ES2657422T3 - Diagnostic method for disorders related to brain damage based on NDKA detection - Google Patents

Abstract

A method of diagnosing a disorder related to brain damage selected from head trauma, ischemic stroke, hemorrhagic stroke, subarachnoid hemorrhage, intracranial hemorrhage, transient ischemic attack and vascular dementia, or the possibility thereof, in a subject suspected of being suffers the same, or prognosis or therapeutic follow-up of said disorder related to brain damage in a subject, which comprises nucleoside diphosphate kinase A, or a variant or mutant thereof, which has at least 90% homology to it , and having substantially the same functional and immunological properties, in a sample of body fluid taken from the subject, in which the nucleoside diphosphate kinase A, or said variant or mutant, is present in the body fluid of subjects affected by a related disorder with brain damage in a larger amount compared to the body fluid of subjects affected by a disorder not related to brain damage, whereby a greater amount of nucleoside diphosphate kinase A, or said variant or mutant, in the body fluid sample is indicative of said disorder related to brain damage.

Description

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homologue of ubiquitin fusion degradation protein 1 (Q92890), also called UFD1 or UFDP1, which has the sequence (SEQ ID NO.10):

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Nucleoside diphosphate kinase A (P15531), also called NDK A, which has the sequence (SEQ ID NO.11):

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Glutathione S tranferase P (P09211), which has the sequence (SEQ ID NO. 12):

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Cathepsin D (P07339), which has the sequence (SEQ ID NO. 13):

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DJ-1 protein (Q99497), which has the sequence (SEQ ID NO. 14):

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Peroxirredoxin 5 (P30044), which has the sequence (SEQ ID NO. 15):

Peptidyl-prolyl cis-trans isomerase A (Cyclophilin A) (P05092), which has the sequence (SEQ ID NO. 16):

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The polypeptides useful in the present invention are not restricted to the above sequences, and include variants and mutants thereof. A variant is defined as a variation of natural origin in the sequence of a polypeptide that has a high degree of homology with the given sequence, and that has substantially the same functional and immunological properties. A mutant is defined as an artificially created variant. A high degree of homology is defined as at least 90%, preferably at least 95%, and much more

30 preferably at least 99% homology. Variants can take place within a single species or

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between different species The above sequences are of human origin, but the invention includes the use of the corresponding polypeptides of other mammalian species, for example, bovines.

Disorders related to brain damage in the context of the present invention include the following:

5 head trauma, ischemic stroke, hemorrhagic stroke, subarachnoid hemorrhage, intracranial hemorrhage, transient ischemic attack and vascular dementia. Other disorders related to brain damage described in the document include corticobasal lymph node degeneration, encephalitis, epilepsy, Landau-Kleffner syndrome, hydrocephalus, cerebral pseudotumor, thalamic diseases, meningitis, myelitis, movement disorders, essential tremor, spinal cord diseases , syringomyelia, Alzheimer's disease (early onset), disease

10 Alzheimer's (late onset), multi-infarcted dementia, Pick's disease, Huntington's disease, Parkinson's, Parkinson's syndromes, frontotemporal dementia, corticobasal degeneration, multisystemic atrophy, progressive supranuclear paralysis, Lewy body disease, amyotrophic lateral sclerosis, Creutzfeldt-Jakob, Dandy-Walker syndrome, Friedreich's ataxia, Machado-Joseph's disease, migraine, schizophrenia, mood disorders and depression. Corresponding disorders are also included in

15 non-human mammals, such as transmissible spongiform encephalopathies (TSE), for example, bovine spongiform encephalopathy (BSE) in cattle or scrapie of sheep.

H-FABP (P05413) and B-FABP (015540) are also useful herein for the diagnosis of disorders related to brain damage or the possibility thereof, especially those other than 20 strokes and CJD.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of an assay for H-FABP (measured in OD units on the axis

25 vertical) for three groups of patients: a control group, a group with acute myocardial infarction (AMI) and a group with acute stroke; Figure 2 shows the results of the sequential determination of H-FABP levels (measured in OD units on the vertical axis) for the group of stroke patients at different time intervals after the stroke;

Figure 3 shows portions of 2-DE maps for healthy and post-mortem CSF, with upwardly directed arrows indicating points corresponding to the RNA-binding regulatory subunit or the DJ-1 protein. The 2-DE map extensions of healthy CSF and CSF of deceased are shown. Forty-five μg of protein was loaded on an IPG gel (pH 3.5-10 NL, 18 cm). The second dimension was a vertical gradient plate gel (9-16% of T). The gel was dyed silver. The points corresponding to the subunit

Regulatory binding to RNA or DJ-1 protein are indicated by upwardly directed arrows (red); Figure 4 shows portions of 2-DE maps for healthy and post mortem CSF, indicating the arrows on the right points corresponding to peroxirredoxin 5. The enlargements of 2-DE maps of healthy CSF and CSF of deceased are shown. Forty-five μg of protein was loaded on an IPG gel (pH 3.5-10

40 NL, 18 cm). The second dimension was a vertical gradient plate gel (9-16% of T). The gel was dyed silver. The point corresponding to Peroxirredoxin 5 is indicated by the arrows on the right (red); Figure 5 shows portions of 2-DE maps for healthy and post-mortem CSF, indicating the pair of arrows on the right points corresponding to peptidyl-prolyl cis-trans isomerase A (cyclophilin A). The

45 extensions of 2-DE maps of healthy CSF and CSF of deceased. Forty-five μg of protein was loaded on an IPG gel (pH 3.5-10 NL, 18 cm). The second dimension was a vertical gradient plate gel (9-16% of T). The gel was dyed silver. The points corresponding to Cyclophilin A are indicated by the pair of arrows (red) on the right; Figure 6 shows the ELISA intensity values for marker polypeptides obtained in a

50 study of stroke patients; Figure 7 shows the detection of UFD1 in plasma samples of said study; Figure 8 is an ROC curve of UFD1 from the data in Figure 7; Figure 9 shows the detection of UFD1 corresponding to Figure 7; Figure 10 shows the detection of RNA-BP in plasma samples of said study;

Figure 11 is an ROC curve of RNA-BP from the data in Figure 10; Figure 12 shows the detection of RNA-BP corresponding to Figure 10; Figure 13 shows the detection of NDK A in plasma samples of said study; Figure 14 is an ROC curve of NDK A from the data in Figure 13; Figure 15 shows the detection of NDK A corresponding to Figure 13;

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and a compound, which improves the intensity and duration of the emitted light, typically, 4-iodophenol or 4-hydroxycinnamic acid.

An amplified immunoassay such as immuno-PCR can be used. In this technique, the antibody binds

5 covalently to an arbitrary DNA molecule comprising PCR primers, whereby the DNA with the antibody bound to it is amplified by the polymerase chain reaction. See E. R. Hendrickson et al., Nucleic Acids Research 1995; 23, 522-529 (1995) or T. Sano et al., In "Molecular Biology and Biotechnology" ed. Robert A. Meyers, VCH Publishers, Inc. (1995), pages 458-460. The signal is read as before.

In one procedure, an enzyme-linked immunosorbent assay (ELISA) can be used to detect the polypeptide.

The use of a rapid turbidimetric immunoassay with rapid microparticles, developed for H-FABP in the case of AMI, M. Robers et al., "Development of a rapid microparticle-enhanced turbidimetric immunoassay for 15 plasma fatty acid-binding protein, an early marker of acute myocardial infarction ", Clin. Chem. 1998; 44: 1564-1567, significantly decreases the test time. Therefore, complete automation in a widely used clinical chemistry analyzer, such as the Hoffmann-La Roche COBAS ™ MIRA Plus system, described by

M. Robers et al. above, or the Abbott Laboratories AxSYM ™ system, should be possible and applied for the routine clinical diagnosis of disorders related to brain damage.

20 Polypeptide concentrations can be measured by other means than the immunoassay. For example, the sample can be subjected to 2D gel electrophoresis and the amount of the polypeptide can be estimated by densitometric scanning of the gel or a transfer thereof. However, it is desirable to perform the test in a rapid manner, so that the patient can be treated promptly.

In principle, any body fluid can be used to provide a sample for diagnosis, but preferably the body fluid is cerebrospinal fluid (CSF), plasma, serum or blood.

According to the invention, a diagnosis of disorders related to brain damage can be made from

30 of the determination of nucleoside diphosphate kinase A, or a variant or mutant thereof, which has at least 90% homology thereto, and which has the same functional and immunological properties, or any combination of nucleoside diphosphate kinase A and one or more of the polypeptides.

The invention also relates to the use of nucleoside diphosphate kinase A and, optionally, one or more of the

35 specified polypeptides that are differentially contained in a body fluid of subjects affected by brain damage and subjects not affected by brain damage, for diagnostic and prognostic applications. This may involve the preparation and / or use of an antibody that recognizes, binds to or has some affinity to the aforementioned polypeptide. The term "antibody" as used herein includes polyclonal antiserum, monoclonal antibodies, antibody fragments such as Fab, and genetically engineered antibodies.

40 modified. The antibodies can be chimeric or of a single species. The above reference to "prognostic" applications includes making a determination of the probable course of a disorder related to brain damage, for example, by measuring the amount of the aforementioned polypeptide in a body fluid sample. The above reference to "therapeutic follow-up" applications includes making a determination of the probable course of a disorder related to brain damage, for example, by measuring the amount of the polypeptide

45 mentioned above in a sample of body fluid (and assessing its level depending on the treatment, the recovery of the disability or not, the size of the lesions, etc.). The above reference to the "therapeutic" applications described herein includes, for example, the preparation of materials that recognize, bind to or have affinity to the aforementioned polypeptides, and that use such materials in therapy. The materials can in this case be modified, for example, by combining an antibody with a drug, whereby

50 directs the drug to a specific region of the patient.

The above reference to "presence or absence" of a polypeptide should be understood to mean simply that there is a significant difference in the amount of a polypeptide that is detected in the affected and unaffected sample. Therefore, the "absence" of a polypeptide in a test sample may include the possibility that

The polypeptide is actually present, but in a significantly smaller amount than in a comparative test sample. According to the invention, a diagnosis can be made based on the presence or absence of a polypeptide, and this includes the presence of a polypeptide in a significantly lower or significantly larger amount with reference to a comparative test sample.

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(PerSeptive Biosystems Voyager STR MALDI-TOF-MS, Framingham, MA, United States) [10] and were identified through a database using the PeptIdent tool (http://www.expasy.ch/sprot/peptident. html).

Table 1:

A-FABP

P15090

E-FABP

Q01469

PGP 9.5

P09936

GFAP

P14136

Prostaglandin D synthase

P41222

Neuromodulin

 P17677

Neurofilament L

P07196

Calcifosine

 Q13938

RNA binding regulatory subunit

O14805

Ubiquitin fusion degradation protein 1 homolog

Q92890

Nucleoside diphosphate kinase A

P15531

Glutathione S transferase P

P09211

Cathepsin D

P07339

H-FABP

P05413

B-FABP

O15540

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EXAMPLE 2

Using two-dimensional gel electrophoresis (2-DE) separation of cerebrospinal fluid (CSF) proteins and mass spectrometry techniques, elevated FABP was found in the CSF of deceased patients, 10 used as a model of massive brain damage. Since H-FABP, a form of FABP present in many organs, is also located in the brain, an enzyme-linked immunosorbent assay (ELISA) was developed to detect H-FABP in plasma samples from stroke versus control. However, with H-FABP also being a marker of acute myocardial infarction (AMI), the levels of troponin I and creatine kinase MB (CK-MB) were tested at the same time to exclude any concomitant damage to the heart. The levels of NSE and S100B are

15 rehearsed simultaneously.

Study population and sample management

The population used for the plasma assessment of the various markers detailed below included a

20 total of 64 patients studied prospectively (Table 2) distributed equally in three groups: (1) a control group that included 14 men and 8 women 65 years (intervals: 34-86 years) without peripheral or central nervous system involvement known; (2) a group of patients with acute myocardial infarction (AMI group), including 14 men and 6 women 65 years (intervals: 29 to 90 years); The diagnosis of AMI was established in all cases by typical electrocardiography modifications and elevated levels of CK-MB (above a value

25 cut-off of 9.3 ng / ml) and troponin I (above a cut-off value of 2 ng / ml); (3) a group of patients with acute stroke (Ictus group), including 14 men and 8 women 65 years (intervals: 30 to 87 years); the diagnosis of stroke was established by a qualified neurologist and was based on the sudden onset of a focal neurological deficit and the subsequent delineation of a lesion compatible with the symptoms in brain images of CT or MRI, with the exception of transient ischemic attacks ( TIA) where a visible injury was not required for the

30 diagnosis The Stroke group separated according to the type of stroke (ischemia or hemorrhage), the location of the lesion (brainstem or hemisphere) and clinical evolution over time (TIA when complete recovery occurred within 24 hours or the stroke established when the neurological deficit was still present after 24 hours).

35 Table 2

Group

Control TO ME Ictus Ictus

Diagnosis

Diagnosis Ischemia Hemorrhage Location Brain stem Hemisphere Type TIA CVA

H-FABP number

22 twenty 22 22

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performed using the Mann-Whitney U test and the longitudinal evaluation of H-FABP concentrations over time were analyzed using repeated measures variance analysis (ANOVA). The reference limits for H-FABP with the aim of distinguishing stroke patients from normal patients were delineated using the receiver's operational characteristic curves (Analyze-It ™ software for Microsoft Excel ™) and,

5 Subsequently, sensitivity, specificity, positive predictive values and negative predictive values at each time point were calculated. Statistical significance was established at p <0.05.

Results

10 The individual results of the H-FABP assay in the three populations, expressed in OD units, are shown graphically in Figure 1 and are detailed in Table 3. The concentration of H-FABP in mean plasma was 0.221 + 0.134 OD in the Control group, 1,079 ± 0.838 OD in the Ictus group and 2,340 ± 0.763 OD in the AMI group. The coefficient of variation found for this ELISA was 5.8% ± 3.8. Using the Kruskall-Wallis test, the three concentrations were significantly different (p <0.001) from each other. The best

15 cut-off value to discriminate between the Control and Ictus groups was set at OD> 0.531 as determined by the ROC curves for the H-FABP level (data not shown). Using this cut-off value, the validity measures of H-FABP for the diagnosis of stroke were as follows: sensitivity was 68.2% with 15 of 22 stroke patients above the cut-off point, specificity it was 100% with the 22 control subjects below the cut-off point, the positive predictive value was 100% and the negative predictive value was 75.9%.

20 Table 3

Group

Control TO ME Ictus

H-FABP

Mean OF Significance 0.2210,134 2,434 0.638 <0.001 1,079 0.838 <0.001

Troponin-I

 Mean OF Significance 0.0 0.1 164.6 205.6 <0.001 0.5 1.3 ns

CK-MB

Mean OF Significance 1.3 0.9 63.8 51.5 <0.001 7.9 21.3 ns

To discriminate, at the biological level, between patients of the AMI and Ictus groups, Troponin I and CK-MB were additionally tested in each group with cut-off values set at 2 ng / ml for the AxSYM Troponin test and 25 3, 8 ng / ml for the CK-MB AxSYM assay (Table 3). As expected, the concentrations of these AMI markers were significantly higher (p <0.01) in the AMI group compared to both the Control and Ictus groups. No difference was found between the last two groups, confirming that Troponin I and CK-MB do not increase as a result of a brain injury and that stroke patients did not suffer a concomitant AMI at the time of their stroke. Taken together, the concentrations of H-FABP, troponin I

30 and CK-MB allowed a correct discrimination between AMI (increase of the three markers) and stroke (increase of H-FABP with normal troponin I and CK-MB) in the 20 patients with AMI and in 15 patients with stroke, with the except for one patient with stroke who showed, together with elevated levels of H-FABP, moderately elevated levels of troponin I and CK-MB in the absence of EKG modifications, all of which is consistent with concomitant non-AMI heart damage.

35 In the Ictus group, seven false negative results were found with H-FABP levels below the cut-off value of OD 0.531 at any time point after the neurological event. Of these seven patients, two had a rapid and complete recovery of their neurological deficits within 24 hours consistent with a transient ischemic attack (TIA), and two had a lacunar stroke in the MR images, one located in the brain stem.

40 Although TIA and lacunar stroke can explain the results of false negatives in most patients, no consistent explanation was found for the three remaining stroke patients with low levels of H-FABP.

Sequential determinations of H-FABP level after stroke showed that 10 of 15 (67%) patients with H-FABP positive stroke had a very early increase in H-FABP levels (<12 hours). In addition, 45 as shown in Figure 2, when all stroke patients were considered, the average H-FABP concentrations decreased steadily after the insult, the highest value being found before 12 hours. The differences between the initial measurement and the measurements of less than 48 hours and later were significant (ANOVA, p <0.05). When H-FABP levels were compared between the different subgroups of the Stroke group, no statistically significant differences were found. H-FABP levels were similar

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Bibliography data:

It was found that PGHD decreased in the CSF of patients suffering from AD (Puchades M. et al., Brain Res. Mol.

5 Brain Res. 2003, 118, 140-146). However, the study was conducted using 2-ED gels and only acid points were analyzed. As these results show in CSF of deceased patients, it is possible that PGHD was deglycosylated in the analyzed samples, resulting in the disappearance of acid points but not a decrease in the total protein level.

10 Using the capillary isoelectric approach, Hiraoka et al. have identified changes in the load microheterogeneity of the PGHD of CSF associated with various neurological disorders (Hiraoka A. et al., Electrophoresis 2001, 22, 3433-3437). It was found that the ratio of basic forms / acid forms increases in AD, in PD with pathological cerebral atrophy, and in multiple sclerosis. It was speculated that these post-translational modifications were related to changes in the N-glycosylation pattern.

15 Pattern of post-translational modifications of PGHD (PTM) in the CSF of a patient with Creutzfeldt-Jakob disease (CJD):

The PTM pattern of PGHD in CSF samples collected from a patient with CJD and a healthy control was compared.

The proteins were separated by 2-DE, electrotransferred on a PVDF membrane and PGHD was detected using a specific antibody, as previously described. CSF samples were collected by lumbar puncture. The control patient had a neurological treatment for benign conditions not related to brain damage. The CSF samples were centrifuged immediately after collection, divided into aliquots, frozen at -20 ° C and stored until analysis.

25 The results are shown in Figure 17, which is a comparison of specific intensities of prostaglandin D2 synthase in mini-2-DE gels prepared with CSF from a patient suffering from Creutzfeldt-Jakob disease

or with a CSF to control a healthy patient. Forty-five μg of protein was loaded on an IPG gel (pH 310 NL, 7 cm). The second dimension was a vertical gradient plate gel (12% T). Immunodetection is

30 performed using a polyclonal rabbit anti-human antibody of PGHD (lipocalin type) (Cayman chemical, Ann Arbor, MI) and Western ECL ™ transfer detection reagents (Amersham Biosciences, Uppsala, Sweden).

The results showed that the PTM pattern of PGHD in the CSF of the patient with CJD is clearly different from the control, with a sharp decrease in the 4 most acidic points (Figure 17). The pattern of the patient with CJD is similar

35 to that observed in the post-mortem CSF. These data support the interest of changes in the PTM pattern of PGHD as a marker of neurological disorders.

EXAMPLE 6

40 This Example provides additional data showing plasma levels of UFDP1 in stroke and control patients. Figure 19 shows the levels of UFDP1 in CSF of a control patient and a deceased patient. Additional data were obtained from two patient and control cohorts, the minors from Geneva, and a more complete panel from the United States. The methodology for this Example and the following Examples 7 and 8 is the same, except that the antibodies used have different specificities for the protein in question. The method in each

One of the studies is similar to that given in Example 4:

An ELISA was performed using black 96-well NeutrAvidin ™ coated plates Reacti-Bind ™ (Pierce, Rockford, IL). The plates were first rinsed in a borate buffer saline solution at pH 8.4 (BBS) (100 mM H3BO3, 25 mM Na2B4O7 (Sigma, St Louis, MO, United States), 75 mM NaCl (Merck, Darmastadt, 50 Germany)) in a NOVAPATH ™ washer (Bio-Rad, Hercules, CA). Then, 50 µl of antibody-biotin-specific conjugate of the relevant biomarker (2 µg / ml) prepared in dilution buffer A at pH 7 (DB, polyvinyl alcohol, 80% hydrolyzed, Weight Mol. 9000-10,000 ( Aldrich, Milwaukee, WI, United States), MOPS (3- [N-morpholino] propane sulfonic acid) (Sigma), NaCl, MgCl2 (Sigma), ZnCl2 (Aldrich), pH 6.90, 30% BSA solution , manufacturing grade (Serological Proteins Inc., Kankakee, IL)) and incubated for one hour at 37 ° C. 55 The plates were then washed 3 times in BBS in the plate washer. Then, 50 µl of antigen or plasma was added and incubated for one hour at 37 ° C. Recombinant protein antigens were diluted to 100, 50, 25, 12.5, 6.25, 3,125, 1.56 ng / ml in dilution buffer A to establish a calibration curve. Plasma samples were diluted to the appropriate dilution in dilution buffer A. After an additional wash step, 50 µl of biomarker-specific alkaline phosphatase-conjugated antibodies were added.

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Claims (1)

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