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Association between TCF4 and schizophrenia does not exert its effect by common nonsynonymous variation or by influencing cis-acting regulation of mRNA expression in adult human brain.

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RESEARCH ARTICLE
Neuropsychiatric Genetics
Association Between TCF4 and Schizophrenia Does
Not Exert its Effect by Common Nonsynonymous
Variation or by Influencing cis-Acting Regulation of
mRNA Expression in Adult Human Brain
Hywel J. Williams, Valentina Moskvina, Rhodri L. Smith, Sarah Dwyer, Giancarlo Russo,
Michael J. Owen, and Michael C. O’Donovan*
MRC Centre for Neuropsychiatric Genetics and Genomics, Department of Psychological Medicine and Neurology,
School of Medicine, Cardiff University, Cardiff, UK
Received 21 March 2011; Accepted 6 July 2011
Large collaborative Genome-wide Association studies of schizophrenia have identified genes and genomic regions that are
associated with the disorder at highly stringent levels of statistical significance. Among these, transcription factor 4 (TCF4) is
one of the best supported although the associated SNP
(rs9960767) is located within intron 3 and has no obvious
function. Seeking the mechanism at TCF responsible for the
association, we examined TCF4 for coding variants, and for cis
regulated variation in TCF4 gene expression correlated with the
associated SNP using an assay to detect differential allelic
expression. Using data from the 1000 genomes project, we
were unable to identify any nonsynonymous coding variants
at the locus. Allele specific expression analysis using human post
mortem brain samples revealed no evidence for cis-regulated
mRNA expression related to genotype at the schizophrenia
associated SNP. We conclude that association between schizophrenia and TCF4 is not mediated by a relatively common nonsynonymous variant, or by a variant that alters mRNA expression
as measured in adult human brain. It remains possible that the
risk allele at this locus exerts effects on expression exclusively in a
developmental context, in cell types or brain regions not
adequately represented in our analysis, or through post-transcriptional effects, for example in the abundance of the protein or
its sub-cellular distribution. Ó 2011 Wiley-Liss, Inc.
How to Cite this Article:
Williams HJ, Moskvina V, Smith RL, Dwyer S,
Russo G, Owen MJ, O’Donovan MC. 2011.
Association Between TCF4 and
Schizophrenia Does Not Exert its Effect by
Common Nonsynonymous Variation or by
Influencing cis-Acting Regulation of mRNA
Expression in Adult Human Brain.
Am J Med Genet Part B 156:781–784.
chromosome 6 [Purcell et al., 2009; Shi et al., 2009; Stefansson et al.,
2009]. Demonstration of strong evidence for association is a crucial
step in implicating susceptibility genes, but genetic association
findings point to genomic locations that harbour susceptibility
variants rather than specific genes per se, it being possible that any
observed association is the result of linkage disequilibrium (LD) to
an as yet unknown functional element close to the gene of interest.
In order to apply the genetic findings for developing an understanding of pathophysiology, it is necessary to determine the
identity of the gene, or other element, whose function is influenced
by the true functional variant. Presently, in no instances in
schizophrenia, and indeed in very few instances in complex diseases
Key words: allelic expression; TCF4; GWAS; eQTL; mRNA
INTRODUCTION
Genome-wide association studies (GWAS) of schizophrenia have
identified a small number of loci at which variants are associated
with the disorder at a level considered genome-wide significant
(GWS) [Dudbridge and Gusnanto, 2008]. To date, the associated
markers implicated in the disorder at this level of support map
within, or in the vicinity of, zinc-finger binding protein 804A
(ZNF804A) [Williams et al., 2010], transcription factor 4 (TCF4)
[Stefansson et al., 2009], neurogranin (NRGN) [Stefansson
et al., 2009] and an extended region spanning the MHC locus on
Ó 2011 Wiley-Liss, Inc.
Grant sponsor: Medical Research Council (UK); Grant sponsor: The
Wellcome Trust; Grant sponsor: NIMH (USA) CONTE: 2 P50; Grant
number: MH066392-05A1.
*Correspondence to:
Michael C. O’Donovan, MRC Centre for Neuropsychiatric Genetics and
Genomics, Henry Wellcome Building, Department of Psychological
Medicine and Neurology, School of Medicine, Cardiff University,
Cardiff CF14 4XN, UK. E-mail: odonovanmc@Cardiff.ac.uk
Published online 2 August 2011 in Wiley Online Library
(wileyonlinelibrary.com).
DOI 10.1002/ajmg.b.31219
781
AMERICAN JOURNAL OF MEDICAL GENETICS PART B
782
as a whole, has the functional variant responsible for the GWS
association been identified. There are several possible explanations
for this. Among these is the requirement to undertake comprehensive variant discovery across often large regions of the genome in
sufficient numbers of individuals to be confident of capturing the
susceptibility variant at least once. Even if the variant is captured,
thereafter, on statistical grounds alone, it will often be difficult to
identify which among many correlated markers best captures what
are often weak (in terms of effect sizes) association signals. Another
approach is to test those markers showing strong evidence for
association for some form of functional impact on the putative gene
responsible for the signal, but a comprehensive evaluation of
functionality requires innumerable functional assays, some under
dynamic conditions (e.g., exposure to hormones).
Given the complexities of assigning function to variants, the
most common approaches are to test genes in associated regions for
(a) nonsynonymous variants in the relevant genes that are associated with disease and then design functional assays based upon the
type of gene and the location of the variant within that gene and (b)
measure mRNA from the candidate gene in extracts from some
relevant tissue and then seek evidence for correlation between
expression levels and an associated variant, the premise being
that if the associated marker tags a functional variant that regulates
gene expression, a so called cis-eQTL, the associated variant should
be associated with expression of the candidate gene. In the present
study, seeking to clarify the mechanism underpinning one of the
most convincing schizophrenia associations to a common allele,
that at the TCF4 locus, we apply both of these tractable approaches.
TCF4 was identified as a susceptibility locus for schizophrenia by
the GWAS and follow up studies of the SGENE consortium
[Stefansson et al., 2009] who observed strong evidence for association to a variant (rs9960767; P ¼ 4.1 109) located within intron
3 of TCF4 (GeneID 6925). The signal most likely points to TCF4 per
se given that that there is no LD between this variant and other
markers that extend beyond the boundaries of this gene, although
LD to an unknown intragenic functional element cannot be
excluded. TCF4 is also a highly plausible candidate for schizophrenia, it being a transcription factor whose functions involve roles in
the development of the nervous system [Blake et al., 2010]. Moreover, point mutations and deletions of the gene are known causes of
Pitt–Hopkins Syndrome, a neurodevelopmental disorder characterized by CNS phenotypes such as severe mental retardation,
microcephaly, and epilepsy [Blake et al., 2010].
METHODS
Seeking Non-Synonymous Variants
Seeking non-synonymous mutations that might be responsible
for association at TCF4, we downloaded data from the 1000
genomes project (http://www.1000genomes.org/page.php) which,
at the time contained data representing 112 haplotypes of
European ancestry. Assuming complete mutation discovery,
this dataset has power of 1.0 to detect a variant with a frequency
of 0.05 (and power of 0.68 to detect an allele with a frequency
of 0.01), and therefore makes in house mutation scanning
redundant for variants that are relatively common in the
population.
Looking for cis-Acting Effects on Expression
To screen TCF4 for evidence for cis-eQTLs, we applied a method
that allows estimation of the relative expression of each copy of the
gene within individual subjects [Bray et al., 2003]. The principle of
the approach is that where individuals are heterozygous for an
unknown cis-eQTL, the copies of the gene will be unequally
expressed, whereas in the absence of such a variant, the transcripts
from each chromosome will be equally expressed. This method
is particularly sensitive for detecting cis-eQTLs as the ‘‘within
subject’’ comparison controls for trans-acting factors (e.g., drug
exposure, disease status) or most sources of noise (total amount of
RNA, brain pH, agonal state, other causes of RNA degradation) that
have to be controlled for in studies of total mRNA.
To measure relative expression levels within subjects, we
identified samples heterozygous for a SNP located within an
exon of TCF4. In those heterozygotes, we then assayed the relative
expression levels of mRNA containing each of the exonic alleles
using the SNaPshot system (Applied Biosystems, Foster City, CA)
using methods we have published before [Bray et al., 2003, 2005].
To identify the presence of cis-eQTLs, we normalized the relative
expression of alleles observed in the cDNA by that observed when
the same assay was applied to genomic DNA from heterozygotes as
this controls for technical biases that can lead to preferential
representation of one allele, even when both alleles are present at
a 1:1 ratio (the allelic ratio expected in people who are heterozygous
and do not have a copy number variant at the site). Analysis of
heterozygous samples was performed as two separate experiments.
In each experiment, cDNA from two separate reverse transcription
reactions were assayed for each heterozygous individual alongside
the corresponding genomic DNA sample. For comparisons of
allelic expression differences stratified by genotype at rs9960767,
it is not necessary to normalize relative allele measurement for
cDNA by that from genomic DNA as any tendency to over-estimate
expression of one allele applies regardless of genotype at rs9960767
(although we note as expected, similar results were obtained if we
did normalize).
Allelic expression analysis was based on the exonic SNP rs8766,
located within the last coding exon of TCF4 transcript
NM_001083962.1.
Statistics
Differences in allelic expression were tested by comparing genomic
ratios with cDNA ratios from the same heterozygous samples.
Group comparisons were analysed by paired t-test, all tests are
two-tailed. To test whether clinical status of the individual from
whom the sample was obtained, or the brain region from which it
was extracted influenced the results, a univariate analysis of
variance test was performed in which the allelic ratio was the
dependant factor, and, whether the sample was gDNA or cDNA
was a fixed factor, and diagnosis and brain region were entered as
covariates. All statistical analyses were performed using SPSS v16.
Brain Samples
mRNA derived from post-mortem brains (frontal, cortical, parietal
or temporal cortex) of 148 unrelated anonymous individuals was
WILLIAMS ET AL.
available for analysis. Samples had been obtained from three
reputable tissue sources (The MRC London Neurodegenerative
Diseases Brain Bank, London, UK; The Stanley Medical Research
Institute Brain Bank, Chevy Chase, MD; The Karolinska Institute,
Stockholm, Sweden). Of these, 78 individuals had received no
psychiatric or neurological diagnosis at the time of death, 22 had
a diagnosis of Alzheimer’s disease, 18 a diagnosis of schizophrenia,
15 a diagnosis of bipolar disorder and 15 a diagnosis of major
depression. The nature of the performed assay (all measures of
relative expression are within individual) means that the disease
status is not expected to confound our analysis. Genomic DNA was
extracted by phenol–chloroform procedures. Total RNA was
extracted using the RNA wizÔ isolation reagent (Ambion, Warrington, UK) and then treated with DNase (Ambion). Reverse
transcription was performed using random decamers (Ambion)
and SuperScript III (Invitrogen, Paisley, UK).
RESULTS
Non-Synonymous Variants
We found no non-synonymous variants in the 1000 genomes
project data at TCF4.
Expression Analysis
In the total sample of successfully genotyped individuals (n ¼ 138),
the genotype frequency distribution for SNP rs8766 was AA (0.49),
AG (0.36), and GG (0.13), 50 individuals being heterozygous and
therefore informative for allelic expression analysis. The assay
showed good reproducibility, with average coefficients of variation
(SD/mean) of 0.02. Samples for which assay quality was poor
(coefficients of variation >0.2) were removed prior to analysis.
No large deviations (>20%) from equal expression were observed
implying that if cis-acting variants exist that substantially affect the
expression of TCF4 in the tissues studied, they are too rare for a
single heterozygote to be observed in 50 informative individuals.
Although we were unable to detect large deviations in expression,
our data were consistent with that of Buonocore and colleagues, in
that overall we observed a modest over expression (3%) of the G
allele of rs8766, although this was not specific to a particular brain
region or diagnostic group (Univariate analysis of variance:
P ¼ 0.72 and 0.86, respectively).
To determine if GWS variant rs9960767 at TCF4 is a cis-eQTL, or
is in strong LD with such a variant, we stratified the allelic
expression results by genotype at that site. For this analysis 48
individuals were successfully genotyped for SNP rs9960767, 41 of
these were homozygous (all ancestral AA genotype) and 7 were
heterozygous (AC genotype). The expectation is that if rs9960767 is
a proxy of (or is itself) an eQTL, subjects that are heterozygous for
this will show greater unequal expression of alleles than homozygotes, since heterozygotes will tend to carry two functionally
distinct eQTL alleles whereas homozygotes will carry two functionally equivalent alleles. However, this pattern was not observed,
heterozygotes and homozygotes showing similar levels of relative
allelic expression defined both directionally (i.e., the amount of
allele G over allele A, t-test P ¼ 0.10; Fig. 1) or in terms of the
magnitude of the deviation from equal relative expression of one
783
FIG. 1. Expression of SNP alleles at rs8766 in samples
homozygous (n ¼ 41) and heterozygous (n ¼ 7) for SNP
rs9960767 defined by direction of effect. Allele G is plotted over
allele A.
allele to another (test of equal variance P ¼ 0.94). The latter is the
more appropriate analysis where there is weak or no LD between a
putative cis-eQTL and the marker used to estimate relative expression because the low and high expression alleles at the eQTL will not
be in phase with a specific allele at the assayed locus, as is the case in
the sample analyzed here (LD between rs8766 and rs9960767;
D0 ¼ 0.20, r2 ¼ 0.005).
DISCUSSION
The aim of the present study was to determine whether the genomewide significant finding at the TCF4 locus could be related to
functional variation in the gene itself, this being a requirement
for concluding that the association is the result of altered function of
this gene per se rather than a co-localized unknown functional
element.
We first looked in silico for nonsynonymous variants at TCF4
that might explain the association at this locus using data from the
1000 genomes project but none was found. Power considerations
suggest that association at this locus is not due to a common coding
variant that is present in the European population at a frequency of
>5%, although in drawing this conclusion, we should note the
caveat that there are still technical difficulties in detecting small
coding insertion/deletion polymorphisms and therefore it is possible that such variants may have been missed by the 1000 genomes
project. We next sought to determine whether the associated SNP
(rs9960767) might be associated by virtue of being itself a cis-eQTL
for TCF4, or in LD with such an eQTL.
Within the TCF4 transcript we chose the SNP rs8766 from
dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP) with which
to measure the relative allelic expression levels, this having been
used to detect cis-acting influences on expression in a previous
study of this locus, but in a sample too small to examine correlation
between that phenomenon and the allele associated with schizophrenia (Buonocore et al., 2010). We next stratified the data with
AMERICAN JOURNAL OF MEDICAL GENETICS PART B
784
respect to genotype at rs9960767, the variant showing strong
evidence for association with schizophrenia. SNP rs8766 tags
each of the two reference transcripts that are described in Entrez
Gene (http://www.ncbi.nlm.nih.gov/gene). These differ in their use
of an alternative in-frame 30 splice site in exon 17 resulting in a
12 base-pair truncation in the shorter isoform (long isoform
a—NP_001077431 and short isoform b—NP_003190).
The allelic expression data did not suggest the existence of
common cis-eQTL alleles at this locus with moderately large effects
on expression, defined here as in our earlier work and that of others
as one allele being expressed 20% more than another. This may
not be surprising given that hemizygosity for TCF4 results in
Pitt–Hopkins syndrome [Blake et al., 2010] and therefore strong
evolutionary constraints on variants that have marked effects on
expression are expected. More importantly for the present study,
heterozygous carriers of the schizophrenia risk allele at TCF4 did
not show evidence for more unequal allelic expression at TCF4 than
those who were homozygous at this locus, which strongly suggests
the schizophrenia associated allele is not an eQTL for TCF4 or in
strong LD with such an eQTL.
Much of what is known about TCF4 relates to its function in the
immune system [Murre, 2005]. In brain, while understanding is still
at an early stage, during brain development, bHLH proteins, of
which TCF4 is one, modulate critical events in neuronal and glial
progenitor cells, controlling the transition from proliferation to
differentiation [Ross et al., 2003]. Given the likely developmental
importance of TCF4, it may be that the associated variant tags an
eQTL, but that effect is only manifest at a particular developmental
stage. We are unable to test that hypothesis, which would require
large numbers of foetal brain samples at different developmental
stages. It should also be noted that our study is unable to address the
role of quantitative post-transcriptional effects, for example in the
abundance of protein or its sub-cellular localization. Finally, in
addition to the two reference transcripts, a large number (n ¼ 38)
of potential transcripts are described in Aceview (http://
www.humangenes.org/) based upon extensive alternative splicing
and promoter use. These remain to be validated, but if they are
genuine TCF4 transcripts, perturbation in splicing or regulation of
a minor transcript is a possible mechanism through which variation
at this locus might impact on disease.
In summary, the results of this study do not support the
hypothesis that the genome-wide significant SNP in the vicinity
of TCF4 exerts its effect either through cis-acting regulation of the
TCF4 transcript or via a common non-synonymous mutation. The
functional mechanism responsible for that association therefore
remains to be uncovered.
ACKNOWLEDGMENTS
We are indebted to all individuals who have participated in, or
helped with, our research. The research was supported by the
Medical Research Council (UK), The Wellcome Trust, and
NIMH (USA) CONTE: 2 P50 MH066392-05A1.
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