This article appeared in a journal published by Elsevier. The attached
copy is furnished to the author for internal non-commercial research
and education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies are
encouraged to visit:
Author's personal copy
Neuroscience Letters 468 (2010) 330–333
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/neulet
Genetic variants of IL-6 and its receptor are not associated with
schizophrenia in Taiwan
Yu-Li Liua,b, Chih-Min Liub, Cathy Shen-Jang Fannc, Wei Chih Yangc, Ya-Hui Chenb,
Li-Jung Tsengb, Shih-Kai Liub, Ming H. Hsiehb, Tzung-Jeng Hwangb, Hung-Yu Chand,
Jiann-Jyh Chend, Wei J. Chene, Hai-Gwo Hwub,e,f,g,∗
aDivision of Mental Health and Addiction Medicine, Institute of Population Health Sciences, National Health Research Institute, Miao-Li, Taiwan
bDepartment of Psychiatry, National Taiwan University Hospital and College of Medicine, National Taiwan University, Taipei, Taiwan
cInstitute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
dTaoyuan Psychiatric Center, Taoyuan, Taiwan
eInstitute of Epidemiology, College of Public Health, National Taiwan University, Taipei, Taiwan
fDepartment of Psychology, College of Science, National Taiwan University, Taipei, Taiwan
gNeurobiology and Cognitive Science Center, National Taiwan University, Taipei, Taiwan
a r t i c l ei n f o
Received 15 September 2009
Received in revised form 30 October 2009
Accepted 7 November 2009
a b s t r a c t
The pathophysiological process of schizophrenia is still unclear. The levels of interleukine-6 (IL-6) and
its receptor, soluble IL-6R, have been reported to be elevated in the plasma and cerebrospinal fluid of
schizophrenic patients. In this study, we tested the association of genetic variants of IL-6 and IL-6R with
schizophrenia. Genotyping of three single nucleotide polymorphisms (SNP) for each IL-6 (IL-6-1, IL-6-2,
and IL-6-3) and IL-6R (rs4845617=IL-6R1, rs4553185=IL-6R2, and rs4379670=IL-6R3) gene was per-
formed in 100 patients with schizophrenia and 113 normal controls. The polymorphisms of IL-6R2 were
genotyped using Tetra-primer ARMS PCR. IL-6R3 polymorphisms were genotyped using restriction frag-
were genotyped using the direct sequencing method. We found a di-nucleotide haplotype block and a
tri-nucleotide haplotype block in the genes of IL-6 and IL-6R, respectively. All six SNPs and their hap-
lotypes failed to show a significant association with schizophrenia. The IL-6-2 SNP showed a nominally
significant association with the positive symptoms of schizophrenia (p=0.0472). We conclude that the
genetic variants of IL-6 and IL-6R are not associated with schizophrenia. In order to verify this result,
further study using a larger sample size and exploring the association between the genotype of IL-6-2
and plasma level of IL-6 is recommended.
© 2009 Elsevier Ireland Ltd. All rights reserved.
Schizophrenia is a complex mental disorder affecting 1% of all pop-
ulations. The disease primarily affects the central nervous system,
but immune alterations have been actively proposed to play a role
in the pathogenesis of schizophrenia [10,16,18]. An autoimmune
involvement in schizophrenia has been suggested by several cel-
lular and humoral cytokine changes in patients . Among these
altered cytokines, interleukine-6 (IL-6; online Mendelian Inheri-
tance in Man (MIM) number *147620) has been most consistently
found to be related to schizophrenia [6,9,14–15,26].
IL-6 is a pleiotropic cytokine released both from peripheral
immune cells and from neurons and microglia of the central
Hospital, No. 7, Chung San South Road, Taipei, 100, Taiwan.
Tel.: +886 2 2312 3456x6785; fax: +886 2 2375 3663.
E-mail address: email@example.com (H.-G. Hwu).
nervous system (CNS) [24,27]. In the CNS, soluble IL-6 receptor
(sIL-6R; MIM number *147880) levels in the CSF have increased
in schizophrenic patients with a marked paranoid-hallucinatory
syndrome . In the periphery, IL-6 has been found to be persis-
tently elevated in the plasma of patients in different ethnic groups
[1,11,19,26]. High IL-6 levels have been found to be related to
duration and treatment resistance in schizophrenia [11,19]. These
results suggested that an elevated plasma level of IL-6 was asso-
ciated with an unfavorable course of schizophrenia with a longer
duration of illness, greater treatment resistance, and more marked
We selected single nucleotide polymorphisms (SNPs) located
and 100 schizophrenic patients to decipher the potential genetic
association of IL-6 and IL-6 receptor in schizophrenia. Endophe-
notypes are recommended to define the role of the gene in the
complex traits of schizophrenia . In this study, we used the sus-
0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.
Author's personal copy
Y.-L. Liu et al. / Neuroscience Letters 468 (2010) 330–333
tained attention endophenotype of schizophrenia as measured by
the continuous performance test (CPT) and the severity of the pos-
itive and negative symptom dimensions to assess the potential
association of these endophenotype indicators with IL-6 and its
receptor in the disease.
We collected the DNA of schizophrenic patients, fulfilling cri-
teria defined by the Diagnostic and Statistical Manual 4th edition
(DSM-VI)  from the Department of Psychiatry of the National
Taiwan University Hospital and normal controls without a history
of any psychotic symptoms or family history of psychotic disorder
from hospital staff and community subjects. All individuals were
screened for the presence of acute infectious disease. The average
age was 33.3±10.6 years for normal controls and 34.5±11.3 years
for patients. The male to female ratios were 50:62 in normal con-
trols and 48:52 in patients. Clinical symptoms were rated using
the schedule for assessment of negative symptoms (SANS)  and
the schedule for assessment of positive symptoms (SAPS) . The
sum score of negative symptoms (SUMN) was the sum of the global
scores of four negative symptom dimensions, including affective
blunting, alogia, avolition-apathy, and anhedonia-asociality. The
sum score of positive symptoms (SUMP) was the sum of the global
behavior and positive formal thought disorder. The patients were
recruited from the outpatient clinics and their psychiatric status
tive symptom score was 1.44±1.18, mean disorganizing symptom
score 1.27±0.97, and mean negative symptom score 1.80±0.96
using the SAPS and SANS scales (rating range 0–5).
tinuous performance task (CPT) in the morning and 10 cc of blood
A CPT machine from Sunrise System, v. 2.20 (Pembroke, MA,
USA), was used to assess sustained attention. The procedure has
been described in detail elsewhere . Briefly, numbers from 0 to
9 were randomly presented for 50ms each, at a rate of one number
per second. Each subject undertook two CPT sessions: the unde-
graded 1–9 task and the 25% degraded 1–9 task. Subjects were
asked to respond whenever the number “9” preceded by the num-
ber “1” appeared on the screen. A total of 331 trials, 34 (10%) of
which were target stimuli, were presented over five minutes for
each session. During the 25% degraded session, a pattern of snow
was used to toggle the background and foreground so that the
image was visually distorted. Each test session began with 2min of
practice (repeated if subjects required). One signal-detection index
of performance on the test, sensitivity (d?), was derived from the
hit rate (probability of response to target trials) and false-alarm
rate (probability of response to nontarget trials) . Sensitivity is
an individual’s ability to discriminate target stimuli from nontar-
get stimuli. In a 1-week test–retest reliability study  of the CPT
reliability of d?were 0.83 and 0.82 for the undegraded and the 25%
degraded 1–9 task, respectively.
The SNP markers were selected according to the potential
moter regions of the loci with average distance of 31kb for IL-6R
(IL-6R1, IL-6R2, and IL-6R3) and 6kb for IL-6. We initially selected
3 SNPs (rs1800797, rs3087236, and rs3087236) of IL-6 and 3 SNPs
database (dbSNP). However, no polymorphisms were found at the
originally selected SNP ID in IL-6 and we found three novel SNPs
(IL-6-1, IL-6-2, IL-6-3) in the region near the originally selected
position of each SNP marker on each gene was genotyped with the
primer pairs as shown in Table 1. The SNP of IL-6R2 was geno-
RFLP with Apo I enzyme, and the others were genotyped by direct
sequencing. All polymerase chain reactions (PCRs) were carried
out according to the protocol of the Pro Taq (Protech Technology,
Taiwan) on a DNA Thermal Cycler ABI 9700. The PCR for IL-6R2
Tetra-primer was performed with an initial denaturation step at
95◦C for 2min, followed by two rounds; 16 cycles of denaturing
at 95◦C for 1min, annealing at 71◦C for 1min and extension at
72◦C for 1min, and 20 cycles with initial 95◦C for 1min, annealing
at 57◦C for 1min and extension at 72◦C for 2min. The PCR prod-
ucts of direct sequencing were purified to remove reaction buffer
and remaining primers with PCR DNA Fragments Extraction Kit
(Geneaid, Taiwan). The sequences of PCR products were directly
determined by BigDye Terminator Cycle Sequencing kit (Applied
Biosystems, CA, USA).
The Hardy–Weinberg equilibrium was assessed using the
ALLELE procedure in SAS/GENETICS release 8.2  for each SNP.
We used Haploview software to construct haplotype blocks con-
stituted by “strong LD” markers . The genotype and allele-type
association analyses were performed by using the CASECONTROL
procedure in SAS/GENETICS release 8.2 with 10000 permutation
resamples between the normal controls and the schizophrenic
patients. The phenotype and genotype association analyses were
Three SNPs of both IL-6 receptor (at chromosome 1q21) and IL-
6 (at chromosome 7p21) were designed with specific primer pairs
for genotyping and were validated in this study (Table 1). The SNPs
at the IL-6 region (IL-6-1, IL-6-2 and IL-6-3) are novel compared
to the IL-6R. The polymorphism of IL-6-1 is 25bps at the 3?-end on
and IL-6-3 is 63bps at the 3?-end on the rs3087236.
No polymorphisms were found at the rs1800797 and the
rs3087236 in these subjects.
The genotype frequency and minor allele frequency of each
SNP in both control and schizophrenia groups are presented in
Table 2. All SNPs were compatible with Hardy–Weinberg’s equilib-
in schizophrenia). However, considering multiple testing, the SNP
IL-6R2 was still compatible with Hardy–Weinberg’s equilibrium.
We found no significant associations of these SNP genotypes with
The intermarker linkage disequilibrium analyses for haplotype
block revealed a three-SNP block of IL-6 with D?of 0.81 and a two-
Fig. 1. Haploview linkage disequilibrium (D?) displays the haplotype structures of
both IL6-R and IL-6 genes. The number in each square is D?×100 between two SNPs.
Author's personal copy
Y.-L. Liu et al. / Neuroscience Letters 468 (2010) 330–333
SNP genotyping on three IL-6 and three IL-6 receptor markers.
Genetic SNP (SNP ID) Gene position (allele type) (exon/intron)Primer Sequence (from 5?- to 3?-end) F: forward R: reverse
−208 (A=1/G=2) (5?-UTR,exon 1) F: CGCCGCTCTGAGTCATGTG
F: (inner primer for T allele): TTCTAGCCCTGTGGCGTAGTTGACCT
R: (inner primer for C allele): CTGCCAAGTATTTAAGAATGATTAATGTG
F: (outer primer (5?–3?)): AGATCTAGAATGCAAGAATCTCCCTGAC
R: (outer primer (5?–3?)): CCCATAGATAAAAGCCTTCTCTCCCT
IL-6R2 (rs4553185) 32850 (C=1/T=2) (intron 6)
IL-6R3 (rs4379670) 61760 (A=1/T=2) (3?-UTR,exon 10)
−636 (C=1/G=2) (promoter)
IL-6-2 (rs3087236+38)5379 (A=1/G=2) (3?-UTR)
IL-6-3 (rs3087236+63) 5404 (G=1/T=2) (3?-UTR)
SNP ID+number: the SNP is located at the number of nucleotides after the SNP ID locus.
Minor allele frequency (MAF) and association analyses of the SNPs of IL-6R and IL-6.
SNP Control Schizophrenia AssociationTest(p-value)
11 1222 MAF 1112 22 MAF Genota
aGenot: genotype frequency association test.
bAllele: allele type frequency association test.
of all compositions in either IL-6 or IL-6R showed no significant
association with schizophrenia (p=0.3495 for IL-6 and p=0.158 for
Table 3 shows the results of association of all SNP genotypes
and the phenotypes of the severity of positive symptom dimension
(SUMP), the severity of negative symptom dimension (SUMN), the
severity of disorganization (SUMDIS) and endophenotype of the
sustained attention indicators (the d?of degraded CPT and the d?
of undegraded CPT). Only the IL-6-2 showed a borderline signifi-
cant association with the positive symptom dimension phenotype
(p=0.0472), where the positive symptom dimension phenotype is
more severe in the recessive model of patients carrying A allele.
In this study, there was a statistically marginally significant
association between the SNP of IL-6-2 genotype and the sever-
ity of the positive symptom dimension of schizophrenia. It has
been reported that high IL-6 levels were related to the duration
and the treatment resistance of schizophrenia [11,19]. The patients
in this study were stable schizophrenics followed in the outpa-
tient department and receiving regular maintenance neuroleptic
treatment. As the IL-6-2 is located within the 3?-UTR, it is possi-
ble that the SNP was responsible for the elevation of IL-6 during
the active pathological process of schizophrenia with prominent
positive symptoms [19,24]. However, we have no plasma level of
Association analyses of quantitative phenotype indicators of schizophrenia and SNPs genotypes of IL-6R and IL-6 using Kruskal–Wallis Test.
Undegraded CPT d?
Degraded CPT d?
aSUMN: the severity of negative symptom dimension.
bSUMP: the severity of positive symptom dimension.
cSUMDIS: the severity of disorganization dimension.
dUndegraded CPT: the sustained attention indicator tested by unmasked CPT.
eDegraded CPT: the sustained attention indicator tested by masked CPT.
fIL-H: IL6 haplotype;
gILR-H: IL6 receptor haplotype.
Author's personal copy Download full-text
Y.-L. Liu et al. / Neuroscience Letters 468 (2010) 330–333
and plasma level of IL-6. Hence, it is worth exploring the associa-
tion between the genotype of IL-6-2 and plasma level of IL-6 in the
Although our results failed to find an association between the
populations have shown that the significant genetic region is pri-
marily located at IL-6R of exon 9 and promoter region [13,23]. One
study even showed an association between the genotype and the
serum soluble IL-6R level in schizophrenia . In a large meta-
analysis, IL-6 was found to be increased in schizophrenia . In
comparison to these results, our IL-6R SNPs were not located in
exon 9 or its promoter region. This may be one of the reasons for
this difference. Another reason may be a different sample compo-
sition resulting from the heterogeneity of schizophrenia. Further
study should consider the genetic region and factors related to
cytokine alterations such as stress, weight gain and different kinds
as well as different dosages of antipsychotic medication in order to
verify the potential etiological relations between the cytokine and
In summary, SNP markers of IL-6 (rs1800797+25 at promoter,
rs3087236+38 at the 3?-UTR, and rs3087236+63 at the 3?-UTR)
and IL-6R (rs4845617 at exon 1 of 5?-UTR, rs4553185 at intron 6,
and rs4379670 at exon 10 of 3?-UTR) showed no significant asso-
ciations with schizophrenia in both single locus and haplotype
ing the association between the genotype of IL-6-2 and plasma
level of IL-6 is recommended. This study suggested that genes
of IL-6 or IL-6 receptor are likely to be environmental mediators
rather than genes that predispose susceptibility to schizophre-
We acknowledge the help from the Department of Medi-
cal Research in National Taiwan University Hospital. This study
was supported by grants from the National Science Coun-
cil, Taiwan (NSC-91-3112-B-002-011; NSC-92-3112-B-002-019;
NSC-93-3112-B-002-012; NSC-94-3112-B-002-020, NSC 95-3112-
B-002-011, NSC 96-3112-B-002-011, NSC 97-3112-B-002-046),
the National Health Research Institute, Taiwan (NHRI-90-8825PP;
NHRI-EX91, 92, 93, 94-9113PP; IRO1 MH59624-01), National Tai-
 K. Akiyama, Serum levels of soluble IL-2 receptor alpha, IL-6 and IL-1 recep-
tor antagonist in schizophrenia before and during neuroleptic administration,
Schizophr. Res. 37 (1999) 97–106.
 American Psychiatric Association, Diagnostic and Statistical Manual, American
Psychiatric Press, Washington, DC, 1994.
 N. Andreasen, The Scale for Assessment of Positive Symptoms (SAPS), Univer-
sity of Iowa, Iowa City, IA, 1984.
 N. Andreasen, The Scale for the Assessment of Negative Symptoms (SANS),
University of Iowa, Iowa City, IA, 1983.
 M.F. Aukes, B.Z. Alizadeh, M.M. Sitskoorn, C. Kemner, R.A. Ophoff, R.S. Kahn,
Genetic overlap among intelligence and other candidate endophenotypes for
schizophrenia, Biol. Psychiatry 65 (2009) 527–534.
 S.L. Buka, M.T. Tsuang, E.F. Torrey, M.A. Klebanoff, R.L. Wagner, R.H. Yolken,
Maternal cytokine levels during pregnancy and adult psychosis, Brain Behav.
Immun. 15 (2001) 411–420.
 W.J. Chen, S.K. Liu, C.J. Chang, Y.J. Lien, Y.H. Chang, H.G. Hwu, Sustained atten-
tion deficit and schizotypal personality features in nonpsychotic relatives of
schizophrenic patients, Am. J. Psychiatry 155 (1998) 1214–1220.
 K.V. Chowdari, K. Mirnics, P. Semwal, J. Wood, E. Lawrence, T. Bhatia, S.N.
Deshpande, K.T.B.R.E. Ferrell, F.A. Middleton, B. Devlin, P. Levitt, D.A. Lewis,
V.L. Nimgaonkar, Association and linkage analyses of RGS4 polymorphisms in
schizophrenia, Hum. Mol. Genet. 11 (2002) 1373–1380.
sIL-2R, IL-6, IL-8 and TNF-alpha in schizophrenic patients, relation with symp-
tomatology and responsiveness to risperidone treatment, Mediators Inflamm.
10 (2001) 109–115.
 R. Freedman, S. Leonard, A. Olincy, C.A. Kaufmann, D. Malaspina, C.R. Cloninger,
D. Svrakic, S.V. Faraone, M.T. Tsuang, Evidence for the multigenic inheritance
of schizophrenia, Am. J. Med. Genet. 105 (2001) 794–800.
 U.H. Frommberger, J. Bauer, P. Haselbauer, A. Fraulin, D. Riemann, M. Berger,
Interleukin-6-(IL-6) plasma levels in depression and schizophrenia: compar-
ison between the acute state and after remission, Eur. Arch. Psychiatry Clin.
Neurosci. 247 (1997) 228–233.
 S.B. Gabriel, S.F. Schaffner, H. Nguyen, J.M. Moore, J. Roy, B. Blumenstiel,
J. Higgins, M. DeFelice, A. Lochner, M. Faggart, S.N. Liu-Cordero, C. Rotimi,
A. Adeyemo, R. Cooper, R. Ward, E.S. Lander, M.J. Daly, D. Altshuler, The
structure of haplotype blocks in the human genome, Science 296 (2002)
 J.C. Galicia, H. Tai, Y. Komatsu, Y. Shimada, K. Akazawa, H. Yoshie, Polymor-
phisms in the IL-6 receptor (IL-6R) gene: strong evidence that serum levels of
soluble IL-6R are genetically influenced, Genes Immun. 5 (2004) 513–516.
 D.L. Garver, R.L. Tamas, J.A. Holcomb, Elevated interleukin-6 in the
cerebrospinal fluid of a previously delineated schizophrenia subtype, Neu-
ropsychopharmacology 28 (2003) 1515–1520.
 T.Kaminska,A. Szuster-Ciesielska,
Michalowska, H. Dubas-Slemp, M. Kandefer-Szerszen, Serum cytokine
level and production of reactive oxygen species (ROS) by blood neutrophils
from a schizophrenic patient with hypersensitivity to neuroleptics, Med. Sci.
Monit. 9 (2003) CS71–CS75.
 A.W. MacDonald 3rd, M.F. Pogue-Geile, T.T. Debski, S. Manuck, Genetic and
environmental influences on schizotypy: a community-based twin study,
Schizophr. Bull. 27 (2001) 47–58.
 G. Muller-Newen, A. Kuster, U. Hemmann, R. Keul, U. Horsten, A. Martens, L.
Graeve, J. Wijdenes, P.C. Heinrich, Soluble IL-6 receptor potentiates the antag-
onistic activity of soluble gp130 on IL-6 responses, J. Immunol. 161 (1998)
 N. Muller, M. Riedel, R. Gruber, M. Ackenheil, M.J. Schwarz, The immune sys-
tem and schizophrenia. An integrative view, Ann. N. Y. Acad. Sci. 917 (2000)
 J. Naudin, J.L. Mege, J.M. Azorin, D. Dassa, Elevated circulating levels of IL-6 in
schizophrenia, Schizophr. Res. 20 (1996) 269–273.
 K.H. Nuechterlein, Vigilance in schizophrenia and related disorders, in: J. Zubin
(Ed.), Handbook of Schizophrenia. Vol.5. Neuropsychology, Psychophysiology
and Information Processing, Elsevier, Amsterdam, 1991, pp. 397–433.
 S. Potvin, E. Stip, A.A. Sepehry, A. Gendron, R. Bah, E. Kouassi, Inflammatory
cytokine alterations in schizophrenia: a systematic quantitative review, Biol.
Psychiatry 63 (2008) 801–808.
 R.D. Strous, Y. Shoenfeld, Schizophrenia, autoimmunity and immune system
dysregulation: a comprehensive model updated and revisited, J. Autoimmun.
27 (2006) 71–80.
Kosten, X.Y. Zhang, Association between interleukin-6 receptor polymorphism
and patients with schizophrenia, Schizophr. Res. 102 (2008) 346–347.
 D.P. van Kammen, C.G. McAllister-Sistilli, M.E. Kelley, J.A. Gurklis, J.K. Yao, Ele-
vated interleukin-6 in schizophrenia, Psychiatry Res. 87 (1999) 129–136.
 S. Ye, S. Dhillon, X. Ke, A.R. Collins, I.N. Day, An efficient procedure for genotyp-
ing single nucleotide polymorphisms, Nucleic Acids Res. 29 (2001) E88–E188.
 X.Y. Zhang, D.F. Zhou, P.Y. Zhang, G.Y. Wu, L.Y. Cao, Y.C. Shen, Elevated
interleukin-2, interleukin-6 and interleukin-8 serum levels in neuroleptic-free
schizophrenia: association with psychopathology, Schizophr. Res. 57 (2002)
 B. Zhao, J.P. Schwartz, Involvement of cytokines in normal CNS development
and neurological diseases: recent progress and perspectives, J. Neurosci. Res.
52 (1998) 7–16.
A. Wysocka, H. Marmurowska-