Method for Detection of Ischemic Strokes

Abstract

The present invention relates to the identification and use of diagnostic markers for ischemic stroke of the lacunar subtype. The invention relates to devices and kits for performing these methods.

Claims

1. A method of diagnosing ischemic stroke in a subject, comprising: detecting the presence and/or amount of at least one marker or at least one antibody against at least one marker in a sample from said subject, wherein the at least one marker is selected from the group consisting of Pellino homolog 1 (Drosophila) (PELI1), TNF receptor-associated protein 1 (TRAP1), Conserved oligomeric Golgi complex subunit 3 (COG3), Homo sapiens solute carrier family 38 member 3 (SLC38A3), Homo sapiens growth arrest-specific 2 like 3 (GAS2L3); and correlating the result to the occurrence or non-occurence of ischemic stroke in said subject.

2. The method of claim 1, wherein the ischemic stroke is an ischemic stroke of lacunar subtype.

3. The method of claim 1, wherein the sample is a sample of blood, serum and/or plasma from said subject.

4. The method of claim 1, wherein the presence and/or amount of at least one antibody against the at least one marker is detected.

5. A device for the detection of ischemic stroke, comprising: a) means for determining the amount and/or presence of at least one marker or at least one antibody against at least one marker in a sample from a subject, wherein the at least one marker is selected from the group consisting of: Pellino homolog 1 (Drosophila) (PELI1), TNF receptor-associated protein 1 (TRAP1), Conserved oligomeric Golgi complex subunit 3 (COG3), Homo sapiens solute carrier family 38 member 3 (SLC38A3), and Homo sapiens growth arrest-specific 2 like 3 (GAS2L3); and b) means for analyzing the amounts and/or presence of the marker(s) or antibodies against the marker(s) against a reference value.

6. Device of claim 5, wherein the ischemic stroke detected is an ischemic stroke of lacunar subtype.

7. A kit adapted for carrying out the method of claim 1, comprising: a) means for determining the amount and/or presence of the at least one marker or antibody against the at least one marker in a sample from a subject; and b) means for analyzing the amounts and/or presence of the at least one marker or antibodies against the at least one marker against a reference value.

8. A method of diagnosing ischemic stroke in a subject, comprising: detecting the presence and/or amount of at least one marker or at least one antibody against at least one marker in a sample from said subject, wherein the at least one marker has more than 80% identity to a protein sequence consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, or SEQ ID NO. 5; and correlating the result to the occurrence or non-occurrence of ischemic stroke in said subject.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0065] Other objects and advantages of the present invention may be ascertained from a reading of the specification and appended claims in conjunction with the drawings and tables therein.

[0066] For a more complete understanding of the present invention, reference is established to the following description made in connection with accompanying drawings in which:

[0067] FIG. 1: Measured data of ischemic strokes without lacunar subtype (cardioembolic and atherothrombotic subtypes; left column), lacunar strokes and stroke mimics for marker BC018950.2 for two protoarrays.

[0068] FIG. 2: Measured data of ischemic strokes without lacunar subtype (cardioembolic and atherothrombotic subtypes; left column), lacunar strokes and stroke mimics for marker NM_006841.3 for two protoarrays.

[0069] FIG. 3: Measured data of ischemic strokes without lacunar subtype (cardioembolic and atherothrombotic subtypes; left column), lacunar strokes and stroke mimics for marker NM_020651.2 for two protoarrays.

[0070] FIG. 4: Measured data of ischemic strokes without lacunar subtype (cardioembolic and atherothrombotic subtypes; left column), lacunar strokes and stroke mimics for marker NM_031431.2 for two protoarrays.

[0071] FIG. 5: Measured data of ischemic strokes without lacunar subtype (cardioembolic and atherothrombotic subtypes; left column), lacunar strokes and stroke mimics for marker NM_174942.1 for two protoarrays.

DETAILED DESCRIPTION OF THE INVENTION

[0072] As already described the method of diagnosing ischemic stroke in a subject, comprising the step of detecting the presence and/or amount of at least one marker or at least one antibody against at least one marker in a sample from said subject, wherein the at least one marker is selected from the group of in total 5 proteins and polypeptides. In a preferred embodiment the markers derived from proteins and polypeptides, which are encoded by the sequences with the Database number shown in the column “Sequence” in table 1. In a more preferred embodiments the markers are derived from proteins and polypeptides, which are encoded by the sequences with the database ID shown in the column “Database ID” in table 1.

[0073] In a preferred embodiment of the invention the presence and/or presence of at least one antibody against the at least one marker is detected. This is preferably done in a protein micro array, e.g. ProtoArray from Invitrogen. In such arrays the protein or polypeptide to be bound by the antibody is printed on a plate, mostly a nitrocellulose coated glass plate. It may be necessary to express the protein or polypeptide as a fusion protein, e.g. GST fusion protein. The plate is then incubated with the sample fluid, wherein the antibody (primary antibody) it so be detected. After a washing step the antibodies bound to the protein or polypeptide are detected by incubating the array with a secondary antibody, which binds the primary antibody. This secondary antibody is labelled with a detectable tag. This can be a fluorescent tag, an enzymatic tag like horseradish peroxidase, a ligand binding tag. The amount of fluorescence is proportional to the amount of primary antibody. It may be necessary to correct the value by the amount of marker present in the array.

[0074] Next to one of the markers also other markers may be measured and analyzed at the same time.

[0075] In a preferred embodiment the method is a method to distinguish ischemic stroke of lacunar subtype against other ischemic strokes and/or stroke mimics, preferably ischemic stroke of the atherothrombic and cardioembolic subtype and/or stroke mimics.

[0076] Preferred markers are shown in the tables 1 or 2. Table 2 also gives the Seq-ID-Nos of the preferred markers for the invention.

[0077] The sequences of the markers are available from public databases. The corresponding protein sequences are listed in the sequence protocol.

[0078] Sample Preparation

[0079] The measurements were preformed using the Invitrogen ProtoArray.

[0080] Samples from 12 controls (stroke mimics) and 13 ischemic stroke patients were obtained. From the stroke patients were 5 of the lacunar subtype, 4 of the cardioembolic subtype and 4 of the atherothrombotic subtype. The stroke mimics can be further classified as epileptic seizure (4 samples), Hypoglicemia (1 sample), brain tumor (3 samples), vertigo (1 sample), radial palsy (1 sample), migraine (1 sample) and syncope (1 sample).

[0081] For the measurement the ProtoArray from Invitrogen was used (www.invitrogen.com/protoarray) following the standard protocol from the manufacturer. 9500 native human proteins were expressed in baculovirus and immobilized on a chip by contact type printing. 3 additional proteins were expressed and included in the array (2 NMDA subunits). The proteins are expressed as GST fusion protein in order to allow quality control by probing the microarray with a GST-antibody.

[0082] 9501 proteins (auto-antibodies against these proteins) were measured, out of which 9411 were unique. Some proteins were tested twice in different chip locations. All 9501 proteins were measured in duplicates.

[0083] For the measurement the array is incubated with the sample, optionally in a probing buffer, typically for 2 hours at 4° C. Any antibodies present in the sample will bind to the different proteins presented in the array. The sample is decanted and the array is washed several times. Then the array is incubated with the secondary antibody. This is typically a fluorescent labelled antibody, e.g. anti-mouse or anti-rabbit Alexa Fluor 647 from Invitrogen). This incubation is also normally done for two hours and at 4° C. The array is then washed several times and dried. Then the microarray is scanned with a fluorescent scanner (GenePix 4000B Fluorescent Scanner; Molecular Devices) and the data was acquired with GenePix Pro software (Molecular Devices) and processed using the ProtoArray Proscpector tool developed by Invitrogen, which performs the normalizing and processing of the data.

[0084] In analysing the measured data either the Minimum (Min) or Average (Avg) of the duplicates was considered.

[0085] The data was further normalized in respect to control proteins and background using either Linear Normalization (LN) or Quantile Normalization (QN).

[0086] This resulted in a total for four combinations of datasets: MinLN, MinQN, AvgLN and AvgQN.

[0087] Negative values were converted to zero. The data was further log-transformed (after adding 1 to account for zeros).

[0088] All analysis was done with all four combinations of datasets. The following results were obtained using the Minimum Quantile Normalized data (MinQN).

[0089] Univariate Analysis

[0090] In this analysis the proteins that are significant on their own were identified using Permutation Test Statistic and Kruskal-Wallis Testing correcting for the small sample size issue.

[0091] Statistical significance is reported in terms of the False Positive Rate (p-value) and the Family Wise Error Rate (FWER).

[0092] Stroke Subtypes Analysis

[0093] For this analysis the data processed and normalized with the quantile normalization method was used.

[0094] In order to obtain the significant proteins the data was analyzed using the Permutation Test Statistic and Kruskal-Wallis Testing. The data of 2 samples were excluded because these samples were redraw samples from 2 patients which were taken one week after hospitalization of the patients. Only samples drawn within 3 hours after onset of the symptoms were included in analysis.

[0095] Graphs were generated for the candidates which were identified by the statistical analysis and were then visually evaluated. The 5 most promising candidate markers were selected visually.

[0096] The analysis of the data in regard of stroke patients of lacunar subtype versus stroke mimics revealed 5 antibodies against proteins as marker of particular significance. These proteins are shown in table 1. The graphs of the data of these proteins are shown in FIGS. 1 to 5.

[0097] In further analysis the data was analyze with regard to different subtypes of ischemic stroke.

[0098] The most significant proteins identified to differentiate the atherothrombic and cardioembolic subtype and stroke mimics from lacunar ischemic stroke are shown in table 1. The corresponding graphs are displayed in FIGS. 1 to 5.

[0099] While the present inventions have been described and illustrated in conjunction with a number of specific embodiments, those skilled in the art will appreciate that variations and modifications may be made without departing from the principles of the inventions as herein illustrated, as described and claimed. The present inventions may be embodied in other specific forms without departing from their spirit or essential characteristics. The described embodiments are considered in all respects to be illustrative and not restrictive. The scope of the inventions are, therefore, indicated by the appended claims, rather than by the foregoing description. All changes which come within the meaning and range of equivalence of the claims are to be embraced within their scope.

TABLE-US-00001 TABLE 1 # Sequence Database ID Description 1 BC018950 BC018950.2 TNF receptor-associated protein 1 (TRAP1) 2 NM_020651 NM_020651.2 Pellino homolog 1 (Drosophila) (PELI1) (IOH26519) 3 NM_031431 NM_031431.2 Conserved oligomeric Golgi complex subunit 3 (COG3) 4 NM_006841 NM_006841.3 Homo sapiens solute carrier family 38, member 3 (SLC38A3) 5 NM_174942 NM_174942.1 Homo sapiens growth arrest-specific 2 like 3 (GAS2L3)

TABLE-US-00002 TABLE 2 Accession Length Number Seq-ID-No. (amino acids) BC018950.2 1 704 NM_020651.2 2 418 NM_031431.2 3 828 NM_006841.3 4 504 NM_174942.1 5 694