Genetic association of cyclic AMP signaling genes with bipolar disorder

Abstract

The genetic basis for bipolar disorder (BPD) is complex with the involvement of multiple genes. As it is well established that cyclic adenosine monophosphate (cAMP) signaling regulates behavior, we tested variants in 29 genes that encode components of this signaling pathway for associations with BPD type I (BPD I) and BPD type II (BPD II). A total of 1172 individuals with BPD I, 516 individuals with BPD II and 1728 controls were analyzed. Single SNP (single-nucleotide polymorphism), haplotype and SNP × SNP interactions were examined for association with BPD. Several statistically significant single-SNP associations were observed between BPD I and variants in the PDE10A gene and between BPD II and variants in the DISC1 and GNAS genes. Haplotype analysis supported the conclusion that variation in these genes is associated with BPD. We followed-up PDE10A's association with BPD I by sequencing a 23-kb region in 30 subjects homozygous for seven minor allele risk SNPs and discovered eight additional rare variants (minor allele frequency <1%). These single-nucleotide variants were genotyped in 999 BPD cases and 801 controls. We obtained a significant association for these variants in the combined sample using multiple methods for rare variant analysis. After using newly developed methods to account for potential bias from sequencing BPD cases only, the results remained significant. In addition, SNP × SNP interaction studies suggested that variants in several cAMP signaling pathway genes interact to increase the risk of BPD. This report is among the first to use multiple rare variant analysis methods following common tagSNPs associations with BPD.

Citation: Transl Psychiatry (2012) 2, e169; doi:10.1038/tp.2012.92

& 2012 Macmillan Publishers Limited All rights reserved 2158-3188/12 http://www.nature.com/tp

Web End =www.nature.com/tp

M-L McDonald1,3, C MacMullen2, DJ Liu1,4, SM Leal1 and RL Davis2

The genetic basis for bipolar disorder (BPD) is complex with the involvement of multiple genes. As it is well established that cyclic adenosine monophosphate (cAMP) signaling regulates behavior, we tested variants in 29 genes that encode components of this signaling pathway for associations with BPD type I (BPD I) and BPD type II (BPD II). A total of 1172 individuals with BPD I, 516 individuals with BPD II and 1728 controls were analyzed. Single SNP (single-nucleotide polymorphism), haplotype and SNP

SNP interactions were examined for association with BPD. Several statistically signicant single-SNP associations were

observed between BPD I and variants in the PDE10A gene and between BPD II and variants in the DISC1 and GNAS genes. Haplotype analysis supported the conclusion that variation in these genes is associated with BPD. We followed-up PDE10As association with BPD I by sequencing a 23-kb region in 30 subjects homozygous for seven minor allele risk SNPs and discovered eight additional rare variants (minor allele frequency o1%). These single-nucleotide variants were genotyped in 999 BPD cases and 801 controls. We obtained a signicant association for these variants in the combined sample using multiple methods for rare variant analysis. After using newly developed methods to account for potential bias from sequencing BPD cases only, the results remained signicant. In addition, SNP SNP interaction studies suggested that variants in several cAMP signaling

pathway genes interact to increase the risk of BPD. This report is among the rst to use multiple rare variant analysis methods following common tagSNPs associations with BPD.

Translational Psychiatry (2012) 2, e169; doi:http://dx.doi.org/10.1038/tp.2012.92

Web End =10.1038/tp.2012.92 ; published online 2 October 2012

Introduction

Bipolar disorder (BPD) affects B2.5% of the United States population aged 18 and older based on data from the population-based National Comorbidity Survey Replication.1 The risk of BPD for a rst-degree relative of an affected individual is 9%, and the concordance rate for monozygotic twins is 4045%.2 Relatives of BPD probands are also at an increased risk of other related psychiatric disorders, such as psychotic, anxiety, substance abuse and impulse control disorders.14 With an overall heritability estimated at 80 90%,2 these and other ndings make clear that susceptibility to BPD has a very strong genetic basis that overlaps with the genetic susceptibility for other neuropsychiatric disorders.

The two most common subtypes of BPD are bipolar disorder I (BPD I) and bipolar disorder II (BPD II). The lifetime risks in the US population is 1% for BPD I and 1.1% for BPD II.1 The clinical course of BPD I and BPD II differ in that BPD I patients exhibit one or more manic episodes while BPD II patients are characterized by recurrent depressive episodes.2,3 Recurrence of manic/depressive episodes and associated disabilities is common5-7 and there exists no

curative treatment. Although BPD I and BPD II have been shown to co-segregate within families,2,8 the diverse clinical symptoms indicate that subtypes of the BPD phenotype exist

Keywords: bipolar disorder; cAMP signaling; DISC1; GNASPDE10A; PDE10A

Genetic association of cyclic AMP signaling genes with bipolar disorder

and/or that phenotypic modiers are involved.9 The identication of BPD I and BPD II susceptibility genes may lead to more effective targets for therapy.

Several recent genome-wide genetic association studies and meta-analyses2,10 have jointly analyzed BPD I and BPD II subjects,1016 or have focused only on BPD I subjects.1719

One motivation for grouping the subtypes is that it remains possible that BPD II is a milder form of BPD I as opposed to being a distinct disorder. However, genetic heterogeneity can reduce the power to detect susceptibility variants when samples of BPD I and BPD II are combined for analysis. For this study, we analyzed BPD I and BPD II separately to increase the likelihood of genetic homogeneity among cases.

A substantial body of evidence indicates a relationship between BPD and cyclic adenosine monophosphate (cAMP) signaling although the specic nature of the relationship remains obscure (reviewed in Gould and Manji20 and Dwivedi and Pandy21). Several studies on peripheral cells or postmortem brain have reported that basal and receptor-activated adenylyl cyclase (AC) activities are increased in BPD patients.22,23 Other studies have reported that the mood stabilizers lithium and carbamazepine interact directly or indirectly to attenuate the receptor-activation of ACs,2429

consistent with the possibility that an increased AC activity or

1Department of Molecular and Human Genetics, Center for Statistical Genetics, Baylor College of Medicine, Houston, TX, USA and 2Department of Neuroscience, The Scripps Research Institute Florida, Jupiter, FL, USACorrespondence: Dr RL Davis, Department of Neuroscience, The Scripps Research Institute Florida, Jupiter, FL 33458, USA.

E-mail: mailto:[email protected]

Web End [email protected]

3Current address: Department of Medicine, Brigham and Womens Hospital, Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115, USA.

4Current address: Department of Biostatistics, Center of Statistical Genetics, University of Michigan, Ann Arbor, MI 48109, USA.

Received 24 August 2012; accepted 23 July 2012

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Table 1 Cyclic AMP signaling pathway genes for which tagSNPs were genotyped

Gene symbol

Gene name Location tagSNPs pre-QC

tagSNPs post-QC

Imputed SNPs

Gene size (kb)

GNAI3 Guanine nucleotide-binding protein (G-protein), alpha inhibiting activity polypeptide 3

1p13 14 12 27 47

PDE4B Phosphodiesterase 4B, cAMP-specic

(phosphodiesterase E4 dunce homolog, Drosophila)

1p31 100 93 361 582

PRKACB Protein kinase, cAMP-dependent, catalytic, beta 1p36.1 21 21 66 160 DISC1 Disrupted in schizophrenia 1 1q42.1 148 137 335 339 PPP1CB Protein phosphatase 1, catalytic subunit, beta isoform 2p23 9 8 30 51 CREB1 cAMP responsive element binding protein 1 2q34 6 5 28 69 GNAI2 Guanine nucleotide-binding protein (G-protein),alpha inhibiting activity polypeptide 2

3p21 4 4 56 33

PRKAR2A Protein kinase, cAMP-dependent, regulatory, type II, alpha 3p21.3-p21.2 3 3 14 97 PDE4D Phosphodiesterase 4D, cAMP-specic

(phosphodiesterase E3 dunce homolog, Drosophila)

5q12 226 219 560 1519

PPP2CA Protein phosphatase 2 (formerly 2A), catalytic subunit, alpha isoform

5q31.1 2 2 10 30

PDE10A Phosphodiesterase 10A 6q26 149 140 333 331 ADCY1 Adenylate cyclase 1 (brain) 7p13-p12 33 30 59 149 PRKAR1B Protein kinase, cAMP-dependent, regulatory, type I, beta 7p22 1 1 NA 22 GNAI1 Guanine nucleotide-binding protein

(G-protein), alpha inhibiting activity polypeptide 1

7q21 30 29 56 85

PRKAR2B Protein kinase, cAMP-dependent, regulatory, type II, beta 7q22 20 20 80 117 ADCY8 Adenylyl cyclase 8 (brain) 8q24 116 108 281 260 PPP1CA Protein phosphatase 1, catalytic subunit, alpha isoform 11q13 3 3 NA 23 PPP1CC Protein phosphatase 1, catalytic subunit, gamma isoform 12q24.1-q24.2

4 3 8 23

GNAO1 Guanine nucleotide10-binding protein, alpha activating activity polypeptide O

16q13 55 49 125 166

AKAP10 Protein kinase A anchoring protein 10 17p11.1 6 6 22 72 NF1 Neurobromin, type1 17q11.2 8 7 94 283 PPP1R1B Protein phosphatase 1, regulatory (inhibitor) subunit 1B 17q12 2 2 2 10 PRKAR1A Protein kinase, cAMP-dependent, regulatory, type I, alpha 17q23-q24 8 8 25 39 PPP4R1 Protein phosphatase 4, regulatory subunit 1 18p11.22 27 26 41 68 PRKACA Protein kinase, cAMP-dependent, catalytic, alpha 19p13.1 1 1 1 26 PDE4C Phosphodiesterase 4C, cAMP-specic

(phosphodiesterase E1 dunce homolog, Drosophila)

19p13.11 8 7 12 47

PDE4A Phosphodiesterase 4A, cAMP-specic

(phosphodiesterase E2 dunce homolog, Drosophila)

19p13.2 3 3 1 49

GNAS GNAS complex locus 20q13.3 31 31 18 71 GNAZ Guanine nucleotide-binding protein(G-protein),alpha Z polypeptide

22q11.22 12 10 40 55

Abbreviation: cAMP, cyclic adenosine monophosphate; NA, not applicable; SNP, single-nucleotide polymorphism.

activation is a biochemical phenotype associated with BPD. Several studies have shown that mRNA levels for Gas, the stimulatory G-protein that couples with AC, are increased in peripheral cells of BPD patients.3034 Post-mortem analyses on brain have demonstrated an increased abundance of the Gas protein, but not Gai or Gao,32,30 and lithium has been

found to decrease the abundance of mRNAs for several G-proteins.35,36 Lithium may also decrease the coupling of receptors to G-proteins.22,26,3739 Post-mortem analyses

have demonstrated a decreased level of the regulatory subunits for protein kinase A (PKA) in BPD,40 with increased PKA activity observed in lymphocytes and platelets from BPD subjects.41,42 Some substrates of PKA have been found to be hyperphosphorylated and increased PKA activity and immunoreactivity have been observed in post-mortem brain43,44 and in peripheral cells41,4547 of BPD patients. Finally, the antidepressant rolipram inhibits cAMP phosphodiesterases (PDE) of the dunce class of PDEs (PDE4),48

implicating this class of enzymes in the general state of depression. PDE4 expression is also altered in both BPD and schizophrenia.49

Overall, the evidence strongly suggests that aberrant cAMP signaling is part of the spectrum of biochemical phenotypes associated with BPD. Because of the well-established roles of cAMP in regulating behavioral processes, we hypothesize that genetic variation in the cAMP pathway genes is associated BPD etiology. To test this hypothesis, and to identify the specic mutations within the signaling system that confer susceptibility to BPD, we focused our association study on the collection of genes comprising the cAMP signaling system, as opposed to testing a variety of genes representing many different signaling systems with unrelated functions in the central nervous system. These analyses detected associations of PDE10A with BPD I and of DISC1 and GNAS with BPD II. In addition, signicant associations were observed for interaction of variants in DISC1 and PDE4 for both BPD I and BPD II.

Materials and methods

Study samples. DNA samples were obtained from the Systemic Treatment Enhancement Program for BPD study

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Table 2 TagSNP associations with BPD I and BPD II at Po0.01

Chromosome SNP Position(GRCh37/hg19)

MAF OR 95% CI P Gene

BPD I

1 rs4384209 66279125 0.23 1.2 (1.11.4) 1.2 10 3 PDE4B

1 rs600674 84701251 0.20 0.8 (0.70.9) 6.5 10 3 PRKACB

1 rs10495310 231918776 0.11 1.3 (1.11.5) 1.8 10 3 DISC1

3 rs2282751 50291785 0.14 1.2 (1.11.4) 6.1 10 3 GNAI2

5 rs17741863 58858302 0.10 1.3 (1.11.5) 4.0 10 3 PDE4D

6 rs9459417 165837992 0.13 0.8 (0.70.9) 8.4 10 3 PDE10A

6 rs470922 165844311 0.31 0.8 (0.70.9) 3.1 10 3 PDE10A

6 rs4709082 166040621 0.39 1.2 (1.11.3) 8.3 10 3 PDE10A

6 rs1033701 166040859 0.28 1.2 (1.11.4) 5.4 10 4 PDE10A

6 rs9459464 166042705 0.42 1.2 (1.11.3) 1.7 10 3 PDE10A

6 rs1079418 166047034 0.31 1.2 (1.11.3) 2.7 10 3 PDE10A

6 rs9459465 166048868 0.23 0.8 (0.70.9) 2.7 10 3 PDE10A

6 rs2983517 166051731 0.27 1.2 (1.11.4) 1.0 10 3 PDE10A

6 rs2983521 166057203 0.24 1.3 (1.11.5) 9.3 10 5 PDE10A

6 rs9365899 166062379 0.31 0.8 (0.70.9) 2.3 10 3 PDE10A

6 rs3008049 166063617 0.32 1.2 (1.11.4) 1.1 10 3 PDE10A

6 rs10214709 166066893 0.24 0.8 (0.71.0) 8.2 10 3 PDE10A

8 rs13278912 132002770 0.16 1.2 (1.11.4) 8.1 10 3 ADCY8

20 rs6064714 57414140 0.15 0.8 (0.70.9) 1.5 10 3 GNAS

20 rs6026565 57439308 0.10 1.3 (1.11.5) 1.7 10 3 GNAS BPD II1 rs2812391 231911976 0.27 1.2 (1.11.5) 5.3 10 3 DISC1

1 rs10495310 231918776 0.11 1.4 (1.11.7) 3.2 10 3 DISC1

1 rs4658954 231979357 0.37 0.8 (0.70.9) 4.0 10 3 DISC1

20 rs6064714 57414140 0.15 0.7 (0.60.9) 1.1 10 3 GNAS

20 rs35113254 57435532 0.23 1.4 (1.21.6) 5.8 10 5 GNAS

20 rs6026565 57439308 0.10 1.4 (1.11.7) 6.9 10 3 GNAS

Abbreviations: BPD I, bipolar disorder I; BPD II, bipolar disorder II; CI, condence interval; MAF, minor allele frequency; OR, odds ratio; SNP, single-nucleotide polymorphism.

(STEP-BPD). STEP-BPD was a national, longitudinal study designed to examine the effectiveness of BPD treatments in over 4000 patients with BPD.50 Assignment of diagnosis was based on information derived from clinical assessment and psychiatric diagnostic interviews using the Affective Disorders Evaluation50 and conrmed by the Mini International Neuropsychiatric Interview.51 The affective disorders evaluation includes a modied version of the mood and psychosis modules of the Structured Clinical Interview from the American Psychiatric Associations, Diagnostic and Statistical Manual of Mental Disorders Fourth Edition (DSM-IV; 1994). The interviews were administered by trained clinical specialists using DSM-IV criteria to establish diagnoses of BPD I, BPD II, BPD NOS, schizoaffective manic or BP type or cyclothymic disorder. Only subjects with BPD I or II diagnoses were included in the analysis. The National Institute of Mental Health-funded genetics repository (STEP-Genetics Repository) that created anonymous immortalized cell lines from participants in the STEP-BPD study provided genomic DNA samples for this study. Genomic DNA from controls selected to be of European-American descent and free of signs of depression was provided by the National Institute of Mental Health Genetic Repository. Controls in the repository were screened with the Composite International Diagnostic Interview to reduce the chances of inadvertently introducing susceptibility alleles for neuropsychiatric disorders into the control population. All subjects involved in both studies gave written, informed

consent. As the subject and control DNA samples were both pre-existing and de-identied, this study was considered exempt human research by the Scripps Institutional and Baylor College of Medicine IRBs.

Single-nucleotide polymorphism (SNP) selection and genotyping. We selected 29 candidate genes representing biochemical functions that have been implicated in cAMP signaling. Cross-talk among signaling pathways is extensive in cellular physiology, which blurs their boundaries, so we selected genes that are widely recognized as comprising the conical cAMP signaling pathway so as to limit our set (Table 1). For these genes, tagSNPs were selected within each gene and anking 10 kb, using HAPMAP data (European-American Utah residents with ancestry from Northern and Western Europe, which were collected by the Center dEtude du Polymorphisme Humaine (CEU)) using an r2X0.8, as a measure of linkage disequilibrium (LD) to capture variation within each gene and a minor allele frequency (MAF) X0.05. A subset of 238 SNPs were selected to control for European substructure from a panel previously shown to distinguish different European populations.52 One-thousand and fty tagSNPs and 238 admixture SNPs were genotyped in 1742 cases and 1757 controls in addition to 58 duplicate samples using the Illumina Golden Gate array (Illumina, Inc., San Diego, CA, USA). Beadstudio software (Illumina) was used for allele detection and genotype calling.

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rs4709082

rs1033701

rs9459464

rs1079418

rs2983517

rs2983521

rs3008049

44 55 42 82 83 57

89 85 34 69 66

33 69 44 54

38 81 23

49 46

27

Figure 2 PDE10A region 1 risk linkage disequilibrium (LD) plot. Shading and values in cells correspond to r2 values between single-nucleotide polymorphism (SNP) pairs. The SNPs within region 1 and with odds ratio (OR) 41.0 were used to dene the boundaries for subsequent sequencing.

Figure 1 PDE10A association with bipolar disorder I (BPD I). The points in panel (a) represent genotyped (black circles) and imputed (open triangles) single-nucleotide polymorphisms (SNPs) and their probability of association with BPD I. Panel (b) illustrates probability as a sequential sliding window with three SNPs per window for the genotyped SNPs (empirical haplotype) and all SNPs (haplotype). The results were adjusted for age and population substructure. Panel (c) shows the structure of the PDE10A gene aligned with panels A and B with exons represented by bars. The direction of transcription, 50-30 is from right to left in this gure.

Data quality control. Data cleaning and analysis were performed using PLINK software.53 Samples missing 42%

of their genotypes were excluded because missingness can be indicative of low-quality DNA and remaining genotypes may have high error rates. SNP markers were excluded if they were not successfully genotyped in 495% of the samples, had a MAF o1% or deviated from Hardy

Weinberg equilibrium with a P-value o1 10 6 in controls.

SNPs with deviation from HardyWeinberg equilibrium with a P-value of o1 10 3 were agged for follow-up and were

also removed before SNP marker loci were imputed. Samples were also removed if they exhibited excess homozygosity or excess heterozygosity (44 s.d. from the median). In addition, identity-by-state statistics were calculated between samples to assess duplicates and to evaluate relatedness among individuals included in the study. Multidimensional scaling (MDS) was used to identify outliers using the population substructure SNPs. Samples were clustered

with data from HAPMAP,54 which was collected from individuals from CEU, Yoruba in Ibadan, Nigeria (YRI) and Japanese in Tokyo, Japan (JPT) and Han Chinese in Beijing, China (CBT). Samples were removed from further analysis if they deviated by o3 s.d. from the median of the MDS component 1 and MDS component 2 within the CEU cluster.

After data cleaning, 1728 controls, 1172 BPD I and 516 BPD II remained available for analysis.

To evaluate potential population substructure/admixture, l, the median of the distribution of test statistics over the median expected under the null of no association55 was calculated before and after sex, age and the rst MDS component were included in the logistic regression model to control for potential confounding.

Association testing. PLINK was used to perform logistic regression analyses under an additive model and was used to test for associations between single tagSNPs and BPD I and BPD II.53 Haplotype analysis was performed using GenABEL56 and Haplo Stats57 using a sliding window of three SNPs. GenABEL was used to evaluate two-way interactions between tagSNPs in the cAMP signaling pathway. Findings were assessed for confounding with age and sex. The rst MDS component calculated from the admixture SNPs for BPD I and for the analysis of BPD II sex was also included in the model to control for population substructure.

Imputation. MACH was used to impute additional SNPs that were not genotyped in genes within the cAMP pathway.58,59 It was not possible to impute additional SNPs for

PRKAR2B and PPP1CA (Table 1) because of the limited number of genotyped SNPs. CEU phase haplotypes from HAPMAP phases 1 and 260 were used as templates to infer SNP markers that were not genotyped. The imputed SNPs with Rsq scores 40.8 were analyzed in PLINK using the probability of each genotype in the analysis, which were analyzed as a dosage.

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Table 3 PDE10A Region 1 SNVs with MAF o0.01

Observed SNP Chromosome Position A1 A2 Controls (N 801) BPD cases (N 999) BPD I cases (N 807)MAF MAF MAF

C4T 6 166042004 T C 0 0.000502 0.0006219 G4A 6 166045644 A G 0.000624 0.001505 0.001863

T4G 6 166046710 G T 0.000625 0.001503 0.001861 delATT 6 166046780_166046778 A T 0 0.001001 0.001239 C4G 6 166052752 G C 0.000624 0.001001 0.001239 insA 6 166054721 A T 0 0.001001 0.001239 A4T 6 166056798 T A 0.000624 0.003504 0.003717

C4T 6 166058713 T C 0 0.000502 0.0006203

Abbreviations: BPD, bipolar disorder; MAF, minor allele frequency; SNP, single-nucleotide polymorphism; SNV, single-nucleotide variant. Position: February 2009 (GRCh37/hg19) build physical position. A1: minor allele. A2: major allele.

Single-nucelotide variant discovery. Thirty BPD I patients homozygous for the PDE10A region 1 risk haplotype were randomly selected for single-nucleotide variant (SNV) variant discovery. An B23 kb region (chr6: 166063714166040354)

was amplied as three long-range PCR products using the Takara LA polymerase (Fisher Scientic, Pittsburgh, PA, USA). Nested PCR products ranging in size from 23 kb were amplied from the long-range PCR products using the Roche high delity and GC rich polymerase systems (Branchburg, NJ, USA). The PCR products were then sequenced using Sanger methodology (Genewiz, South Plaineld, NJ, USA), analyzed for variants using the Sequencher v.5 software (Gene Codes Corporation, Ann Arbor, MI, USA), and compared with a reference sequence (UCSC genome browser, Feb 2009 GRCh37/hg19 build). All coding exons of PDE10A isoforms 1 and 2 (Genbank NM001130690.1 and NM 006661.2) were also sequenced and analyzed to rule out any obvious non-synonomous amino acid or splice site changes that could be disease associated.

SNV genotyping and analysis. All variants from the reference sequence were investigated using dbSNP build 135. Any novel variants, not reported in dbSNP, were analyzed using Taqman SNP genotyping assays (Applied Biosystems, Carlsbad, CA, USA). The following numbers of randomly chosen individuals were analyzed for SNV allele frequencies: 807 BD I, 192 BD II and 801 controls. Taqman assays were performed in a 384-well plate format according to manufacturers instructions on a 7900HT Fast real-time PCR instrument (Applied Biosystems).

Rare variant analysis was restricted to those SNVs with MAF o1%. We previously demonstrated that directly combining sequence and genotype data can lead to inated type I errors in two-stage association studies.61 This experimental design involves sequencing a portion of the cases to discover variants followed by genotyping the identied variants in the remaining sample. We therefore developed a likelihood-based method named SEQCHIP, for integrating sequencing and genotype data from two-stage association studies. It corrects for the genotypes of sequenced BPD I and BPD II cases, so that the corrected sequenced genotypes follow the same distribution as that of the genotyped samples.62 The

corrected genotypes can then be analyzed by existing rare

variant tests, which were shown to have correct type I errors.62 More specically, the association analyses described here utilized the ANRV (aggregated number of rare variants),63 the WSS (weighted sum statistics)64 and the VT (variable threshold)65 tests.

Results

Nine hundred and eighty-eight tagSNPs that met quality control criteria within 29 candidate genes (Table 1) were tested for associations using 1172 BPD I cases, 516 BPD II cases and 1728 controls. There was evidence of population substructure in the sample comparing the controls to the BPD cases (l 1.11). The addition of one MDS component

calculated from the admixture SNPs decreased l to 1.03.

None of the association tests of the tagSNPs with either BPD I or BPD II reached genome-wide signicance (Po5 10 8). Association results for tagSNPs with

Po0.01 with BPD I and BPD II are shown in Table 2. Those SNPs that displayed a nominal signicance level of Po0.05 are shown in Supplementary Table 1. An additional 2675 tagSNPs were imputed in the cAMP signaling pathway genes using the 988 tagSNPs that passed quality control (Table 1). All imputed SNPs were analyzed and results of imputed SNPs with Po0.05 are included in Supplementary Table 2.

Twelve different tagSNPs in the PDE10A gene were associated with BPD I at Po0.01, including rs2983521 with an odds ratio (OR) of 1.3, a 95% condence interval ranging from 1.1 to 1.5, and a P 9.3 10 5 (Table 2). Seven of

the PDE10A tagSNPs, such as rs1033701 (OR 1.2, P 5.4 10 4), that associated with BPD I with ORs ranging from1.2 to 1.3 (Po0.01) were found to be in moderate LD (r240.3). An additional 333 SNPs were imputed in PDE10A (Figure 1, panel a). Our analyses of these imputed SNPs indicated that an additional seven SNPs were associated with BPD I at Po0.01 (Supplementary Table 2). One example is rs2983516, with OR of 1.3 and P 5.4 10 5.

The analysis of PDE10A haplotype windows revealed a series of haplotypes that were associated with BPD I (Figure 1, panel b). Two regions in PDE10A (region 1 at 166.1 Mb and region 2 at 165.8 Mb) were associated with BPDI. Region 1 is large non-coding region within intron 1 that may be important for the regulation of PDE10A expression. Because of this possibility and the large number of associated

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Figure 3 DISC1 association with bipolar disorder II (BPD II). The points in panel (a) represent genotyped. (black circles) and imputed (open triangles) single-nucleotide polymorphisms (SNPs) and their probability of association with BPD II. Panel (b) illustrates probability as a sequential sliding window with three SNPs per window for the genotyped SNPs (empirical haplotype) and all SNPs (haplotype). The results were adjusted for age and population substructure. Panel (c) shows the structure of the DISC1 gene aligned with panels (a) and (b) with exons represented by bars. The direction of transcription, 50-30 is from left to right in this gure.

Figure 4 GNAS association with bipolar disorder II (BPD II). The points in panel (a) represent genotyped. (black circles) and imputed (open triangles) single-nucleotide polymorphisms (SNPs) and their probability of association with BPD II. Panel (b) illustrates probability as a sequential sliding window with three SNPs per window for the genotyped SNPs (empirical haplotype) and all SNPs (haplotype). Panel (c) shows the structure of the GNAS gene aligned with panels (a) and (b) with exons represented by bars. The direction of transcription, 50-30 is from right to left in this gure.

tagSNPs that fell within this region, SNV identication was focused to a 23-kb region on 30 BPD I patients homozygous for the region 1, minor allele risk SNPs (Figure 2).

Thirteen SNVs were identied among these BPD I cases. These SNVs were genotyped in 999 cases (807 BPD I and 192 BPD II) in addition to 801 controls. Subsequent analysis was restricted to the eight novel SNVs with a MAF o1% to exclude common polymorphisms (Table 3). Interestingly, the

SNVs chr6:166054721 insA and chr6: 166046780_ 166046778 delATT were found simultaneously in 2 of the 807 BD I cases genotyped. These SNVs are in complete LD in

all individuals genotyped. As the newly identied variants are individually rare, they were aggregated and jointly analyzed to determine their collective effect. When only genotyped samples were analyzed, the P-values were P(ANRV) 3.9 10 2, P(WSS) 7.1 10 2 and P(VT) 8.0 10 2.

When SEQCHIP was used to integrate both sequence and genotype data, the P-values were P(ANRV) 2.2 10 2,

P(WSS) 2.0 10 3 and P(VT) 3.0 10 2. Using SEQ-

CHIP, genotype and sequence data can be more efciently utilized and a stronger association signal was identied.

One tagSNP in DISC1, rs10495310, was associated with BPD I (Table 2). However, three tagSNPs in DISC1 were associated with BPD II with Po0.01 (Table 2). Two of the

DISC1 tagSNPs, rs2812391 and rs10495310, conferred increased risk of BPD II with ORs of 1.2 and 1.4 for each SNP, whereas the third SNP, rs4658954, conferred decreased risk with an OR of 0.8. The tagSNPs rs2812391 and rs10495310 are in mild LD (r2 0.28), whereas both are

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not in LD with rs4658954 (r2 0.01 and r2 0.02, respec

tively). Furthermore, an additional 335 SNPs were imputed in DISC1 (Figure 2, panel a) and one SNP, rs3082, was associated with BPD II with an OR of 1.3 (P 5.5 10 3).

DISC1 haplotype analysis (Figure 2, panel b) indicated that two regions (region 1 at 231.9 Mb and region 2 at 232.1 Mb) were associated with BPD II. The DISC1 region 1 haplotype consisting of rs10495310, rs4658945 and rs16854967 had an empirical global P 1.7 10 3 for its association with BPD II.

The DISC1 region 2 haplotype consisting of rs12137417, rs9431736 and rs7548491 had an empirical global P 3.9 10 2.

Two tagSNPs in GNAS, rs6064714 and rs6026565, were associated with BPD I at Po0.01 (Table 2). However, GNAS appeared to have a stronger association with BPD II in this study with three tagSNPs associated at Po0.01 (Table 2).

Two of tagSNPs conferred increased risk (rs35113254 and rs6026565) for BPD II, whereas the third (rs6064714) conferred decreased risk. The SNPs rs35113254 and rs6026565 are in moderate LD (r2 0.35) but are both not in

LD with rs6064714 (r2 0.04 and r2 0.01, respectively). The

rs35113254 SNP in GNAS was the most signicantly associated tagSNP representing the cAMP signaling pathway with P 5.8 10 5 with an OR of 1.4 for BPD II. Similarly, an

additional 18 SNPs were imputed in GNAS (Figure 3, panel a) and an additional SNP, rs6026560, was associated with BPD II with an OR of 0.7 (P 1.4 10 4). Haplotype analysis in

GNAS indicated a region around 5.74 Mb that was associated with BPD II (Figure 4, panel b). This region overlaps with the results from imputed and genotyped SNPs.

Single tagSNPs in PDE4B (rs4384209), PRKACB (rs600674), GNAI2 (rs2282751), PDE4D (rs17741863) and ADCY8 (rs13278912) were also associated with BPD I with P o0.01. Additionally, imputed SNPs in PRKACB (four imputed

SNPs), PDE4D (nine imputed SNPs) and ADCY8 (one imputed SNP) were associated with BPD I at Po0.01 (Supplementary Table 2). Numerous haplotype windows had Po0.05, however, only PDE10A, DISC1 and GNAS had notable peaks like those presented in Figures 14 panel(b) for these genes.

Results for all two-way interactions between SNPs in the

cAMP signaling pathway with Po0.0001 are presented in Supplementary Table 4. A total of 29 two-way tagSNP interactions with BPD I and 34 two-way tagSNP interactions with BPD II had Po0.0001. For both BPD I and BPD II, several interactions between PDE4 and DISC1 SNPs were signicant. PDE4D tagSNP rs4699931 and ADCY8 tagSNP rs928136 interaction was associated with an increased ORinteraction of 3.65 for BPD I (P 1.06 10 5), while PDE4B

tagSNP rs12404118 and DISC1 tagSNP rs823162 interaction was associated with an increased ORinteraction of 3.53 for BPD

II (P 5.47 10 5).

Discussion

This study represents the rst genetic association study of BPD I and BPD II using dozens of genes comprising the cAMP signaling pathway. Collectively, our results indicate that some of the analyzed SNPs in several genes of the pathway are in LD with causal variants for BPD I and BPD II. Furthermore, the

SNV discovery aspect of our study has identied new variants in the PDE10A gene of BPD subjects that may be causally related to the disorder. Our genetic studies are consistent with prior biochemical studies implicating cAMP signaling in BPD, and highlight specic genes and SNVs that could be of major importance for these disorders.

We employed three noteworthy strategies for elucidating the genetic basis for susceptibility to BPD. First, our association study utilized 29 candidate genes representing a single-signaling pathway. The hypothesis underlying this strategy was that if one gene in the signaling pathway is involved, then casual variants in other genes of the same pathway are likely involved as well. We recognize that there exist known and obvious intersections and cross-talk between the cAMP signaling pathway and other signaling pathways, but we selected genes that are widely recognized as comprising the canonical cAMP signaling pathway in order to sample a dened and limited set of genes. A molecular pathway hypothesis as contrasted with a single gene hypothesis, may account for the genetic heterogeneity of neuropsychiatric disorders in general and the marked difculty in discovering genetic variants that have prominent roles in these disorders. It can also explain heterogeneity in clinical phenotypes or severity within a disorder, since a variant in an AC gene, for instance, might have largely overlapping but some distinct phenotypic effects compared with a variant in cAMP PDE. The interaction analyses were motivated by the molecular pathway hypothesis as functional variants in two molecular functions of a pathway could produce a more pronounced effect than either single functional variant. For instance, the relatively large OR produced by considering an interaction between PDE4D and ADCY8 for BPD I (Supplementary Table 4) could be due to a functional variant that decreases PDE4D activity synergistically interacting with one that increases ADCY8 activity. Similarly, an interaction between variants in PDE4B and DISC1 for BPD II could be because of two separate functional variants that have synergistic effects on signaling. These ideas remain speculative and depend on the sign and magnitude that the functional variants have on the molecular factors involved.

Second, the choice of candidate molecular pathways considered the known and major role of the signaling pathway in the regulation of behavior in model systems. Genetic studies of learning and memory, circadian rhythms and other behaviors in model systems like Drosophila and the mouse have identied hundreds of genes that have behavioral roles. A good example of this is the cAMP PDE encoded by the Drosophila dunce gene, which is involved in learning and memory in Drosophila,66 but whose human counterparts are involved in mood regulation48 and schizoaffective disorders.67 Candidate genes selected from the human homologs of genes with known behavioral roles in model systems should provide an extremely effective lter in gene discovery for neuropsychiatric disorders.

Third, we followed-up the initial association study with SNV discovery in the vicinity of the associated PDE10A tagSNPs that identied additional variants that may be causally involved in BPD. This required the development of a new method for integrating information from sequencing projects in a cost effective way. Using this method, named SEQCHIP, we

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were able to combine the sequencing data from 30 BPD I patients used for discovery with the data from the follow-up genotyping in a larger subset while accounting for potential bias. This approach was used to identify potentially causal variants that are tagged by the genotyped SNPs. When the data are integrated by SEQCHIP, all rare variant tests have correct type I errors. The genetic information from both sequence and genotype data were efciently utilized, and therefore, more signicant association signals were identied.

Suggestive evidence from other studies is consistent with the possibility that these newly discovered SNVs are involved in BPD. A recent RNA sequencing study employing human prefrontal cortex suggested that two of these SNVs (chr6:166052752 C4G and chr6: 166046780_166046778 delATT) may be associated with novel PDE10A transcripts.68

Another study performing chromatin immunoprecipitation sequencing on neuronal cells from human prefrontal cortex tissue showed that SNVs, chr6:166058713 C4T, chr6: 166056798 A4T and chr6: 166046780_166046778 delATT are associated with an enriched H3K4me3 signal, suggesting that they participate in the transcriptional regulation of PDE10A.69 Although this preliminary evidence must be conrmed in human tissue expressing PDE10A, it does suggest that these variants may have a role in PDE10A protein expression.

Our study highlights the association of PDE10A with BPD I. Of related and high interest is the observation that the PDE10A gene resides at 6q26, within one of the four leading linkage regions for BPD.2 The PDEs degrade cAMP and/or cyclic guanosine monophosphate to their 50-AMP and 50-GMP counterparts and members of this large enzyme family are known to have broad roles in controlling behavioral output in diverse organisms from ies to humans.66,70 The family

member PDE10A codes for an 89-kDA protein capable of hydrolyzing both cAMP and cyclic guanosine monophosphate that is expressed in the brain and preferentially in the basal ganglia.7174 This expression pattern paired with basal gang-lia dysfunction in schizophrenia have led to robust efforts in identifying drugs that target PDE10A for the treatment of schizophrenia.7577 The association of PDE10A with BPD I expands the potential range over which PDE10A drugs might exhibit efcacy.

The gene disrupted in schizophrenia 1 (DISC1) was rst implicated in psychiatric disorders after a t(1;11) translocation was discovered in a large Scottish family cosegregating with schizophrenia, BPD and recurrent major depression.78,79 The

DISC1 region has subsequently been implicated with bipolar spectrum traits in several candidate gene association studies.8085 The selection of tagSNPs has varied from study to study so we were unable to compare the precise haplotype windows between this and other studies. In addition, the majority of studies failed to control for population admixture and in most cases termed a P-value of less than nominal signicance (P-value o0.05) as signicant. Relative to individual tagSNP associations that have been reported,

Perlis et al.86 observed an association between rs1934909 and BPD. We observed an association between this tagSNP and BPD II (OR 0.8, P 0.01) but not BPD I cases (Supple

mentary Table 1). Similarly, Schosser et al.83 observed an

increased prevalence of BPD for carriers of the minor allele of rs2492367. We observed an association between this SNP and BPD II but not BPD I (OR 0.8, P 0.03).

It seems highly likely given the complexity of the DISC1 protein and its multiple molecular roles87 that there exist multiple functional variants linked to the various tagSNPs used in this and other studies. DISC1 interacts with several key proteins in pathways that have important roles in neuronal migration, dendritic organization and neurogenesis in addition to cAMP signaling.67,8792 The binding sites for interacting

proteins are plausible regions for multiple susceptibility regions as are regions of the protein that modify binding. This could underlie the DISC1 and PDE4 tagSNP interactions observed to be signicantly associated with BPD I and BPD II in this study. Furthermore, DISC1 variants in intronic regions may inuence DISC1 mRNA and subsequent protein levels that also could interfere with key pathways. Indeed, Maeda et al.93 demonstrated reduced DISC1 mRNA levels in lymphoblasts from BPD subjects compared with unaffected family controls. Thus, it is plausible that multiple genetic variants in LD with DISC1 tagSNPs exist that alter protein function or mRNA levels that inuence susceptibility to neuropsychiatric disorders. Identifying these variants and characterizing their effect on DISC1 molecular functions may provide useful information for the development of novel therapeutics for bipolar spectrum traits.

The guanine nucleotide-binding protein, a-stimulating poly-peptide 1 (GNAS) encodes the Gas subunit of heterotrimeric

G-proteins with Gas activating AC for increased synthesis of cAMP. Although the exact mode of action of lithium in treating BPD remains elusive, there exists evidence that lithium decreases G-protein subunit mRNA levels.35 Moreover, it has been speculated that lithium functions to stabilize the inactive conformation of G-proteins.94 GNAS variants, like rs6064714 and rs6026565, may be tagging functional variants in BPD I patients that interfere with Gas role in regulating cAMP signaling.

It is of interest that SNPs representing certain genes, such as PDE4B and PDE10A, were found as associated with BPD I but not BPD II. It is possible that this represents an authentic genetic difference between the two BPD subtypes. However, it could also be that the power to detect the association was stronger with the BPD I population, given that the BPD I population was twice the size of the BPD II population. If the data reect authentic genetic differences between the two subtypes, they could be important to the pharmacogenetics of BPD treatment. For instance, perhaps an altered regulation of PDE10A, specic to BPD I, is most related to full-blown mania associated with BPD I. Drugs with PDE10A action may then have a primary effect on mania.

Overall, this study provides insight into the variation that exists in genes in the cAMP signaling pathway and the association of that variation with BPD I and BPD II. Key regions were identied in PDE10A, GNAS and DISC1 that are associated with BPD I and BPD II. The effect size for these associations was modest, as observed in most other genetic association studies for neuropsychiatric disorders. Our study also illustrates the feasibility of performing large candidate gene or GWAS (genome-wide associated studies) studies

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followed by targeted direct sequencing to uncover novel, rare SNVs that may be associated with disease.

Conict of interest

The authors declare no conict of interest.

Acknowledgements. This study was supported by grant R01 MH074791 to RLD.

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