Abstract:
The invention provides an analytical method suitable for detecting the presence of Takotsubo-cardiomyopathy (TTC), especially for differentiating TTC from myocardial infarction, especially from STEMI, by analysing a sample obtained from a patient, preferably a blood sample for the concentration microRNAs miR-16 and miR-26a.

Description:
[0001]    The present invention relates to an analytical method for the analysis of Takotsubo-cardiomyopathy (TTC) in a patient sample, especially for differentiating the TTC from acute myocardial infarction. Accordingly, the method can determine in a sample from a patient analytes specific for cardiomyopathy as well as analytes specific for TTC. Preferably, the method in a sample determines analytes specific for TTC and differentiates TTC from myocardial infarction. Further, the invention relates to the use of compounds as probes in the analytical method, especially for analysing a sample for TTC, and to the use of a kit of parts for the analytical method. 
         [0002]    The present invention is based on the finding that specific analytes indicate the occurrence of TTC in a patient, especially differentiating the occurrence of TTC from the occurrence of acute myocardial infarction. 
       STATE OF THE ART 
       [0003]    TTC as referred to in the present invention, has first been described by Sato et al. (Clinical aspect of myocardial injury: from ischemia to heart failure, Heart Fail. 1990: 56-64) as a transient, reversible, regional systolic dysfunction of the left ventricle. In TTC patients, there often is hypokinesis or akinesis of the apical region despite any obstructive epicardial coronary artery disease, while the basal segments of the left ventricle remain in a hypercontractile state. It is known, e.g. from Dote et al., (Myocardial stunning due to simultaneous multivessel coronary spasms: a review of 5 cases, J. Cardiol. 1991; 21: 203-214) that the clinical symptoms and findings of TTC can mimic those of acute myocardial infarction, e.g. in respect of acute onset of angina, as T-segment elevation or depression noted on the ECG, and arise in cardiac enzyme levels above normal limit. 
       OBJECT OF THE INVENTION 
       [0004]    It is an object of the invention to provide an analytical method for identifying the presence of TTC in patients, especially for differentiating TTC from acute myocardial infarction in patients. 
       GENERAL DESCRIPTION OF THE INVENTION 
       [0005]    The invention achieves the object by providing an analytical method suitable for detecting the presence of TTC, especially for differentiating TTC from myocardial infarction, especially from STEMI, by analysing a sample obtained from a patient, preferably a blood sample, especially a serum sample for the concentration (level) of least one of the following microRNAs (miR): miR-16 (UAGCAGCACGUAAAUAUUGGCG, SEQ ID NO: 1), miR-26a (UUCAAGUAAUCCAGGAUAGGCU, SEQ ID NO: 2), preferably of miR1 (UGGAAUGUAAAGAAGUAUGUAU, SEQ ID NO: 3) and/or of miR133a (UUUGGUCCCCUUCAACCAGCUG, SEQ ID NO: 4), optionally in addition of miR-let7f (UGAGGUAGUAGAUUGUAUAGUU, SEQ ID NO: 5), of miR-410 (AAUAUAACACAGAUGGCCUGU, SEQ ID NO: 6), and/or of miR-640 (AUGAUCCAGGAACCUGCCUCU, SEQ ID NO: 7). The concentration of the at least one microRNA is determined, e.g. by analysing the microRNA and determining its concentration in relation to the concentration of a reference or standard nucleic acid, e.g. a different microRNA, or in relation to the concentration of the respective microRNA (miR) in a sample of a person not suffering from myocardial infarction, e.g. from a healthy person, and/or by adding to the sample a known concentration of a microRNA serving as an internal standard, e.g. a human or a non-human microRNA, or the same microRNA. Examples for a different microRNA suitable as a standard nucleic acid are  C. elegans  cel-miR39 (UCACCGGGUGUAAAUCAGCUUG, SEQ ID NO: 8). The concentration of the microRNA can e.g. be analysed using quantitative RT-PCR or using hybridization to a specific nucleic acid probe molecule with quantitative detection. In the nucleic acid sequence protocol, the sequences of mature microRNAs are given. 
         [0006]    It has been found that an elevated level of each of miR-16 and of miR-26a indicates presence of TTC, also in presence of elevated levels of miR1 and of miR133a, but absence of acute myocardial infarction, especially of acute ST elevation myocardial infarction (STEMI). Elevated levels of miR1 and of miR133a are found in both samples from STEMI patients and in samples from TTC patients, with generally higher elevation levels in STEMI patients compared to TTC patients. 
         [0007]    The elevation of levels of miR16 and of miR26a in samples from TTC patients has been found to be significant over both samples from healthy persons and samples from STEMI patients, with levels of miR16 and of miR26a generally being similar in healthy persons and STEMI patients. 
         [0008]    Preferably, the method includes analysis of the concentration of miR-133a and/or of miR-1 in the sample, because a low concentration of miR-133a and/or of miR-1 indicates TTC, whereas a higher concentration of miR-113a and/or of miR-1 indicates presence of STEMI in patients, or absence of TTC cardiomyophathy. 
         [0009]    As a further indicator, the miR let-7f can be analyzed in the process, with a level of miR let-7f that is elevated in comparison to the level in samples from myocardial infarction patients indicating TTC rather than STEMI. 
         [0010]    Further, the invention relates to the use of nucleic acid molecules as probes in the process, for detecting the concentration of at least one of miR-16, miR-26a, preferably for detecting the concentration of miR-16 and of miR-26a in combination with miR-410 and miR-640 and/or optionally miR let-7f, in a sample, especially for detecting an elevated concentration of the least one of these, preferably in combination with the use of nucleic acid molecules as probes in the process, which are specific for at least one of miR-133a and miR-1, especially for detecting a non-elevated concentration thereof, e.g. a concentration corresponding to the concentration found in healthy persons. 
         [0011]    Such nucleic acid molecules can be selected from primer pairs suitable for amplifying the respective microRNA, or from nucleic acid molecules hybridising specifically to the microRNA, e.g. nucleic acid molecules having the reverse complementary sequence to the microRNA to be analyzed. 
         [0012]    It is an advantage of the present invention that the analytical method provides for data that can be used for diagnosing the presence of TTC, while eliminating acute myocardial infarction from the diagnosis without the need for invasive analysis, like e.g. catheterisation. 
         [0013]    A kit of parts adapted to the analytical method of the invention contains nucleic acid molecules suitable as probes for detecting the at least one microRNA, e.g. specific primer pairs and/or reverse complementary nucleic acid molecules. Optionally, the kit of parts further contains a nucleic acid molecule for use as an internal reference, e.g. a non-human microRNA, and nucleic acid molecules fur use as probes for detecting the internal reference molecule, e.g. specific primer pairs and/or reverse complementary nucleic acid molecules. Accordingly, the analytical method preferably includes the step of adding to a sample a known amount of standard as a dopant, wherein the dopant forms an internal standard and wherein the standard preferably has the form of an RNA, preferably a microRNA, which RNA or microRNA, respectively can comprise or consist of a non-human RNA, e.g. cel-miR-39, and/or which RNA or microRNA, respectively can comprise or consist of the sequence of the analyte, i.e. miR-16, miR-26a, miR-1, miR-133a, miR let-7f, miR-410 and/or miR-640 or combinations of two or more of these. In these embodiments, an aliquot of the sample may be doped with a standard in the form of a RNA and another aliquot may be subjected to the analytical method without addition of a standard. Generally, it is preferred in these embodiments that the detected level of the microRNA is reduced by the amount detected on the basis of the standard added to the sample. Therein, the amount detected on the basis of the standard is the level that is detected for the added standard in addition to the original analyte content of the probe. 
         [0014]    Optionally, the analytical method comprises or consists of detecting the level of the analytes miR-16, miR-26a, miR-1 and miR-133a, and optionally further including miR let-7f, miR-410 and/or miR-640 in a sample, and comparing the levels detected for each microRNA with the level of the respective microRNA determined in a sample from a STEMI patient and/or with the level of the respective microRNA determined in a sample from a healthy person. The level of the respective microRNA determined in a sample from a STEMI patient and/or the level of the respective microRNA determined in a sample from a healthy person can be pre-determined. These embodiments can be performed with or without adding a known amount of standard RNA or microRNA as a dopant, and when adding the dopant, optionally reducing the level of detected analyte by the amount detected on the basis of the added standard (dopant). 
         [0015]    It has been found that in the majority of infarction patients diagnosed with TTC, the level of miR-16 and the level of miR-26a significantly decreases from a first to a second sampling point in time. Therefore, the analytical method preferably comprises detecting the level of the analyte, preferably of miR-16 and/or of miR-26a in a sample obtained at a first sampling point in time and in a sample obtained at a second sampling point in time, wherein the second sampling point in time is at least 10 h to 24 h later than the first sampling point in time. A sampling point in time is the time at which the sample is taken from the patient. 
         [0016]    Optionally, the analytical method can indicate TTC, preferably in combination with indicating absence of STEMI, in a sample obtained from an infarction patient by a significant increase in the level of miR-16, e.g. by a factor of at least 3 in comparison to the level of the same miR in a sample from a healthy person and/or in comparison to the level of the same miR in a in a sample from a STEMI patient, and/or 
         [0017]    by a significant increase in the level of miR-26a, e.g. by factor of at least 5 or 6 in comparison to the level of the same miR in a in a sample from a healthy person and/or in comparison to the level of the same miR in a in a sample from a STEMI patient. 
         [0018]    Optionally additionally, the analytical method indicate TTC, preferably in combination with indicating absence of STEMI, in a sample obtained from an infarction patient by an increase of the concentration of miR-1, e.g. by factor of at maximum 2 or 2.5 in comparison to the level of the same miR in a in a sample from a healthy person and/or by a factor of approx. 2 to 3 in comparison to the level of the same miR in a in a sample from a STEMI patient, of miR-133a, e.g. by factor of at maximum 2 to 4 in comparison to the level of the same miR in a in a sample from a healthy person, 
         [0019]    of miR-410, e.g. by factor of at approx. 3 in comparison to the level of the same miR in a in a sample from a healthy person, 
         [0020]    of miR-640 by factor of approx. 1.5 in comparison to the level of the same miR in a in a sample from a healthy person, and/or 
         [0021]    of miR let-7f by factor of at least 1.5 or at least 2 or at least 3 in comparison to the level of the same miR in a in a sample from a healthy person and/or in comparison to the level of the same miR in a in a sample from a STEMI patient. Optionally, the level of each miR is determined in relation to an internal standard which e.g. is an added known amount of an RNA, preferably a microRNA, of a non-human or a human nucleic acid sequence as described herein. 
         [0022]    For miR-133a, an increase of concentration by a factor of at least 30 in comparison to the level of the same miR in a in a sample from a healthy person from has been determined in samples from STEMI patients. Accordingly, an increase of the concentration of miR-133a by a factor of at least 20 to 30 in comparison to the concentration in a sample from a healthy person is indicative for STEMI, whereas a concentration of miR-133a increased by a factor of at maximum 4 in comparison to the concentration in a sample from a healthy person is indicative of TTC. 
         [0023]    For miR-1, an more important increase of concentration, e.g. by a factor of approx. 10 in comparison to the level of the same miR in a in a sample from a healthy person from has been determined in samples from STEMI patients. Accordingly, an increase of the concentration of miR-1 by a factor of at least 9, preferably of at least 10, in comparison to the concentration in a sample from a healthy person is indicative for STEMI, whereas a less important increase in the concentration of miR-1, e.g. increased by a factor of at maximum 2.5, preferably of at maximum 3, in comparison to the concentration in a sample from a healthy person is indicative of TTC. 
     
    
     
       DETAILED DESCRIPTION OF THE INVENTION 
         [0024]    The invention is now described in greater detail by way of examples with reference to the figures which show 
           [0025]    in  FIG. 1  the relative concentration of miR-16 in samples from healthy persons, from STEMI patients and from TTC-patients, 
           [0026]    in  FIG. 2  the relative concentration of miR-26a in healthy persons, in STEMI-patients and in TTC-patients, 
           [0027]    in  FIG. 3  A) relative concentrations of miR-16 in patients suffering from depression and being free from depression, resp., and in B) relative concentrations of miR-26a in patients suffering from depression and being free from depression, in C) the concentration of miR-16 in the presence or absence of SSRI, and in D) the concentration of miR-26a in patients with and without administration of antidepressant drugs, 
           [0028]    in  FIG. 4  the concentration of miR-410 in healthy persons and in TTC patients, 
           [0029]    in  FIG. 5  the relative concentration of miR-640 in healthy persons and in TTC patients, 
           [0030]    in  FIG. 6  the concentration of miR-133a in healthy persons, in STEMI-patients and in TTC-patients, 
           [0031]    in  FIG. 7  the concentration of miR-1 in healthy persons, in STEMI-patients, and in TTC-patients, 
           [0032]    in  FIG. 8  the concentration of miR let-7f in healthy persons, in STEMI-patients, and in TTC-patients, 
           [0033]    in  FIG. 9  A to E the sensitivity for the specificity of the microRNA indicated for detecting TTC, once in respect of STEMI and once in respect of healthy persons, 
           [0034]      FIG. 10  the concentrations of miR-16 in TTC patient samples at a first point in time  1  and at a later second point in time  2  for different patients, and 
           [0035]      FIG. 11  the concentrations of miR-26a in TTC patient samples at a first point in time  1  and at a later second point in time  2  for different patients. 
       
    
    
       [0036]    In the Figures, * denotes p&lt;0.05, ** denotes p&lt;0.01, and *** denotes p&lt;0.001, n.s. denotes no significance between values. 
       EXAMPLE 
     Analysis of Blood Plasma Samples from Infarction Patients 
       [0037]    Blood plasma samples were collected from 33 TTC-patients, from 28 patients with acute ST elevation myocardial infarction (STEMI), and from 28 healthy persons serving as a control. Blood samples were collected within 24 hours from the onset of chest pain. 
         [0038]    RNA was isolated from 100 μl plasma with the MiRNeasy Isolation Kit (available from Qiagen, Hilden, Germany), according to the manufacturer&#39;s instructions, after adding 5 μl of 1 fmol/μl cel-miR-39 (miR-39 of  Caenorhabditis elegans ) (SEQ ID NO: 8) as an internal standard before starting the isolation procedure. 
         [0039]    MicroRNAs were analysed using reverse transcription-polymerase chain reaction (RT-PCR). Preferably, quantitative RT-PCR was made using the TaqMan microRNA Reverse Transcription Kit (available from Applied Biosystems) using labelled primers, e.g. one primer of a pair was labelled with a fluorochrome, and the other primer of the pair was labelled with a quencher specific for the emission of the fluorochrome. Results were normalized to the amount of the added cel-miR-39, serving as an internal standard. 
         [0040]    Results are given as mean values+/−SEM. Differences between groups were analysed by the student t-test, Excel, or by one-way ANOVA, followed by Bonferroni&#39;s Multiple Comparison Test, applied as a post-hoc test. 
         [0041]      FIG. 1  shows the relative concentration of miR-16 in healthy persons, in STEMI-patients, and in TTC-patients, respectively. It can be seen that the concentration of miR-16 is significantly elevated specifically in TTC-patients, approximately a factor of at least 3 over the concentration in healthy persons and/or in STEMI-patients. 
         [0042]      FIG. 2  shows that the relative concentration of miR-26a is significantly elevated in TTC-patients, e.g. by a factor of about 6, over the concentration of miR-26a in healthy persons or in STEMI-patients. 
         [0043]    These data show that an elevated concentration of each of miR-16 and miR-26a specifically indicates TTC by excluding STEMI or the absence of an infarction (healthy persons). As miR-16 and miR-26a are also known to be correlated with depression, the correlation with depression, exemplified by SSRI, and administration of antidepressant were analyzed. 
         [0044]      FIG. 3A ) shows that the concentration of the miR-16 does not significantly differ in samples obtained from TTC-patients who are free from a depression (no), and in patients suffering from a depression (yes). Correspondingly,  FIG. 3B ) shows the concentration of miR-26a in patients being free from a depression (no), and in patients suffering from a depression (yes). The data show that concentrations of miR-26a do not significantly differ in TTC-patients suffering from or being free from a depression. 
         [0045]    Accordingly,  FIGS. 3A ) and  3 B) show that an elevated concentration of each of miR-16 and miR-26a independent from presence or absence of a depression in the patient specifically indicates TTC, e.g. excluding presence of STEMI. 
         [0046]      FIG. 3C ) shows the relative concentration of miR-16 in the absence of (SSRI) (no) and in the presence of SSRI (yes) in TTC-patients. These data show that the elevated relative concentration of miR-16 independently from SSRI indicates TTC, e.g. eliminating the presence of STEMI. 
         [0047]      FIG. 3D ) shows the relative concentration of miR-26a in the absence of antidepressant drugs from TTC-patients (no), and with application of antidepressant (yes) in TTC-patients. These data show that there is no significant influence of the presence of antidepressant on the specificity of the elevated concentration of miR-26a in TTC-patients. 
         [0048]      FIG. 4  shows the relative concentration of miR-410 in TTC-patients and in healthy persons. The concentration of miR-410 is increased by a factor of approximately 3 in TTC-patients. 
         [0049]      FIG. 5  shows the elevated concentration of miR-640 in TTC-patients by a factor of approximately 1.7 in comparison to healthy persons. 
         [0050]    The data shown in  FIGS. 4 and 5  show that an elevated concentration of miR-410 and/or of miR-640 also indicates presence of TTC. 
         [0051]      FIG. 6  shows the relative concentration of miR-133a and makes it clear that the concentration of miR-133a is not significantly elevated in TTC-patients over the concentration in healthy persons, whereas the concentration of miR-133a is significantly increased, e.g. by a factor of more than 100, in STEMI-patients compared to healthy persons and also compared to TTC-patients. This shows that elevated serum levels of miR-133a rather indicate STEMI but are not significantly indicative of TTC. 
         [0052]      FIG. 7  shows that the elevated concentration of miR-1 is indicative of the presence of STEMI, e.g. by a factor of approximately 10 over the concentration found in healthy persons. The elevated concentration of miR-1 in TTC-patients is not significant. 
         [0053]    The data shown in  FIGS. 6 and 7  show that the concentrations of miR-113a and of miR-1 are not significantly increased in TTC-patients compared to healthy persons, but that a significant increase in the concentration of miR-133a or of miR-1 over the concentration found in healthy persons indicates the presence of STEMI and the absence of TTC. 
         [0054]      FIG. 8  shows that a concentration of the miR let-7f is significantly increased in TTC-patients, whereas its concentration can be decreased in STEMI-patients, showing that an increase of the concentration of miR let-7f is indicative of TTC rather than of STEMI. 
         [0055]      FIG. 9  shows the correlation of specificity and sensitivity of the concentration A) of miR-16, B) miR-26a, C) miR-1, D) miR-133a, and at E) for combined miR-16, miR-26a, miR-1 and miR-133a, each in respect of TTC. This mathematical analysis was done using ROC analysis and shows the high specificity and sensitivity of the analytical method when analyzing the concentration of each of these analytes singly, and especially of the analytical method when analyzing the combination of the analytes miR-16, miR-26a, miR-1 and miR-133a. 
         [0056]    In addition to the analytical data depicted in  FIGS. 1 to 9 ,  FIG. 10  shows concentrations determined by the analytical method at a first point in time ( 1 ) and at a second point in time ( 2 ), which was 10 h to 24 h later in the same TTC patient. Samples from the same patient are indicated for each graph as TT-55, TT-49, TT-52, TT-53, TT-46, TT-56, TT-38, and TT-50, respectively. Except for the samples from patient TT-50, the analytical method determines a significant decrease in concentration of the analyte miR-16 from the first point in time ( 1 ) to the later second timpoint ( 2 ). At present, it cannot be ruled out that the data for patient TT-50 have been mixed up. 
         [0057]    Generally, the data of  FIG. 10  show that an analytical method analyzing a sample taken at a later second point in time from the same patient in addition to analyzing the sample taken at the first point in time confirm the presence of TTC when the concentration of miR-16 remains or preferably is reduced. 
         [0058]      FIG. 11  shows the concentrations of miR-26a for the first point in time ( 1 ) and the second point in time ( 2 ) for the samples as analyzed for  FIG. 10 . Again with the exception of the samples of patient TT-50, the results show that an analytical method analyzing a sample taken at a later, second point in time from the same patient in addition to analyzing the sample taken at the first point in time confirm the presence of TTC when the concentration of miR-26a remains or preferably is reduced.