ORIGINAL RESEARCH ARTICLE
Gene expression and association analyses of LIM
(PDLIM5) in bipolar disorder and schizophrenia
T Kato1, Y Iwayama2, C Kakiuchi1, K Iwamoto1, K Yamada2, Y Minabe3, K Nakamura3, N Mori3, K Fujii4,
S Nanko5and T Yoshikawa2
1Laboratory for Molecular Dynamics of Mental Disorders, RIKEN Brain Science Institute, Wako-shi, Saitama, Japan;
2Laboratory for Molecular Psychiatry, RIKEN Brain Science Institute, Wako-shi, Saitama, Japan;3Department of Psychiatry
and Neurology, Hamamatsu University School of Medicine, Hamamatsu, Japan;4Biwako Hospital, Otsu, Japan;5Department
of Psychiatry and Genome Research Center, Teikyo University School of Medicine, Tokyo, Japan
We previously reported that expression level of LIM (ENH, PDLIM5) was significantly and
commonly increased in the brains of patients with bipolar disorder, schizophrenia, and major
depression. Expression of LIM was decreased in the lymphoblastoid cells derived from
patients with bipolar disorders and schizophrenia. LIM protein reportedly plays an important
role in linking protein kinase C with calcium channel. These findings suggested the role of LIM
in the pathophysiology of bipolar disorder and schizophrenia. To further investigate the role of
LIM in these mental disorders, we performed a replication study of gene expression analysis
and performed genetic association studies. Upregulation of LIM was confirmed in the
independent sample set obtained from Stanley Array Collection. No effect of sample pH or
medication was observed. Genetic association study revealed the association of single
nucleotide polymorphism (SNP)1 (rs10008257) with bipolar disorder. In an independent sample
set, SNP2 (rs2433320) close to SNP1 was associated with bipolar disorder. In total samples,
haplotype of these two SNPs was associated with bipolar disorder. No association was
observed in case–control analysis and family-based association analysis in schizophrenia.
These results suggest that SNPs in the upstream region of LIM may confer the genetic risk for
Molecular Psychiatry (2005) 10, 1045–1055. doi:10.1038/sj.mp.4001719; published online 26 July 2005
Keywords: bipolar disorder; schizophrenia; PDLIM5; association study; gene expression;
enigma homolog (ENH)
The role of genetic factors in bipolar disorder has
been well established from twin, adoption, and family
studies.1Extensive linkage analyses suggested many
candidate loci.2In such loci, genes having functions
related to bipolar disorder were examined as candi-
date genes, and several promising results have been
reported. Among them, association with G72 at 13q34
has been replicated in several studies.3–5
The other strategy to identify candidate genes is
gene expression analysis. Mirnics et al6performed
gene expression analysis using cDNA micoarray and
reported that RGS4 was downregulated in the post-
mortem brains of patients with schizophrenia. They
further examined the association of RGS4 with
schizophrenia and found a positive association.7
Several studies confirmed this finding.8–10A similar
approach to identify candidate genes may also be
effective for bipolar disorder.
We have performed comprehensive gene expression
analysis of the frontal lobes obtained from Stanley
Foundation Brain Bank using oligonucleotide micro-
array.11By analyzing 50 brains, we found that two
genes, LIM and PRPF4B, were commonly altered in
three mental disorders, bipolar disorder, schizophre-
nia, and major depression. Of the two genes,
upregulation of LIM in the postmortem brain was
confirmed by RT-PCR. Subsequently, we also found
that LIM was significantly downregulated in the
lymphoblastoid cell lines from patients with bipolar
disorder. Since we cultured lymphoblastoid cells for
more than 1 month after blood collection, effects of
drugs and secondary effects of other confounding
factors, such as endocrinological abnormalities, can
be ruled out in this analysis.
Next, we performed a replication study of LIM
expression in lymphoblastoid cells.12Reduced ex-
pression was confirmed in the extended samples with
bipolar I disorder (N¼26). We also found that LIM
was significantly downregulated in bipolar II disorder
(N¼10) and schizophrenia (N¼13). Thus, we specu-
lated that regulation of LIM might be genetically
impaired in bipolar disorder and other mental
Received 25 March 2005; revised 11 May 2005; accepted 23 May
2005; published online 26 July 2005
Correspondence: Dr T Kato, MD, PhD, Laboratory for Molecular
Dynamics of Mental Disorders, RIKEN Brain Science Institute, 2-1
Hirosawa, Wako-shi, Saitama 351-0198, Japan.
Molecular Psychiatry (2005) 10, 1045–1055
& 2005 Nature Publishing Group All rights reserved 1359-4184/05 $30.00
LIM encodes an adopter protein connecting protein
kinase C (PKC) e and N-type calcium channel.13
Altered PKC activity in peripheral blood cells of
bipolar patients is reported.14Furthermore, altered
calcium signaling has been postulated as an impor-
tant pathophysiological mechanism of this disorder.15
Thus, it is reasonable to hypothesize that genetic
variation of LIM causes genetically determined dysre-
gulation of LIM, which causes calcium-signaling
abnormalities in bipolar disorder.
LIM is located at 4q22,16for which some linkage
signal has been detected in bipolar disorder17and
schizophrenia.18Only a few studies revealed the loci
in 4q for bipolar disorder19–21or schizophrenia.22–24
Although the support by linkage studies is marginal,
above-mentioned findings by gene expression ana-
lyses seemed strong enough to start genetic associa-
tion analysis of this gene in bipolar disorder.
Here, we performed a replication study of altered
expression levels of LIM in a larger number of samples
of postmortem prefrontal cortex of bipolar disorder
and schizophrenia obtained from the Stanley Array
Collection, and analyzed possible confounding fac-
tors. We further performed association study of LIM in
bipolar disorder and schizophrenia. While LIM was
not associated with schizophrenia, it was associated
with bipolar disorder, which was replicated in a
different sample set. These results suggest that
polymorphisms of LIM may confer a genetic risk for
Subjects and methods
RNA samples extracted from the prefrontal cortices
(Broadmann’s Area 46) were donated by the Stanley
Array Collection. They contain total RNA samples
from 35 individuals in each of three diagnostic groups
(BD, SZ, and controls). Diagnoses was made accord-
ing to the Diagnostic and Statistical Manual of Mental
Disorders, Fourth Edition (American Psychiatric
Association). Detailed information about the diagno-
sis, and summary of demographic variables of each
diagnostic group can be found at the website (http://
Real-time quantitative RT-PCR
In all, 3–5mg of total RNA was used for cDNA
synthesis by olgo(dT) and SuperScript II reverse
transcriptase (Invitrogen). RT-PCR using SYBER/
GREEN I (Applied Biosystems, Foster city, CA,
USA) was performed with an ABI PRISM 7900HT
(Applied Biosystems). The comparative Ct method
was used for quantification according to the manu-
facture’s protocol (Applied Biosystems). Measure-
ment of delta Ctwas carried out at least in triplicate.
Amplification of the single product was confirmed by
monitoring the dissociation curve and by gel electro-
phoresis. We used two control genes (GAPDH and
CFL1) for normalization to control for possible
fluctuations in quantitative values of the target
transcripts. The validity of the use of CFL1 as an
internal control gene in postmortem brain samples
was shown previously. Primer pairs used in this study
were according to the previous report.11Among the
105 samples, four samples showing poor RNA
qualities were not analyzed.
Subjects for genetic analyses: bipolar disorder
The first sample set was collected in the Shiga
University of Medical Science Hospital, University
of Tokyo Hospital, and Laboratory for Molecular
Dynamics of Mental Disorders (called ‘MDMD’ sam-
ples). These include 128 patients with bipolar
disorder (47.8713.6 years old, 50 males and 78
females) and 130 controls (48.8715.3 years old, 65
males and 65 females). They were diagnosed with the
consensus of two senior psychiatrists without using
any structured interviews, or were diagnosed by a
senior psychiatrist after an interview using SCID-IV
(Structured Clinical Interview for DSM-IV). Controls
were selected from students, nurses, office workers,
and doctors in participating institutes, and their
friends. A senior psychiatrist interviewed them and
they did not have major mental disorders. Only a part
of them were interviewed using a structured inter-
view, Mini-International Neuropsychiatric Interview
The replication sample set was collected in the
Tokyo Medical and Dental University, Hamamatsu
University School of Medicine, and Lab. for Molecu-
lar Psychiatry (‘MPS’ samples). These include 240
patients with bipolar disorder (51.2713.1 years old,
132 males and 108 females) and 240 controls
(51.4710.7 years old, 120 males and 120 females).
For the quantification of copy number of LIM gene,
28 patients with bipolar disorder were selected from
Subjects for genetic analyses: schizophrenia
Subjects for the case–control analysis consist of 570
patients with schizophrenia (48.6712.0 years old,
285 males and 285 females) and an equal number of
control subjects (48.4711.8 years old, 285 males and
285 females) collected by the Laboratory for Molecu-
lar Psychiatry. Control subjects were recruited from
hospital staff and their acquaintances. They were
interviewed by an experienced psychiatrist without
using structured interviews and found not to have
psychoses. Most of the controls in the MPS samples
are included in this control group. All were Japanese.
Diagnosis of the patients by DSM-IV criteria was
made by consensus of two psychiatrists based on
unstructured interviews of the patients, chart re-
views, and information from family members and
We presumed that all these subjects were unrelated
to each other, but it cannot be totally ruled out that
some of the patients were related, because the ethics
policy of the Japanese Government requires stringent
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LIM and bipolar disorder
T Kato et al