NOS1 Influences Verbal Intelligence And Working
Memory In Both Patients With Schizophrenia And
Healthy Controls.
Authors
Gary Donohoe D.Clin.Psych, Ph.D.1,2, James Walters MRCPsych, BM3, Derek W.
Morris Ph.D.1, Emma M. Quinn M.Sc.1, Róisín Judge B.A.1, Nadine Norton Ph.D.3,
Ina Giegling Ph.D.4, Annette M. Hartmann Ph.D.4, Hans-Jürgen Möller M.D.5,
Pierandrea Muglia M.D.6, Hywel Williams Ph.D.3, Valentina Moskvina Ph.D.3,
Rosemary Peel M.Sc.1, Therese O’Donoghue M.Sc.1,2, Michael J Owen FRCPsych,
Ph.D.3, Michael C. O’Donovan FRCPsych, Ph.D.3, Michael Gill MRCPsych., M.D.
1,2
, Dan Rujescu M.D.4, Aiden Corvin MRCPsych., Ph.D.1,2.
Affiliations
1
Neuropsychiatric Genetics Research Group, Dept of Psychiatry & Institute of
Molecular Medicine, Trinity College Dublin, Ireland.
2
Trinity Institute of Neuroscience, Trinity College Dublin
3
Department of Psychological Medicine, School of Medicine, Cardiff University,
Heath Park, Cardiff, UK.
4
Molecular and Clinical Neurobiology, Ludwig-Maximilians-University, Munich,
Germany.
5
Department of Psychiatry, Ludwig-Maximilians-University, Munich, Germany.
6
Medical Genetics, GlaxoSmithKline R&D, Verona, Italy.
Address for correspondence
Dr. Gary Donohoe.
Neuropsychiatric Genetics Research Group,
Dept of Psychiatry,
Trinity Health Sciences Building,
St. James’s Hospital,
Dublin 8.
Telephone
+353 1 896 2467
Fax
+353 1 896 3405
E-mail
Gary.Donohoe@tcd.ie
Word count: 4485
Number of Tables: 2
Number of Figures: 2
Supplemental Files: 1
ABSTRACT
Background: Human and animal studies have implicated the gene NOS1 in both
cognition and schizophrenia susceptibility. Objectives: To investigate whether a
potential schizophrenia risk SNP (rs6490121) identified in a recent genome-wide
association study (O’Donovan et al., PMID 18677311) negatively influences
cognition in patients with schizophrenia and healthy controls. Design: A comparison
of both cases and controls grouped according to NOS1 genotype (GG v AG v AA) on
selected measures of cognition in two independent samples. Setting: Unrelated
patients from general adult psychiatric inpatient and outpatient services and unrelated
healthy volunteers from the general population were ascertained. Participants:
Patients with DSM-IV diagnosed schizophrenia and healthy controls from
independent samples of Irish (n=349 cases and n=230 controls) and German (n=232
cases and n=1344 controls) nationality. Method: We tested for association between
NOS1 rs6490121 and cognitive functions known to be impaired in schizophrenia (IQ,
episodic memory, working memory, and attentional control) in the Irish sample. We
then sought to replicate significant results in the German sample. Results: A main
effect of NOS1 genotype on verbal IQ and working memory was observed in Irish
samples where the homozygous carriers of the schizophrenia risk ‘G’ risk allele
performed poorly compared with the other genotype groups. These findings replicated
in the German sample, again with the GG genotype carriers performing below other
genotype groups. Post-hoc Analysis of additional IQ measures (Full scale and
Performance IQ) in the German sample revealed that NOS1 GG carriers
underperformed on these measures also. Discussion: NOS1 is associated with
clinically significant variation in cognitive. Whether this is a mechanism by which SZ
risk is increased (e.g. via an influence on cognitive reserve) is yet to be confirmed.
Introduction
Schizophrenia (SZ) is a disorder of substantial heritability (~80%), but uncertain
underlying pathophysiology (1). Despite some progress, most of the variance in SZ
susceptibility attributable to genetic factors remains to be identified (2). A number of
recent studies, exploiting high-throughput technologies investigating structural and
genomic sequence variation in large SZ samples, have identified more consistent
support for several susceptibility loci (3-5). As genomic data emerges, an important
question is how these variants increase disease liability, and whether they are only
relevant to the neurobiology of schizophrenia, or to neurodevelopment more generally
(6-7).
One putative schizophrenia risk gene is Nitric Oxide Synthase (NOS1, also known as
neuronal NOS or nNOS; OMIM:163731), which maps to chromosome 12q24 and
synthesises nitric oxide (NO) in both the central and peripheral nervous system. NO is
a highly reactive messenger molecule, which diffuses freely across membranes
stimulating guanylyl cyclase and modifying protein structure with multiple roles in
immune, cardiac and neurological function. NO is produced by different nitric
oxidase synthetase (NOS) enzymes including neuronal NOS and transported to
different cellular compartments by adaptor proteins to minimize non-specific
interactions.
Transcription of NOS1 involves 12 alternate untranslated first exons and coding
regions consisting of 28 exons covering 240KB. Following modest linkage support to
the NOS1 region (8-11), four of five published NOS1 candidate gene association
studies suggest evidence of association with SZ (12-15) with the exception being Liou
et al (16). Moreover, in their SZ genome wide association study (GWAS) of 479 cases
and 2937 controls, O’Donovan and colleagues (4) identified a single nucleotide
polymorphism (SNP) at the NOS1 locus (rs6490121) as being one of twelve SNPs
with strong initial statistical evidence for association (p=9.82 X 10-6). The same allele
at this SNP was significantly associated in a replication sample of 1664 cases and
3541 controls of European Ancestry, but not in a sample of mixed European and
Asian ancestry (4).
Functional evidence for involvement of NO in SZ comes from studies showing
abnormal distribution of nitrinergic neurons in frontal and temporal lobes in SZ (2324); increased NO metabolites in the serum of SZ patients (25-27); and post mortem
increased NOS1 mRNA in prefrontal cortex of patients (28). Neurally generated NO
displays many of the properties of a neurotransmitter and is involved in processes of
potential relevance to SZ including synaptogenesis, long term potentiation,
neurotransmitter release, synaptic plasticity, blood flow and cell death (21-22). These
functions are dependent on the delivery and localization of NOS1 to multiple
intracellular locations by specific adaptor proteins. Presynaptically, NOS1 is linked to
Nitric oxide synthase 1 (neuronal) adaptor protein (NOS1AP) (OMIM: 605551) and
forms a complex with Synapsin, which is involved in the assembly of synaptic vesicle
clusters and synaptogenesis. The genes NOS1AP and SYN2 (OMIM: 600755) have
both been implicated in SZ by genetic association studies (17-19; 88-89). Postsynaptically, NOS1 is coupled to N-methyl-D-aspartate (NMDA) receptors through
Post Synaptic Density-95 (PSD95), representing another potential mechanism for
involvement in SZ susceptibility. Molecular pathway analysis of structural variants
implicated in SZ by Walsh et al (20) identified a significant excess of disrupted genes
involving the NO signalling pathway. Taken together, these studies provide support
for the involvement of NO signalling in SZ.
In mouse models, NOS1 knock-outs have repeatedly been associated with variance in
cognition (29-30). Notably, the PCP-induced cognitive and behavioural deficits that
model SZ symptoms (including pre-pulse inhibition, habituation of acoustic startle,
latent inhibition, spatial learning, spatial reference memory, and working memory),
can all be prevented by interfering with the production of NO (31-40). In patients with
SZ, Reif et al. (41) report that two of four genetic markers tested at the NOS1 loci,
were associated with variance in performance on a measure of pre-frontal function
(the Continuous Performance Task) in terms of behavioural performance, P300 peak
amplitude and response latency. Whether this represents a specific or more general
effect on cognition, or impacts cognition in the general population is unknown.
The purpose of this study was to investigate the impact of NOS1 on cognition in SZ
and control populations. We selected a single SNP at NOS1 (rs6490121) from the
initial GWAS study by O’Donovan et al. (4) to which our cases had contributed as
replication samples. We chose this SNP alone as the only SNP in O’Donovan et al.
(4) that surpassed a significance threshold of p<10-5, and replicated with the same
allele “G” in the European sample. We hypothesised that the genotypes carrying the
risk allele would be associated with poorer performance on a selected set of cognitive
domains typically impaired in SZ, namely IQ, episodic and working memory, and
attentional control (42).
Method
Sample characteristics
Irish patient and control samples (Discovery samples): This sample consisted of 349
cases and 230 controls. Ninety one of the case participants were genotyped as part of
the prior GWAS study (4), the remaining case participants and all control samples
were independent of that study. Cases consisted of clinically stable patients with a
DSM-IV diagnosis of SZ (n=294) or schizoaffective disorder (n=55) recruited from
five sites across Ireland. Inclusion criteria required that participants were aged 18 to
65 years, had no history of co-morbid psychiatric disorder, substance abuse in the
preceding six months, or prior head injury with loss of consciousness or a history of
seizures. Diagnosis was confirmed by trained psychiatrists using the Structured
Clinical Interview for DSM-IV Axis 1 Diagnoses (SCID; 43). Additional diagnostic
details and clinical sample characteristics ascertained at time of interview including
symptom severity (SANS/SANS; 44-45) and medication dosage are detailed
elsewhere (46).
The healthy control sample was recruited on the basis of responses to local media
advertisements. Control participants were only included if they were aged between 18
and 65 and satisfied, based on clinical interview, the criteria of having no history of
major mental health problems, intellectual disability or acquired brain injury, and no
history of substance misuse in the preceding six months based on self report. Control
participants were also excluded from the study if they reported having a 1st degree
relative with a history of psychosis. All patients and control assessments were
conducted in accordance with the relevant ethics committees’ approval from each
participating site. All patients and controls were of Irish ancestry (i.e. four
grandparents born in Ireland) and all provided written informed consent.
German patient and control samples (Replication sample)
The German sample consisted of 240 clinically stable patients with a DSM-IV
diagnosis of SZ and 1344 healthy controls, all of whom were genotyped as part of the
previous study (4). Patients were ascertained from mental health services in the
Munich area and all participants provided written informed consent. Inclusion criteria
were a diagnosis of SZ (over 6 month symptom duration) and age 18-65. Exclusion
criteria included a history of head injury or neurological diseases. Detailed medical
and psychiatric histories were collected, including a clinical interview using the
Structured Clinical Interview for DSM-IV (SCID; 43), to evaluate lifetime Axis I and
II diagnoses. Four physicians and one psychologist rated the SCID interviews and all
measurements were double-rated by a senior researcher. In cases, 68% were of strict
German descent (all 4 grandparents born in Germany) and the other 32% were
German Caucasian. Of the 240 participants 216 completed the full WAIS-R
assessment and 232 a further comprehensive neuropsychological battery.
Healthy control participants of German descent (all 4 grandparents German) were
randomly selected from the general population of Munich, Germany, and contacted
by mail. Control patients for this study were only included if aged between 18 and 65.
To exclude subjects with central neurological diseases and psychotic disorders or
subjects who had first-degree relatives with psychotic disorders, several screenings
were conducted before the volunteers were enrolled in the study. First, subjects who
responded were initially screened by phone for the absence of neuropsychiatric
disorders. Second, detailed medical and psychiatric histories were assessed for both
themselves and their first-degree relatives by using a semi-structured interview. Third,
if no exclusion criteria were fulfilled, they were invited to a comprehensive interview
including the Structured Clinical Interview for DSM-IV (SCID I and SCID II; 43,47)
to validate the absence of any lifetime psychotic disorder. Additionally, the Family
History Assessment Module (48) was conducted to exclude psychotic disorders
among their first-degree relatives. A neurological examination was also conducted to
exclude subjects with current CNS impairment. In the case of volunteers older than 60
years, the Mini Mental Status Test (49) was performed to exclude subjects with
possible cognitive impairment. All of the 1344 control participants completed the full
WAIS-R assessment and 364 completed tests from a further extensive
neuropsychological battery.
Cognitive assessment
This study was designed so that identical, or near identical, tests of the phenotypes of
general cognitive function (IQ), episodic memory, working memory, and attention
were used for both the Irish discovery samples and the German replication samples.
Only one verbal and one visuo-spatial measure for each cognitive function was tested
in the Irish samples. Replication in the German sample was then sought for the
cognitive functions showing significant association in the discovery sample.
General cognitive functioning (IQ) was measured in the Irish sample using selected
subtests (Vocabulary, Similarities, Block Design and Matrix Reasoning) from the
Wechsler Adult Intelligence Scale, 3rd edition (WAIS-III; 50), yielding a full scale,
verbal and performance IQ. For the German sample, IQ was indexed by the German
version of the Wechsler Adult Intelligence Scale, revised edition (51) using all
available 11 verbal/performance subtests (Vocabulary, comprehension, information,
digit span, arithmetic, similarities, block design, picture completion, picture
arrangement, object assembly, digit symbol coding). Verbal and visual Episodic
memory was assessed in the Irish samples using the Logical memory and Faces
subtests respectively from the Wechsler Memory Scale, 3rd edition (WMS-III; 52)
and in the German sample using the Logical memory and Visual memory score from
the German version of the Wechsler Memory Scale- Revised (53, 54). Verbal and
spatial Working memory was assessed in the Irish samples using the Wechsler Letter
Number Sequencing task (WMS-III; 52) and the Spatial working memory task from
the Cambridge Automated Neuropsychological Test Battery (CANTAB SWM; 55). In
the German samples, working memory was measured using the composite Digit Span
and Spatial Span score from the WMS-R and the N-back task (NIMH-version, 56).
Attentional control was assessed in the Irish sample using the Continuous
performance task, identical pair’s version (CPT-IP, 57) and in the German sample
using the Continuous performance task 3-7 version (58). Full descriptions of all
memory and attention tasks are provided in Supplemental file 3.
Genotyping of samples
The SNP rs6490121 was genotyped in the German sample and a proportion of the
Irish sample using the Sequenom iPLEX Gold system (further details are provided in
(4)). The call rate for the iPLEX genotyping was >99% and all Irish and German case
and control samples were in Hardy-Weinberg Equilibrium (HWE; p>0.05). The
remainder of the Irish sample (n=529) was genotyped using a Taqman® SNP
Genotyping Assay on a 7900HT Sequence Detection System (Applied Biosystems).
The call rate for the Taqman genotyping was >95% and both case and control samples
were in HWE (p>0.05). Along with the Irish samples, a number of HapMap CEU
DNA samples (www.hapmap.org) were genotyped for rs6490121 for quality control
purposes and were all found to be concordant with available online HapMap data for
this SNP.
Statistical analyses
Association between NOS1 rs6490121 and the cognitive intermediate phenotypes of
general cognitive function, episodic memory, working memory, and attentional
control was tested using a general factorial design in SPSS 14 (SPSS, 2005). In the
original paper identifying this variant there was no difference in genotypic versus
allelic model; as there was no evidence on which to test a specific dominant or
recessive model, and as sample sizes allowed, our analysis was based on a
comparison of all three genotype groups. NOS1 genotype (GG versus AG versus AA)
and diagnosis (cases versus controls) were entered as fixed effects. Scores for each
neuropsychological subtest were entered as the dependent variables, with age and
gender included as covariates as appropriate.
Results
Clinical and demographic characteristics by genotype for each sample appear in table
1. For the Discovery control sample, NOS1 genotypes were not associated with age,
gender, or years in education. For the patient group, AA genotype carriers were
observed to be older than the AG by an average of four years (t=-2.94; uncorrected
p=.026); the GG group did not differ significantly in age from the other genotype
groups. To investigate whether possible differences in symptom severity rating
between genotype groups might influence cognitive performance in cases we
conducted a principal components analysis with varimax rotation of all SAPS and
SANS yielding three factors of positive, negative and disorganised symptoms (see
table 1 and supplemental table 1). We did not observe any differences on any of the
symptom factors associated with genotype. Neither did we observe difference in
medication dosage associated with NOS1 genotype (all p values >0.05). A similar
analysis of PANSS scores in the German sample also failed to identify any
differences in symptom severity associated with NOS1; medication dosage was
similarly non-significant (all p values >.05).
TABLE 1 HERE
NOS1 and general cognitive ability
Analysis of verbal IQ in the Irish sample revealed a significant overall effect of
genotype (see table 2). Tukey post hoc analysis revealed that this effect was driven by
differences between GG and AA groups (t=-6.36; p=.04). An effect size estimate
(partial ή2) suggests that genotype explains ~2% in cases and ~3.5% in controls. In
clinical terms, carriers of the risk GG allele performed on average 5-6 standard score
verbal IQ points below carriers of AG and AA genotypes (see Figure 1). No genotype
by diagnostic group interaction was observed (even though, as expected, cases
performed significantly below controls on this and all other cognitive tests, see Table
2). Although the same general pattern of results were observed for performance and
full scale IQ scores, these differences were not significant (p>.05).
TABLE 2 HERE
Analysis of Verbal IQ in the German sample also revealed a significant overall effect
of genotype (see table 2), with carriers of the risk GG allele performing significantly
below AA and AG carriers. Based on post hoc analysis, the largest difference was
between the GG and AG genotype groups (2.87; p=.012), with the difference between
GG and AA groups just failing to achieve significance (t=2.34; p=.051). In this
sample an interaction effect with affected status was also observed (F=11.51;
p<.00001); as illustrated in Figure 1, the association with genotype appears to be
strongest in cases. An effect size estimate (partial ή2) suggests that genotype explains
~5% of variance in Verbal IQ in cases and 0.2% in controls.
FIGURE 1 HERE
Following this replication of association between NOS1 genotype and Verbal IQ, we
investigated post-hoc whether NOS1 was also associated with the two other indices of
IQ in the German sample – Performance and Full scale IQ. A similar pattern of
differences associated with genotype was observed (see Table 2). Genotype by
diagnosis interactions were observed for both scores (Performance IQ: F=11.52;
p<.0001; Full scale IQ: 8.95; p=.0002), such that the effect of genotype was apparent
in cases only (see Figure 1). For cases the estimate of effect size was 6.9% for
Performance IQ and 6.4% for Full scale IQ.
As an additional secondary analysis, we further sought to determine whether the
overall effect of genotype was comparable when cases and controls were considered
separately. For the Irish samples, Verbal IQ was not significantly associated with
NOS1 when cases and controls were considered individually (P>.05), suggesting
insufficient power to analyse these samples separately. In the German sample, the GG
genotype was associated with significantly poorer verbal IQ in patients (F=5.54;
p=.005) but not controls; this pattern of association in patients but not controls was
also observed for Performance IQ in patients (F=7.84; p=.001) and Full scale IQ in
patients (F=7.26; p=.001).
NOS1 and working memory performance
For working memory, we observed a significant difference associated with genotype
in verbal working memory as measured in the Irish samples by scores on the WMS
Letter number sequencing task (see Table 2). While the association was significant as
a main effect (F=4.02; 0=.02), as figure 2 illustrates, and a statistical interaction of
genotype by diagnosis confirms, this association is clearly been driven by differences
in the control group. For the control group, the GG risk allele carriers performed 3
scaled score points (1 standard deviation based on norms for the test) lower than AA
on this measure of verbal working memory, with the between group difference
significant between GG carriers and both other genotype groups (GG<AG: t=-2.21;
0=.013; GG<AA: t=-3.05; p=.0002). A significant, main effect of genotype was also
observed with the spatial working memory task used (see Table 2), in the absence of
an interaction with diagnosis. Post hoc analysis reveals the difference as largest
between the AG and AA groups (t=.31; p=.02); this is illustrated in Figure 3.
Using two different measures of working memory in the German sample, the GG risk
allele was again associated with poorer performance on both measures (see Table 2).
While association with genotype was observed as a main effect, in the absence of a
statistical interaction with diagnosis, the effect size appeared slightly larger in cases
than controls (N-back partial ή2 =2.2% versus 1.1%).
We again sought to determine whether the overall effect of genotype was comparable
when cases and controls were considered separately. For the Irish samples,
differences on both working memory tasks were associated with NOS1 genotype in
controls (LN sequence: F2,163=9.02; p=.0002; SWM task F2,163=4.99; p=.008) but not
patients (P>.05). When the German case and control samples were considered
separately, the association was seen in the patient group but not the control group on
the Wechsler working memory score (F=5.49; p=.005); for the N-back task the effects
of genotype were no longer apparent when cases and controls were considered
separately.
NOS1 genotype and memory and attentional control
In the Irish sample no differences associated with NOS1 genotype was observed on
either the verbal or nonverbal memory tests used (the Logical memory and CANTAB
PAL tests respectively). Similarly, when the effects of NOS1 on attention were
investigated using the IDED task (scores for block 8) and the CPT-IP task (using d’
values for each of the three conditions) no significant associations were observed. As
no association with NOS1 genotype was observed in the Irish sample (all P>.05) we
did not investigate these aspects of cognition in the German samples.
Discussion
Variants at NOS1 have previously been associated with both increased SZ risk and
variation in cognitive performance. The involvement of NOS1 in cognition is well
established from animal models, with the NMDA-NO-cGMP signaling cascade being
linked to neurotransmitter release and synaptic plasticity in multiple genetic and
pharmacological studies (84). From the known biology of SZ, NOS-driven negative
feedback on NMDA receptor function and inhibition of synaptic reuptake of
dopamine positions it at the crossroads between two messenger systems whose mutual
regulation is a leading hypothesis for SZ (21).
This study shows that a NOS1 SNP, identified as a putative risk variant by a recent SZ
GWAS study, is associated with variation in verbal intelligence and working memory
in two large independent samples. Significant issues for studies addressing the
cognitive effects of previously identified risk variants in SZ have included small
sample size, lack of a suitable replication sample within the same study design, and a
heavy multiple testing burden. One approach to the issue of multiple testing is
statistical (e.g. Bonferroni) correction; however, the expected effects size based on
previous molecular studies of cognition, and the non-independence of measures of
cognition, serve to limit the value of these statistical approaches. Instead, our
approach was to seek independent replication of significant results using two large
independent case and control samples both of which contributed to the original
identification of the NOS1 rs6490121 variant studied here (4). Secondly, only a tightly
selected group of cognitive functions (IQ, memory, and attention), each measuring a
core cognitive deficit in schizophrenia, were selected for analysis on the basis of their
availability in both samples. Finally, this study was based on a single associated allele
and results obtained were in the same expected direction in both samples, with
homozygous G carriers - the associated allele in the original GWAS study performing more poorly.
The utility of the cognitive intermediate phenotypes approach in schizophrenia has
been well articulated as potentially representing less complex genetic architecture
than the broader disease phenotype, often shared by unaffected relatives, and thus
capable of increasing power to detect genetic effects (83). While verbal IQ and
working memory are themselves complex processes, likely to be influenced by many
genetic variants (e.g. DTNBP1; 60-62), the observed impact of NOS1 genotype on
cognition is not simply statistically significant. In both samples the size of the
association for verbal IQ in clinical terms is between 0.3 and 0.5 standard deviations.
For WM memory the differences between genotype groups represent a difference of
approximately 1 standard deviation. By comparison with findings of association
between cognitive performance and other gene variants, the size of effect observed is
considerably higher than that reported in the recent meta-analysis of COMT and IQ
(82), but comparable to reports of the association between Dysbindin and ‘g’ in
patients and controls (60) and spatial working memory in patients (61). While the
overall pattern was the same across the Irish and German samples, the effect size
varied between patients and healthy controls. Such differences may reflect differences
related to sample size or ascertainment strategy (for example, the somewhat better
than expected overall performance of German patients and Irish controls,
respectively).
That NOS1 genotype was associated with both verbal IQ and working memory but not
attentional control is noteworthy. We have previously argued that deficits in working
memory, unlike attentional control, correlate with the general cognitive decline
apparent in schizophrenia (63). Working memory has been characterised as the
common factor in Spearman’s ‘g’, a measure of general cognitive function derived
from the statistical correlation between cognitive tasks. Alternatively, working
memory may simply be another name for ‘g’ given the overlap between working
memory and psychometric tests of ‘g’ (64). This is supported by the evidence that
various measures of working memory correlate with ‘g’ at a level corresponding to
the reliability of the measure used (65-67). Our association with indices of both
(verbal) intelligence and working memory is consistent with this evidence, and
suggests that for NOS1 verbal IQ and working memory may represent overlapping, or
at least psychometrically similar, constructs. Providing empirical support for this
point, co-varying for the influence of either cognitive variable resulted in the
association of the other with NOS1 becoming non-significant (See supplementary
table 2). These findings are consistent with cognitive studies in both animal and
human models of NOS1 where a general rather than specific impact on cognition is
suggested (29-30; 36-39; 41,41).
NOS1 & Cognition: Molecular mechanism
The implicated SNP (rs6490121), located in intron 10 of NOS1, has no obvious
functional impact and may reflect a proxy association with another causal variant(s).
Based on HapMap CEU data, rs6490121 is not in high LD (r2 >0.8) with any other
common SNP at this locus. NOS1 is characterized by complex transcriptional
regulation. Twelve alternative first exons have been identified (exon 1a – 1l; 68).
Exon 1d and 1f, along with the exon 1g transcript, are the most commonly expressed
NOS1 transcripts in the brain (69). A dinucleotide variable number tandem repeat
(VNTR) is located in the core promoter region of exon 1f (70). The short alleles of
this variant, termed NOS1 Ex1f VNTR, are associated with decreased transcriptional
activity of the NOS1 exon 1f promoter, alterations in the neuronal transcriptome
impacting on other putative SZ susceptibility genes (RGS4 and GRIN1) and affects
electrocortical measures of prefrontal functions involved in cognitive control (81).
Interestingly, data from our laboratory indicates that the risk G allele of rs6490121 is
in LD (D = 0.70, r2 = 0.26, HapMap CEU) with the short alleles of NOS1 Ex1f
VNTR, highlighting a putative functional mechanism of cognitive performance
dysregulation at NOS1 for future study.
To explore potential mechanisms by which NO could exert an effect on cognitive
processes we screened experimentally validated Protein-Protein Interactions (PPI’s)
of NOS1 using the PPI databases (STRING (85), DIP, Interact (86) and BIND (87))
and identified 19 confirmed human binary interactions. With potential relevance to SZ
susceptibility are genes involved in pre-synaptic synaptogenesis (NOS1AP and
Syntrophin (SNTA1) (OMIM: 601017)) and post-synaptically through the PSD-95NOS1 receptor complex including direct NOS1 interaction with GRIN1 and GRIN2b.
Elements of PSD-95 signaling cascades have been targeted in SZ genetic association
studies including erbB4/Neuregulin signaling and the NMDA receptor complex which
is involved in long-term potentiation, memory and learning. There is some support
from these studies for involvement of GRIN2B, GRM3, erbB4 and NRG1 in SZ
susceptibility. Negative studies have been reported for PSD-95 (Kawashima et al,
2006; Tsai et al, 2006) and NOS1AP (Zheng et al, 2005; Puri et al, 2006a; Puri et al,
2006b; Fang et al, 2008). DeQuervain & Papassotiropoulus (2006) identified variation
at GRIN2B and GRM3 as genes contributing to the memory formation signaling
cascade involved in episodic memory performance. Additionally, NOS1 activators
Calmodulin and PKCA (protein kinase C alpha type), although not directly associated
with SZ risk, have also been associated with memory in humans (de Quervain &
Papassotiropoulos, 2006; Huentelman et al, 2007). More systematic studies of genes
involved in NOS1 regulation and adaptor proteins linked to synaptic function and
plasticity are warranted and we plan to screen functional variants at genes from these
pathways for additive and epistatic effects on cognition and SZ susceptibility.
NOS1 and schizophrenia: functional implications
The findings reported here raise interesting questions. Is NOS1 a SZ susceptibility
gene? Or perhaps a modifier gene that influences cognitive deficits without altering
disease liability (6; 71)? The present data do not provide evidence one way or another
for association between NOS1 and SZ. Including the additional Irish samples into the
meta-analysis of NOS1 by O’Donovan et al. (4) does not change the significance of
the findings of that study (i.e. NOS1 remains moderately associated with SZ in the
European only samples [p=.005, 2-tailed], but not in the total European and Asian
sample combined). One interpretation of these findings is in terms ‘cognitive reserve’,
the idea that individuals with lower IQ are more vulnerable to, or more likely to
express the features of, neurodevelopmental, neurodegenerative, or acquired brain
injury (72). In SZ, evidence of low intelligence as a risk factor is long established,
with both prospective birth cohort studies (73-75) and conscription studies (76-80)
consistently reporting lower intelligence in children and adolescence who will later
develop SZ. However, differences in education (often used as a proxy measure of premorbid IQ) were only observed as a trend in the Irish samples, and were absent in the
German samples, thus only weakly supporting lowered pre-morbid IQ as the
mechanism of risk associated with NOS1. Instead, given the association between
NOS1 and cognition in both patients and controls presented here, a more
parsimonious explanation is that NOS1 influences cognition generally, an impact
which is observed to some extent in both cases and controls. In terms of the
intermediate phenotype aproach in psychiatric genetics, these findings highlight the
benefits of cognitive studies of putative SZ variants for understanding the genetics of
fundemental cognitive and neurodevelopmental processes. It remains an open
question as to the degree to which these genes are coding for disease specific
processes, either as mediated though or independent of the effects on cognition
reported here.
Acknowledgements
The authors sincerely thank all patients would contributed to this study and all staff
who facilitated their involvement. Recruitment of the patients from Munich, Germany
was partially supported by GlaxoSmithKline (GSK). We are grateful to the Genetics
Research Centre GmbH, an initiative by GSK and LMU. Genotyping of the German
sample was funded by grants from the MRC and Wellcome Trust. Recruitment and
genotyping of the Irish sample was supported by the Wellcome Trust and Science
Foundation Ireland (SFI). Dr. Donohoe was supported by an SFI research frontiers
grant and a NARSAD young Investigator award. Dr. Walters was supported by an
MRC clinical training fellowship.
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List of Tables:
Table 1: Demographic and clincial data for Irish and German samples.
Table 2: Cognitive performance according to NOS1 genotype in both discovery and
replication samples.
Supplemental Table 1: Principal component analysis of positive and negative
symptoms in Irish and German samples.
Supplemental Table 2: Observed correlations of verbal and spatial working memroy
with Verbal, Performance and full scale IQ.
List of Figures:
Figure 1: Differences in verbal IQ associated with NOS1 rs6490121 in the Irish and
German cases and controls. In addition to a main effect of genotype in both samples,
an genotype-diagnosis interaction was observed in the German sample such that the
effect of NOS1 was greater in cases than controls.
Figure 2: Differences in working memory associated with NOS1 genotype. In
addition to a main effect of NOS1 genotype an interaction effect was observed in both
groups with NOS1 showing a larger effect in controls in the Irish sample and a larger
effect in cases in the German sample.