WO2008010660A1

WO2008010660A1 - Marker for diagnosing neurodegenerative diseases

Info

Publication number: WO2008010660A1
Application number: PCT/KR2007/003446
Authority: WO
Inventors: Hyun-Kee Kim; Jin-Woo Kim
Original assignee: Hyun-Kee Kim; Jin-Woo Kim
Priority date: 2006-07-18
Filing date: 2007-07-16
Publication date: 2008-01-24
Also published as: KR100856375B1; KR20080007776A

Abstract

The present invention relates to a marker for diagnosing a neurodegenerative disease, more particularly, a marker and a method for diagnosing a neurodegenerative disease, in particular such as an Alzheimer's disease, with a fibrinogen gamma -A chain precursor or the like, as an effective element.

Description

MARKER FOR DIAGNOSING NEURODEGERNERATIVE DISEASES

TECHNICAL FIELD

BACKGROUND ART

Neurodegenerative diseases are characterized by chronic, progressive and irreversible aggravation of nerve function including cognition and kinesthesia system, which makes people degenerative and completely dependent. For diagnosing the neurodegenerative disease, many people may see the doctor with a help of their family only after their neuro-abnormality disturbs their daily life since a diagnosis is to be dependent on a clinical symptom. However, in this clinical stage, nerve cells are damaged sufficiently to an extent to be beyond a predetermined limit value so that a disease inevitably proceeds. For this reason, there has been a need of a maker based on molecules existing in a biological fluid of neurodegenerative diseases in an initial or pre-clinical stage and the marker may be a disease-variant compound with respect to a medicine development .

Biochemical changes in a brain are well reflected to a CSF (cerebrospinal) , which is faced directly to the extra cellular space of a brain. Furthermore, brain metabolic resultants going to a plasma is highly diluted and thus the CSF is a surer source of a biomarker with respect to a neurodegenerative disease than a plasma (Clinical Neurology and Neurosurgery 107 (2005) 165-173, Niels Andreasen et al)

Biomarkers are often hypothesized but not used in gerontological studies generally due to lack of scientific data thereof. However, they are sometimes good target for a healing trial and further need to have surely very high diagnosis specificity and sensitivity in much diagnosis of elderly people. In many procedures adapted for finding biomarkers, a clinical proteomics field is proper particularly when finding the biomarkers within CSF reflecting a brain protein, which is under a serious neurodegenerative disease and even a health condition. A protein has been found from CSF of a neurodegenerative disease using a proteomics. Among many neurodegenerative diseases, Alzheimer's disease (AD) has been studied most commonly (Appl . Theor. Electrophor. 3. 1992; G. Johnson et al, NeurpReport 13.2002; P, Davisso et al).

Alzheimer's disease is the most common neurodegenerative disease and is the most common type of dementia, and further nearly 10% of population aged older than 65 are suffering therefrom ( S.S.Sisodia (Eds.), Alzheimer's disease, 2^nd ed. , Lippincott Williams & Wilkins, Philadephia, 1999, pp. 95-115; C.H.Kawas et al) . As in other neurodegenerative diseases, a diagnosis of Alzheimer's disease is performed based on a disease progress and a clinical photo thereof, excluding other reasons of dementia. However, depending only on neuro- pathological tests provides a limited diagnosis. Most diagnosis pathological characteristics include an extracellular amyloid plaque and an intracellular neuofibrillary tangle (J Neurol Sci 11: 205-242, 1970; Tomlinson BE et al) .

In particular, though studies of biomarkers that are proper to elderly people the old man having AD has partly advanced, however, further studies and works are needed before these potential markers are to be used in practical clinics. In addition, the previous studies thereof have focused little on the clinical stage in which a disease progress is to be tested under a profound neuropsychological inspection.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a novel disease biomarker in relation to a memory impairment comprising Alzheimer's disease, which is one of the commonest neurodegenerative diseases, by applying clinical proteomics to CSF, focusing on quantity changes of protein biomolecules in association with a disease progress.

To achieve the above object, the present invention provides a composition for diagnosing a neurodegenerative disease comprising, as an effective component, at least one protein selected from a group consisting of a fibrinogen gamma-A chain precursor, a Retinol binding protein, and a Haptoglobin precursor allele 1. The proteins may comprise any amino acid sequences as long as the function and structure thereof are preserved. It is preferable that the fibrinogen gamma-A precursor comprises an amino acid sequence of SEQ ID NO: 1, a mutant or a fragment thereof, and a Retinol binding protein and a Haptoglobin precursor allele 1 comprise amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and mutations or fragments thereof . The neurodegenerative disease in the present invention is preferably Alzheimer's disease.

It is preferable to obtain the proteins from cerebrospinal fluid (CSF) . Additionally, the present invention provides a method for diagnosing a neurodegenerative disease comprising: (a) measuring a level of the fibrinogen gamma-A chain precursor of an experiment sample comprising a fibrinogen gamma-A chain precursor, taken from a patient; and (b) comparing a level of the fibrinogen gamma-A chain precursor of said experiment sample to a level of the precursor of a normal sample taken from other subject or group of subjects, wherein a higher level of precursor of the experiment sample is indicative of having a neurodegenerative disease. On the other hands, the present invention provides a method for diagnosing a neurodegenerative disease comprising: (a) measuring a level of the Retinol binding protein or the Haptoglobin precursor allele 1 of an experiment sample comprising a Retinol binding protein or a Haptoglobin precursor allele 1, taken from a patient; and (b) comparing a level of the Reinol binding protein or the Haptoglobin precursor allele 1 of said experiment sample to a level of the Reinol binding protein or the Haptoglobin precursor allele 1 of a normal sample taken from other subject or group of subjects, wherein a lower level of the Reinol binding protein or the Haptoglobin precursor allele 1 of the experiment sample is indicative of giving a neurodegenerative desease.

In the diagnosing method, the disease is preferably Alzheimer's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiments of the invention and together with the description serve to explain the principle of the invention. In the drawings:

Figs . 1 are comparison photos of 2D gels with which CSF from an AD patients are loaded. In all gels, 50 μg CSF proteins were loaded, and an electrophoresis of 2D extract polyacrylamide gel was performed using 12 %T acrylamide gel and a silver staining was performed to see all proteins; Fig. 2a is an image of a 2D gel electrophoresis using a CSF sample from an AD patient. The spots identified as specifically expressed spots are indicated with arrow marks. These spots were identified as fibrinogen gamma-A chain precursor. The identified spots have about 50 kDa molecular weight and 5.6 pi value;

Figs. 3 are images of a 2D gel electrophoresis gel using CSF samples from normal, MCI and AD patients. The spot found as specifically expressed spots are indicated by arrow marks. (A) This spot is identified as RBP. The identified spot has about 22 kDa molecular weight and 5.24 pi value. (B) These spots are identified as a Haptoglobin precursor allele 1, respectively. These identified spots have about 40 kDa molecular weight and 5.3 pi value; Fig. 4 is a view showing differentially expressed spot of a fibrinogen gamma-A chain precursor. Through the figure, it is found that the expression increases according to progress of disease. This result is reproduced in repeated experiments ; Figs . 5 are photos showing differentially expressions of the Retinol binding protein or the Haptoglobin precursor allele 1 with respect to normal, MCI and AD patients. (A) It is shown that the Retinol binding protein RBP is expressed differentially. The arrow mark means that the expression of RBP increases in the normal control group and then decrease or miss along with the progress of a disease. This fact is confirmed in repeated experiments. (B) It is shown that the Haptoglobin precursor allele 1 is expressed differentially. The arrow mark means that the expression of the Haptoglobin precursor allele 1 increases in the normal control group and then decrease along with the progress of a disease. This fact is confirmed in repeated experiments.

DETAILED DESCRIPTION OF THE INVENTION

The biological markers in CSF may be very helpful when diagnosing Alzheimer' s disease since an initial detection thereof and a loosening of disease progress is important in relation to increasing an average life span. The object of the present invention is to search CSF biomarker candidates with respect to the patients having mild cognitive impairment (MCI) and those having Alzheimer's disease compared with a normal control group according to ages. The present inventors classified the patients into three groups that is a normal, MCI and AD. The AD group is further divided into three sub-groups based on their clinical severity according to clinical dementia ratings (CDR) of known dementia clinical scales. The inventors of the present invention has found that CSF fibrinogen gamma-A chain precursors are induced gradually from the patients having mild cognitive impairment (MCI) and Alzheimer's disease, compared with a normal control group according to ages. Further, it has been shown that the level of CSF fibrinogen gamma-A chain precursor increased in proportion to the progress and severity of Alzheimer's disease. The expression level of the fibrinogen gamma-A chain precursor protein is very low on a normal control group. From this result, the inventors of the present invention have confirmed that the CSF level of fibrinogen gamma-A precursor may be very important biomarker when MCI proceeds to AD.

In addition, in the present invention, it has been found that the expression of retinol-binding protein (RBP) and haptoglobin precursor allele 1 increased in a normal control group, compared with MCI and AD patients and a expression level decreased gradually according to the progress of diseases in case of MCI and AD patients . From this result, inactivated retinol-binding protein and haptoglobin precursor allele 1 can be used as an important marker to a progress of MCI and AD, and as an important indicator of severity of AD. In the following, the present invention will be described in detail through non-limited examples.

Example 1: Selection of patients and grouping thereof.

A total of 30 human subjects to be tested were registered to the Bobath Memorial Hospital. 15 subjects among them (average age=75.7, age range from 60 to 95) are diagnosed as neurodegenerative disease clinically and, the other 15 subjects belong to normal control group according to ages (average age=73.2, age range from 60 to 95). There was no subject having other cancers or lung diseases among the subjects. CSF was obtained from 30 people before their treatments, and at this time, individual agreements on analysis were received from all of the persons, uses of CSF samples were approved from an Ethics Committee. A diagnosis of AD was pursuant to NINCDS-ADRDA standards (Neurology 34 (1984) 939-944; G. McKhann et al) . Degree of disease progresses were evaluated by clinical CDR (Clinical Dementia Rating) (Neurology, 1993; 43: 2412-2414; Morris J. C.) . Since it was difficult to distinguish CDR 0.5 from 1 and CDR 3 from 4 and 5, the inventors of present invention grouped the CDR into three groups of Group 1 (CDR 0.5, and

1), Group 2 (CDR 2) and Group 3 (CDR 4, and 5). The present inventors also grouped the CDR into a control group

(A group having no cognitive abnormality) and a mild cognitive impairment (MCI) group. Normal groups were defined as having no serious cognitive impairment in living their lives and, no AD or other serious neuropathologies . Non-dimentia control groups were composed of patients having spinal pain and headaches and all of control groups could be verified by neurophychological test.

CSF was obtained from individuals by using a lumbar puncture method. Disease stages when performing a lumbar puncture were very different in most cases. CSF of 10 ml obtained above was divided into 1 ml, respectively, and stored at -70^°C until they are to be used.

CSF used as samples were quantitated using a bicinchoninic acid (BCA) assay which is based on an expression of colors according to total amounts of protein, and BSA (Bovine Serum Albumin) was used as a standard protein.

Example 2: 2D Electrophoresis

2.1 Solubilization and Lysis of CSF protein

125 β& of quantitated CSF sample (including about 50μg protein) was deposited at -20^°C for 2 hours using cold ethanol to induce precipitation of proteins that exist in low concentration. After being precipitated, the precipitate was centrifuged at 15,00Og for 15 minutes and was washed with ethanol, and then centrifuged again for 10 minutes at 15,00Og. After repeating such processes twice, the precipitate was dried for 5 minutes in room temperature. Dried sediment was dissolved again in 20 β& of Lysis buffer solution [7M urea, 2M thiourea, 4% CHAPS, 6OmM DTT, 0.5% pharmalytes (pH4-10) ] and was used as a sample for 2^nd dimensional electrophoresis.

2.2 2D Electrophoresis 2.2.1 Rehydration Isoelectric focusing (IEF) of Lysised sample was performed by using a fixed pH gradients (nonlinear pH gradient 4-7) , and IPGphor (Amersham Pharmacia Biotec) for an isoelectric focusing unit was used.

Sample dissolved into Lysis buffer solution was centrifuged at 15,00Og for 10 minutes and the supernatant thereof was loaded on a fixed pH gradient (IPG) strip holder (Amersham Pharmacia biotec, 7cm) , and then IPG strip was put on the strip holder. Here, the IPG strip was covered completely by 800 βi of cover fluid oil such that buffer solution was prevented being crystallized or dried.

Rehydration condition was maintained without flowing current for 12 hours and at this time the temperature 20^°C of the electronic panel had been maintained until the experiment was finished. 2.2.2. Iso electric focusing (IEF)

Iso electric focusing (IEF) was performed for 15 minutes with 50V, for 30 minutes with 100V, for 30 minutes with 200V, for 30 minutes with 500V, for 30 minutes with 1000V, and for 30 minutes from 100OV up to 5000V gradually. Finally, it was performed with 5000V for 1 hour and 30 minutes and thus IEF was performed for a total of 4 hours 15 minutes.

2.2.3 Equalization

The IPG strip of which the IEF had completed was equalized for 15 minutes in the first equalization buffer solution [6M Urea, 2% SDS, 5OmM Tris-HCI (pH8.8) , 20% glycerol, 13OmM DTT] containing DTT and then, equalized for 15 minutes in the second buffer solution [6M Urea, 2% SDS, 5OmM Tris-HCI (pH8.8) , 20% glycerol, 135mM idoacetate] containing idoacetamide. 2.2.4 SDS-PAGE The equalized IPG strip was loaded on the upper part of 12% acrylamide gel after being washed by buffer solution for Electrophoresis electrode [0.025M Tris-Cl (pH8.3, 0.195M glycine, 0.1% (w/v) SDS). Here the empty space over the strip was poured by electrode buffer solution containing 0.5% Agarose to fix the strip. 2.2.5 Silver Staining

In order to perform a mass spectrometer analysis and confirm a trace amount of protein spots, a modified silver staining method (Blum et al, 1987) was performed. After electrophoresis, the gel was kept in immobilization solution [30% (v/v) ethanol, 10% (v/v) acetic acid] for 12 hours or more to fix protein thereof, and washed by washing solution [20% (v/v) ethanol] for 20 minutes, and by third distilled water for 10 minutes. The gel washed was sensitized by 0.02% (w/v) sodium thiosulfate for 1 minute.

The gel sensitized was washed three times each 20 seconds by the third distilled water. The gel was treated with

0.2% (w/v) silver nitrate solution for 45 minutes and then cleaned for 10 seconds by the third distilled water. Development of protein spots were performed by putting the gel into developer [3% potassium carbonate, 0.025% formaldehyde, 0.001% sodium thiosulfate] and observing.

After protein spots were fully developed, protein spots were implanted by moving gel to implantation solution [5% Tris, 2.5% acetic acid]. The gel for MALDI-TOF was kept at

4°C.

Example 3 : In-gel digestion 3.1 Destaining of protein spot A protein spot to be analyzed was cut out to be 1 mm in diameter by using a tip and destained by putting the protein spot into destaining solution which is a mixture of 3OmM potassium ferricyanide and 10OmM sodium thiosulfate at a rate of 1:1 (v/v) . The gel piece destained was washed three times for 15 minutes by the third distilled water.

3.2 Washing of protein spot

In the destained protein spot, the gel piece was cleaned with 5OmM of ammonium bicarbonate and acetonytril and then, was dehydrated with an acetonytril. After this, the gel piece was fully dried by using a vacuum centrifuge.

3.3 Reduction and Alkylation

The gel piece dried was swollen with 25mM Ammonium Bicarbonate solution containing 1OmM DTT, reacted therewith for 45 minutes at 56 °C and thus reduced. Alkylation was performed by letting the gel piece be reacted with 25mM

Ammonium Bicarbonate solution containing 55mM idoacetamide for 30 minutes at normal temperature in dark condition. The gel piece was washed with 5OmM Ammonium Bicarbonate and

Acetonytril and then, dehydrated and dried by using a vacuum centrifuge.

3 . 4 In-gel Digestion After the gel piece being dried, it was rehydrated with trypsin (sequencing grade: Promega, Madison, WI) solution. A trypsin concentration for treating protein was

5 βg/β$, and further the gel piece was put into 25mM Ammonium Bicarbonate solution containing Trypsin and reacted over night at 37^°C. The reaction was terminated by addition of 3% formic acid thereto. In some spots of low concentration, two or three gel plugs were digested together to increase a yield of Peptide. Samples were crystallized on MTB AnchorChip TM 600/384 (Bruker Daltonik GmbH, Leipzig, Germany) by using a dried droplet method.

Supernatant was recovered by sonicating a treating solution containing Trypsin for 10 minutes. The remaining gel piece was put into 50% Acetonitrile containing 1% trifluoroacetic acid (TFA) and was sonicated for 10 minutes, and then the supernatant was recovered again. The recovered supernatant was distilled by using vacuum centrifuge.

The dried Peptide was re-floated in a mixed solution of 0.1% TFA and Acetonytrile with a ratio of 7:3 (v/v) , and then sonicated for 10 minutes. Here, CHCA (α-cyano-4- hydroxycinnamic acid) of 20 βg/β& was used as a matrix, and CHCA matrix was diluted to be 1/10 with mixed solution of Ethanol and Acetone with a ratio of 7:3 (v/v) for using an anchor chip. With respect to the samples for Mass Spectrometry, the prepared CHCA matrix solution was diluted to be 1/10 and loaded to a plate to prepare a sample for MALDI-TOF.

Example 4: Mass Spectrometry

Linear and Reflectron MALDI (Matrix-assisted laser desorption/ionization) -TOF (Time of flight) Mass Spectra were analyzed by using Ultraflex of BRUKER. Cation Spectra were recorded by using aluminum sample holder which has the standard parameter; Nitrogen laser (λ = 337 run) , 39 ns pulse duration and 20 kV for voltage swell. Peptide mass fingerprinting data were collected on positive MS reflector mode within scope of 500-4000 mass to charge ratio (m/z) by using 1000-3000 shot per each sample, and adjusted internally by using Trypsin Autorisis peak. The spectra were analyzed using flexAnalysis (Version 2.2, Bruker Daltonik GmbH), which acts as an interface between the BioTools (Version 2.2, Bruker Daltonik) containing raw spectra and a local copy of the Mascot search engine http://www.matrixscience.com]. The MS data were searched together with respect to a locally stored copy of the NCBInr human protein database

[http://www.ncbi.nim.nih.gov] using the Mascot search engine. The search range reached to a carbamidomethylation of cysteine, an oxidation of methionine, and one missed trypsin cleavage site.

The results from the foregoing examples are as follows.

In the present invention, proteins were analyzed using a MALDI-TOF instrument. The instrument provides an enough resolution of MS spectra to search data-base for protein identification. Figure 1 shows overall protein expression patterns of 2-DE gels of the CSF samples from a normal control, MCI, and AD grouped as 1, 2, and 3 which are sorted along with the progress of Alzheimer's disease. As shown from 5 sorts of CSF, Albumin occupies more than 70% of total proteins (Thompson, E. J., The CSF Proteins; A

Biochemical Approach, Elsevier, New York 1988) . Therefore, when an experiment was performed after removing Albumin and

IgG from CSF sample, most of protein spots containing

Albumin disappeared. In all CSF samples used in this experiment, the proteins containing Albumin and IgG themselves were used for analyzing the difference of comparative expression. As shown in the comparison of the spot pattern on 2-DE gel pattern from CSF in FIG. 1, they are changed gradually on MCI and AD compared to normal controls. To find out MCI and AD related therapeutic target, the present inventors analyzed differential protein expressions from CSFs of normal, MCI and AD patients. For an identification of selected spots, a peptide mass fingerprinting was performed by using Delayed extraction- matrix assisted laser desorption/ionization-time of flight- mass spectrometry: DE-MALDI-TOF-MS . The requirements of their identification were noted first, with a high Mascot score that is higher than 67 points, and second, with the same molecular weight and pi value as the identified protein, and finally with a high coverage over full sequence in the identified protein. Through this experiment, proteins showing a specific expression were identified. Two spots indicated as an arrow mark of 50 kDa of them were all identified as a precursor of a fibrinogen gamma-A (Figure 2) . The detailed data for the identified protein are described in Table 1.

In addition, a protein spot of over-expressed was identified from the normal controls (Figure 3) . Here the spot indicated by an arrow was identified as having about 22kDa and a Retinol binding protein RBP. In another experiment, one identified over expressed 5 protein spots of over-expressed were identified from the normal controls (Figure 2c) . The spots indicated by arrows were identified as having about 40 kDa and a Haptoglobin precursor allele 1. The detailed data for those two identified protein spots is described in Table 2.

In the other hands, a precursor of a fibrinogen gamma- A was observed to gradually increase along with the progress of disease and showed the highest expression in group 3 which is final. Total two spots were found, and the molecular weight thereof was about 5OkDa and pi of 5.6 was noted. A very high reproducibility was also noted in a repeated experiment. However, the two spots were found a little different expression; the right precursor of fibrinogen gamma-A spot showed even at top and was gradually expressed along with the progress of disease, whereas the left precursor of fibrinogen gamma-A spot hardly showed at top, but the highest expression showed in group 3 which is final (Figure 4) .

RBP expression was observed to gradually decrease or miss along with the progress of disease and showed the highest expression in the normal control group (Figure 5-A) . The spots were all found in the normal control group, the molecular weight thereof was about 22 kDa and pi of 5.24 was also noted, and confirmed a very high reproducibility in a repeated experiment (Figure 5-A) . An expression of Haptoglobin precursor allele 1 was observed to gradually decrease along with the progress of disease and showed the highest expression in the normal control group (Figure 5-B) . All of the five spots were found in the normal control group, and the molecular weight thereof was about 40 kDa. And a pi of 5.6 was also noted, and confirmed a very high reproducibility in a repeated experiment (Figure 5-B) .

[Table 1]

* Total MASCOT score: sum of individual matched peptide scores

÷Percentage amino acid (AA) sequence coverage

[Table 2]

* Total MASCOT score: sum of individual matched peptide scores

+Percentage amino acid (AA) sequence coverage

As understood in the foregoing configuration, the present inventors found that fibrinogen gamma-A chain precursor of CSF were gradually induced in patients with mild cognitive impairement (MCI) and AD compared to the normal control group candidate in proportion to age.

Additionally, they found that the fibrinogen gamma-A chain precursor protein in CSF increased along with the progress and severity of AD disease. In their experiment, the expression level of fibrinogen gamma-A chain precursor protein was very low in the subjects of age-matched normal control group. From these results the present inventors confirmed that the CSF level of fibrinogen gamma-A chain precursor may be an important biomarker for the progress of MCI to AD. Moreover, the present inventors found that retinol binding protein and haptoglobin precursor allele 1 expression increased in normal control group, compared to in MCI and AD patients, and the expression level from MCI and AD patients gradually decreased along with the progress of disease, which suggests that non-activated retinol binding protein and haptoglobin precursor allele 1 can be used as an important biomarker for the progress of MCI and AD, and also, an important factor for the severity of AD.

Claims

What is claimed is :

l.A composition for diagnosing a neurodegenerative disease comprising, as an effective component, at least one protein selected from a group consisting of a fibrinogen gamma-A chain precursor, a Retinol binding protein (RBP) , and a Haptoglobin precursor allele 1.

2.A composition according to claim 1, wherein the fibrinogen gamma-A chain precursor comprises an amino acid sequence of SEQ ID NO: 1, and a Retinol binding protein and a Haptoglobin precursor allele 1 comprise amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

3.A composition according to any one of claims 1 to 3, wherein said neurodegenerative disease is Alzheimer's disease.

4.A composition according to claim 1, wherein the proteins are obtained from cerebrospinal fluid (CSF) .

5.A method for diagnosing a neurodegenerative disease comprising: (a) measuring a level of the fibrinogen gamma-A chain precursor of an experiment sample comprising a fibrinogen gamma-A chain precursor, taken from a patient; and (b) comparing a level of the fibrinogen gamma-A chain precursor of said experiment sample to a level of the precursor of a normal sample taken from other subject or group of subjects, wherein a higher level of precursor of the experiment sample is indicative of having a neurodegenerative disease.

6.A method for diagnosing a neurodegenerative disease comprising:

(a) measuring a level of the Retinol binding protein or the Haptoglobin precursor allele 1 of an experiment sample comprising a Retinol binding protein or a Haptoglobin precursor allele 1, taken from a patient; and

(b) comparing a level of the Reinol binding protein or the Haptoglobin precursor allele 1 of said experiment sample to a level of the Reinol binding protein or the Haptoglobin precursor allele 1 of a normal sample taken from other subject or group of subjects, wherein a lower level of the Reinol binding protein or the Haptoglobin precursor allele 1 of the experiment sample is indicative of giving a neurodegenerative desease.

7. A method for diagnosing a neurodegenerative disease as claimed in claim 5 or 6 , wherein the disease is Alzheimer's disease.