Association studies and gene expression analyses of the DISC1-interacting molecules, pericentrin 2 (PCNT2) and DISC1-binding zinc finger protein (DBZ), with schizophrenia and with bipolar disorder^†‡

INTRODUCTION

Several genetic studies including genome-wide linkage scans and association studies have implicated a potential susceptibility region for psychiatric disorders on chromosome 1q, especially involving the Disrupted-In-Schizophrenia 1 (DISC1) gene [Blackwood et al., 2001; Ekelund et al., 2001; Hennah et al., 2003]. DISC1 has been identified as a disrupted gene by a balanced translocation (1; 11)(q42.1; q14.3) that cosegregated with major psychiatric disorders in a large Scottish kindred [St Clair et al., 1990; Millar et al., 2000; Blackwood et al., 2001; Millar et al., 2001].

DISC1 variations have been implicated in the positive symptoms of schizophrenia [DeRosse et al., 2007; Szeszko et al., 2008], and have been reported to influence the prefrontal function [Prata et al., 2008; Szeszko et al., 2008]. DISC1-transgenic mice have been found to exhibit brain abnormalities [Kvajo et al., 2008; Pletnikov et al., 2008] and behavioral phenotypes [Clapcote et al., 2007; Hikida et al., 2007; Li et al., 2007; Pletnikov et al., 2008] reminiscent of schizophrenia. Recent studies report the association of DISC1 polymorphisms with schizophrenia [Hennah et al., 2003; Kockelkorn et al., 2004; Callicott et al., 2005; Sachs et al., 2005; Zhang et al., 2006; Chen et al., 2007; Qu et al., 2007; Saetre et al., 2008] and with bipolar disorder [Hodgkinson et al., 2004; Thomson et al., 2005].

DISC1 is a multifunctional protein capable of interacting with several cytoskeletal and centrosomal proteins via distinct functional domains [Millar et al., 2003; Miyoshi et al., 2003; Morris et al., 2003; Ozeki et al., 2003; Brandon et al., 2004]. Through these interactions, DISC1 functions as a component of the intracellular machinery that integrates multiple functions including intracellular transport, neuronal cell signaling, and neuronal migration and architecture [Hennah et al., 2006; Ishizuka et al., 2006; Porteous et al., 2006]. The impact of DISC1 across several psychiatric diagnostic categories, thus implicates a complex interaction among loci both within the gene itself, and between DISC1 and its multitude of binding partners.

Previously, we reported the fasciculation and elongation protein-zeta 1 [FEZ1; Miyoshi et al., 2003], pericentrin 2 [PCNT2; Miyoshi et al., 2004], and DISC1-binding zinc finger protein [DBZ, also known as zinc finger protein 365 (ZNF365); Hattori et al., 2007] as interacting partners of DISC1. FEZ1, PCNT2, and DBZ bind to overlapping regions of DISC1; in addition, the domain of DISC1 interacting with DBZ is close to the translocation breakpoint in DISC1 [Hattori et al., 2007]. FEZ1 has been found to be associated with schizophrenia in Japanese cohorts [Yamada et al., 2004]. Lipska et al. 2006 reported reduced mRNA levels of FEZ1 in the postmortem brain tissues of schizophrenia patients. We observed elevated expression of PCNT2 in the postmortem brains and in the peripheral blood lymphocytes (PBL) of bipolar disorder patients, compared to healthy controls; however, there was no significant association of PCNT2 with bipolar disorder [Anitha et al., 2008]. So far, there are no reports on the association of DBZ with any psychiatric disorders.

Here, we examined the genetic association of PCNT2 with schizophrenia in a case–control study of Japanese cohorts. We also examined the genetic association of DBZ with schizophrenia and with bipolar disorder. In addition, we compared the mRNA levels of DBZ in the postmortem prefrontal cortices of schizophrenia, bipolar disorder and control groups.

MATERIALS AND METHODS

Association Study

Subjects

This study was approved by the Ethics Committee of Hamamatsu University School of Medicine; patient confidentiality was maintained at all times, and written informed consent was obtained from all the participants. For the association study of PCNT2 and DBZ with schizophrenia, we collected blood samples from 180 schizophrenia patients [age 53.58 ± 12 years (mean ± SD); male/female 99:81] and 201 healthy control subjects (age 40.54 ± 13 years; male/female 98:103); there was no significant difference in sex distribution between the case and control groups (χ² = 1.48; df = 1; P = 0.223). We examined the association of DBZ with bipolar disorder, in blood samples collected from 238 bipolar disorder patients (age 51.28 ± 13.18 years; male/female 131:107) and 240 age- and gender-matched healthy controls (age 51.49 ± 10.73 years, male/female 120:120); the controls samples were independent of the samples used in the association study of PCNT2 and DBZ with schizophrenia. All the subjects were recruited from a geographical area located in central Japan. Best-estimate lifetime diagnoses of patients were made by direct interview with experienced psychiatrists, according to DSM-IV criteria [American Psychiatric Association, 1994]. Control subjects were recruited from hospital staff and company employees documented to be free from any psychiatric problems.

Genomic DNA was extracted from whole blood using QIAamp DNA blood kit (QIAGEN, Maryland, MD).

Marker selection

The genomic structures of PCNT2 (chromosomal location 21q22.3) and DBZ (10q21.2) are based on the UCSC March 2006 draft assembly of human genome (http://www.genome.ucsc.edu). SNPs for association study were selected using the Applied Biosystems (ABI; Foster City, CA) software SNPbrowser 1.0.19; SNPs reported in databases like National Centre for Biotechnology information (NCBI dbSNP: http://www.ncbi.nlm.nih.gov/SNP) and Japanese Single Nucleotide Polymorphisms (JSNP: http://snp.ims.u-tokyo.ac.jp) were also referred to. On the basis of their genomic locations and minor allele frequencies (MAF > 0.1) in Japanese population, 19 SNPs of PCNT2 (Fig. 1) and 10 SNPs of DBZ (Fig. 2), were chosen for the association study, aiming at an average spacing of one common SNP at every 3–5 kb.

Genotyping

Assay-on-Demand™ or Assay-by-Design SNP genotyping products (ABI) were used to score SNPs, based on the TaqMan assay method [Ranade et al., 2001]. Genotypes were determined in ABI PRISM 7900 Sequence Detection System (SDS), and analyzed using SDS v2.0 (ABI).

Statistical analysis

All the genotyping results were tested for Hardy–Weinberg Equilibrium (HWE). The statistical significance of variations in allelic and genotypic distributions between the schizophrenia and control groups was evaluated using Fisher's exact test. Haplotype associations were examined using the COCAPHASE program of UNPHASED v2.403 [Dudbridge, 2003; http://www.litbio.org]. Expectation maximization (EM) algorithm was used to resolve uncertain haplotypes, infer missing genotypes and provide maximum-likelihood estimation of frequencies. LD parameters were estimated using the ldmax option of GOLD v1.1.0 [Excoffier and Slatkin, 1995; Abecasis and Cookson, 2000; http://www.well.ox.ac.uk/asthma/GOLD/]. LD strength was estimated in terms of the squared correlation coefficient [r²; Devlin and Risch, 1995].

RESULTS

DISCUSSION

In this study, we observed SNP- and haplotype- associations of the DISC1-interacting molecule PCNT2 with schizophrenia. SNP04, SNP14, and SNP19 showed significant allelic and genotypic associations. The allelic association of SNP04 and the haplotypic associations involving SNP04 withstand multiple testing correction. Deviation from HWE was observed for SNP04 in schizophrenia patients; heterozygotes of patients were lower than the expected values based on HWE. Since SNP04 showed association, which withstands correction for multiple testing, the deviation from HWE in patients, but not in controls, may be viewed as an additional evidence of association. We observed a strong LD between SNPs 14 and 19. SNP19, located in exon 45, is a non-synonymous SNP with Ser/Arg substitution. Since Ser is a neutral amino acid and Arg is a strong basic amino acid, the substitution may be suggested to exert an influence on the protein structure of PCNT2. To the best of our knowledge, this is the first report of a genetic association study of PCNT2 with schizophrenia.

Abnormalities of brain morphogenesis, especially cortical dysplasia, have been observed in individuals with partial deletion of chromosome 21q22.3 harboring the PCNT2 gene [Yao et al., 2006]. PCNT2, an integral component of the pericentriolar material, interacts with pericentriolar material 1 (PCM1), another member of the DISC1 interactome [Kamiya et al., 2008], for its centrosomal localization, and to accomplish its function in microtubule organization [Balczon et al., 1994; Doxsey et al., 1994; Kubo et al., 1999; Dammermann and Merdes, 2002]. Recently, PCM1 has been implicated in susceptibility to schizophrenia [Gurling et al., 2006]. In the developing cerebral cortex, suppression of PCM1 has been reported to lead to neuronal migration defects [Kamiya et al., 2008]. Thus, centrosomal proteins such as PCNT2 may be suggested to have an important role in cortical development, and therefore, in the pathogenesis of neurodevelopmental disorders like schizophrenia. The PCNT2-binding region of DISC1 overlaps with the region interacting with FEZ1 [Miyoshi et al., 2004], a schizophrenia susceptibility gene that plays a vital role in axonal outgrowth and fasciculation [Yamada et al., 2004].

In our previous study, elevated expression of PCNT2 was observed in the brain and in the PBL of bipolar disorder patients, compared to controls; however, there was no significant difference in PCNT2 expression between control and schizophrenia groups [Anitha et al., 2008]. We suggested that a possible apoptotic process in the brain, resulting from over-expression of PCNT2, may underlie the pathogenesis of bipolar disorder. However, in the subsequent association study, none of the SNPs showed association with bipolar disorder.

Considering the results from the genetic analyses of PCNT2 in bipolar disorder and schizophrenia, it might be suggested that DISC1 and its interacting molecules are involved in psychiatric symptoms that cross diagnostic boundaries. The translocation in the original Scottish family [St Clair et al., 1990] demonstrated that the disruption of DISC1 gene, although sufficient to predispose an individual to psychiatric disorder, was, in itself, insufficient to predict any particular disorder. Although the family members showed predominantly schizophrenic symptoms, they also manifested a wide spectrum of other psychiatric phenotypes. In addition to schizophrenia, DISC1 has also been found to be associated with other neuropsychiatric disorders including bipolar disorder [Hodgkinson et al., 2004; Thomson et al., 2005], major depression [Hashimoto et al., 2006], and autism [Kilpinen et al., 2008]. Accumulating evidence show that malfunction of DISC1 lead to brain structural and functional abnormalities [Faulkner et al., 2008; Kvajo et al., 2008; Pletnikov et al., 2008; Prata et al., 2008; Szeszko et al., 2008]. Thus, it may be suggested that the basis of the shared risk for major neurodevelopmental disorders could be centered on DISC1, while its interaction with other members of the interactome lead to different phenotypic outcomes.

Several other DISC1-interacting molecules, including phosphodiesterase 4B [PDE4B; Numata et al., 2008], 14-3-3ε [YWHAE; Ikeda et al., 2008], nudE nuclear distribution gene E homolog 1 [NDE1; Hennah et al., 2007], nudE nuclear distribution gene E homolog like 1 [NDEL1; Burdick et al., 2008], and activating transcription factor 4 [ATF4; Qu et al., 2008], have been suggested as potential susceptibility genes for schizophrenia. The DISC1 interactome is expanding with the discovery of novel interacting proteins, and this information could be useful in the identification of potential pathways involving DISC1, in the pathology of major psychoses.

In this study, no significant genetic associations were observed for DBZ, the other DISC1-interacting molecule, with schizophrenia or with bipolar disorder; in addition, there was no significant difference between the DBZ mRNA levels of control, schizophrenia and bipolar postmortem brains.

In conclusion, we suggest a possible role of PCNT2 in the pathogenesis of schizophrenia. Abnormalities of PCNT2 may lead to defects in microtubule function; during the development of the central nervous system, this dysfunction might result in alterations in neuronal migration, axonal extension, and neurite outgrowth, subsequently leading to impaired neurodevelopment. Our study is limited by small sample size; therefore, the results should be treated with caution, until replication using a larger sample size. It would also be interesting to study the role of PCNT2 in neurodevelopment.