American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 144B:113 –116 (2007)
Brief Research Communication Schizophrenia Is Not Associated With the Functional Candidate Gene ERBB3: Results From a Case-Control Study Tetsufumi Kanazawa,1,2* Stephen J. Glatt,1,3 Atsushi Tsutsumi,2 Hiroki Kikuyama,2 Jun Koh,2 Hiroshi Yoneda,2 and Ming T. Tsuang1,3,4,5 1
Department of Psychiatry, Center for Behavioral Genomics, University of California, San Diego, La Jolla, California Department of Neuropsychiatry, Osaka Medical College, Osaka, Japan 3 Veterans Medical Research Foundation, San Diego, California 4 Harvard Departments of Epidemiology and Psychiatry, Harvard Institute of Psychiatric Epidemiology and Genetics, Boston, Massachusetts 5 Veterans Affairs San Diego Healthcare System, San Diego, California 2
Increasing evidence has supported the hypothesis of a neurodevelopmental component in the etiology of schizophrenia. Recently, several independent microarray gene expression studies have revealed downregulated expression of myelinrelated genes in the postmortem brains of schizophrenia patients. Complete myelination of the cortex has been observed to occur in late adolescence and early adulthood, which is typically the age of onset of schizophrenia. ERBB3 is a gene which has not only been found to be downregulated in schizophrenia simultaneously in three microarray studies, but also is a strong candidate because of its potential role in neurodevelopment as a receptor of NRG1. Therefore, we performed association analysis of seven nonsynonymous SNPs in this gene. Two SNPs in ERBB3 (rs773123 and rs2271188) were polymorphic in our samples, neither of which showed significant evidence of association with the illness (P ¼ 0.639 and 0.561, respectively). Because replication across such studies is notoriously difficult, the microarray evidence implicating ERBB3 still strongly supports some role of this gene in schizophrenia. However, our failure to find genetic association suggests that the differential expression of ERBB3 in schizophrenia may be environmentally driven, or involve cis- or trans-acting genetic factors beyond the boundaries of the gene itself. ß 2006 Wiley-Liss, Inc.
KEY WORDS:
association; case-control; ERBB3; myelin; gene; schizophrenia
Please cite this article as follows: Kanazawa T, Glatt SJ, Tsutsumi A, Kikuyama H, Koh J, Yoneda H, Tsuang MT. 2007. Schizophrenia Is Not Associated With the Functional Candidate Gene ERBB3: Results From a CaseControl Study. Am J Med Genet Part B 144B:113–116.
*Correspondence to: Tetsufumi Kanazawa, M.D., Department of Psychiatry, University of California, San Diego, 9500 Gilman Drive, Mail Code 0603, La Jolla, CA 92093. E-mail:
[email protected] Received 13 January 2006; Accepted 4 May 2006 DOI 10.1002/ajmg.b.30367
ß 2006 Wiley-Liss, Inc.
Increasing evidence has supported the hypothesis that schizophrenia is a neurodevelopmental disorder [Weinberger, 1987; Rapoport et al., 2005]. This theory suggests that schizophrenia is the behavioral outcome of an aberration in neurodevelopmental processes that begin long before the onset of clinical symptoms and are caused by a combination of environmental and genetic factors. Longitudinal brain-imaging studies comparing schizophrenic and control subjects age 10 or older have generally supported this hypothesis [DeLisi, 1997]. The search for schizophrenia risk genes has proceeded through the evaluation of candidate genes, positionally cloned chromosomal segments, and cytogenetic aberrations. A large number of putative susceptibility loci and genomic regions of interest have been identified; however, no causal genes with a proven mechanism of action have yet been revealed [Singh et al., 2004]. Recently several independent microarray gene expression studies have observed downregulated expression of myelin-related genes in the postmortem brains of schizophrenia patients [Hakak et al., 2001; Tkachev et al., 2003; Aston et al., 2004]. Myelination, which is performed by oligodendrocytes, is a critical component of neuronal development, and thus its potential involvement in the etiology of schizophrenia is plausible. This hypothesis is further supported by the fact that demyelination is strongly associated with white matter hyperintensities, which are frequently seen in the brain of schizophrenia patients. Also the magnetization transfer ratio (a putative index of the degree of myelination) has been found to be significantly decreased in both right and left temporal lobes from schizophrenia patients [Foong et al., 2000]. A similar phenomenon is seen in frontal lobes of schizophrenia patients [Davis et al., 2003]. Furthermore, complete myelination of the cortex has been observed to occur in late adolescence and early adulthood, which is typically the age of onset of schizophrenia. This late myelination is most evident in the frontal and temporal lobes [Bartzokis, 2002; Corfas et al., 2004]. While dysregulation of myelin-related genes in schizophrenia is a common finding in gene expression microarray studies, specific genes have not typically been observed in common across reports. A strong exception is ERBB3, which codes for avian v-erb-b2 erythroblastic leukemia viral oncogene homolog 3. Three novel papers using microarray methods have simultaneously reported that ERBB3 showed altered expressed patterns in postmortem brain tissue samples from schizophrenia patients. Further supporting the functional candidacy of the ERBB3 gene is the fact that its protein, ERBB3, can form a heterodimer with ERBB2 and serve as a receptor for the neuregulin 1 protein, the gene for which (NRG1) is presently considered one of the strongest positional candidates for the disorder. NRG1 is not just a positional
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candidate gene, but also a physiological candidate gene due to its involvement in the glutamate system, which has been repeatedly implicated in the pathology of schizophrenia [Duan et al., 2005]. In addition, numerous processes like neurotransmitter receptor expression, oligodendrocyte development, myelination, and onset of puberty in the human nervous system are considered to be regulated by NRG1. Several traits observed in schizophrenia patients are also thought to occur through the abnormal functioning of NRG1. For example, NRG1 regulates neuronal migration and formation of neuroglia which may influence the occurrence of heterotopias and enlarged ventricles in schizophrenia. NRG1 also influences the onset of puberty which may regulate the observed age of onset of schizophrenia [Corfas et al., 2004]. That these effects of NRG1 might be mediated or moderated by ERBB3 has not yet been evaluated, but the evidence implicating NRG1 polymorphisms and ERBB3 expression in schizophrenia and the known relationships between NRG1 and ERBB3 do suggest that ERBB3 should be considered a strong candidate gene for the illness. In this study, we therefore tested for association between schizophrenia and ERBB3. We examined all seven of the known nonsynonymous single nucleotide polymorphisms (SNPs) in the gene, which covered a span from exon 3 (SNP 1) to exon 28 (SNP 7). One hundred ninety-three schizophrenia patients and 121 normal control subjects were investigated. The schizophrenia group was recruited from the inpatients of mental hospitals or the outpatients of mental health clinics. All hospitals and clinics are located in the western area of Japan,
with all participants in this study being ethnically Japanese. The average age of the schizophrenia group was 52.8 years, consisting of 123 males and 70 females. The controls were unrelated individuals with an average age of 33.5 years and consisted of 60 males and 61 females. There was a significant age difference between the case and control groups (t(312) ¼ 14.69, P < 0.001), with cases being older than controls (mean SD; 52.8 11.8, 33.5 10.4, respectively). To address this age difference, we performed our analyses on the entire case and control groups, but also performed an additional analysis using the 121 youngest cases (mean SD; 45.9 8.8) to serve as a more appropriately age-matched group for comparison. The results of this analysis were no different from those using the full case sample; however, this younger subgroup of schizophrenia patients was still significantly older than the control group (t(240) ¼ 9.98, P < .001). All schizophrenia patients were diagnosed with DSM-IV criteria by two trained psychiatrists. Informed consent was obtained from each subject. The ERBB3 gene is located on chromosome 12q13, and is comprised of 28 exons and various known SNPs. Genomic DNA was extracted from peripheral lymphocytes. Real-time PCR (Lightcycler1) was used for genotyping. Experimental details on the analysis of each SNP are reported in Table I. Deviation of the genotype counts from Hardy–Weinberg equilibrium was tested using a chi-square goodness-of-fit test. The statistical significance of differences in the genotype and allele frequencies between patients and controls was determined by Chi-square tests with a ¼ 0.05.
TABLE I. Investigated SNPs in the ERBB3 Gene SNP no.
Contig position
dbSNP rs#
dbSNP allele
Protein residue
SNP1
18622164
rs984896
G
Gly
SNP2
18629881
rs12320176
T G
Val Ser
SNP3
18633934
rs3891921
A C
Asn Ser
SNP4
18637299
rs17118292
G T
Asn Ile
SNP5
18638304
rs773123
G T
Met Cys
SNP6
18638329
rs2271188
A A
Ser His
SNP7
18638927
rs11171743
G A
Arg Ser
SNP1 SNP2 SNP3 SNP4 SNP5 SNP6 SNP7
G Probe 1 LC Red 640-GGGTCCCTCGCCCCA-Phosphate LC Red 640-ACACTGAGCTTCTCTGGGPhosphate LC Red 640-CAAGCTGTGACACATGTAAGTPhosphate LC Red 640-CCAGATGATGGACTTAAAAGGCTCTGGCTCTAGGA-Phosphate LCRed640- ACA TTG ACA CTT TCT CCT GGA GCT CAG CCT CAG-Phosphate LC Red 640-CTATCTCCGCGTGGCC-Phosphate LC Red640-GTTGCCGATTCATATATTCATAGTCTTCATCTGGAGTTGTGCC- Phosphate
Forward primer
Reverse primer
GGCCATGAATGAATTCTCTACT
AGGAACCATCGGGAACTGA
GCTCATTGCCATTGAGTTATAC
TTATGCCAGTGGTTCACCTA
GGAGTGTGGATCCCTGAG
ACAGGACCCTGCTTCTAC
ATGGTGAATGTAGATTTCTCCC
CCTCTCAGCAACAGAACT
TGG GAA TGG TAG GCG CTA
CTC TAC ACC CAA TGC CAC
GCTGAGCTCCAGGAGAAA
CATAACCGTTGACATCCTCTT
TCCACCCTGTACCCATCA
TCATCTCTTCATACCCTTGCTC
Gly Probe 2 ACATGACGAAGATGGCAAACTTCCCATCGTAGACC-Fluorescein ACTCACCTGTGATCTCCCGTACTGTCCGGAA-Fluorescein AAAGTCATTGAGGACAAGAGTGGACGGCAGAGTTT-Fluorescein CCTGGTTCATGGGCATGTA-Fluorescein TCC TGC ACC GGC TCC TA-Fluorescein CAGACTGTGGCGCTGGGAATGGTAGGC-Fluorescein CAGGACCACCTCCATCT- Fluorescein
Case-Control Study of ERBB3 and Schizophrenia
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TABLE II. Results of Allele Frequency of Both SNP5 and SNP6 Genotype
Allele
Cys/Cys
Cys/Ser
Ser/Ser
Cys
Ser
28 28
26 30
6 3
82 86
38 36
56 31
12 6
167 95
79 45
His/His
55 33 Genotype His/Arg
Arg/Arg
His
Arg
57 60
3 1
0 0
117 121
3 1
116 68
7 2
0 0
239 136
7 2
a
SNP 5 (rs 773123) Normal (n ¼ 121) Male (n ¼ 60) Female (n ¼ 61) Schizophrenia (n ¼ 193) Male (n ¼ 123) Female (n ¼ 70) b
SNP 6 (rs 2271188) Normal (n ¼ 121) Male (n ¼ 60) Female (n ¼ 61) Schizophrenia (n ¼ 193) Male (n ¼ 123) Female (n ¼ 70) a b
Allele
P-value; 0.844, 0.985, 0.712 (All samples, male only, female only). P-value; 0.557, 0.847, 0.642 (All samples, male only, female only).
Two SNPs in ERBB3 (SNP5 and SNP6) were polymorphic in our sample, while the remaining five SNPs evaluated were not polymorphic. Neither SNP5 nor SNP6 showed any significant allele- or genotype-frequency differences between the patient and control groups (Table II). We attempted statistical analysis with regard to gender between case and control, the result still lacks to show the significance. It is not unreasonable to deduce that the lack of significant associations with schizophrenia detected for SNP6 could be due to the low frequency of its minor allele. Given our sample size and the frequency of the alleles of SNP5, we had adequate power (b > 0.80) to detect a SNP having a relatively minor influence on risk for schizophrenia (relative risk ¼ 1.4); yet, no such effect was observed. Thus, although our sample size is still relatively small, conditions were very favorable for detecting a significant association if such a relationship existed between ERBB3 and schizophrenia, we are confident our failure to find any relationship is not due to a lack of power based upon the formula derived by Sham et al. [2000] and implemented in the Genetic Power Calculator (http://pngu.mgh.harvard.edu/purcell/gpc/cc2.html). Besides there was a significant age difference between the groups; however, it is unlikely that our failure to find a significant association is solely attributable to this fact. The mean age difference between the groups, while significant, was not extremely large (12.4 years), so the possibility of a cohort effect is minimal. In addition, the control group’s mean age and age range (20–61) indicates that most of those subjects were beyond the period of risk for developing schizophrenia. Also, these control subjects were screened for any psychiatric symptoms, and none showed any signs, further suggesting that these controls will not develop schizophrenia, despite their relatively young age. In regard to haplotype variation, we have also investigated differences in haplotype frequencies of two polymorphic SNPs between schizophrenia and control samples. The result did not show statistically significant evidence for association (Pearson-chi2 ¼ 0.813, P ¼ 0.937). Instead our evidence simply suggests that a genetic influence of the seven investigated SNPs of ERBB3 on schizophrenia is not supported. ERBB3 can be considered a strong candidate gene for schizophrenia based on its interaction with NRG1 and its replicated pattern of dysregulated expression in postmortem brain tissue from schizophrenia patients in three studies. Because microarray studies are prone to false-positive results,
and because replication across such studies is notoriously difficult, the latter line of evidence implicating ERBB3 strongly supports some role of this gene in schizophrenia. However, the present data suggest that perhaps the dysregulation of ERBB3 in schizophrenic brain is not regulated by a genetic polymorphism in the gene itself; rather we suggest its expression may be regulated by environmental factors, cis- or trans-acting elements, or by a polymorphism in or near the gene that simply is not in strong linkage disequilibrium with the evaluated SNPs. Further evaluation of this relationship in larger casecontrol samples, and in family-based samples, should help clarify the nature of the relationship between ERBB3 and schizophrenia. ACKNOWLEDGMENTS The authors thank Ms. Michiyo Ban, Kazuyo Emura MD, Jun Sakai MD for encouragement and helpful comment, Shahid Salaria PhD, Gursharan Chana PhD, and Sharon ‘‘Doc’’ Chandler PhD for reviewing our manuscript. REFERENCES Aston C, Jiang L, Sokolov BP. 2004. Microarray analysis of postmortem temporal cortex from patients with schizophrenia. J Neurosc Res 77(6): 858–866. Bartzokis G. 2002. Schizophrenia: Breakdown in the well-regulated lifelong process of brain development and maturation. Neuropsychopharmacol 27(4):672–683. Corfas G, Roy K, Buxbaum JD. 2004. Neuregulin 1-erbB signaling and the molecular/cellular basis of schizophrenia. Nat Neurosci 7(6):575–580. Davis KL, Stewart DG, Friedman JI, Buchsbaum M, Harvey PD, Hof PR, Buxbaum J, Haroutunian V. 2003. White matter changes in schizophrenia: Evidence for myelin-related dysfunction. Arch Gen Psychiatry 60(5):443–456. DeLisi LE. 1997. Is schizophrenia a lifetime disorder of brain plasticity, growth and aging? Schizophr Res 23(2):119–129. Duan J, Martinez M, Sanders AR, Hou C, Krasner AJ, Schwartz DB, Gejman PV. 2005. Neuregulin 1 (NRG1 ) and schizophrenia: Analysis of a US family sample and the evidence in the balance. Psychol Med 35(11): 1599–1610. Foong J, Maier M, Barker GJ, Brocklehurst S, Miller DH, Ron MA. 2000. In vivo investigation of white matter pathology in schizophrenia with magnetisation transfer imaging. J Neurol Neurosurg Psychiatry 68(1): 70–74.
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