1 Stanley Division of Developmental
Neurovirology, Johns Hopkins University School of Medicine, Baltimore,
Maryland
2 Howard Hughes Medical Institute, Johns Hopkins
University School of Medicine, Baltimore, Maryland
3> Stanley Foundation Research Program,
Bethesda, Maryland, USA
Correspondence: Dr Robert H. Yolken, Theodore and Vada Stanley Professorship of Neurovirology, Johns Hopkins University, Stanley Division of Developmental Neurovirology, 600 N. Wolfe Street, Blalock IIII, Baltimore, MD 21287-4933, USA. Tel: +1 410 955 3271; fax: +1 410 955 3723; e-mail: yolken{at}welchlink.welch.jhu.edu
Declaration of interest This research was supported by the Theodore and Vada Stanley Foundation.
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ABSTRACT |
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Aim To identify RNA transcripts that are up- or down-regulated in the frontal cortex regions of individuals with bipolar disorder.
Method Serial analysis of gene expression (SAGE) and reverse transcriptase-polymerase chain reaction were used to identify RNA transcripts which are differentially expressed in the frontal cortex of brains obtained postmortem from individuals with bipolar disorder compared with other psychiatric and control conditions.
Results Levels of RNA transcripts encoding the serotonin transporter
protein and components of the NF-B transcription factor complex are
significantly increased in individuals with bipolar disorder compared with
unaffected controls. Increased levels of expression of these RNA transcripts
were also detected in the brains of some individuals with schizophrenia and
unipolar depression.
Conclusion The SAGE technique offers promise for the characterisation of complex human brain diseases.
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INTRODUCTION |
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The recently described technique of serial analysis of gene expression (SAGE) allows the accurate identification of large numbers of RNA transcripts in a practical and efficient manner. This technique has been employed for the characterisation of RNA transcripts that are differentially expressed in tumour cells and cell lines which overexpress p63 tumour suppressor gene product (Velculescu et al, 1995; Madden et al, 1997; Polytak et al, 1997; Zhang et al, 1997). We applied SAGE to the analysis of RNAs expressed in the brains of individuals with bipolar disorder.
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METHOD |
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Serial analysis of gene expression
One microgram of total RNA was converted to complementary cDNA using a
CapFinder cDNA synthesis kit (Clontech, catalogue no. K1052-1, Palo Alto, CA)
and the biotinylated primers required for SAGE. The tagging enzyme
Nlalll was used and BsmFl was used as the anchoring enzyme.
Multitag fragments were selected to clone into a plasmid vector, pZero
(Invitrogen, catalogue no. K2500-1, Carlsbad, CA) for bacterial transformation
by electroporation. Bacterial colonies were randomly picked, amplified by
polymerase chain reaction (PCR) using M13 forward and reverse primers, and the
PCR products sequenced. Tag information was then identified and analysed using
the SAGE computer software.
Reverse transcriptasepolymerase chain reactions
The reverse transcriptasepolymerase chain reaction (RT-PCR)
technique was performed using primers designed specifically for amplifying
genes identified in SAGE. The following cycling conditions were employed in
the DNA Thermal Cycler 480: 94 °C, 1 min; 55 °C, 1 min; and 72 °C,
2 min. The number of cycles for each PCR was adjusted according to the
relative abundance of each RNA in the tag library. Oligo-dT-primed cDNAs from
the frontal cortex regions were used as templates for PCR. The primers
employed for the amplification of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) RNA have been described by Johnston et al
(1997). The primers for the
amplification of serotonin transporter RNA were
5'-CAGAGCTACCCTGTGTGTCCG-3' and
5'-GTGTCTGGGGGAAGTCTTTCG-3'. The primers used for amplification of
NF-B2 were 5'-CACTAGACAGAGCCGGGCCTA-3' and
5'-CACAGCCATATCGAAATCGGA-3'.
Ribonucleic acid encoding additional transcription factors was measured using a semi-nested RT-PCR approach to visualise low-abundance transcripts. Initial reactions consisting of 35 cycles were performed under the conditions described above. For each sample, a 5 µl aliquot of the first PCR was diluted in 25 µl of PCR buffer and re-amplified for an additional 20 cycles employing a semi-nested set of primers. The primers used for these reactions were as follows:
Following amplification, PCR products were separated and visualised as described below.
Amplicon quantitation
The PCR products were resolved by polyacrylamide gel electrophoresis and
stained with SYBR Green I. The gel was converted to a digital image and the
fluorescence generated by the appropriately sized band quantified by a digital
fluoroimager (Vistra II, Molecular Dynamics, Sunnyvale, CA). For each target
amplicon, levels were expressed as the ratio of the fluorescence generated by
the target amplicon to the fluorescence generated in the same sample by
primers directed at the constitutively expressed RNA encoding GAPDH. The
resulting ratios of the diagnostic groups were compared using analysis of
variance and t-test statistics performed by the statistical
data-analysis program Statsoft (Tulsa, OK).
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RESULTS |
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We constructed and identified 1856 sequence tags from the expressed RNAs
from the brain of the individual with bipolar disorder and 4237 sequence tags
from the brain of the unaffected control. We identified four sequence tags
that displayed significant differences in number between the case and the
control brain tissues (Table
1). The levels of RNA transcripts homologous to these tags were
further analysed in tissue obtained postmortem from the same brain region of
19 individuals with bipolar disorder and 15 unaffected controls
(Fig. 1). Serotonin transporter
protein RNA transcripts were detected in 13 of the 19 (68%) individuals with
bipolar disorder compared with 4 of the 15 (27%) unaffected individuals
(P=0.018, Fisher's exact test). Transcripts encoding the nuclear
transcription factor NF-B2 were detected in 12 of the 19 (63%)
individuals with bipolar disorder and 4 of the 15 (27%) control individuals
(P=0.037, Fisher's exact test). Either serotonin transporter or
NF-
B2 transcripts were detected in 17 of the 19 (89%) individuals with
bipolar disorder compared with 5 of the 15 (33%) control individuals
(P=0.001, Fisher's exact test). On the other hand, transcripts
encoding GAPDH were detected in 18 of the 19 individuals with bipolar disorder
and all 15 of the samples from the control individuals. The other two RNA
transcripts identified in the initial SAGE evaluation, encoding RACH1 and
biliary glycoprotein, were not found to be differentially expressed in the
larger group of samples and were not analysed further (data not shown).
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In addition to these sample analyses, RNA transcripts encoding the
serotonin transporter protein and NF-B2 were also quantitated in
samples obtained from the same brain region of individuals with schizophrenia
(n=18), unipolar depression (n=14) and Huntington's disease
who had received neuroleptic medication (n=8) (Fig
2 and
3). These analyses were
performed using a semi-quantitative RT-PCR procedure that expresses the level
of target amplicons in relation to the level of amplicons generated by primers
specific for GAPDH, which acts as a control for overall level of RNA
expression (Nicoletti & Sassy-Prigent,
1995; Zhao et al,
1995). The levels of RNA transcripts encoding the serotonin
transporter protein and NF-
B2 were significantly increased in the
bipolar group compared with the unaffected controls (P < 0.002 and
P < 0.02, respectively). There was no significant correlation
between the levels of these RNA transcripts and a range of ante- and
post-mortem variables including age, gender, medication history, brain pH and
post-mortem interval. Elevated levels of RNA transcripts encoding the
serotonin transporter protein and NF-
B2 were also found in samples
obtained from some individuals with schizophrenia and unipolar depression.
However, as a group, the levels did not differ from those measured in the
unaffected controls to a statistically significant extent (all P >
0.1). Neither of these RNA transcripts were detected in the brains of
individuals with Huntington's disease, despite the fact that these individuals
had received neuroleptic medication.
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The expression of NF-B2 was further analysed in terms of its
biological properties. The gene encodes the p49/p100 subunit of the
NF-
B transcription factor complex. This complex is expressed upon the
induction of a wide variety of cellular and viral genes involved in immune
function and inflammatory response. The NF-
B complex consists of a
number of proteins involved in interrelated transcriptional and signal
transduction pathways. Activation of NF-
B2 is thus often associated
with activation of other transcription factors, including I
B,
NF-
B1 and NF-
B3 (Ten et
al, 1992; Cogswell et
al, 1993; Deloukas &
van Loon, 1993; Liptay et
al, 1994). In fact, we found that in comparison with
unaffected individuals, these factors are also increased in the frontal cortex
of many people with bipolar disorder (Fig.
4).
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We also examined the samples for increased expression of a number of control RNA transcripts. Disease-associated differences were not detected in the expression of a number of housekeeping and metabolically active RNAs including those encoding actin, cytochrome oxidase, myelin basic protein, phosphotyrosine phosphatase, nerve growth factor, autoantigen DFS70, synaptosomal-associated protein (SNAP) 25, and heat shock proteins 70 and 90. In addition, diagnosis-related differences were not found in the levels of RNA transcripts homologous to the following sequence tags identified in equal numbers in the case and control brains employed in the original SAGE analysis: guanylate kinase (GenBank accession no. L76300), c-erbB3 receptor (GenBank accession no. M29366) and RNA helicase (GenBank accession no. D50924) (data not shown).
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DISCUSSION |
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The finding of increased levels of RNA encoding components of the
NF-B transcription complex in brains of many individuals with bipolar
disorder is also of interest. Inducers of the NF-
B complex include
viruses, oxidants, cell adhesion molecules, cytokines and growth factors
(Meyer et al, 1991;
Baeuerle & Thomas, 1994; Beauerle & Baltimore,
1996). The presence of increased levels of RNA encoding
NF-
B in the brains of some individuals with bipolar disorder is thus
consistent with studies indicating that environmental factors may contribute
to the pathogenesis of this disease. The documented occurrence of disease
discordance in monozygotic twins, increased levels of autoreactive antibodies,
and an increased prevalence of disease following winter or spring birth and
maternal fever are in agreement with a possible role of infectious or
inflammatory factors in the aetiopathogenesis of some cases of bipolar
disorder (Bertelsen et al,
1977; Kinney et al,
1993; Haggerty et al,
1997; Torrey et al,
1997). The finding of increased levels of RNA encoding NF-
B
transcription factors in the cortical regions of some individuals with
schizophrenia indicates that such factors may also be operant in some cases of
this disease as well. The interactions between transcription factors in
cortical and other brain regions, genetic susceptibilities and disease
phenotype are the subject of current investigations.
Our study differs from others employing the SAGE technique in that we analysed RNA extracted from post-mortem human brain tissue as opposed to RNA from cell lines or tumour tissues (Velculescu et al, 1995; Madden et al, 1997; Polytak et al, 1997; Zhang et al, 1997). Therefore, the number of tags we were able to characterise was substantially lower than the number required for the complete analysis of RNA expression, a limitation related to the quantity and quality of RNA that can be obtained from post-mortem human brain specimens. In addition, initial analysis was limited to cortical regions from only two representative individuals. Furthermore, owing to the limited sample size, the possibility that RNA expression may have been affected by environmental factors not directly related to the underlying clinical diagnosis cannot be completely excluded. Despite these limitations, we were able to identify RNA transcripts that are differentially expressed in the brains of a population of individuals with bipolar disorder and that correspond to potential genetic and environmental components of this disease. The elucidation of differentially expressed RNA transcripts in the initial SAGE analyses could also be used to identify the differential expression of additional functionally related RNA transcripts. Our studies indicate that SAGE analyses of human brain tissues are potentially useful for the analysis of complex human brain disorders.
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Clinical Implications and Limitations |
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LIMITATIONS
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ACKNOWLEDGMENTS |
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