Composition and kit for diagnosing autism disorder

Abstract

The present invention relates to a composition and a kit for diagnosing autism disorder. The composition, the kit, and a method for providing information according to the present invention can conveniently and effectively diagnose autism disorder in a small amount of blood by using multiple biomarkers in which proteins and metabolites have been combined in order to diagnose autism disorder.

Description

Composition and kit for diagnosing autism spectrum disorder

[0001] The present invention relates to a composition and kit for diagnosing autism spectrum disorder.

[0002]

[0003] Autism is a neurodevelopmental disorder that impairs communication and social interaction. While the cause of this condition is still unclear, some researchers speculate that it is a genetic developmental disorder rather than an emotional one. The incidence is approximately 1 in 1,000, with a male-to-female ratio of approximately 4:1 in the United States. A 2011 survey found that the prevalence of autism in Korea was over 2%, drawing attention. Autism is generally referred to as "autism spectrum disorder" or "autism spectrum disorder," and academics divide this disorder into three categories: first, autism (Kanner's Syndrome), defined by Leo Kanner as a child; second, Asperger's Syndrome, discussed by Hans Asperger; and finally, high-functioning autism, including Savant Syndrome.

[0004] The prevalence of autism continues to rise worldwide. It is one of the diseases for which the pathophysiological cause is unclear, hindering the development of diagnostic and therapeutic drugs. Because the pathophysiological cause of autism remains unclear, research on the condition is typically conducted using clinical or laboratory animal models that exhibit the disease's characteristic and diverse traits. Currently, autism is diagnosed based on behavioral pattern analysis and questionnaires, based on limited behavior or impaired social interaction. Currently, molecular diagnostic techniques for autism are not available in clinical practice. These subjective diagnoses often make it difficult to assess the complexity and severity of symptoms.

[0005] To overcome these problems, molecular diagnostic techniques for diagnosing autism are being studied, but they are still at a minimal level.

[0006]

[0007] The purpose of the present invention is to provide a composition and kit for diagnosing autism disorder that can easily and accurately diagnose autism.

[0008] The purpose of the present invention is to provide a method for providing information for diagnosing autism spectrum disorder by measuring the expression level of specific proteins and metabolites.

[0009]

[0010] 1. A composition for diagnosing autism disorder, comprising at least one substance that specifically binds to the mRNA or protein of mannose-binding protein C, receptor-type tyrosine-protein phosphatase gamma or peptidase inhibitor 16, and a substance that specifically binds or reacts with Acetylcarnitine, PCaa C42:6, PCae C40:1 or PCae C38:6.

[0011] 2. In the above 1, the material is a composition comprising at least one selected from the group consisting of antibodies, aptamers, DNA, RNA, proteins, and polypeptides.

[0012] 3. A kit for diagnosing autism spectrum disorder comprising the composition of the above 1.

[0013] 4. A kit for diagnosing autism spectrum disorder comprising a quantitative means for 3 above: Mannose-binding protein C, Receptor-type tyrosine-protein phosphatase gamma, Peptidase inhibitor 16, Acetylcarnitine, PCaa C42:6, PCae C40:1 or PCae C38:6.

[0014] 5. A method for providing information for diagnosing autism disorder, comprising a step of measuring the expression level of mannose-binding protein C, receptor-type tyrosine-protein phosphatase gamma, peptidase inhibitor 16, acetylcarnitine, PCaa C42:6, PCae C40:1 or PCae C38:6 in a sample isolated from a diagnostic subject.

[0015] 6. In the above 5, the method is selected from the group consisting of blood, serum, plasma, tissue, cells, saliva, or urine.

[0016] 7. A method according to 5 above, wherein the step of measuring the expression level is performed by a mass quantification means.

[0017] 8. A method for determining that, in the above 5, if the expression level is higher than that of the control group, the possibility of developing autism spectrum disorder is higher than that of the control group.

[0018]

[0019] The present invention relates to a composition and a kit for diagnosing autism spectrum disorder. The composition, kit, and information providing method of the present invention can easily and effectively diagnose autism spectrum disorder from a small amount of blood using multiple biomarkers comprising a combination of proteins and metabolites for diagnosing autism spectrum disorder.

[0020]

[0021] Figure 1 shows the chemical structures of (a) Acetylcarnitine, (b) PCaa C42:6, (c) PCae C40:1, and (d) PCae C38:6.

[0022] Figure 2 shows the results of proteins / peptides identified through the above proteomic analysis.

[0023] Figure 3 shows the results of metabolite analysis on the blood of autism patients and their family members to discover autism disorder metabolite markers.

[0024] Figure 4 shows the analysis results of metabolites whose expression increased in the patient from the above metabolite analysis results.

[0025] Figure 5 is a schematic diagram of the experimental process for proteomic and metabolomic analysis in autism patients and their blood samples.

[0026] Figure 6 is a schematic diagram of the sample preparation process for the experiment of Figure 5.

[0027] Figure 7 shows the results of analyzing the correlation between proteins and metabolites analyzed in autism patients and their blood samples.

[0028] Figure 8 is a multi-panel ROC curve that was created by evaluating an ASD model by selecting a group of mutually independent candidate substances among proteins and metabolites from the correlation analysis results of Figure 7.

[0029] Figure 9 is an ROC curve of the excavated proteome and metabolome multi-markers.

[0030]

[0031] Hereinafter, the present invention will be described in detail.

[0032]

[0033] The present invention relates to a composition for diagnosing autism disorder, comprising at least one substance that specifically binds to mRNA or protein of mannose-binding protein C, receptor-type tyrosine-protein phosphatase gamma or peptidase inhibitor 16, and a substance that specifically binds or reacts with Acetylcarnitine, PCaa C42:6, PCae C40:1 or PCae C38:6.

[0034] Mannose-binding protein C, receptor-type tyrosine-protein phosphatase gamma, and peptidase inhibitor 16 are proteins encoded by the MBL2, PTPRG, and PI16 genes, respectively, and Acetylcarnitine, PCaa C42:6, PCae C40:1, and PCae C38:6 are metabolites, and their structures are shown in Figure 1. Specific information on the metabolites can be found in Acetylcarnitine (https: / hmdb.ca / metabolites / HMDB0000201), PCaa C42:6 (https: / hmdb.ca / metabolites / HMDB0008734), PCae C40:1 (https: / hmdb.ca / metabolites / HMDB0013433), and PCae C38:6 (https: / hmdb.ca / metabolites / HMDB0013409).

[0035] Mannose-binding protein C, receptor-type tyrosine-protein phosphatase gamma, and peptidase inhibitor 16 are present in a sample isolated from the diagnostic target, and their mRNA or protein sequences can utilize sequences known to NCBI, etc. The sequences are not limited thereto, but the sequences of the individual species being diagnosed can be used.

[0036] Acetylcarnitine, PCaa C42:6, PCae C40:1, and PCae C38:6 are metabolites present in samples isolated from diagnostic subjects.

[0037] The subject of the diagnosis is an animal that currently has autism, is suspected of having autism, or seeks information for autism diagnosis. The animal may be a mammal, including a human.

[0038] The sample is separated from the diagnostic subject, and examples thereof include blood, serum, plasma, tissue, cells, saliva, or urine, and specifically, it may be blood, serum, or plasma separated from the diagnostic subject, but is not limited thereto.

[0039] The above substance is not limited as long as it can specifically bind to and detect any one of the mRNA and protein of Mannose-binding protein C, Receptor-type tyrosine-protein phosphatase gamma and Peptidase inhibitor 16, and can specifically bind to or react with any one of Acetylcarnitine, PCaa C42:6, PCae C40:1 and PCae C38:6 and detect these metabolites, but may be, for example, one from the group consisting of antibodies, aptamers, DNA, RNA, proteins and polypeptides.

[0040] As used herein, "antibody" means a protein molecule specific for an antigenic site. For the purposes of the present invention, the antibody means an antibody that specifically binds to the marker protein, such as Mannose-binding protein C, Receptor-type tyrosine-protein phosphatase gamma, Peptidase inhibitor 16, Acetylcarnitine, PCaa C42:6, PCae C40:1, or PCae C38:6, and may include all monoclonal antibodies, polyclonal antibodies, and recombinant antibodies.

[0041] The polyclonal antibodies described above can be produced by methods well known in the art, including injecting the protein antigen described above into an animal and collecting blood from the animal to obtain serum containing the antibodies. Such polyclonal antibodies can be produced from any animal species host, including, but not limited to, goats, rabbits, sheep, monkeys, horses, pigs, cows, and dogs.

[0042] Additionally, the antibodies of this invention may also include special antibodies such as chimeric antibodies and humanized antibodies.

[0043] The above "peptide" possesses the advantage of high binding affinity to target substances and is resistant to denaturation even during heat and chemical treatments. Furthermore, its small molecular size allows it to be attached to other proteins to form fusion proteins. Specifically, it can be attached to polymer protein chains, making it suitable for use as a diagnostic kit and drug delivery material.

[0044] The above "aptamer" refers to a type of polynucleotide composed of a special type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure in itself and has the characteristics of being able to bind to a target molecule with high affinity and specificity. As described above, an aptamer can specifically bind to an antigenic substance in the same way as an antibody, but is composed of a polynucleotide that is more stable than a protein, has a simpler structure, and is easy to synthesize, and therefore can be used as a substitute for an antibody.

[0045] The substance that specifically binds to the mRNA may be, but is not limited to, sense and antisense primers or probes.

[0046] In the present invention, "primer" refers to a short genetic sequence that serves as the starting point for DNA synthesis, and is an oligonucleotide synthesized for purposes such as diagnosis and DNA sequencing. The primers are typically synthesized and used in lengths of 15 to 30 base pairs, but this may vary depending on the intended use, and may be modified by methylation, capping, etc. using known methods.

[0047] In the present invention, the term "probe" refers to a nucleic acid capable of specifically binding to mRNA, ranging from a few bases to several hundred bases in length, produced through enzymatic, chemical, or synthetic processes. The presence or absence of mRNA can be confirmed by labeling with a radioisotope or enzyme, and the probe can be designed and modified using known methods.

[0048] A probe or primer that specifically binds to a nucleotide sequence of a gene encoding Mannose-binding protein C, Receptor-type tyrosine-protein phosphatase gamma and Peptidase inhibitor 16, a sequence complementary to the nucleotide sequence or a fragment of the nucleotide sequence is known. Therefore, a person skilled in the art can design the primer or probe based on the sequence according to a conventional method in the art.

[0049] The composition of the present invention is a substance for use in diagnosing autism spectrum disorder in a patient suspected of having autism spectrum disorder, and can be used to determine whether autism spectrum disorder exists by treating a sample isolated from the subject of diagnosis and measuring the expression levels of Mannose-binding protein C, Receptor-type tyrosine-protein phosphatase gamma, Peptidase inhibitor 16, Acetylcarnitine, PCaa C42:6, PCae C40:1 or PCae C38:6 in the sample.

[0050] The present invention relates to a kit for diagnosing autism spectrum disorder comprising the above composition.

[0051] The kit may comprise one or more other component compositions, solutions or devices suitable for an analytical method for measuring the expression level of Mannose-binding protein C, Receptor-type tyrosine-protein phosphatase gamma, Peptidase inhibitor 16, Acetylcarnitine, PCaa C42:6, PCae C40:1 or PCae C38:6.

[0052] The kit of the present invention may include not only a preparation capable of measuring the expression levels of mannose-binding protein C, receptor-type tyrosine-protein phosphatase gamma, peptidase inhibitor 16, acetylcarnitine, PCaa C42:6, PCae C40:1, and PCae C38:6, but also one or more types of compositions, solutions, or devices suitable for analyzing the expression levels. For example, the kit may include a quantitative means for quantifying the concentration of proteins and metabolites. The above concentration quantification means may use high-performance liquid chromatography (HPLC) or a mass spectrometry means such as liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis-mass spectrometry (CE-MS), direct infusion mass spectrometry (DIMS), liquid chromatography-ion trap mass spectrometry, four-transistor infrared (FT-IR), nuclear magnetic resonance (NMR), Raman spectroscopy, malditope mass spectrometry (MALDI-TOF MS), triple-TOF mass spectrometry (Triple TOF MS), quadrupole-TOF mass spectrometry (Q-TOF MS), or a combination thereof, but is not limited thereto, and an analysis method commonly used in the art may be used. Preferably, a mass spectrometry means may be included.

[0053] The above kit can use mass spectrometry or ELISA to analyze proteins and metabolites in the same way. Mass spectrometry can use the mass quantification methods listed above. ELISA can use antibodies that specifically bind to mannose-binding protein C, receptor-type tyrosine-protein phosphatase gamma, peptidase inhibitor 16, acetylcarnitine, PCaa C42:6, PCae C40:1, and PCae C38:6, respectively.

[0054] Additionally, proteins and metabolites can be analyzed using different methods. For example, the expression levels of the proteins mannose-binding protein C, receptor-type tyrosine-protein phosphatase gamma, and peptidase inhibitor 16 can be measured using mRNA, while the expression levels of the metabolites acetylcarnitine, PCaa C42:6, PCae C40:1, and PCae C38:6 can be measured using mass spectrometry.

[0055] The mRNA of the above proteins can be analyzed using RT-PCR or microarray methods.

[0056] Accordingly, the kit may be a kit containing essential elements required for performing the RT-PCR method. For example, it may include a set of primers specific for the mRNA of the mannose-binding protein C, receptor-type tyrosine-protein phosphatase gamma, and peptidase inhibitor 16 genes, as well as a test tube or other appropriate container, a reaction buffer, deoxyribonucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC water, sterile water, etc. In addition, it may include a set of primers specific for a gene used as a quantitative control.

[0057] The above microarray method can be performed according to a method known in the art. For example, it may be a microarray in which cDNA corresponding to a sequence of mRNA or a fragment thereof of a gene encoding the above proteins is attached to a substrate as a probe.

[0058]

[0059] The present invention relates to a method for providing information for diagnosing autism disorder, comprising a step of measuring the expression level of mannose-binding protein C, receptor-type tyrosine-protein phosphatase gamma, peptidase inhibitor 16, acetylcarnitine, PCaa C42:6, PCae C40:1 or PCae C38:6 in a sample isolated from a diagnostic subject.

[0060] The above samples and diagnostic subjects are as described above.

[0061] The method for measuring the expression level of the above Mannose-binding protein C, Receptor-type tyrosine-protein phosphatase gamma, Peptidase inhibitor 16, Acetylcarnitine, PCaa C42:6, PCae C40:1 or PCae C38:6 can be performed by selecting a commonly used method capable of measuring the concentration of protein, protein mRNA and metabolites in a sample.

[0062] For example, as a method for measuring the concentration of a protein in the sample, the amount of protein can be confirmed using an antibody that specifically binds to the protein. Analysis methods for this purpose include an immunoblot test, a sandwich assay, an enzyme linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry, immunoprecipitation assay, complement fixation assay, fluorescence-activated cell sorting (FACS), western blotting, flow cytometry, enzyme-substrate chromogenic assay, antigen-antibody agglutination, and a protein chip, and the like. Preferably, an enzyme linked immunosorbent assay (ELISA) can be used, but is not limited thereto.

[0063] Methods for measuring the mRNA concentration of proteins in the sample include, but are not limited to, reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (Competitive RT-PCR), real-time reverse transcriptase polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting, and DNA chips.

[0064] As a method for measuring the concentration of metabolites in the sample, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis-mass spectrometry (CE-MS), direct infusion mass spectrometry (DIMS), liquid chromatography-ion trap mass spectrometry, four-transistor infrared (FT-IR), nuclear magnetic resonance (NMR), Raman spectroscopy, malditope mass spectrometry (MALDI-TOF MS), triple-TOF mass spectrometry (Triple TOF MS), quadrupole-TOF mass spectrometry (Q-TOF MS) or a combination thereof may be used, but is not limited thereto, and a metabolite analysis method commonly used in the art may be used.

[0065] Preferably, it can be performed using a mass quantification means capable of simultaneously measuring the concentration of proteins and metabolites in the sample, such as liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis-mass spectrometry (CE-MS), direct infusion mass spectrometry (DIMS), liquid chromatography-ion trap mass spectrometry, four-transistor infrared (FT-IR), nuclear magnetic resonance (NMR), Raman spectroscopy, malditope mass spectrometry (MALDI-TOF MS), triple-TOF mass spectrometry (Triple TOF MS), quadrupole-TOF mass spectrometry (Q-TOF MS), or a combination thereof.

[0066] The method of the present invention may further include a step of comparing the expression level with the expression level of a control group.

[0067] For example, if the expression levels of the above proteins and metabolites in a sample of a patient suspected of autism are compared with the expression levels of a normal person (control group), and the expression levels of the patients suspected of autism are higher than those of the control group, it can be determined that the likelihood of developing autism is higher than that of the control group.

[0068] In addition, if the expression levels of the above proteins and metabolites in the samples of patients suspected of having autism are measured to be low or have no statistically significant difference (e.g., p<0.05) compared to the control group, it can be determined that the possibility of developing autism is low.

[0069] In addition, by comparing the expression levels of the above proteins and metabolites in samples from two patients suspected of having autism, it can be determined that a patient with a higher expression level is more likely to develop autism than a patient with a lower expression level.

[0070]

[0071] Hereinafter, the present invention will be described in more detail through examples. However, the following examples and test examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0072]

[0073] Hereinafter, the present invention will be described in detail by way of examples to specifically explain the present invention.

[0074]

[0075] Discovery of multiple biomarkers from protein and metabolite combinations

[0076] 1. Discovery of protein biomarker candidates

[0077] To identify protein biomarker candidates, proteomic analysis was performed on the blood of patients and their family members who were thought to have very similar genetic and environmental factors.

[0078] Proteomic analysis utilized label-free mass spectrometry technology and the newly developed 16-plex TMT technology. Experiments were conducted in 16 sets with 5 fractions per set, and as a result, the number of proteins identified per set increased by approximately two-fold with TMT technology compared to the existing label-free analysis, with an average of ~600 proteins identified, and a total of 11,861 peptides and 2,241 proteins were identified. The results of analyzing the number of blood proteins identified per set and the cumulative number of peptides / proteins are shown in Figure 2.

[0079]

[0080] 2. Discovery of candidate metabolite biomarkers

[0081] Metabolomic analysis was performed using the same blood samples as the proteomic analysis samples. Targeted MRM-MS was used to analyze 180 metabolites. The results identified altered metabolites in the patients compared to their normal family members. The results of the blood metabolomic analysis are shown in Figure 3, and the altered metabolites are shown in Figure 4. The altered metabolites were found to be increased in the autism patients compared to both parents. These altered metabolites were selected as biomarker candidates.

[0082]

[0083] 3. Proteome and metabolome analysis and multi-biomarker panel discovery

[0084] Among the blood samples from 54 families, samples with severe hemolysis were excluded, and proteomic and metabolomic analyses were performed using blood samples from 53 fathers, 53 mothers, 54 patients, 10 siblings, and 57 normal individuals.

[0085] A schematic diagram of the protein experiment is shown in Figure 5, and a schematic diagram of the protein sample preparation process is shown in Figure 6.

[0086] Data were collected using a high-resolution QE Orbitrap mass spectrometer, and a few μL sample was trapped onto an online capillary C18 column using an online nano liquid chromatography system. Then, peptide samples were separated in nanoliter flow using a C18 analytical column (50 cm long, 75 μm inner diameter) and mass spectrometry chromatograms were performed.

[0087] To integrate multi-omics to be used in the autism plasma diagnostic model, protein (peptide) and metabolite correlation analysis was performed on the protein biomarker candidate group and metabolite biomarker candidate group selected from the individual analyses above. Fifty-five peptides and 21 metabolites were used as unique pattern variables, respectively, and correlation analysis was performed on 1,155 pairs. Of these, 577 peptide-metabolite pairs showed a significant correlation (P<0.10). The results of the correlation analysis are shown in Figure 7. In the correlation analysis, independent candidate groups of proteins and metabolites were selected to create an ASD classification model. When these models were evaluated, the cutoff point where the sum of sensitivity and specificity was maximized was 0.068, and when the ROC curve was derived based on this, the AUC value was confirmed to be 0.819 (Figure 8).

[0088]

[0089] 4. Verification of multi-markers of proteins and metabolites

[0090] We conducted experiments to validate the multi-marker combination of proteins and metabolites identified above. Blood samples from 75 families (72 fathers, 74 mothers, 75 patients, 37 siblings, and 42 healthy individuals) were analyzed. Proteomic and metabolomic analyses were performed using PRM-MS technology.

[0091] As a result, we confirmed that the AUC value of the curve was 0.807 when training the Random Forest model using protein markers (Mannose-binding protein C, Receptor-type tyrosine-protein phosphatase gamma, and Peptidase inhibitor 16) and metabolite markers (Acetylcarnitine, PCaa C42:6, PCae C40:1, and PCae C38:6). When a model was created by combining the optimal logistic-regression model variables for the single-omics multi-marker panel, we confirmed that the multi-omics multi-marker panel improved the performance for the training / test set compared to the single-omics multi-marker panel. Surprisingly, we obtained an AUC value of 0.926 for the training set and 0.885 for the test set. This result is shown in Fig. 9.

Claims

1. A composition for diagnosing autism disorder, comprising at least one substance that specifically binds to the mRNA or protein of mannose-binding protein C, receptor-type tyrosine-protein phosphatase gamma or peptidase inhibitor 16, and a substance that specifically binds or reacts with Acetylcarnitine, PCaa C42:6, PCae C40:1 or PCae C38:

6.

2. A composition according to claim 1, wherein the material is at least one selected from the group consisting of antibodies, aptamers, DNA, RNA, proteins, and polypeptides.

3. A kit for diagnosing autism spectrum disorder comprising the composition of claim 1.

4. A kit for diagnosing autism spectrum disorder according to claim 3, comprising a quantitative means for mannose-binding protein C, receptor-type tyrosine-protein phosphatase gamma, peptidase inhibitor 16, acetylcarnitine, PCaa C42:6, PCae C40:1, or PCae C38:

6.

5. A method for providing information for diagnosing autism disorder, comprising a step of measuring the expression level of mannose-binding protein C, receptor-type tyrosine-protein phosphatase gamma, peptidase inhibitor 16, acetylcarnitine, PCaa C42:6, PCae C40:1, or PCae C38:6 in a sample isolated from a diagnostic subject.

6. A method according to claim 5, wherein the sample is selected from the group consisting of blood, serum, plasma, tissue, cells, saliva, or urine.

7. A method according to claim 5, wherein the step of measuring the expression level is performed by a mass quantification means.

8. A method according to claim 5, wherein if the expression level is higher than that of the control group, the likelihood of developing autism spectrum disorder is determined to be higher than that of the control group.

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