No association evidence between schizophrenia and
dystrobrevin-binding protein 1 (DTNBP1) in Taiwanese families
Chih-Min Liu
a,b, Yu-Li Liu
c, Cathy Shen-Jang Fann
d, Wei-Chih Yang
d,
Jer-Yuarn Wu
e, Shuen-Iu Hung
e, Wei J. Chen
f, Ching-Mo Chueh
g, Wei-Ming Liu
h,
Chen-Chung Liu
a, Ming-Hsien Hsieh
a, Tzung-Jeng Hwang
a, Stephen V. Faraone
i,
Ming T. Tsuang
j, Hai-Gwo Hwu
a,f,⁎
a
Department of Psychiatry, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
b
Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
c
Division of Mental Health and Substance Abuse Research, National Health Research Institutes, Taipei, Taiwan
d
Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan
e
National Genotyping Center, Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan
f
Institute of Epidemiology, College of Public Health, National Taiwan University, Taipei, Taiwan
gDepartment of Psychiatry, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan hYuli Hospital, Department of Health, Executive Yuan, Hualien, Taiwan
iMedical Genetics Research Center and Departments of Psychiatry and Neuroscience and Physiology, SUNY Upstate Medical University,
Syracuse, New York, USA
jInstitute of Behavioral Genomics University of California, San Diego, California, and Harvard Institute of Psychiatric Epidemiology and
Genetics, Boston, Massachusetts, USA
Received 3 July 2006; received in revised form 6 February 2007; accepted 9 February 2007 Available online 3 April 2007
Abstract
Several linkage studies have shown significant linkage of schizophrenia to chromosome 6p region, which includes the
positional candidate genes, Dystrobrevin-binding protein 1 (DTNBP1). The aim was to examine the association evidence of the
candidate gene in 693 Taiwanese families with at least two affected siblings of schizophrenia. We genotyped nine SNPs of this gene
with average intermarker distance of 17 kb. Intermarker linkage disequilibrium was calculated with GOLD. Single locus and
haplotype association analyses were performed with TRANSMIT program. We found no significant association between
schizophrenia and DTNBP1 either through single locus or haplotype analyses. We failed to replicate the association evidence
between DTNBP1 and schizophrenia and this gene may not play a major role in the etiology of schizophrenia in this Taiwanese
family sample.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Schizophrenia; DTNBP1; Chromosome 6p; Family-based association study
1. Introduction
Schizophrenia is a serious mental illness affecting 1%
of the general population. Family, twin, and adoption
⁎ Corresponding author. Department of Psychiatry, National Taiwan University Hospital, No. 7 Chung San South Road, Taipei, 100, Taiwan. Tel.: +886 2 2312 3456x6657; fax: +886 2 2331 4775.
E-mail address:[email protected](H.-G. Hwu).
0920-9964/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2007.02.003
studies have shown that schizophrenia is predominantly
genetically determined and has high heritability
(
McGuffin et al., 2003
). A multilocus model is favored
and assumes that there are several genes, each having a
small effect and acting in epistasis, responsible for
schizophrenia (
Risch, 1990
). Using linkage analyses, a
number of positive findings have been reported on
chromosome 6p (
Antonarakis et al., 1995; Arolt et al.,
1996; Bailer et al., 2000; Brzustowicz et al., 1997;
Matthysse et al., 2004; Maziade et al., 2001; Moises
et al., 1995; Schwab et al., 1995, 2000; Straub et al.,
2002b, 1995, 1996; Wang et al., 1995
). Furthermore,
suggestive linkage evidence for chromosome 6p was
reported in our earlier report using Taiwanese family
sample (
Hwu et al., 2000
).
There have been several reports implicating DTNBP1
in the etiology of schizophrenia.
Straub et al. (2002a)
presented the original evidence for a significant
associa-tion between schizophrenia and SNPs within DTNBP1
in a sample of 270 multiply affected Irish pedigrees. In
that study, eight SNPs across the gene, as well as several
three-marker haplotypes, showed significant
over-trans-mission to affected offspring across a range of different
diagnostic categories. Significant genetic association
evidence has also been produced by several
investiga-tions of various ethnic samples using different study
designs (
Fallin et al., 2005; Funke et al., 2004; Gornick
et al., 2005; Kirov et al., 2004; Li et al., 2005; Numakawa
et al., 2004; Schwab et al., 2003; Tang et al., 2003;
Tochigi et al., 2006; van den Oord et al., 2003; Williams
et al., 2004
), however, several others have failed to
replicate the result (
De Luca et al., 2005; Hall et al.,
2004; Holliday et al., 2006; Joo et al., 2006; Morris et al.,
2003
). It has also been reported that the gene is
significantly associated with schizophrenic patients
with positive family history (
Van Den Bogaert et al.,
2003
), and associated with patients with high levels of
negative symptoms (
DeRosse et al., 2006; Fanous et al.,
2005
).
DTNBP1 shows widespread expression in the human
brain (
Weickert et al., 2004
).
Talbot et al. (2004)
found
that the level of presynaptic dysbindin-1 are reduced in
schizophrenia and are related to glutamatergic
altera-tions in intrinsic hippocampal formation connecaltera-tions.
Numakawa et al. (2004)
found that, in neuronal culture,
over-expression of dysbindin induced expression of
SNAP25 and synapsin-I, promoted phosphorylation of
Akt and increased glutamate release. Conversely,
knockdown of dysbindin expression resulted in lower
pre-synaptic protein expression and a decrease in
glutamate release (
Numakawa et al., 2004
).
Bray et al.
(2005)
showed that a defined schizophrenia risk
haplotype tags one or more cis-acting variants that
results in a relative reduction in DTNBP1 mRNA
expression in human cerebral cortex, indicating
varia-tion in the DTNBP1 gene confers susceptibility to
schizophrenia through reduced expression.
Our aim here was to examine the association
evidence between schizophrenia and DTNBP1 in a
large family sample of 693 Taiwanese families with at
least two siblings affected with schizophrenia.
2. Materials and methods
2.1. Subjects
The subjects were recruited from two sample
collection programs; the Multi-dimensional
Psycho-pathology Study of Schizophrenia (MPSS) from 1993 to
2001 (
Hwu et al., 2002
) and the Taiwan Schizophrenia
Linkage Study (TSLS) (
Hwu et al., 2005
) from 1998 to
2002. A total of 693 families with at least two affected
siblings with schizophrenia were used for this study, of
which 86 families were from MPSS and 607 were from
TSLS.
The MPSS families were recruited mainly from the
Department of Psychiatry, National Taiwan University
Hospital and the University-affiliated Taoyuan
Psychia-tric Center. The families came mostly from northern
Taiwan. Data collection was initiated after informed
consent had been obtained from the identified study
subjects and their families. All family members were
personally interviewed by research psychiatrists using
the Psychiatrist Diagnostic Assessment (PDA) (
Hwu,
1999
). The final diagnostic assessment was formulated
by integrating the PDA data and clinical information
obtained from the medical chart records. The final
diagnosis used criteria specified by the Diagnostic and
Statistical Manual of Mental Disorders fourth edition
(DSM-IV) (
APA, 1994
).
The TSLS families were recruited from hospitals all
over Taiwan, except for the above two institutions. Data
collection was initiated after informed consent had been
obtained from the identified study subjects and their
family members. All the family members were
inter-viewed by well-trained assistants using the Mandarin
Chinese version of the Diagnostic Interview for Genetic
Studies (DIGS) (
Chen et al., 1998
). The final diagnostic
assessment was formulated by integration of the DIGS
data and clinical information from the medical chart
records by two board-certified research psychiatrists
independently. Research diagnosis was made based on
DSM-IV criteria. All data schedules and medical
records for subjects with inconsistent diagnoses from
these two independent research diagnosticians were
evaluated further by the senior researcher (H-G Hwu) to
achieve final diagnosis. Detailed information has been
previously published about the recruitment procedures
(
Hwu et al., 2005
). Both projects of sample recruitment
have been approved by the ethics committee of National
Taiwan University Hospital.
The diagnoses were comparable between the two
sample collection programs, because they were both
made based on DSM-IV criteria. Clinical symptoms
were rated using schedule for assessment of negative
symptoms (SANS) (
Andreasen, 1983
) and schedule for
assessment of positive symptoms (SAPS) (
Andreasen,
1984
) with satisfactory reliability. The comparison of
age at onset between the affected individuals of the two
samples was not significant. The severity of positive and
negative symptoms were comparable and the severity of
positive symptoms are with low mean values of 1.31 and
1.63 for MPSS and TSLS sample, respectively, and the
severity of negative symptoms are of 1.92 and 1.65 for
MPSS and TSLS sample, respectively. Under the
sensitive statistical method of generalized estimating
equations (GEE) model, for related affected sibpairs,
with large sample size, the positive symptoms of TSLS
sample were significantly more severe than those of
MPSS sample. On the contrary, the negative symptoms
of TSLS sample were significantly less severe than
those of MPSS sample. We judged that the small
differences, though significant, of clinical symptoms
between the two samples would contribute little to
subsequent genetic analyses.
Through the procedures described above and
else-where, we enrolled a total of 693 multiplex (i.e. at least
two affected siblings) schizophrenic nuclear families,
incorporating a total of 2787 individuals from whom
DNA was available. This sample is a representative
family sample, which includes about 40% of all
multiplex schizophrenic families in Taiwan (
Hwu
et al., 2005
). The family structure detailed by the
number of affected offspring and parent genotyped is
presented in
Table 1
. A total of 1487 individuals were
Table 1
Distribution of families by number of affected offspring and parents genotyped
Affected offspring genotyped
Parents genotyped Total
0 1 2 1 3 5 5 13 2 22 283 325 630 3 2 36 10 48 4 0 2 0 2 Total 27 326 340 693 T able 2 Th e list of prim ers and probes of the nine validate d SNP s used in MALDI-T O F meth od dbS NP ID Forw ard prim er Ba ckward prim er Pr obe rs90 9706 ACGT TGGA TGA AA CCA TCC TGG GAGA TCAG A CGTTGG A TGG TCAA GTCAGTT TCCAA GGG AG AGACA TGCCA AAGG GA TCT rs10 18381 ACGT TGGA TGA A TGA CTGCT GAGA TCTG CC A CGTTGG A TGC CTCAC CTTTC CT AA T A GCC GCC GGTG A TTC AACAGC rs26 19522 ACGT TGGA TGA A T A GCTGGC AGA AGCAGTG A CGTTGG A TGG CTC TT A TGTC T ACC TTTCC GCAG TGAG TGAG AGCTGAC A rs20 05976 ACGT TGGA TGTC CTGAC CTCAAG TGA TCTG A CGTTGG A TGTG TCAGTC TTCAGG GAAA CG GA TT A T AGGT A TGAG CCAC rs26 19528 ACGT TGGA TGCC A TTCTT AAGCTT AGT AGT G A CGTTGG A TGG AG TTTTTG GGA TTGGA TGC TT AA GCTT AG T A G TGCTGA GT A C rs10 1 1313 ACGT TGGA TGA TTCAC AGGCT A CAGAA TGG A CGTTGG A TGC CAAGTT ACTGC ACACAA GC CT A CAGAA TGGA TGTTG C rs26 19539 ACGT TGGA TGA TGCAC CAAGT A GCTT AG AG A CGTTGG A TGA T A ACT AGT CTGAC A TGG TC A T AA TCCT A T T A GCT A TGA T AGT rs38 29893 ACGT TGGA TGA GG TTGTTC TCT ACCT CCTC A CGTTGG A TGG GA TTCT GACTTT TGA GGTC GCC TCT AAA TGA GCTGA AA rs74 2106 ACGT TGGA TGCA AGGA GCAGAC TCAAA TGG A CGTTGG A TGC CGGT AA CTTTGG TGAG TTG GA CTCAA A TGG A TTTC TGG
diagnosed schizophrenic; the mean age was 36.0
(± 9.6) years and 61.1% were male. The mean age at
onset was 22.9 (± 6.8) years. The mean age of the
unaffected subjects was 55.7 (± 15.4) years with
48.4% male.
2.2. SNP selection and validation
A total of 18 SNPs for DTNBP1 were selected from
the previous studies (
Straub et al., 2002a
) and a public
database (
http://www.ensembl.org/Homo_sapiens/
martview
) for initial validation. A sample subset of 31
trios and one independent individual was used to
validate the 18 selected SNPs. Considering the power
of further linkage disequilibrium test, we required SNPs
to have a minor allele frequency above 10% to be
genotyped in the full sample.
2.3. SNP genotyping
All SNP genotypings were performed by the method
of matrix-assisted laser desorption/ionization-time of
flight mass spectrometry (MALDI-TOF MS) (
Rodi
et al., 2002
). Primers and probes flanking the SNPs were
designed by using SpectroDESIGNER software
(Seque-nom, San Diego, CA, USA). A DNA fragment (100–
300 bp) encompassing the SNP site was amplified using
the four-plexed polymerase chain reaction (PCR) with
GeneAmp 9700 thermocycler (Applied Biosystems,
Foster city, CA, USA) under the condition of 95 °C for
15 min, then 45 cycles of denaturing at 95 °C for 20 s,
annealing at 56 °C for 30 s, and extension at 72 °C for
1 min, and finally 72 °C for 3 min according to the
manufacturer's instruction.
To neutralize the un-incorporated deoxynunleotide
triphosphate (dNTP), the PCR reaction mixture was
treated with shrimp alkaline phosphatase (SAP) at 37 °C
for 20 min. The un-incorporated dNTP was converted
to deoxynucleotide diphosphate (dNDP). The reaction
mixture was incubated at 85 °C for 5 min to inactivate
SAP activity. Then primer extension was performed by
adding the probe, Thermo Sequenase (Amersham
Phar-macia, Piscataway, NJ, USA) and appropriate
dide-oxynucleotide triphosphate (ddNTP)/dNTP mixture,
and followed by 55 cycles of denaturing at 94 °C for
5 s, annealing at 52 °C for 5 s, and extension at 72 °C for
Table 3
Detailed description of the validated SNPs for DTNBP1 and the single locus association analysis results dbSNP ID.
(alternative SNP name)a
Locationb Intermarker
distance (kb)
Polymorphismc Minor allele
frequency
Single locus association Chi-square p-value rs909706 (P1583) Intron 1 – T/C 0.40 0.13 0.71 rs1018381 (P1578) Intron 1 3.8 C/T 0.07 0.33 0.56 rs2619522 (P1763) Intron 1 3.4 A/C 0.08 0.11 0.74 rs2005976 (P1757) Intron 3 2.8 G/A 0.08 0.11 0.74 rs2619528 (P1765) Intron 3 1.0 G/A 0.08 0.10 0.75 rs1011313 (P1325) Intron 4 16.4 C/T 0.19 3.09 0.079 rs2619539 (P1655) Intron 5 12.6 C/G 0.39 0.20 0.66 rs3829893 Intron 5 5.2 G/A 0.32 0.10 0.76 rs742106 (P1328) Intron 9 91.2 T/C 0.42 0.63 0.43 a
The SNP number in the study ofStraub et al. (2002a).
b
The SNP location for DTNBP1 was determined based upon the m-RNA accession no. NM_032122.
c
Second allele under oblique line (/) is the minor allele.
Table 4
Intermarker D’ value of the nine SNPs of DTNBP1 calculated by GOLD
rs909706 rs1018381 rs2619522 rs2005976 rs2619528 rs1011313 rs2619539 rs3829893 rs742106 rs909706 – rs1018381 .91 – rs2619522 .91 .99 – rs2005976 .90 .98 .99 – rs2619528 .91 .99 .99 .98 – rs1011313 .88 .83 .78 .78 .78 – rs2619539 .95 .92 .74 .74 .74 .89 – rs3829893 .93 .84 .83 .81 .81 .87 .92 – rs742106 .18 .39 .35 .36 .36 .58 .18 .14 –
5 s. Different extension products were differentiated by
mass through MALDI-TOF. The primers of validated
SNPs used for amplification and probes for extension in
the MALDI-TOF method were listed in
Table 2
.
This genotyping method has been applied to a broad
variety of clinical applications, since it fulfills criteria
such as accuracy of SNP detection, sensitivity to score
SNPs using a small amount of template throughput
capacity, flexibility of the procedure, and
cost-effec-tiveness (
Tost and Gut, 2005
).
2.4. Statistical analysis
We used PEDCHECK version 1.1 (
O'Connell and
Weeks, 1998
) and UNKNOWN version 5.23 (
Terwilli-ger and Ott, 1994
) to check for Mendelian inheritance of
SNPs and the Procedure ALLELE in SAS/GENETICS
release 8.2 (
Institute, 2002
) to test for Hardy–Weinberg
equilibrium. Linkage disequilibrium (LD) among
mar-kers was measured with coefficient D' (
Hedrick, 1987
),
which was used to define haplotype blocks. A graphic
presentation of block pattern was completed using
GOLD software (
Abecasis et al., 2000; Abecasis and
Cookson, 2000
). Both single point and haplotype
association analyses were carried out using TRANSMIT
version 2.5.4 (
Clayton, 1999
). SNPtagger (
Ke and
Cardon, 2003
) was used to screen for minimal sets of
SNPs (haplotype tagging SNPs, htSNPs) to represent
the haplotype block structure. Power estimation was
calculated with PBAT (
Lange et al., 2004
) using the real
family structure available for genotyping. The type I
error was set at 0.01. Two inherited models, additive and
multiplicative, were set up with the parameters of
disease gene frequency 0.05 and population prevalence
0.003 based upon previous epidemiological study (
Hwu
et al., 1989
).
3. Results
At the stage of SNP validation, nine out of the
eighteen SNPs, of which eight are overlapping with the
SNPs studied in the initial association study (
Straub
et al., 2002a
), with average intermarker distance of
17 kb met the validation criterion of minor allele
frequency above 10%.
Table 3
gives a detailed
description of the validated SNPs.
The single SNP association analyses showed no
significant evidence for any SNP of DTNBP1 (
Table 3
).
The intermarker LD assessed by coefficient D' is
presented in
Table 4
. The eight SNPs from rs909706 to
rs3829893 showed intermarker D' above 0.7 and
cons-tituted a haplotype block. Three SNPs, rs2005916
(P1757), rs1011313 (P1325), and rs3829893, were
selected as htSNPs using SNPtagger (
Ke and Cardon,
2003
). The haplotype association analysis using the three
htSNPs showed marginally significant preferential
transmission of G–T–G haplotype to affected
indivi-duals (p = 0.042). The detailed haplotype frequency and
analysis results are shown in
Table 5
. We also analyzed
the 2-SNP, 3-SNP haplotype associations within the
8-SNP haplotype block using moving window strategy and
the results were all negative (data not shown).
4. Discussion
This SNP fine mapping study revealed no significant
association in the single locus analysis. In the haplotype
association analysis, the haplotype G–T–G of the three
SNPs, rs2005916 (P1757), rs1011313 (P1325), and
rs3829893, had preferential transmission to affected
individuals only at marginally significant level, and was
not significant after correction for multiple tests. We
failed to replicate the association evidence between
DTNBP1 and schizophrenia in this Taiwanese family
sample.
As for the negative result in our sample, we would
give the following comments. Though we reported
suggestive linkage evidence to 6p22.3 (
Hwu et al.,
2000
) in a sub-sample of this study, of which 22 families
Table 5
Haplotype association analysis using TRANSMIT Haplotypes of rs2005976-rs1011313-rs3829893 Haplotype frequency Chi-square p-value G–C–G 0.42 1.06 0.30 G–C–A 0.31 0.22 0.64 G–T–G 0.18 4.12 0.042 A–C–G 0.08 0.053 0.82 Table 6
Comparison of allele frequencies of the SNPs between our samples, those ofStraub et al. (2002a)andLi et al. (2005)
dbSNP ID. (alternative SNP name)a Common allele (allele frequency) in our samples Straub et al. (2002a) Li et al. (2005) Scottish Chinese rs909706 (P1583) T (0.60) C (0.62) C (0.61) T (0.63) rs1018381 (P1578) C (0.93) C (0.92) C (0.90) – rs2619522 (P1763) A (0.92) A (0.85) A (0.80) A (0.92) rs2005976 (P1757) G (0.92) G (0.83) G (0.79) G (0.92) rs2619528 (P1765) G (0.92) G (0.83) G (0.80) G (0.93) rs1011313 (P1325) C (0.81) C (0.91) C (0.92) C (0.80) rs2619539 (P1655) C (0.61) C (0.54) G (0.53) C (0.66) rs3829893 G (0.68) – – – rs742106 (P1328) T (0.58) C (0.62) – –
were overlapped, the peak of non-parametric linkage
score was on the marker D6S285, 3.5 Mb from
DTNBP1. This suggested that DTNBP1 might not be
within the candidate region. The single point and
haplotype association analyses in this sub-sample were
also not significant (data not shown).
Ethnic differences might contribute to our failure to
replicate prior studies. The comparison of allele
frequencies of the SNPs between our samples, those
of
Straub et al. (2002a)
and
Li et al. (2005)
was listed in
Table 6
. The allele frequencies of the SNPs in our
Taiwan Chinese sample were similar to those of Han
Chinese (
Li et al., 2005
) and different from those of Irish
sample (
Straub et al., 2002a
) and Scottish sample (
Li
et al., 2005
). However, the risk haplotypes
‘G–G’ of
P1757
–P1765 and ‘G–A–C’ of P1757–P1765–P1325
reported to have significant association evidence in
Li et al. (2005)
were not significant in our sample
(Chi-square = 0.10, df = 1, p>0.05; Chi-square=1.1,
df = 1, p
>0.05, respectively). The risk haplotype of
‘G–T–C–A’ of P1655–P1763–P1578–P1583 reported
to have significant association evidence in another Han
Chinese sample (
Tang et al., 2003
) were also not
significant in our sample (Chi-square = 2.14, df = 1,
p
>0.05). Mutation screening study could not identify
any mutation and/or polymorphism in the 5′ promoter
region or the protein-coding sequences of DTNBP1 in a
Taiwanese Han Chinese sample (
Liao and Chen, 2004
).
These differing results suggest that genetic heterogeneity
exists in the susceptibility genes across different ethnic
groups.
The powers to detect association for each relative risk
of 1.4, 1.5, and 1.6, were 0.50, 0.73, and 0.89,
respectively, in the additive model. The powers for the
same relative risk were 0.52, 0.76, and 0.91, respectively,
in the multiplicative model. These results indicated that
the study has enough power to detect the association with
the relative risk over 1.6. Therefore, the negative results
in our study may not result from inadequate power to
detect gene of moderate genetic effects.
Considering the different statistical strategies across
studies, we also used the moving window strategy in
addition to SNPtagger program to detect all the possible
haplotypes with significant association and the results
were still not significant. We genotyped only one SNP in
the distal genomic region of the gene; therefore, we
cannot exclude completely the possibility of association
with the distal genomic region of DTNBP1 in our
sample.
Another reason for failure to replicate in our sample
may be due to a complex pattern of allelic associations
that are not sufficiently captured by the SNPs employed
in the present study. This is consistent with the
observation that different haplotypes from this gene
seem to be contributing to different populations
(
Schwab et al., 2003
). Our samples come from families
with at least two affected siblings. We failed to replicate
the finding that DTNBP1 is particularly involved in
schizophrenic cases with a familial genetic loading (
Van
Den Bogaert et al., 2003
).
In summary, we failed to replicate the association
evidence between DTNBP1 and schizophrenia and this
gene may not play a major role in the etiology of
schizophrenia in this Taiwanese family sample.
Acknowledgements
We gratefully acknowledge the help from the
Department of Medical Research in National Taiwan
University Hospital and the SNP genotyping work done
by the National Genotyping Center (NGC), National
Science Council, Taiwan. This study was supported by
grants from the National Science Council, Taiwan
(91-3112-B-002-011; 92-3112-B-002-019;
93-3112-B-002-012; 94-3112-B-002-020);
NSC-95-3112-B002-011, and the National Health Research
Institute, Taiwan (NHRI-90-8825PP; NHRI-EX91, 92,
93, 94-9113PP; NHRI-EX-9511PP and National
Insti-tute of Mental Health, USA (IRO1 MH59624-01).
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