LACTOFERRIN FOR USE IN THE DIAGNOSIS OR PROGNOSIS OF ALZHEIMER'S DISEASE, OR IN THE DIAGNOSIS OF PARKINSON'S DISEASE

Abstract

The present invention is the protein of lactoferrin, or an encoding nucleic acid of the same, for use in the diagnosis or prognosis of Alzheimer's disease (AD). The invention is a method of diagnosis or prognosis of AD in a subject, comprising assessing the level of lactoferrin in the saliva or in a saliva sample of said subject and determining whether said level is above or below a value of 7.43 μg/ml, wherein a value below 7.43 μg/ml is indicative of AD or of the prognosis of AD. Another aspect is the protein of lactoferrin, or an encoding nucleic acid of the same, for use in the diagnosis of Parkinson's disease (PD) in a saliva sample of a subject.

Claims

1.-16. (canceled)

17. A method of detecting lactoferrin in a sample obtained from a subject having or at risk of having mild cognitive impairment (MCI) or Alzheimer's disease, the method comprising: detecting a level of lactoferrin protein in a sample collected from the subject by exposing the sample to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and determining whether the level of lactoferrin is above or below a value of 7.43 μg/ml.

18. The method of claim 17, wherein the subject is a mammal.

19. The method of claim 18, wherein the mammal is a human.

20. The method of claim 17, wherein the sample comprises a protease inhibitor.

21. The method of claim 17, further comprising a step of analyzing the sample with mass spectrometry.

22. The method of claim 17, wherein the sample is selected from the group consisting of: saliva, oral mucosa, oral mucous tissue, and tears.

23. A method of detecting lactoferrin in a sample obtained from a subject having or at risk of having mild cognitive impairment (MCI) or Alzheimer's disease, the method comprising: detecting a level of lactoferrin protein in a sample collected from the subject by contacting the sample with an antibody specific for lactoferrin and detecting specific binding of the antibody and lactoferrin, and determining the level of lactoferrin in the sample; wherein the sample is selected from the group consisting of: saliva, oral mucosa, oral mucous tissue, and tears.

24. The method of claim 23, wherein the subject is a mammal.

25. The method of claim 24, wherein the mammal is a human.

26. The method of claim 23, wherein the level of lactoferrin protein is detected using a lactoferrin human enzyme-linked immunosorbent assay (ELISA) kit.

27. The method of claim 23, wherein the sample comprises a protease inhibitor.

28. A kit for detecting lactoferrin, comprising at least one reagent for the quantification of lactoferrin in a sample derived from a subject, and enabling the comparison of the quantification of lactoferrin with a predetermined cut-off value.

29. The kit of claim 28, wherein the at least one reagent is an antibody specific for lactoferrin.

30. The kit of claim 28, further comprising reagents suitable for an enzyme-linked immunosorbent assay (ELISA).

31. The kit of claim 28, wherein the sample is selected from the group consisting of: saliva, oral mucosa, oral mucous tissue, and tears.

32. The kit of claim 28, wherein the subject has, is suspected of having, or is at risk of developing mild cognitive impairment or Alzheimer's disease.

33. The kit of claim 28, wherein the predetermined cut-off is 7.43 μg/ml.

34. The kit of claim 33, wherein a value below 7.43 μg/ml indicates the subject has or is susceptible to developing mild cognitive impairment.

35. The kit of claim 33, wherein a value below 7.43 μg/ml indicates the subject has or is susceptible to developing Alzheimer's disease.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0050] FIG. 1A shows a 75kDa lactoferrin band after SDS-PAGE fractionation present in all samples. Identification of lactoferrin in human saliva from MCI, AD, and healthy controls. Coomassie blue staining PAGE-SDS gel corresponding, to saliva pools. Lane 1, control group; lane 2, MCI group; lane 3, and 4 AD group. Band corresponding to around 75kDa is signed with arrow.

[0051] FIG. 2A shows that saliva levels of lactoferrin, measured by human ELISA kit, were decreased in MCI and AD patients compared with control group.

[0052] FIG. 2B shows that saliva levels of lactoferrin, measured by human ELISA kit, were increased in PD compared with control group.

[0053] FIG. 3A shows a correlation between saliva levels of lactoferrin and cognitive decline in MCI and AD groups. This relation was driven primarily by a significant negative association between stages of disease and lactoferrin levels (R=−0.74; p<0.001).

[0054] FIG. 3B shows a correlation between saliva levels of lactoferrin and MMSE score a measure of cognition available in patients with MCI and AD (R=0.73; p<0.001).

[0055] FIG. 3C shows the ROC curve obtained for the test of saliva lactoferrin levels from the full control group and MCl/AD group. The ROC plot represents sensitivity (true positive rate) versus 1-specificity (false positive rate). The area under the ROC curve AUC=1 (95% CI 1-1).

[0056] FIG. 4A shows the receiver operating characteristic (ROC) curve obtained for the test of saliva lactoferrin levels from the full control group and converter group. The ROC lot represents sensitivity (or true positive rate) versus 1-specificity (or false positive rate). This binary classifier system (ROC curve) yielded a robust area under the curve AUC=0.984 (95% CI 0.932-1). AUC is a measure of how well a parameter can distinguish between two diagnostic groups, with 95% confidence interval (CI) from 0.93 to 1)

[0057] FIG. 4B shows the conversion to either alVICl or AD as predicted by salivary lactoferrin levels. The image shows the average time for phenoconversion to either aMCI or AD depending on abnormally reduced (Positives) or normalihigh (Negatives) lactoferrin levels, based on the minimal Cox proportional hazards model. Dashed line is Negatives Continuous line is Positives.

[0058] FIG. 4C shows a logistical regression analysis using lactoferrin expression values and time in years of onset or phenoconversion, The equation generated by regression analysis was y=0.6289x+1,6954.

[0059] FIG. 5A shows a regression analysis using saliva lactoferrin values and age as accurate measurement to classify both young- and aged-healthy groups. Young non-demented; Elderly non-demented.

[0060] FIG. 5B shows a regression analysis using saliva lactoferrin expression values and age as accurate measurement to classify both aged-group (non-phenoconverters) and phenoconverters to MCI and AD. Phenoconverters; E Elderly non-demented.

EXAMPLES

[0061] 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

[0062] An AD diagnostic training study was carried out enrolling 274 participants at the Neurology Service at the Hospital Universitario 12 de Octubre (Madrid, Spain). Four (4) groups of age-matched subjects according to their cognitive status were defined: aMCI, AD, Parkinson's disease (PD) and cognitively healthy control group (Table 1). For AD patients, diagnosis was established 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 Demerit. 2011; 7; 263-9). PD patients were diagnosed under the criteria of probable PD (Gelb et al., “Diagnostic criteria for Parkinson disease”. Arch Neural. 1999 Jan; 56(1):33-9). A group of MCI patients were also added defined after patients with cognitive impairment that did not fulfill the criteria for dementia (Pedersen, “Mild cognitive impairment as a diagnostic entity”. J Intern Med 2004; 256: 183-94). Disease severity was evaluated using Mini-Mental State Examination (MMSE) scores. 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 Abetal-42 as potential biomarker of Alzheimer's disease: a pilot study”. BMC Neural 2010; 10: 108). Collected samples were immediately placed on ice and pre-cleared by a low spin at 600 ×g for 10 min at 4′C. Aliquoted 0.5 ml samples were stared 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, and clinical characteristics of subjects from first training study. Variable Control aMCI AD PD p value n (F/M) 91 (59/32) 44 (25/19) 80 (49/31) 59 (32/27) ns Age (years) 73.7 ± 6.88 75.16 ± 5.13   76.2 ± 5.33** 69.5 ± 8.6** p < 0.01 MMSE score  29 ± 0.8   26.8 ± 1.16***  19.25 ± 1.76*** NA  p < 0.001 APOE ε4 12.9% 42.1%** 45.9%** NA p < 0.01 carriers M = male; F = female; aMCI = amnestic Mild Cognitive Impairment; AD = Alzheimer's disease; PD = Parkinson's disease; MMSE = mini-mental state examination scores; NA = not applicable; ns = not significant. Data are expressed as mean ± S.D. **p < 0.01 versus control group; ***p < 0.001 versus control group.

Example 2

Measure of Lactoferrin in the Saliva Samples

[0063] Human lactoferrin according to SEQ ID.NO :1 expression levels in saliva were detected in pooled samples from AD patients compared to MCI donors and control subjects. Saliva samples from 4 male subjects from each group (MCI, AD, and elderly non-demented controls) were pooled by mixing equal amounts. 50 pg of each pool were loaded on a SOS-PAGE gel. After SOS-PAGE fractionation a 75kDa band was detected in all samples matching with the lactoferrin molecular weight as confirmed by mass spectrometry analysis (31% coverage). Differences in protein expression were evaluated in using lmageQuant software (GE Healthcare). Upon equal amount of protein loaded, the band intensity analysis showed reduced lactoferrin levels in MCI (14%) and AD (51% and 58%) compared to the healthy control group (FIG. 1A). To validate the presence of lactoferrin in human saliva, this protein was identified by MALDl-TOF/TOF mass spectrometer 4800 Proteornics Analyzer (Applied Biosysterns, Framingham, Mass.) and 4000 Series Explorer™ software (Applied Biosystems) after in gel digestion with trypsin and endopeptidase Asp-N (Thermo Fisher Scientific). The amino acid coverage was 31% for lactoferrin.

[0064] Further confirmation of these differences was obtained averaging the lactoferrin expression levels by a commercial lactoferrin human ELISA kit (Abcam), according to the manufacturers instructions. Pair-wise comparisons between the three groups, using ANOVA followed by a Tuckey-Kramer test, showed a significant reduction in lactoferrin levels in MCI and AD patient groups relative to healthy control group (p<0.05; FIG. 2A). Lactoferrin levels in PD saliva showed significantly higher levels to those observed in the control healthy group (FIG. 26).

Example 3

Saliva Lactoferrin Content as Diagnostic Tool

[0065] Saliva levels of lactoferrin were evaluated throughout the progression of dementia. Correlation between saliva lactoferrin levels and cognitive decline in MCI and AD groups seems evident. This relation was driven primarily by a significant negative association between stages of disease and lactoferrin levels (R=−0.742; p<0.001) (FIG. 3A). The MMSE score was used to following up the progression of dementia. The saliva lactoferrin concentration could also be correlated with MMSE score in patients with MCI and AD, after a highly significant correlation (R=0.731; p<0.001) (FIGS. 3B) to 15 and 10 μg/ml in healthy humans, and less than 7.43 μg/ml in demented humans, including MCI and AD. The Kendall's tau and Spearman rank correlation were used for correlation analyses, respectively.

[0066] Using linear regression analysis, we discovered that patients suffering from AD and aMCI had 6.432 μg (95%Cl: 6.850-6.014; p<0.001) and 5.310 μg (95%Cl: 5.810-4.810; p<0.001) of salivary lactoferrin per ml less than cognitively healthy participants respectively. We used these results from the lactoferrin analysis to build separate linear classifier models that would distinguish the aMCliAD groups from the control group, and we did receiver operating characteristic (ROC) analysis to assess the performance of the classifier models for group classification. A classifier model using the discovered lactoferrin levels from saliva analysis yielded an area under the curve (AUC) of 1 (95% CI 1-1), being the sensitivity 100% (95% CI 96.90° k-100%) and specificity 100% (95% CI 95.95%-100%) for aMCl/AD and healthy control group classification (FIG. 3C). The cut-off value was 7,43 pg/mi (Youden index: 1).

Example 4

Validation of Saliva Lactoferrin as Diagnostic Tool

[0067] The cut-off value of saliva lactoferrin was then validated in two new blinded and independent cohorts enrolling 91 additional participants with the same standardized clinical assessments used in the previous study. Demographic characteristics of participants recruited in two entities: Alzheimer Disease Research Unit, CIEN Foundation, Queen Sofia Foundation Alzheimer Center (Madrid, Spain), and Pablo de Olavide University from Sevilla, Spain, are shown in Table 2.

TABLE-US-00002 TABLE 2 Demographic, characteristics of subjects from validation study. Variable Control aMCI AD p value n (F/M) 40 (25/15) 15 (5/10) 36 (23/13) ns Age (years) 66.78 ± 7.33 68.93 ± 6.12 80.67 ± 8.76*** p < 0.001 F = female; M = male; aMCI = amnestic Mild Cognitive Impairment; AD = Alzheimer's disease; ns = not significant. Data are expressed as mean ± S.D. ***p < 0.001 versus control group.

[0068] Results showed that cut-off value of saliva lactoferrin (7.43 μg/ml) classified correctly all patients (MCl/AD; n=51) and all cognitively healthy subjects (n=40).

Example 5. Saliva Lactoferrin Content as Predictive Tool

[0069] In order to investigate predictive potential of lactoferrin levels in saliva, cognitively healthy control participants, without memory impairment, integrated this group (Table 3).

TABLE-US-00003 TABLE 3 Demographic characteristics of subjects. Subjects No. M/F Age (mean ± SEM) Controls 116 45/71 68.06 ± 1.12 (non demented) M = male, F = female.

[0070] Unstirnulated whole saliva was collected into sterile plastic containers, and lactoferrin levels were determined as described in Example 1. Eight (8) subjects showed significantly reduced levels of lactoferrin in saliva compared to a healthy control group (3.47 ±0.41 pg/mi vs 10.54 ±1.58 pg/m1; p<0.05). The average time far phenoconversion to either MCI or AD was 3.25 years (range 1-5 years). Table 4 shows the presence of an association between time of phenoconversion (onset) and age, being shorter with older subjects.

TABLE-US-00004 TABLE 4 Demographic characteristics of converters. Sub- Lt levels Neurological Other clinical jects Sex Age onset (μg/ml) diagnose diagnose 1 M 82 2 3.01 MCI HT, DM 2 F 70 4 3.17 MCI 3 F 71 5 3.69 MCI HT, HC 4 F 68 5 5.10 MCI/AD 5 F 81 1 1.65 MCI/AD 6 F 77 2 1.89 MCI HT 7 M 83 3 6.18 MCI/AD HT, DM, HD 8 M 88 4 4.45 MCI HT Lt = lactoferrin; AD = Alzheimer's disease; MCI = Mild Cognitive Impairment; M = male, F = female; HD = Hypertension, DM = Diabetes Meilitus; HD = Heard Disease; HC = Hypercholesterol.

Example 6

Mucosa Iactoferrin Contents

[0071] Oral mucosa was collected into sterile plastic containers according to Aagaard (Aagaard et al., “The Human Microbiorne Project strategy for comprehensive sampling of the human microbiome and why it matters”. FASEB J. 2013 March; 27(3):1012-22). Briefly, participants, described in Table 5, drooled into a 50-ml collection tube after allowing saliva to collect in the mouth for ≥1 minute, centrifuged at 6000 xg for 10 min at 4°, and pellets were stored at −80° C.

TABLE-US-00005 TABLE 5 Demographic characteristics of subjects. Subjects No. M/F Age (mean ± SEM) Controls 190 110/80 62 ± 1.23 (non demented) M = male, F = female.

[0072] (Lactoferrin levels were determined as described in Example 1. Six (6) subjects showed significantly reduced levels of lactoferrin compared to a healthy control group (4.28 ±0.50 μg/ml in AD vs. 9.05 ±1.47 μg/ml; p<0 05: Table 6). The average time for phenoconversion to either MCI or AD was 3.83 years (range 4-3 years). Cognitively healthy control participants, without memory impairment. integrated the group shown in

[0073] Table 5.

TABLE-US-00006 TABLE 6 Demographic characteristics of converters. Lt levels Neurological Subjects Sex Age onset (μg/ml) diagnose 1 F 96 4 5.02 AD 2 M 66 4 3.66 MCI 3 M 82 4 2.51 AD 4 M 67 4 5.92 MCI 5 M 84 4 4.17 AD 6 M 85 3 4.13 MCI/AD Lt = lactoferrin; AD = Alzheimer's disease; MCI = Mild Cognitive Impairment; M = male, F = female.

Example 7

Build of a Predictor Model of Phenoconversion to MCl/AD

[0074] The data shown in the fore examples of the lactoferrin Elisa analysis were used to build a separate linear classifier model able to distinguish between AD pathological or non-pathological status. Receiver operating characteristic (ROC) analysis assesses the performance of the classifier models for group classification. A classifier model using the discovered lactoferrin levels from saliva analysis effectively classified Converters and healthy control groups with an area under the curve (AUG) of 0.98 with 95% (0.93-1) confidence interval (Cl: FIG. 4A). This model yielded a sensitivity of 100% and specificity of 98.6%, for classifying the Converters and healthy control groups (FIG. 4A) This ROC curve, a fundamental tool for diagnostic test evaluation, evaluated the accuracy of the test to discriminate diseased cases from normal cases. The ROC can 1:S be understood as a plot of the probability of classifying correctly the positive samples against the rate of incorrectly classifying true negative samples. So the AUC measure of an ROC plot is a measure of predictive accuracy.

[0075] The probability to estimate the average time for phenoconversion to either aMCl or AD depending on abnormally reduced or normal/high lactoferrin levels was determined, using the Cox proportional hazards model (FIG. 4B). Our results show that salivary lactoferrin is an independent prognostic factor that predicts the probability of occurrence of AD, HR: 0.428 (95% Cl 0.324-0,567; p<0.0001), In the present study, AUC=098 indicated a robust discrimination power, being 1 a perfect discrimination. Regression analysis generated an equation to describe this relationship between saliva lactoferrin levels and the time (years) of phenoconversion (onset), y=0.6289x+1.6954, being “y” the saliva lactoferrin levels, and “x” the time in years of phenoconversion (FIG. 4C).