MELANOTRANSFERRIN FOR USE IN THE DIAGNOSIS OF PARKINSON`S DISEASE

Abstract

The present invention is the protein of melanotransferrin, or an encoding nucleic acid of same, for use in the diagnosis of Parkinson's disease (PD). The invention is a method of diagnosis of PD in a subject, for assessing the level of melanotransferrin in the saliva or in a saliva sample of the subject and determining whether the level is above or below a value of 8.6 g/ml, wherein a value below 8.6 g/ml is indicative of PD. Another aspect is a kit having at least one reagent, preferably an antibody, for the quantification of melanotransferrin in the saliva or in a saliva sample of a subject enabling the comparison of the quantification with a predetermined cut-off value.

Claims

1. A method of diagnosis of Parkinson's disease using melanotransferrin in the saliva or in a saliva sample of a subject.

2. A method of diagnosis of Parkinson's disease in a subject showing phenoconversion of a neurological disease, said method comprising: assessing the level of melanotransferrin in the saliva or in a saliva sample of said subject, and determining whether said level is above or below a value of 8.6 g/ml, wherein a value below 8.6 g/ml is indicative of Parkinson's disease.

3. Method according to claim 2, in which said level value is 4 g/ml.

4. Method according to claim 2, in which said subject is a mammal.

5. A method according to claim 4, in which said mammal is human.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0030] FIG. 1A shows that saliva levels of MTf measured by human ELISA kit were decreased in PD patients compared with control group. White bar, control; hatched bar, PD.

[0031] FIG. 1B shows that saliva levels of transferrin measured by specific human ELISA kits remained unchanged in PD compared with control group. White bar, control; Hatched bar, PD.

[0032] FIG. 1C shows that saliva levels of Lactoferrin measured by specific human ELISA kits remained unchanged in PD compared with control group. White bar, control; Hatched bar, PD.

[0033] FIG. 2 shows that saliva levels of MTf measured by human ELISA kit remained unchanged in AD compared with control group. White bar, control; Hatched bar, AD.

[0034] FIG. 3 shows a regression analysis using saliva MTf expression values and age as accurate measurement to classify PD and control groups. Y=0.0374x+1.825; .square-solid. PD; control.

[0035] FIG. 4A shows the ROC (Receiver Operating Characteristic) curve obtained for the test of saliva MTf levels from the PD patients and the control group using the low cutoff (4 g/ml) value. The ROC plot represents sensitivity (true positive rate) versus 1-specificity (false positive rate). The area under the ROC curve (AUC) was 0.99.

[0036] FIG. 4B shows the ROC curve obtained for the test of saliva MTf levels from the PD patients and the control group using the high cutoff (8.6 g/ml) value. The ROC plot represents sensitivity (true positive rate) versus 1-specificity (false positive rate). The area under the ROC curve (AUC) was 0.92.

EXAMPLES

[0037] The following examples are provided for the purpose of showing the present invention in an illustrative yet non-limiting manner.

Example 1. Extraction of Saliva Samples

[0038] Two groups of donors were included in the study: (n=56) PD patients and (n=72) elderly non-demented control, recruited in Hospital 12 de Octubre, Madrid, Spain (Table 1). For PD patients, diagnosis was established according to the criteria of probable PD (Gelb et al., Diagnostic criteria for Parkinson disease. Arch Neurol. 1999 January; 56(1):33-9). A group of AD patients were added defined after patients who had been diagnosed according to the National Institute on Neurological Disorders and Stroke, and the Alzheimer's Disease and Related Disorders Association (NINDS ADRDA) guidelines (McKhann et al., The diagnosis of dementia due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease. Alzheimer's Dement. 2011; 7: 263-9). Subjects' consent was obtained according to the Declaration of Helsinki, and approval was obtained from the Research Ethic Committee of Hospital 12 de Octubre. Unstimulated whole saliva was collected into sterile plastic containers pre-coated with 2% sodium azide solution, as previously described by Bermejo-Pareja (Bermejo-Pareja et al., Saliva levels of Abeta1-42 as potential biomarker of Alzheimer's disease: a pilot study. BMC Neurol 2010; 10: 108). Collected samples were immediately placed on ice and pre-cleared by a low spin at 600 xg for 10 min at 4 C. Aliquoted 0.5 ml samples were stored at 80 C. after treatment with Protease Inhibitor Cocktail (Roche). Protein estimation was analyzed using a BCA protein assay kit (Pierce, Rockford, Ill.) according to the manufacturer's instructions.

TABLE-US-00001 TABLE 1 Demographic characteristics of subjects. Diagnosis No. M/F Age (mean SEM) Controls 72 21/49 73.2 0.9 PD 56 26/30 69.5 1.4 AD 30 10/20 75.7 1.12 PD = Parkinson's disease; M = male, F = female; SEM = standard error of media.

Example 2: Measure of Melanotransferrin in the Saliva Samples

[0039] Human melanotransferrin levels were measured using a commercial melanotransferrin human ELISA kit (Cusabio), according to the manufacturer's instructions. Human lactotransferrin levels were measured using a commercial lactotransferrin human ELISA kit (Abcam), according to the manufacturer's instructions. Human transferrin levels were measured using a commercial transferrin human ELISA kit (Abcam), according to the manufacturer's instructions. Pair-wise comparisons between the two groups, using ANOVA followed by a Mann-Whitney test, showed a significant reduction in melanotransferrin levels in PD patient groups relative to healthy control group (p=0.0001; FIG. 1A).

[0040] Transferrin (FIG. 1B) and lactoferrin (FIG. 1C) levels in PD saliva showed similar to those observed in the control healthy group.

[0041] Melanotransferrin levels in AD saliva showed similar to those observed in the control healthy group (FIG. 2).

Example 3. Saliva Melanotransferrin Content as Diagnostic Tool

[0042] The data shown in the previous examples of the melanotransferrin Elisa analysis were used to build a separate linear classifier model able to distinguish between PD pathological or non-pathological status. Receiver Operating Characteristic (ROC) analysis assesses the performance of the classifier models for group classification.

[0043] This model was applied using the cutoff values, which are 0.75 standard deviation away from the mean in both directions. When the lower cutoff (4 g/ml) value was used, ROC analysis revealed an area under the curve (AUC) of 0.99 with 95% (0.95-1) confidence interval (Cl). This model yielded a sensitivity of 93.8% and specificity of 100%, for classifying the PD and healthy control groups (FIG. 4A).

[0044] When the higher cutoff (8.6 g/ml) value was used, ROC analysis revealed an area under the curve (AUC) of 0.92 with 95% (0.85-1) confidence interval (Cl). This model yielded a sensitivity of 100% and specificity of 86.8%, for classifying the PD and healthy control groups (FIG. 4B).

[0045] Subject classification in groups according to cutoff values is shown in Table 2.

TABLE-US-00002 TABLE 2 Subject classification Cutoff values Control PD total <4 g/ml 0 30 (53.57%) 23.43% >8.6 g/ml 38 (52.77%) 0 29.68% 4-8.6 g/ml 34 (47.22%) 26 (46.42%) 46.87%