P2RX7: Expression Responds to Sleep Deprivation and
Associates with Rapid Cycling in Bipolar Disorder Type 1
Lena Backlund1*, Catharina Lavebratt2,3, Louise Frise ´n1,2,3, Pernilla Nikamo2,3¤, Dzana Hukic Sudic2,3,
Lil Tra ¨skman-Bendz4, Mikael Lande ´n1,5, Gunnar Edman2,3,7, Marquis P. Vawter6, Urban O¨sby2,3,7,
1Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden, 2Neurogenetics Unit, Department of Molecular Medicine and Surgery, Karolinska
Institutet, Stockholm, Sweden, 3Center for Molecular Medicine, Karolinska University Hospital, Stockholm, Sweden, 4Department of Clinical Sciences, University Hospital,
Lund, Sweden, 5Institution of Neuroscience and Physiology, The Sahlgrenska Academy at Gothenburg University, Sweden, 6Functional Genomics Laboratory,
Department of Psychiatry and Human Behavior, School of Medicine, University of California Irvine, Irvine, California, United States of America, 7Department of Psychiatry,
Tiohundra AB, Norrta ¨lje, Sweden
Context: Rapid cycling is a severe form of bipolar disorder with an increased rate of episodes that is particularly treatment-
responsive to chronotherapy and stable sleep-wake cycles. We hypothesized that the P2RX7 gene would be affected by
sleep deprivation and be implicated in rapid cycling.
Objectives: To assess whether P2RX7 expression is affected by total sleep deprivation and if variation in P2RX7 is associated
with rapid cycling in bipolar patients.
Design: Gene expression analysis in peripheral blood mononuclear cells (PBMCs) from healthy volunteers and case-case and
case-control SNP/haplotype association analyses in patients.
Participants: Healthy volunteers at the sleep research center, University of California, Irvine Medical Center (UCIMC), USA
(n=8) and Swedish outpatients recruited from specialized psychiatric clinics for bipolar disorder, diagnosed with bipolar
disorder type 1 (n=569; rapid cycling: n=121) and anonymous blood donor controls (n=1,044).
Results: P2RX7 RNA levels were significantly increased during sleep deprivation in PBMCs from healthy volunteers
(p=2.3*1029). The P2RX7 rs2230912 _A allele was more common (OR=2.2, p=0.002) and the ACGTTT haplotype in P2RX7
(rs1718119 to rs1621388) containing the protective rs2230912_G allele (OR=0.45–0.49, p=0.003–0.005) was less common,
among rapid cycling cases compared to non-rapid cycling bipolar patients and blood donor controls.
Conclusions: Sleep deprivation increased P2RX7 expression in healthy persons and the putatively low-activity P2RX7
rs2230912 allele A variant was associated with rapid cycling in bipolar disorder. This supports earlier findings of P2RX7
associations to affective disorder and is in agreement with that particularly rapid cycling patients have a more vulnerable
Citation: Backlund L, Lavebratt C, Frise ´n L, Nikamo P, Hukic Sudic D, et al. (2012) P2RX7: Expression Responds to Sleep Deprivation and Associates with Rapid
Cycling in Bipolar Disorder Type 1. PLoS ONE 7(8): e43057. doi:10.1371/journal.pone.0043057
Editor: Namni Goel, University of Pennsylvania School of Medicine, United States of America
Received December 8, 2011; Accepted July 16, 2012; Published August 28, 2012
Copyright: ? 2012 Backlund 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: This project was supported by grants from Karolinska Institutet, the Swedish Research Council, The So ¨derstro ¨m-Ko ¨nigska Foundation, The Royal
Physiographic Society in Lund, the Fredrik and Ingrid Thuring Foundation and Psychiatry Southwest, Stockholm. Financial support was also provided through the
regional agreement on medical training and clinical research (ALF) between the Stockholm County Council and Karolinska Institutet. The research was further
supported by the William Lion Penzner Foundation (Department of Psychiatry and Human Behavior, University of California, Irvine), and National Institute of
Mental Health (NIMH) grant R01MH085801 (MPV). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have read the journal’s policy and have the following conflicts: LB has received consultancy fees from Bristol-Myers Squibb
and has been reimbursed by Eli Lilly and Bristol-Myers Squibb for attending conferences and presenting lectures. UO¨has received grant/research support from
Bristol-Myers Squibb and Janssen-Cilag and has been a consultant for or received fees for speaking and attending conferences from AstraZeneca, Bristol-Myers
Squibb, Eli Lilly and Pfizer. GE has been a consultant for AstraZeneca and Janssen-Cilag and his wife is a shareholder of AstraZeneca. ML has been reimbursed by
Eli Lilly, AstraZeneca and Wyeth for lectures, serves on advisory boards for AstraZeneca and Lundbeck, and has received research grants from AstraZeneca. This
does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: email@example.com
¤ Current address: Dermatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden
Rapid cycling (RC) is a severe form of bipolar disorder,
characterized by four or more disease episodes within one year
(DSM-IV) [1,2]. A RC course occurs in 12–24% of bipolar
disorder patients . In addition to more frequent episodes, RC
implies greater functional impairment, increased risk of suicide
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attempts and a higher rate of alcohol abuse [1,4–6]. Due to the
clinical severity of RC, clinicians try to prevent its development by
early active symptom treatment. In bipolar disorder, antidepres-
sants are often necessary to treat bipolar depression, despite the
possible risk for mood switches in vulnerable patients [7–10].
Methods to identify patients at risk for RC would be of great
The heritability of bipolar disorder is estimated to 79–93% .
Several interesting candidate genes have been identified, but
replication of these findings are scarce. This may relate to the
heterogeneity of symptoms and course, comorbidity between
bipolar disorder and other psychiatric disorders and genetic
differences between ethnic groups and environmental effects.
Specific symptoms and sub-phenotypes of bipolar disorder may
represent different biological variants, and genetic association
studies may be helpful to identify these. Some studies using this
strategy have been promising, e.g. both persecutory delusions and
an early onset of illness have been associated with the DAOA and
GRK3 genes . Mood-incongruent psychotic symptoms have
been linked to 1q32.3, 7p13 and 20q13.3  and a favorable
lithium-response to chromosome 10p15 . Previously we have
presented cognitive symptoms in mania associated with the P2RX7
gene . Case-case studies (comparing patients with specific
symptoms to patients without such symptoms) may minimize the
effect of environmental factors (i.e. stress, drug exposure, socio-
economic factors) between patients [16–18]. We have used this
strategy in a previous genetic study of RC in bipolar disorder .
Furthermore, a specific genetic RC vulnerability is suggested by a
familial aggregation of RC , and associations to RC have been
found in the COMT , SLC6A4 [22,23], BDNF [24,25] and
CRY2  genes.
Sleep disturbances and diurnal-related anxiety in bipolar
episodes are associated with disturbed biological rhythm function
. Bipolar patients with RC have a more vulnerable diurnal
system than those without RC [27–29]. Treatment studies also
showing that both social rhythm therapy and psychotherapy
focused on stable sleep-wake cycle is especially beneficial in RC
bipolar patients [30,31]. Most patients with RC have to bee
treated with a combination of lithium and valproic acid. Lithium
has been shown to stabilize the circadian rhythm in bipolar
disorder [32–34]. In mice, lithium is proposed to prolong the
circadian period in the suprachiasmatic nucleus (SCN) .
Valproic acid is also believed to influence the circadian rhythm,
but through other mechanisms . Altogether, this may explain
the usefulness lithium and valproate in combination in RC
The P2RX7 gene
P2RX7 is a candidate gene for bipolar disorder that was first
identified by linkage analysis in a French-Canadian population
. The SNP rs2230912 was associated with bipolar disorder in a
large and detailed family-based study . Other polymorphisms
in the P2RX7 gene have been associated with mood and anxiety
disorders [38–41]. The P2RX7 gene has been suggested to be
involved in the regulation of glutamate activation [42,43] which
has been proposed to to be involved in the circadian rhythm
[44,45]. Absence of P2rx7 in mouse brain have been shown to
decrease depressive-like behavior and attenuate amphetamine-
induced hyperactivity  whereas activation of the P2RX7 gene
may lead to glutamate over-activation and secondary to depressive
We hypothesized that P2RX7 expression in a healthy state is
affected by disturbance in the circadian rhythm and further that
polymorphisms in P2RX7 are associated with RC which is a
subtype of bipolar disorder associated with a more vulnerable
In the expression study informed consent was obtained from
each participant using an approved University of California
Institutional Review Board (IRB) protocol. All these participants
were healthy individuals. The genetic study was approved by the
Regional Ethical Review Board in Stockholm in accordance with
the Helsinki Declaration of 1975. All bipolar participants were in
euthymic phase. In both studies all individuals had full capacity to
consent and the informed consent process was both verbal and
written during a visit to a special trained psychiatric nurse.
Participants in the RNA expression study
Eight healthy volunteers (4 women, 4 men, all with European
descent), aged 24 years on average (SD=5.9, range=19–34
years), were investigated at the sleep research center at University
of California at Irvine Medical Center (UCIMC) for 48 hours and
deprived of sleep for 36 hours after an overnight stay (Figure 1).
During sleep deprivation, wakefulness was maintained by allowing
activities such as walking, reading, watching television, and playing
card games. Subjects were not permitted to consume caffeinated
foods or beverages to stay awake. Subjects’ wakefulness was
ensured by research assistants. Venous blood samples were drawn
at 9 different times, beginning at 7 p.m. and every 6 hours
thereafter. The blood was collected standard acid citrate dextrose
(ACD) tubes (Becton Dickinson, Franklin Lakes, NJ, USA). Within
60 minutes in room temperature (RT) after blood drawl, the
whole blood samples were centrifuged at 1500 rpm for 10 min at
RT and the upper layer was thereafter transferred onto Ficoll-
Paque (Amersham Biosciences, Piscataway, NJ, USA). Peripheral
blood mononuclear cells (PBMC) were separated by density
gradient centrifugation at 2500 rpm at RT for 20 min. The
resulting ‘buffy’ coat layer was added to 10 ml phosphate buffered
saline (PBS) at pH of 7.4 and centrifuged at 1000 rpm for 10 min
at RT. The resulting pellet (52106106cells) was resuspended in
1 ml Trizol and total RNA was extracted using the standard
Trizol isolation protocol (Invitrogen, Carlsbad, CA, USA). The
RNA was resuspended in 100 mL diethyl pyrocarbonate (DEPC)
treated water, analyzed for quality and quantity on a 2100
Bioanalyzer (Agilent, Palo Alto, CA, USA) and concentration was
adjusted to 1 mg/ml.
RNA expression levels were determined using the Affymetrix
oligonucleotide microarray chip (Human Gene- Chip Exon 1.0
ST) and the expression profiling experiments were carried out
following the manufacturer’s technical protocol (Affymetrix, Santa
Clara, CA). In brief, total RNA was first subjected to a ribosomal
RNA removal procedure (RiboMinus Human/Mouse Transcrip-
tome Isolation Kit, Invitrogen) to reduce the 28S and 18S rRNA
population significantly minimizing background and increasing
sensitivity. Reduced RNA was reverse-transcribed to cDNA with
random hexamers tagged with a T7 promoter sequence followed
by second strand cDNA synthesis using DNA polymerase. The
double stranded cDNA was then used for amplification of
antisense cRNA and cleaned (GeneChip Sample Cleanup
Module). A second cycle cDNA synthesis reaction was performed
using random primers to reverse transcribe the cRNA into sense
single stranded DNA which was then fragmented, and hybridized
to the Affymetrix Human Genome Exon Arrays. Arrays were
washed, stained, and scanned on the Affymetrix Fluidics Station
and G7 Affymetrix high-resolution scanner using GCOS 1.3. The
P2RX7 Associates with Rapid Cycling
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CEL files derived from the 1.0 ST chips were analyzed with a
robust multi-array condensation algorithm (RMA) . All the
expression data for P2RX7 is listed in Table S1 in a MIAME
The quality control of each exon array was evaluated by using
ExACT (Affymetrix software) for all CEL files. Based upon
meeting Affymetrix quality control standard and principal
component analysis, all 71 exon arrays were included in the
analysis, that is, 9 samples per healthy volunteer collected day 1 at
7 p.m., day 2 at 1 am, 7 a.m., 1 p.m. and 7 p.m., as well as day 3 at
1 a.m., 7 a.m., 1 p.m. and 7 p.m. where the volunteers were
deprived from sleep after the day 1 to day 2 night. Time point day
1 at 7 p.m. did not have high quality RNA from one of the healthy
volunteers and was therefore not run on an exon array. Each array
was individually normalized using an individual mean center.
Data were analyzed in Partek Genomic Solutions (St. Louis, MO)
and a total of 1,389,155 probes were imported into Partek after
filtering out probes with SNPs. The RNA levels reported are the
average of exon expression (24 probe sets) across the entire RefSeq
transcript (transcript ID 3434726).
Real-time quantitative PCR (qPCR) for validation
cDNA was generated using TaqMan reverse transcription (RT)
reagents (Applied Biosystems). Each 50 ml RT reaction mix
contained 5 ml of 106 Taqman RT buffer; 11 ml of 25 mM
MgCl2; 10 ml of deoxy NTPs; 2.5 ml of Oligo d(T)16primer; 1 ml of
RNase inhibitor; 1.25 ml of Multiscribe reverse transcriptase, and
1 ml of RNA (1 mg/ml). The reaction mixtures were run on a
GeneAmp PCR System 9700 (Applied Biosystems). The thermal
cycling conditions were as follows: 25uC for 10 min for primer
incubation, 48uC for 30 min for reverse transcription, and 95uC
for 5 min for inactivation of reverse transcriptase. All cDNAs were
stored at 220uC. qPCR was performed for P2RX7 exon 3 (near
the hybridization site for the probe (Probe Set 2950343;
Affymetrix, Inc.)), and the housekeeping genes GAPDH and
TFG. Primer sequences were as follows: P2RX7 forward primer:
CTGCTGGTTCACCATCCTAA-39; GAPDH forward: 59-AC-
CCACTCCTCCACCTTTGA-39, reverse: 59-AATTCGTTGT-
CATACCAGGA-39; and TFG forward: 59-TCGTTCAGACT-
CACTTCAATTTC-39. The primers were tested by using a set
of brain cDNA samples (2 individual cDNAs from cerebellum),
genomic DNA, no temple control (NTC), and RT minus (2
individual DLPFC RNA without cDNA) in duplicate and run on
an ABI 7900HT Sequence Detection System (Applied Biosys-
tems). The primer test results showed that all cDNA amplified
well, the NTC and gDNA had a cycle threshold (Ct) greater than
40 and RT minus was 100-fold less than cDNA concentration.
This detection ensured that the primers were not measuring
residual genomic DNA, and the melting and amplification curves
did not show any contribution of primer-dimers to the measure-
The qPCR was performed on the ABI 7900HT in 384-well
plates. The samples were assembled by Robot Biomek3000
(Beckman Coulter) and run with triplicates in one plate per gene.
The reaction was performed in a 12.5 ml total volume with 6.25 ml
of 26Power SybrGreen master mix (Life Technologies); 0.25 ml of
10 mM forward primer; 0.25 ml of 10 mM reverse primer; 2 ml of a
1:10 dilution of cDNA template (corresponding to approximately
4 ng RNA), and water to a total volume of 12.5 ml. The thermal
cycling profiles were as follows: pre-steps at 50uC for 2 min
(incubation) and 95uC for 10 min (activation), followed by
Figure 1. P2RX7 gene expression in peripheral blood mononuclear cells from healthy volunteers. P2RX7 RNA expression was increased
by sleep deprivation in men (filled diamonds) and women (open triangles). Blood draw (x-axis) was at 7 p.m. and thereafter once every 6 hours. Sleep
deprivation was at time points #6, #7, #8 and #9. RNA levels are the normalized average of exon expression across the entire RefSeq transcript.
Diamonds and triangles indicate mean and error bars indicate SEM.
P2RX7 Associates with Rapid Cycling
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45 cycles at 95uC for 15 sec (denaturation) and 60uC for 1 min
(annealing/extension), then by a dissociation step at 95uC for
15 sec, 60uC for 15 sec, and 95uC for 15 sec.
The cycle threshold (Ct) was determined approximately in the
middle of the exponential phase of the amplification. The average
and standard deviation (SD) and CV of triplicate Ct were
calculated and the average value was accepted if the SD was lower
than 0.39. A variation of the GENorm quantification method 
was used to normalize gene expression. GAPDH and TFG were the
selected housekeeping genes based upon in-house data. Each gene
was individually scaled to the lowest Ct, and after scaling, the
mean of the two housekeeping genes was used to normalize
Participants in the genetic study
Patients (n=646) at clinical diagnosis of bipolar disorder type 1
were recruited in Sweden, most of them from specialized
outpatient clinics for affective disorders (n=582; the Huddinge
cohort: n=509, the S:t Go ¨ran cohort: n=73) and some from
ordinary psychiatric outpatient clinics (n=64). Life-time manic
and depressive symptoms were assessed. The bipolar diagnosis was
validated by noting the following manic symptoms: elevated mood,
irritability, over-activity, grandiosity, decreased sleep, talkative-
ness, distractibility, goal-directed behavior, thought disorder, and
embarrassing behavior. The assessment was based on interviews
and medical records focusing on the most severe manic episode
and performed by a psychiatrist specialized in bipolar disorder or
by a trained psychiatric nurse. Manic and depressive symptoms
were assessed using the modules for mania and depression in the
Schedules for Clinical Assessment in Neuropsychiatry (SCAN;
Table 1) , or with the Affective Disorder Evaluation as has
been described elsewhere . Depressive symptoms were
assessed according to DSM-IV. On the basis of these assessment
patients were considered as fulfilling the diagnostic criteria for
bipolar disorder type 1, 2, or not otherwise specified (NOS). The
phenotypes RC, mixed episodes and the age of onset of mania as
well as of depression were also assessed. Patients who did not fulfill
the DSM-IV criteria for bipolar disorder type 1 were excluded
(n=59). According to the DSM-IV definition of bipolar disorder,
individuals were excluded if a manic episode was a result of
alcohol or drug abuse, medication or somatic disease (n=7). Also
excluded were patients with close relatives already included in the
study (n=11). Another nine patients withdrew from the study. A
total of 569 bipolar type 1 patients (42% men) were included in the
study. Within the final study group 508 (88%) patients had been
hospitalized for an affective episode at least once, 172 (30%) had
mixed episodes and 121 (21%) were diagnosed as suffering from
RC. Anonymous ABD controls (ABD; n=1044, 59% men)
recruited from Karolinska University Hospital, Stockholm,
Sweden, were used as controls. They were between 18–70 years
and not allowed to be on sick leave. They were negative for
hepatitis B, C, HIV and syphilis, and requested to wait up to 6
months after being exposed for a risk for blood borne infection
such as major surgical intervention, blood transfusion, accidental
pin-prick, tattoo, piercing, sex with a new partner, or visit to a
malaria endemic country. Furthermore, intravenous user of illicit
drugs and men who had had sex with men were not allowed to
Peripheral blood samples were drawn and genomic DNA was
extracted by standard procedures. Single nucleotide polymor-
phisms (SNPs) were selected from the HapMap database (www.
hapmap.org). All SNPs were genotyped on a 7900HT Fast Real-
Time PCR System Instrument using allele-specific Taqman MGB
probes labeled with fluorescent dyes FAM and VIC (Applied
Biosystems), according to the manufacturer’s instructions. Allelic
discrimination was performed with the ABI PRISM 7900HT SDS
and the SDS 2.2.1 program (Applied Biosystems). Ten percent of
the samples were run as duplicates to check for genotyping errors.
P2RX7 genetic variants with a putative functionality were
studied. Eight non-synonymous SNPs in the P2RX7 gene were
rs28360457 (Arg307Gln), rs1718119 (Thr348Ala), rs2230911
(Thr357Ser), rs2230912 (Gln460Arg), rs3751143 (Glu496Ala)
and rs1653624 (Ile568Asn) as well as one synonymous SNP:
rs1621388 and finally one essential splice site SNP rs35933842
For the array data, differential gene expression between time
points was analyzed by repeated measures ANOVA applying a
Huynh and Feldt-correction with sex and time point * sex
interaction as fixed effects and time-point as a within-subject effect.
For validation of differential expression of P2RX7 by sleep
deprivation, a similar ANOVA model was applied on qPCR data
where time point was before (denoted 0, i.e. expression level being
the mean of time points #1 to #5) or during sleep deprivation
(denoted 1, i.e. time points #6 to #9). P,0.05 was regarded as
significant in the qPCR validation analysis.
The genetic association between P2RX7 and bipolar disorder
type 1 patients with RC versus non-RC bipolar disorder type 1
patients (nonRC) was first investigated in a case-case model.
Second, the association was tested between RC and population-
based controls (ABD) in a case-control analysis . In both the
case-case and the case-control analyses, the allele frequency
difference was tested for the ten P2RX7 SNPs using logistic
regression. Since RC has been reported to be more common in
women, gender was used as covariate . The Hardy-Weinberg
equilibrium (HWE) was evaluated for each SNP using a x2-test.
The measure D9 of linkage disequilibrium (LD) was calculated
between the SNPs using the ABD controls, and haplotype blocks
were constructed according to criteria proposed by Gabriel et al.,
2002 . Haplotype distribution for the RC compared to the
nonRC patients and ABD controls was analysed using the x2-test
whereas the associations of a specific haplotype to case were
calculated using logistic regression with gender as covariate. P-
values reported in the genetic analyses are uncorrected for
multiple testing. The Bonferroni corrected threshold considering
the partial LD between markers p,0.017 (0.017=0.050/3 (3 SNP
Table 1. Clinical characteristics of the sample set that are
Bipolar disorder type 1 569
Men [n (%)] 241 (42)
Women [n (%)]328 (58)
Age at first depression (md, range) 23.5 (4–64)
Age at first mania (md, range)29.0 (6–70)
Hospitalized for affected episodes [n (%)]508 (88)
Mixed episodes [n (%)] 172 (30)
Rapid cycling [n (%)]121 (21)
Non-rapid cycling [n (%)]446 (78)
P2RX7 Associates with Rapid Cycling
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groups (defined by D9.0.80))) was regarded as significant in the
allelic association tests, whereas p,0.05/6=0.0083 (6 haplotypes)
was regarded significant in the analyses of specific haplotypes
[53,54]. The power was .0.75 to detect an association between
RC and rs2230912 for an allelic, a dominant and a co-dominant
model in the case-case design whereas it was .0.82 in the case-
control design. The corresponding power for recessive model was
0.41 for both designs. The power to detect RC association to the
other SNPs was,0.35 (http://pngu.mgh.harvard.edu/ &purcell/
P2RX7 and sleep deprivation
Using array data P2RX7 RNA levels in PBMCs were
significantly increased by total sleep deprivation in healthy
volunteers (p=2.3*1029). Sex did not influence the P2RX7
mRNA levels (p.0.85). That similar finding was found for both
male and female samples constituted a replication since male and
female samples were collected at different occasions (Figure 1).
This was validated using qPCR showing a significant increase of
P2RX7 RNA levels from before sleep deprivation (time points #1
to #5) to during sleep deprivation (time points #6 to #9) (F=7.1,
df=1, P=0.037, partial g2=0.54) where sex and sex*time point
interaction had no effect (p.0.15). The individual qPCR-based
increase in P2RX7 RNA level by sleep deprivation was for the
male samples 119%, 592%, 45% and 151%, and for the female
samples 226%, 27%, 20% and 231%.
P2RX7 and rapid cycling
The major allele rs2230912_A, was more common among RC
patients than among nonRC patients (OR=2.2, p=0.0027, 95%
CI=1.3–3.6; corresponding to OR=0.45 for allele G; Table 3).
Similarly, the allele rs2230912_A was more frequent among RC
patients than among ABD controls (OR=2.2, p=0.0016, 95%
CI=1.4–3.7, corresponding to OR=0.45 for rs2230912_G;
Table 3). Linkage disequilibrium analysis of the ten P2RX7
genotyped SNPs showed that six of them (from rs1718119 to
rs1621388) formed an LD block (Figure 2). A haplotype
association analysis was performed comparing RC with nonRC
patients. Six haplotypes of the P2RX7 block were identified and a
difference in distribution of haplotypes between the RC and
nonRC patients was found (x2=13.0, df=5, p=0.023). The risk
allele rs2230912_A was present in five haplotypes, whereas the
rs2230912_G allele was present in only one haplotype (haplotype
1). An analysis of which specific haplotypes that differed in
frequency between the RC and nonRC patients was performed.
rs1621388), including the protective G-allele from rs2230912,
was less common in RC than in nonRC patients (OR=0.45,
p=0.0031, 95% CI=0.3–0.8; Table 4). As expected, this
haplotype association was supported by the comparison between
RC and ABD controls. This comparison showed that the
ACGTTT haplotype was less common in RC patients compared
to ABD controls (OR=0.49, p=0.0050, 95% CI=0.3–0.8), and
that there was an almost significant difference comparing the
distribution of all haplotypes between RC and ABD controls
(x2=11.0, df=5, p=0.052; Table 4) .
The findings of this study were that P2RX7 expression was
affected by sleep deprivation in healthy volunteers and that a
functional polymorphism in the P2RX7 gene was associated to
rapid cycling (RC) in bipolar type 1 patients in comparison to
other bipolar disorder type 1 patients and anonymous blood
donors. The study of P2RX7 expression was hypothesis-driven
based on (i) previously reported P2RX7 genetic associations to
bipolar disorder [39,39], and (ii) circadian rhythm disturbances
reported in these patients  and (iii) previous findings of mood
dysregulation in mice lacking P2RX7 expression in the brain .
The here reported 1.5-fold increase of P2RX7 expression (mean
level of 24 probe sets over the entire RefSeq transcript) by sleep
deprivation is only slightly lower than the previously reported 2.0
fold increase of CRY2 expression level by sleep deprivation found
in the same healthy volunteers and validated by real-time PCR
. CRY2 participates in the core circadian loop. No difference in
effect of sleep deprivation on P2RX7 expression level was found
between males and females, ensuring robustness in the analysis
since males and females were investigated at different occasions.
Table 2. SNPs analyzed in P2RX7 gene.
RC nonRCcontrols success rate(%)
rs35933842 (A/C*)Intron 1Essential Splice site 0.00450.00560.0053 87.5
rs208294 (A/G*)Exon 5His155Tyr0.39 0.45 0.4286.4
rs7958311 (A/G*) Exon 8Arg270His 0.25 0.280.26 95.8
rs28360457 (A/G*)Exon 9 Arg307Gln 0.0230.015 0.01287.2
rs1718119 (A/G*)Exon 11 Ala348Thr 0.360.390.40 93.6
rs2230911 (G/C*)Exon 11 Thr357Ser0.11 0.069 0.09792.5
rs2230912 (G/A*)Exon 13 Gln460Arg0.0760.150.16 99.0
rs3751143 (C/A*)Exon 13 Glu496Ala0.16 0.160.1592.7
rs1653624 (A/T*) Exon 13Ile568Asn0.0460.033 0.030 92.8
rs1621388 (A/G*) Exon 13Synonymous0.370.400.40 96.8
aData from www.ensembl.org . Minor allele first.
bMinor allele first, data from www.hapmap.org.
*Ancestral allele in CEU population data (CEPH (Utah residents with ancestry from northern and western Europe)) from www.ncbi.nlm.nih.gov.
MAF, minor allele frequency.
P2RX7 Associates with Rapid Cycling
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Since bipolar disorder patients with RC have a more vulnerable
diurnal system than those without RC [27–29] and psychotherapy
focused on stable sleep-wake cycle is especially beneficial in RC
bipolar patients [30,31], RC patients were investigated in the
genetic study. RC was associated with a major allele of P2RX7
(Gln460) previously reported to have lower activity than the
alternative variant (Arg460). Thus, the association between RC
and a P2RX7 genetic variation implicated in P2RX7 activity, and
the sleep deprivation-induced increase of P2RX7 expression level –
sleep deprivation influences circadian rhythm - can be considered
to be in agreement with the clinically well-established knowledge
that RC patients have a more vulnerable diurnal system than those
without RC. A lower P2RX7 activity in RC may result in a less
appropriate P2RX7 response to sleep deprivation which may in
part explain a vulnerable diurnal system and consequently more
All the patients participating in this study of RC were patients
from clinics with catchment responsibility for all patients with
bipolar 1 disorder in the catchment area. Therefore, the
participants may represent patients treated for bipolar 1 disorder
in the general population of the catchment area. Almost all
patients were recruited from specialized affective disorder units, all
the medical records were studied by two investigators and most of
the patients were also interviewed, resulting in a thorough
phenotype assessment process. Cases as well as controls were
ascertained from an ethnically homogeneous population  and
further, controls were recruited from the same catchment area as
the patients, thus reducing bias due to the ethnic variation. The
prevalence of RC in bipolar disorder varies (12–24%) , which
may result from different diagnostic procedures and/or differences
between populations. Patients diagnosed as nonRC might develop
RC after the assessment but nearly 30 percent of bipolar patients
appear with RC in the early course of the disease and the
increased rate of episodes would therefore be more likely in the
first years of illness . This indicates that probably only a
limited number of the patients may change status to a RC course
The P2RX7 gene is highly polymorphic and encodes the
purinergic receptor P2X7 present in microglia, astrocytes and
neurons in several brain regions . This receptor is a ligand-
gated calcium-permeable cat ion channel activated by ATP, which
is involved in Ca2+-dependent signaling pathways . The
expression of P2RX7 is high especially in the sub thalamic nucleus,
hypothalamus and substantia nigra, all structures known to be
associated with bipolar disorder (https://www.nextbio.com/b/
search/ba/p2rx7?type=feature&id=19358).There is strong evi-
dence that P2RX7 promotes excitatory neurotransmitter release at
presynaptic sites from neurons . The SNP rs2230912
polymorphism in P2RX7 results in a glutamine–to–arginine
Figure 2. The strength of linkage disequilibrium (LD) between
pairs of SNPs in ABD controls for P2RX7. The heavy-line frame
shows suggested haplotype blocks. The numbers in the squares
represent the pair-wise D9 value, empty squares stand for D9=1. Pink-
red color indicates a pair-wise LOD$2 with redness proportional to D9.
White-blue square indicates LOD,2.
Table 3. Allelic associations for RC compared to nonRC bipolar patients and for RC compared to ABD controls in the P2RX7 gene.
Bipolar disorder type 1 Blood donors as controls
OR [95% CI]*aa/ab/bbnpb
OR [95% CI]*
rs35933842 (A/C)0/1/110111 0/4/352356 0.87 0.50 0.83 [0.089–7.2]0/10/932 9420.890.70 0.86 [0.11–6.7]
rs208294 (A/G)17/52/42 11170/173/107 3500.12 0.11 0.78 [0.59–1.1] 158/463/3109310.51 0.43 0.91 [0.66–1.2]
rs7958311 (A/G)8/39/63 110 34/151/209 3940.44 0.55 0.88 [0.61–1.2]69/409/562 1040 0.62 0.750.92 [0.68–1.3]
rs28360457 (A/G) 0/5/1051100/11/345 356 0.450.86 1.5 [0.51–4.3]0/22/917 939 0.120.087 2.2 [0.74–5.2]
rs1718119 (A/G)17/52/51 12070/208/166 444 0.37 0.56 0.87 [0.81–1.4]161/433/350 9440.29 0.280.86 [0.79–1.3]
rs2230911 (G/C) 1/24/94 1193/53/372 428 0.0510.0431.6 [1.0–2.7] 8/166/769943 0.44 0.48 1.2 [0.74–1.8]
rs2230912 (G/A)0/18/101 119 7/119/315441 0.00270.00260.45 [0.28–0.77] 26/274/7351035 0.0016 0.0015 0.45 [0.27–0.72]
rs3751143 (C/A)2/34/83 11912/110/304 4260.93 0.86 1.0 [0.69–1.5]20/240/686946 0.550.591.1 [0.76–1.6]
rs1653624 (A/T)0/11/108 1190/28/402 4300.340.44 1.4 [0.71–2.9]2/52/892 9460.210.17 1.5 [0.82–3.1]
rs1621388 (A/G) 17/49/47113 69/205/152 426 0.350.56 0.86 [0.64–1.2]174/471/375 1020 0.370.33 0.88 [0.65–1.2]
aSNP name (minor allele/major allele).
bgender as covariate, not corrected for multiple testing.
cPoint-wise p-value from 10,000 permutations with no covariate (EMP1).
*Odds ratio (OR), the proportion of minor versus major allele among affected (RC)/proportion of minor versus major allele among unaffected (nonRC or controls).
P2RX7 Associates with Rapid Cycling
PLOS ONE | www.plosone.org6 August 2012 | Volume 7 | Issue 8 | e43057
Table 4. Haplotype association for RC compared with nonRC patients and RC patients compared with blood donor controls, in the P2RX7 gene.
RC compared with nonRC
OR [95% CI]b
RC compared with controls
Fcases/Fcontrols, percent with that haplotype in the RC group/non-RC/ABD controls with that haplotype, with successful haplotyping.
anot corrected for multiple testing.
bOdds ratio (OR), the ratio specific haplotype versus all other haplotypes among RC patients/ratio specific haplotype versus all other haplotypes among nonRC patients.
P2RX7 Associates with Rapid Cycling
PLOS ONE | www.plosone.org7August 2012 | Volume 7 | Issue 8 | e43057
change (Gln460Arg), which is likely to affect P2RX7 dimerization
and protein–protein interactions . The Arg-variant (allele G)
resulted in enhanced P2X7 pore activity in human monocytes
. The gene is located on chromosome 12q24 at the center of a
strong bipolar disorder linkage peak . P2RX7 has previously
been associated with bipolar disorder, depression, anxiety
disorders [38,40,41,61], and cognitive symptoms in mania 
and is believed to play a role in antidepressant action and
causation of bipolar disorder by influencing neurotransmission
, neuroprotection , and neuroinflammatory responses
. In agreement with our finding that the relatively rare RC
phenotype had a higher frequency of the allele rs2230912_A
compared to the rest of the bipolar disorder type 1 patients,
previous studies have shown that bipolar disorder, depression and
anxiety disorders were associated with the rs2230912_G allele
[40,41]. However, frequency of rs2230912_G was similar in
nonRC and anonymous ABD controls in this study.
P2RX7 may be involved in the diurnal rhythm regulation
through indirect regulation of glutamate levels. Activated P2X7
channels have been proposed to mediate release of cytosolic
glutamate . In general, glutamate concentration increases
rapidly and progressively during wakefulness and REM sleep, and
decreases during non-REM sleep. Levels of glutamate receptors
are altered between sleep and waking periods to keep the
concentration of extracellular glutamate within a homeostatic
range across sleep–waking states [66,67]. However, alternatively
the increased P2RX7 expression seen upon sleep deprivation might
be a stress response to the sleep deprivation.
Bipolar disorder and unipolar depression patients often show a
disturbed phase in their circadian rhythm. Further, sleep
deprivation is an effective short-term treatment for depression.
The biological basis of the anti-depressive effect of sleep
deprivation is not clear, but there is evidence that sleep deprivation
resets the circadian rhythm. The P2RX7 through P2X7 receptors
induces a higher permeability for calcium, leading to an increased
intracellular calcium level and activated cytokines in the limbic
dopaminergic pathways [68,69]. We found the putatively low-
activity P2RX7 rs2230912 allele A variant to be associated with
RC in bipolar disorder which supports earlier findings of P2RX7
associations to affective disorder [37,39,40,59]. We found no
association between RC and anyone of the other SNPs analyzed.
However, due to lack of power true single SNP association to RC
for any of the SNPs without detected association cannot be
excluded. The findings may lead to a better understanding of the
biology behind RC in bipolar disorder but further studies are
needed for validation. The result also illustrates the potential of
studying defined subtypes of bipolar disorder and of applying a
compliant format. Raw and normalized data for each 71
samples624 probesets covering 18 exons is listed. The 71 samples
consist of 8 individuals (subjects) sampled at 9 time points.
Expression data for P2RX7 listed in a MIAME
We thank all the patients who participated in this study. The authors would
also like to thank the MSc Emarndeena Haji Cheteh, the study
coordinators Inger Ro ¨mer Ek and Martina Wennberg, and research
nurses Agneta Carlswa ¨rd-Kjellin and Stina Stadler for skilful assistance.
Conceived and designed the experiments: LB CL LF PN UO¨MPV MS.
Performed the experiments: CL PN MPV DSH. Analyzed the data: LB CL
PN DSH GE MPV. Contributed reagents/materials/analysis tools: LB LF
UO¨LTB ML MPV MV MS. Wrote the paper: LB CL PN LTB ML MS
UO¨GE MPV DZH MS.
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