Markers for mood disorders

Abstract

Disclosed is a method for diagnosing a mood disorder or susceptibility to a mood disorder, including depressive disorders and bipolar disorder, from a biological sample taken from a subject. The method includes detecting markers of monoamine oxidase-A (MAO-A) in the biological sample; determining MAO-A concentration from the markers; and correlating the MAO-A concentration in the biological sample to a control group which does not have a mood disorder in order to diagnose or determine susceptibility to the mood disorder in the subject. Also disclosed is a method of detecting peripheral markers of MAO-A for the diagnosis of a mood disorder or susceptibility to a mood disorder. Also provided are polypeptide markers.

Claims

1. A method for diagnosing major depressive disorder or susceptibility to major depressive disorder from a blood sample taken from a subject, said method comprising the steps of: a) subjecting the blood sample to protease-induced hydrolysis of monoamine oxidase-A (MAO-A) using trypsin, chymotrypsin, or a combination thereof, to generate polypeptides derived from MAO-A; b) detecting the polypeptides that are one or more markers of MAO-A in said blood sample; c) determining MAO-A concentration from said one or more markers; and d) comparing said MAO-A concentration in said blood sample to a control group which does not have major depressive disorder in order to diagnose major depressive disorder or susceptibility to major depressive disorder in the subject; wherein the one or more markers of MAO-A are one or more polypeptides comprising: AAREVLNGLGK (SEQ ID NO:5); wherein the one or more markers do not comprise full length MAO-A; and wherein: i) MAO-A concentration in the blood sample that is higher than the MAO-A concentration in the control group indicates a presence of major depressive disorder or susceptibility to major depressive disorder in the subject; ii) MAO-A concentration in the blood sample that is higher than the average MAO-A concentration in the control group indicates a presence of major depressive disorder or susceptibility to major depressive disorder in the subject; or iii) MAO-A concentration in the blood sample that is higher than a MAO-A concentration value previously determined for the control group indicates a presence of major depressive disorder or susceptibility to major depressive disorder in the subject.

2. The method of claim 1, wherein the protease is trypsin.

3. The method according to claim 1, wherein the subject is symptomatic for major depressive disorder and has previously been symptomatic for major depressive disorder.

4. The method according to claim 1, wherein the subject is asymptomatic for major depressive disorder and has not previously been symptomatic for major depressive disorder.

5. The method according to claim 1, wherein the subject is asymptomatic for major depressive disorder and has previously been symptomatic for major depressive disorder.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) These and other features of the invention will become more apparent from the following description in which reference is made to the following drawings:

(2) FIG. 1 is a schematic representation of a MAO-A protein sequence (SEQ ID NO: 8) with targeted transitions as marked;

(3) FIG. 2 is an extracted ion chromatogram for SRM-MS transitions for peptide AAREVLNGLGK (SEQ ID NO:5).

(4) FIG. 3 is an extracted ion chromatogram for SRM-MS transition peaks for 5 transitions for peptide NEHVDYVDVGGAYVGPTQNR (SEQ ID NO:1).

(5) FIG. 4 is an extracted ion chromatogram for SRM-MS transition peaks for 5 transitions from peptide AAREVLNGLGK (SEQ ID NO:5) derived from plasma specimens.

(6) FIG. 5 shows mean peak area (counts) for analyte AAREVLNGLGK (SEQ ID NO:5) (Transition 564.3.fwdarw.811.3) from the plasma of depressed patients versus healthy controls, measured using SRM-MS methods. Dots represent scores for individual subjects.

(7) FIG. 6 shows peak areas for two transitions for each of the MAO-A peptides ILRLSK (SEQ ID NO:2) and FVGGSGQVSER (SEQ ID NO:3), depressed vs controls.

(8) FIG. 7 shows the standard curve for AAREVLNGLGK (SEQ ID NO:5) as measured using SRM-MS and a labeled internal standard.

DESCRIPTION OF THE INVENTION

(9) The following description is of a preferred embodiment by way of example only and without limitation to the combination of features necessary for carrying the invention into effect.

(10) The present invention relates generally to diagnosis of psychiatric disorders and markers therefor. More specifically, the invention relates to diagnostic and prognostic markers for mood disorders. The present invention also relates to identifying subjects that have increased concentration of one or more MAO-A markers in serum compared to a control group, for example a control group that does not have depressive disorder.

(11) Mood disorders as used herein include, but are not limited to, depressive disorders, such as major depressive disorder (MDD), dysthymia and depressive disorder not otherwise specified, and bipolar disorder (or manic-depression). MDD can be further subcategorized as being atypical depression, melancholic depression, psychotic major depression, catatonic depression, postpartum depression and seasonal affective disorder. Depressive disorders not otherwise specified include recurrent brief depression and minor depressive disorder. Bipolar disorder can also be subcategorized into bipolar I, bipolar II, cyclothymia and bipolar disorder not otherwise specified.

(12) The present invention provides a method for diagnosing a mood disorder or susceptibility to a mood disorder from a biological sample taken from a subject. This method includes detecting one or more markers of monoamine oxidase-A (MAO-A) in the biological sample. The present invention also provides that the MAO-A concentration in the sample is determined from the one or more markers. Finally, the MAO-A concentration in the biological sample is correlated to a control group which does not have a mood disorder or susceptibility to a mood disorder in order to diagnose the mood disorder or susceptibility to the mood disorder in the subject.

(13) Markers of MAO-A can include, but are not limited to, polypeptides corresponding to all or portions of the MAO-A protein. However, certain peptides are preferred as described herein. The markers or polypeptides identified herein may be employed in methods as described. Further, the markers or polypeptide may be useful in additional ways, for example in the generation of antibodies for immunological testing and assays and/or as controls in mass spectroscopy, immunological and other research methods and protocols.

(14) In a preferred embodiment, these markers are identified/detected from a biological sample using mass spectrometry or immunological methods as are known in the art. In particular, Liquid Chromatography/Selected Reaction Monitoring-Mass Spectrometry (LC/SRM-MS) is used to quantify specific MAO-A peptide fragments (transitions) that can be used as markers to diagnose the mood disorder or susceptibility to a mood disorder. Transition peptides of the present invention include peptides shown in FIG. 1, namely NEHVDYVDVGGAYVGPTQNR (SEQ ID NO:1); ILRLSK (SEQ ID NO:2); FSVTNGGQER (SEQ ID NO:3); YVINAIPPTLTAK (SEQ ID NO:4); AAREVLNGLGK (SEQ ID NO:5); DVPAVEITHTFWER (SEQ ID NO:6) and FVGGSGQVSER (SEQ ID NO:7). Preferably, the transition peptides are selected from NEHVDYVDVGGAYVGPTQNR (SEQ ID NO:1); YVINAIPPTLTAK (SEQ ID NO:4); AAREVLNGLGK (SEQ ID NO:5); and FVGGSGQVSER (SEQ ID NO:7). Most preferred is AAREVLNGLGK (SEQ ID NO:5).

(15) In a further embodiment, antibodies directed to these transition peptides can be developed to extract, detect and/or quantify differences in MAO-A levels in subjects being tested or suspected of having a mood disorder compared to a control group. At the protein level various techniques exist to identify changes in protein levels. These include, but are not limited to, immunoblotting, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).

(16) Comparisons of mass-spectometry spectra from suspected mood disorder subjects and controls, for example comparisons of peptide fragment sequence information can be carried out using spectra processed in MATLB with script called Qcealign (see for example WO2007/022248, herein incorporated by reference) and Qpeaks (Spectrum Square Associates, Ithaca, N.Y.), or CIPHERGEN PEAKS 2.1 software. The processed spectra can then be aligned using alignment algorithms that align sample data to the control data using minimum entropy algorithm by taking baseline corrected data (see for example WIPO Publication WO2007/022248, herein incorporated by reference). The comparison result can be further processed by calculating ratios. Protein expression profiles can be discerned.

(17) The present invention also provides a method for detecting peripheral markers of MAO-A for the diagnosis of a mood disorder or for determining susceptibility to mood disorder. The method includes obtaining a biological sample from a subject symptomatic with the mood disorder or asymptomatic for the mood disorder and has been symptomatic for the mood disorder. The present invention also provides subjecting the biological sample to SRM-MS or one or more immunological methods. With respect to mass spectroscopy, the transitions in MAO-A are identified from the MS. Finally, the transitions are correlated to a control group that does not have a mood disorder in order to identify a peripheral marker of MAO-A.

(18) A raw SRM-MS dataset may be obtained from a biological sample from a subject symptomatic with a mood disorder or asymptomatic for the mood disorder, but who has previously been symptomatic for the mood disorder. The dataset is processed to identify transition peaks and determine peak area. An example of suitable software to process the dataset is MultiQuant software. A t smoothing window can be set prior to peak area integration. Significance analysis can be conducted using statistical analysis software, such as R and SPSS.

(19) For each transition (feature) measured in a given biological sample in replicate SRM-MS experiments, numerical measures such as mean peak area and peak area coefficient of variation (CV) are calculated. Using each feature, within-group mean peak area and CV can be calculated for the mood disorder and control groups from the individual sample mean peak area values.

(20) Fold-change can be derived for each feature from the ratio of mean peak area in the mood disorder samples to mean peak area in control samples. Positive fold-changes represent increased expression in the mood disorder group.

(21) Without wishing to be limiting or bound by theory in any manner, those peptides for which at least two transition features displayed a fold-change of 1.20 or greater and a within-group CV of 0.20 or less would be suitable candidates for use as markers for the diagnosis of the mood disorder.

(22) The results provided herein show that one or more MAO-A markers are elevated in the plasma of patients with mood disorder. The results also suggest that the levels of peripheral polypeptide sequences described herein correlate with depression and provide an index of MAO-A levels in the brain. Thus the present invention also contemplates determining if a subject exhibits one or more MAO-A markers and if the level of the one or more MAO-A markers is elevated compared to a control group, for example, a control group that does not have mood disorder, in order to determine which subjects should be subjected to continued screening and/or monitoring, counseling, additional psychological testing, one or more genetic or other tests that predict, determine or diagnose mood disorder(s) or susceptibility thereto, and/or family screening. The present application also contemplates treating a patient with elevated MAO-A levels using therapies known in the art in order to improve mood, and/or prevent or reduce susceptibility to a mood disorder or the symptoms associated therewith.

(23) The present invention also contemplates one or more antibodies that are capable of binding to any one of the amino acid or polypeptide sequences described herein. In a preferred embodiment, the antibody is a monoclonal antibody. Also contemplated are nucleotide sequences comprising the one or more antibodies describe herein.

(24) Antibodies, including monoclonal antibodies can be prepared using a wide variety of techniques known in the art including, for example, hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art, for example, but not wishing to be considered limiting in any manner, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981). The term monoclonal antibody refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and is not meant to be limited by the method by which it is produced.

(25) Methods for producing and screening for specific antibodies using hybridoma technology are routine and also well known in the art. As an example, but not to be considered limiting in any manner, an animal capable of eliciting an immune response to an antigen (for example, mice) can be immunized with an antigen, for example a polypeptide as described herein, a fragment or variant thereof, a fusion protein, or a cell expressing an antigen, polypeptide or fragment or variant thereof. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable cells, for example, mylenoma cells or the like. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

(26) Accordingly, the present invention provides methods of generating polyclonal and monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse or other animal immunized with a polypeptide of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.

(27) Other well known methods for producing antibodies also may be employed. Such methods include but are not limited to Epstein Barr Virus (EBV) transformation protocols, for example, in Current Protocols in Immunology, Coligan et al., Eds., 1994, John Wiley & Sons, NY, which is hereby incorporated in its entirety by reference.

(28) The present invention also contemplates the production of antibody fragments which recognize the polypeptides as described herein, fragments thereof or specific epitopes therein. Such antibody fragments may be generated by known techniques. For example, Fab and F(ab)2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab)2 fragments). F(ab)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.

(29) Antibodies that bind to an antigen can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make antibodies that bind to an antigen include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference.

(30) As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab and F(ab)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988), herein incorporated by reference.

(31) Examples of additional techniques which may be contemplated herein include those which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988). Methods for producing chimeric antibodies are also known in the art and may be employed if desired. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety.

(32) The present invention also contemplates a kit comprising one or more components, such as, but not limited to one or more primary antibodies that are capable of binding to any amino acid or polypeptide sequence described herein, one or more secondary antibodies that are capable of binding the primary antibody, one or more solutions or reagents for immunological analysis, for example, blocking or binding solution or the like, one or more polypeptides as described herein, said polypeptide optionally conjugated to a non-protein carrier, polypeptide carrier, heterologous amino acid sequence, support, dish, multi-well plate or the like, purification media for example, but not limited to remove abundant plasma proteins from samples that are collected, centrifugation media, immunoabsorption columns, resin, buffers, enzymes, one or more supports, dishes, multiwell plates, instructions for using any component or practicing any method as described herein, or any combination thereof.

EXAMPLES

Example 1: Sample Preparation Using Trypsin as a Protease

(33) Frozen EDTA-plasma samples were thawed and immunodepleted using a MARS-14 (Agilent Technologies, USA) depletion column. The depleted plasma fraction was quantified for total protein by Bio-Rad DC Protein Assay. Twenty micrograms of total protein was reduced and then alkylated. The sample pH was adjusted to pH 8 with 1M NH.sub.4HCO.sub.3 and 1 microgram of trypsin enzyme was added to the sample and incubated for 16 hours at 37 C. The digested peptides were acidified with formic acid to a final concentration of 1% formic acid. A portion of each sample digest was pooled into one tube to be used for SRM method optimization. The remaining aliquots of the acidified digests were frozen at 80 C. until needed for SRM.

Example 2: Liquid Chromatography/Selected Reaction Monitoring-Mass Spectrometry (LC/SRM-MS)

(34) An Eksigent nano-LC was used for chromatographic separation. The following gradient was used: 5% B-30% B in 60 minutes where solvent A is 98% water:2% acetonitrile and solvent B is 2% water:98% acetonitrile both with 0.1% formic acid. One microgram of tryptic peptides was loaded onto the column. The nano-LC was coupled to a 5500 Q TRAP hybrid triple quadrupole/linear ion trap mass spectrometer (AB SCIEX, USA) through a nanoflow electrospray ionization source equipped with a 15 m ID emitter tip. Tryptic peptides and SRM transitions were generated by MRMPilot 2.0 software (AB SCIEX, USA) based on common chemical rules of peptide fragmentation. The specificity of each peptide was verified using BLAST alignment against the NCBI-NR human protein database. The SRM method contained retention times to increase the number of transitions that can be monitored in one LC separation. Each patient sample was analyzed in triplicate using this SRM method.

Example 3: Preprocessing and Analysis of SRM-MS Dataset

(35) The raw SRM-MS dataset was processed with MultiQuant software version 1.1 to identify transition peaks and determine peak area. A 3 point Gaussian smoothing procedure was applied prior to peak area integration. Significance analysis was conducted using R software version 2.10.0. For each SRM transition (feature) measured in a given sample, within patient peak area mean and coefficient of variation (CV) over replicate experiments were calculated. For each feature, within-group mean peak area and CV were calculated for the MDD and control groups using within-patient mean peak area values from each group. Fold-change was derived for each feature from the ratio of mean peak area in MDD patient samples to mean peak area in control samples; positive fold-changes indicated increased expression in the MDD group. Peptides for which at least two transition features displayed a fold-change of 1.20 or greater and a within-group CV of 0.20 less (in both MDD and control groups) were retained.

(36) These investigations identified transitions that produced a reliable and reproducible signal in human plasma. The targeted peptides are shown in FIG. 1. These peptides include:

(37) TABLE-US-00001 (SEQIDNO:1) NEHVDYVDVGGAYVGPTQNR; (SEQIDNO:2) ILRLSK; (SEQIDNO:3) FSVTNGGQER; (SEQIDNO:4) YVINAIPPTLTAK; (SEQIDNO:5) AAREVLNGLGK; and (SEQIDNO:6) DVPAVEITHTFWER.

Example 4: Peripheral MAO-A Levels Correspond to Levels Seen in Brain

(38) Three MDD and two control plasma samples were analysed. These samples had previously undergone PET imaging and were shown to have relative changes similar to those seen in earlier studies. These specimens were then analysed in triplicate SRM-MS assays for multiple MAO-A transitions with the peptides: NEHVDYVDVGGAYVGPTQNR (SEQ ID NO:1); YVINAIPPTLTAK (SEQ ID NO:4); and AAREVLNGLGK (SEQ ID NO:5).

(39) These peptides all show some overlap with MAO-B, but with sufficiently different sequences for the purposes of the present experiment (see Tables 1-3).

(40) TABLE-US-00002 TABLE1 NEHVDYVDVGGAYVGPTQNR(SEQIDNO:1) Align- Protein Gene ments Accession Entryname Status names Length Identity Score E-Value names P21397 AOFA_HUMAN Amineoxidase 527 100.0% 152 6.0x10.sup.-15 MAOA [flavin-containing] A P27338 AOFB_HUMAN Amineoxidase 520 75.0% 103 1.0x10.sup.-7 MAOB [flavin-containing] B

(41) TABLE-US-00003 TABLE2 AAREVLNGLGK(SEQIDNO:5) Align- Gene ments Accession Entryname Status Proteinnames Length Identity Score E-Value names P21397 AOFA_HUMAN Amineoxidase 527 100.0% 76 5.0x10.sup.-4 MAOA [flavin-containing] A P27338 AOFB_HUMAN Amineoxidase 520 63.0% 49 5.7 MAOB [flavin-containing] B

(42) TABLE-US-00004 TABLE3 YVINAIPPTLTAK(SEQIDNO:4) Align- Gene ments Accession Entryname Status Proteinnames Length Identity Score E-Value names P21397 AOFA_HUMAN Amineoxidase 527 100.0% 97 6.0x10.sup.-7 MAOA [flavin-containing] A P27338 AOFB_HUMAN Amineoxidase 520 90.0% 69 8.0x10.sup.-3 MAOB [flavin-containing] B

(43) A summary of the results from this experiment is given in Table 4 and sample peaks are given in the Figures. Results show generally consistent fold changes with what has been seen in the brain. Coefficients of variation were all below 20% and triplicate replications of SRM findings were also within acceptable limits of consistency.

(44) TABLE-US-00005 TABLE 4 Rep 1 Rep 2 Rep 3 Transition Fold- Fold- Fold- Fold- Peptide (charge/ion) Change CV Change Change Change NEHVDYVDVGGAYVGPTQNR 2/y7 1.28 0.16 1.34 1.43 1.09 (SEQ ID NO: 1) 2/y11 1.14 0.07 1.07 1.15 1.19 2/b7 1.05 0.02 1.04 1.07 1.05 2/b8 1.17 0.19 1.15 1.38 1.01 2/b9 1.36 0.10 1.22 1.48 1.38 AAREVLNGLGK (SEQ ID NO: 5) 2/y7 1.69 0.05 1.57 1.71 1.78 YVINAIPPTLTAK (SEQ ID NO: 4) 2/y9 1.32 0.12 1.18 1.36 1.45

Example 5

(45) In a further study, PET scans revealed that the brains of 3 clinically depressed patients showed higher levels of MAO-A than seen in the brains of healthy controls. When plasma samples from these same individuals were depleted to remove the fourteen most abundant plasma proteins, digested with enzyme and analyzed using SRM-MS. We found more than threefold higher MAO-A levels in the depressed patients than in the healthy controls using the AAREVLNGLGK (SEQ ID NO: 5) transition (564.3.fwdarw.811.3, see FIG. 6). Other peptides also showed higher levels in depressed than controls, including ILRLSK (SEQ ID NO: 2) and FVGGSGQVSER (SEQ ID NO: 7) (FIG. 7) and the identity of all three peptides were confirmed using synthetic peptides of the same sequences. Without wishing to be bound by theory or limiting in any manner, the results presented herein suggest that the polypeptides can be employed as peripheral markers for an index of brain MAO-A concentration.

Example 6

(46) In a further study, plasma MAO-A concentration (i.e. concentration of the AAREVLNGLGK (SEQ ID NO: 5) fragment) was determined in a single subject who was asymptomatic for depression at the time of testing but had recovered from a past major depressive episode. The subject's relative plasma MAO-A concentration was determined to be approximately 8-fold greater than the mean plasma MAO-A concentration determined for healthy subjects and was interpreted to be an outlier. The subject went on to a recurrence of a major depressive episode within the following year as assessed in follow-up with the structured clinical interview for DSM-IV conducted by a trained rater (psychology PhD).

(47) One or more currently preferred embodiments have been described by way of example. It will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.