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Association of the putative susceptibility gene transient receptor potential protein melastatin type 2 with bipolar disorder.

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American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 141B:36 – 43 (2006)
Association of the Putative Susceptibility Gene,
Transient Receptor Potential Protein Melastatin Type 2,
With Bipolar Disorder
Chun Xu,1 Fabio Macciardi,2,5,6 Peter P. Li,1,4,5 Il-Sang Yoon,7 Robert G. Cooke,3,5 Bronwen Hughes,1
Sagar V. Parikh,3,5 Roger S. McIntyre,3,5 James L. Kennedy,2,5,6 and Jerry J. Warsh1,3,4,5,6*
1
Laboratory of Cellular and Molecular Pathophysiology, Centre for Addiction and Mental Health, University of Toronto, Toronto,
Ontario, Canada
2
Laboratory of Neurogenetics, Centre for Addiction and Mental Health, University of Toronto, Toronto, Ontario, Canada
3
Mood Disorders Program, Centre for Addiction and Mental Health, University of Toronto, Toronto, Ontario, Canada
4
Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada
5
Department of Psychiatry, University of Toronto, Toronto, Ontario, Canada
6
Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada
7
Department of Neurosciences, University of California, San Diego, California
Disturbed intracellular calcium (Ca2þ) homeostasis has been implicated in bipolar disorder (BD).
Reduced mRNA levels of the transient receptor
potential Ca2þ permeable channel melastatin type
2, TRPM2, in B lymphoblast cell lines (BLCL) from
bipolar I disorder (BD-I) patients showing elevated basal intracellular Ca2þ ([Ca2þ]B), an index
of altered intracellular Ca2þ homeostasis, along
with its location within a putative BD susceptibility locus (21q22.3), implicates the involvement
of this gene in the Ca2þ abnormalities and the
genetic diathesis to BD. We tested this hypothesis
by examining the association of selected single
nucleotide polymorphisms (SNPs) and their haplotypes, spanning the TRPM2 gene, with BD and
BLCL [Ca2þ]B, in a case control design. The 50
TaqMan SNP assay was used to detect selected
SNPs. BLCL [Ca2þ]B was determined by ratiometric fluorometry. SNP rs1618355 in intron 18
was significantly associated with BD as a whole
(P < 7.0 105; odds ratio (OR) ¼ 2.60), and when
stratified into BD-I (P < 7.0 105, OR ¼ 2.48) and
BD-II (P ¼ 7.0 105, OR ¼ 2.88) subgroups. In
addition, the alleles of the individual SNPs forming a seven marker at-risk haplotype were in
excess in BD (12.0% in BD vs. 0.9% in controls;
P ¼ 2.3 1012). A weak relationship was also
detected between BLCL [Ca2þ]B and TRPM2 SNP
rs1612472 in intron 19. These findings suggest
genetic variants of the TRPM2 gene increase risk
for BD and support the notion that TRPM2 may be
involved in the pathophysiology of BD.
ß 2005 Wiley-Liss, Inc.
Grant sponsor: Canadian Institutes of Health Research; Grant
number: MOP 12851; Grant sponsor: Ontario Mental Health
Foundation.
*Correspondence to: Jerry J. Warsh, M.D., Ph.D., Head,
Laboratory of Cellular and Molecular Pathophysiology, Centre
for Addiction and Mental Health, 250 College Street, Room R20,
Toronto, ON, Canada, M5T 1R8. E-mail: jerry_warsh@camh.net
Received 24 May 2005; Accepted 22 July 2005
DOI 10.1002/ajmg.b.30239
ß 2005 Wiley-Liss, Inc.
KEY WORDS:
intracellular calcium homeostasis; calcium permeable channel;
single nucleotide polymorphisms
(SNPs); haplotype
INTRODUCTION
Genome-wide linkage scan and candidate gene studies
(for review see [Sklar, 2002]), together with meta-analyses
[Segurado et al., 2003], have provided important clues as to
chromosomal regions that may harbor candidate genes, which
contribute to the vulnerability to bipolar disorder (BD),
inconsistencies, design and methodological limitations, notwithstanding. Among those genes implicated in genome
scanning studies, the transient receptor potential protein of
the melastatin type (TRPM2), a calcium (Ca2þ) permeable ion
channel, is an attractive candidate in regards to some of the
pathophysiological disturbances implicated in BD. Straub et al.
[1994] first described linkage to BD at 21q21, obtaining a LOD
score of 3.41 with D21S171, a marker only 29.6 kb from
TRPM2, in an extended BD pedigree. Confirmation was
subsequently reported by a number of independent studies,
supporting that the 21q21-22 region harbors BD susceptible
loci [Straub et al., 1994; Detera-Wadleigh et al., 1997; Smyth
et al., 1997; Aita et al., 1999; Curtis, 1999; Kwok et al., 1999;
Berrettini, 2000; Kelsoe et al., 2001; Liu et al., 2001; Ewald
et al., 2003].
Building evidence on the physiological functions of TRPM2
and disturbances in its expression in BD also support its
potential involvement in the pathophysiology of BD. TRPM2
has been shown to regulate Ca2þ entry in response to several
novel intracellular messengers including adenine diphosphate-ribose, nicotinamide adenine dinucleotide, and reactive oxygen species, such as H2O2 [Perraud et al., 2004].
TRPM2 is highly expressed in human brain including cerebral
cortex, hippocampus, amygdala, caudate nucleus, putamen
[Nagamine et al., 1998], B-lymphoblast cell lines (BLCLs)
[Yoon et al., 2001], and other tissues (reviewed in [Perraud
et al., 2004]). Of particular interest, TRPM2 expression was
found significantly lower in BLCLs from BD-I patients with
high basal intracellular Ca2þ concentration ([Ca2þ]B) compared with BD-I patients showing normal [Ca2þ]B, with BD-II
patients, with major depressive disorder patients and healthy
subjects [Yoon et al., 2001]. Sustained elevations of intracellular Ca2þ levels reduce TRPM2 mRNA levels in BLCLs [Yoon,
2002] and TRPM2 isoforms promote (long form) or suppress
Association of TRPM2 With Bipolar Disorder
(short form) susceptibility to cell death [Zhang et al., 2003].
These observations further implicate altered expression,
structure, and/or function of this protein in BD.
Such evidence of positional and biological candidacy prompted us to scrutinize more closely the potential role of TRPM2
variants in modifying susceptibility to BD and in the
occurrence of altered intracellular Ca2þ homeostasis also
observed in this disorder [Warsh et al., 2004]. Towards this
end, we examined the relationship between DSM-IV diagnostic
categories of BD-I and II disorder, and allelic and haplotype
frequencies in the TRPM2 gene. Seven single nucleotide
polymorphisms (SNPs) located in the promoter and selected
intronic regions (8, 16, 18, 19, 20, and 27), the positions of which
are representative of the overall SNP distribution in the
TRPM2 gene, were studied. Association with elevated [Ca2þ]B,
an index of altered intracellular Ca2þ homeostasis, which may
define a pathophysiologically more homogeneous subgroup of
BD (reviewed in [Warsh et al., 2004]), was also examined. We
report here evidence in a case-control study of 446 Caucasian
subjects of an allelic and haplotype association within the
TRPM2 gene and BD.
MATERIALS AND METHODS
Sample Collection
Patients and healthy subjects were recruited from the
greater Toronto area as previously described [Emamghoreishi
et al., 1997]. Because potential participants were assessed
in the context of studies of intracellular Ca2þ homeostasis
abnormalities in mood disorders, subjects were excluded from
the study if they had a history of recent (<3 months) substance
abuse or current physical illness. Psychiatric diagnoses were
confirmed using the Structured Clinical Interview for DSM-IV
Axis I Disorders (SCID-I), patient edition [First et al., 1995a],
administered by a research psychiatrist or trained psychiatric
research assistant, complemented by review of available
medical records. Healthy subjects had no personal or family
(first degree relatives) history of psychiatric disorder, based on
structured interview with the SCID-I non-patient version
[First et al., 1995b], and no abnormalities on systems review, or
where indicated, physical examination.
Demographic information including ethnicity, age of onset of
primary diagnosis, duration of illness, number of episodes, and
number of hospitalizations were obtained during the interview
of the probands, facilitated by life charting and review of
available medical records. Ethnicity was determined at interview by systematic questioning of the proband in regards to
ancestral ethnic and geographic origins for the prior 1–3
generations, to the extent of the participant’s knowledge of
family origins. Ethnicity was classified based on ancestral
region of origin as identified within current country boundaries. Controls were matched to cases on the basis of age,
gender, and ethnicity. The study was approved by the Research
Ethics Board of the Centre for Addiction and Mental Health,
and after a complete description of the study, all subjects
provided written informed consent.
DNA Analysis
Blood was drawn from each subject into anticoagulant (ACD)
containing tubes and the leukocyte fraction transformed with
Epstein–Barr virus as previously described [Emamghoreishi
et al., 1997]. BLCLs were frozen at approximately 15–
18 passages and stored over liquid nitrogen in aliquots of
approximately 5 106 cells. For genotyping, BLCL aliquots
were rapidly thawed and washed in phosphate buffered saline
(PBS). After re-suspending in 200 ml PBS, genomic DNA was
extracted using a QIAamp1 Blood Kit (Qiagen, Mississauga,
Ontario, Canada) according to the manufacturer’s instruc-
37
tions. In a subset of the healthy controls recruited in parallel
for other psychiatric genetic studies in this center, DNA was
extracted from EDTA anticoagulated blood samples using a
high salt method [Lahiri and Nurnberger, 1991].
SNP Selection
A total of seven SNPs were chosen to explore the association
between the TRPM2 gene and BD in this preliminary study.
These were selected using the Assay-On-Demand database
(www.allsnps.com, Applied Biosystems, Inc., Foster City, CA),
which provides the physical location and allele frequency
data in four ethnic populations, the NCBI dbSNP database
(www.ncbi.nlm.nih.gov/SNP), and SNPper (http://snpper.
chip.org/). Criteria for choice of SNPs used were: (1) minor
allele frequencies >19% in the Caucasian population; and (2)
location within the promoter region, and exonic and intronic
sites that could potentially impact on TRPM2 expression and
function. The NetGen2 server service for predicting splice-sites
(http://www.cbs.dtu.dk/services/NetGene2/ [Hebsgaard et al.,
1996] was used to scrutinize the TRPM2 genomic sequence for
SNPs located at or proximal to splice-sites.
SNP Analysis and Allele Determination
Genotyping was performed using the 50 nuclease allelic
discrimination TaqMan assay in a 96-well format. To each well,
5 ml of PCR master mix (TaqMan1 Universal PCR Master Mix),
5–10 ng of DNA template, 0.5 ml of 20 Assay-On-Demand
SNP Genotyping Assay Mix (sequence-specific primers and
probes) were added. The amplification reaction was carried out
on an ABI PRISM1 7300 Sequence Detection System (Applied
Biosystems, Inc.) using the following cycle parameters: initial
denaturation 958C for 10 min, followed by 40 cycles of
denaturing at 928C for 15 sec, and annealing and primer
extension at 608C for 1 min. After amplification, end point plate
reading to discriminate alleles was performed using the
proprietary Sequence Detection System software (Applied
Biosystems, Inc.). In addition to positive control samples with
known genotyping information, four negative control samples
without genomic DNA, as well as randomly selected samples
from six individuals, were assayed in duplicate on each 96-well
plate. SNP genotyping was performed blind to subject diagnosis, characteristics, and sample replication.
Basal Intracellular Calcium Determination
The [Ca2þ]B was determined in BLCLs at 13–17 passages
using fura2-AM and ratiometric fluorometry, as previously
described [Emamghoreishi et al., 1997].
Statistical Analysis
Dependent measures including [Ca2þ]B, age, and age of
onset of illness are expressed as means SD. Hardy–Weinberg Equilibrium (HWE) analysis, linkage disequilibrium
(LD), and haplotype block structure were examined using
Haploview, version 3.2 [Barrett et al., 2005] (http://
www.broad.mit.edu/mpg/haploview/) based on the normal
control subjects. The D0 for all pairs of SNPs was calculated
and the haplotype blocks were estimated using the solid spine
of LD method [Barrett et al., 2005]. Association between the
haplotypes and diagnostic groups was tested using COCAPHASE within the UNPHASED suite of programs (http://
www.hgmp.mrc.ac.uk/fdudbrid/software/unphased/) [Cordell
and Clayton, 2002], as well as PowerMarker3 (version 3.09;
http://statgen.ncsu.edu/powermarker/) [Zaykin et al., 2002],
which obviates the effects of multiple testing.
Disease association of individual SNP allele, genotype or
haplotype frequencies with BD patients, as a group, compared
38
Xu et al.
with healthy individuals, was assessed using 2 2 or 2 3
contingency tables, two-tailed w2 tests, or Fisher exact tests.
Differences in dependent measures ([Ca2þ]B, age, age of onset)
were analyzed by univariate ANOVA with genotype or
haplotype, and diagnosis as factors, followed by post hoc
comparisons of group means using the Tukey’s test. Statistical
analyses were performed using the SPSS statistical software
package (version 10, SPSS, Inc., Chicago, IL.). Differences with
two-tailed probability values of P 0.05 were taken as
statistically significant. P-values for tests of allelic and
genotype association were conservatively corrected for multiple testing using the Bonferroni method.
Assessment of Potential Population
Stratification Effects
The statistical genetics programs FSTAT (http://www.
unil.ch/izea/softwares/fstat.html) and GENEPOP, version 3.3
[Raymond and Rousset, 1995] were used to detect potential
genetic substructure using unlinked SNP markers located in
different human chromosome regions in this study material. In
these analyses, the number of ethnic subgroups in the sample
was set for four Caucasian subpopulations (Table IB). The
default parameter settings were used for the GENEPOP
analyses.
Assessment of Statistical Power
The adequacy of statistical power was evaluated using two
different methods: the first employed binomial power calculations (http://calculators.stat.ucla.edu/powercalc/) to determine
the power of the w2 tests. The sample size of 178 BD patients
and 268 controls used in this study gave a power of 88% with
relative risk of 2.0 (two-sided test). The second method was
performed with the genetic power calculator (statgen.iop.kcl.ac.uk http://statgen.iop.kcl.ac.uk/gpc/cc2.html) using a variance components analysis with the following parameters:
disease prevalence, 0.01; D0 between disease and SNP alleles,
0.8; alpha, 0.05. The power to detect association was estimated
as 98%, based on the total sample of 446 subjects with disease
and marker allele frequency (A) ¼ 0.30 and 94%, with A ¼ 0.20,
since these frequencies are the minor allele frequencies of the
tested SNPs/markers.
RESULTS
Demographic Characteristics
Selected demographic characteristics of the subjects studied
are detailed in Table IA and Table IB. There was no difference
in age (F ¼ 2.18, df ¼ 2,442, P ¼ 0.114) or gender (w2 ¼ 2.21,
df ¼ 2, P ¼ 0.33) among comparison groups. Age of onset
was significantly lower in BD-II (16.5 6.0 years) compared
with BD-I patients (20.4 8.2 years; t ¼ 3.05, df ¼ 171,
P ¼ 0.003).
TABLE IA. Demographic Characteristics of Bipolar and Healthy
Subjects in Caucasian Population: Sex, Age, and Age of Onset
Diagnostic group
Control
BD-I
BD-II
a
N
268
124
54
Sex (F/M)
159/109
82/42
36/18
Age
Age of onset
a
38.7 9.1
37.9 10.9
35.5 8.9
NA
20.4 8.2
16.5 6.0b
Age and age of onset are expressed as the mean SD.
Significantly earlier in BD-II compared with BD-I patients (t ¼ 3.05
df ¼ 171, P ¼ 0.003).
b
LD, SNP Allele, Haplotype
Frequency, Haplotype Blocks
Figure 1 shows the physical location of the SNP in the
putative promoter region and the six SNPs, which span
87,074 bp and approximately 63% of the TRPM2. The use of
SNPs with high minor allele frequencies (mean 0.27) enhances
the power to detect LD [Ohashi and Tokunaga, 2002]; the
observed frequencies in present study concur with those
values reported in the Assay-On-Demand SNP database
(www.allsnps.com) (Table II).
The genotype frequencies of each of the seven SNPs
examined did not deviate significantly from HWE in healthy
controls (Table II). As shown in Figure 1, two potentially
associated haplotype blocks were identified across the 87-kb
region and designated as blocks 1 and 2, respectively (see
Fig. 1), based on D0 and r2 in the healthy control population.
Moderate LD was evident between promoter and intron 8 SNPs
(D0 ¼ 0.88), spanning a 31.4 kb region, and strong LD among
four of the SNPs (intron 16, 18, 19, 20), spanning a 14.2 kb
region. Of these seven SNPs, strong pairwise LD was observed
between intron 16 and 18 SNPs (D0 ¼ 0.96) and between intron
19 and 20 SNPs (D0 ¼ 0.98) (Fig. 1), suggesting that there has
been little historic recombination in this region over time. The
values of D0 between the SNPs in introns 8 and 16, at a 22.6 kb
distance, and between SNPs in introns 20 and 27, at a distance
of 18.8 kb, were much lower (D0 ¼ 0.33 and 0.28, respectively).
Thus, these SNPs are not in complete LD. Finally, blocks 1 and
2 showed some degree of multivariate D0 , suggesting that a
putative recombination event between these blocks should be
old, if any.
Association Between SNPs and Haplotypes,
and Diagnostic Groups and Phenotypes
Statistically significant increased allele 2 (minor allele)
frequencies were observed for intron 18 (P < 7.0 105) and 19
(P ¼ 0.00022) SNPs in BD as compared with those in healthy
controls (44.7 vs. 23.7%, OR ¼ 2.60; 38.2% vs. 25.1%, OR ¼ 1.85,
respectively, Table III). Moreover, the allele 2 frequencies of
intron 18 and intron 19 SNPs were also significantly increased
in both BD-I and BD-II patient groups as compared with
controls (Table III).
Since the use of haplotypes may provide greater statistical
power in association studies of common genetic variation than
can be obtained with individual SNPs [Gabriel et al., 2002], a
seven-locus-haplotype analysis was performed. A number of
susceptible haplotypes were detected: the T-T-T-C-T-T-A
haplotype is one among these showing greatest statistical
significance (P ¼ 2.3 1012) with a frequency of 12.0% in the
BD cohort and 0.9% in controls (Table IV). To pinpoint the
susceptibility or protective area more precisely, we also
analyzed the block-based haplotypes from adjacent SNPs in a
sliding-window fashion. Statistically significant associations
were observed between most of the haplotypes and BD (Pvalues range from 9.5 1010 to 2.5 1014) compared with
controls (data not shown). The best result was obtained for the
4-locus-haplotype analysis in block 2 with an omnibus test
showing a P ¼ 2.3 1015, with the largest difference between
cases and controls (1.3% in control vs. 15.5% in BD) observed
for the T-C-T-T haplotype. Genotype and allele distribution did
not differ significantly for the seven TRPM2 SNPs between
females and males in both control and BD groups (data not
shown).
Tests of Population Stratification
No significant differences were found for five unlinked SNPs
in different genes among Caucasian subpopulations from
the four ethnically defined groups (FSTAT: Fst ¼ 0.002) and
Association of TRPM2 With Bipolar Disorder
39
TABLE IB. Demographic Characteristics of Bipolar and Healthy Subjects in Caucasian Population: Case and Control Ethnicity
Caucasian sub-groups
British Islesa
European–Otherb
Recent admixture of Europeansc
Uncertain European ancestryd
BD (as a Group)
BD-I
BD-II
Control
Total
51 (28.7%)
26 (14.6%)
67 (37.6%)
34 (19.1%)
36 (29.0%)
23 (18.5%)
38 (30.6%)
27 (21.8%)
15 (27.8%)
3 (5.6%)
29 (53.7%)
7 (13.0%)
69 (25.7%)
63 (23.5%)
69 (25.7%)
67 (25.0%)
120
89
136
101
a
‘‘British Isles’’ includes: British, English, Irish (North and South), Welsh, Scottish or admixtures within these groups.
‘‘European–Other’’ includes: non-admixed North European (4BD, 1HC), West European (10BD, 11HC), East European (5BD, 21HC), Russian (0BD, 3HC),
Mediterranean (6BD, 27HC), and French Canadian (1BD, 0HC).
c
‘‘Recent admixture of Europeans’’ includes: admixture with each parent of different regional/ethnic European origin.
d
‘‘Uncertain European ancestry’’ includes: probands who lack knowledge of one or the other of the maternal and/or paternal grandparent.
b
(GENEPOP: Fst ¼ 0.002). These results suggest the four
categorized Caucasian populations used in the present study
show relative homogeneity, at least in the context of the genetic
analyses of TRPM2 employed here.
BLCL Basal Calcium Concentrations
Mean BLCL [Ca2þ]B values differed significantly among
diagnostic groups (F ¼ 4.26, df ¼ 2,296, P ¼ 0.015), due to
significantly higher [Ca2þ]B in BD-I patients (61.7 9.1 nM,
N ¼ 160; P ¼ 0.023, post hoc Tukey test), but not in BD-II
patients (59.4 7.5, N ¼ 73) compared with that in the healthy
subjects (58.4 7.5, N ¼ 64). Moreover, no statistically significant differences were evident in BLCL [Ca2þ]B values
Fig. 1. Genomic structure with seven SNP positions and haplotype
blocks identified in the human TRPM2 gene as described by Nagamine et al.
(1998). Top: Depicts the distance in kb between selected SNPs. Middle: The
32 exons coding for TRPM2 are represented by filled squares. Circles
represent SNP locations. Exon and intron sizes are not drawn to exact scale.
Bottom: The locations of seven SNPs and LD structure. Two blocks have
among different ethnic groups (F ¼ 1.002, df ¼ 3,185, P ¼
0.393) (data not shown). In addition, [Ca2þ]B was significantly
higher (F ¼ 4.64, df ¼ 2,115, P ¼ 0.012) in BD-I patients with
the TRPM2-intron 19 SNP T/T genotype (62.6 9.8 nM)
compared to those with T/C (61.2 8.5 nM; P ¼ 0.008) and
with C/C (54.4 6.5 nM; P ¼ 0.035) genotypes. However, no
association was observed between basal [Ca2þ]B and the other
six TRPM2 SNPs or haplotypes studied here in Caucasian BD
patients as a whole, or in BD-I or BD-II subgroups.
DISCUSSION
The results of the SNP allelic and haplotype analysis
obtained in this study provide strong support for the candidacy
been identified. Block 1 consists of promoter and intron 8 SNPs; Block 2
covers the region between intron 16 and 20 SNPs. The regions of high LD
(D0 > 95) are shown in dark gray. Regions with lower LD are shown in light
gray with the intensity decreasing with decreased D0 value. Blocks defined
by solid spine of LD based on normal controls.
SNPs are ordered centromeric to telomeric on chromosome 21q.
Major/ minor allele. The nucleotide position of each SNP is relative to the start of exon 1 based on the NCBI location.
HWE analysis was performed as described in the Statistical Methods.
d
Frequencies for each SNP are based on the entire sample of current study and compared with published data shown in italics. http://myscience.appliedbiosystems.com/index.jsp).
e
Abbreviations: CC, Caucasian; AA, African American; Ch, Chinese; NA, not available. Allelic and genotype frequencies for seven SNPs did not differ significantly between Caucasian and Asian populations.
c
b
0.21
0.25
0.26
0.32
0.33
0.26
0.21
0.62
0.59
0.19
0.70
0.35
0.18
0.95
5851 T/C
25580 T/C
48210 T/C
52878 A/C
59401 T/C
62421 T/C
81223 A/G
4889 bp upstream of E1
284 bp downstream of E8
14 bp downstream of E16
14 bp upstream of E19
788 bp upstream of E20
1806 bp upstream of E21
206 bp upstream of E28
Promoter
Intron-8
Intron-16
Intron-18
Intron-19
Intron-20
Intron-27
rs734336
rs2838556
rs1785437
rs1618355
rs1612472
rs933151
rs749909
a
NA
0.09
NA
NA
NA
NA
NA
NA
0.15
NA
NA
NA
NA
NA
0.45
0.36
0.35
0.42
0.46
0.28
0.11
0.20
0.22
0.22
0.23
0.20
0.23
0.19
0.25
0.26
0.24
0.30
0.45
0.22
0.13
0.22
0.25
0.26
0.33
0.32
0.26
0.22
Ch
AA
Entire sample
HWEc P-value
Locationa
SNP ID
Distance (bp) from exon
Nucleotide
changeb
Current study
d
CCe
Asian
Frequencies
CC
Published
Japanese
Xu et al.
TABLE II. Location and Frequencies of SNPs in TRPM2
40
of TRPM2 as a potential vulnerability gene that contributes to
the risk for BD. In addition, a statistically significant relationship between a genetic variant (intron 19 SNP) of TRPM2 gene
and elevated BLCL [Ca2þ]B in BD-I patients also provides some
support for a link between variation in TRPM2 and disturbances of intracellular Ca2þ homeostasis in BD. Increased
frequencies were found for several of the TRPM2 SNP alleles
examined in the BD population compared with those in healthy
controls, including allele 2 of the intron 18 SNP and of the
intron 19 SNP. The relatively high OR of 2.60, obtained for the
intron 18 SNPs also suggests an important biological effect
increasing the risk of developing BD, in the context of a
polygenic complex disorder.
Haplotype block analysis has been used to successfully map
mutations associated with complex diseases [Tishkoff and
Verrelli, 2003] as it affords the advantage of being able to test
associations between genomic segments encoding multiple
genes and traits [Gabriel et al., 2002], in contrast to individual
polymorphisms. The two haplotype blocks, identified in the
present study, spanned 31.4 kb between promoter and intron
8 SNPs, and 14.2 kb between intron 16 and 20 SNPs,
respectively (Fig. 1), the latter region containing the intron
18 putative BD-risk SNP rs1618355. This agrees with the
suggested average block length of 22 kb and range in length
from <1 to 175 kb as reported by Dawson et al. [2002]. Strong
LD was observed in the region between intron 16 and intron 20,
which corresponds with the haplotype block 2 region in which
two significantly associated SNPs (intron 18 and 19 SNPs)
were located. The block 2 identified here has similar properties
of the block generated by the Applied Biosystems SNPbrowserTM software, version 1.0, which provides a visual representation of putative haplotype block boundaries based on
Caucasian, African American, Chinese, and Japanese ethnic
populations.
Of note, the statistically significant association observed
between the risk haplotypes of the TRPM2 gene and BD
(Table IV) further strengthens the notion that certain TRPM2
variants confer susceptibility to BD. The statistically significant higher frequency of the T-T-T-C-T-T-A haplotype (BD:
12.0% vs. controls: 0.9%) suggests an important role of this
haplotype in conferring susceptibility to BD. In addition, some
haplotypes were significantly less common in BD, which may
reflect their effect as a putative ‘‘protective’’ factor; for example,
the frequency of the T-T-T-A-T-T-A haplotype was significantly more common in control (52%) compared with BD (26%)
(P ¼ 5.6 1015, Table IV). However, the nature of ‘‘susceptibility’’ and ‘‘protective’’ haplotypes in the TRPM2 locus needs
to be explored further in regard to this gene’s role as a
candidate in the pathogenesis of BD.
Lack of allele frequency information and the low allele
frequency of the exonic SNPs from 390 SNPs reported in the
dbSNP database ruled out their use, therefore, the two
associated SNPs (intron 18 and 19) were then selected, as they
lie proximal to exon–intron boundaries. The intron 18 SNP is
only 14 bp upstream from the predicted splice site of exon 19, as
estimated with 91% confidence using splice-site prediction
program, NetGene2 [Hebsgaard et al., 1996]. This proximity
might influence RNA splicing [Bracco and Kearsey, 2003] or
serve as a marker for another variant(s) that directly affects
the function of the TRPM2 gene. Furthermore, the potential for
regulation of gene expression by intronic transcribed noncoding RNAs has recently been recognized [Mattick, 2004].
Finally, these SNPs may be close to and in LD with other yet
unidentified functional variants.
The use of a continuously distributed phenotype in LD
analysis has been shown to be a more powerful strategy for
detecting genetic susceptibility to complex diseases than the
simple classification of individuals into ‘‘affected’’ and ‘‘nonaffected’’ categories (e.g., [Leckman et al., 2001; Rowe et al.,
87 (24.4)
69 (27.8)
18 (11.7)
107 (20.0)
97 (27.2)
75 (30.2)
22 (20.4)
121 (22.7)
109 (30.6)
75 (30.2)
34 (31.5)
119 (22.5)
159 (44.7)
108 (43.5)
51 (48.2)
119 (23.7)
136 (38.2)
90 (36.3)
46 (42.6)
133 (25.1)
113 (31.7)
77 (31.0)
36 (33.3)
118 (22.2)
92 (25.8)
65 (26.2)
27 (25.0)
98 (18.4)
269 (75.6)
179 (72.2)
90 (83.3)
427 (80.0)
259 (72.8)
173 (69.8)
86 (79.6)
413 (77.3)
247 (69.4)
173 (69.8)
74 (68.5)
411 (77.5)
197 (55.3)
140 (56.5)
57 (52.8)
383 (76.3)
220 (61.8)
158 (63.7)
62 (57.4)
397 (74.9)
243 (68.3)
171 (69.0)
72 (66.7)
414 (77.8)
264 (74.2)
183 (73.8)
81 (75.0)
434 (81.6)
Allele 2
N (%)
1.54 (1.12–2.13)
1.57 (1.10–2.25)
1.48 (0.91–2.40)
1.92 (1.43–2.58)
1.58 (1.13–2.21)
1.75 (1.12–2.75)
1.85 (1.38–2.47)
1.70 (1.23–2.35)
2.21 (1.44–3.40)
2.60 (1.94–3.48)
2.48 (1.79–3.43)
2.88 (1.87–4.743)
1.52 (1.12–2.07)
1.50 (1.07–2.10)
1.59 (1.01–2.50)
1.28 (0.94–1.74)
1.48 (1.05–2.08)
0.87 (0.52–1.45)
1.29 (0.94–1.78)
1.54 (1.08–2.18)
0.80 (0.46–1.38)
OR (95% CI)
a
Allele 1 is the major allele and allele 2 is the minor allele.
*Bonferroni corrected P-values for the number of SNPs (N ¼ 7) tested.
Promoter-SNP
BD (N ¼ 178)
BD-I (N ¼ 124)
BD-II (N ¼ 54)
Control (N ¼ 267)
I-8-SNP
BD (N ¼ 178)
BD-I (N ¼ 124)
BD-II (N ¼ 54)
Control (N ¼ 267)
I-16-SNP
BD (N ¼ 178)
BD-I (N ¼ 124)
BD-II (N ¼ 54)
Control (N ¼ 265)
I-18-SNP
BD (N ¼ 178)
BD-I (N ¼ 124)
BD-II (N ¼ 54)
Control (N ¼ 254)
I-19-SNP
BD (N ¼ 178)
BD-I (N ¼ 124)
BD-II (N ¼ 54)
Control (N ¼ 265)
I-20-SNP
BD (N ¼ 178)
BD-I (N ¼ 124)
BD-II (N ¼ 54)
Control (N ¼ 266)
I-27-SNP
BD (N ¼ 178)
BD-I (N ¼ 124)
BD-II (N ¼ 54)
Control (N ¼ 266)
Diagnosis
Allele 1a
N (%)
6.19
5.48
2.07
10.13
7.09
6.11
17.31
10.36
13.61
41.77
30.97
24.45
7.43
5.48
4.01
2.43
5.18
0.27
2.43
5.88
0.65
w2
2.2 104
9.0 103
1.6 103
3.0 105
1.3 103
2.3 104
0.013
0.019
0.151
NS
NS
NS
0.011
0.055
NS
<7.0 105
<7.0 105
7.0 105
<1.0 105
<1.0 105
1.0 105
0.002
0.008
0.014
0.042
NS
NS
NS
NS
NS
NS
NS
NS
Corrected
P-value*
0.006
0.019
0.045
0.119
0.023
0.602
0.119
0.015
0.420
P-value
102 (57.31)
73 (58.9)
29 (53.7)
177 (66.5)
82 (46.1)
56 (45.2)
26 (48.1)
157 (59.0)
71 (39.9)
50 (40.3)
21 (38.9)
151 (57.0)
58 (32.6)
40 (32.3)
18 (33.3)
148 (58.3)
82 (46.1)
56 (45.2)
26 (48.1)
155 (58.5)
97 (54.5)
62 (50.0)
35 (64.8)
159 (59.6)
101 (56.7)
63 (50.8)
38 (70.4)
170 (63.7)
1/1
N (%)
60 (33.7)
37 (29.8)
23 (42.6)
80 (30.1)
79 (44.4)
59 (47.6)
20 (37.0)
100 (37.6)
78 (43.8)
58 (46.8)
20 (37.0)
95 (35.8)
81 (45.5)
60 (48.4)
21 (38.9)
93 (36.6)
83 (46.6)
61 (49.2)
22 (40.7)
101 (38.1)
65 (36.5)
49 (39.5)
16 (29.6)
95 (35.6)
67 (37.6)
53 (42.7)
14 (25.9)
87 (32.6)
1/2
N (%)
16 (9.0)
14 (11.3)
2 (3.7)
9 (3.4)
17 (9.6)
9 (7.3)
8 (14.8)
9 (3.4)
29 (16.3)
16 (12.9)
13 (24.1)
19 (7.2)
39 (21.9)
24 (19.4)
15 (27.8)
13 (5.1)
13 (7.3)
7 (5.6)
6 (11.1)
9 (3.4)
16 (9.4)
13 (10.5)
3 (5.6)
13 (4.9)
10 (5.6)
8 (6.5)
2 (3.7)
10 (3.7)
2/2
N (%)
7.85
9.74
3.35
11.47
7.80
11.97
16.12
10.19
15.52
41.05
31.44
30.35
8.20
6.30
6.60
3.28
5.74
0.71
2.46
6.14
0.94
w2
0.020
0.008
0.188
0.003
0.020
0.003
3.2 103
6.0 103
4.3 103
1.2 109
1.5 107
2.6 107
0.017
0.043
0.037
0.194
0.057
0.702
0.292
0.046
0.624
P-value
TABLE III. Genotype and Allelic Association of TRPM2 SNPs Between Bipolar Patients (BD-I and BD-II) and Healthy Controls in Caucasian Population
NS
NS
NS
0.063
NS
0.042
7 103
NS
7 103
2.0 108
3.0 106
3.6 106
NS
NS
NS
NS
NS
NS
NS
NS
NS
Corrected
P-value*
Association of TRPM2 With Bipolar Disorder
41
42
Xu et al.
TABLE IV. Association of Haplotypes With BD in Caucasian Population
Haplotype
T-T-T-C-T-T-A
T-T-T-C-T-T-G
C-C-C-G-C-C-A
T-T-T-A-C-T-A
T-T-C-C-C-C-A
T-T-T-A-T-T-G
T-T-C-C-C-C-G
T-T-T-A-T-T-A
Remaining hapa
BD (%)
(2N ¼ 356)
12.0
2.9
6.4
5.2
8.8
6.4
2.2
26.3
29.7
Control (%)
(2N ¼ 536)
0.9
1.0
1.8
2.6
8.9
8.5
0.8
52.3
23.3
w2
49.24
14.39
6.57
3.94
0.26
1.60
0.38
61.04
—
P-value*
12
2.3 10
1.5 103
0.010
0.047
0.610
0.206
0.538
5.6 1015
—
Effect
Susceptible
Susceptible
Susceptible
Susceptible
Protective
Global P-value of 1.5 1017 (1.05 1016 after correcting for multiple testing of the number of SNPs [N ¼ 7]).
*P-values were calculated using COCAPHASE as described in the Materials and Methods.
a
Haplotypes with an estimated frequency <0.8% in control are designed as ‘‘remaining haplotypes.’’
2001]). This strategy was applied in this study, examining the
association of TRPM2 SNP alleles and haplotypes in BD with
[Ca2þ]B, a putative index of intracellular Ca2þ homeostasis,
which has been found elevated in peripheral blood cells and
BLCLs from BD patients [Emamghoreishi et al., 1997; Warsh
et al., 2004]. Although no association was evident between the
individual TRPM2 SNP genotypes evaluated and BLCL
[Ca2þ]B, significantly higher BLCL [Ca2þ]B was found in BDI patients stratified on TRPM2-intron 19 SNP genotype. This
suggests the possibility of a putative link between TRPM2
genetic variation and the alterations in Ca2þ homeostasis
implied by elevated BLCL [Ca2þ]B, although the relationship
may typify only a discrete subpopulation of BD-I patients
within this heterogeneous disorder. Genetic variation in
TRPM2 that is filtered through a network of gene/environment
interactions [Petronis, 2003] and complex proteomic networks
to modulate intracellular Ca2þ dynamics, could also confound
detection of stronger associations with the latter.
In order to ensure the quality of this case-control study of BD,
we followed suggested standards for population-based association investigations of complex disorders [Schulze et al., 2003].
Original samples were used along with a quality control that
included replicated samples in each 96-well plate. Moreover, a
nucleotide-specific genotyping method was used based on
TaqMan1 probes and 50 nuclease chemistry, a method that
provides improved mismatch discrimination [Livak, 1999].
Genotype frequencies were in HWE and two-tailed P-values
were estimated. To rule out potential population stratification
and to minimize the risk of false-positive results in the present
population-based association study, we subdivided Caucasian
populations into four subgroups (British Isles, European–
Other, Recent admixture of Europeans, and Uncertain
European ancestry, Table IB) based on geographical patterns
of origin and ancestry (e.g., Northern, Eastern, Southern
European descendants). No statistical differences in allele,
genotype, and haplotype distributions for seven TRPM2 SNPs
were observed (data not shown). Notwithstanding the limited
number of unlinked SNPs used, the lack of statistical support
for population stratification in the Caucasian population
investigated here, as tested with FASTAT and GENEPOP,
argues against a major contribution of this phenomenon to the
statistically significant differences in frequency of the intron
18 and intron 19 SNPs, and their haplotypes, between patients
and controls. However, the possibility cannot be completely
excluded. Secondly, possible interactive effects of environmental factor(s) or other gene(s) with TRPM2 that impact the
pathophysiology of BD, cannot be excluded, although heritability is uninformative in distinguishing group differences
related to interactive effects that influence the expression of a
complex trait [Mountain and Risch, 2004].
In summary, we have conducted the first systematic screen
of SNPs within the TRPM2 gene in a population based
association study of BD and comparison healthy subjects.
SNPs were used to capture haplotype diversity within the gene
of interest. Potential susceptibility alleles were identified for
several SNPs and the haplotypes of the TRPM2 gene, for BD as
a whole, as well as for BD-I and BD-II patient subgroups.
Moreover, a relationship was also suggested between the
specific TRPM2-intron 19 genotypes and elevated [Ca2þ]B in
BLCLs from BD-I patients. The present results clearly support
the importance of more intensive investigations of TRPM2 and
its potential role in modifying risk for BD and disturbed
intracellular Ca2þ homeostasis abnormal in BD-I patients.
ACKNOWLEDGMENTS
Kin Po Siu, Arvind Kamble, and Karen Woo assisted with the
transformation and culture of cell lines. Wendy Hiscox and
Hester Tims assisted with the structured interview of patients
and Damodar Godse with database development. The facilitation of subject recruitment by the staff of the Mood Disorders
Program of CAMH and the Department of Psychiatry,
University of Toronto is gratefully acknowledged. Supported,
in part, by grants from the Canadian Institutes of Health
Research, MOP 12851 and Ontario Mental Health Foundation
to J.J.W.
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