of CHRNA5), which was earlier associated with
both ND3 and lung cancer.7,8 As we studied females
only (as in our original study design10), our replica-
tion is limited to that gender. However, no gender
effect was found by Saccone et al.1 in their mixed
sample with regard to any of the SNPs in this gene
cluster.
We extended the logistic regression model to
include additional variables associated with severity
of ND in our univariate analyses (Supplementary
Table 2), the following variables serving as predictors:
(1) background variables: religious observance, par-
ental smoking and life-time experience of trauma; and
(2) cognitive variables: performance speed and im-
pulsivity factors, which are composite variables
produced by confirmatory factor analysis. The analy-
sis indicated that all the single-test reaction time
measures are loaded on one factor (performance
speed) and most of the single-test false-alarm mea-
sures loaded on a second factor (impulsivity). The
score for each factor was calculated by regression
method score: (3) personality variables: TPQ-novelty-
seeking and reward dependence scales; and (4)
genetic variables: SNP rs578776 (the two SNPs that
were nominally significant on univariate analysis
were omitted due to multicolinearity and lower
association with the phenotype); percentage of life-
time smoking was forced into the model as a
controlling variable. Three variables remained in the
model (Table 1): parental smoking (P=0.01; odds
ratio = 3.21), lifetime experience of trauma (P=0.006;
odds ratio = 2.77) and SNP rs578776. Carrying one
minor allele of rs578776 lowered the probability of
being in the ND group by 3.44 (P=0.002) and carrying
two minor alleles lowered the probability by 8.33
(P=0.02). No other direct or interactive effects
remained in the model. Nagelkerke pseudo R2 of the
model was 0.25 (P<0.0001) and it correctly classifies
70% of the cases.
Our findings replicate the association of
the CHRNA5–CHRNA3–CHRNB4 cluster with ND
using the phenotype of Saccone et al.,1 support the
notion that at least two independent genetic risk
factors for ND are found in this gene cluster and
show the importance of an integrated approach that
takes into account background, personality, life
experience and cognitive factors in addition to
genetic variables.
L Greenbaum, A Rigbi, O Teltsh and B Lerer
Biological Psychiatry Laboratory, Department of
Psychiatry, Hadassah-Hebrew University Medical
Center, Jerusalem, Israel
E-mail: lerer@cc.huji.ac.il
References
1 Saccone SF, Hinrichs AL, Saccone NL, Chase GA, Konvicka K,
Madden PA et al. Hum Mol Genet 2007; 16: 36–49.
2 Bierut LJ, Stitzel JA, Wang JC, Hinrichs AL, Grucza RA, Xuei X
et al. Am J Psychiatry 2008; 165: 1163–1171.
3 Thorgeirsson TE, Geller F, Sulem P, Rafnar T, Wiste A, Magnusson
KP et al. Nature 2008; 452: 638–642.
4 Berrettini W, Yuan X, Tozzi F, Song K, Francks C, Chilcoat H et al.
Mol Psychiatry 2008; 13: 368–373.
5 Schlaepfer IR, Hoft NR, Collins AC, Corley RP, Hewitt JK, Hopfer
CJ et al. Biol Psychiatry 2008; 63: 1039–1046.
6 Wang JC, Grucza R, Cruchaga C, Hinrichs AL, Bertelsen S, Budde
JP et al. Mol Psychiatry 2008 (e-pub ahead of print, doi: 10.1038/
mp.2008.42).
7 Hung RJ, McKay JD, Gaborieau V, Boffetta P, Hashibe M, Zaridze D
et al. Nature 2008; 452: 633–637.
8 Amos CI, Wu X, Broderick P, Gorlov IP, Gu J, Eisen T et al. Psychol
Med 1999; 29: 299–308.
9 Kendler KS, Neale MC, Sullivan P, Corey LA, Gardner CO, Prescott
CA. Psychol Med 1999; 29: 299–308.
10 Greenbaum L, Kanyas K, Karni O, Merbl Y, Olender T, Horowitz A
et al. Mol Psychiatry 2006; 11: 312–322.
11 Yakir A, Rigbi A, Kanyas K, Pollak Y, Kahana G, Karni O et al.
Eur Neuropsychopharmacol 2007; 17: 339–351.
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Matsunami N et al. PLoS Genet 2008; 4: e1000125.
Supplementary Information accompanies the paper on the Molecular
Psychiatry website (http://www.nature.com/mp)
Histone methylation and
decreased expression of
TrkB.T1 in orbital frontal
cortex of suicide completers
Molecular Psychiatry (2009) 14, 830–832;
doi:10.1038/mp.2009.35
Decreased expression of full length TrkB and TrkB.T1
have been implicated in suicide and major depression
Table 1 Logistic regression model for predicting nicotine dependence
Variable B s.e. Wald d.f. P-value OR 95% CI
Constant 1.25 0.56 4.94 1 0.02 3.57
Percentage of lifetime smoking 3.10 2.02 2.34 1 0.12 22.25 0.41–1185.75
Parental smoking 1.17 0.46 6.44 1 0.01 3.22 1.30–7.95
Life experience of trauma 1.02 0.37 7.53 1 0.006 2.77 1.33–5.75
rs578776-CT 1.22 0.39 9.82 1 0.002 3.44 0.13–0.63
rs578776-TT 2.04 0.88 5.31 1 0.02 8.33 0.02–0.73
Abbreviations: CI, confidence interval; OR, odds ratio.
Nagelkerke pseudo R2 of the model, 0.25 (P<0.0001).
Letters to the editor
830
Molecular Psychiatry

and this may be due to an epigenetic mechanism.1–3
Methylation of certain histone residues is thought to
decrease transcription at DNA close to the histone
complex. Residues studied to date include lysine 4, 9,
27 and 36, all of which are present on the histone
H3 protein. Of particular interest to the current
study is H3 lysine 27. This residue has been shown
to have increased methylation at the P3 and P4
BDNF promoters in rats who underwent chronic
defeat stress (an animal model of depression)
compared with a control group.4 As the molecular
mechanism of H3 lysine 27 methylation related
to decreased transcription is known,5 and
histone methylation analysis in postmortem
brain is experimentally feasible,6 we opted to test
whether H3 lysine 27 methylation at a TRKB
promoter locus was increased in orbital frontal cortex
of suicide completers as compared with control
subjects.
Ten controls and 20 suicide completes were
included in this study. Subjects were recruited at
the Montreal morgue and underwent full psycho-
logical autopsy procedures, as described earlier.7
All subjects were Caucasians of French–Canadian
descent. No subjects were used with a history of
psychotic symptoms, including bipolar disorder and
schizophrenia. One subject of 30 years was female.
There were no significant differences across groups
in pH (t28 = 0.87, P=0.40; Mean Control = 6.49þ 0.08,
Mean Suicide = 6.58þ0.06), age (t28 = 0.83, P=0.41;
Mean Control = 38.7þ 4.3 years, Mean Suicide =
35.2þ2.6 years), or PMI (t28 = 0.50, P=0.62; Mean
Control = 24.9þ1.9 h, Mean Suicide = 26.3þ 1.8h),
nor were any correlations between histone methyl-
ation status and clinical variables significant.
Sixteen of 20 suicides and eight of 10 controls in this
study are identical to those used in our previous
study.3
For histone immunoprecipitation, we followed
procedures laid out expertly here.6 DNA was extrac-
ted from BA10 and cerebellum from all subjects.
We first tested that the IP reaction had worked by
using positive (GRIN2A) and negative (HBB) control
primers described by Huang et al.6 Each fraction of
isolated DNA (input and bound) underwent quanti-
tative PCR analysis for each subject.
RNA extraction was performed using Qiagen
RNeasy kits, and cDNA conversion was performed
using random hexamer primers (Invitrogen, Carlsbad,
CA, USA) according to the manufacturer’s instruc-
tions (Roche Molecular Biochemicals, Indianapolis,
IN, USA). RNA Integrity Numbers ranged from
6.0 to 8.0.
A mixture of 10ml, containing DNA 5 ml LightCycler
480 SYBR Green I Master and 0.1 mM of the TRKB
sense 50-CCCTAGCACACATGAACACG-30 and TRKB
antisense primers 50-ATGTAGCCATTCCCAGATCG-30
(112 bp product), were loaded into LightCycler capil-
laries (Roche Molecular Biochemicals). In separate
experiments we evaluated the glutamine synthetase
promoter. Sense: 50-GTGCCTTTAGCCACCACAAT-30,
antisense 50-TGGGATGTTTCAGACTGGTG-30. Quality
control for primer specificity included ensuring the
presence of a single melting peak followed by
pictorial analysis of standard curve and specificity.
Reactions were repeated in triplicate. To determine the
relative concentrations of TRKB promoter immuno-
precipitated, a standard curve of 10-fold serial
dilutions of a mixture of each of the sample DNA
was used to plot the relative Ct value on the y
axis and the amount of DNA used on the x axis. To
calculate the fold-change, the relative amount of the
bound fraction was divided by the relative amount of
input for each subject. Primers directed against
TrkB.T1 mRNA were; Forward: 50-GGATAAGCCAA
CAGCAGTCC-30; Reverse: 50-GGATAAGCCAACAG
CAGTCC-30.
We have previously shown that TrkB.T1 is down-
regulated in the frontal cortex of suicide completers
in BA 10.3 There, we found a downregulation in two
different TrkB probe sets that are specific to the
TrkB.T1 isoform. We performed RT-PCR in all
samples in the current study to determine if the
decrease of TrkB.T1 could be validated (t28 = 2.26,
P=0.035).
Methylation analysis of H3 lysine 27 was per-
formed in both orbital frontal cortex (BA10)
and cerebellum. After immunoprecipitation using
antibodies directed against methylated lysine 27 (H3),
we performed DNA RT-PCR on a region of the TrkB
promoter on both input and bound fractions from
both cerebellum and BA 10, in triplicate for each
subject and brain region. In cerebellum, we found
no significant difference in methylation at this
residue (t28 = 0.646, P=0.53) between suicides and
controls. In BA 10, we found a significant
difference in methylation state at Lysine 27 (H3)
between suicides and controls (t28 = 2.60, P=0.015).
Methylation was increased at Lysine 27 in suicide
brains.
We next asked whether any segment of DNA,
known to contain an astrocyte-expressed gene
such as TrkB.T1, would be associated with increased
methylation at lysine 27 H3 in BA 10. To this end,
we performed RT-PCR in BA10 for the glutamine
synthetase promoter. We found no significant associa-
tion between histone methylation and the suicide
phenotype in BA 10 (t28 = 0.67, P=0.51).
We next asked whether expression level of TrkB.
T1 inversely correlated with the level of H3 lysine
27 methylation in BA 10. We found consistent
trends of significant Pearson’s values of 0.34,
0.14, 0.16 for TrkB.T1 probe sets 214680_at,
221795_at, and 221796, respectively. In subjects with
low levels of TrkB.T1 (0.5 s.d. below expression level
in controls; Pearson =0.61, P=0.046, N=11 suicide
completers); however, we found a statistically
significant correlation between increased H3 lysine
27 methylation and TrkB.T1 expression level. This
finding suggests that H3 lysine 27 may need to be
more heavily methylated before any effect on trans-
cription is observed.
Letters to the editor
831
Molecular Psychiatry