binding proteins FABP3, FABP4, FABP5 and FABP7 in a sample prepared from a human living body, or Includes a nucleic acid probe, nucleic acid primer, nucleic acid aptamer, antibody or peptide probe for detecting at least one of the FABP3 gene expression product, the FABP4 gene expression product, the FABP5 gene expression product and the FABP7 gene expression product in the sample , Inspection kit.
- the present invention has the effect of providing an inspection method and an inspection kit for inspecting for the presence or absence of a predisposition to autism spectrum disorder.
- Distribution chart showing distribution of FABP4 concentration in serum in each of age group of 4-6 years and age group of 7-8 years in Example 2 of the present invention (upper figure), and age group of 4-6 years And a table (lower table) that calculated the sensitivity, specificity, positive predictive value, and negative predictive value in each of the age groups of 7-8 years.
- the test method according to the present invention is a method for testing for the presence or absence of a predisposition to autism spectrum disorder, and fatty acids in a sample prepared from a human living body (hereinafter sometimes referred to as “biological sample”)
- Binding protein FABP3 (sometimes referred to as fatty acid binding protein 3: FABP3)
- FABP4 (sometimes referred to as fatty acid binding protein 4: FABP4)
- FABP5 (sometimes referred to as fatty acid binding protein 5: FABP5)
- FABP7 fatty acid binding protein7: which may be referred to as FABP7
- ⁇ Autism spectrum disorder Specific examples include pervasive developmental disorder (PDD), autism, and Asperger's syndrome. Symptoms may include communication problems, language development problems, discrepancies, repeated behavior, and interpersonal relationships.
- PDD pervasive developmental disorder
- autism and Asperger's syndrome.
- Symptoms may include communication problems, language development problems, discrepancies, repeated behavior, and interpersonal relationships.
- test refers to the identification of a disease or condition based on the signs and symptoms of a patient made by a physician.
- “inspection” or “examination” in this specification means an autism spectrum disorder in a human subject to be examined (sometimes referred to as “subject”) that does not require identification (diagnosis) by a doctor. Refers to testing for the presence or absence of predisposition to.
- the test result obtained by the test method of the present invention can be a material for diagnosis made by a doctor.
- the test of the present invention provides important information.
- To check for a predisposition to autism spectrum disorder means to test the possibility of developing autism spectrum disorder in the future, regardless of whether or not it has already developed autism spectrum disorder. It is a concept that also includes On the other hand, checking whether or not an autism spectrum disorder has occurred refers to testing whether or not an autism spectrum disorder has occurred.
- the age of the subject is preferably as low as possible.
- the amount of FABP4 in the blood is used as an index, it is preferably 8 years old or less, more preferably 7 years old or less, and further preferably 6 years old or less. .
- a subject who has a predisposition to autism spectrum disorder or is determined early to develop the disease can receive medical treatment from an early stage.
- the biological sample is collected from the subject. There are no particular limitations on the type of biological sample, and it is sufficient that it contains at least one of the subject's protein and nucleic acid (mRNA as a gene expression product).
- the biological sample include a cell sample, a tissue sample, and a body fluid sample, and among them, a body fluid sample is preferable.
- the body fluid sample include a blood sample, a lymph fluid sample, a cerebrospinal fluid sample, and the like.
- a blood sample and a lymph fluid sample are preferable, and among the blood samples, a peripheral blood sample and an umbilical cord blood sample are particularly preferable.
- the peripheral blood sample can be easily collected, for example, by puncturing a fingertip or the like, so that the burden on the subject is small, and in addition, the peripheral blood sample sufficiently contains the molecular marker that is the subject of the test method of the present invention.
- An umbilical cord blood sample can be easily collected from the umbilical cord at the time of birth, and has an advantage that ultra-early diagnosis is possible.
- the biological sample is also collected from a healthy person who does not have an autism spectrum disorder in order to use it as a control sample.
- the control sample is preferably the same type as that of the subject (collected from the same site).
- control data is prepared in advance when performing the test method of the present invention, it is not necessary to collect a biological sample from a healthy person.
- the collected biological sample may be subjected to an operation for extracting a protein or nucleic acid or an operation for removing an unnecessary component as necessary, and then subjected to an inspection.
- an operation for extracting a protein or nucleic acid or an operation for removing an unnecessary component as necessary for example, when a blood sample is used, it is preferable to use serum or plasma prepared from the collected blood for the test.
- the obtained biological sample may be stored by a method suitable for the type of biological sample, such as cryopreservation as necessary.
- a biological sample can be stored to measure a molecular marker that is an object of an inspection method at a desired time.
- a sample as it is collected may be stored, or a sample (for example, serum or plasma) prepared after collection may be stored.
- the inspection method of the present invention includes the inspection steps shown in the following a) or b).
- Step a) Examining the amount of at least one of fatty acid binding proteins FABP3, FABP4, FABP5 and FABP7 in the biological sample
- Step b) The expression level of at least one of the FABP3 gene, FABP4 gene, FABP5 gene, and FABP7 gene in the biological sample is examined.
- step a) or step b) is selected based on conditions such as the type of biological sample or the type of subject (age, disease to be examined), etc., but step a) is preferred.
- the amount of FABP4 is preferably examined, and in the case of step b), the expression level of the FABP4 gene is preferably examined.
- Step a) the amount of at least one protein of the fatty acid binding proteins FABP3, FABP4, FABP5 and FABP7 in the biological sample, more specifically, the amount of protein contained per unit amount of the biological sample (for example, Protein concentration).
- the concept of measuring the amount of protein contained in a unit amount of a biological sample includes both quantitative and qualitative measurements. More specifically, for example, a data comparison at the time of acquisition before concentration conversion using a calibration curve or the like, or a result in a format indicating whether the amount of protein exceeds a certain threshold value. This also includes the presentation of
- the method for measuring the amount of at least one protein of FABP3, FABP4, FABP5 and FABP7 is not particularly limited.
- an immunological technique using an antibody specific to FABP3, FABP4, FABP5 or FABP7 Liquid column chromatography, mass spectrometry and the like.
- Examples of the method using an antibody include an ELISA (Enzyme-Linked Immuno Immuno Sorbent Assay) method, a quantitative Western blotting method, and an immunoprecipitation method, and the ELISA method is preferably used.
- the type of ELISA method is not particularly limited, but is a so-called antigen measurement system (measurement of the amount of antigen contained in a biological sample), ELISA by direct adsorption method, ELISA by competition method, ELISA by sandwich method, and micro flow Examples include ELISA specialized for the measurement of a small amount of sample using a road system or microbeads.
- the antibody specific to FABP3, FABP4, FABP5 or FABP7 may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.
- human FABP3, FABP4, FABP5 and FABP7 amino acid sequences are available from public databases such as NCBI.
- NCBI the registration number of FABP4 is CAG33184 / NP_001433.
- An example of FABP4 has the amino acid sequence shown in SEQ ID NO: 1. Based on the above information, those skilled in the art can easily determine an amino acid sequence suitable as an antigen for producing antibodies specific to FABP3, FABP4, FABP5 and FABP7.
- antibody is intended to mean a form including all classes and subclasses of immunoglobulins and functional fragments of antibodies.
- the antibody is a concept including both a natural antibody of a polyclonal antibody and a monoclonal antibody, and additionally includes an antibody produced using a gene recombination technique, and a functional fragment of the antibody.
- the “functional fragment of an antibody” refers to one having a partial region of the above-described antibody and having an antigen-binding ability (synonymous with a binding fragment).
- Natural antibodies can be derived from any species including, but not limited to, humans, mice, rats, goats, rabbits, camels, llamas, cows, chickens, sharks, and fish.
- the antibody produced using gene recombination technology is not particularly limited, but chimeric antibodies such as humanized antibodies and primatized antibodies obtained by genetic modification of natural antibodies, synthetic antibodies, recombinant antibodies, Mutagenized antibodies and graft-bound antibodies (for example, antibodies to which other proteins and radioactive labels are conjugated or fused), and antibodies already produced using genetic recombination techniques are described above. These also include antibodies that have been modified in the same manner as when genetically modifying natural antibodies. Specific examples of functional fragments of antibodies include F (ab ′) 2 , Fab ′, Fab, Fv (variable fragment of antibody), sFv, dsFv (disulphide stabilized Fv), and dAb (single domain antibody). (George et al, Exp. Opin. Ther. Patents, Vol. 6, No. 5, p.441-456, 1996).
- the binding fragment includes an antibody fragment mutated in a range that maintains reactivity with the target protein as a concept of the binding fragment.
- the above-described mutation introduction is performed using a known technique such as a gene modification technique, which is appropriately selected by those skilled in the art.
- Step b) is a step of measuring the expression level of at least one of the FABP3 gene, FABP4 gene, FABP5 gene and FABP7 gene in the sample using the biological sample.
- the FABP3 gene is a generic term for nucleic acids encoding FABP3
- the FABP4 gene is a generic term for nucleic acids encoding FABP4
- the FABP5 gene is a generic term for nucleic acids encoding FABP5.
- the FABP7 gene is a general term for nucleic acids encoding FABP7.
- the method for measuring the expression level of at least one of the FABP3 gene, the FABP4 gene, the FABP5 gene and the FABP7 gene is not particularly limited, but a desired nucleic acid (eg, mRNA as a transcription product) using a nucleic acid amplification technique such as a PCR method. ) May be included.
- a nucleic acid amplification technique such as a PCR method.
- examples of the method using the nucleic acid amplification technique include quantitative RT-PCR, and examples of the method for directly detecting mRNA include the Northern blot method.
- it may be a method for measuring the expression level of a gene using a nucleic acid chip such as a microarray. Note that “measuring the expression level of a gene” can be used interchangeably with “measuring the amount (concentration, etc.) of a gene expression product (described later)”.
- cDNA may be prepared using mRNA contained in a biological sample as a template.
- Amplification of FABP3 gene, FABP4 gene, FABP5 gene and FABP7 gene as mRNA can be performed based on base sequence information available from public databases such as NCBI.
- NCBI the registration number of the FABP4 gene is NM_001442.
- An example of the FABP4 gene has the base sequence shown in SEQ ID NO: 2. Based on the above information, those skilled in the art can easily design appropriate primers for amplifying the FABP4 gene, FABP5 gene or FABP7 gene.
- the determination of the presence or absence of predisposition or onset of autism spectrum disorder is the amount of protein of at least one of FABP3, FABP4, FABP5 and FABP7 obtained by the test step shown in the above step a) or step b) Alternatively, the expression level of at least one of the FABP3 gene, the FABP4 gene, the FABP5 gene and the FABP7 gene is compared between the subject and the control. In one example of the determination, when the amount of at least one of FABP3, FABP4, FABP5, or FABP7 (which may be a protein amount or a gene expression amount) in a biological sample of the subject is significantly lower than the control, Is determined to have or be predisposed to autism spectrum disorder.
- the amount is significantly low may be a result of quantitative measurement or a result of qualitative measurement.
- comparison of relative amounts actual It is not necessary to calculate the amount, and it is a concept that also determines whether it is higher or lower than a certain standard.
- the above-described inspection of the control sample may be performed simultaneously with the inspection of the subject's sample, or may be performed separately. That is, the numerical value of the control sample compared with the numerical value of the subject may be a value obtained by a test performed when the test sample is different from the test sample. Further, the test of the control sample does not need to be performed by the person who performs the test of the subject. For example, the test value of the control sample that has already been acquired and accumulated in a database or the like can be used as the threshold value.
- the numerical value of the individual sample of a healthy person may be directly used, or the average value obtained when the numerical value of the sample of a certain number of healthy persons is used as a population is used. May be.
- a cutoff value may be set in advance, and the numerical value of the subject may be compared with this cutoff value. For example, the amount of protein of at least one of FABP3, FABP4, FABP5, and FABP7 in a biological sample of a subject, or the expression level of at least one of FABP3 gene, FABP4 gene, FABP5 gene, and FABP7 gene in the sample is cut off. When it is above, it can be determined that the subject is less likely to develop an autism spectrum disorder.
- the amount of at least one protein of FABP3, FABP4, FABP5 and FABP7 in the sample prepared from the subject, or the expression level of at least one of the FABP3 gene, FABP4 gene, FABP5 gene and FABP7 gene in the sample is determined from the cut-off value. If it is low, it can be determined that the subject has a possibility and risk of developing an autism spectrum disorder.
- “Cutoff value” refers to both diagnosis sensitivity (prevalence of disease diagnosis) and specificity of diagnosis (no disease diagnosis rate) when the presence or absence of a predisposition or onset of disease is determined based on this value. Indicates a sufficiently high value. For example, a value showing a high positive rate in an individual who develops autism spectrum disorder and a high negative rate in an individual who does not develop autism spectrum disorder can be set as a cutoff value .
- diagnosis sensitivity is a ratio (true value) indicating a positive (abnormal value) when a test is performed on a population having a predisposition to a specific disease or developing a specific disease. Positive rate).
- diagnosis specificity refers to a ratio (a ratio of true negative) indicating a negative (normal value) when a test is performed on a population not suffering from a specific disease.
- positive predictive value refers to the proportion of individuals who actually have a disease among subjects who showed a positive result in the test, and negative predictive value represents the proportion of subjects who showed a negative result in the test. Means the percentage of individuals who do not actually have the disease.
- the calculation method of the cut-off value a method known in the technical field can be used. For example, the amount of at least one of the fatty acid binding proteins FABP3, FABP4, FABP5 and FABP7 in a sample prepared from an individual who has developed autism spectrum disorder and an individual who has not developed autism spectrum disorder, or The expression level of at least one of the FABP3 gene, FABP4 gene, FABP5 gene and FABP7 gene in the sample is calculated, and the diagnostic sensitivity and diagnostic specificity at the calculated values are obtained. Based on the values obtained above, a commercially available suitable analysis software is used to create a ROC (Receiver-Operating-Characteristic) curve.
- ROC Receiveiver-Operating-Characteristic
- a value when the diagnostic sensitivity and diagnostic specificity are as close to 100% as possible is obtained from the curve, and the value can be used as a cutoff value.
- at least one protein of fatty acid binding proteins FABP3, FABP4, FABP5 and FABP7 in a sample prepared from a large number of healthy subjects or at least one of FABP3 gene, FABP4 gene, FABP5 gene and FABP7 gene in the sample It is also preferable to use “average value + 2 standard deviation” of the expression level as a cut-off value, and if this value is used, autism spectrum disorder develops or has a risk of onset with good sensitivity and specificity Can be determined.
- the cut-off value Indicates the risk of the onset of all autism spectrum disorders, ie the presence or absence of predisposition, or the presence or absence of onset.
- the cut-off value may be determined by classifying data for each fixed age and creating an ROC curve at each age.
- the present invention is a test kit for examining the presence or absence of predisposition or onset of autism spectrum disorder, wherein at least one of fatty acid binding proteins FABP3, FABP4, FABP5 and FABP7 in a sample prepared from a human living body.
- One or a nucleic acid probe, a nucleic acid primer, a nucleic acid aptamer, an antibody for detecting at least one of the expression product of the FABP3 gene, the expression product of the FABP4 gene, the expression product of the FABP5 gene and the expression product of the FABP7 gene in the sample A test kit comprising a peptide probe is provided.
- the expression product refers to mRNA transcribed from the FABP3 gene, FABP4 gene, FABP5 gene or FABP7 gene.
- the test kit of the present invention also includes a detection kit that detects the mRNA as a form of cDNA obtained by reverse transcription.
- the nucleic acid probe refers to a nucleic acid probe that specifically binds to any of the expression products, and more specifically includes a TaqMan probe, an Invador probe, and the like.
- the nucleic acid primer refers to a nucleic acid primer that can specifically amplify the mRNA as the expression product or cDNA obtained by reverse transcription of the mRNA, and more specifically, a primer used in a nucleic acid amplification method such as RT-PCR method. Is mentioned.
- the nucleic acid aptamer refers to a nucleic acid construct composed of a nucleic acid that specifically binds to any one of the fatty acid binding proteins FABP3, FABP4, FABP5, and FABP7 contained in a biological sample.
- the peptide probe refers to a peptidic probe that specifically binds to any of the fatty acid binding proteins FABP3, FABP4, FABP5, or FABP7. Specific examples include peptide sequences that specifically bind to FABP3, FABP4, FABP5, or FABP7.
- the nucleic acid probe, nucleic acid primer, and nucleic acid aptamer included in the kit may include a non-natural nucleic acid (PNA or the like) in addition to the natural nucleic acid.
- a peptide probe may be configured to include non-natural amino acids in addition to natural amino acids.
- the test kit according to the present invention further includes various reagents and instruments (polymerase, PCR buffer, each dNTP, pipette, etc.) used for nucleic acid amplification methods such as PCR, and various reagents and instruments for preparing a sample, if necessary. (Test tubes, buffers, etc.), various reagents and instruments for analyzing nucleic acid amplification fragments (electrophoresis gel materials, pipettes, etc.), instruction manuals for test kits, samples used as controls for measurement, and measurement results May be provided with at least one of control data and the like used when analyzing.
- the instruction manual for the test kit includes the above-mentioned [1. The contents of the inspection method according to the present invention described in the column of “Inspection method” are recorded.
- diagnosis of autism spectrum disorder which has so far been based on the subjectivity of doctors, can be diagnosed based on tests that introduce biological standards. Therefore, improvement of diagnostic technology is expected.
- early diagnosis since early diagnosis is possible, early medical treatment can be effectively performed on the patient.
- the test of the present invention provides important information. Can be provided.
- iPS cells artificial pluripotent stem cells prepared from a human having a gene mutation that causes a stop codon in the region encoding protein FABP4 are, for example, 1) autism spectrum disorder It is useful as a screening model for therapeutic drugs of 2), a disease model cell for confirming the effect of a therapeutic drug candidate for autism spectrum disorder, 3) a disease model cell for elucidation of disease onset mechanism and pathology, etc. .
- the inspection result obtained by performing the inspection method described in the column of “Inspection method” can be used as one of diagnostic materials for diagnosis by a doctor.
- the above [1.
- a subject who has obtained a test result indicating that there is a problem can be treated with the result of diagnosis by a doctor as necessary.
- a medical treatment performed by a doctor or an expert other than the doctor can be cited.
- each member constituting the inspection system according to an embodiment of the present invention is realized by “calculation means such as a CPU executing a program code stored in a recording medium such as a ROM or a RAM.
- the case of “functional block” will be described as an example, but it may be realized by hardware that performs similar processing. It is also possible to realize a combination of hardware that performs a part of the processing and the above-described arithmetic means that executes program code for controlling the hardware and the remaining processing.
- the hardware for performing a part of the processing and the arithmetic means for executing the program code for performing the control of the hardware and the remaining processing It can also be realized in combination.
- the calculation means may be a single unit, or a plurality of calculation means connected via a bus inside the apparatus or various communication paths may execute the program code jointly.
- An example of the inspection system according to the present invention includes at least an input reception unit 11A, a measurement unit 11, a storage unit 12, a CPU 13, and a display unit 14 as functional blocks.
- the input receiving unit 11A is an interface that receives an input, and is configured as an interface that connects an inspection system and an external device, or may be configured as an input device such as a keyboard or a mouse in some cases.
- the operating conditions of the inspection system are input via the input receiving unit 11A.
- the measurement unit 11 measures 1) a receiving unit for receiving the biological sample collected from the subject, and 2) measures at least one amount of fatty acid binding proteins FABP3, FABP4, FABP5 and FABP7 in the biological sample, And a measuring device for obtaining a signal value for measuring the expression level of at least one of the FABP3 gene, FABP4 gene, FABP5 gene and FABP7 gene in the biological sample.
- a measuring apparatus is an apparatus that obtains a signal value for measuring the amount of at least one of fatty acid binding proteins FABP3, FABP4, FABP5 and FABP7, preferably the amount of FABP4, in a biological sample according to an ELISA method.
- the storage unit 12 includes a measurement value receiving unit 12a that receives a signal value for measuring the concentration of the above-described biomarker such as FABP3 measured by the measurement apparatus.
- the storage unit 12 may further include a reference value storage unit 12b that stores a reference value (cutoff value) for determining whether or not there is a predisposition to autism spectrum disorder or whether or not it has developed (for example, Configured as memory).
- the CPU 13 includes a calculation unit 13a that obtains a concentration value from a signal value for measuring the concentration of the above-described biomarker such as FABP3.
- the CPU 13 further serves as a calculation unit 13b that generates information for determining whether or not there is a predisposition to the onset of autism spectrum disorder or whether or not there is an onset, and a determination unit 13c that receives evaluation information from the calculation unit 13b and performs determination. It has a function.
- the display unit 14 has a function of displaying the measurement value obtained by the calculation process by the calculation unit 13a or a function of the determination result display unit 14 for displaying the determination result by the determination unit 13c (for example, a liquid crystal display Device or printing device).
- This functional block is realized by the CPU 13 executing a program stored in the storage unit 12 and controlling peripheral circuits such as an input / output circuit.
- step 1 a biological sample is set in the receiving unit of the measuring unit 11, and the concentration of FABP4 in the biological sample is automatically measured using the measuring device of the measuring unit 11.
- the method for measuring the concentration of FABP4 is as described in detail in, for example, the steps a) and b) in the description of the ⁇ Inspection Step> section above.
- step 2 the measured value receiving unit 12 a receives (stores) the measured value of the concentration of FABP 4 measured in step 1.
- the calculation unit 13a obtains a measurement value from the signal value for measuring the concentration of FABP4 stored in the measurement value receiving unit 12a by calculation processing, and stores the reference value stored in the reference value storage unit 12b. Evaluation information for determining whether or not there is a predisposition to autism spectrum disorder or onset from a value (for example, a cutoff value) is generated by the calculation unit 13b.
- the determination unit 13c determines whether or not there is a predisposition to the onset of autism spectrum disorder or whether or not it has developed.
- step 5 the determination result generated by the determination unit 13 c for the presence or absence of a predisposition to the onset of autism spectrum disorder is displayed on the display unit 14.
- the steps (steps 3 to 4) from the generation of the evaluation information to the determination can be executed by operating an evaluation program stored in the storage unit 12, for example.
- the evaluation program performs the determination described in the ⁇ determination> column. More specifically, for example, the measured value of the concentration of FABP4 stored in the measured value receiving unit 12a is compared with the cutoff value stored in the reference value storing unit 12b, and the comparison result is generated as evaluation information. . And when the measured value of the concentration of FABP4 is lower than the cut-off value, it is determined that the subject has a possibility of developing an autism spectrum disorder and a risk.
- the present invention includes any of the following inventions as an example.
- a test method comprising a test step of examining the expression level of at least one of the FABP3 gene, the FABP4 gene, the FABP5 gene and the FABP7 gene in the sample.
- test method according to 1) or 2) wherein the sample is a blood-derived sample.
- the amount of fatty acid binding protein FABP3, the amount of fatty acid binding protein FABP4, the amount of fatty acid binding protein FABP5, the amount of fatty acid binding protein FABP7, the expression level of FABP3 gene, the expression level of FABP4 gene When at least one selected from the expression level of the FABP5 gene and the expression level of the FABP7 gene is lower than that of a healthy subject, it is determined that there is a predisposition to or develops an autism spectrum disorder 1) The inspection method according to any one of 1 to 5).
- a test kit for examining the presence or absence of a predisposition or development of an autism spectrum disorder wherein at least one of fatty acid binding proteins FABP3, FABP4, FABP5 and FABP7 in a sample prepared from a human living body, or Includes a nucleic acid probe, nucleic acid primer, nucleic acid aptamer, antibody or peptide probe for detecting at least one of the FABP3 gene expression product, the FABP4 gene expression product, the FABP5 gene expression product and the FABP7 gene expression product in the sample , Inspection kit.
- Example 1 Preparation of biological sample
- a healthy person aged 5 to 19 127 people were examined as a group of regular development children (control, ie, control group). All of the above subjects were used as test subjects, and biological samples were prepared. All subjects did not receive any drugs.
- peripheral blood was collected from the above-mentioned subjects by peripheral puncture of the antecubital vein, medial rib vein, or dorsal vein. Further, the collected peripheral blood was centrifuged using a centrifuge to separate serum from the cell sediment. The obtained serum was stored at ⁇ 80 ° C. until used for testing.
- the serum fatty acid binding protein FABP4 concentration obtained by the above method was measured by an ELISA method using a commercially available ELISA kit (HUMAN AFABP ENZYME IMMUNOASSAY KIT, SPI bio).
- the measurement was carried out using a sample obtained by diluting 30 ⁇ L of the subject's serum in a buffer solution (EIA buffer attached to the ELISA kit) 10-fold, and for each group of the autistic children group and the control group, 4 to 4 Divided into age groups of 5 years old, 6-7 years old, 8-9 years old, 10-11 years old, 12-13 years old, 14-15 years old, 16-17 years old and 18-19 years old, FABP4 protein concentration in each age group The average value (referred to as average concentration) was calculated.
- Example 2 (Preparation of biological sample) Autistic children group (Autism (native)) of 152 patients (152 of whom 11 are women) who have been diagnosed as having autism by a doctor The same procedure as in Example 1 was performed except that 119 healthy persons (27 females, 119 females) aged 4 to 18 years were used as regular development children (control, ie, healthy control group). It was.
- the serum fatty acid binding protein FABP4 concentration obtained by the above-described method was measured using the same measurement method as the measurement method in Example 1. The measurement was carried out using a sample obtained by diluting 30 ⁇ L of the subject's serum 10-fold in a buffer solution (EIA buffer attached to the ELISA kit), and for each group of autistic children and healthy controls, 4 It was divided into age groups of -6 years, 7-8 years, 9-10 years, 11-12 years, 13-14 years, 15-16 years and 17-18 years, and the average value of FABP4 protein concentration in each age group ( (Referred to as average concentration).
- the average concentration of the autistic children group of the same age group and the average concentration of the healthy control group are compared for each age group (Age), and the average concentration of the healthy control group is compared with the average concentration of the autistic children group.
- Age age group
- the average concentration of the healthy control group is compared with the average concentration of the autistic children group.
- the concentration of fatty acid binding protein FABP4 was 16 ng / ml, and in the age group of 7-8 years old, the concentration of fatty acid binding protein FABP4 was The cut-off value was 15.7 ng / ml. Based on these cutoff values, positive and negative numbers were counted to calculate sensitivity, specificity, positive predictive value, and negative predictive value. In addition, Area Under the Curve and Youden index values were obtained. The results are shown in FIG. Although not shown, the Mann-Whitney test (Mann-Whitney test) shows that there is no difference in the concentration of FABP4 between the male and female individuals in the healthy control group in each of the age groups 4-6 and 7-8 years old. ) To confirm.
- BMI Body Mass Index
- the upper diagram in FIG. 4 is a distribution diagram showing the distribution of FABP4 concentration in serum in each of the age groups of 4 to 6 years and age groups of 7 to 8 years.
- the number of positive and negative persons was counted for the presence or absence of disease in each of the autism group and the healthy subject group.
- all 15 out of 15 autistic groups were positive.
- 5 were positive and 21 were negative.
- 21 out of 23 autism groups were positive and 2 were negative.
- out of 16 healthy control groups 7 were positive and 9 were negative.
- the lower table in FIG. 2 is a table of sensitivity, specificity, positive predictive value, and negative predictive value calculated based on these results.
- the present invention can be used for testing for the presence or absence of a predisposition to autism spectrum disorder.
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Abstract
Description
すなわち、本発明は以下の何れかの発明を包含する。
1)自閉症スペクトラム障害の素因の有無または発症の有無を検査する方法であって、ヒトの生体より調製した試料における脂肪酸結合タンパク質FABP3、FABP4、FABP5およびFABP7のうちの少なくとも一つの量を調べるか、当該試料におけるFABP3遺伝子、FABP4遺伝子、FABP5遺伝子およびFABP7遺伝子のうちの少なくとも一つの発現量を調べる検査工程を含む、検査方法。
2)自閉症スペクトラム障害の素因の有無または発症の有無を検査する検査キットであって、ヒトの生体より調製した試料における脂肪酸結合タンパク質FABP3、FABP4、FABP5およびFABP7のうちの少なくとも一つ、または当該試料におけるFABP3遺伝子の発現産物、FABP4遺伝子の発現産物、FABP5遺伝子の発現産物およびFABP7遺伝子の発現産物の少なくとも一つを検出するための核酸プローブ、核酸プライマー、核酸アプタマー、抗体またはペプチドプローブを含む、検査キット。
本発明に係る検査方法は、自閉症スペクトラム障害の素因の有無または発症の有無を検査する方法であって、ヒトの生体より調製した試料(以下「生体試料」と称する場合がある)における脂肪酸結合タンパク質FABP3(fatty acid binding protein3:FABP3と称する場合がある)、FABP4(fatty acid binding protein4:FABP4と称する場合がある)、FABP5(fatty acid binding protein5:FABP5と称する場合がある)およびFABP7(fatty acid binding protein7:FABP7と称する場合がある)のうちの少なくとも一つの量を調べるか、当該試料におけるFABP3遺伝子、FABP4遺伝子、FABP5遺伝子およびFABP7遺伝子のうちの少なくとも一つの発現量を調べる検査工程を含む方法である。
本明細書中の「自閉症スペクトラム障害」としては、具体的には広汎性発達障害(PDD)、自閉症、およびアスペルガー症候群等が挙げられる。症状として、コミュニケーションの障害、言語発達の障害、こだわりや繰り返し行動および対人関係成立の障害等を呈する。
本明細書中の「診断する」または「診断」とは、医師によってなされる、患者の徴候および症状に基づく疾患または病態の同定を指す。一方、本明細書中の「検査する」または「検査」とは、医師による同定(診断)を必須としない、検査対象のヒト(「被験者」と称する場合もある)における、自閉症スペクトラム障害の素因の有無または発症の有無の検査を指す。本発明の検査方法によって得られた検査結果は、医師によってなされる診断の一材料になりうる。また、例えば、脂肪酸結合タンパク質FABPに対し親和性の強い脂肪酸を含む化合物が見出され、さらに、当該化合物をヒトへ投与することが、補充療法につながる場合、本発明の検査は重要な情報を提供し得る。なお、自閉症スペクトラム障害の素因の有無を検査するとは、自閉症スペクトラム障害を既に発症しているか否かとはかかわり無く、将来的に自閉症スペクトラム障害を発症する可能性を検査することも含む概念である。一方、自閉症スペクトラム障害の発症の有無を検査するとは、自閉症スペクトラム障害を発症しているか否かについて検査することを指す。
被験者の年齢は低いほど好ましく、例えば血中のFABP4の量を指標とする場合には8歳以下であることが好ましく、7歳以下であることがより好ましく、6歳以下であることがさらに好ましい。より年齢が低い時期に検査を受けることで、自閉症スペクトラム障害の素因を有する、または当該疾患を発症していると早期に判定された被験者は、早期から療育を受けることができる。
生体試料は、被験者から採取される。生体試料の種類としては、特に限定されず、被験者のタンパク質および核酸(遺伝子発現産物としてのmRNA)の少なくとも一方を含んでいればよい。生体試料としては、例えば、細胞試料、組織試料、体液試料が挙げられ、中でも体液試料が好ましい。体液試料としては、血液試料、リンパ液試料、髄液試料等が挙げられるが、血液試料およびリンパ液試料が好ましく、血液試料のうち、特に末梢血試料および臍帯血試料が好ましい。末梢血試料は、例えば指先への穿刺等によって容易に採取が可能であるから被験者の負担が少なく、加えて、本発明の検査方法の対象となる分子マーカーを充分に含んでいる。臍帯血試料は出生時に臍帯から容易に採取が可能であり、超早期診断が可能であるという利点を有している。
本発明の検査方法は、以下のa)またはb)に示す検査工程を含む。
工程a)上記生体試料における脂肪酸結合タンパク質FABP3、FABP4、FABP5およびFABP7のうちの少なくとも一つの量を調べる、
工程b)上記生体試料におけるFABP3遺伝子、FABP4遺伝子、FABP5遺伝子およびFABP7遺伝子のうちの少なくとも一つの発現量を調べる。
工程a)は、上記生体試料における脂肪酸結合タンパク質FABP3、FABP4、FABP5およびFABP7のうちの少なくとも一つのタンパク質の量、より具体的には、生体試料の単位量あたりに含まれるタンパク質の量(例えば、タンパク質の濃度)を測定する工程である。但し、生体試料の単位量あたりに含まれるタンパク質の量を測定するという概念には定量的測定および定性的測定の両方が含まれ、濃度測定の他、対照と比較可能な形式でタンパク質の量を提示することが含まれる。より具体的には、例えば、検量線等を用いて濃度換算する以前の、取得した時点でのデータ比較、またはタンパク質の量が、ある一定の閾値を超えているか否かを示す形式での結果の提示等も含まれる。
工程b)は、上記生体試料を用いて、当該試料中のFABP3遺伝子、FABP4遺伝子、FABP5遺伝子およびFABP7遺伝子のうちの少なくとも一つの発現量を測定する工程である。なお、FABP3遺伝子とはFABP3をコードしている核酸の総称であり、FABP4遺伝子とはFABP4をコードしている核酸の総称であり、FABP5遺伝子とはFABP5をコードしている核酸の総称であり、FABP7遺伝子とはFABP7をコードしている核酸の総称である。
自閉症スペクトラム障害の素因の有無または発症の有無の判定は、上述の工程a)または工程b)に示す検査工程により得られた、FABP3、FABP4、FABP5およびFABP7のうちの少なくとも一つのタンパク質量、またはFABP3遺伝子、FABP4遺伝子、FABP5遺伝子およびFABP7遺伝子のうちの少なくとも一つの発現量を、被験者と対照とで比較することによって行う。
判定の一例では、対照と比較して被験者の生体試料におけるFABP3、FABP4、FABP5またはFABP7(タンパク質量でも遺伝子の発現量であってもよい)のうちの少なくとも一つの量が有意に低い場合、被験者は、自閉症スペクトラム障害の素因を有しているまたは発症していると判定される。なお、量が有意に低い(少ない)とは、定量的測定による結果であっても定性的測定による結果であってもよく、具体的な数値の比較はもちろん、相対的な量の比較(実際に量を算出する必要は無く、ある基準より高いか低いかを判断する)も含む概念である。
また、本発明は、自閉症スペクトラム障害の素因の有無または発症の有無を検査する検査キットであって、ヒトの生体より調製した試料における脂肪酸結合タンパク質FABP3、FABP4、FABP5およびFABP7のうちの少なくとも一つ、または当該試料におけるFABP3遺伝子の発現産物、FABP4遺伝子の発現産物、FABP5遺伝子の発現産物およびFABP7遺伝子の発現産物の少なくとも一つを検出するための核酸プローブ、核酸プライマー、核酸アプタマー、抗体またはペプチドプローブを含む、検査キットを提供する。
後述する参考例1に示すような、タンパク質FABP4をコードする領域にストップコドンが生じる遺伝子の変異を有するヒトから作製した人工多能性幹細胞(iPS細胞)は、例えば、1)自閉症スペクトラム障害の治療薬のスクリーニング用途、2)自閉症スペクトラム障害の治療薬候補の効果を確認するための疾患モデル細胞、3)疾患発症メカニズムおよび病態の解明のための疾患モデル細胞、等として有用である。
上記〔1.検査方法〕の欄で説明した検査方法を行うことによって得られた検査結果は、医師による診断を行う際の診断資料の一つとして利用することができる。また、上記〔1.検査方法〕の欄で説明した検査方法を行うことによって、自閉症スペクトラム障害の発症の危険性が有り(すなわち素因が有り)という検査結果が得られた被験者、または自閉症スペクトラム障害の発症が有りという検査結果が得られた被験者については、必要に応じて医師による診断の結果を伴った上で、治療を行うことができる。ここで、治療の一例としては、医師または医師以外の専門家が行う療育を挙げることができる。
上述したような本発明に係る検査方法を実行するために用いられる検査システム、および当該検査システムを用いた検査方法もまた、本発明の範囲内である。下記実施形態では、本発明の一実施形態としての検査システムを構成する各部材が、「CPUなどの演算手段がROMやRAMなどの記録媒体に格納されたプログラムコードを実行することによって実現される機能ブロックである」場合を例にして説明するが、同様の処理を行うハードウェアによって実現してもよい。また、処理の一部を行うハードウェアと、当該ハードウェアの制御や残余の処理を行うプログラムコードを実行する上記演算手段とを組み合わせて実現することもできる。さらに、上記各部材のうち、ハードウェアとして説明した部材であっても、処理の一部を行うハードウェアと、当該ハードウェアの制御や残余の処理を行うプログラムコードを実行する上記演算手段とを組み合わせて実現することもできる。なお、上記演算手段は、単体であってもよいし、装置内部のバスや種々の通信路を介して接続された複数の演算手段が共同してプログラムコードを実行してもよい。
本発明は、一例として、以下の何れかの発明を包含する。
1)自閉症スペクトラム障害の素因の有無または発症の有無を検査する方法であって、ヒトの生体より調製した試料における脂肪酸結合タンパク質FABP3、FABP4、FABP5およびFABP7のうちの少なくとも一つの量を調べるか、当該試料におけるFABP3遺伝子、FABP4遺伝子、FABP5遺伝子およびFABP7遺伝子のうちの少なくとも一つの発現量を調べる検査工程を含む、検査方法。
2)検査工程において、脂肪酸結合タンパク質FABP4の量、またはFABP4遺伝子の発現量を調べる、1)に記載の検査方法。
3)上記試料が血液由来の試料である、1)または2)に記載の検査方法。
4)対象となる上記タンパク質の量を調べる、1)~3)のいずれかに記載の検査方法。5)上記ヒトは8歳以下である、1)~4)のいずれかに記載の検査方法。
6)上記検査工程において、上記試料における脂肪酸結合タンパク質FABP3の量、脂肪酸結合タンパク質FABP4の量、脂肪酸結合タンパク質FABP5の量、脂肪酸結合タンパク質FABP7の量、FABP3遺伝子の発現量、FABP4遺伝子の発現量、FABP5遺伝子の発現量、およびFABP7遺伝子の発現量から選択される少なくとも一つが健常者と比較して低いときに、自閉症スペクトラム障害の素因が有るまたは発症している、と判定する、1)~5)の何れかに記載の検査方法。
7)上記検査工程において、機能ブロックとして少なくとも入力受付部、測定部、格納部、CPU、および表示部、を備えている検査システムを用いて自閉症スペクトラム障害の素因が有るまたは発症している、と判定する6)に記載の検査方法。
8)上記自閉症スペクトラム障害は自閉症である、1)~7)に記載の検査方法。
9)自閉症スペクトラム障害の素因の有無または発症の有無を検査する検査キットであって、ヒトの生体より調製した試料における脂肪酸結合タンパク質FABP3、FABP4、FABP5およびFABP7のうちの少なくとも一つ、または当該試料におけるFABP3遺伝子の発現産物、FABP4遺伝子の発現産物、FABP5遺伝子の発現産物およびFABP7遺伝子の発現産物の少なくとも一つを検出するための核酸プローブ、核酸プライマー、核酸アプタマー、抗体またはペプチドプローブを含む、検査キット。
自閉症スペクトラム障害の新たな分子マーカーの候補を探索する目的で、自閉症スペクトラム障害の小児のDNAサンプルを用いてFABP4遺伝子上の変異のスクリーニングを行った。その結果、自閉症スペクトラム障害の267家系のうち一家系では、タンパク質FABP4をコードする遺伝子領域のエキソン3に位置する294番目のGがAに置換している変異があり、その結果FABP4のアミノ酸配列において98番目のトリプトファンを指定するコドンがストップコドンに置換されていることを見出した。
(生体試料の調製)
医師により自閉症を発症していると診断されている6歳から19歳までの男性116名を自閉症児童群(Autism(native))とし、健常者である5歳から19歳までの127名を定型発達児童群(control、すなわち対照群)として検査を行った。上記の全員を検査の対象者とし、生体試料を調製した。なお、対象者はすべて、薬物などは投与されていない。
上述の方法で得られた血清中の脂肪酸結合タンパク質FABP4濃度は、市販のELISAキット(HUMAN AFABP ENZYME IMMUNOASSAY KIT、SPI bio)を用いたELISA法により測定した。測定は、対象者の血清30μLを緩衝液(ELISAキットに付属のEIA buffer)に10倍希釈したものを試料とし、そして、自閉症児童群および対照群のそれぞれの群について、さらに、4~5歳、6~7歳、8~9歳、10~11歳、12~13歳、14~15歳、16~17歳および18~19歳の年齢群に分け、各年齢群のFABP4タンパク質濃度の平均値(平均濃度と称する)を算出した。さらに、同じ年齢群の自閉症児童群の平均濃度と、対照群の平均濃度とを、各年齢群(Age)同士について比較し、対照群の平均濃度に対し自閉症児童群の平均濃度が有意な差が見られるかどうか検討した。結果を図1の(上)に示した。なお、自閉症児童群および対照群のそれぞれについて各年齢群に属する人数を、図1の(下)に示した。
図1に示されるように、7歳以下の自閉症児童群の脂肪酸結合タンパク質FABP4濃度の平均値は、対照群の平均値と比較して有意に低いことを見いだした。6~7歳の群について、より詳細に解析するために、6~7歳の年齢群における個人毎の脂肪酸結合タンパク質FABP4濃度の値について、対照群対自閉症児童群(Control vs Autism)の分布図を作成し、Mann-Whitney test(マン・ホイットニー検定)を用いた検定を行った。脂肪酸結合タンパク質FABP4の濃度16ng/mlをカットオフ値とし、人数を計数して感度、特異度、陽性的中率、および陰性的中率を計算した。結果を図2に示した。
図1および図2の結果から明らかなように、6~7歳の年齢群において、脂肪酸結合タンパク質FABP4の濃度は、対照群と比較して自閉症児童群が著しく低く、有意な差が見られた。したがって、末梢血中の脂肪酸結合タンパク質FABP4濃度の値が自閉症スペクトラム障害の早期診断マーカーとして有用であると結論付けた。
(生体試料の調製)
医師により自閉症を発症していると診断されている、4歳から18歳までの152名(152名中、女性は11名)の患者を自閉症児童群(Autism(native))とし、4歳から18歳までの119名(119名中、女性は27名)の健常者を定型発達児童群(control、すなわち健常対照群)とした以外は、実施例1における方法と同様に行った。
上述の方法で得られた血清中の脂肪酸結合タンパク質FABP4濃度は、実施例1における測定方法と同様の測定方法を用いて測定した。測定は、対象者の血清30μLを緩衝液(ELISAキットに付属のEIA buffer)に10倍希釈したものを試料とし、そして、自閉症児童群および健常対照群のそれぞれの群について、さらに、4~6歳、7~8歳、9~10歳、11~12歳、13~14歳、15~16歳および17~18歳の年齢群に分け、各年齢群のFABP4タンパク質濃度の平均値(平均濃度と称する)を算出した。さらに、同じ年齢群の自閉症児童群の平均濃度と、健常対照群の平均濃度とを、各年齢群(Age)同士について比較し、健常対照群の平均濃度に対し自閉症児童群の平均濃度が有意な差が見られるかどうか検討した。結果を図3の上の図に示した。なお、自閉症児童群および健常対照群のそれぞれについて各年齢群に属する人数を、図3の下の表に示した。
図3に示されるように、4~6歳および7~8歳の年齢群それぞれにおける脂肪酸結合タンパク質FABP4濃度の平均値は、健常対照群の平均値と比較して有意に低いことを見いだした。4~6歳および7~8歳の群について、より詳細に解析するために、4~6歳および7~8歳の年齢群における個人毎の脂肪酸結合タンパク質FABP4濃度の値について、健常対照群対自閉症児童群(Control vs Autism)の分布図を作成し、Mann-Whitney test(マン・ホイットニー検定)を用いた検定を行った。ROC(Receiver Operating Characteristic)曲線を作成し、4~6歳の年齢群では、脂肪酸結合タンパク質FABP4の濃度16ng/mlをカットオフ値とし、7~8歳の年齢群では、脂肪酸結合タンパク質FABP4の濃度15.7ng/mlをカットオフ値とした。これらのカットオフ値に基づき、陽性および陰性の人数を計数して感度、特異度、陽性的中率、および陰性的中率を計算した。また、Area Under the CurveおよびYouden indexの値をそれぞれ求めた。結果を図4に示した。なお、図示しないが、4~6歳および7~8歳の年齢群のそれぞれにおいて、健常対照群における男女の個体間でFABP4の濃度に差がないことを、Mann-Whitney test(マン・ホイットニー検定)を用いた検定によって確認している。なお、図示はしないが、4~6歳および7~8歳のそれぞれの年齢群において、健常対照群と自閉症群との間でBMI(Body Mass Index: BMI)に差が無いことを、Mann-Whitney test(マン・ホイットニー検定)を用いた検定によって確認している。
図4の上の図は、4~6歳の年齢群および7~8歳の年齢群のそれぞれにおける血清中のFABP4濃度の分布を示す分布図である。
Claims (8)
- 自閉症スペクトラム障害の素因の有無または発症の有無を検査する方法であって、
ヒトの生体より調製した試料における脂肪酸結合タンパク質FABP3、FABP4、FABP5およびFABP7のうちの少なくとも一つの量を調べるか、当該試料におけるFABP3遺伝子、FABP4遺伝子、FABP5遺伝子およびFABP7遺伝子のうちの少なくとも一つの発現量を調べる検査工程を含む、検査方法。 - 検査工程において、脂肪酸結合タンパク質FABP4の量、またはFABP4遺伝子の発現量を調べる、請求項1に記載の検査方法。
- 上記試料が血液由来の試料である、請求項1または2に記載の検査方法。
- 対象となる上記タンパク質の量を調べる、請求項1~3の何れか一項に記載の検査方法。
- 上記ヒトは8歳以下である、請求項1~4の何れか一項に記載の検査方法。
- 上記検査工程において、上記試料における脂肪酸結合タンパク質FABP3の量、脂肪酸結合タンパク質FABP4の量、脂肪酸結合タンパク質FABP5の量、脂肪酸結合タンパク質FABP7の量、FABP3遺伝子の発現量、FABP4遺伝子の発現量、FABP5遺伝子の発現量、およびFABP7遺伝子の発現量から選択される少なくとも一つが健常者と比較して低いときに、自閉症スペクトラム障害の素因が有るまたは発症している、と判定する、請求項1~5の何れか一項に記載の検査方法。
- 上記自閉症スペクトラム障害は自閉症である、請求項1~6の何れか一項に記載の検査方法。
- 自閉症スペクトラム障害の素因の有無または発症の有無を検査する検査キットであって、
ヒトの生体より調製した試料における脂肪酸結合タンパク質FABP3、FABP4、FABP5およびFABP7のうちの少なくとも一つ、または当該試料におけるFABP3遺伝子の発現産物、FABP4遺伝子の発現産物、FABP5遺伝子の発現産物およびFABP7遺伝子の発現産物の少なくとも一つを検出するための核酸プローブ、核酸プライマー、核酸アプタマー、抗体またはペプチドプローブを含む、検査キット。
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