Molecular Psychiatry (2011) 16, 860–866 & 2011 Macmillan Publishers Limited All rights reserved 1359-4184/11 www.nature.com/mp ORIGINAL ARTICLE 3 (NRG3) as a susceptibility in a subtype with florid delusions and relatively spared cognition B Morar1, M Dragovic´2, FAV Waters2, D Chandler1, L Kalaydjieva1,3 and A Jablensky2,3 1Centre for Medical Research/Western Australian Institute for Medical Research, The University of Western Australia, Perth, WA, Australia and 2Centre for Clinical Research in Neuropsychiatry and School of Psychiatry and Clinical Neurosciences, The University of Western Australia, Perth, WA, Australia

Linkage of 10q22-q23 to schizophrenia and the recently reported association of (NRG3) polymorphisms with high ‘delusion factor’ scores led us to attempt replication and further refinement of these findings in a sample of 411 schizophrenic patients and 223 nonpsychiatric control subjects. Using quantitative cognitive traits, patients were grouped into a cluster with pervasive cognitive deficit (CD) and a cluster with relatively spared cognition (CS). We found a significant association between rs6584400 and schizophrenia, with a trend for rs10883866. Post hoc analysis revealed that this result was mainly due to the CS cluster, characterized by elevated scores on Schneiderian first-rank symptoms, salience of complex delusions and positive thought disorder—thus closely related to the ‘delusion factor’. In addition, both rs6584400 and rs10883866 were associated with the degraded-stimulus continuous performance task in which ‘risk’ alleles were associated with better than average performance in patients and worse performance in controls. This suggests that NRG3 may be modulating early attentional processes for perceptual sensitivity and vigilance, with opposite effects in affected individuals and healthy controls. The two single-nucleotide polymorphisms are in close proximity to the alternative first exons of the NRG3-a, -b and -d isoforms, of which the human brain-specific NRG-b appears to be the most interesting candidate. Molecular Psychiatry (2011) 16, 860–866; doi:10.1038/mp.2010.70; published online 15 June 2010 Keywords: NRG3; schizophrenia; cognitive endophenotypes; degraded-stimulus continuous performance task

Introduction and Han Chinese families8,9 and by the detection of complex 10q22-q23 rearrangements in several The Neuregulin family of growth and differentiation families with cognitive and behavioral abnormal- factors (NRG1–4)1,2 and their cognate ErbB receptor ities.10 Further support was provided by reports of tyrosine kinases have multiple roles in brain devel- association between single-nucleotide polymorph- opment and neuronal and glial maturation and isms (SNPs) in NRG3 and schizophrenia in Scottish11 function.3,4 By far, the best studied member of the and Han Chinese12 samples and, recently, by compre- family is NRG1, a large with numerous hensive association mapping of the linked 10q22-q23 functional domains and an astonishing diversity of region in Ashkenazi case–control and family-based isoforms,4 in which variation in the 50 region has samples.1 The latter study found no evidence of been implicated in schizophrenia susceptibility, association with the broad phenotype of the Diag- with the expression of some brain-specific isoforms nostic and Statistical Mannual of Mental Disorders, differentially regulated by associated upstream Fourth Edition (DSM-IV) schizophrenia; however, polymorphisms.5–7 factor analysis disaggregation of the clinical pheno- The involvement of Neuregulin 3 (NRG3)in type2 identified the association between a ‘delusion psychiatric illness was first suggested by linkage of factor’ and sequence variants (rs10883866 and schizophrenia to 10q22-q23 in Ashkenazim rs6584400) in intron 1 of the NRG3 gene. Another recent study, focusing on rare copy-number variants Correspondence: Professor A Jablensky, School of Psychiatry and in familial schizophrenia, reported a 73.6 kb duplica- Clinical Neurosciences, University of Western Australia, Medical tion in the first intron of NRG3 segregating to all Research Foundation Building, Level 3, 50 Murray Street, Perth, affected family members, thus highlighting this gene WA 6000, Australia. as ‘particularly worthy of follow-up’.13 Our linkage E-mail: [email protected] 3 analysis in the Western Australian Family Study of These authors contributed equally to this study. 14 Received 9 September 2009; revised 5 May 2010; accepted 10 May Schizophrenia ranked the 10q22-q23 region among 2010; published online 15 June 2010 its most significant findings, in which the linked NRG3 and schizophrenia B Morar et al 861 marker D10S1686 is located B1.9 Mb from NRG3.In graphic features and cognitive profiles of the schizo- this study, we aimed at replicating the Chen et al.1 phrenia cases, CD/CS clusters and control subjects are findings in a schizophrenia case–control sample of given in Supplementary Table 2. Anglo-Irish background, and at investigating the potential effects of the associated variants on neuro- Genotyping cognitive endophenotypes. All participants were genotyped for the SNPs that showed the strongest evidence of association in the Chen et al.1 study, rs10883866 and rs6584400. These Materials and methods two SNPs were genotyped with 50-exonuclease allelic Subjects discrimination assays (Taqman SNP genotyping as- A total of 411 schizophrenic patients were recruited says) according to the protocol of Applied Biosystems from admissions to a major psychiatric hospital and (Applied Biosystems, Foster City, CA, USA). After community-based mental health services in Western quality control and cleaning of the genotyping data, Australia. Diagnostic assessment included structured the number of samples available for the statistical interviews using the Schedules for Clinical Assess- analyses was 395 schizophrenia cases (171 CD, 144 ment in Neuropsychiatry15 or the Diagnostic Inter- CS, 80 non-CD/non-CS) and 221 controls for view for Psychoses,16 a review of case records, and a rs10883866, and 391 cases (169 CD, 143 CS, 79 non- structured developmental history. Research diagnoses CD/non-CS) and 220 controls for rs6584400. (lifetime) according to the International Classification of Diseases, Tenth Revision (ICD-10) and DSM-IV Statistical analyses criteria were assigned by two senior clinicians who Statistical analyses were performed using reviewed all relevant information, including the PLINKv1.06.18 We used the w2 goodness-of-fit test video record of the diagnostic interview. The non- (one degree of freedom) for the initial single-marker psychiatric control participants (N = 223) were re- allelic association analysis with schizophrenia in the cruited from the registry of Red Cross blood donors or entire case–control sample, and for the post hoc by random sampling from local telephone directories. analyses with the CD and CS clusters. Analysis of Inclusion of controls was based on a psychiatric allelic association with the quantitative cognitive screening interview and required absence of psycho- traits was conducted using linear regression as tic illness in the individual and in first-degree implemented in PLINK. In addition, association relatives, as well as absence of a history of substance between the marker, disease status and the cognitive dependence, brain trauma or neurological disease. All phenotypes was investigated using a generalized participants were unrelated and were of European linear model framework with SPSSv17 (SPSS Inc., descent ( > 75% Anglo-Irish). The study was approved Chicago, IL, USA). The scores for each cognitive test by the Human Research Ethics Committees of the (standardized IQ points for the general intelligence University of Western Australia and the North tests; raw scores for the memory and attention tests) Metropolitan Area Health Service in Perth, Western were entered as dependent variables, NRG3 geno- Australia. types and diseases status as fixed factors, and where The 634 subjects in our sample underwent extensive appropriate, age, sex and education as covariates. neurocognitive testing with a battery of neurocognitive tests examining general cognitive ability (premorbid and current IQ), learning and episodic verbal memory, Results executive function, speed of information processing Association with schizophrenia and focused sustained attention.14 A form of latent The initial statistical analysis tested for allelic structure analysis (Grade of Membership, GoM;17 association of each SNP with the binary phenotype Supplementary Methods) was used to identify a of schizophrenia. Unlike the Ashkenazi sample,1 we parsimonious number of multivariate patterns of test did obtain evidence of association between rs6584400 responses (‘pure types’) and to simultaneously tag and schizophrenia, with an odds ratio 1.45 (95% each individual’s degree of affinity to those patterns confidence interval (CI) 1.03–2.04) and P = 0.03 for the with a set of grade of membership scores (constrained minor allele (A), whereas SNP rs10883866 showed a to range from 0 to 1).14 Grouping the subjects by their trend in the same direction without reaching nominal highest membership scores for a given pure type significance, odds ratio 1.36 (0.95–1.94), P = 0.10 yielded two relatively homogeneous clusters: one of (Table 1). the cases with pervasive cognitive deficit (CD, N = 180), and another, comprising cases with relatively Association with case clusters based on cognitive spared cognition (CS, N = 148). The remainder fell into patterns a non-CD/non-CS subgroup (N = 83), which consisted To obtain a closer comparison to the findings of almost exclusively of patients aged X45 years with association with the ‘delusion factor’,1 we further circumscribed mild deficits (mainly reduced informa- examined the association between the two poly- tion processing speed) attributable to aging. These morphisms and the two major schizophrenia case non-CD/non-CS subjects were included only in the clusters in our sample. Previous analyses14,19,20 have schizophrenia case–control comparisons. Demo- shown CD cases to be characterized by higher scores

Molecular Psychiatry NRG3 and schizophrenia B Morar et al 862 Table 1 Allelic association of NRG3 SNPs with schizophrenia and with case clusters CD and CS

NCBI SNP Genomic MAF Schizophrenia controls CD controls CS controls reference position NCBI Cases Controls w2 P-value OR w2 P-value OR w2 P-value OR build 36.3 (95% CI) (95% CI) (95% CI)

rs10883866 (C/G)a 83 633 619 0.142 0.109 2.760 0.097 1.356 (0.946–1.944) 1.895 0.169 1.351 (0.879–2.077) 3.193 0.074 1.493 (0.960–2.320) rs6584400 (G/A)a 83 646 506 0.169 0.123 4.631 0.031 1.452 (1.032–2.041) 2.560 0.110 1.397 (0.926–2.106) 5.937 0.015 1.671 (1.103–2.533)

Abbreviations: CD, cognitive deficit; CI, confidence interval; CS, cognitively spared; MAF, minor allele frequency; OR, odds ratio; SNP, single-nucleotide polymorphism. a Major allele/minor allele. Significant results are shown in bold typeface.

on poverty of thought, flat or incongruous effect, paucity of complex delusions, and poor social functioning, whereas CS patients presented with higher scores on Schneiderian first-rank symptoms, florid and often complex delusions, and positive thought disorder. The CD and CS case clusters were tested for association with rs6584400 and rs10883866. The results indicated that the association with schizophrenia in the overall case–control sample was in fact due to association with the CS cluster: rs6584400 odds ratio 1.67 (1.10–2.53), P = 0.02, and a trend for rs10883866 odds ratio 1.49 (0.96–2.32), P = 0.07. No significant results were obtained in the analysis of the CD cluster (Table 1). The parallels between the characteristics of our CS cluster and the Chen et al.1 ‘delusion factor’, also based mostly on first-rank symptoms,2 suggest a common interpretation of the genetic association Figure 1 Mean CPT-DS/dL test scores in schizophrenia cases and controls, stratified by genotypes at rs10883866. findings in the two studies, namely, a contribution A very similar trend is seen for rs6584400 (not shown). of NRG3 sequence variants to a subtype of schizo- phrenia with relatively preserved cognitive function but prone to florid and complex delusions. b = À0.622 (95% CI À1.053 to À0.192) for rs10883866, Association with individual cognitive traits and P = 0.006, b = À0.548 (95% CI À0.934 to À0.163) To dissect further the contribution of NRG3 sequence for rs6584400). The CPT-DS test requires attentional polymorphisms to the schizophrenia phenotype, we focus on rapidly paced, partially degraded visual examined the allelic association of the two SNPs with stimuli (blurred digits). The task engages medial quantitative traits indexing performance in several superior frontal and inferior temporal regions;23 it cognitive domains, with a differential contribution to demands vigilance, prompt perceptual analysis and the CD/CS clusters.14 We found no effect on general efficient signal/noise discrimination. CPT-DS perfor- intelligence or verbal memory (Supplementary Table 1). mance is stable over time, not state dependent and This is in agreement with our previous finding that relatively uninfluenced by antipsychotic medication. high scores on Schneiderian21 first-rank symptoms With an estimated heritability at 0.57, it meets the are correlated with relatively better performance on criteria for an endophenotype.24 cognitive tasks.20 By contrast, there was a significant The effects of the associated minor (‘risk’) alleles association of the minor alleles of the SNPs (G for of the two polymorphisms on different measures of rs10883866 and A for rs6584400), in both cases and cognitive performance were examined further using controls with the discrimination (signal detection) generalized linear models. This analysis confirmed index dL of the degraded-stimulus continuous the effect of the schizophrenia ‘risk’ alleles on performance task (CPT-DS).22 Under the additive significantly improved CPT-DS performance in model, assumed in the allelic association test, patients (P = 0.013 for rs10883866; P = 0.043 for the minor alleles were associated with better perfor- rs6584400) and significantly worse performance in mance in schizophrenic patients (P = 0.007, b = 0.687 controls (P = 0.012 for rs10883866; P = 0.023 for (95% CI 0.195–1.178) for rs10883866, and P = 0.025, rs6584400) (Figure 1; Table 2). The effect size (and b = 0.523 (95% CI 0.070–0.976) for rs6584400) and group mean) for CPT-DS reaction time is significant poorer performance in healthy controls (P = 0.006, for rs6584400 in the schizophrenia group, and

Molecular Psychiatry NRG3 and schizophrenia B Morar et al 863 Table 2 Test performance (mean and s.d.) of SZ patients, including case clusters CD, CS and healthy controls

rs10883866 rs6584400

SZ cases CC (n = 290) CG þ GG (n = 105) P-value d GG (n = 271) GA þ AA (n = 120) P-value d

NART IQ 96.9 (10.2) 97.9 (10.1) 0.377 0.10 97.1 (10.3) 97.1 (9.8) 0.996 0.00 SILS IQ 89.6 (13.8) 91.6 (13.3) 0.142 0.15 89.8 (13.6) 91.1 (13.6) 0.395 0.10 RAVLT iw 20.2 (5.9) 20.4 (6.1) 0.997 0.03 20.4 (6.0) 20.0 (6.1) 0.508 À0.07 RAVLT dw 5.6 (2.9) 5.7 (3.0) 0.875 0.03 5.7 (2.9) 5.4 (3.1) 0.252 À0.10 CPT-IP/dL 3.12 (2.0) 3.33 (1.8) 0.623 0.11 3.14 (2.0) 3.28 (1.9) 0.833 0.07 CPT-DS/dL 4.45 (1.6) 5.22 (1.7) 0.013 0.47 4.50 (1.6) 5.10 (1.7) 0.043 0.36 CPT-DS/RT 520 (76) 504 (65) 0.147 À0.23 523 (76) 498 (65) 0.026 À0.35

CD cases CC (n = 124) CG þ GG (n = 47) P-value d GG (n = 117) GA þ AA (n = 52) P-value d

NART IQ 90.3 (8.8) 91.5 (9.4) 0.423 0.13 90.2 (8.9) 91.2 (9.3) 0.502 0.11 SILS IQ 79.2 (10.1) 82.1 (10.0) 0.082 0.29 79.5 (9.9) 81.4 (10.7) 0.251 0.18 RAVLT iw 16.4 (4.1) 16.1 (4.0) 0.693 À0.07 16.5 (4.2) 15.9 (4.0) 0.558 À0.15 RAVLT dw 4.0 (2.1) 3.8 (2.1) 0.699 À0.10 4.0 (2.1) 3.6 (2.2) 0.240 À0.19 CPT-IP/dL 1.98 (1.5) 2.21 (1.4) 0.338 0.16 1.99 (1.5) 2.14 (1.4) 0.525 0.1 CPT-DS/dL 3.73 (1.6) 4.74 (2.0) 0.027 0.56 3.79 (1.6) 4.49 (2.1) 0.121 0.37 CPT-DS/RT 538 (87) 509 (75) 0.22 À0.36 539 (88) 505 (74) 0.117 À0.42

CS cases CC (n = 105) CG þ GG (n = 39) P-value d GG (n = 98) GA þ AA (n = 45) P-value d

NART IQ 102.0 (7.9) 102.6 (7.3) 0.373 0.08 102.5 (7.5) 101.8 (7.6) 0.861 À0.09 SILS IQ 98.2 (9.4) 98.8 (10.3) 0.955 0.06 98.3 (9.2) 98.8 (10.4) 0.952 0.05 RAVLT iw 23.3 (4.8) 23.5 (4.6) 0.625 0.04 23.4 (4.7) 23.3 (4.7) 0.475 À0.02 RAVLT dw 7.1 (2.6) 7.1 (2.6) 0.469 0.00 7.2 (2.6) 6.9 (2.7) 0.241 À0.11 CPT-IP/dL 3.88 (1.6) 4.08 (1.4) 0.743 0.13 3.88 (1.6) 4.08 (1.5) 0.677 0.13 CPT-DS/dL 5.31 (1.2) 5.64 (1.3) 0.354 0.26 5.30 (1.3) 5.63 (1.2) 0.381 0.26 CPT-DS/RT 503 (61) 490 (56) 0.278 À0.22 506 (60) 485 (57) 0.087 À0.36

Controls CC (n = 176) CG þ GG (n = 45) P-value d GG (n = 171) GA þ AA (n = 49) P-value d

NART IQ 105.6 (8.2) 105.9 (7.1) 0.814 0.04 105.7 (8.1) 105.1 (7.6) 0.632 À0.08 SILS IQ 109.2 (8.1) 107.7 (7.9) 0.344 À0.19 109.4 (8.0) 107.4 (8.1) 0.206 À0.25 RAVLT iw 27.7 (5.2) 26.6 (5.2) 0.183 À0.21 27.8 (5.2) 26.5 (5.1) 0.156 À0.25 RAVLT dw 9.0 (3.0) 8.7 (2.6) 0.392 À0.11 9.1 (3.0) 8.6 (2.5) 0.421 À0.18 CPT-IP/dL 4.92 (1.7) 4.60 (1.8) 0.483 À0.18 4.93 (1.7) 4.57 (1.8) 0.328 À0.21 CPT-DS/dL 5.89 (1.1) 5.17 (1.0) 0.012 À0.69 5.89 (1.1) 5.24 (1.0) 0.023 À0.62 CPT-DS/RT 481 (68) 477 (40) 0.806 À0.07 482 (68) 471 (46) 0.507 À0.19

Abbreviations: CPT-DS/dL, degraded-stimulus continuous performance task, discrimination index (covaried for age, sex and education); CPT-IP/dL, identical pairs continuous performance task, discrimination index (covaried for age, sex and education); d, effect size (Cohen’s d); IQ, intelligence quotient; NART, National Adult Reading Test, estimated premorbid/ prior IQ; RAVLT dw, Rey Auditory Verbal Learning Test, delayed word recall (covaried for age and sex); RAVLT iw, Rey Auditory Verbal Learning Test, immediate word recall (covaried for age and sex); RT, reaction time, in millisecond (covaried for age and sex); SILS, Shipley Institute of Living Scale, estimated current IQ; SZ, schizophrenia. Generalized linear models. The bolded figures indicate comparisons attaining significance levels p0.05. indicates faster response times in the risk allele the cognitively more impaired CD cases, but a trend in carriers compared with non-carriers, consistent with the same direction was also present in the CS cases. the improved CPT performance in this group. We Effect sizes (Cohen’s d) for these associations are in examined and excluded differential test response bias the medium to high range (0.37–0.62). between cases and controls as an explanation of these observations (data not shown). Furthermore, none of the covariates had a significant effect on the CPT-DS Discussion discrimination index dL, (age was only associated with CPT-DS reaction time). At the case cluster level, Our study replicates previously reported findings a considerable performance gain (improved CPT-DS of association between SNPs within NRG3 and the discrimination index) was statistically significant for clinical phenotype of schizophrenia, and extends

Molecular Psychiatry NRG3 and schizophrenia B Morar et al 864 these results by showing that the association with with ‘supranormal’ expression of a gene product that rs6584400 and rs10883866 is mainly driven by a boosts cognitive performance in individuals with subset of schizophrenia cases characterized by preexisting deficiency, but affects adversely indivi- relatively spared cognition, florid delusions and duals functioning at ‘peak’ level by tipping them over Schneiderian first-rank symptoms (no association was into the descending arm of the inverted U. A caveat to found with cases exhibiting pervasive CD). A novel this interpretation is that the leap from genetic finding of potential interest, revealed by analysis of association with a neurocognitive endophenotype to individual cognitive traits, is the significant statistical functional effects of the associated variants on a interaction of the two associated SNPs with signal/ specific gene/protein function is problematic in the noise discrimination on the CPT-DS, in which cases case of NRG3, whose biological roles remain unclear. and controls carrying the minor (‘risk’) alleles show NRG3 is abundantly expressed in the developing and exactly opposite test performance trends: improve- adult brain.28,29 The encoded transmembrane protein ment (normalization) in cases and worsening (dete- differs from other members of the NRG family rioration) in controls. This observation is strongly (including NRG1) in a number of structural and suggestive of an inverted U-curve dose–response functional characteristics, which suggest distinct pattern, a well-known phenomenon described pre- nonoverlapping functions.29 The extracellular part viously in studies of prefrontal cortex neuronal contains an epidermal (EGF)-like excitability in response to varying concentrations of domain (31% identity with the NRG1 EGF-like dopamine associated with the val/met polymorph- domain). It does not contain Ig-like or kringle isms in the COMT gene.25 Inverted-U response domains, and instead has a unique Ala/Gly-rich patterns have also been reported for the effects of region with multiple sites for O-linked glycosylation. dopamine D1 receptor stimulation on spatial working The putative intracellular domain (ICD) shows only memory,26 and for calcineurin overexpression on the 13% sequence identity to the NRG1 ICD. Finally, transition from short- to long-term memory.27 The NRG3 binds to the ErbB4 receptor , but seemingly paradoxical impact of the minor alleles of is not an ErbB2- and ErbB3- ligand.29 rs10883866 and rs6584400 on CTP-DS in schizophre- Similar to NRG1, extensive alternative splicing and nia cases and healthy controls is therefore compatible different promoter usage result in multiple NRG3

5′ NRG3 gene (RefSeq NM_01010848) 3′

83,625,05 84,736,915

gnf1h05312_at gnf1h10765_at rs6584400 rs10883866

12 3 45 6 7891011121314151617 NRG3-a 8kb 13kb

ATG NRG3-b 6kb ATG NRG3-d 5kb 7kb ATG LD block NRG3-c

NRG3-e

NRG3-f

NRG3-g

NRG3-h-u

Figure 2 Genomic structure and nomenclature of NRG3 transcripts (as listed in NCBI AceView) and relative positions of the associated SNPs. The NRG3 reference sequence NM_01010848 corresponds to isoform NRG3-a. Exons are numbered sequentially based on their genetic position. Exons are shown as shaded boxes and introns as horizontal lines (not to scale). The numbers under the horizontal lines indicate the distances between the two SNPs and the alternative 50 exons of isoforms a, b and d. The position of the translation initiation codons is as in AceView. The approximate locations of probes gnf1h05312_a and gnf1h10765_at used in the NRG3 expression profiling (see main text) allow conclusions on the differential expression of isoforms a, b and d.

Molecular Psychiatry NRG3 and schizophrenia B Morar et al 865 isoforms, with eight alternative transcripts currently of the North Metropolitan Health Area mental health listed in the NCBI AceView. The associated SNPs services in Western Australia for assistance in patient rs10883866 and rs6584400 are located B13 kb apart recruitment. The study was supported by Grant and close to the alternative first exons of NRG3-a, -b #37580400 and #37580900 of the National Health and -d (Figure 2), pointing to possible involvement in and Medical Research Council of Australia to Profes- the regulation of the expression or the alternative sor A Jablensky and Professor L Kalaydjieva, with splicing of these isoforms. Our examination of NRG3 funding contribution from the North Metropolitan expression data (BioGPS30) and bioinformatics analy- Health Area, Perth, Western Australia. sis indicate that the NRG3-a transcript (detected by URLs used in this study: reporter gnf1h05312) is widely expressed with no http://www.cbil.upenn.edu/cgi-bin/tess/tess evidence of tissue specificity, whereas transcripts http://asp.ii.uib.no:8090/cgi-bin/CONSITE/consite NRG3-b and NRG3-d (reporter gnf1h10765) show http://www.umd.be/HSF/ markedly higher expression in fetal and adult brain, http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/ especially in the amygdala, prefrontal cortex and http://genome.ucsc.edu/ cingulate cortex. NRG-b was originally described by 31 http://blast.ncbi.nlm.nih.gov/Blast.cgi Carteron et al. as a fetal brain-specific isoform, http://biogps.gnf.org/#goto = welcome containing novel primate-specific exons. Our bioin- http://www.spss.com/worldwide/ formatics analysis showed that the first two alter- native exons of NRG3-b and NRG3-d (numbered exons 2, 3 and 5 in Figure 2) are absent from the References expressed sequence tag (EST) databases of chimpan- zee, rhesus monkey and rodents, and thus appear to 1 Chen PL, Avramopoulos D, Lasseter VK, McGrath JA, Fallin MD, Liang KY et al. Fine mapping on chromosome 10q22-q23 be human specific. Open reading frame and protein implicates Neuregulin 3 in schizophrenia. Am J Hum Genet homology analysis indicated that translation of 2009; 84: 21–34. NRG3-b starts from exon 2 (unlike the NRG3-d 2 McGrath JA, Avramopoulos D, Lasseter VK, Wolyniec PS, Fallin transcript, where it does not start until exon 10), MD, Liang KY et al. Familiality of novel factorial dimensions of producing a 499 amino-acid protein, whose unique 53 schizophrenia. Arch Gen Psychiatry 2009; 66: 591–600. 3 Falls DL. : functions, forms, and signaling strategies. amino-acid N-terminal sequence has no similarity to Exp Cell Res 2003; 284: 14–30. other currently known eukaryotic . These 4 Carpenter G. ErbB-4: mechanism of action and biology. Exp Cell data make NRG3-b a particularly interesting isoform Res 2003; 284: 66–77. with possibly novel neuronal functions that may be 5 Stefansson H, Sigurdsson E, Steinthorsdottir V, Bjornsdottir S, Sigmundsson T, Ghosh S et al. and susceptibility to modulated by the associated polymorphisms or by schizophrenia. Am J Hum Genet 2002; 71: 877–892. another correlated variant. Bioinformatics analyses do 6 Law AJ, Lipska BK, Weickert CS, Hyde TM, Straub RE, Hashimoto not predict any changes, caused by the associated R et al. Neuregulin 1 transcripts are differentially expressed in SNP alleles, in transcription factor-binding sites or schizophrenia and regulated by 50 SNPs associated with the splicing regulation. SNPs rs10883866 and rs6584400 disease. Proc Natl Acad Sci USA 2006; 103: 6747–6752. 7 Tan W, Wang Y, Gold B, Chen J, Dean M, Harrison PJ et al. are however located within an linkage disequili- Molecular cloning of a brain-specific, developmentally regulated brium (LD) block, spanning the alternative NRG3 neuregulin 1 (NRG1) isoform and identification of a functional promoters and first exons, and part of intron 1. The promoter variant associated with schizophrenia. J Biol Chem 2007; association signal is thus likely to flag a functional 282: 24343–24351. 8 Fallin MD, Lasseter VK, Wolyniec PS, McGrath JA, Nestadt G, variant elsewhere in this region that should be further Valle D et al. Genomewide linkage scan for schizophrenia investigated. susceptibility loci among Ashkenazi Jewish families shows Overall, our results are compatible with the evidence of linkage on chromosome 10q22. 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