Genetic analysis of the calcineurin pathway identifies members of the EGR gene family, specifically EGR3, as potential susceptibility candidates in schizophrenia Kazuo Yamada*, David J. Gerber†, Yoshimi Iwayama*, Tetsuo Ohnishi*, Hisako Ohba*, Tomoko Toyota*, Jun Aruga‡, Yoshio Minabe*§, Susumu Tonegawa†¶, and Takeo Yoshikawa*ʈ** Laboratories for *Molecular Psychiatry and ‡Comparative Neural Development, RIKEN Brain Science Institute, Saitama 351-0198, Japan; †Howard Hughes Medical Institute and RIKEN–MIT Neuroscience Research Center, The Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA 02139; §Department of Psychiatry and Neurology, Kanazawa University School of Medicine, Ishikawa 920-8641, Japan; and ʈCore Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan Contributed by Susumu Tonegawa, December 7, 2006 (sent for review September 22, 2006) The calcineurin cascade is central to neuronal signal transduction, cineurin is particularly enriched in the CNS, where it plays a critical and genes in this network are intriguing candidate schizophrenia role in the regulation of a diverse array of neuronal functions (5, 6). susceptibility genes. To replicate and extend our previously re- Interestingly, calcineurin is positioned downstream of dopaminer- ported association between the PPP3CC gene, encoding the cal- gic signaling (7) and is involved in NMDA receptor-mediated cineurin catalytic ␥-subunit, and schizophrenia, we examined 84 synaptic plasticity (8) and could therefore provide an important SNPs from 14 calcineurin-related candidate genes for genetic as- functional link between these two neurotransmitter systems. To sociation by using 124 Japanese schizophrenic pedigrees. Four of further explore the involvement of calcineurin dysfunction in these genes (PPP3CC, EGR2, EGR3, and EGR4) showed nominally schizophrenia, we have tested for genetic association of a subset of significant association with schizophrenia. In a postmortem brain calcineurin-related candidate genes with schizophrenia in Japanese study, EGR1, EGR2, and EGR3 transcripts were shown to be down- population samples as an extension of the prior study (4). Our regulated in the prefrontal cortex of schizophrenic, but not bipolar, results support the association of PPP3CC with schizophrenia and patients. These findings raise a potentially important role for EGR provide genetic and biological evidence for the involvement of genes in schizophrenia pathogenesis. Because EGR3 is an attractive altered early growth response (EGR) transcription factor signaling candidate gene based on its chromosomal location close to PPP3CC in schizophrenia pathogenesis. within 8p21.3 and its functional link to dopamine, glutamate, and neuregulin signaling, we extended our analysis by resequencing Results the entire EGR3 genomic interval and detected 15 SNPs. One of Family-Based Association Screening. In this study, we examined the ,these, IVS1 ؉ 607A3G SNP, displayed the strongest evidence for following 14 calcineurin-related genes: PPP3CA, PPP3CB disease association, which was confirmed in 1,140 independent PPP3CC, PPP3R1, PPP3R2, RYR3 (ryanodine receptor 3), EGR1, case-control samples. An in vitro promoter assay detected a pos- EGR2, EGR3, EGR4, FKBP5 (FK506 binding protein 5), FKBP1A sible expression-regulatory effect of this SNP. These findings sup- (FK506 binding protein 1A), CDK5 (cyclin-dependent kinase 5), port the previous genetic association of altered calcineurin signal- and PPP1R1B (DARPP32). We analyzed a total of 84 SNPs in 124 ing with schizophrenia pathogenesis and identify EGR3 as a Japanese schizophrenia pedigrees consisting of 374 individuals. The compelling susceptibility gene. potential functional interactions of these genes are illustrated in supporting information (SI) Fig. 4. The results of the genetic ͉ ͉ ͉ genetic association immediate early gene postmortem brain enhancer analyses are shown in SI Table 3 along with the SNP information. We detected nominally significant transmission disequilibrium for chizophrenia is a devastating psychiatric disease with a complex PPP3CC (8p21.3), EGR2 (10q21.3), EGR3 (8p21.3), and EGR4 Sgenetic etiology. Despite a high heritability of Ϸ80%, the major (2p13.2), providing confirmatory evidence for the previously re- genetic components underlying disease susceptibility and pathology ported genetic association of PPP3CC and schizophrenia in patient have remained elusive, although significant progress in this area has samples from the United States and South Africa (4) (Table 1). The been made in recent years (1). Along with genetic studies, a large adjacent SNPs in the gene might be in linkage disequilibrium (LD), body of pharmacological evidence suggests that alterations in the so applying a Bonferroni correction for the multiple markers would regulation of or interactions between the dopamine and glutamate be overly conservative (Bonferroni-corrected P ϭ 0.0756). Apply- GENETICS neurotransmitter systems may comprise important contributing ing a permutation procedure for multiple test correction yielded a factors in the pathophysiology of schizophrenia (2). Recently, we globally significant P value for the multiple markers tested in the have provided evidence suggesting that variation in the PPP3CC gene, encoding the calcineurin A ␥-subunit, contributes to schizo- phrenia susceptibility based on behavioral characterization of Author contributions: K.Y., D.J.G., S.T., and T.Y. designed research; K.Y., Y.I., T.O., H.O., T.T., CNB1 knockout mice (3) and collaborative human genetic associ- J.A., and Y.M. performed research; K.Y. and Y.I. analyzed data; K.Y. and T.Y. wrote the ation studies (4). These studies provided support for the idea that paper; D.J.G. revised the manuscript; and T.T. and Y.M. collected human samples. alterations in calcineurin function contribute to schizophrenia The authors declare no conflict of interest. pathogenesis. Abbreviations: LD, linkage disequilibrium; PDT, pedigree disequilibrium test; DLPFC, dor- solateral prefrontal cortex; EGR, early growth response; NFAT, nuclear factor of activated Calcineurin (protein phosphatase 2B) is a member of the serine/ T cells. threonine protein phosphatase family and the only known phos- ¶To whom correspondence may be addressed at: The Picower Institute for Learning and 2ϩ phatase that can be activated by Ca and calmodulin. Calcineurin Memory, Massachusetts Institute of Technology, 46-5285, 77 Massachusetts Avenue, functions as a heterodimer consisting of a catalytic subunit, cal- Cambridge, MA 02139. E-mail: [email protected]. cineurin A (CNA), and regulatory subunit, calcineurin B (CNB). **To whom correspondence may be addressed. E-mail: [email protected]. Three mammalian isoforms of CNA [CNA␣ (PPP3CA), CNA␤ This article contains supporting information online at www.pnas.org/cgi/content/full/ (PPP3CB) and CNA␥] and two of CNB [CNB1 (PPP3R1) and 0610765104/DC1. CNB2 (PPP3R2)] have been identified. The expression of cal- © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610765104 PNAS ͉ February 20, 2007 ͉ vol. 104 ͉ no. 8 ͉ 2815–2820 Downloaded by guest on September 25, 2021 Table 1. Family-based association analysis of SNPs in calcineurin-related genes PDT Gene (chromosomal location) rs ID Our SNP ID Distance, kb SUM AVE Over-/undertransmitted allele Under PDT, SUM gives more weight to larger families, whereas AVE places equal weight on all families. NA, not applicable. *These results are consistent with those of our prior study (4). first screening (adjusted P ϭ 0.0353). In addition to the support for EGR3 are located in separate LD islands on the genome (Fig. 1). PPP3CC association, this study provides evidence for the role of In addition, the association signal appears to be divided into two EGR family genes as potential predisposing factors to schizophre- major peaks roughly corresponding to the two gene regions (Fig. 1). nia. Of the three nominally associated EGR genes, EGR3 lies close Therefore, the two genes at 8p21.3, PPP3CC and EGR3, are likely to PPP3CC on the short arm of chromosome 8 within a 252-kb to confer independent risk for schizophrenia. genomic interval, which raises the question of whether these two genes contribute to schizophrenia onset independently. We ad- Transcript Expression in Postmortem Brains. To examine whether dressed this issue by dividing the families into two groups according altered expression of PPP3CC or EGR genes could be associated to the transmission status of the PPP3CC risk allele in probands and with schizophrenia pathophysiology, we performed quantitative reanalyzing the data. The results supported independent roles for RT-PCR assays for mRNA levels of the five genes in the the two genes, because the significance of association between dorsolateral prefrontal cortex (DLPFC; Brodmann’s area 46) of EGR3 and schizophrenia increased in families in which the PPP3CC schizophrenia, bipolar disorder, and control brains. The DLPFC risk allele was not transmitted [the most significant P ϭ 0.0004 for has been implicated in the pathology of schizophrenia by a SNP M228 with pedigree disequilibrium test (PDT) software number of studies. Interestingly, the expression of EGR1, EGR2, (PDT-AVE)]. Next, we scrutinized the 564-kb genomic region of and EGR3 was down-regulated in a schizophrenia-specific man- 8p21.3 that encompasses both genes by genotyping 49 SNP markers ner, whereas PPP3CC mRNA levels