Association study of Neuregulin-1 gene polymorphisms in a north Indian schizophrenia sample

Article (PDF Available)inSchizophrenia Research 144(1-3) · January 2013with72 Reads
DOI: 10.1016/j.schres.2012.12.017 · Source: PubMed
Background: Neuregulin-1 (NRG1) gene polymorphisms have been proposed as risk factors for several common disorders. Associations with cognitive variation have also been tested. With regard to schizophrenia (SZ) risk, studies of Caucasian ancestry samples indicate associations more consistently than East Asian samples, suggesting heterogeneity. To exploit the differences in linkage disequilibrium (LD) structure across ethnic groups, we conducted a SZ case-control study (that included cognitive evaluations) in a sample from the north Indian population. Methods: NRG1 variants (n=35 SNPs, three microsatellite markers) were initially analyzed among cases (DSM IV criteria, n=1007) and controls (n=1019, drawn from two groups) who were drawn from the same geographical region in North India. Nominally significant associations with SZ were next analyzed in relation to neurocognitive measures estimated with a computerized neurocognitive battery in a subset of the sample (n=116 cases, n=170 controls). Results: Three variants and one microsatellite showed allelic association with SZ (rs35753505, rs4733263, rs6994992, and microsatellite 420M9-1395, p≤0.05 uncorrected for multiple comparisons). A six marker haplotype 221121 (rs35753505-rs6994992-rs1354336-rs10093107-rs3924999-rs11780123) showed (p=0.0004) association after Bonferroni corrections. Regression analyses with the neurocognitive measures showed nominal (uncorrected) associations with emotion processing and attention at rs35753505 and rs6994992, respectively. Conclusions: Suggestive associations with SZ and SZ-related neurocognitive measures were detected with two SNPs from the NRG1 promoter region in a north Indian cohort. The functional role of the alleles merits further investigation.


Association study of Neuregulin-1 gene polymorphisms in a north Indian
schizophrenia sample
Prachi Kukshal
, Triptish Bhatia
, A.M. Bhagwat
, Raquel E. Gur
, Ruben C. Gur
, Smita N. Deshpande
Vishwajit L. Nimgaonkar
, B.K. Thelma
Department of Genetics, University of Delhi South campus, Benito Juarez Road, New Delhi 110 021, India
C.B. Patel Research Centre, Vile Parle (West), Mumbai, India
Department of Psychiatry, Dr. RML Hospital, New Delhi 110 001, India
Department of Psychiatry, Neuropsychiatry Section, University of Pennsylvania, Philadelphia, PA, USA
Department of Psychiatry and Human Genetics, Western Psychiatric Institute and Clinic, University of Pittsburgh School of Medicine and Graduate School of Public Health, 3811 O'Hara
Street, Pittsburgh, PA 15213, USA
abstractarticle info
Article history:
Received 31 August 2012
Received in revised form 20 November 2012
Accepted 17 December 2012
Available online 26 January 2013
SNP and microsatellite markers
Background: Neuregulin-1 (NRG1) gene polymorphisms have been proposed as risk factors for several common
disorders. Associations with cognitive variation have also been tested. With regard to schizophrenia (SZ) risk,
studies of Caucasian ancestry samples indicate associations more consistently than East Asian samples,
suggesting heterogeneity. To exploit the differences in linkage disequilibrium (LD) structure across ethnic
groups, we conducted a SZ casecontrol study (that included cognitive evaluations) in a sample from the
north Indian population.
Methods: NRG1 variants (n=35 SNPs, three microsatellite markers) were initially analyzed among cases (DSM IV
criteria, n=1007) and controls (n=1019, drawn from two groups) who were drawn from the same geographical
region in North India. Nominally signicant associations with SZ were next analyzed in relation to neurocognitive
measures estimated with a computerized neurocognitive battery in a subset of the sample (n=116 cases, n=170
Results: Three variants and one microsatellite showed allelic association with SZ (rs35753505, rs4733263,
rs6994992, and microsatellite 420M9-1395, p 0.05 uncorrected for multiple comparisons). A six marker haplo-
type 221121 (rs35753505-rs6994992-rs1354336-rs10093107-rs3924999-rs11780123) showed (p=0.0004)
association after Bonferroni corrections. Regression analyses with the neurocognitive measures showed nominal
(uncorrected) associations with emotion processing and attention at rs35753505 and rs6994992, respectively.
Conclusions: Suggestive associations with SZ and SZ-related neurocognitive measures were detected with two
SNPs from the NRG1 promoter region in a north Indian cohort. The functional role of the alleles merits further
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
Schizophrenia (MIM 181500, SZ) is a common, lifelong disorder
with a life time prevalence of 0.8% among Indian adults (Faraone et
al., 2002; Saha et al., 2005). The relatively high heritability of SZ has
motivated intensive gene mapping efforts (Shirts and Nimgaonkar,
2004; Talkowski et al., 2007; Chen et al., 2009; Talkowski et al., 2010;
Greenwood et al., 2012). Meta-analysis of 32 genome-wide linkage
studies of SZ suggested l inkage on chromosome 8p (1633 Mb)
(Ng et al., 2009) for 22 European-ancestry samples. Recently, genome-
wide association studies (GWAS) have identied several relatively
common single nucleotide polymorphisms (SNPs) that are associated
with SZ (Potkin et al., 2009; Shi et al., 2009; McClay et al., 2010; Ripke
et al., 2011; Shi et al., 2011).
Several studies have focused on the signaling protein NRG1 and its
receptor ERRB4. A variety of NRG1 isoforms (estimated n=30) are pro-
duced by alternative splicing (Tan et al., 2007; Liu et al., 2011). They are
expressed in varying proportions at relatively high levels in a variety of
peripheral tissues as well as the brain. In the brain, NRG1 is considered
to be a pleiotropic growth factor with an integral role in its develop-
ment, organization, and function (Li et al., 2006). NRG1 plays key roles
in several neurotransmitter systems, including (N-methyl-
acetylcholine, as well as gamma-Aminobutyric acid (Fischbach and
Rosen, 1997; Ozaki et al., 1997; Rieff et al., 1999; Cameron et al., 2001).
Several NRG1 SNPs have been reported to be associated with SZ,
albeit at nominal levels of signicance (Farrer et al., 2001; Falls,
2003a,b; Hashimoto et al., 2004; Bertram et al., 2005; Harrison and
Schizophrenia Research 144 (2013) 2430
Corresponding author. Tel.: +91 11 24118201; fax: +91 11 24112761.
E-mail address: (B.K. Thelma).
0920-9964/$ see front matter © 2013 Elsevier B.V. All rights reserved.
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Weinberger, 2005; Gardner et al., 2006). Stefansson et al. (2002) rst
reported linkage to a locus on Chromosome 8 in an Icelandic sample and
subsequently a replicated association with a 7-marker risk haplotype in a
Scottish sample (Stefansson et al., 2003). Meta-analysis of 26 published
casecontrol and family-based association studies showed association of
SNP8NRG221132, 420M9-1395, 478B14-848 and suggested population
stratication for SNP8NRG221533 (Gong et al., 2009). Another meta-
analysis of 13 studies reported association with six markers between
two adjacent, but distinct haplotypes blocks in Caucasian and Asian
ancestry samples (Li et al., 2006). Another group found non-signcant
association of SNP8NRG221533 after taking study design and ancestry
into account (Munafo et al., 2006, 2008). Only one study has been
reported from South Asia. This study from Pakistan investigated two
SNPs in 100 cases and 70 adult controls. It suggested nominal association
with the exonic SNP rs3924999 (Naz et al., 2011).
Impairment in several cognitive domains has been reported in SZ
(Heinrichs et al., 1997; Goldberg and Green, 2002; Buchanan et al.,
2005; Snitz et al., 2006; Gur et al., 2007; Reichenberg and Harvey,
2007; Barch and Smith, 2008; Ranganath et al., 2008; Tandon et al.,
2009; Yokley et al., 2012). NRG1 SNPs may also be associated with cog-
nitive dysfunction, particularly attention (Yokley et al., 2012); spatial
memory and social behavior (O'Tuathaigh et al., 2007). The NRG1
SNP8NRG221533 (rs35753505) has most widely been evaluated in
relation to cognition (Kurnianingsih et al., 2011). A role for NRG1 in SZ
has also been supported by animal studies using NRG1 and ErbB4
mutant mice (Gerlai et al., 2000; Stefansson et al., 2002; Bao et al., 2003;
Corfas et al., 2004; Steinthorsdottir et al., 2004; Gu et al., 2005; Rimer et
al., 2005), which exhibit behaviors similar to those of established rodent
models of SZ (Lipska, 2004).
NRG1 polymorphisms have been proposed as risk factors for several
other common disorders, including Alzheimer's disease (Chaudhury et
al., 2003; Go et al., 2005); epilepsy (early myoclonic encephalopa thy;
Backx et al., 2009), stroke (Shyu et al., 2004; Xu et al., 2004) breast cancer
(Raj et al., 2001), multiple sclerosis (Cannella et al., 1999; Viehover et al.,
2001), bipolar disorder (Thomson et al., 2007; Goes et al., 2008; Prata et
al., 2009; Walker et al., 2010; Moon et al., 2011) and Hirschsprung Disease
(Garcia-B arcelo et al., 2009; Tang et al., 2011).
In sum, NRG1 likely plays a key role in brain development and neuro-
transmitter function. With regard to SZ risk, the results from Caucasian
ancestry samples appear to be more consistent whereas the results
from the Asian samples are variable, suggesting locus heterogeneity. In
order to exploit the differences in LD structure across ethnic groups,
we investigated a north Indian population using a casecontrol design.
NRG1 SNP associations with cognitive variation were further tested in
a sub-group of this sample.
2. Methods
2.1. Recruitment and diagnostic assessment
The recruitment and assessment of the sample has been described in
prior studies (Bhatia et al., 2008). Briey, patients with a clinical diagno-
sis of SZ or schizoaffective disorder were referred from the outpatient
department of Dr. Ram Manohar Lohia Hospital, as well as other private
and public psychiatric facilities in Delhi, India. All patients (n=1007)
were assessed using the Hindi versions of the Diagnostic Interview for
Genetic Studies (DIGS) and the Family Interview for Genetic Studies
(FIGS) (Nurnberger et al., 1994; Deshpande et al., 1998; http:// This information was synthesized with
available medical records and presented to board certied psychiatrists
who assigned consensus diagnoses.
The control samples (n=1019) included non-psychotic adults
(n= 521) who were recruited from the same communities in which
the patients resided. Care was taken not to include multiple related
individuals as controls. At the time of recruitment, detailed family infor-
mation was obtained with the use of a semi-structured questionnaire
and care was taken to avoid recruitment of 1st and 2nd degree related-
ness in our casecontrol cohort. We also included a control group com-
prising neonatal blood samples from live births at Lok Nayak Hospital,
New Delhi; this group could not therefore be screened for psychotic ill-
ness (n=498). DIGS and FIGS were administered on mothers of all neo-
natal controls to evaluate for psychotic illness in the parents and other
rst or second degree relatives. Neonatal blood was not taken if any
family member was reported to have psychotic illness. No information
apart from gender was provided about these anonymous samples.
All participants (except the neonatal control samples) provided
written informed consent. Written informed consent was obtained
from mothers for the neonata l sample. The study was appr oved by
Institutional Ethical Committee at Dr. Ram Mano har Lohia (RML)
Hospital, New Delhi, and the Institutional review board at the University
of Pittsburgh, USA.
2.2. Cognitive evaluation
The Hindi version of the Penn Cognitive Neuropsychiatric Battery
(CNB) (Gur et al., 2001; Bhatia et al., 2011) was administered to a subset
(n=256) of participants comprising cases (n= 116) and adult controls
(n=140). The following cognitive domains were assessed: abstraction
and mental exibility, attention, face memory, spatial memory, working
memory, spatial ability, sensorimotor and emotional processing. The
CNB evaluates accuracy, speed and efciency for each domain. As
these indices are correlated for any one domain, we analyzed the accu-
racy measures for parsimony.
2.3. Selection of polymorphisms
A total of 35 SNPs and three microsatellite markers were tested. We
selected markers based on prior reported associations and based on
local LD (r
>0.8; Indian, GIH data in Hapmap, We
also focused on SNPs in exonic regions and in the 5 sequences, the latter
because it has been proposed that regulatory variants in NRG1 may be
particularly involved in pathogenesis (Law et al., 2006).
2.4. Genotype assays
DNA was extracted and used for genotyping the SNPs based on primer
extension reaction chemistry in the MALDI-TOF mass spectrometry plat-
form ( using iPLEX® Gold reagents. An ABI
3730 machine was used for fragment analysis of the uorescent labeled
microsatellite markers. Quality checks were performed by using dupli-
cates and CEPH samples in each plate.
2.5. Statistical analysis
Hardy Weinberg equilibrium (HWE) was examined for each SNP. All
SNPs conforming to HWE estimates (p>0.01) were included in the asso-
ciation analyses. LD values (r
) were estimated for the genotyped data
using the Tagger algorithm in Haploview version 4.1 (Barrett et al.,
2005; Heterogeneity be-
tween neonatal and adult control groups with regard to allele frequencies
was also tested using Haploview software. Casecontrol associations for
individual SNPs were evaluated using the Trends test in PLINK (http://
pngu.mgh.har rcell/plink /). Associations with microsatellite
markers were assessed using CLUMP software (Sham and Curtis, 1995; SNPs which
showed association either for allelic or model-wise tests were included
for haplotype analysis, using PLINK and UNPHASED (Dudbridge, 2003,
2008). Power was estimated using Quanto software (Gauderman and
Morrison., 2006; gxe/).
Multivariate analyses were used to test associations between individ-
ual SNPs and accuracy for cognitive domains using the Statistical Package
for Social Sciences (SPSS Version 16, Linear
25P. Kukshal et al. / Schizophrenia Research 144 (2013) 2430
regression analyses were conducted separately for each cognitive do-
main to test associations between cognitive variables and two SZ associ-
ated SNPs. The normalized cognitive domain scores adjusted for age were
the outcome variables and genotypes for individual SNPs, gender and
diagnosis were used as covariates for these analyses.
3. Results
3.1. Demographic data
Men constituted 56.8% of the cases and 55.74% of the controls. There
were no signicant casecontrol differences with regard to gender in
the two groups. There was a signicant difference in the ages of the
cases and the controls (mean±standard deviation, SD; adult controls:
43.03±14.0; cases: 29.9±8.95).
3.2. Quality control for genotype assays
All the SNPs were in HWE (p >0.01). Of the 2044 participants in
the study, genotypes from individuals with less than 90% genotype
calls were excluded from all analysis (n =18). Therefore, a total of
2026 participants were analyzed (n= 1007 cases, n= 1019 controls).
Overall, the call rate was over 97% for the SNPs and over 95% for the
microsatellite markers.
3.3. LD patterns
LD between pairs of SNPs was estimated for the control individuals
using r
values (Supplementary Fig. 1). Overall, the patterns of LD
resembled those observed in Caucasian ancestry individuals (www. The SNPs genotyped were generally not in tight LD with
the following notable exceptions: rs6988339 and rs10691392 (r
0.9) and rs6994992 and rs4733263 (r
3.4. Casecontrol comparisons
The adult and the neonatal control samples did not differ signicantly
with regard to genotype or allele frequencies for any of the polymor-
phisms (Supplementary Table I). Test of heterogeneity performed for
each SNP did not show signicant differences between the two groups
of controls (Supplementary Table I). Since the distribution of the poly-
morphisms was comparable in the two groups, the control groups were
pooled for all further analysis.
Three polymorphisms were nominally associated with SZ risk
(pb 0.05 uncorrected for multiple comparisons, Table 1; Supplementary
Table II) of which rs6994992 and rs4733263 are in LD: rs35753505
(p=0.04; OR =1.15(95% condence intervals, CI, 1.011.31), rs473
3263 (p=0.04; OR =1.14(95% CI, 1.011.31)), rs6994992 (p =0.026;
OR= 1.15(95% CI, 1.021.3)), but none withstood Bonferroni corrections.
One microsatellite marker 420_M9-1395 (p= 0.016) located in 5
region also showed nominal association (Table 1; Supplementary
Table II). Four more SNPs showed genotypic association. TT genotype
of rs3924999 a Val>Leu missense polymorphism in exon 11 and GG
genotype in rs11780123 in 3 region showed association (0.02 and
0.01 respectively) under a Dominant model. TT genotype of rs1354336
and rs10093107 showed association (0.009 and 0.008 respectively)
under Recessive model (Table 1).
3.5. Haplotypic association
Using the 6 associated SNPs in linkage equilibrium namely
rs35753505, rs6994992, rs1354336, rs10093107, rs3924999 and
rs11780123 (Table 1), two to six SNP sliding window haplotypes
(frequency>5%) were constructed and global p values were tabulated
(Table 2a). Out of 74 haplotypes constructed using Plink, 18 haplotypes
were s ignicantly different. A ve-marker haplotype comprised of
rs6994992rs1354336rs10093107rs3924999rs11780123 (p=
0.0004) and a six-marker haplotype with rs35753505rs6994992
rs1354336rs10093107rs3924999rs11780123 (p= 0.0004) remained
signicant after Bonferroni corrections (alpha value 0.05/74= 0.0006;
Table 2b). Notably, most of the associations were driven by the two
promoter SNPs rs35753505 and rs6994992 (Tables 2a and 2b;
Supplem entary Table II).
3.6. Cognitive variables
Computerized neurocognitive data were available for cases and adult
controls (n = 256). In this group, there were no signicant gender differ-
ence (61.2% and 60% males, respectively for controls and cases). The mean
age of the controls (47.97, SD 15.0) was signicantly higher (F= 126.532;
) than those of cases (31.0, SD 9.32). Therefore, the cogni-
tive measures were adjusted for age. We analyzed eight neurocognitive
domains, namely abstraction and mental exibility, attention, face mem-
ory, spatial memory, working memory, spatial ability, sensorimotor and
emotion processing (Gur et al., 2007). Of the three allelic associated
SNPs only rs35753505 and rs6994992 were used for further cognitive
analysis as rs4733263 was in LD with rs6994992. Following linear regres-
sion analysis, an association between rs35753505 and emotion process-
ing was noted (p=0.031). At rs6994992, an association with attention
was noted (p=0.047; Table 3). There was no signicant interaction be-
tween SNP genotype and casecontrol status at either locus (data not
Table 1
Association of NRG1with SZ.
MAF Trends test Dominant Recessive Additive
SNP BP MA Fca Fco CHISQ p(df=1) CHISQ p(df =1) CHISQ p(df=1) CHISQ p(df=2)
31593683 T 0.37 0.34 4.43 0.04 6.14 0.01 6.176 0.05
rs4733263 31610016 G 0.50 0.47 4.17 0.04 3.04 0.08 4.185 0.12
31615123 T 0.50 0.47 4.99 0.03 3.22 0.07 3.419 0.064 4.992 0.08
420 M9-1395# 31,665,413..
31,665,691 ()
(CA)14 0.003 0.0005 18.70 0.02 (df = 8)
rs1354336 31713684 T 0.13 0.13 0.002 0.96 6.745 0.009 8.667 0.01
rs10093107 32145991 T 0.43 0.46 3.58 0.06 7.076 0.008 7.201 0.03
rs3924999 32572900 T 0.43 0.40 1.71 0.19 5.81 0.02 8.026 0.02
rs11780123 32750870 G 0.14 0.16 3.57 0.06 6.49 0.01 10.19 0.01
BP: genomic location (base pairs). MA: Minor allele; MAF: Minor allele frequency; Fca: Minor allele frequency in cases; Fco: Minor allele frequency in unaffected control aliases:
SNP8NRG221533; ** SNP8NRG243177; # Microsatellite Marker rs4733263 and rs6994992 are in LD (r
Signicant p values (p b 0.05) are marked in bold.
26 P. Kukshal et al. / Schizophrenia Research 144 (2013) 2430
3.7. Power analysis
The sample has >85% power to detect associations with SZ risk having
an OR of 1.5 or greater, for SNPs having minor allele frequencies (MAF)
greater than 5%, assuming alpha=0.05, uncorrected for multiple
4. Discussion
Three SNPs namely rs35753505, rs4733263 and rs6994992 showed
modest allelic association and four addition SNPs rs1354336, rs100
93107, rs3924999 and rs11780123 namely showed genotypic (dominant
or recessive) association and one microsatellite marker (420M9-1395)
showed modest allelic association with SZ in our north Indian sample
(Table 1), though it should be noted that the associations did not remain
signicant following Bonferoni corrections for multiple comparisons.
rs6994992 was also reported to be associated with SZ in Caucasian sam-
ples (Hall et al., 2006; Law et al., 2006). Notably, the risk allele at this
SNP (T) is associated with increased type IV NRG1 messenger RNA levels
(Law et al., 2006), lower prefrontal (and temporal) activation and devel-
opment of psychotic symptoms in individuals at high risk for SZ (Hall et
al., 2006). This SNP maps upstream of the NRG1 type IV 5-exon
(St einthorsdottir et al., 2004; Law et al., 2006; Tan et al., 2007; Shamir
and Buonanno, 2010). It is associated with diminished activation in medi-
al prefrontal cortex and at the right temporo-occipital junction (Hall et al.,
2006). The other associated polymorphisms may also have functional
effects, as associations with cortical volumes have been reported at
420_M9-1395 (Addington et al., 2007) and rs6994992 (Mata et al.,
2009, 2010). 420M9-1395 and rs35753505 may inuence brain develop-
ment (Addington et al., 2007). In addition, reduction of white matter
fractional anisotropy was associated with rs35753505 in the anterior cin-
gulum (Wang et al., 2009; Kurnianingsih et al., 2011). As the associated
polymorphisms are localized to the 5 region, it is possible that variation
in the promoter region of NRG1 elevates risk for SZ. Indeed, post-
mortem studies reveal altered NRG1 mRNA levels in the prefrontal cortex
of SZ patients (Harrison and Law, 2006), as well as the hippocampal
region (Law et al., 2006) and neuroimaging studies reveal changes in sub-
cortical white matter myelination in the frontal lobe (Konrad and
Winterer, 2008). NRG1variants likely modulate brain activation during
episodic memory processing in key areas for memory encoding and
retrieval, with SZ risk alleles showing hyper activation in areas associated
with elaborate encoding strategies (Krug et al., 2010). Exonic SNP
rs3924999, a missense variant present in NRG1 (Val> Leu in exon 11)
increased the risk of schizophrenia (Walss-Bass et al., 2006). Genotypic
association of this SNP has been observed for antisaccades and smooth
pursuit eye movements (Schmechtig et al., 2010) and lower prepulse
inhibition, an endophenotype of schizophrenia (
Hong et al., 2008). This
suggests an impact of NRG1 polymorphism on the neural mechanisms un-
derlying visuospatial sensorimotor transformations, a mechanism that
has been found to be impaired in patients with schizophrenia and their
We also found signicant 5- and 6-marker haplotypic association
withstanding Bonferroni correction (Table 2b) and the associations
seems to be primarily attributable to the promoter SNPs rs6994992
(Tables 2a and 2b). However, a signicantly associated truncated
Table 2a
Sliding window haplotype analysis of nominally associated SNPs.
Name Map information 2-mhap 3-mhap 4-mhap 5-mhap 6-mhap
rs35753505 31593683 0.09 0.28 0.39 0.01 0.01
rs6994992 31615123 0.05 0.04 0.02 0.02
rs1354336 31713684 0.17 0.24 0.09
rs10093107 32145991 0.13 0.11
rs3924999 32572900 0.05
rs11780123 32750870
2-mhap: Global p-values of successive two marker haplotypes generated using UNPHASED.
Data are presented in the same format for each adjacent pair of markers down this column.
3-mhap: Global p-values of three marker haplotypes generated using UNPHASED. Data
are presented in the same format for each set of three adjacent markers down this column.
Similarly, 4-, 5- and 6-mhap denote haplotypes incorporating the respective number of
Haplotypes with a frequency lower than 5% were not included in the analysis;
Signicant p values (p b 0.05) are marked in bold.
Table 2b
Signicant haplotypes of associated SNPs.
SNPs Haplotype Freq. OR chi Sq. P
2 SNP window
rs35753505-rs6994992 22 0.35 1.15 4.45 0.0348
rs35753505-rs6994992 11 0.51 0.87 5.15 0.0233
rs6994992-rs1354336 21 0.45 1.17 5.51 0.0189
rs6994992-rs1354336 11 0.43 0.86 5.43 0.0198
rs10093107-rs3924999 12 0.26 1.17 4.12 0.0424
rs3924999-rs11780123 21 0.36 1.15 4.41 0.0358
3 SNP window
rs35753505-rs6994992-rs1354336 221 0.33 1.17 4.81 0.0284
rs35753505-rs6994992-rs1354336 111 0.42 0.86 5.41 0.0201
rs6994992-rs1354336-rs10093107 112 0.18 0.83 4.12 0.0424
rs6994992-rs1354336-rs10093107 211 0.26 1.25 8.00 0.00468
rs10093107-rs3924999-rs11780123 121 0.22 1.24 6.36 0.0116
4 SNP window
rs35753505-rs6994992-rs1354336-rs10093107 1112 0.18 0.83 4.19 0.0408
rs35753505-rs6994992-rs1354336-rs10093107 2211 0.18 1.26 6.27 0.0123
rs6994992-rs1354336-rs10093107-rs3924999 2112 0.13 1.45 10.50 0.00122
rs1354336-rs10093107-rs3924999-rs11780123 1121 0.20 1.21 4.44 0.035
5 SNP window
rs35753505-rs6994992-rs1354336-rs10093107-rs3924999 22112 0.09 1.57 10.10 0.00148
rs6994992-rs1354336-rs10093107-rs3924999-rs11780123 21121 0.11 1.57 12.80 0.000352
6 SNP window
rs35753505-rs6994992-rs1354336-rs10093107-rs3924999-rs11780123 221121 0.08 1.75 12.60 0.000396
Freq: Frequency of Haplotypes (>5%);
Allele 2 in haplotypes represent minor allele.
Haplotypes signicant after Bonferroni corrections (alpha value 0.05/74=0.0006).
27P. Kukshal et al. / Schizophrenia Research 144 (2013) 2430
haplotype (Table 2b) suggests contributions of the 3 marker
(rs11780123) also. Of note, functional imaging studies have report in-
tragenic epistasis between 5 and 3 markers in NRG1 (Nicodemus et
al., 2010; Moon et al., 2011).
The associations between rs35753505 and rs6994992 and cognitive
functions noted here are consistent with prior reports in Caucasian sam-
ples; e.g., (Yokley et al., 2012)and(O'Tuathaigh et al., 2007), though
there are some reports of non-signicant associations (Crowley et al.,
2008). In healthy participants, rs35753505 was not associated with
working memory or task performance (Krug et al., 2008; Kircher et al.,
2009a), but was associated with semantic verbal uency (Kircher et
al., 2009b) and sustained attention (Stefanis et al., 2007). rs6994992,
originally identied as part of the so-called deCODE haplotype, could
be specically related to disruption of normal frontal and temporal
lobe function, premorbid intelligence levels and the emergence of psy-
chotic symptoms (Harrison and Law, 2006; Li et al., 2006). Individuals
with the TT genotype at this SNP also had reduced white matter density
and structural connectiv ity (McIntosh et al., 2008), impaired frontal and
temporal lobe activation (Hall et al., 2006), and cognition (Hall et al.,
2006; Stefanis et al., 2007; Sprooten et al., 2009), including reduced spa-
tial working memory capacity (Stefanis et al., 2007) and emotion pro-
cessing (Keri and Kelemen, 2008).
There are some limitations in the present study. First, several associ-
ations did not withstand Bonferroni corrections for multiple compari-
sons. Thus, the effects of NRG1 polymorphisms in this, the largest
Indian sample analyzed to date are likely to be modest. Second, the sam-
ple included adult, as well as neonatal controls, though there was no sig-
nicant difference in allele frequencies between these two groups. There
is a modest (~1%) probability that some of the neonatal controls will be
diagnosed with SZ in later life (estimated n=approximately 5). Such
misdiagnosis would tend to diminish observed associations. Finally,
population substructure as a potential source for the association could
not be evaluated in the sample using Principle Components Analysis
(PCA) or Multi Dimensional Scaling (MDS), as ancestry informative or
genome wide markers were not evaluated.
In conclusion, nominal associations with SZ were noted with three
NRG1 polymorphisms. Two of the associated SNPs were also associat-
ed with cognitive variation in the combined casecontrol sample.
These associations are consistent with prior reports, predominantly
in Caucasian samples. As the associated polymorphisms and haplo-
types are localized to the 5NRG1 sequences, they may reect subtle
alterations in gene expression. Further investigations of NRG1 function
in the brain, as well as functional studies of the associated polymor-
phisms are warranted.
Supplementary data to this article can be found online at http://
Role of funding source
We received nanci al support for this project from National Institutes of Health (under
Training Program for Psychiatric Genetics in India, Grant# 5D43 TW006167-02). Additional
NIH support for VLN is acknowledged through grant MH66263.
Prof. B.K. Thelma and Prof. V. L. Nimgaonkar designed the study and wrote the protocol.
Prof. Deshpande and her team provided the research samples; Prof. R.E. Gur and Prof. R.C.
Gur provided the neurocognitive battery and Dr. Triptish Bhatia did the cognitive analysis.
literature searches did the genetic analysis and wrote the rst draft of the manuscript.
All authors contributed to and have approved the nal manuscript.
Conict of Interest
The authors have no conicts of interest to declare.
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Signicant associations between cognitive variables and NRG1 SNPs.
Outcome variable Covariates Unstandardized
t p value 95% condence
interval for B
B Std. error B Lower Upper
Emotion processing (Constant) 0.948 0.264 3.588 0.0004 0.428 1.468
Gender 0.075 0.121 0.037 0.622 0.535 0.163 0.313
Diagnostic status 0.558 0.119 0.279 4.671 4.90×10
0.793 0.322
rs35753505 0.26 0.119 0.161 2.17 0.031 0.49 0.02
Attention (Constant) 0.901 0.298 3.029 0.003 0.314 1.488
Gender 0.192 0.142 0.09 1.348 0.179 0.089 0.473
Diagnostic status 0.645 0.133 0.324 4.834 2.73×10
0.908 0.382
rs6994992 0.237 0.118 0.164 1.997 0.047 0.47 0.003
Signicant p values (pb0.05) are marked in bold.
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30 P. Kukshal et al. / Schizophrenia Research 144 (2013) 2430
    • "Other NRG1 polymorphisms, primarily in the HAP ICE region, have also been linked to cognitive endophenotypes. The 'risk' T allele of rs6994992 (SNP8NRG243177) has been associated with reduced spatial working memory and prepulse inhibition in military conscripts (Roussos et al. 2011; Stefanis et al. 2007), reduced attention and general cognitive ability in those with schizophrenia (Cho et al. 2015; Kukshal et al. 2013), and lower IQ in the prodromal period (Keri et al. 2009). Approximately 21.4 kbp upstream of this variant, the 'risk' C allele of rs35753505 "
    [Show abstract] [Hide abstract] ABSTRACT: Clinical and pre-clinical evidence has implicated neuregulin 1 (NRG1) as a critical component in the pathophysiology of schizophrenia. However, the arrival of the genome-wide association study (GWAS) era has yielded results that challenge the relevance of NRG1 in schizophrenia due to the absence of a genome-wide significant NRG1 variant associated with schizophrenia. To assess NRG1’s relevance to schizophrenia in the GWAS era, we provide a targeted review of recent preclinical evidence on NRG1’s role in regulating several aspects of excitatory/inhibitory neurotransmission and in turn schizophrenia risk. We also present a systematic review of the last decade of clinical research examining NRG1 in the context of schizophrenia. We include concise summaries of genotypic variation, gene-expression, protein expression, structural and functional neuroimaging as well as cognitive studies conducted during this time period. We conclude with recommendations for future clinical and preclinical work that we hope will help prioritize a strategy forward to further advance our understanding of the relationship between NRG1 and schizophrenia.
    Full-text · Article · Jun 2016
    • "Previous studies have implicated NRG1 in patients with schizophrenia (Stefansson et al., 2002; Williams et al., 2003; Yang et al., 2003; Munafo et al., 2006; Pitcher et al., 2011 ). Recent report from Indian populations implicated NRG1 with schizophrenia (Kukshal et al., 2013; Thirunavukkarasu et al., 2014). Studied variant (rs17603876) is in LD with rs12155594, associated with transition to psychosis (Bousman et al., 2013 ). "
    [Show abstract] [Hide abstract] ABSTRACT: Schizophrenia is a severe psychiatric disorder with lifetime prevalence of ~ 1% worldwide. A genotyping study was conducted using a custom panel of Illumina 1536 SNPs in 840 schizophrenia cases and 876 controls (351 patients and 385 controls from North India; and 436 patients, 401 controls and 143 familial samples with 53 probands containing 37 complete and 16 incomplete trios from South India). Meta-analysis of this population of Indo-European and Dravidian ancestry identified three strongly associated variants with schizophrenia: STT3A (rs548181, p = 1.47 × 10− 5), NRG1 (rs17603876, p = 8.66 × 10− 5) and GRM7 (rs3864075, p = 4.06 × 10− 3). Finally, a meta-analysis was conducted comparing our data with data from the Schizophrenia Psychiatric Genome-Wide Association Study Consortium (PGC-SCZ) that supported rs548181 (p = 1.39 × 10− 7). In addition, combined analysis of sporadic case–control association and a transmission disequilibrium test in familial samples from South Indian population identified three associations: rs1062613 (p = 3.12 × 10− 3), a functional promoter variant of HTR3A; rs6710782 (p = 3.50 × 10− 3), an intronic variant of ERBB4; and rs891903 (p = 1.05 × 10− 2), an intronic variant of EBF1. The results support the risk variants observed in the earlier published work and suggest a potential role of neurodevelopmental genes in the schizophrenia pathogenesis.
    Full-text · Article · Jan 2015
    • "The most consistent and strong association has been detected for a core region including five SNPs and two microsatellites located at the 5 0 end of the NRG1 gene (Stefansson et al. 2002). Overall, dozens of SNPs of NRG1 have been associated with schizophrenia, both at 5 0 (Kukshal et al. 2013; Benzel et al. 2007; Corvin et al. 2004; Hall et al. 2004; Petryshen et al. 2005; Stefansson et al. 2002; Zhao et al. 2004) and 3 0 regions (Bakker et al. 2004; Benzel et al. 2007; Lachman et al. 2006; Li et al. 2004; Petryshen et al. 2005). Recently, genome-wide association studies (GWAS) for schizophrenia have also reported a nominal association between several SNPs in NRG1 gene and schizophrenia, with p values spanning from 1.59 9 10 -5 for rs4316112 to 1.38 9 10 -3 for rs10095694 (Athanasiu et al. 2010; Shi et al. 2009 ). "
    [Show abstract] [Hide abstract] ABSTRACT: Neuregulin 1 (NRG1) and v-erb-a erythroblastic leukemia viral oncogene homolog 4 (ErbB4) have been extensively studied in schizophrenia susceptibility because of their pivotal role in key neurodevelopmental processes. One of the reasons for the inconsistencies in results could be the fact that the phenotype investigated has mostly the diagnosis of schizophrenia per se, which is widely heterogeneous, both clinically and biologically. In the present study we tested, in a large cohort of 461 schizophrenia patients recruited in Scotland, whether several SNPs in NRG1 and/or ErbB4 are associated with schizophrenia symptom dimensions as evaluated by the Positive and Negative Syndrome Scale (PANSS). We then followed up nominally significant results in a second cohort of 439 schizophrenia subjects recruited in Germany. Using linear regression, we observed two different groups of polymorphisms in NRG1 gene: one showing a nominal association with higher scores of the PANSS positive dimension and the other one with higher scores of the PANSS negative dimension. Regarding ErbB4, a small cluster located in the 5′ end of the gene was detected, showing nominal association mainly with negative, general and total dimensions of the PANSS. These findings suggest that some regions of NRG1 and ErbB4 are functionally involved in biological processes that underlie some of the phenotypic manifestations of schizophrenia. Because of the lack of significant association after correction for multiple testing, our analyses should be considered as exploratory and hypothesis generating for future studies.
    Full-text · Article · Aug 2014
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