KR101574766B1 - Biomarker Composition for Diagnosing Alzheimer's disease Including ABCF1(ATP-binding cassette sub-family F member 1 isoform a) in CSF and the Kit Including the Same - Google Patents

Abstract

The present invention relates to a biomarker composition for early diagnosis of Alzheimer's disease dementia, which is the most frequent of degenerative dementia, and more particularly, to a biomarker composition for early diagnosis of Alzheimer's disease dementia, The present invention relates to a biomarker composition capable of early diagnosis and clinical course estimation of Alzheimer's dementia including a protein detection agent, and a diagnostic kit comprising the same.
The present invention provides a biomarker composition for diagnosing Alzheimer's dementia, which comprises a drug for the diagnosis of Alzheimer's dementia, which comprises a substance for detecting protein ABCF1 in cerebrospinal fluid (ABCF1). In addition, the present invention provides a diagnostic kit for diagnosing Alzheimer's dementia comprising the biomarker composition.

Description

TECHNICAL FIELD [0001] The present invention relates to a biomarker composition comprising CSF protein AFC1 for early diagnosis of Alzheimer's disease, and a diagnostic kit comprising the CSF protein and AFC-binding cassette subfamily F member 1 isoform a the Kit Including the Same}

The present invention relates to a biomarker composition for early diagnosis of Alzheimer's disease dementia, which is the most frequent of degenerative dementia, and more particularly, to a biomarker composition for early diagnosis of Alzheimer's disease dementia, isoform a) provides a biomarker composition capable of early diagnosis and clinical course estimation of Alzheimer ' s dementia through protein quantification, and a diagnostic kit comprising the same.

The key pathologic proteins of Alzheimer's disease (AD), beta amyloid (beta amyloid 1-42, Aβ42) and tau protein are widely used as substrates for AD biomarkers in cerebrospinal fluid (CSF). Through active research, the concentration of these proteins in CSF (cerebrospinal fluid) has been useful for early and diverse diagnoses of AD (Shaw et al., 2010 Ann Neurol; Vanderstichele et al., 2006; Welge et al., 2009 J Neural Transm.).

However, these can not predict the clinical course or response to the drug, and the concentration in the cerebrospinal fluid (CSF) fluctuates with each measurement, making it less reliable (Carrillo MC et al., 2013 Alzheimer's &Dementia; Niklas Mattsson, 2010 Int J AD). It is difficult to accurately and early diagnose AD (Alzheimer's disease). Cerebrospinal fluid is the closest specimen to the brain and can be obtained in a relatively easy way. Proteins present in cerebrospinal fluid, while maintaining relatively stable levels compared to proteins in blood, best reflect changes in the brain.

It has been suggested that changes in cerebrospinal fluid concentrations of various proteins related to infection, nerve function and Aβ metabolism may be useful for the early diagnosis of AD (Alzheimer's disease) through the study of CSF proteomics (Britschgi M et al., Mol Cell Proteomics, 2011; Hu WT et al., 2010 Acta Neuropathol; Korolainen et al., 2010 J Neurochem). However, candidate biomarkers useful for supplementing the limitations of CSF concentration of A [beta] 42 and tau proteins have not been found yet.
Proteomics research can be approached through large-scale screening without any preconceptions, so new biomarkers can be identified that reflect not only the presence of disease, but also pathological progression. Most previously reported CSF proteomics studies have been performed through two-dimensional (2D) gel displays (reviewed in Korolainen et al., 2010 J Neurochem). The 2D gel display method fails to distinguish the various proteins located within the same spot and displays only a limited range of (10-200 kDa) proteins (Gygi et al., 2000; Zurbig and Jahn, Electrophoresis 2012) .

With the recent development of mass spectrometry (MS) -based technologies, a more robust distinction has become possible by population (Zurbig and Jahn, Electrophoresis 2012). Recent research reports detailing a portion of cerebrospinal fluid using LC-MS / MS (high-resolution liquid chromatography coupled with tandem mass spectrometry) have identified new biomarkers that were not found in previous literature (Ringman et al Jahn H et al., 2011 Plos One; Perrin et al., 2011 Plos one; Vafadar-Isfahani et al., JAD 2012).
However, in many of these studies, the diagnosis of Alzheimer's disease (AD) is based not only on the measurement of Aβ42 and tau protein in cerebrospinal fluid, but also pathological diagnosis through post-mortem examination, There is a considerable likelihood that patients without them will be included. In addition, various stages of clinical progression of AD (Alzheimer's disease) have not been specifically considered. These findings suggest the clinical utility of the new biomarkers discovered in these reports. In addition, since most of the studies are performed on Western patients, there is a possibility that there is a limit to the direct application to Korean patients' samples.

In the present invention, in order to develop a useful cerebrospinal fluid biomarker related to AD (Alzheimer's disease), an AD (Alzheimer's disease) pathologic diagnosis of AD (Alzheimer's disease) through brain imaging and Aβ42 and tau protein measurement in cerebrospinal fluid Large-scale proteomic analysis including only patients with dementia stage was performed.

In order to solve the above problems, the present invention provides a biomarker composition for early diagnosis of Alzheimer's disease including proteins in cerebrospinal fluid that can reflect the effects of drugs and clinical course estimation, and a diagnostic kit comprising the same The purpose is to provide.

In order to achieve the above object, the present invention provides a biomarker composition for diagnosing Alzheimer's dementia, which comprises an agent for detecting protein ABCF1 in cerebrospinal fluid for diagnosis of Alzheimer's disease.

In addition, the present invention provides a diagnostic kit for diagnosing Alzheimer's dementia comprising the biomarker composition.

In the early diagnosis of Alzheimer's disease, the levels of beta amyloid protein 1-42, tau protein, and tau phosphorus tau protein in cerebrospinal fluid were found to be the most useful biomarkers using body fluids. However, they are variable at the time of measurement and are often helpful for diagnosis, but they are not known to directly reflect pathological progress. Research on the treatment of Alzheimer's disease is now very active. Therefore, it is anticipated that the therapeutic agent will soon be clinically administered. At this time, development of a surrogate 'biomarker' is urgent to confirm whether the reaction of the drug, that is, the progress of the disease by the drug, is inhibited or prevented.

A novel biomarker composition capable of performing such a role in the detection of ABCF1, an ATP-binding cassette sub-family F member 1 isoform (a) contained in cerebrospinal fluid, It can be used as a surrogate biomarker for early diagnosis and clinical course estimation of Alzheimer ' s dementia, and it will be possible to develop a simplified diagnostic kit including the same.

A comparison of the cerebrospinal fluid of patients with Alzheimer's disease dementia and normal control subjects of the same age group revealed that the ABCF1, ATP-binding cassette sub-family F member 1 isoform a protein levels were significantly higher in patients with early Alzheimer's dementia (P value = 0.0005). In addition, the cerebrospinal fluid level of this protein is higher than that of conventional cerebrospinal fluid biomarkers such as tau protein, phosphorylated tau protein, beta-amyloid protein 1-42 / tau protein, and beta amyloid protein 1-42 / Respectively.

ABCF1, a member of the ATP-binding cassette sub-family F member 1 isoform a protein, is known to be involved in protein expression in endoplasmic reticulum. Recent studies have shown that the role of endoplasmic reticulum is important in the pathogenesis of Alzheimer's disease.

Therefore, the increase in the level of ABCF1, ATP-binding cassette subfamily F member 1 isoform a in cerebrospinal fluid of Alzheimer's disease can be used as a useful biomarker for early diagnosis of Alzheimer's disease , And may be useful in predicting pathological progression of Alzheimer's disease.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

≪ Example 1: Participants >

For the multi-centered study of biomarkers in AD (Alzheimer's disease), the clinical dementia rating scale (CDR) 0.5 or 1 (n = 13) and the control group (n = 11) cerebrospinal fluid samples were collected. All protocols have been approved by the review committee of individual institutions and meet the principles set out in the Helsinki Declaration. All AD (Alzheimer's disease) patients underwent a comprehensive evaluation including neurological examination, blood test, neuropsychological test and brain MRI within 6 months before CSF (cerebrospinal fluid) test. All AD patients were classified as "probable AD with high level of AD pathology" according to the criteria of the National Institute of Aging-Alzheimer's Association (NIA-AA) (McKhann et al., 2011). Additionally, fluorodeoxyglucose Decreased cerebral metabolism was confirmed in all patients undergoing positron emission tomography (FDG-PET) (n = 13), and increased amyloid accumulation was observed in patients receiving Pittsburgh Compound-B (PIB) -PET n = 9).

In all AD patients, a significant brain slice was found in the temporal lobe of the MRI. Control participants received brain MRI (n = 8) or computed tomography (n = 3) within 6 months of study entry and then agreed to undergo cerebrospinal fluid testing. One day before the CSF examination, normal cognitive function was confirmed by neurological examination, basic blood test, simple cognitive function assessment (MMSE) and clinical dementia rating scale (CDR). In addition, for the diagnostic accuracy of the final subject, CSF concentrations of the conventional AD biomarkers Aβ42, pTau181 and tTau were considered for diagnosis. That is, all AD subjects and control groups were admitted to the AD or normal thresholds for the ratio of Aβ42 / pTau181 (> 0.163, AD subjects) and Aβ42 / tTau (> 0.758 subjects, AD subjects) established in our experiment (Park et al. Dementia and neurocognitive disorders). There was no family history of dementia, except for four of the relatives who had AD. All of the AD patients were screened for mutations in the APP , PSEN1 , and PSEN2 genes (Park HK et al., 2008, JKMS) except one with cognitive symptoms after the age of 70 without family history. No known mutations in the cause of AD were found in all test subjects.

< Example  2 : CSF preparation  >

Cerebrospinal fluid (CSF) was extracted via a lumbar puncture performed between 8 am and 10 am after overnight fasting. The initial 2 ml was used for cell count and general chemistry. Subsequent 3 to 5 ml of CSF were collected in polypropylene tubes. After centrifugation at 4000 g for 10 min at 4 ° C, each sample was purified to 800 μL into a 1.0 mL polypropylene tube with screw cap. Until analysis, the tubes were frozen at -80 ° C. If cerebrospinal fluid was obtained from other organs, it was shipped to a BioMarker Core Lab (Bucheon Hospital, Soonchunhyang University) in a box with dry ice, and all analyzes were performed in one place.

< Example  3: LC - MS / MS Lt; RTI ID = 0.0 &gt;

The cryopreserved cerebrospinal fluid (CSF) in Example 2 above was thawed to freshness just prior to proteome analysis. A certain amount of CSF (10 μg) was loaded separately into the individual wells of 1-D 12% SDS-PAGE without mixing or intensive manipulation. The CSF protein separated by SDS-PAGE was stained with colloidal CBB (coomassie brilliant blue) and then cleaved from the gel. The gel pieces were extracted with 50% acetonitrile containing 50 mM NH ₄ HCO ₃ and centrifuged until CBB (coomassie brilliant blue) was completely removed. They were then dehydrated in 100% acetonitrile and vacuum dried in a speedVac for 20 minutes. For decomposition, the gel pieces in 50mM NH ₄ HCO ₃ 56 ℃ 45 bungan 10 mM dithiothreitol was reduced using the toll (dithiothreitol), 55mM iodine acetamide in after 30 minutes in the dark at 50mM NH ₄ HCO ₃ ( iodoacetamide. Finally, each gel fragments were treated with sequencing grade modified trypsin (Promega, USA) at 37 ° C overnight in 50 mM NH ₄ HCO ₃ buffer (pH 7.8). After digestion, trypsinolytic peptides were extracted with 5% formic acid in 50% acetonitrile solution at room temperature for 20 minutes. The supernatant was collected and dried on a SpeedVac. Samples were desalted with 0.1% formic acid using C18 ZipTips (Millipore, USA) prior to MS analysis and concentrated.

< Example  4 : LC - MS / MS  Analysis>

The trypsin digestion peptide was loaded onto a fused silica microcapillary column (12 cm x 75 μm) with C18 reverse phase resin (5 μm, 200 Å). LC separation was carried out under a linear gradient according to the following: 3-40% solution B (0.1% formic acid in 100% acetonitrile) gradient at a flow rate of 250 nL / min for 60 minutes. The column was directly connected to an LTQ linear ion-trap mass spectrometer (Finnigan, USA) with a nanoelectrospray ion source. The electrospray voltage was set to 1.95 kV, and the threshold to be converted from MS to MS / MS is 500. The normalized collision energy for MS / MS was 35% of the main radio frequency amplitude (RF) and the activation duration was 30 ms. All spectra were obtained in a data-dependent scan mode. Five consecutive MS / MS scans were performed on the strongest of the five strong peaks of the entire MS scan after each full MS scan. The peak number of repetitions for dynamic exclusion was 1 and the repetition duration was 30s. The duration of dynamic exclusion was 180 s and the width of exclusion mass was ± 1.5 Da. The list size of dynamic exclusion was 50.

< Example  5: Database searching  >

Tandem mass spectra were obtained from the US National Biotechnology Center (http://www.ncbi.nlm.nih.gov/) nonredundant human database containing the reverse protein sequence database using the SEQUEST search engine (BioWorksBrowser ™ , version Rev. 3.3.1 SP1, Thermo Fisher Scientific Inc., CA). The search parameters are as follows: complete trypsin digestion, two false disruptions, precursor ion tolerance (± 3 amu), fragment ion mass tolerance (± 1 amu), cysteine (+57 Da: carbamidomethylation) and methionine Fixed modifications to the residues. To compare the protein expression of individual samples, a label-free protein quantification method was used to count the number of spectra.

< Example  6: Statistical Analysis>

To identify the candidate CSF (cerebrospinal fluid) biomarkers among the total 724 experimental proteins, AD (Alzheimer's disease) consisting of 13 and 11 samples and the control group were compared in the following two steps. First, the candidate set is a protein set whose expression is expressed in an average AD group or more, and is expressed differently by t-test as compared with the control group. The next step is to further analyze whether each candidate protein is primarily associated with the five known AD biomarkers A? 42, pTau181, tTau, A? 42 / pTau181, and A? 42 / tTau. More specifically, the Pearson correlation coefficients of each known biomarker for the protein expression pattern and 24 samples were measured and the significance was assessed by t-test.

< Results  >

The mean cognitive function test score of AD patients was 21.3 ± 4.9, which was significantly reduced compared to the control group of 28.4 ± 1.2 (Table 1). Clinical dementia rating and CDR-SOB (sum of box) scores of AD patients were 3.9 ± 1.6 and 0.8 ± 0.3, respectively. The level of dementia in the subjects with AD was very early or early, with a CDR of 0.5 (5 patients) or 1 (8 patients).

[Table 1] Clinical and experimental characteristics of participants

When protein expression was compared, expression of ABCF1 (ATP-binding cassette subfamily F member 1 isoform a) was increased in AD (Alzheimer's disease) between AD and the control group (see Table 2).

[Table 2] CSF protein showing significant difference between AD and NC on LC-MS

To better understand the significance of the candidate biomarkers, we looked at the associations and correlations with known AD biomarkers. Regardless of the level of significance, the direction of change in the candidate biomarkers in relation to the level of A [beta] 42 was reversed (Table 3).

[Table 3] Correlation between LC-MS analysis and CSF AD biomarker

(Correlation coefficient = -1.945, p value = 0.0696), pTau181 (correlation coefficient = 2.848, p value = 0.0116), and the ABCF1 (ATP-binding cassette subfamily F member 1 isoform a) ) And tTau (correlation coefficient = 2.974, p value = 0.0090), respectively.

In the present invention, a novel proteomic approach using LC-MS / MS was performed to discover a new CSF (cerebrospinal fluid) biomarker in early AD (Alzheimer's disease) dementia. Extensive studies have identified proteins that are significantly different in AD and in the control group among the 724 proteins analyzed. (ABCF1, ATP-binding cassette sub-family F member 1 isoform a), which is highly correlated with CSF Aβ42, pTau181, and tTau concentrations and their ratio.

The direction of association with A [beta] 42 concentration was in the opposite direction of association with pTau181 and tTau protein. This is consistent with the fact that the down-regulation of CSF Aβ42 in AD (Alzheimer's disease) is always reversed with the change in pTau181-tTau (up). Therefore, these results suggest that the candidate biomarkers are closely related to AD (Alzheimer's disease) pathology.

ABCF1, a member of the ATP-binding cassette subfamily F member 1 isoform a, is a protein present in the cytoplasm and in the endoplasmic reticulum. It is a congenital immune response (Lee MN et al., Nat Immunol 2013) (Paytubi et al., J Biol Chem 2009). Modified stress-induced protein translation and activated unfolding protein responses have recently been proposed as key pathologic causes in AD (Halliday and Mallucci, Neuropharmacology 2013). Thus, looking at the biological role, it is strongly suggested that the increase in ABCF1 concentration in cerebrospinal fluid of AD patients is closely related to AD pathophysiology.

The embodiments presented above are illustrative and those skilled in the art will be able to make various modifications and alterations to the disclosed embodiments without departing from the technical spirit of the present invention. The scope of the present invention is not limited by these variations and modifications.

Claims (2)

A method for detecting Alzheimer &apos; s dementia by detecting protein AFP1 contained in cerebrospinal fluid, comprising detecting a protein AFP-1 (ABCF1, ATP-binding cassette sub-family F member 1 isoform a) A biomarker composition for diagnosis of dementia. A diagnostic kit for diagnosing Alzheimer &apos; s dementia comprising the biomarker composition for diagnosing Alzheimer &apos; s dementia of claim 1.