Schizophrenia is a common psychiatric disorder involving approximately 1% of the population worldwide. Family, twin, and adoption studies suggest genetic factors contribute to this illness (Lang et al. 2007; McGuffin et al. 1995). Meta-analysis including 18 genome scans revealed strong evidence at chromosomal regions 22q, 8p, and 13q as the susceptibility loci (Badner and Gershon 2002), and another meta-analysis of 20 genome-wide scans suggested regions of chromosomes 2q, 5q, 3p, 11q, 6p, 1q, 22q, 8p, 20q, and 14p as the significant loci (Lewis et al. 2003). Chromosomal abnormalities in patients with schizophrenia may provide useful information regarding the susceptible loci (Bassett et al. 2000). Disrupted in schizophrenia 1 (DISC1) gene isolated from a large Scottish family with t(1;11)(q42.1;q14.3) and high risk of schizophrenia in velo-cardio-facial syndrome (VCFS) with a 22q11 deletion are good examples (Arinami 2006; Millar et al. 2000; Murphy 2002). Some linkage and association studies support that schizophrenia could be associated with DISC1 and genes at 22q11 (Chubb et al. 2008; Liu et al. 2002; O’Donovan et al. 2003; Shifman et al. 2002).

Microarray technologies have now become practical tools for detection of submicroscopic copy number changes. Using custom bacterial artificial chromosome (BAC) microarray (4,219 BACs at 0.7-Mb resolution), we analyzed 59 patients with schizophrenia. Chromosomal abnormalities found in this study are presented.

Materials and methods


A total of 59 subjects (31 men and 28 women) with schizophrenia were recruited in this study. Forty-one had family history. Diagnosis was made for each patient according to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) criteria on the basis of unstructured interviews and information from medical records. Participants were excluded if they had organic brain diseases, including head injury and infection, or if they met criteria for alcohol/drug dependence. After written informed consent, genomic deoxyribonucleic acid (DNA) from lymphoblastoid cell line (LCL) of all patients was isolated using DNA isolation systems [Quick Gene-800 (Fujifilm, Tokyo, Japan) and/or NA-3000 (Kurabo, Osaka, Japan)]. Micorarray comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) analysis were performed using materials from LCL. Peripheral blood lymphocytes were reevaluated in ID394, MZ102, and MZ127, but could not be obtained for reexamination in ID67, ID345, or ID391. Only parents of ID345 subjects were available for familial analysis. Other parents or sibs could not be evaluated. Experimental protocols were approved by the Committee for Ethical Issues at Yokohama City University School of Medicine.

Microarray CGH analysis

Comparative genomic hybridization analysis was performed using our custom BAC microarray containing 4,219 BAC clones, as previously described (Saitsu et al. 2008). In brief, after complete digestion using DpnII, subject’s DNA was labeled with Cy-5 dCTP (Amersham Biosciences, Piscataway, NJ), and reference DNA was labeled with Cy-3 deoxycytidine triphosphate (dCTP) (Amersham Biosciences) using the DNA random primer Kit (Invitrogen). Prehybridization, probe hybridization, washing, and drying steps for arrays were preformed on a Tecan hybridization station HS400 (Tecan Japan, Kawasaki, Japan). Arrays were scanned by GenePix 4000B (Axon Instruments, Union City, CA, USA) and analyzed using GenePix Pro 6.0 (Axon Instruments). The signal intensity ratio between patient and control DNA was calculated from the data of the single-slide experiment using the ratio of means formula (F635 mean − B635 median/F532 mean − B532 median) according to GenePix Pro. 6.0. The standard deviation was calculated from the data of all clones. We regarded the signal ratio as abnormal if it ranged out of ±3 standard deviations (SD). Clones showing abnormal copy number were checked to see whether they were in the position of previously registered copy number variations using the Human Genome Variation Database ( (Iafrate et al. 2004). Unregistered changes were considered for further confirmation. Genome position was based on the UCSC genome browser Human Mar. 2006 (hg18) assembly.

Fluorescence in situ hybridization

To confirm status of clones with a possibly abnormal copy number, FISH was performed, as previously described (Shimokawa et al. 2005). BAC DNA was labeled with SpectrumGreen-11-deoxyuridine triphosphate (dUTP) or SpectrumOrange-11-dUTP (Vysis, Downers Grove, IL, USA) by nick translation and denatured at 70°C for 10 min. Probe-hybridization mixtures (15 μl) were applied on chromosomes, incubated at 37°C for 16–72 h, then washed and mounted in antifade solution (Vector, Burlingame, CA, USA) containing 4'-6'-diamidino-2-phenylindole (DAPI). Photographs were taken on an AxioCam MR CCD fitted to Axioplan2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany). In ID394 and MZ102, we counted 100 interphase nuclei to validate the number of cells with X aneuploidy, as well as 30 metaphases.

Results and discussion

Six patients showed chromosomal abnormalities (10%, 6/59) (Table 1). As we could not obtain materials from most of their parents and sibs, heritability of the abnormalities could not fully be investigated. According to our experiences of microarray CGH analysis of more than 200 Japanese patients associated with mental-retardation-related disorders, all chromosomal abnormalities described here were never detected. Thus, it is less likely that the changes are polymorphisms.

Table 1 Summary of six patients with (sub)microscopic chromosomal rearrangements

In ID67, arr cgh 5p12p12(RP11-1037A10 → RP11-929P16) × 1, 12pterp12.1(GS-124K20 → RP11-12D15) × 3 was found. A 23.2-Mb copy number gain from 12pter to 12p12.1 (chr12: 0–23,176,547 bp) was detected (Fig. 1a). G-banded chromosomal analysis revealed that 12pter-12p12.1 was translocated to 13p11 (Fig. 1a). The 12p12.1 translocation breakpoint was localized between two BAC clones, RP11-35A22 and RP11-349E13, by FISH (chr12: 23,176,547–23,861,227 bp) (data not shown). Additionally, a 1.7-Mb submicroscopic deletion at 5p12 from RP11-1037A10 to centromeric sequence gap (chr5: 44,778 009–46,437 323 bp) was also found in this patient (Fig. 1a). The 12p trisomy is recognized as multiple congenital anomalies/mental retardation (MCA/MR) syndrome characterized by dysmorphic face, heavy birth weight, foot deformities, hypotonia, and mental retardation (Allen et al. 1996). A previous study suggested that partial duplication of 12pter-p13.2 is sufficient for recognizable phenotype of 12p trisomy (Rauch et al. 1996). The 23.1-Mb duplicated region contained at least 229 genes. Dysmorphic facial features of 12p trisomy (Rauch et al. 1996) were not recognized in this patient. It is interesting that ID67 also had a 1.7-Mb deletion at 5p12, containing two genes, MRPS30 (the mitochondrial ribosomal protein S30 gene) and HCN1 (the hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 gene). It is worth noting linkage findings within the vicinity of this region in Costa Rican schizophrenia samples (Cooper-Casey et al. 2005). HCN1 is an intriguing candidate gene. The general Hcn1 loss in mice led to a defect in the learning of motor tasks, and specific deletion of the gene in forebrain neurons resulted in an unexpected enhancement of spatial learning and memory (Herrmann et al. 2007; Nolan et al. 2003). ID67 (a 72-year-old male) developed psychotic symptoms (delusions, hallucinations, and psychomotor excitement) at age 20 years. He had received electroconvulsive therapy many times and continuous sleep therapy until antipsychotic medication (chlorpromazine) was introduced at age 23 years. Since the onset of the illness, he has spent most of his life in psychiatric hospitals because of exacerbations of psychotic episodes and marked deterioration of social functions. Intelligent quotient (IQ) at 72 years was 72. He had no family history of major psychosis within the first-degree relatives.

Fig. 1
figure 1

Results of microarray comparative genomic hybridization (CGH) in ID67 (a) and MZ127 (b). Chromosomes 5 (upper) and 12 (lower) are displayed (a). The karyotype is arr cgh 5p12p12(RP11-1037A10 → RP11-929P16) × 1, 12pterp12.1(GS-124K20 → RP11-12D15) × 3. Partial karyotype clearly shows a 12pter-p12.1 segment is translocated to 13p11. Chromosome 11 is presented (b) . The karyotype is arr cgh 11p13p13(RP11-51J14) × 3. RP11-51J14 at 11p13 is duplicated

In MZ127, arr cgh 11p13p13(RP11-51J14) × 3 was recognized. Duplication of RP11-51J14 at 11p13 (chr11: 33,302,231–33,302,660 bp) was confirmed by FISH using LCL and peripheral blood lymphocytes (Fig. 1b). According to the genome browser, the size of RP11-51J14 is 430 bp, indicating that the reference sequence is somehow odd and may contain a deletion overlapping with RP11-51J14 as FISH signals of RP11-51J14 are strong enough to detect on a microscope, suggesting that its size is at least >10 kb. HIPK3 (the homeodomain interactive protein kinase 3 gene) was corresponding to this clone. HIPK3 is a Fas-associated death-domain (FADD)-interacting kinase involved in apotosis (Curtin and Cotter 2003), remaining unknown in relation to schizophrenia. MZ127 (42-year-old woman) presented with epilepsy at age 12 years and has had recurrent depression and slight mania since age 29 years. She began to exhibit auditory hallucination, not synchronizing with mood swing, and was diagnosed as schizophrenia at 40 years. Her mother and sister suffered from major depression and schizophrenia, respectively. Her father committed suicide induced by depression.

In ID345, arr cgh 17p12p12(RP11-78J16 → RP11-103P10) × 1 was found, as previously described (Ozeki et al. 2008). The deletion from RP11-246F16 to RP11-103P10 (chr17: 14,061,460–15,374,745 bp) is 1.4 Mb, compatible with the common deletion found in approximately 85% of hereditary neuropathy with liability to pressure palsies (HNPP; OMIM #162500) (Stogbauer et al. 2000). The deletion was also identified in his father’s chromosomes from peripheral blood lymphocytes. He suffered from auditory hallucination and delusion of persecution and received antipsychotic treatment at age 19. Neurological examination did not reveal any manifestations of HNPP (Ozeki et al. 2008). Pareyson et al. (1996) reported that about 25% of individuals with HNPP deletion are asymptomatic. The peripheral myelin protein 22 gene (PMP22) may be a candidate that is not only expressed in the peripheral nervous system but also in the central nervous system (Ohsawa et al. 2006), this being supported by linkage studies of psychotic bipolar disorder (Park et al. 2004) and schizophrenia (Owen et al. 2004). No family history regarding psychiatric disorders was observed in ID345.

Entire X chromosome copy number aberration was suspected in two patients, ID394 and MZ102 (data not shown). FISH analysis using RP11-65B15 at Xq23 revealed mosaic monosomy of chromosome X: mos45,X[41]/46,XX[59] in ID394 and mos45,X[84]/46,XX[16] in MZ102. X aneuploidy is well known to be seen in elderly normal females (Stone and Sandberg 1995). ID394 and MZ102 were 67 and 38 years old, respectively. The fraction of cells with X monosomy was very high (84% and 41%) in lymphoblastoid cell lines of these patients. Reevaluation of peripheral blood lymphocytes showed mos 45,X[7]/46,XX[98] in ID394 and mos 45,X[4]/46,XX[96] in MZ102. These findings may support involvement of X-chromosomal abnormalities in schizophrenia (Kumra et al. 1998; Kunugi et al. 1999), but mosaic X monosomy is also found in age-matched normal controls (Toyota et al. 2001). ID394 (a 67-year-old woman) developed psychotic symptoms (paranoid delusion and hallucinations) at age 31 years when she delivered her second child. Since then, she had been admitted to a psychiatric hospital three times (each for a few months). She quit her job as a pharmacist after the onset of the illness and has lived as a housewife. She has been managed by antipsychotic medications without major exacerbation for the past decade. The second child developed schizophrenia-like symptoms, including social withdrawal and lack of volition. MZ102 (a 38-year-old woman) exhibited psychomotor excitement and was diagnosed as having schizophrenia at age 23 years. Her father showed psychotic disorder, and her uncle had schizophrenia. In ID391, arr cgh Ypterq11.23(GS-98C4 → RP11-214M24) × 3, Yq11.23qter(RP11-263C17 → RP11-80F8) × 1 was identified. FISH analysis using BACs, RP11-74L17 at PAR1, RP11-375P13 at Yp11.2, RP11-655E20 at Yq11.2, and RP11-80F8 at Yq12 revealed the isodicentric Y chromosome [46,X,idic(Y)(q11.2)] (data not shown). Previously, two cases of idic(Yp) were reported in schizophrenia, although idic(Yp) is one of the most common rearrangements in the Y chromosome (Nanko et al. 1993; Yoshitsugu et al. 2003). ID391 (a 29-year-old man) developed hallucinations and abnormal sense of self at age 21 years, when he was admitted to a psychiatric hospital for 3 months. Since then, his illness has been well controlled by antipsychotic medication. He quit university after the onset of illness and has not obtained a job, suggesting deterioration of functioning. His younger sister (apparently without the Y chromosome) has schizophrenia. Thus, contribution of sex chromosomal abnormalities found in this study is less likely.

Four microarray CGH studies of schizophrenia were reported: 1,440 BAC microarray for 30 patients, 2,460 BAC microarray for 35 patients, a tiling-path microarray consisting of ~36,000 BACs for 93 patients, and high-resolution microarrays (85,000–2,100,000 oligos) for 150 patients (Kirov et al. 2008; Moon et al. 2006; Walsh et al. 2008; Wilson et al. 2006). We could not replicate any similar abnormalities, though microarray platforms were all different in terms of clones and genome coverage. In this study, (sub)microscopic rearrangements were detected in 10% of patients. Similarly, 15% of patients analyzed by high-resolution microarrays were found to possess submicroscopic chromosomal changes (Walsh et al. 2008). Various kinds of recurrent and unique submicroscopic changes were found in 10–17% of idiopathic mental retardation and 7% of autism by microarray CGH analysis (Miyake et al. 2006; Sebat et al. 2007; Zahir and Friedman 2007). Importantly, a 22q13 deletion (in autism) involving Sh3 and multiple ankyrin repeat domains 3 (SHANK3), whose point mutation was related to autism (Durand et al. 2007), strongly supports this approach as one of the most powerful and straightforward strategies in neuropsychiatric disorders.

In conclusion, microarray technologies could provide good opportunity to identify chromosomal copy number changes in relation to mental and psychiatric disorders, and genome-wide copy number survey should be considered in genetic studies of these disorders.