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Analysis of single nucleotide polymorphisms in genes in the chromosome 12Q24.31 region points to P2RX7 as a susceptibility gene to bipolar affective disorder

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American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 141B:374 –382 (2006)
Analysis of Single Nucleotide Polymorphisms in Genes
in the Chromosome 12Q24.31 Region Points to P2RX7
as a Susceptibility Gene to Bipolar Affective Disorder
Nicholas Barden,1* Mario Harvey,1 Bernard Gagné,1 Eric Shink,1 Monique Tremblay,1
Catherine Raymond,1 Michel Labbé,1 André Villeneuve,1 Denis Rochette,2 Lise Bordeleau,1
Herbert Stadler,3 Florian Holsboer,4 and Bertram Müller-Myhsok4
1
Neuroscience, CHUL Research Centre and Université Laval, Quebec Canada
Complexe Hospitalier de la Sagamie, Chicoutimi, Quebec, Canada
3
Affectis Pharmaceuticals AG, Munich, Germany
4
Max-Planck Institute of Psychiatry, Munich, Germany
2
Previous results from our genetic analyses using
pedigrees from a French Canadian population
suggested that the interval delimited by markers
on chromosome 12, D12S86 and D12S378, was the
most probable genomic region to contain a susceptibility gene for affective disorders. Association studies with microsatellite markers using a
case/control sample from the same population
(n ¼ 427) revealed significant allelic associations
between the bipolar phenotype and marker NBG6.
Since this marker is located in intron 9 of the
P2RX7 gene, we analyzed the surrounding genomic region for the presence of polymorphisms in
regulatory, coding and intron/exon junction
sequences. Twenty four (24) SNPs were genotyped
in a case/control sample and 12 SNPs in all
pedigrees used for linkage analysis. Allelic, genotypic or family-based association studies suggest
the presence of two susceptibility loci, the P2RX7
and CaMKK2 genes. The strongest association was
observed in bipolar families at the non-synonymous SNP P2RX7-E13A (rs2230912, P-value ¼
0.000708), which results from an over-transmission of the mutant G-allele to affected offspring.
This Gln460Arg polymorphism occurs at an amino
acid that is conserved between humans and
rodents and is located in the C-terminal domain
of the P2X7 receptor, known to be essential for
normal P2RX7 function.
ß 2006 Wiley-Liss, Inc.
KEY WORDS: bipolar disorder; association analysis; linkage disequilibrium; purinergic receptor
Please cite this article as follows: Barden N, Harvey M,
Gagné B, Shink E, Tremblay M, Raymond C, Labbé M,
Villeneuve A, Rochette D, Bordeleau L, Stadler H,
Holsboer F, Müller-Myhsok B. 2006. Analysis of Single
This article contains supplementary material, which may be
viewed at the American Journal of Medical Genetics website
at http://www.interscience.wiley.com/jpages/1552-4841/suppmat/
index.html.
*Correspondence to: Dr. Nicholas Barden, Neuroscience,
CHUQ pavillon CHUL, 2705 Blvd. Laurier, Québec, QC, G1V
4G2, Canada. E-mail: barden@crchul.ulaval.ca
Received 22 November 2005; Accepted 8 February 2006
DOI 10.1002/ajmg.b.30303
ß 2006 Wiley-Liss, Inc.
Nucleotide Polymorphisms in Genes in the Chromosome 12Q24.31 Region Points to P2RX7 as a Susceptibility Gene to Bipolar Affective Disorder. Am J Med
Genet Part B 141B:374–382.
INTRODUCTION
Mood disorders, including bipolar (BP) and major depressive
disorders (MDD) are the most common psychiatric disorders
with a combined lifetime prevalence of around 15% [Blazer
et al., 1994]. Patients cycle through episodes of depression and
euthymia (MDD) or mania (BP) demonstrating profound
changes in affect and mood, cognition, neurovegetative function, and psychomotor activity. Mood disorders are complex
traits involving both environmental and genetic factors.
Although several chromosomal regions, including 4p16,
12q23-24, 13q32-33, 18p11, 18q21-23, 21q22, and Xq26, show
genetic linkage with either BP, MDD, or both, only a few
candidate genes have been pointed to [Hattori et al., 2003;
Sjoholt et al., 2004; Hoefgen et al., 2005]. Candidate gene
approaches, focusing on neurotransmitter systems (e.g.,
norepinephrine, serotonin, and excitatory amino acids), stress
hormone regulation (hypothalamic-pituitary-adrenocorticalaxis, HPA-axis) or factors involved in the postulated failure of
adult neurogenesis in depression have had limited success
[Zhang et al., 2005]. Our previous genetic studies in families
from the Saguenay-Lac-St.-Jean (SLSJ) region of Quebec led to
the identification of a susceptibility locus for both BP and MDD
in the region of chromosome 12q24 with a parametric LOD
score value of 3.35 under a recessive model and an MLS score
value of 5.05 [Morissette et al., 1999; Shink et al., 2005a]. These
results have received support from other studies in the 12q2324 region that suggested linkage with mood disorders including both BP and MDD [Abkevich et al., 2003; Curtis et al.,
2003]. One marker, NBG6, gave a MLOD value equal to 3.35
with associated P-value less than 0.0001 [Shink et al., 2005b].
This marker has been located within intron 9 of the P2RX7
gene and for this reason we investigated genes within 100 kb
up- and down-stream of this marker.
In this study, we report association studies on three
consecutive genes P2RX7, P2RX4, and CAMKK2 genes in a
case/control sample from the SLSJ region. All of these genes
share common features including expression in brain and a
role in Ca2þ-dependent signaling pathways. We screened for
mutations and genotyped SNPs with minor allele frequency
greater than 0.01 in case/control and pedigree samples. Taken
together, results of allelic and genotypic association as well as
haplotype analysis and family-based association point to
P2RX7 as a susceptibility gene for bipolar disorder.
P2RX7 and Bipolar Disorder
METHODS
Ascertainment and Diagnosis
Pedigrees. We ascertained 485 individuals from 41
families of the SLSJ region of Quebec. All Individuals were
interviewed using a French translation of the Structured
Clinical Interview for DSM-IIIR/IV [Spitzer et al., 1987] and a
best estimate diagnosis established by panel including at least
two psychiatrists. Diagnoses among the whole genotyped
individuals were distributed as follows: 105 bipolar I disorder
or schizoaffective disorder, bipolar type (BPI), 42 bipolar II
disorder (BPII), 54 recurrent major depression (MDD), and 57
single episode major depression.
Case/control sample. Diagnostic and ascertainment
procedures were as previously described [Morissette et al.,
1999; Shink et al., 2005b]. The SLSJ case sample was composed
of 213 unrelated individuals with attachments to the founding
population of SLSJ and included BPI (n ¼ 182, mean age at
onset 28 11 (mean SD), 60% female) and BP II (n ¼ 31,
mean age at onset 27 11 (mean SD), 55% female) diagnosed
subjects. The control sample contained 214 individuals also
coming from the SLSJ region, most of whom had previously
participated in non-psychiatric genetic projects and provided
informed written consent to participate in other genetic
studies. No detailed genealogic information on this sample is
available and, while only individuals drawn from clearly
different pedigrees were sampled, we cannot rule out more
distant degrees of relationship. All protocols were approved by
and conducted in strict adherence to the ‘Comité d’éthique de la
recherche clinique du CHUQ’. No screen to exclude control
subjects with a history of psychiatric illness was done since
there is no real gain in power to screen controls for association
studies of diseases with lifetime risk estimated at 1% for BP
[Owen et al., 1997]. Genders were distributed similarly in both
samples according to the Fisher’s exact test (P-value ¼ 0.08)
and no attempt was made to match the controls and cases by
age. Using a critical level of 0.05, power calculations done with
PAWE [Gordon et al., 2002, 2003] showed that our case/control
sample had a power of 74% to detect allelic association
assuming an odds ratio (OR) of 1.7 and a diallelic polymorphism with minor allele frequency of 10% in the general
population. Power was 64% for genotypic association under
the same conditions.
Genomic DNA preparation. Blood samples from each
individual were collected in 10-ml K3 EDTA Vacutainer tube
(Becton-Dickinson) and genomic DNA was isolated using a
Puregene DNA Isolation kit (Gentra Systems). DNA was
solubilized in 500 ml of DNA Hydration Solution and the final
concentration was adjusted to 300–400 mg/ml by spectroscopy
at 260 nm.
Mutation analyses. Single nucleotide polymorphisms
and other variations were searched in coding sequences and
exon–intron boundaries of genes using direct sequencing. The
starting sample was composed of 16 unrelated BP affected
individuals selected to maximize the number of different
haplotypes and according to their link with families that gave
positive genetic linkages on chromosome 12q24.
Genotyping of SNPs. SNPs were genotyped by resequencing of the amplification products. The sequencing traces for
each individual are automatically typed for the corresponding
SNP using a home-developed program, GENO.pl and genotyping results compiled in a 4D database. Deviation from Hardy–
Weinberg equilibrium (HWE) was assessed for each SNP in
both case and control groups with the exact test from Genepop
software package (http://wbiomed.curtin.edu.au/genepop).
Statistics. Testing for allelic and genotypic association
with the BPI or BPII phenotype, as well as the computation of
odds ratios and 95% confidence intervals, was tested with the
Fisher’s exact test from the SISA webserver (http://home.-
375
clara.net/sisa/). Haplotypes, either within block or inter-block,
were estimated with the expectation-maximization (EM)
algorithm [Excoffier and Slatkin, 1995] implanted in the
cocaphase module of UNPHASE Version 2.40 [Dudbridge,
2003]. Since the EM algorithm has limited precision to
estimate haplotype frequencies <1%, such haplotypes were
excluded using the–droprare option. P-values for individual
haplotypes were calculated with the–individual option. The
family-based association tests were done using the FBAT
software version 1.5.5 [Rabinowitz and Laird, 2000], in which
alleles transmitted to affected offspring are compared with the
expected distribution of alleles among offspring. The affected
status is defined as BP I and II, all other individuals are
considered as unaffected or unknown. We tested for the null
hypothesis of no association and no linkage, and used a biallelic test. Since we may not estimate disease prevalence we
used the–o option with the FBAT command, thus estimating
an offset value to minimize the variance.
RESULTS
DNA from 16 unrelated BPI (n ¼ 12) and BPII (n ¼ 4)
individuals, who were selected among families and trios from
the SLSJ region to maximize the number of different potential
haplotypes, was sequenced from both strands over the genes
P2RX7, P2RX4, and CAMKK2. We identified 126 variations, of
which 39 were found in untranslated sequences, 34 in coding
sequences, and 53 in intronic regions (see supplementary
information on-line, Table S1). The P2RX7-associated SNPs
accounted for 56% (71) of total identified SNPs in this genomic
region (Fig. 1). Thirteen of these polymorphisms lead to nonsynonymous mutations (nsSNP) causing amino acid changes
and one described a deletion of seven amino acids (del488-494).
Selection of SNPs for genetic analysis was mostly based on the
putative SNP functionality. We typed 24 polymorphisms
identified with minor allele frequency higher or equal to 5%
distributed in regulatory sequences (10), coding sequences
(13), and exon/intron junctions (1) of these three adjacent genes
(Table I). The case/control sample was composed of 213 BP
patients and 214 controls that gave 71% power to detect
significant allelic association, at P-value ¼ 0.05, for polymorphisms of minor allele frequency of 0.05 and showing a relative
risk of 2. Accordingly, our analysis strategy was to determine
positive associations under the P-value 0.05 without correction
for multiple testing and further confirm the positive results in
an independent sample. The deviation from Hardy–Weinberg
equilibrium was calculated in both case and control groups. We
observed disequilibrium with one polymorphism, P2RX7E11B, which gave a heterozygote deficiency in the control
group and an excess among cases. The hypothesis of allelic or
genotypic association with BP was evaluated with a Fisher’s
exact test. Since we may not anticipate the correct inheritance
model, the genotypic analysis was done under additive,
dominant and recessive models. Only three markers, P2RX7I07E, P2RX7-E11B, and P2RX4-UTR3A gave significant allelic
and/or genotypic association (Table IIA). The pairwise LD
measures between P2RX7-I07E and P2RX7-E11B revealed
that both markers are closely linked together, however,
considering the HW disequilibrium at P2RX7-E11B locus we
should exclude these positive results. The P2RX4-UTR3A
polymorphism is about 75 bp downstream of the P2RX4 gene
and is unlikely to interfere with gene function, but it might
be linked to other functional polymorphisms. We tried to
reduce phenotypic heterogeneity by excluding the 31 BPII
patients, and observed only slight decrease in significance
(not shown) probably caused by the loss of power. Considering
both D0 > 0.33 and r2 > 0.1 as a threshold for meaningful LD
[Kruglyak, 1999; Nakajima et al., 2002], P2RX4-UTR3A is
found in LD with six distant SNPs in the case group, P2RX7-
376
Barden et al.
TABLE I. Description of Single Nucleotide Polymorphisms Analyzed
SNP ID
P2RX7-UTR5F
P2RX7-UTR5G
P2RX7-UTR5A
P2RX7-UTR5B
P2RX7-UTR5E
P2RX7-E02A*
P2RX7-I04B
P2RX7-E05A*
P2RX7-I07E
P2RX7-E08A*
P2RX7-E11B*
P2RX7-E11C*
P2RX7-E13A*
P2RX7-E13B*
P2RX7-E13C*
P2RX7-UTR3A
P2RX7-UTR3B
P2RX4-UTR5A
P2RX4-UTR5B
P2RX4-E07A
P2RX4-UTR3A*
CAMKK2-E09A*
CAMKK2-E01B*
CAMKK2-E01A*
dbSNPa
Allele(modification)
Minor allele frequency
Positionb
rs523977
rs520396
rs494986
rs2393799
rs684201
ss35031323
rs208293
rs208294
rs504677
rs7958311
rs1718119
rs6489795
rs2230912
rs3751144
rs3751143
ss35031375
rs1653625
ss35031381
ss35031382
rs11065500
rs11065501
rs4980999
rs3817190
ss35031413
T-C
T-G
C-A
C-T
G-A
T-C(Val76Ala)
C-T
T-C(Tyr155His)
C-T
G-A(Arg270His)
G-A(Ala348Thr)
C-G(Thr357Ser)
A-G(Gln460Arg)
C-T(Pro474Pro)
A-C(Glu496Ala)
C-A
A-C
C-A
A-G
A-G(Ser242Gly)
C-G
C-T(Arg363Cys)
T-A(Thr85Ser)
C-T(Ser10Asn)
0.19
0.10
0.05
0.23
0.05
0.06
0.25
0.49
0.35
0.25
0.37
0.10
0.18
0.10
0.23
0.46
0.07
0.16
0.31
0.15
0.31
0.15
0.38
0.08
12138634
12139014
12139445
12139521
12139852
12162198
12169689
12169762
12174698
12174864
12184612
12184640
12191705
12191748
12191813
12192393
12192394
12216833
12216944
12236155
12241479
12260605
12281586
12281810
All SNPs were genotyped in the case/control sample, whereas only those with asterisk (*) were genotyped in pedigrees.
a
Available in dbSNP build 125.
b
Position relative to the contig NT_009775.15 from the NCBI.
UT5G (r2 ¼ 0.131, D0 ¼ 0.764, distance ¼ 102.5 kb), P2RX7E02A (r2 ¼ 0.112, D0 ¼ 0.857, distance ¼ 79.3 kb), P2RX7-E08A
(r2 ¼ 0.168, D0 ¼ 0.483, distance ¼ 66.7 kb), P2RX7-E13C
(r2 ¼ 0.132, D0 ¼ 0.952, distance ¼ 49.7 kb), P2RX4-UTR5B
(r2 ¼ 0.106, D0 ¼ 0.688, distance ¼ 24.7 kb), and CAMKK2E01A (r2 ¼ 0.168, D0 ¼ 0.945, distance ¼ 40.4 kb). This irregular pattern of LD has been noted in many studies on small
genomic regions with densely selected markers [Taillon-Miller
et al., 2000; Abecasis et al., 2001; Nakajima et al., 2002], and is
to be expected when recombination becomes rare relative to
other factors influencing LD, such as mutation rate, genetic
drift, and gene conversion [Ardlie et al., 2001]. Consequently,
considering P2RX4-UTR3A as a true positively associated
locus, the causal disease mutation(s) might be found anywhere
within the three adjacent genes.
To support the single marker association study and define
more accurately a susceptibility locus, we then carried out
haplotype analysis. The LD measures, especially the very low
r2 values, reflect large variation in marker allele frequencies,
and consequently suggest important haplotype diversity
(Fig. 2). Considering this, we firstly segmented the region in
haplotype blocks to minimize estimated haplotype and
increase power to detect positive associations (Fig. 2). We
defined four blocks, and estimated haplotypes using the EM
algorithm implemented in cocaphase program. We identified
global significant P-values for blocks 2 and 4 with P-value ¼
0.0410 and 0.0390, respectively (Table IIB). The haplotype
block 2 defines the last six exons of the P2RX7 gene while block
4 encloses the associated SNP P2RX4-UTR3A, hence supporting the prior single marker analysis.
Although useful for statistical analysis, it is however
inappropriate to arbitrarily segment a continuous chromosomal region. To circumvent this without loss of power, we
treated the selected region as a continuum by performing
haplotype analysis using a sliding window method (in
cocaphase program) with a small window of seven SNPs
(Table IIC). We considered only estimated haplotypes showing
frequencies greater than or equal to 0.03 in either group, thus
decreasing the degree of freedom and giving greater power to
detect effects in common haplotypes. This method showed two
significantly associated haplotypes, which are close to those
aforementioned. The 7-SNP positively associated haplotype
window bordered by P2RX7-I04B and P2RX7-E13A overlaps
blocks 1 and 2 and is characterized by a haplotype strictly
observed in the case group (P-value ¼ 0.0006) bearing the
mutated allele for the non-synonymous SNP P2RX7-E08A
(Table IIC). The other 7-SNP positively associated window
bordered by markers P2RX7-UTR3A and CAMKK2-E01B
comprises blocks 3 and 4 and the associated marker P2RX4UTR3A (Table IIC). This haplotype analysis supports the
single marker result at the locus P2RX4-UTR3A and raises the
possibility of a susceptibility locus in the P2RX7 gene.
However, according to the LD structure, we may not answer
the question as to whether these loci are linked between each
other, linked with other loci or independently involved.
The selection of this region for a case/control SNP-based
association studies was based on linkage results in pedigrees
from the SLSJ region. We therefore genotyped in the 41
pedigrees that were used for linkage studies [Shink et al.,
2005a] all 11 non-synonymous SNPs (with minor allele
frequency >0.01) and the associated SNP P2RX4-UTR3A that
are found in the region delineated by the positive haplotype
results. We used the family-based approach for association
(FBAT), which uses data from all family members and avoids
complications due to population admixture often found in case/
control studies. Allelic association was first tested under the
null hypothesis of no association and no linkage, and we
generated data under three inheritance models: additive,
dominant, and recessive, as well as using either or both bipolar
I and bipolar II as affected status (see supplementary Table
S2). Considering that BP disorder is a complex trait and having
no estimation of disease prevalence, we used the–o option
giving an offset value in order to minimize the variance of the
statistic. Table IIIA summarizes the results by showing
significant associations (P-value <0.05). We observed two
positively associated polymorphisms, P2RX7-E13A and
P2RX7 and Bipolar Disorder
377
TABLE IIA. Allelic and Genotypic Associations
SAMPLE
SNP
SAMPLE
Fisher’s
(P-value) OR (CI 95%) Genotype
Allele
N
A
P2XR7-I07E
C
T
272
156
278
140
0.387
0.88
(0.66–1.17)
P2XR7-E11B
G
A
271
155
269
157
0.9343
1.02
(0.77–1.35)
P2XR4-UTR3A
C
G
313
115
279
147
0.018
1.43
(1.07–1.92)
C/C
C/T
T/T
G/G
G/A
A/A
C/C
C/G
G/G
N
A
92
88
34
93
85
35
114
72
17
87
104
18
78
113
22
91
96
25
Fisher’s
(P-value)
OR (CI 95%)
Model
0.0262
0.50 (0.27–0.92)
Recessive
0.0172
1.34 (0.90–1.98)
Additive
0.008
1.70 (1.16–2.51)
Dominant
SNP-based association study. Positive results from the SNP-based association study conducted in the case/control sample from the SLSJ region (BP ¼ 213,
controls ¼ 214).
TABLE IIB. Haplotype Analysis Within Blocks
Frequency
Block
Marker
Allele
Case
Control
Chi2 (P-value)
LRS (global
P-value)
0.0410
2
P2RX7-I07E
P2RX7-E08A
P2RX7-E11B
P2RX7-E11C
P2RX7-E13A
P2RX7-E13B
P2RX7-E13C
P2RX7-UTR3A
C-A-G-C-A-C-A-C
C-G-G-C-A-C-C-C
T-G-A-C-G-C-A-A
T-G-A-C-A-C-A-A
C-G-G-G-A-T-A-A
C-G-G-C-A-C-A-C
C-G-A-C-A-C-A-A
C-A-G-C-G-C-A-C
T-G-G-C-A-C-A-C
0.26
0.21
0.18
0.13
0.09
0.04
0.04
0.01
0
0.22
0.21
0.16
0.18
0.11
0.05
0.02
0
0.02
0.159
0.979
0.551
0.095
0.346
0.249
0.101
0.012
0.133
4
P2RX4-UTR3A
CAMKK2-E09A
CAMKK2-E01B
CAMKK2-E01A
C-C-T-C
C-C-A-C
G-C-T-C
C-T-T-C
G-C-A-T
G-C-A-C
0.26
0.26
0.19
0.13
0.09
0.07
0.34
0.22
0.13
0.17
0.06
0.07
0.012
0.314
0.065
0.179
0.235
0.411
0.0390
SNP-based association study. Positive results from the SNP-based association study conducted in the case/control sample from the SLSJ region (BP ¼ 213,
controls ¼ 214).
TABLE IIC. Haplotype Analysis With a Sliding Window Strategy
Frequency
7-SNP window
(markers)
Allele
Case
Control
Chi2 P-value
C-C-C-A-G-C-A
0.16
0.15
0.523
C-C-C-G-G-C-A
C-T-C-A-G-C-A
C-T-C-G-G-C-A
C-T-C-G-G-G-A
C-T-T-G-A-C-G
T-C-C-A-G-C-A
T-C-T-G-A-C-A
T-C-T-G-A-C-G
0.06
0.08
0.19
0.07
0.13
0.03
0.11
0.05
0.06
0.08
0.19
0.09
0.14
0
0.16
0.02
0.864
0.911
0.959
0.391
0.824
0.0006
0.060
0.048
A-A-G-G-C-T-T
0.11
0.13
0.362
A-C-A-A-C-C-A
A-C-A-A-C-C-T
A-C-A-A-C-T-T
A-C-A-A-G-C-A
A-C-A-A-G-C-T
A-C-G-A-C-C-A
A-C-G-A-C-C-T
C-C-A-A-C-C-T
0.14
0.18
0.02
0.13
0.19
0.09
0.05
0.03
0.16
0.2
0.03
0.10
0.13
0.06
0.07
0.07
0.685
0.487
0.544
0.106
0.073
0.163
0.327
0.012
LRS (global P-value)
0.0178
P2RX7-I04B—
P2RX7-E13A
0.0249
P2RX7-UTR3B—
CAMKK2-E01B
SNP-based association study. Positive results from the SNP-based association study conducted in the case/control sample from the SLSJ region (BP ¼ 213,
controls ¼ 214).
378
Barden et al.
Fig. 1. Gene structure of P2RX7, P2RX4, and CAMKK2. a: The P2RX7 gene codes for 13 exons. All coding, untranslated sequences and 1,800 pb upstream
of the start codon were analyzed. The P2RX4 gene codes for 12 exons. The CAMKK2 gene has two protein variants from differential usage of the last coding
exon, resulting in distinct 30 -untranslated sequences. b: The P2RX7 gene product has four major domains: a short intracellular N-terminal domain, an
extracellular loop (ECL) separated by two trans-membrane domains (TM), and a long intracellular C-terminal domain. Eight SNPs were identified in the
extracellular loop, one in the second hydrophobic domain (TM2) and eight in the intracellular C-terminus. Residues Gly150, Glu186, and Arg276 are mostly
conserved between P2X family members and Leu191, Thr357, Gln460, Glu496, and Arg578 among P2RX7 ortholog genes. The P2RX4 gene product shares
the same structural organization as P2RX7. CAMKK2 encodes for two isoforms of 587 and 533 amino acids and is composed of a kinase domain (KD), ATP
binding site (ABS) and two overlapping domains, the calmodulin binding domain and the autoinhibitory domain (CaMBD/AID). The CAMKK2 gene extends
over 60 kb, and is divided into 20 exons.
Fig. 2. Linkage disequilibrium (LD) measures and haplotype block structure across the 167 kb P2RX7-adjacent region using the Haploview program
[Barrett et al., 2004]. Haplotype blocks are defined by successive SNPs given a mean jD0 j > 0.80.
P2RX7 and Bipolar Disorder
379
TABLE IIIA. Allelic Analysis
Allele
Frequency
Model
Z
P-value
Offset
P2RX7-E13A
G
A
0.146
0.854
Additive
3.387
0.000708
0.311360
CAMKK2-E01B
A
T
0.419
0.581
Additive
2.421
0.01548
0.275688
Marker
Family-based association study. Description of positive results (P-value <0.05) from the family-based association
study.
TABLE IIIB. Genotypic Analysis
Genotype
Frequency
Z
P-value
P2RX7E-13A
A/A
A/G
G/G
0.722
0.265
0.014
2.028
0.688
3.157
0.04253
0.49170
0.00156
CAMKK2-E01B
A/A
A/T
T/T
0.127
0.583
0.290
1.973
0.254
1.549
0.04846
0.79963
0.12131
Marker
Family-based association study. Description of positive results (P-value <0.05) from the family-based association
study.
CAMKK2-E01B, with the additive model. Both are nonsynonymous SNPs, and describe an over-transmission of the
mutant allele (minor allele) to affected offspring. If the
phenotype was restricted to BPI only, results remained
significant but decreased slightly (not shown) probably due to
loss of power. It is noteworthy that the marker P2RX4-UTR3A
is not overtransmitted. When we tested for the null hypothesis
in large pedigrees that show linkage on chromosome 12, the
locus P2RX7-E13A remained positively associated but with
lesser significance (P-value ¼ 0.008, not shown). This indicates
that the strong positive association (nominal P-value ¼
0.000708, P-value of 0.0252 Bonferroni corrected for a total
of 36 SNPs tested both in the case-control and the FBAT
analysis) is not a reflection of a pedigree-specific effect but has a
broader incidence in the SLSJ population. The P2RX7-E13A
locus defines a missense mutation leading to amino acid
change Gln460Arg (Table IIIA). This residue of the C-terminal
domain is located in an SH3-like domain and is conserved
between humans and rodents. In contrast, the other associated
polymorphism in the CAMKK2 gene, Thr85Ser, is not
conserved, and is not a phosphorylation site. The analysis
of genotype distribution gave support to allelic results
(Table IIIB). We noted a significant over-transmission of the
homozygote mutant/mutant-genotype to affected children.
This is also in agreement with the previous linkage findings
where positive LOD scores on 12q were observed under a
recessive model (Morissette et al., 1999; Shink et al., 2005a].
This conclusion is strengthened by TDT analysis in the SLSJ
families used for the linkage finding and which gave a
significant result for P2RX7-E13A, with the G-allele, which is
positively associated in the case-control studies, being transmitted preferentially in 71% of all informative meioses
(P ¼ 0.04). Using only one sib sampled at random from each
family we estimated the relative risk in these families to be 2.5.
We also performed haplotype analysis in order to reveal any
other loci that might be hidden by single marker analysis, but
did not observe any significantly associated haplotypes except
for those bearing the P2RX7-E13A locus (data not shown).
DISCUSSION
Several groups have pointed to the long arm of chromosome
12 as a susceptibility locus for bipolar disorders. However,
extensive genetic studies on 12q did not demonstrate a
consensus region but suggested three major and overlapping
susceptibility loci extending over 35 Mb (see Figure 3). Our
linkage analyses revealed two loci on chromosome 12q. The
major locus is located at 12q24.31 where non-parametric
analysis, based on a broad affection status (bipolar, schizoaffective disorders, and recurrent major depression), gave a
maximum MLS score of 5.05 at the marker D12S378. Both
linkage and association studies have already highlighted this
narrow critical region [Dawson et al., 1995; Degn et al., 2001;
Curtis et al., 2003]. While linkage studies from Danish
pedigrees also demonstrated positive linkages more telomeric
on 12q24 with a maximum LOD score at D12S1639 [Ewald
et al., 2002], other studies pointed to regions more centromeric
on 12q23-24 [Craddock et al., 1994; Ekholm et al., 2003]. A
recent study on two pedigrees that cosegregated mood
disorders and Darier’s disease delimited another critical region
of interest at 12q23-q24.1 between D12S1127 and D12S1646
[Green et al., 2005]. A more conservative analysis of these data
described a full region extending over 26 cM. Accordingly, the
long arm of chromosome 12 may include more than one gene
involved in the susceptibility to mood disorders. Bipolar
disorder is described as a complex genetic trait with influenced
by several genes with common natural variants. Consequently,
it is quite likely that more than one gene associated to bipolar
disorders will be found on chromosome 12q. Recent candidate
gene analyses on 12q have pointed to three genes with positive
association to bipolar disorder [Glaser et al., 2005a,b; LyonsWarren et al., 2005]. Considerable distances between each of
these genes strongly supports the assumption of several BPassociated genes or more speculatively, a mood-regulating
gene cluster.
In this report, we present the analysis of three genes in the
vicinity of the associated marker, NBG6. These three genes,
P2RX7, P2RX4, and CAMKK2, are potentially implicated in
Ca2þ signaling and neurotransmitter release. Changes in
intracellular concentration of Ca2þ have been observed in
patients suffering from mood disorders [Wasserman et al.,
2004]. The mutation screening among BP affected individuals
covered 26 kb, mostly in coding and regulatory sequences. This
genomic region showed a mutation rate of 5 103 (71/9789),
which is greater than the mean genome mutation rate of
8 104 [Reich et al., 2002]. Interestingly, the P2RX7 gene
380
Barden et al.
Fig. 3. Positive genetic linkage findings in bipolar disorders on 12q23-24. The relevant studies are shown, together with the location of the maximum
LOD score in support of linkage. At left, the positively associated candidate genes are listed. Their approximate genomic position is relative to the human
genome build 35. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
revealed several non-synonymous polymorphisms (16), but
after confirmation in a larger sample (50 affected individuals)
many of these had a minor allele frequency lower than 0.01. It
is possible that our extensive mutation screening within this
genomic region revealed variations usually missed by other
more conservative mutation screens. Considering that this
region is associated with bipolar disorder (results presented
here), the observation of such mutation variability [Splendore
et al., 2000] could underline the possible contribution of many
relatively rare polymorphisms in alteration of protein function
rather than a few common variants [Wright et al., 2003] a fact
that decreases the power to detect associated SNPs in small
case/control samples. While the functional relevance of all
these mutations is unknown, the high variability is intriguing,
suggesting that P2RX7 is not essential for survival as indicated
by P2RX7 knockout transgenic mice [Solle et al., 2001].
Regardless, our case/control association studies revealed
positive association with polymorphisms in P2RX4. These
polymorphisms are putatively non-functional and most likely
in LD with another functional SNP in P2RX7, P2RX4, or
CAMKK2.
Fewer susceptibility alleles should be found among family
members compared to unrelated case/control samples and a
family-based approach with large pedigrees would give more
power to detect specific alleles. In fact, this strategy allowed us
to detect a strong association between BP disorder and the
functional polymorphism P2RX7-E13A, which leads to change
of amino acid Gln460 to Arginine. Retrospectively, we could
ask why P2RX7E13A was not associated in the case/control
sample. Firstly, single marker analysis gave an odds ratio of
1.26 at this locus (frequency in cases ¼ 0.200, frequency in
controls ¼ 0.165, see supplementary Table S3), and our sample
was thus underpowered to detect an association. However,
haplotype analysis pointed to P2RX7-E13A since the haplotype
unique to the case group has the P2RX7-E13A mutant-G allele,
which is the only variation from the most frequent haplotype.
The family-based association study also pointed to a second
associated polymorphism in the CAMKK2 gene. This mutation
causes the change Thr85Ser. Thr85 is not conserved between
mammals and is not a predicted phosphorylation site. This
locus is in meaningful LD (r2 ¼ 0.170, D0 ¼ 0.695) with the
strongly associated marker P2RX7-E13A, and could explain
the positive result. However, another genotyped marker with a
similar frequency, P2RX7-E11B, is also in useful LD with
P2RX7-E13A (r2 ¼ 0.358, D0 ¼ 0.847) but has not been associated to BP. Therefore, we cannot exclude the CAMKK2 gene
as an additional susceptibility gene, as hinted by microarray
gene expression analysis with mouse models that showed both
methamphetamine and valproate influence the expression of
CAMKK2 [Ogden et al., 2004].
ATP-gated P2X receptors are cation-selective ion channels
with high calcium permeability that open on binding of
extracellular ATP [Khakh, 2001; North, 2002]. The P2RX7E13A susceptibility mutation described here is located in the
intracellular C-terminal domain, which is known to be
essential for protein functions such as large pore formation,
intracellular signaling, membrane blebbing and receptor
trafficking [Kim et al., 2001; Wilson et al., 2002]. Two
mutations, E496A (rs3751143) and I568N (rs1653624) in this
region have been reported as critical for P2RX7 function [Gu
et al., 2001; Wiley et al., 2003]. Chimeric protein experiments
between human and rodent P2RX7 have demonstrated that
amino acids 347–595 are responsible for the functional
differences between these native receptors [Rassendren et al.,
1997] indicating that non-synonymous polymorphisms in the
C-terminal domain (conserved or non-conserved) are likely to
be dysfunctional. Little is known about the functional effect of
the Q460R mutation. However, the Q460 residue is conserved
between humans and rodents and is part of an SH3-like
domain [Denlinger et al., 2001]. This residue could thus be
involved in P2RX7 dimerization or in other protein–protein
interactions involving SH3 domain-containing proteins.
P2RX7 and Bipolar Disorder
Although conserved missense mutations are not always
disease-predisposing alleles [Saunders et al., 2006], the likelihood that these variations would be more frequently observed
associated to diseases is suggested by extrapolation from
monogenic diseases [Botstein and Risch, 2003] and also by
some complex disorders [Bonifati et al., 2003].
Expression of P2X7 receptor is mostly restricted to immunerelated cells, such as monocyte/macrophage, NK-cells, T- and Bcells [Collo et al., 1997; Gu et al., 2000; Chakfe et al., 2002]. In
brain, both microglia and astrocytes are stimulated, by ATPinduced P2X7 receptor activation, to produce cytokines, chemokines and growth factors. These effects of P2RX7 are thought to
promote inflammatory response by activating and recruiting
immune cells. On the other hand, the activation of astrocytes to
produce growth and trophic factors enhances neuronal survival
and promotes neurogenesis [Walter et al., 2004]. There is also
evidence for the presence of P2X7 receptors in presynaptic
terminals, however this is controversial since the P2RX7-KO
mice showed the same anti-P2X7 labeling characteristics as the
control animals [Sim et al., 2004]. While there is some support
for a neurodegenerative role of P2RX7 [Le Feuvre et al., 2002],
recent studies demonstrated its neuroprotective effect [Wang
et al., 2003; Suzuki et al., 2004; Walter et al., 2004].
Consequently, the role of P2RX7 during an immune response
would not be limited to the activation of immune associatedcells, but would extend to the modulation of the response
intensity, thus preventing extended or uncontrolled inflammatory response. The P2X7 receptor has already been associated to
polygenic inflammatory diseases such as systemic lupus
erythematosus [Nath et al., 2004; Elliott et al., 2005].
The concept of an inter-relationship between the psychological state and immune status can be traced back several years
and it is now well accepted that the nervous, endocrine, and
immune systems are so closely linked that they should be
regarded as a single network. Recently it has been suggested
that immune activation could be present in depressed patients.
Several works reported increased concentrations of proinflammatory cytokines and their receptors in depressed
individuals [Song et al., 1994; Maes, 1995; Kim et al., 2002].
Consequently, harmful or sustained inflammatory responses
could be associated to depressive behavior, and it is likely to
propose a role for the inflammatory mediator P2X7 receptor.
In conclusion, the association studies presented here,
especially the family-based study, clearly demonstrate the
presence of a BP susceptibility locus close to the already
associated marker NBG6. Although the strongest result has
been observed with the P2RX7 gene, we may not exclude the
CAMMK2 gene as a second putative candidate gene. Further
genetic work will probably answer whether these loci are
linked, interact together or are totally independent. The
presence of several non-synonymous SNPs in the P2RX7 gene
suggests a role for different mutant haplotypes and recent
molecular studies on P2RX7 haplotypes lead to the assumption
that many mutations would trigger a similar phenotype, that is
a dysfunctional P2X7 receptor.
ACKNOWLEDGMENTS
We acknowledge the participation of individuals and family
members in this work.
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