NORMAL-PRESSURE HYDROCEPHALUS DIAGNOSIS COMPOSITION AND DIAGNOSIS MARKER DETECTION METHOD, USING LEVEL OF EXPRESSION OF CHI3L1 IN BLOOD

Abstract

The present invention relates to a normal-pressure hydrocephalus diagnosis composition and diagnosis marker detection method which use the level of expression of chitinase 3-like 1 (CHI3L1) in blood and, more specifically, to a normal-pressure hydrocephalus diagnosis composition and diagnosis kit which comprise a preparation for measuring the expression level of CHI3L1 protein or of mRNA encoding the protein, and to a normal-pressure hydrocephalus diagnosis method using same. According to the present invention, the expression level of CHI3L1 is significantly increased in patients with normal-pressure hydrocephalus. Thus, the present invention is excellent since it can rapidly and accurately diagnose normal-pressure hydrocephalus by analyzing the expression level of CHI3L1.

Claims

1.-9. (canceled)

10. A method for diagnosing and treating a normal-pressure hydrocephalus in a subject, the method comprising the steps of: (a) providing a sample of the subject; (b) measuring the expression level of CHI3L1 protein or mRNA encoding the same in the sample; (c) comparing the expression level of the protein or mRNA with a normal subject and determining that the subject with increased expression level compared to the normal subject has a normal-pressure hydrocephalus; and (d) administering an effective amount of an agent that modulates the expression of CHI3L1 (Chitinase 3-Like 1) protein or mRNA encoding the same to the subject determined to have normal-pressure hydrocephalus.

11. The method of claim 10, wherein the sample is one selected from the group consisting of blood, plasma, and serum.

12. The method of claim 10, wherein the CHI3L1 protein comprises an amino acid sequence defined by SEQ ID NO: 1.

13. The method of claim 10, wherein the mRNA comprises a nucleotide sequence defined by SEQ ID NO: 2.

14. The method of claim 10, wherein the step of measuring the expression level of a CHI3L1 (Chitinase 3-Like 1) protein or an mRNA encoding the same is conducted using an antibody that specifically binds to CHI3L1 protein.

15. The method of claim 10, wherein the step of measuring the expression level of a CHI3L1 (Chitinase 3-Like 1) protein or an mRNA encoding the same is conducted using a probe or primer set that specifically binds to mRNA encoding CHI3L1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0095] FIG. 1 is a result of analyzing the expression level of the CHI3L1 protein marker using ELISA. (AD: Alzheimer's disease, MCI: mild cognitive impairment, NPH: normal-pressure hydrocephalus, PD: Parkinson's disease)

[0096] FIG. 2 shows the results of analyzing the expression level of the LCN2 and PTX3 protein markers using ELISA. (AD: Alzheimer's disease, MCI: mild cognitive impairment, NPH: normal-pressure hydrocephalus, PD: Parkinson's disease)

[0097] FIG. 3 shows the CHI3L1 ROC curve for normal-pressure hydrocephalus compared to the whole group.

[0098] FIG. 4 shows the CHI3L1 ROC curve for normal-pressure hydrocephalus compared to the normal control group.

MODE FOR CARRYING OUT INVENTION

[0099] Hereinafter, the present invention will be described in detail.

[0100] However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

[0101] Method

[0102] 1. Patient Sample

[0103] Healthy volunteer samples were used as a control group. Participants were recruited from patients who visited the dementia clinic of Chilgok Kyungpook National University Hospital (Daegu). Extensive neuropsychological tests including clinical dementia rating (CDR) and mini mental state examination (MMSE) were performed in all test groups (normal and patient groups).

[0104] Patients were classified into Alzheimer's disease (AD), mild cognitive impairment (MCI), normal-pressure hydrocephalus (NPH), Parkinson's disease (PD), and normal subjects according to the cause of dementia.

[0105] Clinical records, diagnosis, treatment modalities, and blood samples were collected with the consent of the patients.

[0106] Plasma samples were collected in sodium heparin tubes within the next morning from patients who fasted more than 8 hours the day before, followed by centrifugation for 15 minutes with a 2000 rpm centrifuge, and then the supernatant was separated and stored frozen at −80° C. until used in the experiment.

TABLE-US-00001 TABLE 1 The characteristics of Participants Controls AD IPD NPH MCI Characteristics (N = 66) (N = 110) (N = 13) (N = 29) (N = 14) Gender, 30 (45.5) 40 (40) 5 (38.5) 13 (44.8) 5 (35.7) male (%) Age (year) 64.5 ± 10.5 64.8 ± 9.5 62.2 ± 9.9  70.6 ± 5.8 67.0 ± 9.3 K-MMSE 28.3 ± 2.0  17.4 ± 6.0 27.4 ± 71.9 18.6 ± 6.0 24.9 ± 3.4 CDR 0.1 ± 0.2  1.1 ± 0.6 0.3 ± 0.2  0.9 ± 0.5  0.5 ± 0.2

[0107] 2. Sandwich ELISA of CHI3L1

[0108] CHI3L1 levels in participants' plasma samples were measured using the Sandwich Elisa Duo-set (R & D Systems; Minneapolis, Minn., cat no. DC3L10) method as follows. Primary antibody (Rat Anti-Human CHI3L1 capture Antibody, R & D Systems; Minneapolis, Minn.) was diluted in PBS and attached to 96-well ELISA plate overnight at room temperature, and washed three times with PBS-T (phosphate buffered saline with 0.05% Tween 20). Blocking was reacted with PBS containing 1% BSA (bovine serum albumin) for 1 hour at room temperature and washed three times with PBS-T. Standard (human recombination CHI3L1 protein) is 15.6 to 1000 pg/ml. All samples were put in 100 μl in each well, and reacted for 2 hours at room temperature and washed three times with PBS-T. 100 ul of a secondary antibody (Biotinylated Goat Anti-CHI3L1 detection Antibody, R & D Systems; Minneapolis, Minn.) was added to each well and reacted at room temperature for 2 hours, washed three times with PBS-T, and then reacted for 20 minutes with the addition of horse radish peroxidase-conjugated streptavidin, followed by washing three times with PBST. Finally, 100 μl of each mixture of 3,3′, 5,5′tetramethylbenzidine (TMB) and peroxidase solution H.sub.2O.sub.2 in a 1:1 ratio were added, 2N H.sub.2SO.sub.4 was added thereto to stop the reaction, and the absorbance was measured at 450 nm. All experiments were repeated and analyzed using the average value, and individual protein concentrations were analyzed using Bradford assay. Plasma CHI3L1 concentrations were calibrated with each individual protein concentration and analyzed.

[0109] 3. Statistical Analysis

[0110] Comparison of plasma CHI3L1 levels in normal, MCI, AD, PD, and NPH patient groups was performed by one-way analysis of variance (ANOVA) with a Turkey-HSD (Thukey-HSD) test of post-hoc comparison. In addition, clinical data from various groups were added as predictors, and age, gender, and education year were added as covariates in the covariance model analysis. Covariance analysis (ANCOVA) is performed when covariance is observed. In addition, a spearman analysis of the correlations was performed for each group. This was done using linear regression on covariates with all available data related to CHI3L1 levels and MMSE, CDR and UPDRS values. Statistical analysis was performed using SPSS 18.0 software (SPSS Inc; Chicago, Ill.), sigmaplot 10.0 (SPSS Inc) and MATLAB 7.0 (The Mathworks; Natick, Mass.). Statistical significance value (p) was set to <0.05 and all result values were expressed as mean±SD.

Example 1: Confirmation of Increased Expression of CHI3L1 in Serum of Patients with Normal-Pressure Hydrocephalus

[0111] The concentration of CHI3L1 in the plasma of normal-hydrocephalus patients was measured. As shown in FIG. 1, the mean value of plasma CHI3L1 was 59.3 ng/mL (standard deviation 35.1 ng/mL) in 66 healthy volunteers. The CHI3L1 level in patients with normal-hydrocephalus (average 132.6 ng/mL, standard deviation 71.7 ng/mL, n=29) was very high compared to the Alzheimer's disease (86.4 ng/mL, standard deviation 68.1 ng/mL, n=110), Parkinson's disease (65.4 ng/mL, standard deviation 35.8 ng/mL, n=13), and patients with mild cognitive impairment (63.9 ng/mL, standard deviation 50.3 ng/mL, n=14).

[0112] Therefore, this confirmed that CHI3L1 can be used as a marker for diagnosing normal-pressure hydrocephalus, for the data of the present invention showed a significant difference in the level of CHI3L1 protein expression in the plasma samples of patients with normal-pressure hydrocephalus compared to other patients with degenerative brain diseases.

Example 2: Comparison of Expression Levels of Various Markers Including CHI3L1 in Blood of Normal-Pressure Hydrocephalus

[0113] In this patient group, various expression levels of biomarkers in plasma {(lipocalin-2 (LCN-2, R & D Systems DLCN20, Minneapolis, Minn.), pentraxine-3 (PTX-3, R) disease & D Systems DPTX30, Minneapolis, Minn.), CHI3L1)} were measured and compared by ELISA for patients with normal-pressure hydrocephalus and Alzheimer's disease. Experimental methods used the same method as the sandwich ELISA for the CHI3L1.

[0114] As shown in FIG. 1, as a result of the post hoc analysis on the level of CHI3L1, it was confirmed that the expression level of CHI3L1 was significantly increased in the plasma of patients with normal-pressure hydrocephalus compared to patients with Alzheimer's disease (p=0.001). There was also a significant increase in normal-pressure hydrocephalus compared to the normal control group (p<0.001), Parkinson's disease group (p=0.007), and mild cognitive impairment group (p<0.001).

[0115] However, as shown in FIG. 2, lipocalin-2 and pentlaccin-3 did not show significant results with normal-pressure hydrocephalus.

[0116] The expression concentration levels of lipocalin-2 in all groups of plasma were compared. As shown in FIG. 2, the mean value of plasma lipocalin-2 was 46.3 ng/mL (standard deviation 28.5 ng/mL) in 66 healthy volunteers. Lipocalin-2 levels were measured in patient groups with normal-pressure hydrocephalus (mean 52.5 ng/mL, standard deviation 30.6 ng/mL, n=29), Alzheimer's disease (48.9 ng/mL, standard deviation 23.6 ng/mL, n=110), Parkinson Disease (35.6 ng/mL, standard deviation 14.7 ng/mL, n=13) and mild cognitive impairment (43.0 ng/mL, standard deviation 22.8 ng/mL, n=14). The ANOVA and post hoc analysis of each group showed no statistical significance.

[0117] The expression concentration levels of pentraxine-3 in the plasma of all groups were compared. As shown in FIG. 2, the mean value of plasma pentlaccin-3 was 0.8 ng/mL (standard deviation 0.7 ng/mL) in 66 healthy volunteers. Pentraxine-3 levels were measured in patient groups with normal-pressure hydrocephalus (mean 1.0 ng/mL, standard deviation 1.0 ng/mL, n=29), Alzheimer's disease (1.0 ng/mL, standard deviation 0.8 ng/mL, n=110), Parkinson Disease (0.9 ng/mL, standard deviation 0.5 ng/mL, n=13) and mild cognitive impairment (0.9 ng/mL, standard deviation 0.6 ng/mL, n=14). The ANOVA and post hoc analysis of each group showed no statistical significance.

[0118] Therefore, according to the result of Example 2, it was confirmed that the CHI3L1 protein of the present invention can diagnose and predict normal-pressure hydrocephalus more accurately than other biomarkers.

Example 3: Evaluation of the Diagnostic Efficiency of CHI3L1

[0119] To evaluate the diagnostic efficiency of CHI3L1, ROC curves were drawn to measure AUC/sensitivity/specificity. The AUC value can be determined as the area under the graph, and the higher the AUC value is, the more accurate the corresponding diagnostic model is. When the total area is 1, the closer the model efficiency is to 1, the more accurate the model is. Sensitivity may refer to a rate of positively determining a subject having an actual disease when using a specific diagnostic model and specificity may refer to a rate of negatively determining a subject having no actual disease when using a specific diagnostic model.

[0120] As shown in FIG. 3, when the ROC curve for the normal-pressure hydrocephalus was plotted against the level of CHI3L1, the AUC value (area below the curve) was 0.773, and the sensitivity was 77.4% and specificity was 68% at a cutoff value of 76.5 ng/mL.

[0121] As shown in FIG. 4, when the ROC curve (recipient manipulation characteristic curve) for normal-pressure hydrocephalus was plotted for the level of CHI3L1, the AUC value (the sub-curve region) was 0.840, and the sensitivity was 80.6% and the specificity was 71.6% at a cutoff value of 73.3 ng/mL.

[0122] Based on Example 3, it was confirmed that the accuracy of the AUC value of 0.7 or more was very high, and the sensitivity and specificity were also very good, of 70% or more, based on FIG. 3 and FIG. 4. Therefore, the CHI3L1 protein of the present invention is clinically useful for specifically diagnosing normal-pressure hydrocephalus as compared to other degenerative diseases.

[0123] According to the results of this Example, CHI3L1 expression levels were significantly different in the normal-pressure hydrocephalus group than in the control group (normal subject), Alzheimer's disease, Parkinson's disease, and mild cognitive impairment. It was found that plasma levels of CHI3L1 were significantly associated with normal-pressure hydrocephalus. Therefore, it is thought that CHI3L1 in plasma could be used as a potential biomarker for clinical diagnosis and prediction of normal-pressure hydrocephalus.

INDUSTRIAL APPLICABILITY

[0124] According to the present invention, the expression level of CHI3L1 is significantly increased in patients with normal-pressure hydrocephalus. Therefore, by analyzing the expression level of CHI3L1, it is possible to diagnose the normal-pressure hydrocephalus quickly and accurately, and is highly available in industry.