Calcineurin is a calcium-dependent protein phosphatase that plays an important role in cellular responses and calcium signal transduction.1 Several studies have suggested that calcineurin is one of the key molecules in signal transduction in the brain and that dysfunction of calcineurin signaling could be linked to schizophrenia.2, 3 Calcineurin is a heteromeric protein complex consisting of a catalytic subunit (calcineurin A) and a regulatory subunit (calcineurin B).1, 4 PPP3CC encodes the calcineurin γ-catalytic subunit and is located on chromosome 8p21.3 within a few cM of markers reported to be linked to schizophrenia.5, 6, 7,10 Gerber et al.8 reported genetic associations of the PPP3CC gene with schizophrenia in populations from the United States and South Africa. However, only one replication study has been published, and these associations were not confirmed in 457 Japanese schizophrenic patients and 429 control subjects.9 HapMap data indicated that a haplotype block spans almost the entire PPP3CC region in Japanese and European populations. The single nucleotide polymorphism (SNP) haplotype reported to be associated with schizophrenia by Gerber et al.8 is located in the haplotype block. Therefore, we examined the associations in a large case–control study of 1645 schizophrenic patients and 1673 control subjects. This sample size has a power >0.98 to replicate the haplotypic association with the same magnitude as that reported by Gerber et al.,8 assuming an α value=0.05, two-tailed, a haplotype relative risk of 1.23, and haplotype frequency of 0.26 or effect size of 0.1. The haplotype frequency in the Japanese population was reported by Kinoshita et al.9
All subjects were of Japanese descent and were recruited from the main island of Japan. The study included 1645 unrelated patients with schizophrenia (mean age±s.d., 48.2±14.5 years; 899 men and 745 women) diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). Control subjects were 1673 mentally healthy unrelated subjects (age, 47.9±14.3, 886 men and 787 women) without self-reported family histories of mental illness within second-degree relatives. The subjects studied by Kinoshita et al.9 were not included in the present study. The present study was approved by the Ethics Committees of the University of Tsukuba, Niigata University, Fujita Health University, Nagoya University, Okayama University and Teikyo University and all participants provided written informed consent. To rule out population stratification between patients and controls in the present study, 35 SNPs that are not in linkage disequilibrium (LD) with each other were genotyped in all samples and analyzed with the STRUCTURE program 2.0.5 No stratification was observed.
We genotyped five SNPs. SNP1 (rs10108011, CC21 in Gerber et al.8) and SNP2 (rs2461491, CCS3 in Gerber et al.8) were selected because Gerber et al.8 reported nominally significant allelic association of these SNPs with schizophrenia. SNP4 (rs2449340), SNP3 (rs2461490) and SNP5 (rs1116085) were genotyped to distinguish common haplotypes with frequencies ⩾5% in the haplotype block based on HapMap data of the Japanese population. SNPs were genotyped with the TaqMan SNP Genotyping Assay (Applied Biosystems, Foster City, CA, USA) and ABI PRISM 7900HT Sequence Detection System (Applied Biosystems).
Deviation from predicted Hardy–Weinberg frequency was examined by χ2-test. Individual allelic and genotypic associations were examined by Fisher's exact test. LD between polymorphisms and haplotypic associations were evaluated with Haploview software version 3.32.6 To deal with multiple testing, allelic associations were evaluated by permutation tests implemented in Haploview. The genotype distributions were evaluated by the Cochran–Armitage test without correction for multiple testing.
The genotype and allele distributions of the five SNPs in the patient group and control group are shown in Table 1. Distributions of these SNPs did not differ significantly from Hardy–Weinberg equilibrium. All five SNPs showed nominally significant allelic associations with schizophrenia and permutation tests revealed significant allelic associations of SNP1 (P=0.012), SNP3 (P=0.005) and SNP4 (P=0.013) with schizophrenia. The genotype distributions suggest that the minor allele of each SNP is likely to have an additive effect in the susceptibility to schizophrenia. These five SNPs are in LD; however, the LD is not complete. Therefore, these associations were not caused by a single SNP in the present study. As shown in Table 2, there were only two common haplotypes constructed from these SNPs. The most common haplotype in the control group was observed less frequently in the patient group (P=0.034) and the second most common haplotype in the control group tended to be more frequently in the patient group (P=0.079). A global P-value for the 5 SNP haplotype was 0.055.
Gerber et al.8 found associations of the G allele of SNP1 (CC21) and the A allele of SNP2 (CCS3) with schizophrenia by transmission disequilibrium test. In the present study, we found that the G allele of SNP1 and the A allele of SNP2 occurred more frequently in the patient group than in the control group (P=0.003, odds ratio (OR)=1.15 for SNP1; P=0.02, one-sided, OR=1.10 for SNP2). Gerber et al.8 found that the most common haplotype was overtransmitted to patients in their US population. According to the data reported by Kinoshita et al.,9 only two SNPs (SNP1 and SNP2 in the present study) are sufficient to distinguish the associated haplotype reported by Gerber et al.8 from other haplotypes in the Japanese population. When haplotypes were constructed with SNP1 and SNP2, the haplotype that was the most common haplotype associated with schizophrenia in the US population was the second most common haplotype in our Japanese population. The second most common haplotype was more frequent in the patient group than in the control group (P=0.036, two-sided) in the present study (Table 2). Therefore, the present study replicates the allelic and haplotypic associations found in the US population described by Gerber et al.,8 although the ORs of the haplotype were lower in the present study (OR=1.12) than in the study by Gerber et al.8 (OR=1.23).
Kinoshita et al.9 failed to replicate these associations. The OR of the G allele of SNP1 for schizophrenia reported by Kinoshita et al.9 was 1.11, which is similar to that (OR=1.15) in the present study, although the ORs of the A allele of SNP2 and the second most common haplotype for schizophrenia were 1.0 in the Kinoshita et al.9 study and 1.09 and 1.10 in the present study, respectively. Recently, Yamada et al.7 reported a nominally significant association of SNP2 (CCS3, rs2461491) and a trend towards association of SNP1 (CC21, rs10108011) with schizophrenia in Japanese family based-association analysis.
The present study replicated the allelic and haplotypic associations of PPP3CC with schizophrenia. Thus, an association between genetic variations of PPP3CC and schizophrenia appears to exist in US and Japanese populations. However, the ORs of 1.10–1.15 observed in the present study indicate that the associations are weak and will be difficult to replicate without large sample sizes. Further studies are needed to evaluate wither alterations in calcineurin signaling contribute to the pathogenesis of schizophrenia.
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Horiuchi, Y., Ishiguro, H., Koga, M. et al. Support for association of the PPP3CC gene with schizophrenia. Mol Psychiatry 12, 891–893 (2007). https://doi.org/10.1038/sj.mp.4002019
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