CACNA1C Risk Variant and Amygdala Activity in Bipolar Disorder, Schizophrenia and Healthy Controls

Abstract

Several genetic studies have implicated the CACNA1C SNP rs1006737 in bipolar disorder (BD) and schizophrenia (SZ) pathology. This polymorphism was recently found associated with increased amygdala activity in healthy controls and patients with BD. We performed a functional Magnetic Resonance Imaging (fMRI) study in a sample of BD and SZ cases and healthy controls to test for altered amygdala activity in carriers of the rs1006737 risk allele (AA/AG), and to investigate if there were differences across the diagnostic groups.
Rs1006737 was genotyped in 250 individuals (N = 66 BD, 61 SZ and 123 healthy controls), all of Northern European origin, who underwent an fMRI negative faces matching task. Statistical tests were performed with a model correcting for sex, age, diagnostic category and medication status in the total sample, and then in each diagnostic group.
In the total sample, carriers of the risk allele had increased activation in the left amygdala. Group-wise analyses showed that this effect was significant in the BD group, but not in the other diagnostic groups. However, there was no significant interaction effect for the risk allele between BD and the other groups.
These results indicate that CACNA1C SNP rs1006737 affects amygdala activity during emotional processing across all diagnostic groups. The current findings add to the growing body of knowledge of the pleiotropic effect of this polymorphism, and further support that ion channel dysregulation is involved in the underlying mechanisms of BD and SZ.

Full text

CACNA1C
Risk Variant and Amygdala Activity in Bipolar
Disorder, Schizophrenia and Healthy Controls
Martin Tesli
1,2
*, Kristina C. Skatun
2
, Olga Therese Ousdal
1,2
, Andrew Anand Brown
2,7
,
Christian Thoresen
2
, Ingrid Agartz
1,4,5
, Ingrid Melle
1,2
, Srdjan Djurovic
1,2,3
, Jimmy Jensen
1,6
,
Ole A. Andreassen
1,2
1Institute of Clinical Medicine, University of Oslo, Oslo, Norway, 2Division of Mental Health and Addiction, Oslo University Hospital, Oslo, Norway, 3Department of
Medical Genetics, Oslo University Hospital, Oslo, Norway, 4HUBIN Project, Psychiatry Section, Department of Clinical Neuroscience, Karolinska Institutet and Hospital,
Stockholm, Sweden, 5Department of Psychiatric Research, Diakonhjemmet Hospital, Oslo, Norway, 6Department of Psychiatry and Psychotherapy, Charite
´
Universita
¨tsmedizin, Berlin, Germany, 7Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom
Abstract
Objectives:
Several genetic studies have implicated the CACNA1C SNP rs1006737 in bipolar disorder (BD) and schizophrenia
(SZ) pathology. This polymorphism was recently found associated with increased amygdala activity in healthy controls and
patients with BD. We performed a functional Magnetic Resonance Imaging (fMRI) study in a sample of BD and SZ cases and
healthy controls to test for altered amygdala activity in carriers of the rs1006737 risk allele (AA/AG), and to investigate if
there were differences across the diagnostic groups.
Methods:
Rs1006737 was genotyped in 250 individuals (N = 66 BD, 61 SZ and 123 healthy controls), all of Northern
European origin, who underwent an fMRI negative faces matching task. Statistical tests were performed with a model
correcting for sex, age, diagnostic category and medication status in the total sample, and then in each diagnostic group.
Results:
In the total sample, carriers of the risk allele had increased activation in the left amygdala. Group-wise analyses
showed that this effect was significant in the BD group, but not in the other diagnostic groups. However, there was no
significant interaction effect for the risk allele between BD and the other groups.
Conclusions:
These results indicate that CACNA1C SNP rs1006737 affects amygdala activity during emotional processing
across all diagnostic groups. The current findings add to the growing body of knowledge of the pleiotropic effect of this
polymorphism, and further support that ion channel dysregulation is involved in the underlying mechanisms of BD and SZ.
Citation: Tesli M, Skatun KC, Ousdal OT, Brown AA, Thoresen C, et al. (2013) CACNA1C Risk Variant and Amygdala Activity in Bipolar Disorder, Schizophrenia and
Healthy Controls. PLoS ONE 8(2): e56970. doi:10.1371/journal.pone.0056970
Editor: Hossein Fatemi, University of Minnesota, United States of America
Received June 29, 2012; Accepted January 17, 2013; Published February 20, 2013
Copyright: ß2013 Tesli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was supported by grants to the TOP study group from the University of Oslo (http://www.uio.no/english/), the Research Council of Norway
(http://www.forskningsradet.no/servlet/Satellite?c=Page&cid=1177315753906&p=1177315753906&pagename=ForskningsradetEngels%2FHovedsidemal)
(#167153/V50, #163070/V50) and the SouthEast Norway Health Authority (http://www.helse-sorost.no/omoss/english/Sider/page.aspx) (#2004123, #2008-080).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: m.s.tesli@medisin.uio.no
Introduction
Despite the high heritability estimates for bipolar disorder (BD),
the molecular genetic and neurobiological mechanisms for this
disorder remain poorly understood. However, recent genome-
wide association (GWA) studies with substantially more statistical
power than former psychiatric genetic studies have provided
evidence for new and promising candidate genes [1]. One of the
most consistent findings is related to CACNA1C (calcium channel,
voltage-dependent, L type, alpha 1C subunit) gene variants. The
CACNA1C SNP rs1006737 was first found to be associated with
BD in a combined analysis of two GWA study datasets
(P = 3.15610
26
) [2]. When adding a third sample, this CACNA1C
SNP was significantly associated with BD in the combined sample
of 4,387 BD cases and 6,209 healthy controls (P = 7.0610
28
,
OR = 1.181) [3]. Subsequent studies also reported association
between CACNA1C and schizophrenia (SZ) [4,5] and major
depressive disorder (MDD) [4,6], thereby supporting the hypoth-
esis of genetic overlap between these severe psychiatric disorders.
CACNA1C is located on chromosome 10 and encodes an alpha-1
subunit of a voltage-dependent calcium (Ca
v
1.2) channel. Ca
v
1.2
channels can be found in cardiac smooth muscle, neuronal and
endocrine cells, where they have a variety of functions including
excitation-contraction coupling, endocrine secretion and regula-
tion of neuronal Ca
2+
transients, enzyme activity and transcription
[7,8]. Mutations in CACNA1C have been found to lead to Timothy
syndrome, a lethal disorder consisting of somatic symptoms like
cardiac arrhythmia, and psychiatric symptoms like autism and
cognitive disability [9].
Emotional dysregulation is part of the clinical phenotype of BD
and SZ, manifesting as mood swings as well as affect flattening.
Morphometric studies point to structural abnormalities within the
medial temporal lobe, which may cause altered responsivity to
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emotional stimuli among these patients. In particular, the
amygdala has been extensively studied in BD. In a recent meta-
analysis of 321 patients with bipolar I disorder and 442 healthy
controls, amygdala volume was found to be greater in patients
treated with lithium compared to controls and patients not treated
with lithium [10]. A meta-analysis comprising 65 fMRI studies of
1074 healthy volunteers and 1040 BD cases, found evidence for
amygdala over-activation in euthymic BD patients compared with
healthy controls [11].
With regards to the potential involvement of BD risk genes in
limbic system dysregulation, there are reports of an association
between CACNA1C SNP rs1006737 and amygdala activity. In a
study of 64 healthy individuals, carriers of the risk allele (AA/AG)
had increased activity in the right amygdala in a monetary reward
paradigm [12]. Another group reported enhanced activity in AA/
AG individuals compared to GG individuals in the right amygdala
during a fearful faces paradigm (N = 41 BD patients, 25 relatives
and 50 healthy controls) [13]. A third study found healthy controls
with the AA genotype to have trend significantly greater right
amygdala activity than those with AG/GG in a negative faces
matching paradigm [14]. Thus, there is some evidence supporting
the hypothesis that CACNA1C SNP rs1006737 affects amygdala
activity during different paradigms related to limbic system
functioning.
Interestingly, two recent meta-analyses reported that CACNA1C
was one out of three common genes for BD and SZ [1,15]. These
findings on the molecular genetic level are consistent with
similarities in clinical and cognitive characteristics [16], and the
continuum hypothesis for severe psychiatric disorders [17].
However, to the best of our knowledge CACNA1C has not been
investigated with fMRI in individuals suffering from SZ. But a
recent study of healthy controls found increased activity in the
prefrontal cortex during executive cognition in CACNA1C risk
allele carriers, a finding which could imply inefficient prefrontal
functioning as a genetically conditioned mechanism underlying SZ
[14].
Taken together, it remains unclear whether the effect of this
gene on amygdala activity during emotional processing is general
or confined to one or more diagnostic categories.
The primary aim of the current study was to test for altered
amygdala activity in carriers of the CACNA1C SNP rs1006737 risk
allele. Secondarily, we aimed to determine the specificity of such
associations, by testing for potential differences between healthy
controls and patients with BD or SZ. Therefore, we measured
fMRI amygdala blood-oxygen-level dependence (BOLD) respons-
es during a faces matching paradigm [18] in genotyped BD and
SZ cases, as well as healthy controls.
Materials and Methods
Sample characteristics
The total number of individuals in this study was 250, including
66 BD cases, 61 SZ cases and 123 healthy control subjects. All
participants were of Northern European origin (96% were born in
Norway with Norwegian parents, 4% had one or both parents
from another Northern European country), part of the ongoing
TOP (Thematic Organized Psychosis) Study, and included from
2003 to 2009.
To be included in the study, patients had to be between 18 and
65 years, have a DSM-IV diagnosis of a bipolar spectrum or
schizophrenia spectrum disorder, and be willing and able to
provide written informed consent. Exclusion criteria were an IQ
score below 70 and reporting a history of head injury or
neurological disorder. In the healthy control group, we also
excluded subjects if they or their close relatives had a lifetime
history of a severe psychiatric disorder (SZ, BD and major
depression). Subjects with a history of a medical condition
potentially interfering with brain function (hypothyroidism,
uncontrolled hypertension and diabetes), and an illicit drug
abuse/addiction diagnosis were also excluded.
Patients were recruited from psychiatric in- and out-patient
hospital units in the Oslo area, and had been diagnosed with
bipolar I disorder (N = 30), bipolar II disorder (N = 32), bipolar
disorder not otherwise specified (N = 4), schizophrenia (N = 48),
schizoaffective disorder (N = 9) or schizophreniform disorder
(N = 4), according to DSM-IV using the Structural Clinical
Interview for DSM-IV (SCID) [19]. 23 out of 30 (76.7%) patients
with bipolar I disorder had a SCID-verified lifetime history of
psychosis, and the corresponding numbers were 3 out of 32 (9.4%)
for bipolar II disorder and 2 out of 4 (50%) for bipolar disorder not
otherwise specified. Diagnostic evaluation was performed by
trained psychologists and psychiatrists, of whom all participated
regularly in diagnostic meetings supervised by professors in
psychiatry. Reliability measures of the diagnostic assessment in
the TOP study were performed, and the overall agreement for the
DSM-IV diagnostic categories tested was 82% and the overall
Kappa 0.77 (95% CI: 0.60–0.94). Information on education, age
of onset, number of relapses, medication status, alcohol and illegal
substance abuse was obtained during an initial clinical interview. A
three-hour neuropsychological test battery, including Wechsler
Abbreviated Scale of Intelligence (WASI), was carried out by
trained clinical psychologists [16].
On the day of scanning, patients underwent an abbreviated re-
interview including Young Mania Rating Scale (YMRS) [20],
Inventory of Depressive Symptoms (IDS) [21] and Positive and
Negative Syndrome Scale (PANSS) [22]. For patients lacking data
for this re-interview, we used corresponding data from the clinical
interview.
The healthy control subjects were randomly recruited from the
same catchment area as the patients, and underwent an initial
interview where demographic and clinical information was
obtained. Clinical assessment of the patients and healthy controls
participating in the TOP study is described in details in a previous
report [23].
Carriers of the risk allele did not differ significantly from the GG
homozygotes with respect to demographical variables within the
total sample or any of the diagnostic groups (Table S1). Further,
the clinical characteristics did not differ between genotype groups,
except that risk allele carriers in the total patient sample had
significantly lower Global Assessment of Functioning-symptom
score (GAF-S) than those with the GG genotype (P = 0.01).
However, this was not seen in the subgroups, and was probably
due to a higher frequency of the risk allele in the SZ group (43/61
(70.5%)) compared to the BD group (34/66 (51.5%)). For further
details on demographic and clinical characteristics, see Table S1.
Ethics Statement
The Norwegian Scientific-Ethical Committees and the Norwe-
gian Data Protection Agency approved the study. All subjects have
given written informed consent prior to inclusion into the project.
Genotyping
Genomic DNA was extracted from whole blood. CACNA1C
SNP rs1006737 was genotyped in the 250 subjects participating in
this study using Affymetrix Gene Chip Genome-Wide SNP 6.0
array (AffymetrixInc, Santa Clara, CA, USA), as described in
details elsewhere [24,25]. There was no deviation from Hardy–
Weinberg equilibrium (HWE) in the controls (P = 0.84) or in the
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cases (P = 0.57), using PLINK (version 1.07;http://pngu.mgh.
harvard.edu/purcell/plink/) [26].
Experimental paradigm
A widely used and validated paradigm was employed to elicit
amygdala reactivity [18]. In this task participants select which of
two stimuli (displayed at the bottom of the screen) matches a target
stimulus (displayed at the top). The images displayed were either
human faces expressing anger or fear (faces matching task) or
geometrical shapes (the sensorimotor control task). Participants
completed 4 blocks of the faces matching task, where each block
consisted of 6 emotion-specific face trios derived from a standard
set of facial affect pictures [27]. Interleaved between these blocks,
participants completed 5 blocks of the sensorimotor control task.
Each trial (faces or shapes) was presented for 5.4 seconds with no
inter-stimulus interval, for a total block length of 32.6 seconds.
The total paradigm lasted 310 seconds. E-prime software (version
1.0 Psychology Software Tools, Inc, Pittsburgh, PA, USA)
controlled the presentations of the stimuli using VisualSystem
(NordicNeuroLab, Bergen, Norway). Response times and accura-
cy were recorded through MR-compatible ResponseGrips (Nor-
dicNeuroLab, Bergen, Norway). Behavioural data was missing for
13 individuals.
Image acquisition
MRI scans were acquired on a 1.5 T Siemens Magnetom
Sonata scanner (Siemens Medical Solutions, Erlangen, Germany)
supplied with a standard head coil. Volumes (n = 152, 24 axial
slices, 4 mm thick with 1 mm gap) covering the whole brain were
acquired in the axial plane, using a BOLD EPI sequence
(TR = 2040 ms, TE = 50 ms, flip angle = 90u, matrix 64664,
FOV 1926192 mm). The first seven volumes were discarded.
Prior to BOLD fMRI scanning, a sagittal T1-weighted 3D
Magnetization Prepared Rapid Gradient Echo (MPRAGE) scan
(TR = 2000 ms, TE = 3.9 ms, flip angle = 7u, matrix 1286128,
FOV 2566256 mm) was collected for better localization of
functional data.
fMRI data analyses
All fMRI volumes were preprocessed and analysed with
Statistical Parametric Mapping (SPM2) (http://www.fil.ion.ucl.
ac.uk/spm) implemented in MATLAB7.1 (The Mathworks Inc,
Natick, Massachusetts). All of the functional images were realigned
to the first image in the time series to correct for head motion [28].
All subjects moved less than 2.5 mm in any direction during the
scan. Subsequently, the mean functional image and the anatomical
image were coregistered to ensure that they were aligned. The
images were spatially normalized to the stereotactical MNI
template [28], and resampled at 26262 mm voxels. The images
were smoothed using a 6 mm full width-half maximum (FWHM)
isotropic kernel. Data were high-pass filtered using a cutoff value
of 128 s. The fMRI data for all subjects were first analysed using a
single-subject fixed-effect model. The model was built by
convolving boxcar functions for the onsets of the two different
conditions (faces and figures) with a canonical hemodynamic
response function (HRF). Individual contrast images were created
by subtracting ‘‘figures’’ from ‘‘faces’’. The contrast images for
faces versus figures for each subject were entered into a random
effects statistical model. These data were analysed with a region of
interest (ROI) approach and a pre-defined anatomical mask
(bilateral amygdala) derived from the Wake Forest University
PickAtlas for SPM2 [29]. The six movement parameters were
included in the first-level analyses as regressors without interest.
Statistical analysis
For the overall sample, we used an ANCOVA model
comparing amygdala activity in risk-allele carriers (AA/AG) with
the corresponding activity in carriers of the protective allele (GG),
using sex, age, diagnostic category (BD, SZ, healthy control) and
medication status as covariates. Medication status was dichot-
omised for each of the categories Antipsychotics, Lithium,
Antidepressants, Anticonvulsives and Hypnotics. We tested for
potential specific effects in each diagnostic group (BD, SZ and
healthy controls) and phenotypic subcategory (bipolar I disorder,
bipolar II disorder, psychotic and non-psychotic features within
the BD group, schizophrenia patients within the SZ group), for
genotype6diagnosis interactions, and for effect of diagnostic
category on amygdala activity. Findings were regarded as
significant if they obtained a small volume corrected Family-Wise
Error (FWE) P-value below 0.05 within the respective ROI
(amygdala).
Statistical analyses of behavioural, sociodemographic and
clinical data were performed with IBM SPSS Statistics version
19.0.
Results
Behavioural results
Genotype group and diagnosis did not influence the accuracy
rate significantly. The mean response time (RT) was significantly
longer for individuals in the BD group (RT = 1255 milliseconds
(ms)) and SZ group (RT = 1231 ms) than those in the healthy
control group (RT = 1065 ms) (P,0.001), but did not differ
significantly with respect to genotype group. For details, see Table
S1.
fMRI results
The main fMRI results are presented in Table 1 and Figure 1.
In the total sample (N = 250), carriers of the CACNA1C SNP
rs1006737 risk allele (AA/AG) showed significantly increased
activation in the left amygdala (x = 224, y = 22, z = 214;
Z = 3.47; cluster-size = 72), with an FWE-corrected P-value of
0.026. The risk allele was also significantly associated with
enhanced activity in the left amygdala (x = 224, y = 0, z = 214;
Z = 3.35; cluster-size = 91) in the BD group (FWE-corrected
P = 0.041). There were also AA/AG associated increased activa-
tions in the right amygdala in the total sample (x = 26, y = 0,
z=216, Z = 2.65, cluster-size = 32) and in the BD group (x = 22,
y=0, z=220, Z = 2.54, cluster-size = 61), but these did not reach
FWE-corrected significance level. There were no FWE-corrected
significant findings in SZ individuals or healthy controls, but the
activation patterns had the same direction in all diagnostic groups
for the most significant voxel in the total sample (Figure S1), and
there were only nominally significant results in amygdala for the
AA/AG.GG contrast and none for the opposite contrast
(GG.AA/AG) in any of the groups (Table 1). There was no
significant genotype6diagnosis interaction effect between BD and
the other groups. Furthermore, there was no significant effect of
diagnosis or medication status on amygdala activity. Carriers of
the rs1006737 risk variant did not reach FWE-corrected
significance level in any of the phenotypic subcategories, and
there was no significant interaction effect of the risk allele between
bipolar I and II disorder or between psychotic and non-psychotic
BD subjects (Table S2 and Table S3).
CACNA1C Increases Amygdala Activity
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Discussion
The main finding in this study was an enhanced amygdala
activation in carriers of the CACNA1C SNP rs1006737 risk allele.
This is in line with two previous studies [12,13]. One of these
studies found that individuals with the risk allele have increased
activity in the right amygdala during a reward paradigm (N = 64)
[12], and the other reported increased activity in the right
amygdala during a fear-face paradigm (N = 116) [13]. In the
current study, we found significantly enhanced activation in the
left amygdala during a negative faces paradigm. But there was also
nominally significantly increased activity in the right amygdala in
our sample, although this finding did not remain significant after
FWE-correction (Table 1).
As increased amygdala activity in CACNA1C risk allele carriers
has been reported for both reward and aversive paradigms, it is
uncertain whether these responses reflect different psychological
responses and neurobiological pathways. These findings might also
represent a general effect, irrespective of emotional valence. The
latter interpretation is in accordance with recent data suggesting
that amygdala is occupied with relevance detection in general,
rather than with only fear-related or reward-related information
[30,31].
In the present study we found increased activity in the left
amygdala, while two other studies reported increased activity in
the right amygdala [12,13]. With regards to the laterality of
enhanced amygdala activity in BD, a meta-analysis reported
evidence for this effect being most pronounced in the left
hemisphere [11], which is in line with our findings. But more
and larger studies are needed to address the question of amygdala
laterality in BD. Further, despite several hypotheses on amygdala
laterality in general, it is still unclear whether the left and right
amygdalae are involved in different psychological mechanisms
[32].
When performing group-wise analyses, we found an increased
amygdala activity in risk allele carriers within the BD group, but
not in the SZ or healthy control group. However, there was no
significant diagnosis6genotype interaction, a phenomenon that
was probably due to the same direction of the activation pattern
through all diagnostic groups (Figure S1). Further, there were no
interaction effects for the risk allele between the phenotypic
subcategories within the BD spectrum (Table S2). Thus, we can
Table 1. Results for CACNA1C SNP rs1006737 (AA+AG.GG) effect on amygdala activation in a sample of bipolar disorder and
schizophrenia cases and healthy controls.
Hemisphere Group x y z Cluster size Z P uncorrected P FWE corrected
Left Total sample 224 22214 72 3.47 0.000 0.026
BD 224 0 214 91 3.35 0.000 0.041
SZ – – – – – n.s. –
CTR 224 22212 16 2.51 0.006 0.284
Right Total sample 26 0 216 32 2.65 0.004 0.217
BD 22 0 220 61 2.54 0.006 0.278
SZ 24 24216 26 2.24 0.013 0.449
CTR 28 4 216 2 1.96 0.025 0.598
Abbreviations: BD, bipolar disorder; SZ, schizophrenia; CTR, healthy controls; FWE, Family-wise error rate; n.s., non-significant.
Only nominally significant results (Nominal P = ,0.05) are shown.
doi:10.1371/journal.pone.0056970.t001
Figure 1. Increased amygdala activity in
CACNA1C
SNP rs1006737 risk allele carriers. Carriers of the CACNA1C SNP rs1006737 risk allele A
have significantly increased activity in the left amygdala in the total sample (x= 224, y = 22, z = 214; FWE P = 0.026) and BD subgroup (x = 224, y = 0,
z=214; FWE P = 0.041), and non-significantly increased activity in the right amygdala in the total sample (x = 26, y = 0, z = 216) and BD subgroup
(x = 22, y = 0, z = 220) compared with GG homozygotes during a negative faces paradigm. Abbreviations: BD, bipolar disorder; FWE, family-wise error.
Threshold for significance in Figure 1 is set to Nominal P,0.05 within the ROI.
doi:10.1371/journal.pone.0056970.g001
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not conclude with diagnostic specificity for the current effect of this
polymorphism.
Interestingly, recent GWA studies found the presently investi-
gated SNP to be significantly associated with BD (P = 1.7610
25
;
OR = 1.11) as well as SZ (P = 1.2610
26
; OR = 1.11) [1,15]. This
suggests susceptibility of similar effect sizes on the clinical
phenotype level, a finding which is in accordance with the current
lack of evidence for specificity at the brain activity level. Two
recent studies might shed further light on the genotype/phenotype
relationship between the CACNA1C polymorphism and BD and
SZ. One of these studies found that healthy risk allele carriers had
elevated hippocampus activity during emotional processing, and
increased activity in the prefrontal cortex during executive
cognition [14]. As the former phenomenon has been reported in
BD and the latter in SZ, these results correspond to the findings of
a pleiotropic effect of this SNP. Another recent study reported
impaired working memory in risk allele carriers among SZ cases
and healthy controls, but not in BD cases, potentially implying
differential specificity in different diagnostic groups [33] for this
neurocognitive phenotype.
This phenomenon of unspecific effects at some levels and
differential diagnostic specificity at other levels could be explained
by different, although overlapping, overall genetic architecture
between BD and SZ patients [34]. In such a model, the cumulative
effect of all risk variants, including gene-gene and gene-environ-
ment interactions, could condition the role of the current
CACNA1C SNP in the pathophysiological processes related to
these disorders.
Genetic variations in CACNA1C have been shown to imply
additional psychiatric manifestations to those observed in BD and
SZ. Patients with the above-mentioned Timothy syndrome are
characterized by symptoms like autism and cognitive disability. In
a proposed model of a spectrum of psychiatric disorders with
autism in the neurodevelopmental end and MDD in the affective
end [17], it is possible that polymorphisms in or around the
CACNA1C gene could increase the risk of developing less severe
conditions than those observed in Timothy syndrome, like BD and
SZ. This is further supported by the fact that rs1006737 is situated
in one of the introns of CACNA1C, thus probably affecting
pathophysiological pathways related to these disorders by regulat-
ing the expression of the protein, and not by altering the structure,
as is the case with the de novo mutations in Timothy syndrome,
which are located in one of the exons [9]. In this respect, it is
noteworthy that a recent post mortem brain expression study
found healthy carriers of the CACNA1C risk-associated SNP to
express higher levels of CACNA1C mRNA than carriers of the
protective allele [14].
Hence, genetically conditioned calcium channel dysregulation
resulting from CACNA1C risk variants might be a common
mechanism increasing the risk for developing several neuropsy-
chiatric disorders, by affecting various brain structures, including
amygdala.
Taken together, the current findings provide evidence that the
CACNA1C SNP rs1006737 is associated with increased amygdala
activity across different diagnostic groups. These findings add to
the growing body of knowledge on the pleiotropic effect of this
polymorphism.
Supporting Information
Table S1 Demographic data and clinical characteriza-
tion for individuals genotyped for rs1006737 and
participating in a negative faces functional MRI study.
Abbreviations: BD, bipolar disorder; SZ, schizophrenia; CTR,
controls; SD, standard deviation; WASI, Wechsler Abbreviated
Scale of Intelligence; IDS, Inventory of Depressive Symptoms;
YMRS, Young Mania Rating Scale; PANSS, Positive and
Negative Syndrome Scale; GAF-S, Global Assessment of Func-
tioning–symptom score; GAF-F, Global Assessment of Function-
ing–function score; ms, milliseconds. aMean age at fMRI
scanning. bLast six months.
(DOC)
Table S2 Results for the effect of CACNA1C SNP
rs1006737 (AA
+
AG
.
GG) interaction with diagnostic
category on amygdala activation. Abbreviations: BD, bipolar
disorder; SZ, schizophrenia; CTR, healthy controls; BDI, bipolar I
disorder; BDII, bipolar II disorder; FWE, Family-wise error rate;
n.s., non-significant. Only nominally significant results (Nominal
P=,0.05) are shown.
(DOC)
Table S3 Results for CACNA1C SNP rs1006737
(AA
+
AG
.
GG) effect on amygdala activation in pheno-
typic subcategories. Abbreviations: BD, bipolar disorder; BDI,
bipolar I disorder; BDII, bipolar II disorder; FWE, Family-wise
error rate; n.s., non-significant. Only nominally significant results
(Nominal P = ,0.05) are shown.
(DOC)
Figure S1 Parameter estimates for diagnostic and
genotype groups for CACNA1C SNP rs1006737 effect on
amygdala activation. Parameter estimates for the contrast
AA+AG.GG in the voxel with highest activation (x = 224,
y=22, z = 214) in the total sample (N = 250) in different
diagnostic and genotype groups. Prot = GG genotype group.
Risk = AA+AG genotype group. Abbreviations: BD, bipolar
disorder; SZ, schizophrenia; CTR, healthy controls.
(DOC)
Acknowledgments
We thank patients and controls for their participation in the study, and the
health professionals who facilitated our work. We also thank Thomas D.
Bjella for assistance with the database and Christine Lycke and Niels Petter
Sigvartsen for assistance with fMRI data collection and analyses.
Author Contributions
Conceived and designed the experiments: MT OAA SD IM IA JJ.
Performed the experiments: MT KCS OTO CT. Analyzed the data: MT
KCS CT OAA JJ. Contributed reagents/materials/analysis tools: JJ SD
OAA IA IM. Wrote the paper: MT OTO JJ OAA AAB KCS.
References
1. Sklar P, Ripke S, Scott LJ, Andreassen OA, Cichon S, et al. (2011) Large-scale
genome-wide association analysis of bipolar disorder identifies a new susceptibility
locus near ODZ4. Nat Genet 43: 977–983. ng.943 [pii];10.1038/ng.943 [doi].
2. Sklar P, Smoller JW, Fan J, Ferreira MA, Perlis RH, et al. (2008) Whole-genome
association study of bipolar disorder. Mol Psychiatry 13: 558–569. 4002151
[pii];10.1038/sj.mp.4002151 [doi].
3. Ferreira MA, O’Donovan MC, Meng YA, Jones IR, Ruderfer DM, et al. (2008)
Collaborative genome-wide association analysis supports a role for ANK3 and
CACNA1C in bipolar disorder. Nat Genet 40: 105 6–1058. ng.209
[pii];10.1038/ng.209 [doi].
4. Green EK, Grozeva D, Jones I, Jones L, Kirov G, et al. (2010) The bipolar
disorder risk allele at CACNA1C also confers risk of recurrent major depression
and of schizophrenia. Mol Psychiatry 15: 1016–1022. mp200949 [pii];10.1038/
mp.2009.49 [doi].
CACNA1C Increases Amygdala Activity
PLOS ONE | www.plosone.org 5 February 2013 | Volume 8 | Issue 2 | e56970

5. Nyegaard M, Demontis D, Foldager L, Hedemand A, Flint TJ, et al. (2010)
CACNA1C (rs1006737) is associated with schizophrenia. Mol Psychiatry 15:
119–121. mp200969 [pii];10.1038/mp.2009.69 [doi].
6. Wray NR, Pergadia ML, Blackwood DH, Penninx BW, Gordon SD, et al.
(2012) Genome-wide association study of major depressive disorder: new results,
meta-analysis, and lessons learned. Mol Psychiatry 17: 36–48. mp2010109
[pii];10.1038/mp.2010.109 [doi].
7. Catterall WA (2011) Voltage-gated calcium channels. Cold Spring Harb Perspect
Biol 3: a003947. cshperspect.a003947 [pii];10.1101/cshperspect.a003947 [doi].
8. Zuccotti A, Clementi S, Reinbothe T, Torrente A, Vandael DH, et al. (2011)
Structural and functional differences between L-type calcium channels: crucial
issues for future selective targeting. Trends Pharmacol Sci 32: 366–375. S0165-
6147(11)00034-4 [pii];10.1016/j.tips.2011.02.012 [doi].
9. Splawski I, Timothy KW, Sharpe LM, Decher N, Kumar P, et al. (2004)
Ca(V)1.2 calcium channel dysfunction causes a multisystem disorder including
arrhythmia and autism. Cell 119: 19–31. 10.1016/j.cell.2004.09.011
[doi];S0092867404008426 [pii].
10. Hallahan B, Newell J, Soares JC, Brambilla P, Strakowski SM, et al. (2011)
Structural magnetic resonance imaging in bipolar disorder: an international
collaborative mega-analysis of individual adult patient data. Biol Psychiatry 69:
326–335. S0006-3223(10)00913-3 [pii];10.1016/j.biopsych.2010.08.029 [doi].
11. Chen CH, Suckling J, Lennox BR, Ooi C, Bullmore ET (2011) A quantitative
meta-analysis of fMRI studies in bipolar disorder. Bipolar Disord 13: 1–15.
10.1111/j.1399-5618.2011.00893.x [doi].
12. Wessa M, Linke J, Witt SH, Nieratschker V, Esslinger C, et al. (2010) The
CACNA1C risk variant for bipolar disorder influences limbic activity. Mol
Psychiatry 15: 1126–1127. mp2009103 [pii];10.1038/mp.2009.103 [doi].
13. Jogia J, Ruberto G, Lelli-Chiesa G, Vassos E, Maieru M, et al. (2011) The
impact of the CACNA1C gene polymorphism on frontolimbic function in
bipolar disorder. Mol Psychiatry 16: 1070–1071. mp201149 [pii];10.1038/
mp.2011.49 [doi].
14. Bigos KL, Mattay VS, CallicottJH, Straub RE, Vakkalanka R, et al. (2010) Genetic
variation in CACNA1C affects brain circuitries related to mental illness. Arch Gen
Psychiatry 67: 939–945. 67/9/939 [pii];10.1001/archgenpsychiatry.2010.96 [doi].
15. Ripke S, Sanders AR, Kendler KS, Levinson DF, Sklar P, et al. (2011) Genome-
wide association study identifies five new schizophrenia loci. Nat Genet 43: 969–
976. ng.940 [pii];10.1038/ng.940 [doi].
16. Simonsen C, Sundet K, Vaskinn A, Birkenaes AB, Engh J, et al. (2011)
Neurocognitive dysfunction in bipolar and schizophrenia spectrum disorders
depends on history of psychosis rather than diagnostic group. Schizophr Bull 37:
73–83. sbp034 [pii];10.1093/schbul/sbp034 [doi].
17. Craddock N, Owen MJ (2010) The Kraepelinian dichotomy - going, going…
but still not gone. Br J Psychiatry 196: 92–95. 196/2/92 [pii];10.1192/
bjp.bp.109.073429 [doi].
18. Hariri AR, Tessitore A, Mattay VS, Fera F, Weinberger DR (2002) The
amygdala response to emotional stimuli: a comparison of faces and scenes.
Neuroimage 17: 317–323. S1053811902911791 [pii].
19. Spitzer RL, Williams JB, Gibbon M, First MB (1992) The Structured Clinical
Interview for DSM-III-R (SCID). I: History, rationale, and description. Arch
Gen Psychiatry 49: 624–629.
20. Young RC, Biggs JT, Ziegler VE, Meyer DA (1978) A rating scale for mania:
reliability, validity and sensitivity. Br J Psychiatry 133: 429–435.
21. Rush AJ, Gullion CM, Basco MR, Jarrett RB, Trivedi MH (1996) The
Inventory of Depressive Symptomatology (ID S): psychometric properties.
Psychol Med 26: 477–486.
22. Kay SR, Fiszbein A, Opler LA (1987) The positive and negative syndrome scale
(PANSS) for schizophrenia. Schizophr Bull 13: 261–276.
23. Welander-Vatn AS, Jensen J, Lycke C, Agartz I, Server A, et al. (2009) No
altered dorsal anterior cingulate activation in bipolar II disorder patients during
a Go/No-go task: an fMRI study. Bipolar Disord 11: 270–279. BDI680
[pii];10.1111/j.1399-5618.2009.00680.x [doi].
24. Athanasiu L, Mattingsdal M, Kahler AK, Brown A, Gustafsson O, et al. (2010)
Gene variants associated with schizophrenia in a Norwegian genome-wide study
are replicated in a large European cohort. J Psychiatr Res 44: 748–753. S0022-
3956(10)00036-1 [pii];10.1016/j.jpsychires.2010.02.002 [doi].
25. Djurovic S, Gustafsson O, Mattingsdal M, Athanasiu L, Bjella T, et al. (2010) A
genome-wide association study of bipolar disorder in Norwegian individuals,
followed by replication in Icelandic sample. J Affect Disord 126: 312–316.
S0165-0327(10)00343-5 [pii];10.1016/j.jad.2010.04.007 [doi].
26. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, et al. (2007)
PLINK: a tool set for whole-genome association and population-based linkage
analyses. Am J Hum Genet 81: 559–575. S0002-9297(07)61352-4 [pii];10.1086/
519795 [doi].
27. Tottenham N, Tanaka JW, Leon AC, McCarry T, Nurse M, et al. (2009) The
NimStim set of facial expressions: judgments from untrained research
participants. Psychiatry Res 168: 242–249. S0165-1781(08)00148-0
[pii];10.1016/j.psychres.2008.05.006 [doi].
28. Friston KJ, Holmes AP, Poline JB, Grasby PJ, Williams SC, et al. (1995) Analysis
of fMRI time-series revisited. Neuroimage 2: 45–53. S1053-8119(85)71007-5
[pii];10.1006/nimg.1995.1007 [doi].
29. Maldjian JA, Laurienti PJ, Kraft RA, Burdette JH (2003) An automated method
for neuroanatomic and cytoarchitectonic atlas-based interrogation of fMRI data
sets. Neuroimage 19: 1233–1239. S1053811903001691 [pii].
30. Ousdal OT, Jensen J, Server A, Hariri AR, Nakstad PH, et al. (2008) The human
amygdala is involved in general behavioral relevance detection: evidence from an
event-related functional magnetic resonance imaging Go-NoGo task. Neuroscience
156: 450–455. S0306-4522(08)01172-X [pii];10.1016/j.neuroscience.2008.07.066
[doi].
31. Sander D, Grafman J, Zalla T (2003) The human amygdala: an evolved system
for relevance detection. Rev Neurosci 14: 303–316.
32. Blond BN, Fredericks CA, Blumberg HP (2012) Functional neuroanatomy of
bipolar disorder: structure, function, and connectivity in an amygdala-anterior
paralimbic neur al system. Bipolar Disord 14: 340–355. 10.1111/j.1399-
5618.2012.01015.x [doi].
33. Zhang Q, Shen Q, Xu Z, Chen M, Cheng L, et al. (2012) The effects of
CACNA1C gene polymorphism on spatial working memory in both healthy
controls and patients with schizophrenia or bipolar disorder. Neuropsychoph ar-
macology 37: 677–684. npp2011242 [pii];10.1038/npp.2011.242 [doi].
34. Hamshere ML, O’Donovan MC, Jones IR, Jones L, Kirov G, et al. (2011)
Polygenic dissection of the bipolar phenotype. Br J Psychiatry 198: 284–288.
198/4/284 [pii];10.1192/bjp.bp.110.087866 [doi].
CACNA1C Increases Amygdala Activity
PLOS ONE | www.plosone.org 6 February 2013 | Volume 8 | Issue 2 | e56970