Progressive supranuclear palsy diagnostic marker

Abstract

To provide a biomarker correlating with the presence of progressive supranuclear palsy (PSP).SOLUTION: A method for collecting data for evaluating whether a subject may suffer from progressive supranuclear palsy, the method comprising measuring an amount of miR-6088 in a biological sample derived from the subject, the amount being collected as data that positively correlates with the possibility that the subject suffers from progressive supranuclear palsy.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to biomarkers that can be used in the diagnosis of progressive supranuclear palsy. [Background technology]

[0002] Progressive supranuclear palsy (PSP) is a type of Parkinson's syndrome (PS), which also includes corticobasal degeneration (CBD), multiple system atrophy (MSA), and Parkinson's disease (PD).

[0003] PSP is a progressive neurodegenerative disease of unknown cause, and no fully effective treatment has yet been found. Pathologically, PSP is characterized by the formation of abnormally phosphorylated tau aggregates in the brain, and although this is thought to be involved in the pathology, the full picture has not yet been elucidated.

[0004] According to epidemiological studies in Japan, the prevalence of PSP is estimated to be approximately 20 per 100,000 people. However, an analysis of forensic autopsies confirmed PSP pathology in 4.6% of autopsied brains from people aged 60 or older, suggesting that the number of PSP cases is potentially much higher.

[0005] PSP is typically characterized by gait disturbance accompanied by a tendency to fall and impaired vertical eye movement. However, no clear biomarkers are known for PSP, and clinical diagnosis is made based on clinical symptoms and imaging findings, with a definitive diagnosis ultimately being made by pathological diagnosis of the brain at autopsy.

[0006] Advances in clinical research have revealed that PSP can have a wide variety of clinical manifestations, including not only Richardson's syndrome, which exhibits a typical clinical picture, but also subtypes characterized by tremor and muscle rigidity similar to PD, subtypes characterized by cerebellar symptoms similar to MSA, subtypes characterized by speech impairment and freezing of gait, and subtypes characterized by clinical manifestations similar to CBD. As such, PSP exhibits a wide variety of clinical manifestations, making it difficult to differentiate and diagnose from other PS diseases, especially in the early stages of the disease. Currently, the only way to assess disease progression or treatment efficacy is by assessing the scale of clinical signs (Non-Patent Documents 1-5). While disease-modifying therapies using anti-tau antibodies are being developed, the difficulty of diagnosis is thought to be one of the factors that hinders the execution of clinical trials and drug development.

[0007] Patent Document 1 describes that a peptide fragment of chromogranin B, which appears as a specific peak in mass spectrometry of a cerebrospinal fluid sample, shows a negative correlation with progressive supranuclear palsy. Patent Document 2 describes that measurement data of the amount of a molecular species bound by an antibody that recognizes an epitope of an amyloid precursor protein and has an apparent molecular weight of 100 kDa in a cerebrospinal fluid sample derived from a subject positively correlates with the possibility of suffering from progressive supranuclear palsy. Patent Documents 1 and 2 are patent applications with the same inventors as the present application. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Litvan et al., Neurology, 1996, 47(1):1-9. [Non-patent document 2] Williams et al., Brain, 2005, 128:1247-1258 [Non-patent document 3] Williams et al., Movement Disorders, 2007, 22(15):2235-2241 [Non-patent document 4] Kanazawa et al., Mov. Disord., 2009, 24(9):1312-8. [Non-patent document 5] Golbe and Ohman-Strickland, Brain, 2007, 130:1552-1565 [Patent documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 2023-005573 [Patent Document 2] Japanese Patent Application Publication No. 2023-163821 Summary of the Invention [Problem to be solved by the invention]

[0010] In light of the above problems, there is a strong need for new diagnostic tools to assess or determine whether a subject has PSP, and in particular, for new biomarkers that correlate with the presence of PSP. [Means for solving the problem]

[0011] In a study to identify biomarkers characteristic of PSP patients, the present inventors identified microRNA-6088 (also called miR-6088 or hsa-miR-6088) as a substance that exhibits PSP-specific changes in patient serum. The levels of miR-6088 in serum samples from PSP patients were significantly higher than those in serum samples from non-PSP individuals, and it was found that this could be used as a biomarker for PSP.

[0012] The present disclosure includes at least the following embodiments. [1] A method for collecting data to assess the likelihood that a subject has progressive supranuclear palsy, comprising measuring the amount of miR-6088 in a biological sample derived from the subject, wherein the amount is collected as data that positively correlates with the likelihood that the subject has progressive supranuclear palsy. [2] 1. A method for determining whether a subject has or is likely to have progressive supranuclear palsy, comprising: (1) obtaining a measurement of the amount of miR-6088 in a biological sample from the subject; and (2) comparing the measured value of said quantity with a reference value; and determining that the subject is suffering from or is likely to be suffering from progressive supranuclear palsy when the measured value of the amount is higher than the reference value. [3] A method for collecting data to estimate the duration of progressive supranuclear palsy in a patient, the method comprising measuring the amount of miR-6088 in a biological sample derived from the patient, the amount being collected as data that positively correlates with the duration of the patient's disease. [4] A method for collecting data to estimate the rate of disease progression in a patient with progressive supranuclear palsy, the method comprising measuring the amount of miR-6088 in a biological sample derived from the patient, wherein the amount is collected as data that negatively correlates with the rate of disease progression in the patient. [5] The method according to any one of [1] to [4], wherein the biological sample is a blood sample selected from the group consisting of whole blood, plasma, and serum samples. [6] The method according to [5], wherein the miR-6088 to be measured comprises a contiguous nucleotide sequence consisting of at least 18 bases contained in SEQ ID NO:1. [7] The method according to [5], wherein the measured miR-6088 comprises the nucleotide sequence of SEQ ID NO: 2.

[0013] Embodiments of the present disclosure make it possible to collect data useful for diagnosing PSP from patient-derived samples. Data collection methods according to embodiments of the present disclosure provide new tools that can replace or add to existing tools for diagnosing PSP, and by providing a data collection method useful for assessing or determining the presence, duration, rate of disease progression, or efficacy of treatment, they may contribute to improving the diagnosis and treatment of PSP and promote drug development. Furthermore, embodiments of the present disclosure make it possible to determine whether a subject has progressive supranuclear palsy or is likely to have it. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 shows the results of RT-PCR analysis of miR-6088 levels in serum samples from individuals with progressive supranuclear palsy (PSP) and non-PSP control individuals (CTL). Error bars indicate standard deviation (SD). [Figure 2] FIG. 2 shows a receiver operating characteristic (ROC) analysis based on the amount of miR-6088 in serum samples in a population including multiple PSP and non-PSP individuals, respectively. [Figure 3] FIG. 3 is a two-dimensional scatter plot in which the horizontal axis represents the duration of PSP disease (years) and the vertical axis represents the amount of miR-6088 in serum samples, with the values ​​for each PSP individual plotted on a plane. [Figure 4] Figure 4 is a two-dimensional scatter plot in which the horizontal axis represents the annual rate of change of PSP-RS (PSPRS / y) and the vertical axis represents the amount of miR-6088 in serum samples, with the values ​​for each PSP individual plotted on a plane. DETAILED DESCRIPTION OF THE INVENTION

[0015] In one aspect, the present disclosure provides a method for collecting data for assessing the likelihood that a subject has progressive supranuclear palsy, the method comprising measuring the amount of miR-6088 in a biological sample derived from the subject, wherein the amount is collected as data that positively correlates with the likelihood that the subject has progressive supranuclear palsy.

[0016] The subject in this disclosure is a human. The biological sample derived from the subject may be a blood sample or a biopsy sample, with a blood sample being preferred. Blood samples are advantageous because they are easier to collect than cerebrospinal fluid samples and impose a significantly smaller burden on the subject. The blood sample may be a whole blood, plasma, or serum sample. While the amounts of miR-6088 in whole blood, plasma, and serum may be correlated, plasma or serum is preferred from the standpoint of measurement stability, with serum samples being particularly preferred. Those skilled in the art will understand that blood samples such as serum may contain disease-specific microRNAs (miRNAs) secreted by neurons, other brain cells, or disease-related cells other than these cells (e.g., via exosomes or other extracellular vesicles). Methods for collecting and processing plasma and serum samples are well known to those skilled in the art.

[0017] Pre-microRNAs (pre-miRNAs), which are generated by processing miRNA gene transcripts (pri-miRNAs) in the nucleus, form hairpin structures due to base pairs formed within the molecule. After being exported from the nucleus, they undergo further processing to become mature single-stranded miRNAs. miR-6088 in this disclosure can refer to pri-miR-6088, a transcript of the human MIR6088 gene corresponding to NCBI gene ID 102464836; pre-miR-6088 and mature miR-6088, which are generated by processing of pri-miR-6088; or other RNA molecular species generated during the processing of pri-miR-6088 and pre-miR-6088. Pri-miR-6088 has a nucleotide sequence corresponding to SEQ ID NO: 1 (since it is RNA, it has uracil (u) bases instead of thymine (t)). In the present embodiment, the miR-6088 detected or measured may be RNA containing a continuous base sequence consisting of at least 18, 19, 20, or 21 bases contained in SEQ ID NO: 1. In the present embodiment, the miR-6088 detected or measured may be RNA containing the nucleotide sequence consisting of SEQ ID NO: 2, which corresponds to mature miR-6088, and may be mature miR-6088.

[0018] In embodiments of each aspect of the present disclosure, the amount of miR-6088 can be measured by methods known to those skilled in the art, including microarrays, reverse transcription quantitative PCR (RT-qPCR), and RNA sequencing (RNA-seq), with RT-qPCR being preferred. Detection of miR-6088 by RT-qPCR can be performed by assay methods known to those skilled in the art. For example, detection by RT-qPCR may involve first-strand cDNA synthesis from mature miR-6088 using a hairpin primer. Alternatively, first-strand synthesis for RT-qPCR detection can be performed by adding a polyA tail to mature miR-6088 using polyA polymerase, followed by an oligo-dT primer containing a 5' adapter sequence. Detection by RT-qPCR may also be performed to detect pri-miR-6088 rather than mature miR-6088.

[0019] In the present disclosure, "positively correlated with the likelihood of suffering from progressive supranuclear palsy" means that the quantity (level) of the biomarker in question is significantly higher in individuals suffering from progressive supranuclear palsy compared to individuals not suffering from the disease. Therefore, the higher the level of a biomarker that shows a positive correlation with progressive supranuclear palsy, the higher the likelihood that the individual is suffering from progressive supranuclear palsy. As will be apparent to those skilled in the art, in the present disclosure, "significantly increased" or "significantly decreased" can mean "statistically significantly increased" or "statistically significantly decreased," which can be determined by statistical tests known by those skilled in the art, such as Student's t-test.

[0020] In one embodiment, provided is a method for determining whether a subject suffers from or is likely to suffer from progressive supranuclear palsy.The method includes: (1) obtaining the measurement value of the amount of miR-6088 in the biological sample from said subject; and (2) comparing said measurement value with a reference value; if said measurement value is higher than said reference value, determine that said subject suffers from or is likely to suffer from progressive supranuclear palsy.On the other hand, if the measurement value of miR-6088 amount is lower than or equal to reference value, determine that said subject does not suffer from or is unlikely to suffer from progressive supranuclear palsy.

[0021] In the above (1), obtaining a measurement value of the amount of miR-6088 can be performed by measurement methods known to those skilled in the art, including microarray, reverse transcription quantitative PCR (RT-qPCR), and RNA sequencing (RNA-seq), and is preferably performed by RT-qPCR.

[0022] In (1) or (2) above, the measured amount of miR-6088 can be normalized by the expression level of other genes in the same biological sample, or by the amount of protein, lipid, etc. contained in the biological sample, or can be normalized and then used for comparison. The gene used for normalization is preferably a gene, such as a housekeeping gene, that shows a constant expression level regardless of whether the subject has progressive supranuclear palsy. Normalization can be performed, for example, by dividing the expression level of miR-6088 by the expression level, protein amount, or lipid amount of the gene used for normalization in the same biological sample. Measurement of protein and lipid amounts can be performed using methods known to those skilled in the art.

[0023] The reference value used in (2) above may be a cutoff value obtained by ROC (Receiver Operating Characteristic) curve analysis of values ​​measured in biological samples derived from multiple PSP individuals and non-PSP control individuals. ROC curve analysis and determination of cutoff values ​​are well known to those skilled in the art. Most typically, sensitivity (i.e., true positive rate) is plotted on the upward vertical axis, and (1-specificity) (i.e., false positive rate) is plotted on the horizontal axis extending from the bottom of the vertical axis to the right. The cutoff value is the point with the shortest distance to the point in the upper left corner of the graph (the ideal point where sensitivity = 1 and specificity = 1), or the point where the Youden Index (sensitivity + specificity - 1) is maximized.

[0024] However, it should be understood that, in general, the reference values ​​(threshold levels) of biomarkers can be made looser or stricter depending on the purpose of each individual test (for example, whether the purpose is initial screening to broadly select a group of patient candidates, or whether the purpose is diagnosing each individual patient, etc.), and that the reference values ​​in the present disclosure can also be determined in advance for each individual application by a person skilled in the art based on their ordinary skills, and can be varied as appropriate.

[0025] In one embodiment, the level of miR-6088 in a serum sample (test sample) from a subject is measured, and if it is higher than a predetermined reference value, it is determined that the subject is suffering from, or is likely to be suffering from, progressive supranuclear palsy.

[0026] In one embodiment, the level of miR-6088 in a serum sample (test sample) from a subject is measured, and the higher the measured level, the more likely the subject is to suffer from progressive supranuclear palsy.

[0027] In another aspect, there is provided a method for collecting data for estimating the duration of progressive supranuclear palsy in a patient, the method comprising measuring the amount of miR-6088 in a biological sample derived from the patient, wherein the amount is collected as data that positively correlates with the duration of the patient's disease.

[0028] The disease duration is understood by those skilled in the art to mean the time elapsed since the onset of the disease in a patient of interest. Therefore, in this embodiment, a method for collecting data for estimating the time elapsed since the onset of progressive supranuclear palsy in a patient of progressive supranuclear palsy is provided.

[0029] In the present disclosure, "positively correlated with the disease duration of a patient" means that the quantity (level) of the biomarker in question is higher in patients with a longer disease duration. Therefore, the higher the level of a biomarker that shows a positive correlation with progressive supranuclear palsy, the longer the disease duration of the individual can be evaluated.

[0030] The duration of disease can be estimated using data obtained by the method of the embodiment and the relationship between the amount of miR-6088 and the duration of disease. Such a relationship can be predetermined based on the amount of miR-6088 in biological samples from multiple patients and the known duration of disease. Such a relationship can be a function with the amount of miR-6088 as the independent variable and the duration of disease as the dependent variable. The function can also be a linear function with a positive slope. Deriving a linear function representing the relationship between the amounts of miR-6088 and the duration of disease in multiple patients can be performed using methods known to those skilled in the art, including regression analysis. The duration of disease in a patient can be estimated based on such a function and the amount of miR-6088 in the patient.

[0031] In yet another aspect, there is provided a method for collecting data for estimating a rate of disease progression in a patient with progressive supranuclear palsy, the method comprising measuring the amount of miR-6088 in a biological sample derived from the patient, wherein the amount is collected as data that negatively correlates with the rate of disease progression in the patient.

[0032] The rate of disease progression is understood by those skilled in the art to represent the change in the severity of symptoms of a disease of interest per unit time. Thus, in this embodiment, a method for collecting data for estimating the change in the severity of progressive supranuclear palsy per unit time in a patient with progressive supranuclear palsy is provided.

[0033] The severity of progressive supranuclear palsy symptoms can be assessed using the Progressive Supranuclear Palsy Rating Scale (PSP-Rating Scale; also abbreviated as PSP-RS or PSPRS) (Golbe and Ohman-Strickland, Brain, 130(6), 1552-1565, 2007). Therefore, the change in the PSP-RS per unit time, e.g., PSP-RS / year (PSPRS / y), can be used as an index of the rate of progression of progressive supranuclear palsy. It has been reported that the average progression of the PSP-RS was 11.3 points per year (Golbe and Ohman-Strickland, Brain, 130(6), 1552-1565, 2007).

[0034] In the present disclosure, "negatively correlated with the rate of disease progression in a patient" means that the quantity (level) of the biomarker of interest is higher in patients with a slower rate of disease progression. Therefore, the higher the level of the biomarker, the slower the rate of disease progression in that patient can be assessed.

[0035] The duration of disease can be estimated using data obtained by the method of the embodiment and the relationship between the amount of miR-6088 and the rate of disease progression. Such a relationship can be predetermined based on the amount of miR-6088 in biological samples from multiple patients and the known rate of disease progression. Such a relationship can be a function with the amount of miR-6088 as the independent variable and the rate of disease progression as the dependent variable. The function can also be a linear function with a negative slope. Deriving a linear function representing the relationship between the amounts of miR-6088 and the rate of disease progression in multiple patients can be performed using methods known to those skilled in the art, including regression analysis. The rate of disease progression in a patient can be estimated based on such a function and the amount of miR-6088 in the patient. [Example]

[0036] Specific embodiments will be described in detail below with reference to examples, but these are merely examples and the invention of the present disclosure is not limited to these examples.

[0037] Example 1: Exploratory Study In a study to discover and identify biomarkers for PSP, miRNAs were extracted from serum samples collected from five PSP patients (mean age 74.4 years; including two pathologically diagnosed cases) and five control individuals (mean age 73.4 years; age- and sex-matched controls without neurodegenerative disease), as shown in Table 1 below, and analyzed using a miRNA microarray (3D-Gene™ Human miRNA oligo chip, Toray Industries, Inc.). 2,566 miRNAs were analyzed, and miR-6088 was identified as one of the miRNAs showing a significant difference (p<0.10) between the two groups using Student's t-test. [Table 1]

[0038] Example 2: Validation Study In a study to verify the usefulness of miR-6088 as a PSP marker, miRNA was extracted from serum samples collected from 30 PSP patients (mean age 72.9 years) and 30 non-PSP control individuals (mean age 72.8 years), as shown in Table 2 below. The relative expression levels of miR-6088 (SEQ ID NO: 2) were quantified by reverse transcription quantitative PCR (RT-qPCR) using Thermo Fisher's TaqMan® assay, and differences between the groups were analyzed. A significant difference (p<0.05) in the expression levels of miR-6088 between the two groups was confirmed by Student's t-test or ANOVA analysis (Figure 1). Receiver operating characteristic (ROC) analysis was performed using the obtained data, and the expression level (5.02) corresponding to the point on the ROC curve with the smallest distance from the upper left corner of the plot plane was determined as the cutoff value. This resulted in an area under the curve (AUC) of 0.701, a sensitivity of 70.0%, and a specificity of 70.0% (Figure 2). These results are comparable to the sensitivity of 84.4% and specificity of 34.2% based on existing clinical diagnostic criteria for early-stage MDS (Mov Disord 2019;34:1144-1153). [Table 2]

[0039] [Example 3: Estimation of disease duration] The correlation between the duration of disease and the amount of miR-6088 in serum samples of PSP patients in Example 2 was analyzed by regression analysis (FIG. 3). The amount of miR-6088 (miR6088) and the duration of disease showed a positive correlation (miR6088 = -2.441 + 5.017 * duration of disease; R 2 =0.268, p<0.01), indicating that the duration of disease could be estimated from the collected data on miR-6088 levels.

[0040] Example 4: Estimation of disease progression rate The PSP rating scale (PSP-RS) and its annual rate of change (PSP-RS / y) were determined for the PSP patients in Example 2, and the correlation between these and the amount of miR-6088 in serum samples was analyzed by regression analysis. The amount of miR-6088 (miR6088) did not show a significant correlation with the PSP-RS itself (not shown), but showed a negative correlation with the annual rate of change of the PSP-RS (PSPRS / y) (miR6088 = 21.828 - 0.912 * PSPRS / y; R 2 =0.315, p=0.0574; Figure 4), demonstrating that the collected miR-6088 abundance data could be used to estimate the rate of disease progression, and suggesting that miR-6088 may have a protective effect against PSP.

[0041] These results demonstrate that miR-6088 can be useful as a diagnostic marker for progressive supranuclear palsy, and that miR-6088 measurement data can be collected as data that positively correlates with the possibility that a subject has progressive supranuclear palsy, data that positively correlates with the duration of PSP disease in PSP patients, and / or data that negatively correlates with the rate of disease progression in PSP patients.Furthermore, these results demonstrate that by comparing the measured amount of miR-6088 in a sample derived from a subject with a reference value, it can be determined whether the subject has progressive supranuclear palsy or is likely to have it.

Claims

1. A method for collecting data to assess the likelihood that a subject has progressive supranuclear palsy, comprising measuring the amount of miR-6088 in a biological sample derived from the subject, wherein the amount is collected as data that positively correlates with the likelihood that the subject has progressive supranuclear palsy.

2. 1. A method for determining whether a subject has or is likely to have progressive supranuclear palsy, comprising: (1) obtaining a measurement of the amount of miR-6088 in a biological sample from the subject; and (2) comparing the measured value of said quantity with a reference value; and determining that the subject is suffering from or is likely to be suffering from progressive supranuclear palsy when the measured value of the amount is higher than the reference value.

3. A method for collecting data to estimate the duration of progressive supranuclear palsy in a patient, the method comprising measuring the amount of miR-6088 in a biological sample derived from the patient, the amount being collected as data that positively correlates with the duration of the patient's disease.

4. A method for collecting data to estimate the rate of disease progression in a patient with progressive supranuclear palsy, the method comprising measuring the amount of miR-6088 in a biological sample derived from the patient, wherein the amount is collected as data that negatively correlates with the rate of disease progression in the patient.

5. The method according to any one of claims 1 to 4, wherein the biological sample is a blood sample selected from the group consisting of a whole blood, plasma, and serum sample.

6. The method of claim 5, wherein the miR-6088 to be measured comprises a continuous nucleotide sequence consisting of at least 18 bases contained in SEQ ID NO:

1.

7. The method of claim 5, wherein the miR-6088 to be measured comprises a nucleotide sequence consisting of SEQ ID NO:2.

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