Neuregulin 1 transcripts are differentially expressed
in schizophrenia and regulated by 5? SNPs
associated with the disease
Amanda J. Law*†, Barbara K. Lipska‡, Cynthia Shannon Weickert‡, Thomas M. Hyde‡, Richard E. Straub‡,
Ryota Hashimoto‡, Paul J. Harrison*, Joel E. Kleinman‡, and Daniel R. Weinberger‡
*Department of Psychiatry, University of Oxford, Warneford Hospital, Oxford OX3 7JX, United Kingdom; and‡Clinical Brain Disorders Branch,
Intramural Research Program, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892-1385
Communicated by Gerald D. Fischbach, Columbia University College of Physicians and Surgeons, New York, NY, March 13, 2006 (received for review
September 16, 2005)
Genetic variation in neuregulin 1 (NRG1) is associated with schizo-
phrenia. The disease-associated SNPs are noncoding, and their
functional implications remain unknown. We hypothesized that
differential expression of the NRG1 gene explains its association to
the disease. We examined four of the disease-associated SNPs that
gene for their effects on mRNA abundance of NRG1 types I–IV in
human postmortem hippocampus. Diagnostic comparisons re-
vealed a 34% increase in type I mRNA in schizophrenia and an
interaction of diagnosis and genotype (SNP8NRG221132) on this
transcript. Of potentially greater interest, a single SNP within the
risk haplotype (SNP8NRG243177) and a 22-kb block of this core
haplotype are associated with mRNA expression for the novel type
IV isoform in patients and controls. Bioinformatic promoter anal-
yses indicate that both SNPs lead to a gain?loss of putative binding
sites for three transcription factors, serum response factor, myelin
transcription factor-1, and High Mobility Group Box Protein-1.
These data implicate variation in isoform expression as a molecular
genetics ? mRNA ? human postmortem brain ? hippocampus ? ErbB
been identified (1). Genomewide linkage studies and metaanalyses
locus (2–8). Extensive fine-mapping of the 8p locus, haplotype-
association analysis, and linkage disequilibrium (LD) tests subse-
roles in neurodevelopment and plasticity (9).
The NRG1 gene spans 1.2 Mb (6) and gives rise to many
structurally and functionally distinct isoforms, through alternative
promoter usage. These isoforms are divided into three classic
groups (9): type I (previously known as acetylcholine receptor
inducing activity, heregulin, or neu differentiation factor), type II
(glia growth factor) and type III (cysteine-rich domain containing),
which are based on distinct amino termini. All isoforms have a
bioactive EGF-like domain that is responsible for activation of
ErbB receptor tyrosine kinases (ErbB2–ErbB4). Additional NRG1
NRG1 types IV–VI in the human brain (10). No biological infor-
mation is available presently for these novel isoforms.
In the original report of association with schizophrenia in
an Icelandic population, Stefansson and colleagues (6) identi-
fied a ‘‘core at-risk haplotype’’ consisting of five SNPs
(SNP8NRG221132, SNP8NRG221533, SNP8NRG241930,
SNP8NRG243177, and SNP8NRG433E1006) and two microsatel-
lites covering the 5? end of the NRG1 gene and extending into the
second intron (hereafter referred to as the ‘‘deCODE haplotype’’).
Separate follow-up studies in Scottish, Irish, mixed United King-
dom, and Dutch populations confirmed the genetic association
between schizophrenia and NRG1 by using markers within the
chizophrenia is a complex, heritable psychiatric disorder. Re-
cently, several putative schizophrenia susceptibility genes have
same core haplotype (11–14) or with overlapping markers in the 5?
region (15, 16). Studies in four Asian populations also showed a
strong association between schizophrenia and NRG1 polymor-
results, not withstanding two negative studies (21, 22), provide
strong evidence that NRG1 is a schizophrenia-susceptibility gene.
Additional support for NRG1’s role in schizophrenia comes from
the phenotype of NRG1 and ErbB4 mutant mice (6, 23–25), which
exhibit behaviors similar to those of established rodent models of
Exactly how genetic variation in NRG1 impacts on disease
susceptibility remains uncertain because the SNPs associated with
schizophrenia are all noncoding, being either intronic, synonymous
exonic substitutions, or upstream of the transcription start site. It is
possible that an as-yet unknown (rare) coding mutation(s) exists,
but it is more probable that the noncoding SNPs themselves, or
other SNPs with which they are in LD, are functionally associated
with the disease. One plausible explanation is that the NRG1 SNPs
are regulatory and affect disease susceptibility by altering expres-
sion (via altering transcriptional activity, alternative splicing, or
protein and ultimately its function. Support for this hypothesis
comes from data demonstrating increased NRG1 type I mRNA in
the prefrontal cortex in schizophrenia (27) and from altered gene
in other brain diseases (1, 28, 29).
The aim of this study was to address whether disease-associated
polymorphic loci in the 5? upstream region of NRG1 modulate
NRG1 mRNA expression and contribute to its association with
schizophrenia. We performed a series of hypothesis-driven exper-
iments aimed at confirming previously published positive and
negative expression data in schizophrenia (i.e., elevated type I,
NRG1, and no change in type II or III (27) and to test specifically
the hypothesis that disease-associated SNPs in the original risk
haplotype influence expression of the novel type IV isoform based
on their physical proximity to its 5? regulatory region.
To test for differential expression of NRG1 in schizophrenia, we
examined mRNA abundance for NRG1 types I–IV in the human
hippocampus, a region prominently implicated in the pathogenesis
of schizophrenia (30) and in the neurobiology of NRG1 (31).
included 4 SNPs from the deCODE core haplotype (6), with each
SNP being tested individually for associations with NRG1 mRNA
levels in patients and controls. LD between SNPs was examined,
Conflict of interest statement: No conflicts declared.
lin 1; PMI, postmortem interval; SRF, serum response factor.
†To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2006 by The National Academy of Sciences of the USA
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April 25, 2006 ?
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with NRG1 mRNA abundance. Our results support our primary
hypotheses and indicate that the region of the gene implicated by
the core at-risk haplotype impacts on specific NRG1 isoforms and
interacts with their expression in schizophrenia.
NRG1 Isoform mRNA Expression in Schizophrenia. Normalized hip-
pocampal NRG1 type I mRNA expression levels were increased by
1; F (1, 85) ? 8.65; P ? 0.004). Similar significant findings were
observed in the less well matched full cohort (84 controls vs. 44
schizophrenics; data not shown). No significant differences were
II [F (1, 85) ? 0.10; P ? 0.74]; type III, [F (1, 85) ? 0.36; P ? 0.54];
type IV, [F (1, 68) ? 1.6; P ? 0.20}; 17 individuals were not
available for the type IV study because of a shortage of RNA].
Expression ratios for all NRG1 isoforms were calculated to inves-
tigate relative expression abnormalities, given previous reports of
altered isoform ratios in the dorsolateral prefrontal cortex in
schizophrenia (27). Type I NRG1 mRNA expression was increased
5.20; P ? 0.02], I?III [F (1, 85) 5.0; P ? 0.02], and I?IV [F (1, 68) ?
10.11; P ? 0.002]. No changes in any other isoform ratio were seen.
No significant differences were observed between diagnostic
groups for any of the housekeeping genes PBGD, [F (1, 85) ? 0.27;
P ? 0.6]; TBP, [F (1, 85) ? 0.16; P ? 0.21], and GUSB, [F (1, 68) ?
0.001; P ? 0.98]. Covariation for pH, postmortem interval (PMI),
and age was used in all analyses (see Supporting Text, which is
published as supporting information on the PNAS web site).
Effects of 5? SNPs on NRG1 Expression. The effect of SNPs on NRG1
type I-III isoform expression were examined in the whole cohort
(n ? 84 controls and n ? 44 schizophrenics). For type IV analysis,
24 individuals were not available for analysis because of a shortage
of the SNPs examined showed any effect on expression of type II
or type III isoforms.
Genetic Variation and Type I NRG1 mRNA. A genotype ? diagnosis
interaction [F (3, 122) ? 9.21; P ? 0.003] was found for
SNP8NRG221132 and type I NRG1 mRNA. There was no main
was significant in the controls alone (post hoc t test; P ? 0.003; Fig.
2) with individuals carrying the rare A (2) allele expressing higher
levels than individuals homozygous for G (1?1 genotype). The G
allele constitutes the risk allele in the deCODE haplotype (6, 11).
However, this pattern was not seen in schizophrenic patients who
tended in the opposite direction (Fig. 2). Schizophrenic patients
I NRG1 mRNA compared with control subjects homozygous for
the same allele (t test; P ? 0.001). To confirm these findings, we
genotyped SNP8NRG221132 in brain tissue from an earlier study
of a separate cohort, in whom NRG1 mRNA expression for types
I–III had been measured by identical quantitative RT-PCR meth-
ods (controls n ? 13, schizophrenics n ? 16; n ? 22 African
was found to be increased in the dorsolateral prefrontal cortex of
effect of SNP8NRG221132 genotype in the whole sample [F (3,
25) ? 15.17; P ? 0.0005] on prefrontal type I NRG1 mRNA, with
subjects carrying the A allele (n ? 4) again showing higher type I
NRG1 levels compared with homozygous G cases (n ? 25, data not
shown). A genotype ? diagnosis interaction also was found in this
cohort [F (3, 25) ? 16.26; P ? 0.0005], with schizophrenic patients
homozygous for the G allele (n ? 14) having greater expression of
type I NRG1 mRNA than control subjects (n ? 11) with the same
genotype (P ? 0.02). No other SNPs examined in the study were
associated with type I NRG1 expression.
Genetic Variation and Type IV NRG1 mRNA. Because ?10% of
individuals in the cohort were homozygous for the rare risk allele
(T) at SNP8NRG243177, a complete analysis was conducted based
on the three genotype groups (C?C, C?T, and T?T). We found a
main effect of genotype for SNP8NRG243177 on type IV NRG1
mRNA abundance in the whole sample [F (5, 98) ? 3.15; P ? 0.04;
Fig. 3A]. The data suggest an allele dose effect with individuals
heterozygous for the (T) risk allele (6, 11) having 21% more type
IV NRG1 mRNA than homozygous C?C individuals and individ-
types I–IV normalized to PBGD. (A–C) n ? 53 normal control subjects (NC) and
falling between the 25th and 75th percentiles. Bars outside the box represent
the SD. Bar inside represents the mean. Significant differences were found
between controls and patients for type I NRG1 expression.**, significant
differences (P ? 0.01)
NRG1 types I–IV mRNA expression in the hippocampus of schizo-
normal controls (NC) and schizophrenics (SZ). A significant interaction of
genotype and diagnosis was observed on normalized type I mRNA expression
expression in controls, with A allele carriers having increased levels compared
with homozygous G individuals (P ? 0.003). Schizophrenic patients homozy-
gous for the G allele had higher levels of type I mRNA compared with controls
with the same genotype (P ? 0.001). Two individuals were excluded from
analysis because of genotyping failure. Box represents the proportion of the
distribution falling between the 25th and 75th percentiles. Bars outside the
box represent the SD. The bar inside represents the mean.
Association between SNP8NRG122132 and type I NRG1 mRNA in
www.pnas.org?cgi?doi?10.1073?pnas.0602002103Law et al.
mRNA expression than homozygous C?C individuals. However,
the two homozygote groups to be significantly different in terms of
type IV abundance (post hoc t test; P ? 0.05; Fig. 3A). This effect
appeared more pronounced in the schizophrenia group alone (Fig.
3B), although no hint of a diagnosis ? genotype interaction was
observed. Of note, normal control individuals homozygous for the
T allele also showed the relatively greatest type IV NRG1 mRNA
expression (Fig. 3B).
Significant diagnosis ? genotype interactions were found be-
tween five of the six haplotype-tagging SNPs (htSNPs) and type IV
mRNA abundance (rs4268090, rs4298458, rs4452759, rs4733263,
and rs4476964) [range F (3, 100) ? 3.35–5.82; P ? 0.033–0.018]. A
trend for a main effect of genotype was observed for the htSNP,
rs4268090 [F (3, 100) ? 3.55; P ? 0.06]. Carrying the T (2) allele
at this SNP was associated with higher levels of type IV mRNA
compared with subjects homozygous for the C (1?1) allele.
Haplotype Analysis. Results of LD tests between pairs of all 10 5?
SNPs in African American and Caucasian individuals can be found
PNAS web site. The four markers chosen from the deCODE
Supporting Text). The frequencies for the four common haplotypes
comprised of the four deCODE SNPs are shown in Table 3, which
4 contains the specific alleles that form part of the deCODE
To test whether this four SNP risk haplotype (hap4) was asso-
ciated with NRG1 mRNA levels, we used SNPHAP (www-
gene.cimr.cam.ac.uk?clayton?software) to assign a diplotype (hap-
lotype pair) to each individual. We then compared hap4 carriers
non-hap4 individuals (diplotypes hap1?hap1, hap1?hap2, hap1?
of carrying the risk haplotype on NRG1 mRNA abundance.
ANOVA revealed a main effect of hap4 on type IV mRNA
abundance in the entire sample [F (3, 89) ? 3.38; P ? 0.04]. Hap4
carriers had 27% more type IV mRNA compared with non-hap4
individuals (Fig. 4A). This effect appeared more pronounced in the
schizophrenic patients, where a 53% increase in type IV NRG1
mRNA was seen in hap4 carriers compared to noncarriers; in
controls, only a 14% increase was observed (Fig. 4B). However, no
as a factor in the analysis because of the small number of African
American individuals carrying hap4. Hap4 showed no effect on the
expression of any of the other NRG1 isoforms.
Promoter Analysis Based on Transcription Factor Binding Sites. As a
SNPs in the NRG1 gene, we performed an analysis of putative
transcription factor binding sites by using MATINSPECTOR software
(Genomatix, Munich), a computational suite for promoter infor-
matics. Two SNPs in the haplotype were indicated to be contained
within transcriptional regulatory elements; notably, these elements
were the two SNPs that, as described above, were found to impact
upon expression of NRG1 isoforms, SNP8NRG221132, which is
associated with type I NRG1, and SNP8NRG243177, which is
associated with type IV NRG1. SNP8NRG221132 is within a
predicted transcription factor binding domain for serum response
factor (SRF), with the risk allele (G) abolishing SRF binding.
for myelin transcription factor 1. Carrying the risk allele (T) results
in a predicted loss of binding to both of these transcription factors
243177 and type IV NRG1 mRNA expression.
(A) In the whole cohort, a main effect of
genotype was observed (ANOVA; P ? 0.04)
An allele dose-dependent effect is sug-
NRG1 mRNA (P ? 0.05). (B) Data parsed by
diagnosis. No genotype ? diagnosis interac-
tion was observed.
Association between SNP8NRG-
type IV NRG1 mRNA. Individuals were divided
according to diplotype into two groups, non-
hap4 carriers (haplotypes 1?1; 1?2, 1?3, 2?2,
3?3, and 2?3) and hap4 carriers (haplotypes
1?4, 2?4, 3?4, and 4?4). A main effect of dip-
lotype was observed on normalized type IV
NRG1 mRNA levels (ANOVA; P ? 0.04). (A)
Individuals carrying the hap4 risk haplotype
had increased levels compared with individu-
als not carrying hap4. (B) Effect of diplotype
on type IV NRG1 mRNA levels in controls and
failure of genotyping at one or more of the
SNPs or low probability (?93%) of diplotype
assignment according to SNPHAP.
Association between diplotypes con-
Law et al.
April 25, 2006 ?
vol. 103 ?
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and the acquisition of the transcription factor binding site for High
in the study mapped to transcription factor binding domains.
Analysis of Negative SNP Controls. The two negative control SNPs
(rs10954867 and rs7005288) showed no association with any
NRG1 isoform in either controls or schizophrenic patients (all
P ? 0.2).
Regional Distribution of Hippocampal NRG1 mRNA in Schizophrenia.
Because no information is available regarding the distribution of
NRG1 in the human hippocampus, and this data was not provided
from the quantitative RT-PCR experiments, we examined NRG1
mRNA in the hippocampus in schizophrenia by using in situ
hybridization with a ‘‘pan’’ NRG1 probe (Supporting Text; see also
Fig. 5, which is published as supporting information on the PNAS
We have investigated the expression of NRG1 type I–IV mRNA
in the human hippocampus and examined the effects of schizo-
phrenia and disease-associated polymorphisms in the 5? up-
stream region on expression of these transcripts. We hypothe-
sized that the genetic association of NRG1 with schizophrenia is
mediated by altered expression of the gene based on the location
and noncoding nature of the disease-associated polymorphisms
and the fact that extensive sequencing of NRG1 has failed to
identify pathogenic coding mutations (6). We report three
principal findings: (i) up-regulation of type I expression in the
hippocampus in schizophrenia, (ii) association of type I expres-
sion with a single SNP residing in the original deCODE risk
haplotype, and (iii) association of type IV expression with a
single SNP and a four-marker haplotype representing the 5?
upstream region of the original at-risk haplotype associated with
schizophrenia. We provide evidence of association between
disease linked-variation in NRG1 and altered NRG1 isoform
expression in the brain, and we propose that altered transcript
regulation is a potential molecular mechanism behind the ge-
netic association of NRG1 with schizophrenia.
Our finding of increased type I mRNA NRG1 expression in the
hippocampus in schizophrenia replicates the finding in the dorso-
lateral prefrontal cortex of a smaller and separate brain series (27).
found in two separate brain regions in schizophrenia. In addition,
we also replicate the finding that type II and type III isoform
expression is unaltered in schizophrenia, suggesting that these
isoforms may not be directly relevant to the pathophysiology of the
disease. However, we did observe increases in the relative abun-
dance of type I to type II–IV, suggesting that the contribution of
indirectly compromised in patients with schizophrenia. At present,
it is unclear whether type I up-regulation in schizophrenia is
primary or secondary to other abnormalities in NRG1 isoform
When the four individual SNPs representing the 5? region of the
deCODE at-risk haplotype were tested for association with type I
SNPs. A diagnosis ? genotype interaction was observed at a single
SNP, SNP8NRG221132, and post hoc tests showed an effect of
genotype only in control subjects on type I mRNA abundance. In
a second independent cohort of brains in which increased type I
mRNA expression previously had been reported in the schizophre-
entire sample and again a genotype by diagnosis interaction. This
main effect of the genotype was not seen in the first cohort;
however, we note that the main effect in the entire sample is driven
primarily by the controls. The observations that the main effect of
risk haplotype had no effect on type I NRG1 expression, raise the
possibility that SNP8NRG221132 influences type I expression
support for the functional relevance of SNP8NRG221132 comes
from the bioinformatic promoter analysis that predicts the risk
allele (G) leads to a loss of binding for SRF. SRF is a transcription
factor that regulates the expression of genes encoding cytoskeletal
proteins, such as cofilin and actin (32), both of which have been
linked directly to NRG1s role in actin dynamics (33). The loss of
SRF binding in controls homozygous for the risk allele, therefore,
may be related to lower levels of type I mRNA transcription, as
reported here. The direct functional consequences of this SNP for
type I NRG1 transcriptional control remain difficult to predict
because the SNP resides 1 Mb upstream from the transcriptional
start site of type I. However, SNP8NRG221132 could conceivably
reside in a regulatory element of the gene, as is seen in other key
developmental genes where genomic regions harboring cis-
regulatory elements can be located as far as 1 Mb from the
transcription unit (34).
The interrelationship between SNP8NRG221132, type I NRG1
expression, and schizophrenia is somewhat more difficult to inter-
pret, because in contrast to the effect seen in controls, we did not
see a similar genotype effect in the patients. This issue is discussed
in Supporting Text, Discussion: Genetic Association and Type I
Expression in Schizophrenia).
In contrast to the type I finding, which is not manifestly related
to genetic variation in NRG1 associated with schizophrenia, the
association between both SNP8NRG243177 and the four-marker
at-risk haplotype with expression of a novel isoform of NRG1, type
IV, suggests that we may have identified a genetic mechanism and
ceptibility for schizophrenia. The risk allele of SNP8NRG243177
and the deCODE haplotype predicted higher levels of type IV
NRG1 expression in our entire sample. Analysis of the three
genotype groups for SNP8NRG243177 revealed that individuals
homozygous for the risk allele had the highest levels of type IV
expression, with evidence of an allele dose-dependant effect. This
observation appeared more pronounced in the patients, although
trends in the same direction were found in the normal controls
and no diagnosis ? genotype interaction was observed.
SNP8NRG243177 is the most 3? of the SNPs in the four-marker
deCODE haplotype and is located ?1.2 kb upstream of the
transcriptional start of type IV. Because none of the other single
SNPs in this haplotype were associated with type IV NRG1
expression, our results suggest that SNP8NRG243177 is a func-
tional polymorphic variant that regulates type IV NRG1 mRNA
levels or is in strong LD with a nearby functional mutation.
SNP8NRG243177 for gene regulation comes from the bioinfor-
matic prediction that this SNP determines a putative transcription
factor binding domain for SRF, myelin transcription factor 1, and
High Mobility Group Box Protein-1. Of note, SRF and myelin
transcription factor 1 play critical roles in neuronal migration,
synaptic plasticity, and oligodendrocyte proliferation and survival,
respectively, providing a striking molecular convergence with cur-
rent hypotheses regarding the neurobiology of schizophrenia and
the potential role of NRG1 (35). However, we do not know which,
if any, of these changes in transcription factor binding sites might
mediate the association between SNP8NRG243177 and type IV
Group Box Protein-1, an abundant chromatin-binding protein,
sample, acquisition of two High Mobility Group Box Protein-1-
binding motifs (i.e., homozygosity for the risk allele) was associated
with significantly elevated type IV NRG1 expression, whereas
acquisition of one (i.e., heterozygosity for the risk allele) was not.
This observation suggests (i) that this binding site may potentiate
www.pnas.org?cgi?doi?10.1073?pnas.0602002103 Law et al.
type IV NRG1 transcription (and that SRF binding is necessary for
optimal levels of type IV transcription) and 2) that this effect may
levels in schizophrenia suggest that altered type IV, unlike type I,
is not a general characteristic of the disease state, per se. Indeed, if
altered NRG1 type IV expression is part of the genetic architecture
an effect at the general population level, assuming that the at-risk
haplotype is relevant for, at most, 10% of cases. Furthermore, our
finding that the deCODE risk haplotype is associated specifically
with type IV NRG1 expression argues that the clinical association
with NRG1 is based on this molecular effect.
We further report association of type IV NRG1 mRNA in
schizophrenia with five additional htSNPs, which span a 17-kb gap
between the four SNPs from the deCODE haplotype. To our
knowledge, these SNPs have not been tested for association with
schizophrenia in the same clinical samples in which the deCODE
SNPs were positive. We genotyped these SNPs to address the
information regarding genetic diversity in our sample. None of
these SNPs showed main effects, and their association with NRG1
type IV expression in schizophrenia is likely via LD with
In our sample, the deCODE risk haplotype, which we termed
hap4, was present in both Caucasian and African American pop-
degree of LD across this region of the gene suggests that, at least
in Caucasians, it has undergone very little recombination (37).
Furthermore, the region is highly conserved between species,
including chimpanzee, dog, mouse, and rat, suggesting that this
regulation of NRG1 (38). We found no evidence to suggest that the
frequency of the deCODE haplotype was higher in our patient
population compared with controls, but our sample is too small to
meaningfully test for association with clinical phenotype. Of note,
we observed that the frequency of hap2 was somewhat greater in
the African American patients (34%) compared with African
American controls (25%), suggesting that in different ethnic
groups, different haplotypes in the same region of the gene may be
associated with schizophrenia. However, because of the small
sample size involved, conclusions are limited. Interestingly, hap2 in
the African American sample contains the same allele at
SNP8NRG243177 as hap4.
In the original report by Stefansson et al. (6), association in
Icelandic families was mapped to a seven-marker haplotype span-
ning a 270-kb LD block starting at SNP8NRG221132 and ending
with a synonymous SNP in exon two (SNP8NRG433E1006) and
two microsatellites in the second intron (478N14-848 and 420M9-
13950). Evidence of association to this region of the gene in other
samples has been primarily to SNPs at the 5? end of this haplotype,
encompassing the SNPs typed in this study. Thus, although we
cannot exclude the possibility that the causative mutation(s) ac-
typed SNPs, we tend to doubt this possibility for three reasons: (i)
the exon 2 SNP and the microsatellites typed in the deCODE
haplotype have not shown single point (pairwise) association with
schizophrenia in any study (6, 18, 19, 39), in contrast to the four
SNPs tested here, (ii) the physical location of SNP8NRG243177
(i.e., ?1,200 bases upstream from the exon 1 start site) makes it a
region for type IV, and (ii) this SNP is in a putative functional
transcription factor binding domain.
The known biological functions of NRG1 (9) fit well with current
hypotheses regarding the neurobiology of schizophrenia (35), in-
cluding regulation of synaptogenesis, in vivo synaptic transmission,
long-term potentiation, activity-dependent synaptic plasticity, and
neuronal migration as well as neurotransmitter function (NMDA,
GABA, ?-7, and dopamine) and oligodendrocyte biology, all of
which are proposed to interact or be altered in schizophrenia (30,
40). Of particular relevance is the recent finding that NRG1
down-regulates NMDA-receptor currents in prefrontal cortical
pyramidal neurons and slices (41). These data suggest that in-
creased expression of NRG1 type I or IV would translate into
neurotransmitter hypotheses of schizophrenia.
Finally, it should be noted that we have performed a number of
tests in this study, and correction for multiple testing was not
performed. Correction for random effects, such as Bonferroni
correction, would be an excessively conservative approach, partic-
location of the SNPs) of four SNPs and a single haplotype com-
prised of these SNPs. Because the SNPs are in moderate LD, the
degree of independence between markers is low and, therefore,
correcting for multiple testing would result in a high type II error
rate. The prior probability and the predictable association between
the deCODE haplotype and expression of NRG1 isoforms (espe-
cially type IV, which is its immediate physical neighbor) combined
with the LD between SNPs in this haplotype makes statistical
correction for these comparisons inappropriate. Nevertheless, our
finding regarding type IV expression and the deCODE haplotype
and SNP8NRG243177 requires independent replication.
In summary, we provide evidence of splice variant-specific
alterations of NRG1 gene expression in schizophrenia and dem-
onstrate that disease-associated polymorphisms in a 5? regulatory
region of NRG1 are associated with differential NRG1 isoform
expression. We suggest that the mechanism behind the clinical
association of NRG1 with schizophrenia is altered transcriptional
regulation, which modifies, probably to a small degree and in an
neural development and plasticity. Such alterations may compro-
to the genetic risk architecture for the disease.
Materials and Methods
Human Postmortem Tissue. Postmortem hippocampal tissue was
collected at the Clinical Brain Disorders Branch, National Institute
of Mental Health, from 84 normal controls (22 females?62 males,
mean age 40.5 ? (SD) 15.4 years, PMI 30.7 ? 13.9 h, pH 6.59 ?
0.32) and 44 schizophrenic patients (15 females?29 males, 24
African Americans?20 Caucasians, mean age 49.7 ? 17.2 years,
PMI 36.3 ? 17.7 h, pH 6.48 ? 0.28). This whole cohort was used
for the analysis of effects of genetic variation on NRG1 isoform
expression. The different genotype groups in this cohort did not
differ on any of the potential variables that affect gene expression
in the human postmortem brain (i.e., age, PMI, pH, and age).
Because the diagnostic groups in the whole cohort were not
perfectly matched for these variables, we selected a subcohort of 53
controls (17 females?36 males, 31 African Americans?17 Cauca-
13.7, pH 6.53 ? 0.24) and 38 schizophrenic individuals (12 fe-
males?26 males, 18 African Americans?19 Caucasians?1 Hispanic
0.26), matched for these potential confounding variables. This
levels. Details of brain collection, neuroleptic medication history,
and RNA extraction are described in Supporting Text.
Oligonucleotide and Primer Design. Primer and probe designs for
NRG1 types I–III were as described in ref. 27. Details of type IV
Law et al.
April 25, 2006 ?
vol. 103 ?
no. 17 ?
Quantitative Real-Time RT-PCR. NRG1 mRNA expression levels
were measured by quantitative RT-PCR by using an ABI Prism
7900 sequence detection system with a 384-well format (Applied
Biosystems) as described (see Supporting Text). Our primary data
analysis is based on normalization of NRG1 mRNAs to an endog-
enous control gene, because it accounts for variability in the initial
concentration and quality of total RNA and in the conversion
efficiency of the reverse transcription reaction (42). Optimal nor-
malization of a target mRNA to an endogenous control gene
requires that the two transcripts have similar expression levels and
that the control gene expression levels do not differ between the
comparison groups. Based on this finding, PBGD was considered
the most reliable for normalization here. Similar results were,
however, obtained with normalization to TBP or GUSB (data not
NRG1 Genotype Determination. DNA was extracted from cerebel-
lar tissue by using a standard protocol supplied by PUREGENE
(Gentra Systems). We genotyped four SNPS from the de-
CODE core haplotype (SNP8NRG221132, SNP8NRG221533,
SNP8NRG241930, and SNP8NRG243177) and selected six addi-
tional SNPs (rs10096573, rs4268090, rs4298458, rs4452759,
rs4733263, and rs4476964; Table 5 and Fig. 6, which are published
as supporting material on the PNAS web site) from HAPMAP
(www.hapmap.org) based on designation of these as htSNPS by
using HAPLOVIEW (www.broad.mit.edu?mpg?haploview). The ad-
ditional markers define the common haplotypes in an LD block
containing a 22-kb region upstream of the first exon in type IV
NRG1, which includes the four most 5? SNPs of the deCODE core
risk haplotype. These six htSNPs were chosen to maximize genetic
coverage because they are highly informative tags for the common
haplotypes in this region of the gene.
Two SNPs at the 3? end of NRG1 were selected from the dbSNP
database as negative control genotypes (rs10954867 and
rs7005288). These SNPs previously have not been associated with
schizophrenia, are not in known regulatory domains, and were
included in the analysis as a control for random statistical effects.
Genotyping was performed by using the Taqman 5? exonuclease
allelic discrimination assay (details available upon request). Geno-
type reproducibility was routinely assessed by regenotyping all
samples for selected SNPs and was generally ?99%. LD between
5? SNPs was determined by using the program LDMAX/GOLD (43).
The program SNPHAP written by David Clayton (version 1.0) was
used to calculate haplotype frequencies and to assign diplotypes to
variables were performed for all subjects by using Spearman’s
correlations. Correlations of mRNA levels with neuroleptic med-
ication (lifetime neuroleptic exposure, daily dose, and final neuro-
leptic dose) were investigated in the schizophrenic cohort. Primary
planned comparisons between diagnostic groups were made by
using univariate ANCOVA for each mRNA with diagnosis as the
independent variable and age, pH, and PMI as covariates. Effects
of genetic variation on NRG1 mRNA expression were examined by
Primary comparisons examined the effects of four SNPs and the
core haplotype on type I–IV expression in patients and controls.
Secondary, post hoc analyses included examination of the 6
htSNPS, where warranted. Where there was a significant geno-
type ? diagnosis interaction, individual group post hoc tests were
examined as part of the standard ANOVA readout. Analysis of the
effects of race was restricted to African American and Caucasian
individuals because of the small sample size in other ethnic groups.
The genotype groups did not differ on any of the demographic
variables, and no correlations were seen between NRG1 isoform
expression, age, pH, or PMI in the different genotype groups;
therefore straight ANOVAs are reported for the effects of geno-
type, but the statistical results were not changed when covariates
were included. To increase power for statistical analyses of SNPs
with minor allele frequencies ?10%, we grouped individuals het-
erozygous and homozygous for the rare allele. Examination of all
three genotype groups was conducted when the minor allele
frequency was ?10%. All experiments were conducted blind to
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