Embed Size (px)
Citation preview
RESEARCH ARTICLE
Association Study of PDE4B Gene Variants inScandinavian Schizophrenia and Bipolar DisorderMulticenter Case–Control SamplesAnna K. K€ahler,1,2,3* Mona K. Otnæss,1,3 Katrine V. Wirgenes,1,3 Thomas Hansen,4 Erik G. J€onsson,5
Ingrid Agartz,1,5,6 Ha�
kan Hall,5 Thomas Werge,4 Gunnar Morken,7 Ole Mors,8 Erling Mellerup,9,10
Henrik Dam,9 Pernille Koefod,9,10 Ingrid Melle,1,3 Vidar M. Steen,11,12 Ole A. Andreassen,1,3
and Srdjan Djurovic1,2,3
1Institute of Psychiatry, University of Oslo, Oslo, Norway2Department of Medical Genetics, Oslo University Hospital – Ulleval, Oslo, Norway3Department of Psychiatry, Oslo University Hospital – Ulleval, Oslo, Norway4Research Institute of Biological Psychiatry, H:S Sct. Hans Hospital, Roskilde, Denmark5Department of Clinical Neuroscience, HUBIN Project, Psychiatry Section, Karolinska Institutet and Hospital, Stockholm, Sweden6Department of Psychiatric Research, Diakonhjemmet Hospital, Oslo, Norway7Østmarka Psychiatric Department, St Olavs Hospital and Institute of Neuroscience, Norwegian University of Technology and Science,
Trondheim, Norway8Centre for Psychiatric Research, Aarhus University Hospital, Risskov, Denmark9Center of Psychiatry, Rigshospitalet, Copenhagen, Denmark10Department of Neuroscience and Pharmacology, University of Copenhagen, Laboratory of Neuroscience, Copenhagen, Denmark11Dr. Einar Martens Research Group for Biological Psychiatry, Department of Clinical Medicine, University of Bergen, Bergen, Norway12Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway
Received 6 October 2008; Accepted 19 February 2009
The phosphodiesterase 4B (PDE4B), which is involved in cogni-
tive function in animal models, is a candidate susceptibility gene
for schizophrenia (SZ) and bipolar disorder (BP). Variations in
PDE4B have previously been associated with SZ, with a suggested
gender-specific effect. We have genotyped and analyzed 40 and 72
tagging single nucleotide polymorphisms (tagSNPs) in SZ and
BP multicenter samples, respectively, from the Scandinavian
Collaboration on Psychiatric Etiology (SCOPE), involving 837
SZ cases and 1,473 controls plus 594 BP cases and 1,421 partly
overlapping controls. Six and 16 tagSNPs were nominally asso-
ciated (0.0005 �P� 0.05) with SZ and BP, respectively, in the
combined samples or in gender-specific subgroups. None of
Additional Supporting Information may be found in the online version of
this article.
Grant sponsor: Research Council of Norway; Grant numbers: #167153/
V50, #163070/V50, #175345/V50; Grant sponsor: Eastern and Western
Norway Health Authority; Grant number: #123–2004; Grant sponsor:
Ulleva�l University Hospital; Grant sponsor: University of Oslo; Grant
sponsor: Copenhagen Hospital Corporation Research Fond; Grant
sponsor: Danish National Psychiatric Research Foundation; Grant
sponsor: Danish Agency for Science, Technology and Innovation
(Centre for Pharmacogenomics); Grant sponsor: Danish Medical
Research Council; Grant sponsor: Lundbeck Foundation; Grant
sponsor: The Stanley Medical Research Institute; Grant sponsor:
Wallenberg Foundation; Grant sponsor: HUBIN Project; Grant sponsor:
Swedish Research Council; Grant number: K2007-62X-15078-04-1; Grant
number: K2007-62X-15078-04-3; Grant number: K2008-62P-20597-01-3.
*Correspondence to:
Anna K. K€ahler, Section for Psychosis Research, Building 49, Department
for Research and Development, Division of Psychiatry, Oslo University
Hospital – Ulleval, Kirkeveien 166, N-0407 Oslo, Norway.
E-mail: [email protected]
Published online 6 April 2009 in Wiley InterScience
(www.interscience.wiley.com)
DOI 10.1002/ajmg.b.30958
How to Cite this Article:K€ahler AK, Otnæss MK, Wirgenes KV,
Hansen T, J€onsson EG, Agartz I, Hall H,
Werge T, Morken G, Mors O, Mellerup E,
Dam H, Koefod P, Melle I, Steen VM,
Andreassen OA, Djurovic S. 2010. Association
Study of PDE4B Gene Variants in
Scandinavian Schizophrenia and Bipolar
Disorder Multicenter Case–Control Samples.
Am J Med Genet Part B 153B:86–96.
� 2009 Wiley-Liss, Inc. 86
Neuropsychiatric Genetics
these findings remained significant after correction for multiple
testing. However, a number of tagSNPs found to be nominally
associated with SZ and BP were located in a high LD region
spanning the splice site of PDE4B3, an isoform with altered
brain expression in BP patients. Four tagSNPs were associated
with SZ in women, but none in men, in agreement with the
previously reported gender-specific effect. Proxies of two
nominally associated SNPs in the SZ sample were also associated
with BP, but the genotypic effect (i.e., homozygosity for the
minor allele), pointed in opposite directions. Finally, four
SNPs were found to be associated with Positive And Negative
Syndrome Scale (PANSS) positive symptom scores in a
subgroup of SZ patients (n¼ 153) or SZ female patients
(n¼ 70). Further studies are needed to evaluate the implicated
PDE4B region of interest, for potential involvement in SZ and BP
susceptibility. � 2009 Wiley-Liss, Inc.
Key words: candidate gene; genetic association; psychotic
disorder; PDE4B isoform; PDE4B3
INTRODUCTION
Phosphodiesterase 4B (PDE4B) belongs to a family of four PDE4
genes, all coding for phosphodiesterases that hydrolyze the second
messenger cyclic adenosine monophosphate (cAMP) [Houslay
and Adams, 2003]. The PDE4B gene encodes at least four different
isoforms, each with a unique N-terminal region [Cheung et al.,
2007; Murdoch et al., 2007].
PDE4B was first suggested as a risk factor for schizophrenia (SZ)
through the study of a Scottish family with a balanced t(1;16)
translocation that directly disrupts PDE4B on chromosome 1p31
[Millar et al., 2005]. This translocation was inherited by two
cousins, one diagnosed with SZ and the other with a psychotic
disorder. Subsequent case–control genetic association studies of
Scottish [Pickard et al., 2007], Japanese [Numata et al., 2008b], and
Caucasian and African American [Fatemi et al., 2008] samples have
reported an association between PDE4B and SZ. In the study by
Pickard et al. [2007], PDE4B variants were only associated with
SZ in women.
The distinction of SZ and bipolar disorder (BP) as separate
biological entities is currently debated, and an etiological overlap
has been suggested [Moller, 2003; Owen et al., 2007]. PDE4B is
an interesting candidate gene for both SZ and BP. PDE4 genes are
orthologous to the dunce gene in Drosophila melanogaster, and
dunce mutants show impaired learning and memory [Davis et al.,
1995], which is among the most consistently reported neurocog-
nitive deficits in both SZ [Barch, 2005] and BP [Martinez-Aran
et al., 2004; Simonsen et al., 2008]. Also, the selective PDE4-
inhibitor Rolipram, has been shown to have antidepressant effects
in humans [Zhu et al., 2001], as well as antipsychotic-like behav-
ioral effects in mice [Kanes et al., 2007] and rats [Siuciak et al.,
2007]. The expression of PDE4B isoforms in postmortem brain
tissue from patients with SZ or BP has been shown to differ
compared with controls [Fatemi et al., 2008]. Furthermore, all
four reported PDE4B isoforms have been demonstrated to interact
with Disrupted-in-schizophrenia-1 (DISC1) [Millar et al., 2005;
Murdoch et al., 2007], a protein encoded by DISC1 which has been
identified as a susceptibility gene for SZ and BP in several studies
[Chubb et al., 2008].
We investigated the potential involvement of PDE4B in SZ and
BP, using gene-wide genotyping of tagging single nucleotide poly-
morphisms (tagSNPs) in Scandinavian multicenter case–control
samples.
MATERIALS AND METHODS
Sample DescriptionThe schizophrenia case–control sample. The SZ association
study was based on three independent case–control samples from
Norway, Sweden, and Denmark, included in the Scandinavian
Collaboration on Psychiatric Etiology (SCOPE). A total of 837 SZ
spectrum cases (SZ (n¼ 734), schizoaffective disorder (SZA)
(n¼ 87), schizophreniform disorder (SZPH) (n¼ 16)), and
1,473 control subject samples were successfully genotyped. The
Norwegian patients had been diagnosed with SZ (n¼ 124), SZA
(n¼ 31), or SZPH (n¼ 8) disorder, according to DSM-IV using
Structural Clinical Interview for DSM-IV (SCID), the Danish
patients with SZ (n¼ 388) or SZA (n¼ 31) according to ICD-10,
and the Swedish patients with SZ (n¼ 224), SZA (n¼ 25), or
SZPH (n¼ 8), according to DSM-III-R/DSM-IV. There is high
concordance between the ICD-10 and DSM systems (pairwise
concordance rate (CR)> 0.70, k> 0.70) [Jakobsen et al., 2006].
The patient and control samples are described in more detail
elsewhere [Hansen et al., 2007; Kahler et al., 2008]. Since the vast
majority of the patients included in the SZ spectrum sample were
diagnosed with SZ, further analysis of diagnostic subgroups were
not performed due to low statistical power, and for simplicity,
we generally refer to schizophrenia/SZ as the clinical phenotype
throughout the text.
The bipolar case–control sample. The BP association study was
based on two independent case–control samples from Norway and
Denmark. A total of 594 BP cases and 1,421 control samples were
successfully genotyped. The Norwegian patients had been diag-
nosed with bipolar disorder type I (BPI) (n¼ 125), bipolar disorder
type II (BPII) (n¼ 80), and BP not otherwise specified (NOS)
(n¼ 13), according to DSM-IV using SCID. The Danish patients
had been included all over Denmark (1996–1998) (n¼ 161), or in
the Copenhagen area by the Danish Psychiatric Biobank
(2002–2007) (n¼ 215). The first patient group had been diagnosed
with SCAN [Wing et al., 1998] interviews fulfilling a best estimate
diagnosis of bipolar affective disorder (n¼ 81) and BPI (n¼ 80),
according to the ICD-10-DCR [WHO, 1993] and the DSM-IV,
respectively. The latter group was clinically diagnosed with bipolar
affective disorder according to ICD-10-DCR [WHO, 1993]. We
generally refer to Bipolar disorder/BP as the clinical phenotype
throughout the text. The Norwegian healthy controls (n¼ 220) are
described in more detail elsewhere [Hansen et al., 2007; Kahler et al.,
2008], and a subset (n¼ 152) are overlapping with controls in the
SZ case–control sample. The Danish controls were distinct from
those in the SZ case–control sample, but recruited as previously
described (n¼ 1,133) [Hansen et al., 2007], or included as selected
controls screened for psychiatric disease in a previous study
(n¼ 68) [Mellerup et al., 2001].
K€AHLER ET AL. 87
The Norwegian Scientific-Ethical Committees, the Norwegian
Data Protection Agency, the Danish Ethical Committees, the
Danish Data Protection Agency, the Ethical Committee of the
Karolinska Hospital, the Stockholm Regional Ethical Committee
and the Swedish Data Inspection Board approved the respective
parts of the study. All patients have given written informed consent
prior to inclusion into the project.
SNP Selection and GenotypingTo evaluate if PDE4B variants are associated with SZ and BP, a
structured gene-wide approach was used, by genotyping tagSNPs.
The tagSNPs were selected at the HapMap website
(www.hapmap.org), based on the CEU population, using pair-wise
tagging, with r2� 0.8 [de Bakker et al., 2005] (www.hapmap.org;
HapMap Data Release 21 for the SZ study, and Release 22 for the BP
study). The assumed northern and western European ancestry of
the CEU population has recently been genetically confirmed [Lao
et al., 2008]. PDE4B (NM_001037341) is a large gene, spanning
582.1 kb, with�450 SNPs with minor allele frequency (MAF)� 5%
(HapMap Data Release 23a). As a first screen of the most common
SNPs in the SZ sample, a MAF� 20% was used as the cut-off when
choosing tagSNPs. The tagSNPs genotyped in the BP sample were
picked independently in a separate genotyping project, using
MAF� 5% to cover most of the common variation.
Genomic DNA was extracted from whole blood, and both
the SZ and BP samples were genotyped as part of two larger
genotyping projects, using the GoldenGate 1536plex assay
(Illumina, Inc., San Diego, CA) on the Illumina BeadStation 500GX
at the SNP Technology Platform, Uppsala University, Sweden
(www.genotyping.se), accredited by the Swedish accreditation
agency SWEDAC, and approved according to a quality system
based on the international SS-EN ISO/IEC 17025 standard. There
were only two duplicate errors in 85,674 duplicate genotype calls
(reproducibility of 99.998%) and five duplicate errors in 124,684
duplicate genotypes calls (reproducibility of 99.996%) for the SZ
and BP genotyping projects, respectively.
The actual tagging efficiency of successfully genotyped tagSNPs
was calculated using HapMap Data Release 21 at the Tagger website
(www.broad.mit.edu/mpg/tagger/server.html).
Statistical AnalysisAll SNPs were tested for departure from Hardy Weinberg-equilib-
rium in cases and controls separately, using the exact chi-square
test implemented in PLINK (version 1.04; http://
pngu.mgh.harvard.edu/purcell/plink/) [Purcell et al., 2007]. Po-
tential SNPs with P< 0.001 in controls were considered in Hardy
Weinberg disequilibrium (HWD) and excluded.
To estimate the level of heterogeneity between the three Scandi-
navian subpopulations in the SZ case–control sample, an overall
fixation index FST has previously been calculated for a larger SNP
set, using the control samples from Norway, Denmark, and Sweden,
showing no evidence of population stratification [Kahler et al.,
2008]. In addition, for the present study we calculated the gene-
based FST for PDE4B, in both the SZ and BP control sample sets,
as implemented in Arlequin 3.1 [Excoffier et al., 2005].
Allelic and genotypic single SNP association tests, as well as
a sliding-window haplotype analysis, were performed with
UNPHASED (version 3.0.13) [Dudbridge, 2008]. To account for
potential population stratification, the population status was
included as a confounder with discrete levels. Pairwise LD (D’ and
r2) and LD blocks were estimated in Haploview 4.1 [Barrett et al.,
2005], the latter using the solid spine definition for the most
extensively genotyped BP sample, acknowledging that such an
estimation is limited when based on tagSNPs. Haplotype effects
were examined by global and individual haplotype association
tests, including 2-, 3-, and 4-marker sliding window haplotypes.
Estimated haplotypes with a frequency below 0.05 in both cases and
controls were considered rare and excluded from the association
tests. We set the nominal significance threshold to P¼ 0.05. For
nominally associated SNPs, the P-values and odds ratios (ORs)
for each individual genotype compared to the other genotypes
pooled together, was calculated. Also, ORs for the risk allele and the
individual genotypes with the largest effect size were determined
in each population separately, using UNPHASED. Each test was
corrected for the multiple SNPs or haplotypes assessed, using
10,000 permutations.
Because a gender-specific effect of PDE4B has previously been
reported [Pickard et al., 2007], the above analyses were also
performed on data subdivided on the basis of gender.
Association With Clinical SymptomsThe diagnosis of SZ is based on the presence of positive symptoms
(e.g., delusions and hallucinations), also frequently observed in BP
during manic episodes, and/or negative symptoms (e.g., flattening
of affect and lack of volition and drive), commonly observed in BP
during depressive episodes. A subset of Norwegian SZ patients
(n¼ 153; 54.2% men, 45.8% women), and BP patients (n¼ 128;
39.8% men, 60.2% women), were symptomatically evaluated with
the Positive And Negative Syndrome Scale (PANSS) [Kay et al.,
1987]. Symptom scores were tested for potential association with
the SNPs nominally associated with SZ or BP diagnosis. Assess-
ments were interview based, and performed by experienced MDs
or psychologists. The SZ sample was moderately symptomatic with
PANSS positive and negative sum scores being 15.3 5.8 and
15.1 6.1, respectively. The BP sample was less symptomatic, with
PANSS positive and negative sum scores being 9.7 2.8 and 10.7 4.0,
respectively. Due to departure from normal distribution, the
genotype–phenotype association analysis was performed using the
non-parametric Kruskal–Wallis test, implemented in SPSS
(version 16.0). The genotype distribution for each SNP was used
as grouping variable, and PANSS positive and negative sum score as
dependent variable. The nominal level of significance was set to
P¼ 0.05, and P-values were Bonferroni corrected for the number
of SNPs assessed in each tested group.
RESULTS
Genotyping and tagSNP CoverageForty out of 44 selected tagSNPs in PDE4B (NM_001037341) were
included in the SZ case–control study, based on probability of
88 AMERICAN JOURNAL OF MEDICAL GENETICS PART B
successful assay design. All 40 PDE4B tagSNPs had a call rate
>96.7%, and the total genotyping rate was 99.51%. Genotype
counts for all SNPs are given in Supplementary Table I. No tagSNPs
had genotype distributions in HWD in controls (P> 0.001);
lowest P in cases was 0.001 for rs12136401. The 40 tagSNPs had
86% coverage with r2 � 0.8 (mean r2 ¼ 0.93 and minimum
r2¼ 0.40), of the 318 SNPs included in the Hapmap data at the
Tagger website (MAF� 20%).
Seventy-three out of 76 selected tagSNPs in PDE4B were suc-
cessfully genotyped in the BP case–control sample. TagSNP
rs6692281 was excluded because only one minor allele was present.
The remaining tagSNPs had a call rate> 94.8%, and the total
genotyping rate was 99.66%. Genotype counts for all SNPs are
given in Supplementary Table II. None of the tagSNPs had genotype
distributions in HWD in either cases or controls (P> 0.001). The
72 tagSNPs had 92% coverage with r2� 0.8 (mean r2¼ 0.94 and
minimum r2 ¼ 0.26), of the 449 SNPs included in the Hapmap data
at the Tagger website (MAF� 5%).
Population StratificationThe gene-based FSTs were 0.00004 and �0.00008, for PDE4B in the
SZ and BP sample, respectively, showing no evidence of stratifica-
tion between the control populations in each of the two case–control samples.
Single tagSNP Association DataSNPs nominally associated in the genotype- and/or allele-based test
for the SZ and BP case–control sample, are presented in Table I.
Gender-specific results are given for the tagSNPs associated with
disease only in females or males. An overview of the ORs for the
most associated individual genotypes for the tagSNPs in Table I are
given in Table II.
Schizophrenia sample. There were nominally significant asso-
ciations between two (rs596662 and rs1892346; r2¼ 0.04) out of 40
independent tagSNPs and SZ in the combined case–control sample,
in both genotype- and allele-based tests. The major alleles conferred
risk with an effect size larger (rs596662) or similar (rs1892346) in
the separate c2-tests for the Danish sample (Table I). However, for
both SNPs, the homozygotes for the minor allele gave the largest
individual genotypic effect (with OR< 1; Table II).
When genders were analyzed separately, there were nominally
significant associations between rs12088813, rs3009872, rs1937450,
and rs2455032 (previous ID: rs9436312) and SZ in females. All but
one was nominally associated both in allele- and genotype-based
tests (P< 0.046), but not in males (P> 0.61), with the major alleles
conferring a risk effect in each of the three case–control samples,
as well as in the combined sample (Table I). These SNPs are present
in a high LD region, flanking the splice site for the PDE4B3 isoform
(see Supplementary Figure 1). For all four SNPs the major allele
homozygotes were nominally associated with an increased risk
for SZ (ORs 1.42–1.48; Table II). However, none of the tagSNPs
remained significantly associated with SZ after correction for
multiple testing (10,000 permutations) within each sample set
analyzed (P� 0.17). No tagSNPs were associated when analyzed
only in males.
Bipolar disorder sample. There were nominally significant
associations between 11 out of the 72 successfully genotyped
tagSNPs and BP, in allele- or genotype-based tests (0.0005�P� 0.05). TagSNPs rs7552762 and rs12080701 are in complete
LD (r2¼ 1.0). For the strongest associated tagSNP, rs17452121, the
best fitting model was recessive (homozygote G/G: OR(95%CI)¼4.43(1.78–11.02), P¼ 0.00052). Three tagSNPs nominally associ-
ated with BP (rs17452121, rs2186122, and rs1937451) are located
close to the splice site for the PDE4B3 isoform, present in an
estimated three-tagSNP LD block. Five additional tagSNPs were
nominally associated only in the male (three) or female (two)
subgroups (Table I). However, none of the tagSNPs remained
significantly associated with BP after correction for multiple
testing (10,000 permutations), within each sample set analyzed
(P� 0.069).
Overlapping association signals in the schizophrenia and
bipolar disorder samples. All of the six nominally associated
tagSNPs in the SZ sample have either been genotyped themselves
(rs2455032) or by proxies in the BP sample. Two of these proxies,
rs11208776 and rs2186122 (r2 ¼ 0.93 and r2 ¼ 1.0 for SZ tagSNPs
rs1937450 and rs3009872, respectively, in HapMap CEU), were
nominally associated with BP in the total sample (Table I). All four
tagSNPs were present in a 48 kb region spanning the PDE4B3 splice
site (see Supplementary Figure 1). However, increased risk for
SZ was associated with being homozygous for the major allele, while
in contrast the homozygotes for the minor allele displayed increased
risk for BP.
Haplotype Association DataSchizophrenia sample. In the total sample set, the association
signal was not strengthened by consideration of 2-, 3-, and 4-SNP
haplotypes. Two 2-SNP haplotypes, including either rs1892346
or rs596662 and a tagSNP in high LD (D’> 0.81), were nominally
associated (P< 0.042).
In the female population, several haplotypes were nominally
associated, with larger effect sizes when combining several alleles
(Table III). The strongest overall and individual haplotype associ-
ation results were attained when combining tagSNPs rs2455032-
rs1354060-rs6588186 (Pglobal ¼ 0.0032; Phaplotype G-A-T ¼ 0.0080,
OR¼ 1.62 (CI (95%): 1.12–2.35)). The G-A-T haplotype was
present in 9.8% and 6.1% in cases and controls, respectively. None
of these associations remained significant after correction for
multiple testing (P� 0.090).
Bipolar disorder sample. In the total sample set, the best 2-SNP
result was obtained for haplotypes combining rs17452121 and
rs2186122, although the association was similar to the single SNP
results (Pglobal¼ 0.030, Pindividual,A-T ¼ 0.010). The 3-SNP results
did not strengthen the significance. The best overall 4-SNP result
was obtained with SNPs (rs558325-rs1040716-rs11803904-
rs12142015) that were not associated in the single tagSNP analysis
(Pglobal ¼ 0.0057), but none of the individual haplotypes displayed
association with disease (0.15� P� 0.96). In females, one 2-SNP
haplotype was nominally associated, but none of the 3- or 4-SNP
haplotypes. In males, four of the five nominally associated
2-SNP haplotypes are combinations of SNP rs11208816 and
the four closest upstream SNPs, with the strongest individual
K€AHLER ET AL. 89
TAB
LEI.
Nom
inal
lySi
gnifi
can
tPD
E4B
SNPs
inSi
ngl
eM
arke
rAn
alys
esof
Scan
din
avia
nSc
hizo
phre
nia
and
Bip
olar
Dis
orde
rC
ase–
Con
trol
Sam
ples
SNP
Tota
l
nu
mbe
r
case
s/
con
trol
s
Min
or
alle
le
Ris
k
alle
leH
WE
tota
ls
am
ple
Case
freq
uen
cy
Con
trol
freq
uen
cy
Gen
otyp
e
test
str
ati
fie
da
Alle
lete
sts
tra
tifi
ed
aAl
lele
test
sa
mp
le-s
ep
ara
ted
Tota
lsa
mpl
eD
enm
ark
Nor
way
Swed
en
PO
RP
OR
PO
RP
OR
Tota
lsc
hizo
pren
iasa
mpl
e
rs1
89
23
46
83
5/1
,47
3T
A0
.63
0.5
88
0.5
56
0.0
45
0.0
14
1.1
7
(1.0
3–
1.3
2)
0.0
33
1.2
0
(1.0
1–
1.4
1)
n.s
.1
.06
n.s
.1
.18
rs5
96
66
28
32
/1,4
65
CA
0.0
80
.65
40
.62
40
.03
60
.02
71
.16
(1.0
2–
1.3
1)
0.0
07
1.2
6
(1.0
7–
1.5
0)
n.s
.1
.07
n.s
.1
.00
Schi
zoph
ren
iafe
mal
es[m
ales
]vs
.co
ntr
olsb
rs1
20
88
81
33
48
/62
3C
A0
.09
0.7
72
0.7
24
0.0
19
[n.s
.]0
.02
5[n
.s.]
1.2
8
(1.0
3–
1.6
0)
n.s
.1
.17
n.s
.1
.43
0.0
89
1.4
7
rs3
00
98
72
34
8/6
24
CT
0.0
30
.61
10
.57
10
.04
3[n
.s.]
0.0
88
[n.s
.]1
.18
n.s
.1
.14
n.s
.1
.35
n.s
.1
.15
rs1
93
74
50
34
8/6
23
TG
0.2
70
.58
60
.53
90
.04
6[n
.s.]
0.0
32
[n.s
.]1
.23
(1.0
2–
1.4
9)
0.0
67
1.2
6n
.s.
1.2
9n
.s.
1.1
1
rs2
45
50
32
34
7/6
25
TG
0.9
10
.66
70
.61
00
.01
8[n
.s.]
0.0
14
[n.s
.]1
.28
(1.0
5–
1.5
6)
n.s
.1
.21
0.0
63
1.5
4n
.s.
1.2
7
Tota
lbi
pola
rsa
mpl
e
rs7
55
27
62
59
4/1
40
3G
G0
.13
0.1
16
0.0
98
0.0
20
0.0
59
1.2
4
(1.0
0–
1.5
5)
n.s
.1
.25
n.s
.1
.21
rs1
20
80
70
15
94
/14
18
GG
0.1
30
.11
60
.09
80
.01
90
.05
91
.24
(1.0
0–
1.5
5)
n.s
.1
.26
n.s
.1
.20
rs1
12
08
77
65
93
/14
15
AA
0.2
80
.47
30
.43
7n
.s.
0.0
45
1.1
5
(1.0
0–
1.3
3)
n.s
.1
.10
0.0
43
1.3
2
(1.0
2–
1.7
1)
rs6
42
14
82
59
1/1
41
4A
A0
.35
0.4
50
0.4
16
n.s
.0
.04
71
.15
(1.0
0–
1.3
3)
n.s
.1
.14
n.s
.1
.20
rs1
74
52
12
15
94
/14
19
GG
0.6
10
.10
10
.08
50
.00
50
.04
91
.27
(1.0
1–
1.6
0)
0.0
51
1.3
1
(1.0
2–
1.6
9)
n.s
.1
.15
rs2
18
61
22
58
8/1
40
7A
A0
.35
0.4
52
0.4
09
0.0
53
0.0
17
1.1
9
(1.0
3–
1.3
7)
0.0
53
1.1
8n
.s.
1.2
2
rs1
93
74
51
59
4/1
42
0T
T0
.18
0.1
79
0.1
52
0.0
42
0.0
34
1.2
2
(1.0
2–
1.4
7)
n.s
.1
.20
n.s
.1
.30
rs1
21
40
10
75
93
/14
21
GA
0.2
90
.87
50
.85
20
.00
80
.02
31
.27
(1.0
3–
1.5
6)
0.0
42
1.2
9
(1.0
0–
1.6
6)
n.s
.1
.22
rs1
27
31
76
45
90
/14
05
GA
0.2
40
.73
10
.73
60
.01
6n
.s.
1.0
2n
.s.
1.1
1n
.s.
1.1
7
rs5
22
03
75
93
/14
16
GG
0.9
60
.40
20
.39
40
.04
7n
.s.
1.0
7n
.s.
1.0
4n
.s.
1.1
5
rs2
14
47
19
59
3/1
41
9G
T0
.04
0.6
08
0.6
00
0.0
44
n.s
.1
.02
n.s
.1
.03
n.s
.1
.02
Bip
olar
fem
ales
[mal
es]
vs.
con
trol
sb
rs1
74
24
88
53
29
/74
1A
G0
.92
0.8
69
0.8
37
0.0
38
[n.s
.]0
.03
8[n
.s.]
1.3
3
(1.0
1–
1.7
5)
n.s
.1
.28
n.s
.1
.45
rs1
12
08
79
33
28
/74
0T
T0
.76
0.3
49
0.3
05
0.0
76
[n.s
.]0
.04
6[n
.s.]
1.2
3
(1.0
0–
1.5
0)
n.s
.1
.14
0.0
45
1.4
8
(1.0
1–
2.1
8)
rs1
21
42
07
0[2
64
/67
9]
CT
0.1
00
.60
00
.58
6n
.s.[
0.0
04
9]
n.s
.[n
.s.]
1.0
7n
.s.
1.2
0n
.s.
0.8
1
(Con
tinu
ed)
90 AMERICAN JOURNAL OF MEDICAL GENETICS PART B
finding for the rs937605-rs524897 combination (Pglobal¼ 0.035;
Pindividual,C-T¼ 0.0056, OR(95% CI)¼ 1.93(1.24–2.99)).
Association With Clinical SymptomsAll tagSNPs nominally associated in the SZ- or the BP case–control
sample were also analyzed for their possible association with
symptom scores, except for SNPs with a genotype count �5
(five BP-SNPs and one SZ-SNP). SNPs with gender-specific asso-
ciation signals were only investigated for association with PANSS
scores in the relevant gender subsample.
TagSNP rs596662 was associated with positive symptoms in the
total sample (P¼ 0.003; Table IV), and all of the three SNPs that
were analyzed in females were associated with positive symptoms
(0.001� P� 0.004). None of the tagSNPs nominally associated
with BP were associated with positive or negative symptoms in
the BP sample. However, when investigating PANSS associations
for the two tagSNPs (rs2186122 and rs11208776) that serve as
proxies for the female SZ single tagSNP associations (rs3009872 and
rs1937450, respectively), we found that both were nominally
associated with negative symptoms in the female BP subgroup
(Table IV). This association was stronger in a subsample (n¼ 36) of
BP women with a history of at least one psychotic event (P¼ 0.007
and P¼ 0.002, respectively).
DISCUSSION
To investigate the potential involvement of PDE4B in the etiology of
both SZ and BP, we have performed a gene-wide association study.
We provide important additional genotyping data, from a homog-
enous Scandinavian sample, but did not find statistically significant
associations after correction for multiple testing. However, the
nominal associations found between PDE4B markers and SZ and BP
in this study, are hypothesis generating and of interest for future
studies. Firstly, there is a cluster of nominally associated tagSNPs
flanking the PDE4B3 isoform splice site, indicating a region
of interest, which might harbor functionally relevant variants.
Secondly, we provide additional data in line with the previously
reported potential gender-specific effects of PDE4B variation on
disease susceptibility. Thirdly, we provide novel data suggesting an
effect of PDE4B on specific SZ symptoms.
It has been suggested that there are common biological mech-
anisms and/or susceptibility genes for SZ and BP [Rzhetsky et al.,
2007; Hennah et al., 2008]. The gene coding for the DISC1 protein,
which biologically interacts with PDE4B, has been associated with
both diseases [Mackie et al., 2007; Hennah et al., 2008]. Also, the
binding of the dephosphorylated form of the PDE4B1 to DISC1,
as well as the influence of drug-induction on their interaction
[Millar et al., 2005], has contributed to the discussion whether these
two genes link SZ and BP [Sawa and Snyder, 2005].
This is to our knowledge the first study reporting PDE4B tagSNPs
nominally associated with both SZ and BP. All of the tagSNPs
nominally associated with SZ in women, as well as eight of the
tagSNPs nominally associated with BP in the total or gender
subsamples are located in a high LD region (D’-based), flanking
the start-site of the isoform PDE4B3 (Supplementary Figure 1). It is
interesting to note that the three genotyped SZ and BP tagSNPs
TAB
LEI.
(Con
tin
ued
)
SNP
Tota
l
num
ber
case
s/
con
trol
s
Min
or
alle
le
Ris
k
alle
leH
WE
tota
ls
am
ple
Case
freq
uen
cy
Con
trol
freq
uen
cy
Gen
otyp
e
test
str
ati
fie
da
Alle
lete
sts
tra
tifi
ed
aAl
lele
test
sa
mp
le-s
ep
ara
ted
Tota
lsa
mpl
eD
enm
ark
Nor
way
Swed
en
PO
RP
OR
PO
RP
OR
rs7
41
59
30
[26
5/6
78
]T
T0
.32
0.1
68
0.1
34
n.s
.[0
.04
0]
n.s
.[0
.08
4]
1.2
9n
.s.
1.2
8n
.s.
1.3
2
rs1
12
08
81
6[2
64
/67
6]
TT
0.0
10
.49
10
.42
6n
.s.[
0.0
17
]n
.s.[
0.0
08
]1
.33
(1.0
8–
1.6
3)
0.0
06
1.4
1
(1.1
0–
1.8
2)
n.s
.1
.13
aB
oth
gen
otyp
e-an
dal
lele
-bas
edte
sts
for
the
tota
lSc
andi
anvi
ansa
mpl
ear
est
rati
fied
byin
clud
ing
coun
try
ofsa
mpl
eor
igin
asco
nfo
unde
r.b
Asso
ciat
ion
data
isgi
ven
form
ales
inbr
acke
tsan
dfo
rfem
ales
wit
hout
brac
kets
.For
thos
eSN
Psas
soci
ated
only
infe
mal
esin
the
tota
lSca
ndi
nav
ian
sam
ple,
the
OR
san
das
soci
atio
nre
sult
sfo
reac
hco
untr
yse
para
rate
lyar
egi
ven
forf
emal
eson
lyan
dvi
ceve
rsa.
K€AHLER ET AL. 91
TAB
LEII
.N
omin
ally
Sign
ifica
nt
PDE4
BSN
PG
enot
ypes
inSi
ngl
eM
arke
rAn
alys
esof
Scan
din
avia
nSc
hizo
phre
nia
and
Bip
olar
Dis
orde
rC
ase–
Con
trol
Sam
ples
SNP
Gen
otyp
efr
eque
nci
esa
Gen
otyp
e
Com
bin
edsa
mpl
ebD
enm
ark
Nor
way
Swed
en
Case
sCo
ntr
ols
Pc
OR
cP
OR
PO
RP
OR
Tota
lsc
hizo
phre
nia
sam
ple
rs1
89
23
46
0.3
40
/0.4
96
/0
.16
40
.30
6/0
.50
0/
0.1
94
T/T
0.0
40
0.7
9(0
.63–
0.9
9)
n.s
.0
.76
n.s
.0
.81
n.s
.0
.83
rs5
96
66
20
.42
6/0
.45
7/
0.1
18
0.4
00
/0.4
48
/0
.15
2C/
C0
.01
20
.72
(0.5
5–
0.9
3)
0.0
05
0.5
9(0
.40–
0.8
5)
n.s
.0
.63
n.s
.1
.05
Schi
zoph
ren
iafe
mal
esvs
.co
ntr
ols
rs1
20
88
81
30
.59
8/0
.34
8/
0.0
55
0.5
06
/0.4
37
/0
.05
78
A/A
0.0
06
1.4
6(1
.11–
1.9
1)
n.s
.1
.33
n.s
.1
.41
0.0
28
1.8
6(1
.07–
3.2
6)
rs3
00
98
72
0.3
76
/0.4
68
/0
.15
50
.29
8/0
.54
7/
0.1
55
T/T
0.0
15
1.4
2(1
.07–
1.8
8)
0.0
24
1.5
3(1
.06–
2.2
1)
n.s
.1
.26
n.s
.1
.29
rs1
93
74
50
0.3
51
/0.4
71
/0
.17
80
.27
5/0
.52
8/
0.1
97
G/G
0.0
13
1.4
4(1
.08–
1.9
2)
0.0
16
1.5
8(1
.09–
2.3
0)
n.s
.1
.14
n.s
.1
.4
rs2
45
50
32
0.4
61
/0.4
12
/0
.12
70
.36
8/0
.48
5/
0.1
47
G/G
0.0
04
1.4
8(1
.13–
1.9
4)
n.s
.1
.36
0.0
43
1.8
7(1
.02–
3.4
3)
n.s
.1
.49
Tota
lbi
pola
rsa
mpl
ers
75
52
76
20
.78
9/0
.19
2/
0.0
20
0.8
10
/0.1
84
/0
.00
57
G/G
0.0
05
3.5
3(1
.40–
8.9
0)
0.0
04
4.2
6(1
.47–
12
.37
)n
.s.
2.0
4d
rs1
20
80
70
10
.78
8/0
.19
2/
0.0
20
0.8
10
/0.1
84
/0
.00
56
G/G
0.0
05
3.5
6(1
.41–
8.9
9)
0.0
03
4.3
2(1
.49–
12
.54
)n
.s.
2.0
3d
rs1
12
08
77
60
.28
2/0
.49
1/
0.2
28
0.3
10
/0.5
07
/0
.18
4A/
A0
.04
71
.28
(1.0
0–
1.6
3)
n.s
.1
.21
rs1
74
52
12
10
.81
8/0
.16
2/
0.0
20
0.8
35
/0.1
59
/0
.00
56
G/G
0.0
00
54
.43
(1.7
8–
11
.02
)0
.00
24
.07
(1.5
9–
10
.38
)N
AN
A
rs2
18
61
22
0.2
96
/0.5
05
/0
.19
90
.34
3/0
.49
6/
0.1
61
A/A
0.0
46
1.3
0(1
.00–
1.6
7)
n.s
.1
.27
rs1
93
74
51
0.6
80
/0.2
83
/0
.03
70
.71
5/0
.26
7/
0.0
18
T/T
0.0
19
2.0
1(1
.11–
3.6
5)
n.s
.1
.92
n.s
.2
.32
e
rs1
21
40
10
70
.78
1/0
.18
9/
0.0
30
0.7
30
/0.2
45
/0
.02
5A/
G0
.00
20
.68
(0.5
3–
0.8
7)
0.0
34
0.7
3(0
.54–
0.9
8)
0.0
18
0.5
9(0
.38–
0.9
2)
rs1
27
31
76
40
.51
5/0
.43
1/
0.0
54
0.5
47
/0.3
77
/0
.07
6G
/G0
.02
00
.61
(0.4
0–
0.9
3)
0.0
02
0.3
8(0
.20–
0.7
3)
n.s
.1
.06
rs5
22
03
70
.33
2/0
.53
1/
0.1
37
0.3
67
/0.4
79
/0
.15
5C/
G0
.01
41
.28
(1.0
5–
1.5
6)
0.0
19
1.3
2(1
.05–
1.6
7)
n.s
.1
.19
rs2
14
47
19
0.3
52
/0.5
11
/0
.13
70
.37
4/0
.45
3/
0.1
73
T/G
0.0
20
1.2
6(1
.04–
1.5
4)
n.s
.1
.19
0.0
41
.48
(1.0
2–
2.1
6)
Bip
olar
fem
ales
[mal
es]
vs.
con
trol
srs
17
42
48
85
0.7
70
/0.2
01
/0
.02
90
.69
7/0
.28
7/
0.0
16
G/A
0.0
11
0.6
6(0
.48–
0.9
1)
0.0
14
0.6
2(0
.43–
0.9
1)
n.s
.0
.78
rs1
12
08
79
30
.41
5/0
.47
3/
0.1
13
0.4
88
/0.4
14
/0
.09
9C/
C0
.02
40
.73
(0.5
6–
0.9
6)
0.0
97
0.7
7(0
.56–
1.0
5)
(Con
tinu
ed)
92 AMERICAN JOURNAL OF MEDICAL GENETICS PART B
closest to this start-site, are nominally associated with SZ and BP,
respectively.
A decrease in isoform PDE4B3 expression in cerebellum has
previously been shown in postmortem tissue from patients with BP
compared with controls. Furthermore, reduced expression of
isoforms PDE4B2 and PDE4B4 was found in the cerebellar tissue
from SZ patients [Fatemi et al., 2008]. A non-isoform-specific
increase in PDE4B expression in monocytes has been reported in
patients with BP compared with healthy controls [Padmos et al.,
2008]. In the latter study, the expression was fourfold higher in
patients treated with lithium or antipsychotics, compared with
unmedicated patients. The differences in isoform-specific expres-
sion in BP and SZ might indicate a complex role of this enzyme in
the susceptibility to psychiatric disease.
Among the SNPs nominally associated with SZ and located close
to the PDE4B3 splice site, two were in complete or high LD in the
hapmap CEU population with tagSNPs, which were genotyped
and nominally associated with disease in the BP sample. However,
the association showed opposing direction in SZ and BP. Specifi-
cally, an increased risk for SZ was nominally associated with being
homozygous for the major allele, while in contrast the homozygotes
for the minor allele were nominally associated with increased risk
for BP. Possible reasons for this discrepancy could be either a true
difference in PDE4B isoform related susceptibility, or false positive
results.
The association results for PDE4B and SZ in the present study,
as well as in a previous report [Pickard et al., 2007], contribute to
the hypothesis that there are differences between men and women
in the effect of this gene on SZ. This potential gender-effect is based
on the nominal association of several tagSNPs with SZ in the
female subgroup in both studies, and non-significant results for all
tagSNPs in the male subgroups. However, these data should be
interpreted with caution, since a sex-genotype-interaction has not
been formally tested for [Patsopoulos et al., 2007]. A potential
gender-effect for PDE4B in SZ susceptibility needs to be confirmed
in a larger sample in order to properly detect possible interactions.
In the BP case–control analysis the gender-specific associations
were not as consistent as for SZ, with different tagSNPs being
nominally associated with BP in either the female or male
subgroup.
PDE4B is inhibited by Rolipram, which has antidepressant and
potential antipsychotic effects [Zhu et al., 2001; Kanes et al., 2007;
Siuciak et al., 2007]. In the present study, one PDE4B tagSNP was
associated with positive symptom scores in the total SZ sample.
Furthermore, three PDE4B SNPs were associated with positive
symptoms scores among SZ women. These results withstand
Bonferroni correction, based on the number of tests for association
between selected tagSNPs and the clinical phenotypes. The SNPs
nominally associated with SZ susceptibility might therefore be
functionally linked to increased positive symptoms. The two BP
tagSNPs which act as proxies for two of the SZ tagSNPs were
nominally associated with negative symptom scores in the female
subsample, although not significant after Bonferroni correction.
Interestingly, when these two SNPs were tested for symptom score
association in a smaller subsample (n¼ 36) of BP women with
a history of at least one psychotic event, the association with
negative symptom scores was stronger. Positive symptoms, but to
TAB
LEII
.(C
onti
nue
d)
SNP
Gen
otyp
efr
eque
nci
esa
Gen
otyp
e
Com
bin
edsa
mpl
ebD
enm
ark
Nor
way
Swed
en
Case
sCo
ntr
ols
Pc
OR
cP
OR
PO
RP
OR
rs1
21
42
07
00
.34
1/0
.56
9/
0.0
90
0.3
51
/0.4
62
/0
.18
7T/
C[0
.00
4]
1.5
4(1
.15–
2.0
8)
0.0
15
1.5
4(1
.09–
2.1
8)
n.s
.1
.56
rs7
41
59
30
0.7
06
/0.2
53
/0
.04
20
.74
6/0
.23
9/
0.0
15
T/T
[0.0
08
]3
.17
(1.3
0–
7.7
4)
n.s
.2
.48
NA
NA
rs1
12
08
81
60
.26
1/0
.49
6/
0.2
42
0.3
43
/0.4
62
/0
.19
5C/
C[0
.00
6]
0.6
3(0
.45–
0.8
7)
0.0
03
0.5
4(0
.36–
0.8
1)
n.s
.0
.86
NA,
data
not
avai
labl
edu
eto
no
con
trol
sha
veth
ete
sted
gen
otyp
e.aH
omoz
ygot
em
ajor
alle
le/h
eter
ozyg
ote/
hom
ozy
gote
min
oral
lele
.b
All
pati
ents
and
con
trol
sar
ean
alyz
edin
com
bin
atio
n,
wit
hsa
mpl
eor
igin
asa
con
foun
der.
c P-va
lues
and
OR
sar
egi
ven
for
the
gen
otyp
egi
vin
gth
ehi
ghes
tri
sk(a
sin
dica
ted
byth
elo
wes
tP-
valu
e,or
ifth
esa
me
P-va
lues
,th
ela
rges
tef
fect
size
)w
hen
com
pare
dto
the
poo
led
othe
rtw
oge
not
ypes
.d
Four
case
s,an
dtw
oco
ntr
ols.
eN
ine
case
s,an
dfo
urco
ntr
ols.
K€AHLER ET AL. 93
a lesser degree negative symptoms, vary during the course of illness.
Thus, the genetic association to PANSS scores could be spurious.
However, the present study sample was reasonable stable receiving
mostly outpatient treatment, which makes such type 1 errors less
likely, and genetic associations to subgroups of SZ based on PANSS
scores have been reported earlier [DeRosse et al., 2006].
At the time of choosing the tagSNPs in this study, the three
other association studies investigating PDE4B and SZ had not been
published [Pickard et al., 2007; Fatemi et al., 2008; Numata et al.,
2008a]. Therefore, the overlap between SNPs investigated in the
present and published PDE4B studies is limited. For an overview of
the marker positions, and associated SNPs, for the previous and
present studies, see supplementary figure 1. Two of the previous
studies investigated Caucasian samples: 26 SNPs were analyzed in
a Scottish sample (386 SZ cases, 368 BP cases, 455 controls) [Pickard
et al., 2007], and 27 SNPs were analyzed in an American Caucasian
sample (644 cases, 407 controls) [Fatemi et al., 2008]. The nominal
associations in the study by Pickard et al. were only found in
females, and do not overlap with the associated SNPs in either the
present or the Fatemi et al. study. A hapmap-based perfect proxy
(r2¼ 1 in the CEU population) for the most nominally significant
SNP by Pickard et al., was investigated by us, but failed to replicate.
We found one SNP previously associated with SZ by Fatemi et al. to
be nominally associated with BP, in the genotype- but not allele-
based test. In contrast, this SNP was not associated with SZ either
in our study or in the study by Pickard et al. Four of the seven
associated SNPs in the study by Fatemi et al., were genotyped and
not associated to SZ in our study. The two most associated SNPs
were linked to the same extent in both studies, but single and
haplotype test results were non-significant.
There are several possible explanations for the conflicting
association findings for PDE4B and psychiatric disorders. First,
the present study is larger than the previously studied SZ and BP
samples, reducing the risk of type II error in comparison with
previous reports. Still, the power of the allelic test in our study,
calculated using the Genetic Power Calculator (pngu.mgh.harvard.
edu/�purcell/gpc; settings: D’¼ 1 between disease and tagSNP, an
additive model with OR (homozygote risk allele)¼ 1.5; a ¼ 0.05;
MAF for disease and tagSNP¼ 5–25%), is limited to 33–84% and
28–75% for the SZ and BP sample, respectively. Second, our study
TABLE III. Nominally Significant PDE4B Haplotype Analyses in a Scandinavian Female Schizophrenia Case–Control Sample
No. SNP Positiona LD (r2/D’)
Pglobal association Pindividual haplotypeb
Single Two Three Four Two Three Four8 rs11208769 66092413 0.34/0.96 0.32 0.10 0.15 0.063 0.031 0.026 0.0189 rs12088813 66119721 0.49/1.00 0.025 0.077 0.041 0.091 0.021 0.014 0.03310 rs3009872 66123421 0.82/0.96 0.087 0.050 0.10 0.054 0.035 0.033 0.03111 rs1937450 66190861 0.71/0.98 0.032 0.033 0.029 0.0056 0.030 0.015 0.02312 rs1392816 66193209 0.34/0.59 0.28 0.070 0.0092 0.0033 0.028 0.023 0.01513 rs2455032 66208160 0.66/0.92 0.014 0.0053 0.0032 0.0081 0.0090 0.0080 0.01314 rs1354060 66223425 0.21/0.99 0.25 0.25 0.32 0.11 0.1315 rs6588186 66259030 0.07/1.00 0.89 0.19 0.09216 rs11208796 66259486 0.11
Position, linkage equilibrium (LD) data, and P-values for global and individual association tests for nine SNPs located in a region of high LD (D’-based) flanking the PDE4B3 splice site are shown.aThe PDE4B3 isoform splice site is located between marker 10 and 11.bLowest P-value from a score test for a difference in risk between one haplotype and all the others pooled together.
TABLE IV. Nominally Significant PDE4B SNPs in Single Marker Analyses of Positive and Negative Syndrome Scale (PANSS) Scores in
Norwegian Schizophrenia and Bipolar Disorder Case–Control Samples
SNP Sample #Cases
PANSS score test results
PPositive score Pnegative score Padjusteda
rs596662 SZ total 153 0.003 0.28 0.02rs3009872 SZ females 70 0.001 0.67 0.005rs1937450 SZ females 70 0.004 0.94 0.02rs2455032 SZ females 70 0.004 0.97 0.02rs2186122 BP females 77 0.33 0.051 —rs11208776 BP females 77 0.14 0.017 0.24
aThe P-values were adjusted for the number of SNPs, and samples tested for each SNP: for the SZ sample 5 SNPs were tested; for the BP sample 12 SNPs were tested, two of these in two samples.
94 AMERICAN JOURNAL OF MEDICAL GENETICS PART B
is based on homogenous samples (as measured by the FST),
originating from Norway, Denmark, and Sweden, which makes
them well suited for genetic studies, with lower risk of type I error
risk due to population stratification. Third, even when comparing
Caucasian samples, suggested locus heterogeneity has previously
been reported for candidate genes within psychiatric genetics, such
as DISC1 [Hennah et al., 2008]. Therefore significant associations to
several tagSNPs in the same gene in several samples might impose
involvement of the investigated gene in disease susceptibility,
despite tagSNP heterogeneity.
We cannot exclude that the present results might be due to type I
error, and we have limited power to detect signals in the lower
frequency range. However, the nominally associated tagSNPs in our
study are not randomly distributed over the large gene. Rather,
several SNPs are located in a high LD region flanking the PDE4B3
splice site. Further studies should therefore examine if there are
variants in the PDE4B gene region close to the isoform PDE4B3
splice site that are involved in SZ and BP susceptibility. Further-
more, investigations for potential epistatic interaction with DISC1
would be desirable.
ACKNOWLEDGMENTS
We thank patients and controls for their participation in the
study, and the health professionals who facilitated our work.
We also wish to thank Morten Mattingsdal, Marie J Skogstad,
Knut-Erik Gylder, Thomas Bjella, Eivind Bakken, and Bente G
Bennike, for skilful technical and administrative assistance. We also
thank Tomas Axelsson and Per Lundmark (SNP Technology
Platform, Uppsala University and Uppsala University Hospital,
Sweden), who were in charge of the Illumina-based genotyping at
the platform in Uppsala, and the University of Oslo Bioportal for
providing a platform for running the statistical software Unphased.
The study was supported by grants from: the Research Council of
Norway (#167153/V50,#163070/V50, #175345/V50), Eastern and
Western Norway Health Authority (#123–2004), Ulleva�l University
Hospital and the University of Oslo to support the Thematic
Organized Psychosis Research (TOP) Study group and the Bergen
group; the Copenhagen Hospital Corporation Research Fond, the
Danish National Psychiatric Research Foundation, the Danish
Agency for Science, Technology and Innovation (Centre for
Pharmacogenomics) and the Danish Medical Research Council,
the Lundbeck Foundation; the Stanley Medical Research Institute;
and the Wallenberg Foundation, the HUBIN project and the
Swedish Research Council (K2007-62X-15078-04-1, K2007-62X-
15078-04-3, K2008-62P-20597-01-3).
REFERENCES
Barch DM. 2005. The cognitive neuroscience of schizophrenia. Annu RevClin Psychol 1:321–353.
Barrett JC, Fry B, Maller J, Daly MJ. 2005. Haploview: Analysis andvisualization of LD and haplotype maps. Bioinformatics 21:263–265.
Cheung YF, Kan Z, Garrett-Engele P, Gall I, Murdoch H, Baillie GS,Camargo LM, Johnson JM, Houslay MD, Castle JC. 2007. PDE4B5,a novel, super-short, brain-specific cAMP phosphodiesterase-4variant whose isoform-specifying N-terminal region is identical to that
of cAMP phosphodiesterase-4D6 (PDE4D6). J Pharmacol Exp Ther322:600–609.
Chubb JE, Bradshaw NJ, Soares DC, Porteous DJ, Millar JK. 2008. TheDISC locus in psychiatric illness. Mol Psychiatry 13:36–64.
Davis RL, Cherry J, Dauwalder B, Han PL, Skoulakis E. 1995. The cyclicAMP system and Drosophila learning. Mol Cell Biochem 149–150:271–278.
de Bakker PI, Yelensky R, Pe’er I, Gabriel SB, Daly MJ, Altshuler D. 2005.Efficiency and power in genetic association studies. Nat Genet37:1217–1223.
DeRosse P, Funke B, Burdick KE, Lencz T, Ekholm JM, Kane JM,Kucherlapati R, Malhotra AK. 2006. Dysbindin genotype and negativesymptoms in schizophrenia. Am J Psychiatry 163:532–534.
Dudbridge F. 2008. Likelihood-based association analysis for nuclearfamilies and unrelated subjects with missing genotype data. Hum Hered66:87–98.
Excoffier L, Laval G, Schneider S. 2005. Arlequin ver. 3.0: An integratedsoftware package for population genetics data analysis. Evol Bioinfor-matics Online 1:47–50.
Fatemi SH, King DP, Reutiman TJ, Folsom TD, Laurence JA, Lee S, Fan YT,Paciga SA, Conti M, Menniti FS. 2008. PDE4B polymorphismsand decreased PDE4B expression are associated with schizophrenia.Schizophr Res 101:36–49.
Hansen T, Olsen L, Lindow M, Jakobsen KD, Ullum H, Jonsson E,Andreassen OA, Djurovic S, Melle I, Agartz I, Hall H, Timm S, WangAG, Werge T. 2007. Brain expressed microRNAs implicated in schizo-phrenia etiology. PLoS ONE 2:e873.
Hennah W, Thomson P, McQuillin A, Bass N, Loukola A, Anjorin A,Blackwood D, Curtis D, Deary IJ, Harris SE, Isometsa ET, Lawrence J,Lonnqvist J, Muir W, Palotie A, Partonen T, Paunio T, Pylkko E,Robinson M, Soronen P, Suominen K, Suvisaari J, Thirumalai S,Clair DS, Gurling H, Peltonen L, Porteous D. 2008. DISC1 association,heterogeneity and interplay in schizophrenia and bipolar disorder. MolPsychiatry DOI: 10.1038/mp.2008.22.
Houslay MD, Adams DR. 2003. PDE4 cAMP phosphodiesterases: Modularenzymes that orchestrate signalling cross-talk, desensitization andcompartmentalization. Biochem J 370:1–18.
Jakobsen KD, Frederiksen JN, Parnas J, Werge T. 2006. Diagnostic agree-ment of schizphrenia spectrum disorders among chronic patients withfunctional psychoses. Psychopathology 39:269–276.
Kahler AK, Djurovic S, Kulle B, Jonsson EG, Agartz I, Hall H, OpjordsmoenS, Jakobsen KD, Hansen T, Melle I, Werge T, Steen VM, Andreassen OA.2008. Association analysis of schizophrenia on 18 genes involved inneuronal migration: MDGA1 as a new susceptibility gene. Am J MedGenet Part B 147B:1089–1100.
Kanes SJ, Tokarczyk J, Siegel SJ, Bilker W, Abel T, Kelly MP. 2007.Rolipram: A specific phosphodiesterase 4 inhibitor with potential anti-psychotic activity. Neuroscience 144:239–246.
Kay SR, Fiszbein A, Opler LA. 1987. The positive and negativesyndrome scale (PANSS) for schizophrenia. Schizophr Bull 13:261–276.
Lao O, Lu TT, Nothnagel M, Junge O, Freitag-Wolf S, Caliebe A, Balasca-kova M, Bertranpetit J, Bindoff LA, Comas D, et al. 2008. Correlationbetween genetic and geographic structure in Europe. Curr Biol18:1241–1248.
Mackie S, Millar JK, Porteous DJ. 2007. Role of DISC1 in neural develop-ment and schizophrenia. Curr Opin Neurobiol 17:95–102.
Martinez-Aran A, Vieta E, Colom F, Torrent C, Sanchez-Moreno J,Reinares M, Benabarre A, Goikolea JM, Brugue E, Daban C, Salamero
K€AHLER ET AL. 95
M. 2004. Cognitive impairment in euthymic bipolar patients: Implica-tions for clinical and functional outcome. Bipolar Disord 6:224–232.
Mellerup E, Bennike B, Bolwig T, Dam H, Hasholt L, Jorgensen MB, PlengeP, Sorensen SA. 2001. Platelet serotonin transporters and the transportergene in control subjects, unipolar patients and bipolar patients. ActaPsychiatr Scand 103:229–233.
Millar JK, Pickard BS, Mackie S, James R, Christie S, Buchanan SR,Malloy MP, Chubb JE, Huston E, Baillie GS, Thomson PA, Hill EV,Brandon NJ, Rain JC, Camargo LM, Whiting PJ, Houslay MD,Blackwood DH, Muir WJ, Porteous DJ. 2005. DISC1 and PDE4B areinteracting genetic factors in schizophrenia that regulate cAMP signaling.Science 310:1187–1191.
Moller HJ. 2003. Bipolar disorder and schizophrenia: Distinct illnesses ora continuum? J Clin Psychiatry 64Suppl 6: 23–27 discussion 28.
Murdoch H, Mackie S, Collins DM, Hill EV, Bolger GB, Klussmann E,Porteous DJ, Millar JK, Houslay MD. 2007. Isoform-selective suscepti-bility of DISC1/phosphodiesterase-4 complexes to dissociation byelevated intracellular cAMP levels. J Neurosci 27:9513–9524.
Numata S, Iga JI, Nakataki M, Tayoshi S, Taniguchi K, Sumitani S,Tomotake M, Tanahashi T, Itakura M, Kamegaya Y, Tatsumi M,Sano A, Asada T, Kunugi H, Ueno SI, Ohmori T. 2008a. Gene expressionand association analyses of the phosphodiesterase 4B (PDE4B) gene inmajor depressive disorder in the Japanese population. Am J Med GenetPart B DOI: 10.1002/ajmg.b.30852.
Numata S, Ueno SI, Iga JI, Song H, Nakataki M, Tayoshi S, Sumitani S,Tomotake M, Itakura M, Sano A, Ohmori T. 2008b. Positive associationof the PDE4B (phosphodiesterase 4B) gene with schizophrenia in theJapanese population. J Psychiatr Res 43:7–12.
Owen MJ, Craddock N, Jablensky A. 2007. The genetic deconstruction ofpsychosis. Schizophr Bull 33:905–911.
Padmos RC, Hillegers MH, Knijff EM, Vonk R, Bouvy A, Staal FJ, de RidderD, Kupka RW, Nolen WA, Drexhage HA. 2008. A discriminatingmessenger RNA signature for bipolar disorder formed by an aberrant
expression of inflammatory genes in monocytes. Arch Gen Psychiatry65:395–407.
Patsopoulos NA, Tatsioni A, Ioannidis JP. 2007. Claims of sex differences:An empirical assessment in genetic associations. JAMA 298:880–893.
Pickard BS, Thomson PA, Christoforou A, Evans KL, Morris SW, PorteousDJ, Blackwood DH, Muir WJ. 2007. The PDE4B gene confers sex-specificprotection against schizophrenia. Psychiatr Genet 17:129–133.
Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D,Maller J, Sklar P, de Bakker PI, Daly MJ, Sham PC. 2007. PLINK: A tool setfor whole-genome association and population-based linkage analyses.Am J Hum Genet 81:559–575.
Rzhetsky A, Wajngurt D, Park N, Zheng T. 2007. Probing geneticoverlap among complex human phenotypes. Proc Natl Acad Sci USA104:11694–11699.
Sawa A, Snyder SH. 2005. Genetics. Two genes link two distinct psychoses.Science 310:1128–1129.
Simonsen C, Sundet K, Vaskinn A, Birkenaes AB, Engh JA, Hansen CF,Jonsdottir H, Ringen PA, Opjordsmoen S, Friis S, Andreassen OA. 2008.Neurocognitive profiles in bipolar I and bipolar II disorder: Differencesin pattern and magnitude of dysfunction. Bipolar Disord 10:245–255.
Siuciak JA, Chapin DS, McCarthy SA, Martin AN. 2007. Antipsychoticprofile of rolipram: Efficacy in rats and reduced sensitivity in micedeficient in the phosphodiesterase-4B (PDE4B) enzyme. Psychophar-macology (Berl) 192:415–424.
WHO. 1993. The ICD-10 classification of mental and behavioural disor-ders. diagnostic criteria for research. Geneva:World Health Organization.
In: Wing JK, Sartorius N, U?st€un TB, editors. 1998. Diagnosis and clinicalmeasurement in psychiatry. A reference manual for SCAN. Geneva:-World Health Organization.
Zhu J, Mix E, Winblad B. 2001. The antidepressant and antiinflammatoryeffects of rolipram in the central nervous system. CNS Drug Rev7:387–398.
96 AMERICAN JOURNAL OF MEDICAL GENETICS PART B
