Patent Grant
8163475
The invention relates to diagnostic and prognostic methods for psychotic disorders such as bipolar disorder, schizophrenia, and other disorders characterized by abnormal expression of metabolic genes.
Psychotic disorders such as bipolar disorder (BPD) are among the top ten causes of disability worldwide. BPD, in particular, is responsible for a national annual economic burden of over $40 billion (estimated in 1991). While the etiology of BPD and other psychotic disorders such as schizophrenia remain largely unknown, recent findings point to a disturbed mitochondrial energy metabolism in such subjects.
BPD causes dramatic mood swings, affects between 1 to 3% of the population in the US and is associated with high risk of suicide. In the case of BPD, recent studies have shown decreased hippocampal (HIP) and dorsolateral prefrontal cortex (PFC) levels of creatine kinase mRNA, as well as decreased levels of high-energy phosphates in the frontal and temporal lobes of BPD patients, providing support for the idea that mitochondrial energy metabolism plays an important role in the etiology of the disease. Previously, a down-regulation in nuclear mRNA coding for mitochondrial electron transport proteins in post-mortem hippocampal tissue from patients with BPD had been reported.
BPD, along with other psychotic disorders such as schizophrenia, are diagnosed based on the course of symptoms and family history, but the etiology of such disorders remains elusive. Previously, no clinical tests existed to verify diagnosis. Thus, there is a need for improved diagnostic and prognostic techniques for psychotic disorders.
The present invention features methods for diagnosing subjects with a psychotic disorder and prognostic methods for monitoring the progression or improvement of a subject having a psychotic disorder.
Accordingly, in a first aspect the invention features a method for diagnosing a psychotic disorder (e.g., bipolar disorder, schizophrenia, or any psychotic disorder described herein) or propensity thereto in a subject including the steps of (a) obtaining a cellular sample, for example, a fluid sample (e.g., a blood sample) or tissue sample, from the subject; (b) subjecting a cell from the sample to stress, for example, nutrient stress (e.g., glucose stress), oxygen stress, temperature stress, or osmotic stress; and (c) measuring expression in the cell of at least one (e.g., 2, 3, 4, 5, 7, 10, 15, 25, 50, or 100) nucleic acid(s) or polypeptide(s) listed in Table 3, FIGS. 1A(I)-1A(IV), or
In a second aspect, the invention features, a method for diagnosing a psychotic disorder (e.g., bipolar disorder, schizophrenia, or any psychotic disorder described herein) or propensity thereto in a subject, including the steps of (a) obtaining a cell sample, for example, a fluid sample (e.g., a blood sample) or tissue sample, from the subject; (b) subjecting a cell from the sample to stress, for example, nutrient stress (e.g., glucose stress), oxygen stress, temperature stress, or osmotic stress; and (c) measuring the level of expression in the cell of at least one (e.g., 2, 3, 4, 5, 7, 10, 15, 25, 50, or 100) mitochondrial energy metabolism nucleic acid(s) or polypeptide(s), where an alteration (e.g., a decrease) in the level of expression as compared to the expression in a cell from a sample obtained from a control subject is indicative of the subject having a psychotic disorder or propensity thereto. In one embodiment, the cell sample includes a lymphocyte. In another embodiment, step (b) subjecting includes culturing the cell.
The invention also features prognostic methods for monitoring a psychotic disorder (e.g., bipolar disorder, schizophrenia, or any psychotic disorder described herein) in a subject having the disorder. The method including the steps of (a) obtaining a cell sample from the subject; (b) subjecting a cell from the sample to stress, for example, nutrient stress (e.g., glucose stress), oxygen stress, temperature stress, or osmotic stress; (c) measuring the level of expression in the cell of (i) at least one (e.g., 2, 3, 4, 5, 7, 10, 15, 25, 50, or 100) mitochondrial energy metabolism nucleic acid(s) or polypeptide(s) or (ii) at least one (e.g., 2, 3, 4, 5, 7, 10, 15, 25, 50, or 100) nucleic acid(s) or polypeptide(s) from in Table 3, FIGS. 1A(I)-1(A)(IV), or
By “subject” is meant either a human or non-human mammal.
By “control subject” is meant a subject that does not have a psychotic disorder.
By “stress,” in the context of stressing cells, is meant any condition resulting in a physiological strain on the cells as compared to standard cell culture conditions, as are known in the art. In some embodiments, these conditions include a reduced concentration of an essential nutrient (e.g., decreased glucose or sucrose concentrations), either increased or decreased oxygen conditions (e.g., as described herein), either increased or decreased temperature (e.g., as described herein), or either increased or decreased osmolarity (e.g., as described herein).
By “biological sample” is meant any sample of biological origin or containing, or potentially containing, biological particles. In certain embodiments, biological samples are cellular samples.
By “blood component” is meant any component of whole blood, including host red blood cells, white blood cells (e.g., lymphocytes), and platelets. Blood components also include the components of plasma, e.g., proteins, lipids, nucleic acids, and carbohydrates.
By “cellular sample” is meant a sample containing cells or components thereof. Such samples include tissue samples (e.g., samples taken by biopsy from any organ or tissue in the body) and naturally occurring fluids (e.g., blood, lymph, cerebrospinal fluid, urine, cervical lavage, and water samples), portions of such fluids, and fluids into which cells have been introduced (e.g., culture media, and liquefied tissue samples). The term also includes a lysate. Any means for obtaining such a sample may be employed in the methods of the invention; the means by which the sample is obtaining is not critical to the invention.
By “alteration in expression” is meant a change in expression level of a nucleic acid or polypeptide. This difference may be either an increase or a decrease in expression when compared to a control or baseline (e.g., a previous measurement). In certain embodiments, the increase or decrease is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. An increase may further be at least 125%, 150%, 200%, 300%, or 500%.
By “psychotic disorder” is meant a mental disorder characterized by psychosis which may involve cognitive problems, delusions, or hallucinations. Psychotic disorders include, without limitation, bipolar disorder, schizophrenia, schizoaffective disorder, schizophreniform disorder, shared psychotic disorder, and brief psychotic disorder.
By “a bipolar disorder” is meant a mood or affective disorder characterized by pathological mood swings from mania to depression. The diagnostic criteria for a bipolar disorder (e.g., bipolar I: mania and depression; bipolar II: hypomania and depression; bipolar III: cyclothymic disorders; bipolar IV: hypomania or mania precipitated by antidepressant drugs; bipolar V: depressed patient with bipolar relatives; and bipolar VI: mania without depression) are known to the skilled artisan, and are described in the Diagnostic and Statistical Manual of Mental Disorders, DSM-IV, 1994, American Psychiatric Association.
By “schizophrenia” is meant a severe brain disorder characterized by unusual thoughts or perceptions that include hallucinations, delusions, and thought disorder. Other symptoms may include a loss or a decrease in the ability to initiate plans, speak, express emotion, or find pleasure in everyday life. Schizophrenia may include cognitive deficits such as problems with attention, memory, and the ability to plan and organize.
By “nuclear encoded mitochondrial energy metabolism nucleic acid molecule” is meant a polynucleotide, or fragment thereof, that naturally occurs in the nucleus and encodes a polypeptide that localizes to the mitochondria or that functions in mitochondrial energy metabolism.
By “nuclear encoded mitochondrial energy metabolism polypeptide” is meant a protein, or fragment thereof, that functions in mitochondrial energy metabolism and is encoded by a nucleic acid molecule that naturally occurs in the cell nucleus. In some embodiments, the polypeptide functions in oxidative phosphorylation. Specifically excluded by this definition are mitochondrial genome encoded polypeptides.
By “antipsychotic” is meant any pharmaceutical therapy capable of reducing or treating at least one symptom of a psychotic disorder. Antipsychotic include, without limitation, acetophenazine maleate, chlorpromazine hydrochloride, chlorprothixene, chlorprothixene hydrochloride, clozapine, fluphenazine decanoate, fluphenazine enathate, fluphenazine hydrochloride, haloperidol decanoate, haloperidol, haloperidol lactate, lithium carbonate, lithium citrate, loxapine hydrochloride, loxapine succinate, mesoridazine besylate, molindone hydrochloride, perphenazine, pimozide, proclorperazine maleate, proclorperazine, proclorperazine edisylate, promazine hydrochloride, risperidone, thioridazine, thioridazine hydrochloride, thiothixene, thiothixene hydrochloride, and trifluoperazine hydrochloride.
Other features and advantages of the invention will be apparent from the following Detailed Description, the drawings, and the claims.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
FIGS. 1A(I) through 1D(IV) show the differential effects on expression of electron transport chain genes in lymphocytes taken from BPD patients as compared to controls cultured under normal and low glucose (stress) conditions. FIGS. 1A(I) through 1A(IV) show probe sets of the electron transport chain with a p<0.05 in low glucose BPD over controls (FIG. 1A(I)), normal glucose BPD over controls (FIG. 1A(II)), low over normal glucose controls (FIG. 1A(III)), and low over normal glucose BPD (FIG. 1A(IV)). FIG. 1A(I) shows that seventeen transcripts were downregulated and none were upregulated. FIG. 1A(II) shows that two transcripts (NADH dehydrogenase Fe—S protein 2 and COX VIIa 2 like) were downregulated and three transcripts (NADH dehydrogenase 1 alpha 5, COX IV-1 (213758_at), and COX VIIa 2) were upregulated. FIG. 1A(III) shows eight transcripts were upregulated and none were downregulated. FIG. 1A(IV) shows that six transcripts were downregulated and none were upregulated. Red indicates up-regulation, blue indicates down-regulation, and yellow indicates that no criteria were met. FIGS. 1B(I) through 1B(IV) show comparisons of regulated electron transport transcripts to all regulated transcripts (n=9399 non-redundant probe sets) in low glucose BPD over controls (FIG. 1B(I)), normal glucose BPD over controls (FIG. 1B(II)), low over normal glucose controls (FIG. 1B(III)), and low over normal glucose BPD (FIG. 1B(IV)). Redundant probe sets were masked; transcripts had to be present in at least 50% of all samples. FIGS. 1C(I) through 1C(IV) show that, of all probe sets on the array that were expressed in at least 50% of all samples (n14245), 114 coded for proteins involved in the electron transport chain. Expression levels of each individual probe set were compared between low glucose BPD and low glucose controls (FIG. 1C(I)), normal glucose BPD and normal glucose controls (FIG. 1C(II)), low and normal glucose controls (FIG. 1C(III)) and low and normal glucose BPD ((FIG. 1C(IV)). The solid green line marks equal regulation, the dashed red line shows the actual average regulation of all transcripts. FIGS. 1D(I) through 1D(IV) show real-time Q-rt-PCR analysis for low glucose BPD (n=15) versus controls (n=14; FIG. 1D(I)), high glucose BPD (n=16) versus controls (n=15; FIG. 1D(II)), high glucose versus low glucose controls (FIG. 1D(III)), and high glucose versus low glucose BPD (FIG. 1D(IV)). Four genes were used in the Q-rt-PCR verification: OSCP subunit of ATP synthase (ANOVA: p=0.006); ATP synthase subunit c (ANOVA: p=ns); ATP synthase subunit g (ANOVA: p=0.04); and cytochrome c oxidase IV isoform 1 (ANOVA: p=0.06). For each set, the averages of all four genes (ANOVA p<0.01) are also shown. Factorial ANOVA5 and Fisher's post hoc protected t-tests; *p≦0.05; **p≦0.01.
Previous work has identified numerous changes in expression levels of genes in the brains of subjects suffering from bipolar disorder as compared to normal control subjects. While such changes in expression provide a basis for developing diagnostic and prognostic assays for psychotic disorders such as BPD or schizophrenia, one of the challenges in developing a convenient and flexible assay has been identifying whether corresponding expression changes take place in non-neuronal as well as neuronal tissues. As outlined below, we have observed differential gene expression in lymphocytes of individuals diagnosed with BPD as compared to normal controls when the lymphocytes are subjected to stress. In particular, we identified genes involved in mitochondrial function as being differentially regulated in lymphocytes from BPD patients. Based on this discovery, the present invention features diagnostic and prognostic methods that include taking a cell sample from a patient, subjecting the cell from the sample to stress, followed by determining nucleic acid or polypeptide expression in the sample, where an alteration (e.g., a decrease) in expression (e.g., in the nucleic acids or polypeptides identified herein or nucleic acids or polypeptides involved in mitochondrial function) in a cell from the subject as compared to expression in a cell from a control subject indicates that the subject either has or has an increased propensity toward developing a psychotic disorder such as BPD or schizophrenia.
Psychotic Disorders
The diagnostic methods of the invention can be used with any psychotic disorder, including bipolar disorder (BPD) and schizophrenia. Other exemplary psychotic disorders include schizoaffective disorder, schizophreniform disorder, shared psychotic disorder, and brief psychotic disorder. As different psychotic disorders (e.g., BPD and schizophrenia) often share symptoms and a given patient may be diagnosed differently by different physicians or at different institutions, the diagnostic methods of the invention can accordingly be used with any psychotic disorder.
Identification of Differentially Regulated Genes in Psychotic Disorders
Previous work has identified genes differentially regulated in hippocampal tissue taken from deceased subjects with a bipolar disorder or schizophrenia (“diseased subjects”), as compared to tissue taken from deceased subjects free of mental illness (“control subjects”) (see U.S. patent application publication 2004/0248286, hereby incorporated by reference). Briefly, RNA from the hippocampal tissue was prepared, and expression levels of transcripts from diseased subjects was compared to that of control subjects. Differential expression of forty-three genes shown in Table 1 below between subjects with bipolar disorder as compared to control subjects were observed.
Eighteen of the genes (42%) identified above encode mitochondrial proteins, including subunits of the membrane-bound respiratory enzyme complexes that carry out oxidative phosphorylation in the mitochondrial inner membrane. The changes in gene expression observed in hippocampi from patients with bipolar disorder included a decrease in expression of one gene encoding a component of mitochondrial respiratory complex I, NADH dehydrogenase; a decrease in one gene encoding a component of complex IV, cytochrome c oxidase; and a decrease in five genes encoding components of complex V, ATP synthases.
Functional descriptions of each of the genes in Table 1 are described in Table 2 below.
Homo sapiens beta 2; beta-
Using a different statistical threshold (p<0.02), an additional two hundred sixty three genes were identified that are differentially expressed in patients having a bipolar disorder. Table 3 provides an inclusive list of the three hundred six genes identified as regulated in patients having bipolar disorder (p level<0.02; fold induction>1.2), their Genebank accession numbers, fold change, and p value.
S. cerevisiae VTI1)
Expression of any polynucleotide, the corresponding polypeptide, or any combination thereof identified in Tables 1, 2, or 3, in FIGS. 1A(I)-1A(IV), or in
Stressing Cells
The diagnostic methods of the invention feature a step of stressing cells in a sample taken from a subject. Any technique for stressing cells known in the art may be used; such techniques include nutrient stress, oxygen stress, temperature stress, osmotic stress, or a combination thereof.
Nutrient stress can be achieved by subjecting cells to a lower availability of a vital nutrient such as glucose or sucrose as compared to standard cell culture conditions. For example, in the lymphocyte culture using RPMI-1640 media described herein, glucose is normally present at 2 g/l. Here, glucose depravation can accordingly be provided by culturing cells at reduced glucose concentrations (e.g., less than 2, 1.5, 1, 0.75, 0.5, 0.25, 0.1, or 0.05 g/l glucose). Nutrient stress, in any cell culture media system, can be achieved by a similar reduction of a vital nutrient.
Oxygen stress can be induced by either increasing or decreasing the oxygen available to cultured cells (e.g., pO2 is generally 10-80 mm in normal tissues). Oxygen stress can be induced by decreasing the pO2 to an amount lower than is normally observed, e.g., less than 40, 30, 20, 10, 5, 2, or 1 mm pO2 or increasing the pO2 above the normal levels, e.g., greater than 80, 90, 100, 110, 120, 130, 150, 170 mm pO2. In another example, standard culture conditions typically include a 5% CO2:20% O2:75% N2 atmosphere. By altering oxygen concentration, e.g., cultured in a reduced oxygen environment, where oxygen levels are less than 19%, 15%, 10%, 5%, 2%, or 1%, or in an increased oxygen environment, e.g., at least 21%, 23%, 25%, 28%, 30%, or 35% oxygen, the cells can be stressed.
Stress can also be induced by culturing cells at increased or decreased temperature. Typically, cells are cultured at 37° C. Low temperature stress can be induced by culturing at a temperature less than 35, 34, 32, 30, 28, 25, 22, or 20° C. Increased temperatures can involve culturing cells at, e.g., at least 39, 40, 42, 44, 46, 48, or 50° C.
Stress can also be induced by culturing cells at altered osmolarity, either by increasing or decreasing salt levels as compared to control samples. The salt which is increased or decreased will depend on the particular type of cell being cultured and the culture medium being used. Any biologically compatible salt known in the art can be added or any salt normally found in culture media can be removed to generate osmotic stress. In one example, using a lymphocyte culture as described below which employs RPMI-1640 media, the concentration of sodium chloride, which is normally 6 g/l, can be increased (e.g., at least 7, 8, 9, 10, 12, 15, or 20 g/l) or decreased (e.g., less than 5.5, 5, 4, 3, 2, 1, 0.5, 0.25 g/l) to produce osmotic stress.
An appropriate duration of a stress depends on the severity of the particular stress employed, and can be determined by one of skill in the art. Typically, the stress can be employed for at least 6, 12, 18, or 24 hours or at least 2, 3, 5, 6, 7, 10, 14, or 21 days. If multiple stresses are simultaneously employed (e.g., nutrient and temperature stress), either the length or severity of each individual stress required for diagnosis of a psychotic disorder can be reduced.
Measuring Gene or Protein Expression
Expression levels of particular nucleic acids or polypeptides can be correlated with a particular disease state, and thus are useful in diagnosis. Expression levels can be measured using any technique known in the art. The skilled artisan will understand that the particular method employed for measuring expression is not critical to the invention.
In one embodiment, a patient having a psychotic disorder (e.g., BPD or schizophrenia) will show an alteration in the expression of at least one of the nucleic acids listed in Table 1, Table 3, in FIGS. 1A(I)-1A(IV), or in
In one embodiment, oligonucleotides or longer fragments derived from any of the nucleic acid sequences described herein (e.g., those listed in Table 1, Table 3, in FIGS. 1A(I)-1A(IV), or in
In another embodiment, an alteration in the expression of a nucleic acid sequence described herein (e.g., those listed in Table 1, Table 3, in FIGS. 1A(I)-1A(IV), or in
In another embodiment, an antibody that specifically binds a polypeptides encoded by a nucleic acid described herein (e.g., listed in Table 1, Table 3, in FIGS. 1A(I)-1A(IV), or in
In yet another embodiment, hybridization with PCR probes that are capable of detecting at least one of the polynucleotide sequences listed in Table 1, Table 3, in FIGS. 1A(I)-1A(IV), or in
In yet another approach, humans can be diagnosed for a propensity to develop a psychotic disorder (e.g., BPD or schizophrenia) by direct analysis of the sequence of at least one of the nucleic acids listed in Table 1 or Table 3.
Quantitative Real Time PCR
Q-rt-PCR can be performed using any method known in the art. In one embodiment, cDNA was synthesized from 1 μg of total RNA with the Invitrogen SuperScript First-Strand Synthesis System for Q-rt-PCR (Invitrogen, Calif.), using oligo dT as the primer. A primer set for each gene was designed with the help of Primer 3 (available from the Massachusetts Institute of Technology, Cambridge, Mass.). Amplicons were designed to be between 100 and 200 base pairs in length. Melt curve analysis and polyacrylamide gel electrophoresis were used to confirm the specificity of each primer pair. The real-time Q-rt-PCR reaction was performed in the MJ RESEARCH DNA ENGINE OPTICON (MJ Research, Waltham, Mass.; Opticon Monitor Data Analysis Software v 1.4), with the DyNAmo SYBR Green Q-rt-PCR Kit (Finnzymes, Finland), according to the company protocol, in 25 μl volume, with 2.5 μl of 1:5 diluted cDNA samples and 0.3 μM primers. PCR cycling conditions were as follows: initially, samples were heated at 95° C. for 10 minutes, followed by 49 cycles of 94° C. for 30 seconds, 55° C. for 30 seconds, 72° C. for 30 seconds. Data were collected between 72° C. and 79° C., depending on amplicon melting temperature. A melt curve analysis was performed at the end of each Q-rt-PCR experiment. Dilution curves were generated for each primer in every experiment by diluting cDNA from a control sample 1:3 twice, yielding a dilution series of 1.00, 0.333, and 0.111. The log of the dilution value was plotted against the cycle threshold (CT) value. Blanks were run with each dilution curve to control for cross contamination. Dilution curves, blanks, and samples were run in duplicate. Reported values were normalized to the average of three internal standards, which are not regulated in the gene array analysis or in the Q-rt-PCR analysis (see Table 5).
Microarray Analysis
The methods of the invention can employ microarrays for determining expression of nucleic acids or polypeptides. Such techniques are known in the art and are described in US 2004/0248286. Any appropriate array technology known in the art can be used in the diagnostic and prognostic methods of the invention.
Monitoring a Subject with a Psychotic Disorder
In addition to diagnostic methods, the invention also features methods for monitoring the progression of a psychotic disorder in a subject. Such methods include obtaining a cell sample from the subject, subjecting a cell from the sample to stress, and measuring the expression of a polypeptide or polynucleotide in the cell. A second measurement of expression is subsequently performed using the same steps following a time interval (e.g., at least 1, 2, 5, 7, 14, or 28 days, or at least 1, 2, 3, 4, 5, 6, 8, 10, 12, or 24 months). The two measurements are then compared, where a change in expression is indicative of disease progression or improvement. In one example, an increase in a gene associated with mitochondrial function or electron transport is taken as an indication of the severity of the disorder decreasing.
Such monitoring methods can be performed in conjunction with administration of a therapy (e.g., pharmaceutical therapy such as those described herein) to the subject and, thus, can be used to determine if a particular therapy is having the desired effect on gene expression, which can be indicative of the severity of the psychotic disorder. In one example, the first measurement is taken prior to commencement of a therapy. Therapy is begun following the first measurement, and a second measurement is performed six months following the commencement of therapy. A change in the second measurement as compared to the first measurement can thus be taken as indication of the effectiveness of the therapy.
The following example is intended to illustrate, rather than limit, the invention.
We isolated lymphocytes from 20-30 ml of blood taken from normal controls and patients diagnosed with BPD according to the criteria of DSM IV (DSM-IV, Diagnostic and Statistical Manual of Mental Disorder, Fourth Ed., American Psychiatric Association, Washington, D.C., 1994). The Structured Clinical Interview for DSM IV Axis I Disorders and the Brief Psychiatric Rating Scale were used to verify diagnoses. For specifics on test subjects see
Gene expression levels were calculated with the RMA algorithm (Irizarry et al., Biostatistics 4:249-264, 2003) and compared using the comparison analysis of the dChip program, which computes P values based on the t distribution, with the degrees of freedom set according to the Welch-modified 2-sample t. test. Only samples that met quality control criteria provided by the GeneChip Operating Software (Affymetrix) and DNA-Chip Analyzer (dChip 2006) (Li and Wong, Proc. Natl. Acad. Sci. USA 98:31-36, 2001) were incorporated into the analysis (
All genes differently expressed between two groups (p<0.05; >50% ‘present’ call; four groups: (I) low glucose: BPD over control; (II) normal glucose: BPD over control; (III) control: low over normal glucose; (IV) BPD: low over normal glucose) were subjected to a classification analysis using the Gene Ontology database gene product attributes (GO), calculated with the dChip software. Multiples of same transcripts were masked for classification analyses.
Similar results were obtained with log 2-transformed and natural scale data. Analysis of variance filtering was carried out in using dChip software. Permuted and adjusted P values for mitochondrial genes were obtained with the MAPPFinder program (Doniger et al., Genome Biol. 4:R7, 2003). We used 271 groupings (MAPPs) of individual genes for this analysis, grouped in a manner that avoided duplication of the same genes in independent groups. MAPPFinder calculates a nonparametric statistic based on 2000 permutations of the data, randomizing the gene associations for each sample to generate a distribution of z scores for each MAPP, which are then used to assign permuted P values. In addition, the Westfall-Young adjustment, which calculates the family wise error rate for each sample and accounts for multiple testing, is used for multiple testing. This adjustment gives the adjusted P value. Fisher exact test was used to examine the statistical difference between the percentage of regulation of mitochondrial transcripts vs. the percentage of regulation of all of the transcripts.
Families of genes, such as genes of the mitochondrial respiratory chain or genes specific for B or T cells, were compared between NC and BPD samples with 2-tailed, paired t tests using the natural expression values. For example, for the mitochondrial respiratory chain, the expression level of each of the 114 individual transcripts in an experimental group was divided by the average expression level of each transcript in all of the groups. False discovery rates were calculated in the dChip program by estimating the empirical false discovery rate for a group of genes (i.e., the 114 mitochondrial transcripts) using 2000 random permutations.
In the comparison of control and BPD lymphocytes in low glucose medium, the GO categories that had more hits for downregulated genes than would be expected by chance included ‘mitochondrion’ (p=0), ‘cytochrome-c oxidase activity’ (p=0.0007), ‘mitochondrial electron transport chain’ (p=0.001) and ‘ubiquinol-cytochrome-c reductase activity’ (p=0.0001). Further analyses revealed that 18 probe sets of electron transport transcripts, out of 114 on the array (see Table 7 for GenBank and Entrez Gene number of all 114 transcripts), were significantly lower expressed in BPD lymphocytes under glucose deprivation (FIG. 1A(I)), while none were expressed at higher levels. The 18 probe sets represented 15 individual mRNA transcripts, composing 19% of all electron transport probe sets on the array (35/114 probe sets were duplicate probe sets), while on average only 8.2% of probe sets were lower in BPD lymphocytes under glucose deprivation (FIG. 1B(I)). This difference was significant in Fisher exact test. Furthermore, the entire group of electron transport transcripts was shifted significantly in BPD toward lower expression levels (FIG. 1C(I) and Table 6).
No differences between BPD and control lymphocytes were observed either under normal glucose concentrations (FIGS. 1A(II), 1B(II), 1C(II)), or in fresh, uncultured lymphocytes (
0.040
0.035
0.043
0.025
0.011
0.000
0.026
0.036
0.026
0.000
0.010
0.014
0.018
0.014
0.041
0.019
0.011
0.036
0.045
0.001
0.001
0.001
0.014
−1.15
0.017
1.18
0.031
1.14
0.007
1.11
0.036
1.11
0.030
−1.12
0.032
−1.1
0.001
−1.07
0.042
1.16
0.021
To determine whether a shift between B and T cells had taken place in any of the comparisons, the expression levels of 54 B-cell-specific transcripts and 77 T-cell-specific transcripts were examined (
Almost all BPD patients were on medication (see
All patents, patent applications, and publications mentioned in this specification are herein incorporated by reference, to the same extent as if each independent patent, patent application, or publication was specifically and individually indicated to be incorporated by reference.
This application claims benefit of U.S. Provisional Application No. 60/801,313, filed May 18, 2006, and U.S. Provisional Application No. 60/928,151, entitled “Methods for Diagnosis and Prognosis of Psychotic Disorders,” filed May 7, 2007, Inventor Christine Konradi. Each of these applications is hereby incorporated by reference.
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