Differential Expression of Disrupted-in-Schizophrenia (DISC1) in Bipolar Disorder

Differential Expression of Disrupted-in-Schizophrenia (DISC1) in Bipolar Disorder Kazuhisa Maeda, Evaristus Nwulia, Jennifer Chang, Rishi Balkissoon, Koko Ishizuka, Haiming Chen, Peter Zandi, Melvin G. McInnis, and Akira Sawa Background: The disruption of the disrupted-in-schizophrenia (DISC1) gene segregates with major mental illnesses in a Scottish family. Association of DISC1 with schizophrenia has been reported in several ethnic groups, and now recently with mood disorder. Methods: A family-based association study of DISC1 and bipolar disorder (BP) in 57 bipolar pedigrees was conducted. Then, we examined possible association of bipolar disorder with DISC1 mRNA expression in human lymphoblasts. We also studied the correlation of several clinical features with the levels of DISC1 mRNA expression. Results: Haplotype analysis identified one haplotype (HP1) that was overtransmitted to the BP phenotype (p ⫽ .01) and a second haplotype that was undertransmitted (HP2). There was a gender influence in the transmission distortion, with overtransmission of HP1 to affected females (p ⫽ .004). A significant decrease in DISC1 mRNA expression was observed in lymphoblasts from affected HP1 group compared to those from unaffected subjects with the HP2 (p ⫽ .006). Further, a higher number of manic symptoms correlated with lower levels of DISC1 expression (p ⫽ .008). Conclusions: These results suggest that decreased mRNA levels of DISC1 expression, associating with the risk haplotype, may be implicated in the pathophysiology of bipolar disorder. Key Words: DISC1, bipolar disorder, manic, gene expression, lymphoblasts

T

here is accumulating evidence that Disrupted-In-Schizophrenia-1 (DISC1) may play a substantial role in pathophysiology of schizophrenia. The DISC1 gene was originally identified from the breakpoint of a chromosomal (1;11)(q42.1; q14.3) translocation in a large Scottish family (Millar et al 2000). In the pedigree, the chromosomal abnormality clearly segregates with occurrence of major mental illness, including schizophrenia (SZ), bipolar disorder (BP), and major depression (Blackwood et al 2001). The linkage of the DISC1 mutation to both SZ and mood disorders in the Scottish pedigree is consistent with observations of a genetic overlap between SZ and BP, as evidenced by the identification of similar loci in both disorders (Berrettini 2000a, 2000b). Linkage and association studies have implicated DISC1 and the 1q42 region in SZ from other populations (Callicott et al 2005; Cannon et al 2005; Ekelund et al 2001, 2004; Hennah et al 2003; Hwu et al 2003; Thomson et al 2005; Zhang et al 2006). There are now at least 4 independent findings of modest linkage evidence to the 1q42 region in BP (Curtis et al 2003; Detera-Wadleigh et al 1999; Macgregor et al 2004; McInnis et al 2003). Recent case-control studies of BP reported an association between DISC1 and the disease (Hodgkinson et al 2004; Thomson et al 2005). Furthermore, a genomewide linkage study in schizoaffective disorder results in the most remarkable linkage at 1q42 close to the DISC1 locus (Hamshere et al 2005). Within the central nervous system, DISC1 is highly expressed in the hippocampus and the cerebral cortex (Austin et al 2003, 2004;

From the Department of Psychiatry and Behavioral Sciences (KM, EN, JC, RB, KI, HC, PZ, MGM, AS), Neuroscience (AS), and Program in Cellular Molecular Medicine (AS), Johns Hopkins University School of Medicine; and the Department of Mental Health (PZ), Johns Hopkins University School of Public Health, Baltimore, Maryland. Address reprint requests to Akira Sawa, M.D., Ph.D., Johns Hopkins University School of Medicine, Department of Psychiatry, 1800 E. Jefferson Street, CMSC 8-117, Baltimore, MD 21287; E-mail: [email protected]. Received January 26, 2005; revised July 28, 2005; revised March 23, 2006; accepted March 23, 2006.

0006-3223/06/$32.00 doi:10.1016/j.biopsych.2006.03.032

Ozeki et al 2003; Schurov et al 2004). Emerging studies indicate that DISC1 is also expressed in several peripheral tissues, but the extent of DISC1 expression is not yet fully characterized (Millar et al 2000; Ozeki et al 2003). DISC1 is likely to play important roles in neurodevelopment and synaptic modulations by participating in neuronal migration, dendritic organization, neurogenesis, and cAMP signaling (Brandon et al 2004; Kamiya et al 2005; Millar et al 2005; Miyoshi et al 2003; Morris et al 2003; Ozeki et al 2003; Sawa and Snyder 2005) all of which may be relevant to the neuropathophysiology of psychiatric disorders including SZ and BP (Coyle and Duman 2003). Here we report the levels of DISC1 expression in lymphoblasts in BP subjects and unaffected family controls. Association studies with DISC1 identify a haplotype overtransmitted to affected BP females, who showed lower levels of DISC1 mRNA expression. Furthermore, we examined a correlation of clinical features of affected subjects to the levels of DISC1 mRNA expression.

Methods and Materials Subjects The research subjects for this study were taken from a large ongoing linkage study in BP (McInnis et al 2003). Briefly, these multiplex pedigrees (the Hopkins/Dana pedigrees) were ascertained on the presence of a treated BPI proband with two affected first-degree relatives. Affection status included BPI, BPII, recurrent major depression, and schizoaffective disorder, manic type. The recent genome-wide scan of these pedigrees identified a susceptibility locus on 1q41. The peak marker, D1S549, was 12 Mb centromeric from the DISC1 gene, however the region of significance is broad and encompasses the DISC1 gene. Similarly, the 1q42 peak identified by Macgregor et al (2004) was centromeric from DISC1. From the larger collection, there were 57 BP pedigrees selected for genotyping. The selection included pedigrees with either both parents present, or pedigrees that included an unaffected sibling as well as two affected siblings. The total sample consisted of 297 genotyped subjects and is described in detail in Table 1. All subjects were collected in an outpatient setting, or in the home of the study subject, most were clinically euthymic at the time of interview and sampling, none were described as being in a current episode at the BIOL PSYCHIATRY 2006;60:929 –935 © 2006 Society of Biological Psychiatry

930 BIOL PSYCHIATRY 2006;60:929 –935

K. Maeda et al

Table 1. Subjects (57 Families) in this Study: Originally Ascertained for Genetic Linkage Analyses

BPI BPII RUP SAM Unaffected Uncertain Total

Total

Male

Female

62 50 35 5 64 81 297

29 24 12 3 26 46 140

33 26 23 2 38 35 157

BP, bipolar; RUP, recurrent unipolar disorder; SAM, schizoaffective manic.

time of interview and sampling. The unaffected sibling served as the “control.” All subjects gave informed consent (reviewed and approved by the institutional review board at Johns Hopkins University, Baltimore, Maryland) to participate in genetic studies of bipolar disorder. Direct Sequencing DNA from 10 bipolar subjects was chosen to identify polymorphisms in the DISC1 gene. They were selected from pedigrees with the strongest evidence for linkage to chromosome 1q42, based on the output from GENEHUNTER. Each coding exon of DISC1 with 50 bp of flanking intron was amplified using primers described in Table 2A. Exon 2 was too large to be amplified by a single

polymerase chain reaction (PCR), and was subsequently sequenced in three reactions. Most of PCR conditions were the following: 80 ng of genomic DNA, .4 ␮M of each primer, 400 ␮M dNTPs, 1.5 mM MgCl2, 1 unit TaqDNA polymerase (invitrogen), 2.5 ␮l of a PCR buffer (200 mM Tris-HCl, 500 mM KCl) and up to 25 ␮l with sterile water. An initial denaturation step of 94°C for 7 min was followed by 40 cycles of 94°C for 45 sec, 55°C for 30 sec and 72°C for 30 sec. Detailed information for amplification is available upon request. The resulting PCR products were purified by spin column (QIAGEN, Valencia, California) and were preformed with BigDye Terminator cycle sequencing kit (Applied Biosystems, Foster City, California). The purified products were sequenced using ABI PRISM 370 DNA sequencer (Applied Biosystems). The sequences were aligned using Sequencher (Gene Codes Corporation, Ann Arbor, Michigan). Genotyping All samples were genotyped using a 5= exonuclease assay (TaqMan, Applied Biosystems) and the ABI 7900HT sequence detection system (Applied Biosystems). For six single nucleotide polymorphisms (SNPs) (rs1538975, rs1954175, rs1407598, rs1000731, rs821653, rs3524), we used Assays-on-Demand kits (Applied Biosystems), and for the remaining six SNPs we ordered custom made, Assays-by-Design kits (Applied Biosystems). The sequence information of the primers and probes in this assay is described in Table 2B. Assays (25 ␮l) were carried out on about 10

Table 2. Primer Information Used in the Study A. Primer Sequencing for Direct Sequencing Exon

Product Size

Forward Primer Sequence (5=–3=)

Reverse Primer Sequence (5=–3=)

331 458 496 485 283 253 250 394 242 249 285 248 388 252 291

GAC TCG CTG AGG AGA AGA AAG TTC TCC AGA TGC AGT TCC AGC CAG CCC CTA CTG TGA CCT CTG T GAA CGT GGA GAA GCA GAA GG GAA ACT AAT GCA TCT GGC ATT GA CAC CGG GGT TAT CTA TTT TGC AT CAT GAG GAT TTC AGC TTC TGC GGT GCA TAT GGC CAA TTC TC GTG GAA GGT TCA CTT TTT GCA G AAT CTC TGA CCT GGC TGT TCC TCT TCC ATG TGT GTG GAT GCT G TCA ATC CTT TGG CTT TGA GCT T AGC CAG GTA GAC AAG CTA TCG GTC CAC GGC ACT AAC AAG TGA T AGA GGG CCA CGA TCA CCT TC

GGT TGT TAA CAG AGG CAC GC TAT CCA TGG CTG CAA ACT CTT AAG ACT GAA GGG CCG AGA GAG AC GGA AGT CAG TTG AGC CCA GA TGG GAC ATG ATG ACA AAA CAA T TGA GGG GAA AAT GGT GAC AAT A GCA AGA CCC TGT CTC AAA GAA CCA ATG AAC AGG TCA AAG AGG TGC AGA AGC CAG GTA TCC TAA G ACG ATG TGC TGG TAG CTG TCA T ACT TTG CCG GGG AAC AGT TG CTG GCC AGC CTT TTT CAT CG GAA TAA GGG TCC CCT CTG GAG CCA TCT TCT GAG GCA TGA AAA AC ATC TCC GTA ATT GAT TCA GGC A

1 2 part 1 2 part 2 2 part 3 3 4 5 6 7 8 9 10 11 12 13

B. Primers and Probes in the TaqMan Assays Forward Primer Sequence (5=–3=) ACGAGGACCCGCGATG CAGGCCGCAAGGAACAG CCCTCAACTTGTCACTTAAAGAAATCAC CAACGTGCTGTAGGAAACCATTT GACTTGGAAGCTTGTCGATTGC TGCTGAATTTCCTTCTAAATGTCACTCA

Reverse Primer Sequence (5=–3=)

VIC Probe Sequence (5=–3=)

FAM Probe Sequence (5=–3=)

ACCCGTGTAGCCAAGAGACT GGTCCATGTCTGGTAAAGAATGCA AGTCAAACGAAAACTTCATAGGCTTCT CAGCCCTTCTCTCTCTGATGTTAAT GCTTCCCCTGGCTTCCT AATTCACCCCAACTTCTCTCTAATGG

TCTCTCTCAGCCCTTC ACGCTCTGGCCTGG AAAATGGGAATATAAAGGTACTTA CTCGGTGAGGTCTTTA CTGTAGGCACTGGATAA ATTCTTGGGAATGTC

TCTCTCGGCCCTTC CACGCTCTAGCCTGG AATGGGAATATAAAGGTGCTTA TCGGTGAAGTCTTTA CTGTAGGCTCTGGATAA CATTCTTGAGAATGTC

C. Primers for RT-PCR Exons 4–5 8–9

Forward Primer Sequence (5=–3=)

Reverse Primer Sequence (5=–3=)

ACGCGTCGACCCAGCCAGCTCTTAGCAGTTTC GCACCCTGAGGAAGAAAGTT

AACAGCCTCGGCTCCATTCTC TCTCTTCTCAGTCTGTTGTAATCT

RT-PCR, reverse transcriptase polymerase chain reaction.

www.sobp.org/journal

BIOL PSYCHIATRY 2006;60:929 –935 931

K. Maeda et al ng genomic DNA according to manufacturer’s instructions. PCR reactions were done on GeneAmp PCR system 9700 (Applied Biosystems) and fluorescence signals were detected on ABI 7900HT (Applied Biosystems). The reliability of the TaqMan assay has been consistently established with ⬎ 99.5% concordance with independent methods (De la Vega et al 2005). Genetic Analysis and Statistics Inter-marker LD and haplotype block structure were examined using the program Haploview (www.broad.mit.edu/mpg/haploview/index.php). Association tests were then performed using the computer program FBAT (Family-Based Association Test) (Horvath et al 2001). FBAT was chosen because it provides valid tests of association in the presence of linkage even when using multiple affected siblings from families of variable structure. In addition to performing tests of association for individual markers, FBAT allows for tests of association with haplotypes that may be phase ambiguous. Power calculations using the PBAT program associated with FBAT (Horvath et al 2001) were performed assuming an additive model and disease prevalence between 1% and 2%, and using the 57 families with 2 affected offspring. Assuming a disease allele frequency of .2, there was 80% power to detect a locus with a relative risk (RR)1 (heterozygote) of 1.9 and a RR2 (homozygote) of

2.9, and corresponding attributable fraction of .27. Allowing for a disease allele frequency of .1 there was 80% power to detect a locus with RR1 of 2.1 and RR2 of 3.2 with an attributable fraction of .18. Expression Analysis Lymphoblasts from patients were established with infection of Epstein Barr virus, and frozen down into aliquots (Sawa et al 1999). Cells were maintained in RPMI1640 media with 10% fetal bovine serum, and used within 2 weeks after thaw (4 cycles of cell division) for expression analyses. Expression of DISC1 was initially verified by reverse transcriptase (RT)-PCR with two sets of primers. To avoid the contamination of the amplification from genomic DNA, each pair was chosen from two independent exons (Sawa et al 1997). The primer sequences are described in Table 2C. Expression levels of DISC1 in lymphoblasts were measured by the RNA counter kit (TrimGen, Sparks, Maryland). This kit employs the isometric primer extension technology, and has been used in other expression analyses (Akiyoshi et al, in press). A portion of DISC1 including both exons 6 and 7 (nucleotides 1627–1647 in the National Center for Biotechnology Information [NCBI] AF22980) was used for the probe. The use of two independent exons avoids contaminated signals from genomic DNA. The amplified region was

A TEL

Human Breakpoint

CEN rs1538975

2 3

1

rs1407598

rs1954175

rs3738401 (Arg/Gln)

4 5 6

NOVEL (Pro/Leu)

78

rs2273890

rs1000731

9

rs821653 rs3524

10

rs6675281 (Leu/Phe)

11 12

rs821616 (Ser/Cys)

13

rs3737597

B SNP ID

Location

Amino acid change

1 2

rs1538975 rs3738401

228,863,377 228,865,300

intron 1/2 Exon 2

Arg/Gln

3

Novel SNP rs1954175 rs2273890

228,865,369

Exon 2

Pro/Leu

228,890,415 228,966,053

intron 3/4 intron 7/8

228,981,830 228,989,104

intron 8/9 Exon 9

Leu/Phe

228,998,496 229,161,892 229,179,608

intron 9/10 intron 10/11 Exon 11

Ser/Cys

229,193,684 229,207,839

intron 11/12 Exon 13

4 5 6 7 8 9 10 11 12

rs1407598 rs6675281 rs1000731 rs821653 rs821616 rs3524 rs3737597

Figure 1. Schematic presentation of SNPs in DISC1 gene. (A) Exon/intron structure of DISC1 gene with the location and identification of SNPs studied here is depicted. NOVEL indicates the SNP we identified by the direct sequencing. The SNPs that lead to amino acid variation are indicated. (B) The SNPs analyzed in this study. The identification number, location, and possible changes of deduced amino acid of each SNP are summarized. The physical location (bp) is based on build 34 of National Center for Biotechnology Information (NCBI). (C) Sequences of HP1 and HP2. DISC1, Disrupted-in-Schizophrenia-1; HP, haplotype; SNP, single nucleotide polymorphism.

C

www.sobp.org/journal

932 BIOL PSYCHIATRY 2006;60:929 –935

A

K. Maeda et al

B

RT-PCR (semi-quantitative)

RNA counter DISC1

β-actin 1.5

Test 1 Test 2 Test 3 Test 4

1.0

O.D. (405nm)

O.D. (405nm)

1.5

0.5 0

0

2

4

6

8

10

Total RNA ( ng )

12

1.0

0.5 0

0 25 50 100

200

500

750

Total RNA (ng)

Figure 2. DISC1 expression in human lymphoblasts at mRNA levels. (A) Expression of DISC1 at mRNA level in human lymphoblasts. Independent portions of DISC1 (between exons 4 and 5 as well as exons 8 and 9) were amplified by RT-PCR from both HeLa cells and human lymphoblasts (LB). Both amplifications suggest higher levels of DISC1 expression in HeLa cells than in lymphoblasts. (B) Measurement of mRNA levels by RNA counter kit. The reproducibility was tested with four independent tests for ␤-actin. The linearity of the signals was tested with both ␤-actin and DISC1. At least, the signals were linear up to the use of 12 ng and 750 ng total RNA for ␤-actin and DISC1, respectively. DISC1, Disrupted-in-Schizophrenia-1; HP, haplotype; RT-PCR, reverse-transcriptase coupled polymerase chain reaction.

selected because this includes no genetic variability or SNP of which we are aware. ␤-actin was used for an internal control of expression. No difference was observed in the levels of ␤-actin among groups in the present study (data not shown). The reproducibility of the results in this method was tested with ␤-actin, and the linearity of the signals was tested with both ␤-actin and DISC1 (Figure 2B). The signals were linear up to the use of 12 ng and 750 ng total RNA for ␤-actin and DISC1, respectively. Western blotting was carried out as described (Ozeki et al 2003). Clinical Data Analysis Using the statistical software, STATA (StataCorp LP, College Station, Texas), clinical features of affected subjects with DISC1 expression data were analyzed. The broad affection status model (including BPI, BPII, recurrent major depression, and schizoaffective disorder manic type) was used for definition of affection. Multiple linear regression was used to model DISC1 expression levels as a dependent variable in relation to various clinical features of the affected subjects.

Results Family-Based Association Study Sequencing of the exons from 10 probands taken from the pedigrees with most evidence of linkage identified one novel SNP (Figure 1A, 1B). This SNP was typed in the subset of 57 BP pedigrees. Using the algorithm of Gabriel et al (2002), as implemented in Haploview, there were at least 3 haplotype blocks in this gene spanning approximately 200 kb. Analysis of the individual markers identified no significant association with BP disorder. Haplotype analyses using FBAT and all 12 markers identified two haplotypes with frequencies of .11 (HP1) and .10 (HP2) (Figure 1C). All families were informative using the 12 SNP haplotype; there were 19 informative families carrying the HP1 haplotype, of which 14 had an affected female and 15 an affected male for the sex specific analyses. We tested several individual SNPs and various combinations of SNPs for possible haplotypes, and obtained the significance only from the total 12 marker haplotype. HP1 was overtransmitted to the affected subjects (p ⫽ .01) and HP2 was undertransmitted. The overtransmission of HP1 was exclusively to the affected females (p ⫽ .004). www.sobp.org/journal

DISC1 Expression in Human Lymphoblasts First, we examined expression of DISC1 in human lymphoblasts. DISC1 mRNA was addressed by reverse-transcriptase coupled PCR (RT-PCR). Two sets of primer pair were chosen from exons 4 and 5 as well as exon 8 and 9 of DISC1 gene (Table 2C). The primer selection over two independent exons can exclude contaminated signals from genomic DNA. In both amplifications, the specific signals were obtained from both HeLa cells and human lymphoblasts with a consistently lower intensity from lymphoblasts (Figure 2A). DISC1 protein in lymphoblasts was confirmed in Western blotting by using a well-characterized anti-DISC1 antibody (C2) (Ozeki et al 2003; Sawamura et al 2005) (data not shown). Second, we examined the relationship of DISC1 haplotypes to their mRNA expression levels. Expression of DISC1 in lymphoblasts from affected subjects with the risk haplotype (HP1) (12 cases) was compared with that from unaffected family members with the undertransmitted protective haplotype (HP2) (11 controls). Levels of DISC1 are significantly lower in bipolar subjects with HP1 than unaffected relatives with HP2 (p ⫽ .006: t-test) (Figure 3A, 3C). The consistent gender influence, as observed in the over-transmission to affected females, exists with significant suppression of DISC1 expression in affected females (p ⫽ .001: t-test) (Figure 3B, 3C). In the two-way analysis of variance (ANOVA), the haplotype, not the gender, was significantly associated with DISC1 expression. Clinical Data Analysis The clinical data analyses were conducted by using affected subjects in which DISC1 expression was examined as above. Of all the clinical features, the number of manic symptoms recorded as the number of Research Diagnostic Criteria (RDC) symptoms for mania, correlates most with the decreased levels of DISC1 expression (coefficient of correlation, ⫺.41; p ⫽ .008) (Table 3). Earlier age of first manic episode also tends to correlate with lower DISC1 expression. In addition, age of first hospitalization seems to correlate negatively with DISC1 expression when controlling for other factors. Other features, including psychosis, show no significant correlation when controlling for other factors.

Discussion There are two main findings in this study: first, decreased levels of DISC1 expression are observed in affected lymphoblasts with the

BIOL PSYCHIATRY 2006;60:929 –935 933

K. Maeda et al

B

Total

1.2 1 0.8 0.6 0.4

1.4

1 0.8 0.6 0.4 0.2

0.2 0

Female

1.2

DISC1 expression (DISC1/ -actin)

DISC1 expression (DISC1/ -actin)

1.4

DISC1 expression (DISC1/ -actin)

A

NON-HP2

1.2 1 0.8 0.6 0.4 0.2

0

BP-HP1 p=0.006*

Male

NON-HP2

BP-HP1 p =0.001**

0

NON-HP2

BP-HP1 p =0.24

C DISC1 Expression NON-HP2 Mean SD

BP-HP1 Mean

SD

p value

All

0.77

0.31

0.44

0.20

0.006*

Female

0.83

0.23

0.41

0.08

0.001**

Male

0.74

0.37

0.49

0.31

0.240

DISC1 risk haplotype for BP, in comparison with lymphoblasts from control subjects with the protective haplotype. Second, decreased DISC1 expression is correlated with some of clinical features of BP, including an earlier age of first manic episode and higher number of manic symptoms. These findings support the hypothesis that DISC1 contributes to susceptibility to mood disorder as well as SZ, consistent with the finding that patients with SZ and mood disorder were identified in the original Scottish family that showed linkage to DISC1 (Blackwood et al 2001). Potentially, the modest linkage in the Hopkins/Dana pedigrees at 1q42 (McInnis et al 2003) is driven, at least in part, by these findings at DISC1, however the association is not strong. We tested 12 SNPs across the DISC1 gene, but none were found to be associated with BP. These 12 SNPs are unlikely to capture the full haplotype diversity at DISC1. However, there was one haplotype incorporating all 12 SNPs that was overtransmitted, especially among females, and one that was undertransmitted. Although the specific haplotype associations were nominally significant, they do not represent a global p value, nor would they survive correction for multiple testing. On the other hand, the associations were consistent with the expression data showing differences with the two haplotypes. The susceptibility conferred by DISC1 was not limited to cases with psychotic episodes as part of their illness. Comparison of the haplotypes in the present study with those positively associated with BP and/or SZ is shown in Figure 4. We hypothesize that loss of DISC1 function may be implicated in the risk for major mental illness such as BP or SZ. In cell and animal models, mutant C-terminal truncated DISC1 (mutDISC1), that could occur in the Scottish patients, functions in a dominant-negative function, disturbing neurite outgrowth, neuronal migration, neurogenesis, and dendritic arborization (Kamiya et al 2005). MutDISC1 is below detection levels in lymphoblasts from the patients of the Scottish pedigree by Western blotting, reflecting haploinsufficiency or instability of mutDISC1 protein (Millar et al 2005; Sawa and Snyder 2005). The phenotype induced by expression of mutDISC1

Figure 3. DISC1 haplotype and gene expression: gender difference. (A) Comparison of DISC1 expression between affected subjects with the overtransmitted risk haplotype (BP-HP1) and unaffected with the undertransmitted haplotype (NONHP2).DecreasedDISC1expressionwasobservedinBP-HP1comparedwith NONHP2(p⫽.006).(B,C)ComparisonofDISC1expressionbetweenBP-HP1and NONHP2 in female and male subjects, respectively. Marked decrease in DISC1 expression was observed in BP-HP1 in female (p ⫽ .001; t-test), but no significant difference in DISC1 expression was observed between two groups in males. The data from individual cell lines were plotted as dots in this figure A and B. DISC1, Disrupted-in-Schizophrenia-1; BP, bipolar disorder; HP, haplotype.

is consistent with suppression of endogenous DISC1 with RNA interference in mouse developing cerebral cortex (Kamiya et al 2005; Sawa and Snyder 2005). Our observation of association between decreased DISC1 expression and the risk haplotype of BP supports our hypothesis. Loss of DISC1 function may underlie aberrant neuronal circuit formation and/or affect adult neurogenesis, which are plausible underlying mechanisms in the psychopathology of bipolar disorder. It is not clear why the risk haplotype contributes to its decrease expression; potentially some of the SNPs examined in this study may directly influence gene transcription or stability of mRNA. Another scenario, which may be more probable, is that causative genetic variation(s) for the decreased expression segregate with the risk haplotype. Thus further association study with denser SNPs will clarify the genetic-functional link. In the present study, we observe evidence suggestive of a gender effect on the transmission distortion as well as association of decreased DISC1 expression with the risk haplotype. The risk haplotype was significantly more frequently transmitted to affected females. The lymphoblasts derived from affected females showed a significant decrease in DISC1 expression in the t-test. This gender effect was modest, as the significance identified in the initial comparisons using the t-test was not sustained in the two-way ANOVA, where only haplotype (and not gender) was associated Table 3. Linear Regression of DISC1 Expression in Relation to Severity Measures of Mania in the Clinical Data of Study Subjects MEASURES Number of Manic Symptoms Age of 1st Episode Number of Manic Episodes Age of 1st Hospitalization

COEFFICIENT

STD ERROR

Z

p

⫺.41 .24 ⫺.03 ⫺.27

.15 .13 .01 .12

⫺2.64 1.92 ⫺1.92 ⫺2.2

.008 .06 .06 .03

DISC1, disrupted-in-schizophrenia gene.

www.sobp.org/journal

934 BIOL PSYCHIATRY 2006;60:929 –935 A.

K. Maeda et al

HEP3

B.

HEP1

Block1

Block2

Block3

HEP4

Block4

C.

Ser704Cys

D.

Region1

Region3

Region2

E.

DISC1 1

2 3

4

56

78

Arg264Gln

9

Leu607Phe

10

11

12 13

Ser704Cys

Figure 4. Polymorphisms of the DISC1 gene that are reportedly associated with major mental illnesses. The 415kb DISC1 gene with scaling based on the May 2004 University of California at Santa Cruz (UCSC) Genome Browser (http://genome.ucsc.edu/). Exons are indicated by vertical bars and are numbered. Common mis-sense mutations are shown below exons 2, 9, and 11. Statistically significant (p ⬍ .05) association findings for DISC1 are depicted above as determined by previous studies (A-D) and the present study (E). Vertical bars in sections A-E represent individual markers (obtained using the UCSC Genome Browser) while horizontal regions represent statistically significant haplotypes. (A) Hennah et al (2003). HEP3, HEP1, and HEP4 haplotypes were found to exhibit transmission distortion in SZ patients particularly when the sample was subdivided by sex. (B) Hodgkinson et al (2004). Individual markers comprising Block2-4 as well as haplotypes in Block1, Block2, Block3, and Block4 were found to exhibit case-control associations in patients with SZ, schizoaffective disorder, and BP.(C) Callicott et al (2005). The Ser704Cys SNP was found to exhibit transmission distortion in schizophrenia and 3-marker sliding haplotypes in the region (C), particularly those containing the Ser704Cys SNP are over-transmitted to SZ patients. (D) Thomson et al (2005). Haplotypes in Region1, Region2, and Region3 show case-control association in SZ and BP particularly when the sample was subdivided by sex. Region1 extends into the TRAX gene (not shown here) as indicated by the arrow. (E) Maeda et al (the present study). A 12-SNP haplotype in the region (E) exhibit transmission distortion in patients with BP, particularly in female cases. DISC1, Disrupted-in-Schizophrenia-1; SZ, schizophrenia; BP, bipolar disorder; HP, haplotype.

with the decrease in expression. Of interest, in the previous study of Finnish schizophrenics, a unique haplotype, different from the risk haplotype (HP1) in this study, was significantly under-transmitted only to affected females (Hennah et al 2003). While it is not at all clear what the gender effect (if present) may be, these results may indicate the importance of studying the gender-preferential or gender-specific impact of DISC1 in major mental illnesses. Human lymphoblasts are becoming an increasingly popular cellular system for study of gene expression or other cellular functions. The advantage of lymphoblasts is that we can obtain them more easily than neuronal tissues; the disadvantage is that the gene expression or other functions in this peripheral tissue may not be correlated with that of the brain. It is unlikely that experimental results from established lymphoblasts are influenced by adventitious factors, dietary or drug treatment differences, because media changes associated with preparation of the lymphoblasts for more than one month would wash away all such drug and dietary effects. Previously, we reported abnormalities in lymphoblasts from patients with Huntington’s disease (HD) (Nagata et al 2004; Sawa et al 1999, 2005). HD symptoms are brain preferential, but its causative gene product, Huntingtin, is expressed in almost all tissues. In our studies, lymphoblasts derived from HD patients show increased stress-induced mitochondrial depolarization, apoptotic cell death, and autophagy-like vacuole formations, which are correlated with increased glutamine repeats in Huntingtin. These previous studies suggest that genetic determinants for brain pathology can be reflected in alteration of cellular functions in peripheral lymphoblasts. Gene expression varies in lymphoblasts and particular genes with substantial individual variation have been reported (Cheung et www.sobp.org/journal

al 2003). It is not clear whether our finding of variable expression in DISC1 represents normal variation, or if we have indeed identified two distinct lymphoblast populations with meaningful variation in DISC1 expression. Furthermore, it is not clear yet whether the variation in expression is secondary to a putative risk haplotype or the presence of BP disorder. Nor can specific conclusions be drawn regarding the sex effect. Although it is not so likely, the medication that the affected group was taking might affect the gene expression status of the lymphoblasts, even after immortalization of cells. Loci that influence gene expression have recently been identified (Morley et al 2004), so we may have identified a potential phenotype of low DISC1 expression. More substantial studies of the family set are warranted in the effort to identify loci that may be influencing DISC1 expression. It is a very strong possibility that a number of loci influence the expression of a gene, the expression of which ultimately influences the manifestation of illness. This is possibly one of several reasons why genes are so difficult to identify in diseases of complex inheritance. This study, as is often the case in complex disorders, raises more questions than it answers. Hypotheses generated from these initial experiments may be tested in larger scale analyses, including BP, SZ, and control populations. In summary we have obtained supportive evidence of an association between DISC1 and BP and more importantly have demonstrated the feasibility of using lymphoblasts as a model system for gene expression and possibly for study in complex disorders. We appreciate departmental supports, especially Dr. Raymond DePaulo. We thank Ms. Yukiko Lema for preparation of figures and typing. We thank Dr. Naoya Sawamura, Ms. Yuqing Huo, Ms.

K. Maeda et al Amanda Rebert, and Ms. Ariel Lyons-Warren for technical assistance. This work was supported by United States Public Health Service (USPHS) grants MH-069853 (to AS), MH-59533 (to MGM), foundation grants from National Alliance for Research on Schizophrenia and Depression (NARSAD) (to AS), Stanley (to AS), S-R (to AS), and funds from the departments (to AS). KM is currently at Tottori University, Department of Neuropsychiatry, Yonago, Japan. MGM is currently at University of Michigan, Ann Arbor, Michigan. Supplementary material cited in this article is available online. Akiyoshi T, Nakamura M, Koga K, Nakashima H, Yao T, Tsuneyoshi M, et al (in press): Gli1, down-regulated in colorectal cancers, inhibits proliferation of colon cancer cells involving Wnt signalling activation. Gut. Austin CP, Ky B, Ma L, Morris JA, Shughrue PJ (2004): Expression of Disrupted-InSchizophrenia-1, a schizophrenia-associated gene, is prominent in the mouse hippocampus throughout brain development. Neuroscience 124:3–10. Austin CP, Ma L, Ky B, Morris JA, Shughrue PJ (2003): DISC1 (Disrupted in Schizophrenia-1) is expressed in limbic regions of the primate brain. Neuroreport 14:951–954. Berrettini WH (2000a): Are schizophrenic and bipolar disorders related? A review of family and molecular studies. Biol Psychiatry 48:531–538. Berrettini WH (2000b): Susceptibility loci for bipolar disorder: overlap with inherited vulnerability to schizophrenia. Biol Psychiatry 47:245–251. Blackwood DH, Fordyce A, Walker MT, St Clair DM, Porteous DJ, Muir WJ (2001): Schizophrenia and affective disorders–cosegregation with a translocation at chromosome 1q42 that directly disrupts brain-expressed genes: clinical and P300 findings in a family. Am J Hum Genet 69:428–433. Brandon NJ, Handford EJ, Schurov I, Rain JC, Pelling M, Duran-Jimeniz B, et al (2004): Disrupted in Schizophrenia 1 and Nudel form a neurodevelopmentally regulated protein complex: implications for schizophrenia and other major neurological disorders. Mol Cell Neurosci 25:42–55. Callicott JH, Straub RE, Pezawas L, Egan MF, Mattay VS, Hariri AR, et al (2005): Variation in DISC1 affects hippocampal structure and function and increases risk for schizophrenia. Proc Natl Acad Sci U S A 102:8627– 8632. Cannon TD, Hennah W, van Erp TG, Thompson PM, Lonnqvist J, Huttunen M, et al (2005): Association of DISC1/TRAX haplotypes with schizophrenia, reduced prefrontal gray matter, and impaired short- and long-term memory. Arch Gen Psychiatry 62:1205–1213. Cheung VG, Conlin LK, Weber TM, Arcaro M, Jen KY, Morley M, et al (2003): Natural variation in human gene expression assessed in lymphoblastoid cells. Nat Genet 33:422– 425. Coyle JT, Duman RS (2003): Finding the intracellular signaling pathways affected by mood disorder treatments. Neuron 38:157–160. Curtis D, Kalsi G, Brynjolfsson J, McInnis M, O’Neill J, Smyth C, et al (2003): Genome scan of pedigrees multiply affected with bipolar disorder provides further support for the presence of a susceptibility locus on chromosome 12q23-q24, and suggests the presence of additional loci on 1p and 1q. Psychiatr Genet 13:77– 84. De la Vega FM, Lazaruk KD, Rhodes MD, Wenz MH (2005): Assessment of two flexible and compatible SNP genotyping platforms: TaqMan SNP Genotyping Assays and the SNPlex Genotyping System. Mutat Res 573:111–135. Detera-Wadleigh SD, Badner JA, Berrettini WH, Yoshikawa T, Goldin LR, Turner G, et al (1999): A high-density genome scan detects evidence for a bipolar-disorder susceptibility locus on 13q32 and other potential loci on 1q32 and 18p11.2. Proc Natl Acad Sci U S A 96:5604 –5609. Ekelund J, Hennah W, Hiekkalinna T, Parker A, Meyer J, Lonnqvist J, et al (2004): Replication of 1q42 linkage in Finnish schizophrenia pedigrees. Mol Psychiatry 9:1037–1041. Ekelund J, Hovatta I, Parker A, Paunio T, Varilo T, Martin R, et al (2001): Chromosome 1 loci in Finnish schizophrenia families. Hum Mol Genet 10:1611–1617. Gabriel SB, Schaffner SF, Nguyen H, Moore JM, Roy J, Blumenstiel B, et al (2002): The structure of haplotype blocks in the human genome. Science 296:2225–2229. Hamshere ML, Bennett P, Williams N, Segurado R, Cardno A, Norton N, et al (2005): Genomewide linkage scan in schizoaffective disorder: significant evidence for linkage at 1q42 close to DISC1, and suggestive evidence at 22q11 and 19p13. Arch Gen Psychiatry 62:1081–1088.

BIOL PSYCHIATRY 2006;60:929 –935 935 Hennah W, Varilo T, Kestila M, Paunio T, Arajarvi R, Haukka J, et al (2003): Haplotype transmission analysis provides evidence of association for DISC1 to schizophrenia and suggests sex-dependent effects. Hum Mol Genet 12:3151–3159. Hodgkinson CA, Goldman D, Jaeger J, Persaud S, Kane JM, Lipsky RH, et al (2004): Disrupted in Schizophrenia 1 (DISC1): Association with Schizophrenia, Schizoaffective Disorder, and Bipolar Disorder. Am J Hum Genet 75:862– 872. Horvath S, Xu X, Laird NM (2001): The family based association test method: strategies for studying general genotype–phenotype associations. Eur J Hum Genet 9:301–306. Hwu HG, Liu CM, Fann CS, Ou-Yang WC, Lee SF (2003): Linkage of schizophrenia with chromosome 1q loci in Taiwanese families. Mol Psychiatry 8:445–452. Kamiya A, Kubo K, Tomoda T, Takaki M, Youn R, Ozeki Y, et al (2005): A schizophrenia-associated mutation of DISC1 perturbs cerebral cortex development. Nat Cell Biol 7:1067–1078. MacgregorS,VisscherPM,KnottSA,ThomsonP,PorteousDJ,MillarJK,etal(2004):A genomescanandfollow-upstudyidentifyabipolardisordersusceptibilitylocus on chromosome 1q42. Mol Psychiatry 9:1083–1090. McInnis MG, Lan TH, Willour VL, McMahon FJ, Simpson SG, Addington AM, et al (2003): Genome-wide scan of bipolar disorder in 65 pedigrees: supportive evidence for linkage at 8q24, 18q22, 4q32, 2p12, and 13q12. Mol Psychiatry 8:288 –298. Millar JK, Pickard BS, Mackie S, James R, Christie S, Buchanan SR, et al (2005): DISC1 and PDE4B are interacting genetic factors in schizophrenia that regulate cAMP signaling. Science 310:1187–1191. Millar JK, Wilson-Annan JC, Anderson S, Christie S, Taylor MS, Semple CA, et al (2000): Disruption of two novel genes by a translocation co-segregating with schizophrenia. Hum Mol Genet 9:1415–1423. Miyoshi K, Honda A, Baba K, Taniguchi M, Oono K, Fujita T, et al (2003): Disrupted-In-Schizophrenia 1, a candidate gene for schizophrenia, participates in neurite outgrowth. Mol Psychiatry 8:685– 694. Morley M, Molony CM, Weber TM, Devlin JL, Ewens KG, Spielman RS, et al (2004): Genetic analysis of genome-wide variation in human gene expression. Nature 430:743–747. Morris JA, Kandpal G, Ma L, Austin CP (2003): DISC1 (Disrupted-In-Schizophrenia 1) is a centrosome-associated protein that interacts with MAP1A, MIPT3, ATF4/5 and NUDEL: regulation and loss of interaction with mutation. Hum Mol Genet 12:1591–1608. Nagata E, Sawa A, Ross CA, Snyder SH (2004): Autophagosome-like vacuole formation in Huntington’s disease lymphoblasts. Neuroreport 15:1325–1328. Ozeki Y, Tomoda T, Kleiderlein J, Kamiya A, Bord L, Fujii K, et al (2003): Disrupted-in-Schizophrenia-1 (DISC-1): mutant truncation prevents binding to NudE-like (NUDEL) and inhibits neurite outgrowth. Proc Natl Acad Sci U S A 100:289 –294. Sawa A, Nagata E, Sutcliffe S, Dulloor P, Cascio MB, Ozeki Y, et al (2005): Huntingtin is cleaved by caspases in the cytoplasm and translocated to the nucleus via perinuclear sites in Huntington’s disease patient lymphoblasts. Neurobiol Dis 20:267–274. Sawa A, Oyama F, Cairns NJ, Amano N, Matsushita M (1997): Aberrant expression of bcl-2 gene family in Down’s syndrome brains. Brain Res Mol Brain Res 48:53–59. Sawa A, Snyder SH (2005): Genetics. Two genes link two distinct psychoses. Science 310:1128 –1129. Sawa A, Wiegand GW, Cooper J, Margolis RL, Sharp AH, Lawler JF Jr, et al (1999): Increased apoptosis of Huntington disease lymphoblasts associated with repeat length-dependent mitochondrial depolarization. Nat Med 5:1194 –1198. Sawamura N, Sawamura-Yamamoto T, Ozeki Y, Ross CA, Sawa A (2005): A form of DISC1 enriched in nucleus: altered subcellular distribution in orbitofrontal cortex in psychosis and substance/alcohol abuse. Proc Natl Acad Sci U S A 102:1187–1192. Schurov IL, Handford EJ, Brandon NJ, Whiting PJ (2004): Expression of disrupted in schizophrenia 1 (DISC1) protein in the adult and developing mouse brain indicates its role in neurodevelopment. Mol Psychiatry 9:1100–1110. Thomson PA, Wray NR, Millar JK, Evans KL, Hellard SL, Condie A, et al (2005): Association between the TRAX/DISC locus and both bipolar disorder and schizophrenia in the Scottish population. Mol Psychiatry 10:657– 668, 616. Zhang F, Sarginson J, Crombie C, Walker N, Stclair D, Shaw D (2006): Genetic association between schizophrenia and the DISC1 gene in the Scottish population. Am J Med Genet B Neuropsychiatr Genet 141: 155–159.

www.sobp.org/journal