Journal of Affective Disorders 136 (2012) 902–908

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Journal of Affective Disorders

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Research report

Cholecystokinin system genes: Associations with panic and otherpsychiatric disorders

Julia Wilson⁎, David Markie, Alison FitchesDepartment of Pathology, University of Otago, Dunedin, New Zealand

a r t i c l e i n f o

⁎ Corresponding author at: Department of PatholoMedicine, University of Otago, PO Box 913, DunedinTel.: +64 3 479 9190; fax: +64 3 479 7136.

E-mail address: julia.wilson@otago.ac.nz (J. Wilson

0165-0327/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.jad.2011.09.011

a b s t r a c t

Article history:Received 15 June 2011Received in revised form 12 September 2011Accepted 12 September 2011Available online 5 October 2011

Background: The cholecystokinin (CCK) system has long been hypothesised to have a role in thepathogenesis of panic attacks. Previous research into genetic variation within the CCK gene andthe genes for its two receptors, CCKAR and CCKBR, has producedmixed results.Weaimed to clarifythis association by investigating multiple variants within each gene and multiple phenotypes as-sociated with panic that may have confounded the previous studies' findings.Methods:Variants were selected for the three genes based on HapMap CEU data. Individuals froma family based cohort (n=563) were genotyped for these variations and this data was analysedin FBAT.Results: CCKBR showed the strongest association with panic, having multiple variants withpb0.05 (lowest: p=0.007). In CCKAR, some evidence was found for an association with panic,though further analysis suggested that the co-morbid bipolar–panic phenotype was most strong-ly associated. No variants in CCKwere associated with panic but broader anxiety phenotypes didshow associations.Limitations: Small sample size prevented thorough investigation of phenotypes, particularly puredisorders, and no correction was made for the multiple phenotypes analysed.Conclusions: Our findings support the involvement of variation in the CCK system, particularlyCCKBR, in the pathogenesis of panic. Our data suggest that variation in CCK may be involved inseveral anxiety phenotypes and CCKAR may be involved in the development of panic co-morbidwith bipolar disorder. These latter findings require further investigation and highlight the impor-tance of clearly defined phenotypes when investigating psychiatric genetics.

© 2011 Elsevier B.V. All rights reserved.

Keywords:Panic disorderAnxiety disordersGeneticsCholecystokinin

1. Introduction

Panic is a pattern of anxiety characterised by acute attacks,with panic disorder (PD) being the most severe form (Carsonet al., 2000). There is strong evidence for a genetic contributionto PD, with heritability estimated at over 40% (Hettema et al.,2001). A meta-analysis of family studies found an approxi-mately five-fold increased risk of developing PD among first-degree relatives of a PD proband compared to other relatives(Hettema et al., 2001). Additionally twin studies have consis-

gy, Dunedin School o9054, New Zealand

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tently found higher rates of concordance betweenmonozygot-ic, than dizygotic twins, such that amonozygotic twin of some-one with PD may be nine times more likely to have PD than adizygotic twin (Chantarujikapong et al., 2001). Multiple genesare expected to be involved, with some predisposing individ-uals to higher anxiety levels in general and other genes predis-posing to specific anxiety disorders. This is further complicatedby gene–gene and gene–environment interactions.

Cholecystokinin (CCK) is a peptide that acts as a signallingmolecule in the gut and central nervous system (CNS) (asreviewed by Bourin et al., 1998). Administration of CCK inducespanic attacks in both patients with PD and healthy controls, al-though higher doses are required in controls (Eser et al., 2005;Zwanzger et al., 2004). As with naturally occurring panic at-tacks, CCK induced panic can be attenuated by acute treatment

Table 1Number of subjects (N females in parentheses) with each phenotype withinstudy sample.

Co-morbidity No panic Any Panic PD PA PS

Any mood 284 (162) 116 (79) 54 (37) 44 (31) 12 (6)Depression 150 (93) 33 (25) 11 (10) 17 (13) 4 (1)Any BP 134 (69) 83 (54) 43 (27) 27 (18) 8 (5)BP I or II 115 (56) 75 (48) 40 (24) 24 (16) 8 (5)No BP 301 (162) 44 (32) 13 (12) 24 (17) 6 (2)No mood 151 (69) 11 (7) 2 (2) 7 (4) 2 (1)Other anxiety 90 (46) 65 (46) 41 (30) 17 (11) 4 (3)Agoraphobia 7 (3) 33 (24) 5 (4) 4 (4) 0 (0)Social phobia 44 (16) 34 (25) 29 (20) 7 (4) 2 (2)Spec. phobia 48 (29) 39 (28) 25 (19) 12 (8) 2 (1)OCD 5 (3) 8 (6) 5 (4) 1 (0) 2 (2)No other anxiety 345 (185) 62 (40) 15 (9) 34 (24) 10 (4)Any substance 94 (22) 44 (21) 23 (13) 15 (6) 4 (1)No substance 341 (209) 83 (65) 33 (26) 36 (29) 10 (6)Total 435 (231) 127 (86) 56 (39) 51 (35) 14 (7)

PD=panic disorder; PA=panic attacks; PS= limited symptompanic attacks;BP=bipolar disorder; Spec.= specific; OCD=obsessive compulsive disorder;Substance includes any substance abuse or dependence disorder;subsyndromal forms of anxiety and substance disorders were not included.

903J. Wilson et al. / Journal of Affective Disorders 136 (2012) 902–908

with benzodiazepines (Zwanzger et al., 2003) or chronic treat-ment with antidepressants (Bradwejn and Koszycki, 1994;Shlik et al., 1997; vanMegen et al., 1997). Hence, abnormalitiesin the CCK system are hypothesised to be involved in the path-ogenesis of PD.

Various active forms of CCK are produced by cleavage of a115 amino acid precursor protein (encoded by the gene CCK),the most common being the eight amino acid sulphated form(Harro, 2006). CCK has two known receptors, CCKA and CCKB,encoded by the genes CCKAR and CCKBR respectively. TheCCKB receptor is the most common form in the brain and isfound in high levels in the striatum, cerebral cortex, olfactorybulb and olfactory tubercle (as reviewed by Harro, 2006).Compared to controls, patients with PD have been found tohave lower levels of CCK in cerebrospinal fluid (Lydiard,1994) and enhanced CCK induced calcium mobilisationfrom T cells (Akiyoshi et al., 1996). In addition, transgenicmice that over-express CCKBR show higher levels of anxietythan wild-type mice and, similar to anxiety in humans, thiscan be attenuated with benzodiazepines (Chen et al., 2006).

There have been a number of previous studies investigatingpossible associations between CCK system genes and panic. Atleast five of these found no significant associations (Hamiltonet al., 2001; Hattori et al., 2001b; Ise et al., 2003; Kato et al.,1996; Yamada et al., 2001); however, other studies havefound associations with single nucleotide polymorphisms(SNPs), haplotypes and/or repeat length polymorphisms inCCK system genes (Gratacos et al., 2009; Hattori et al., 2001a;Hosing et al., 2004; Kennedy et al., 1999; Koefoed et al., 2010;Maron et al., 2005; Miyasaka et al., 2004; Wang et al., 1998).These mixed results suggest that there is an association withinthis system, but the specific functional variants have yet to befound or that population specific gene–environment interac-tions are altering the associations.

In the present study we investigated whether variation inthe CCK system genes (CCK, CCKAR and CCKBR) was associatedwith panic in a New Zealand cohort. Furthermore, we exploredwhether other phenotypes often co-morbid with panic wereassociated with CCK system gene variation, the findings ofwhich may help explain the variability of past research.

2. Materials and methods

2.1. Subjects

Recruitment of participants for the South Island BipolarStudy (SIBS) has been described elsewhere (Joyce et al., 2004).Briefly, participants were individuals from the South Island ofNew Zealand with bipolar I or bipolar II (bipolar probands)and their family members. All participants gave informed con-sent, a blood sample for DNA extraction, and detailed medicalinformation in the form of the Diagnostic Interview for GeneticStudies (DIGS) (Nurnberger et al., 1994). Diagnoses werebased on DSM-IV criteria (American Psychiatric Association,1994). This study was conducted with the approval of theMulti-region Ethics Committee, New Zealand.

The present research investigated 795 individuals (563with direct genotype and phenotype information), forming139 pedigrees (200 nuclear families), from the SIBS. Pedigreesvaried in size and composition, the smallest having threemembers (at least two with genotype/phenotype information)

and the largest having twelve members (up to eight withgenotype/phenotype information) and three generations.The majority of the sample is of UK/Irish or European descent,but also included 25 individuals with one or both parents ofother or unknown ethnicity. The age of participants averaged45 years and ranged from 17 to 94 years.

Anxiety, particularly PD, has been previously found to behighly co-morbid with bipolar disorder (MacKinnon et al.,1997) and this has also been found in the SIBS, making this co-hort useful for the investigation of these disorders (Doughty etal., 2004). The primary phenotype analysed in this study wasany panic spectrum disorder (Any Panic), including panic disor-der (PD), multiple panic attacks (PA) and multiple limitedsymptom panic attacks (PS). Since the cohort was selected onthe basis of bipolar disorder, a control analysis was undertakento ensure any associations seen were not with bipolar disorder.For this analysis, individuals with any type of bipolar disorderthat did not have co-morbid panic were coded as affected (BPno Panic). As panic is commonly co-morbid with mood andother anxiety disorders (Scott et al., 2006), additional analyseswere undertaken to investigate which phenotypes were mostcommonly associatedwith each gene. These phenotypes includ-ed: bipolar disorder I or II (BPD); any panic co-morbid with anybipolar disorder (Panic+BP); PD, PA, any phobia or obsessivecompulsive disorder (OCD) (Anxiety); any panic co-morbidwith any phobia (Panic+Phobia); any panic co-morbid withsubstance abuse or dependence disorders (Panic+Substance);and subgroups of panic. Sub-syndromal forms of OCD or phobicdisorders were not included as affected.

The numbers of affected participants for the various phe-notypes are described in Table 1. No patterns were noted be-tween affection status and ethnicity, but the average age ofonset did vary for the different disorder groups. Any Panichad the oldest onset age (just under 25 years), followed bythe mood disorders (approximately 24 years), while theonset of substance disorders was 21.5 years and other anxi-ety disorders was just over 14 years. The majority of thosewith any panic co-morbid with any mood disorder developedthe mood disorder first (approximately 60%), while 20%

904 J. Wilson et al. / Journal of Affective Disorders 136 (2012) 902–908

experienced panic first and the remainder first experiencedboth disorders within the same year.

2.2. Genetic analysis

Phase II data for CCK, CCKAR and CCKBR were downloadedfrom the HapMap Project (http://www.hapmap.org) during2007 and analysed using Haploview software (http://www.broad.mit.edu/mpg/haploview/). The HapMap CEU populationdata was used, as this was the most similar population to ourstudy group. The CEU population consists of Utah residentswith Northern/Western European ancestry and the Phase IIdata for this group is expected to cover over 90% of commongenetic variation (The International HapMap Consortium,2005). To determinewhich SNPs to genotype, blocks were cus-tom designed so that one block encompassed the entire geneand any SNPs within 1 kb of the 5′ untranslated region. Onlyhaplotypes with a frequency of at least 1% were included.SNPs with a rare allele frequency of at least 3% that were de-fined as tag SNPs by Haploview were genotyped and analysed,with one exception — rs2947029 in CCKBR was selected inplace of rs2880898. For haplotype analysis, blockswere createdusing the default setting/confidence intervals analysis (Gabrielet al., 2002). Haplotypes for CCKwere formed from rs4602358,rs10865918 and rs747455. Haplotypes for CCKARwere formedfrom rs7665027, rs2000978 and rs1800855. Haplotypes forCCKBR were formed from rs906895, rs292180, rs2941025,rs2929183, rs2941023, rs2947025, rs2880898 and rs2947028.

Various methodologies were used to genotype tag SNPs andsequencing was used to confirm the accuracy of genotyping forsome SNPs (Supplementary data). TaqMan hydrolysis probeswere purchased from Applied Biosystems and used accordingto manufacturer's instructions. For all other reactions, primerswere custom designed and optimised for MgCl2 concentration,annealing temperature and number of cycles. PCR conditionsconsisted of: 1.0–3.0 mM MgCl2 in 1x PCR buffer, 100 μM ofeach dNTP, 200 nM of each primer, 0.5 U Taq polymerase (Invi-trogen), and 100 ng DNA template, in a total volume of 20 μl.The basic programme was: denature at 94 °C for 2 min, dena-ture at 94 °C for 30 s, anneal at 55–64 °C for 30 s, extend at72 °C for 45 s, repeat these three steps for 28–45 cycles followedby a final extension of 72 °C for 5 min. Allele specific primerswere designed to the SNP, optimised and, when possible, multi-plexed with other sets of allele specific primers. Primer pairs forrestriction fragment length polymorphism (RFLP) or sequenc-ing assays were designed 50–200 bp from the SNP. Restrictionenzymes were obtained from NEB and digests were conductedovernight according to manufacturer's instructions. Sequencingreactions were carried out using BigDye Terminator v3.1 (Ap-plied Biosystems), according to the manufacturer's instructionsand visualised using an ABI Prism 310 Genetic Analyzer and310 Data Collection Software Version 3.1.0.

2.3. Statistical analysis

All genotyped SNPswere analysed to ensure that the result-ing allele frequencies did not significantly differ from Hardy–Weinberg equilibrium. Since our cohort is family based, smalldiscrepancies that may have been the result of a single familywere ignored. If large inconsistencies were found, the genotyp-ing accuracy was confirmed by sequencing.

Transmission disequilibrium test (TDT) was conductedusing the FBAT statistical programme with the empirical cor-rection to account for possible linkage in extended pedigrees(Laird et al., 2000). Separate pedigree files were constructedfor each phenotype analysed. Haplotype analysis utilisedthe tag SNPs identified by the confidence intervals analysisof the HapMap data. Unadjusted p-values are presented,with a threshold of pb0.05 for nominal significance andpb0.017 when adjusted to account for the three genes inves-tigated (Neale and Sham, 2004). We have not corrected forthe multiple variants tested within each gene, as these vari-ants are not independent; consequently, the overall patternof association within each gene should be considered moreimportant than individual SNPs or haplotypes.

3. Results

Locations of tag SNPs from the custom designed blocks(encompassing the whole gene) are shown in Fig. 1, with fur-ther details included in the Supplementary data. Most SNPsdisplayed similar rare allele frequencies to those of the Hap-Map CEU population and few, if any Mendelian errors (Sup-plementary data). Six SNPs (two tag SNPs) were not inHardy–Weinberg equilibrium; however, most of theseshowed small deviations from the predicted values, generallydue to higher numbers of homozygous individuals, caused bythe family-based nature of the cohort. There was one excep-tion: rs1042047 in CCKBR had an excess over predicted of ho-mozygous rare individuals (84 vs 51) and homozygouscommon (288 vs 255) and also had a higher number ofMendelian errors than any other SNP. This SNP was initiallygenotyped by TaqMan hydrolysis probe; when sequenced,another SNP (rs8192471) was noted four base pairs away,which is assumed to have disrupted probe annealing andtherefore altered the genotyping call. A subset of familieswas re-genotyped by sequencing and analysed for associa-tion with Any Panic. Since no association was found in the16 analysable families, no further sequencing was conductedand this SNP was excluded from further evaluation.

3.1. Associations with Any Panic

No statistically significant associationswere foundwith CCKSNPs or haplotypes and Any Panic. For CCKAR, one SNP and thetwomost common haplotypes were associated with Any Panicat pb0.05 (Table 2). Of the 14 SNPs analysed in CCKBR, eightshowed a significant association with Any Panic, includingone SNP with a p-value below the adjusted threshold(rs2947028, p=0.009). Additionally, one of the three CCKBRhaplotypes common enough to analyse was also significantlyassociated with Any Panic, with p=0.007 (Table 2). No SNPsor haplotypes in CCK or CCKBR showed a significant associationin the control analysis, bipolar disorder without panic (BP noPanic). One SNP and one haplotype in CCKAR did show an asso-ciation with BP no Panic, at pb0.05.

3.2. Phenotypes most commonly associated with each gene

A variety of different co-morbid conditions were presentin the SIBS cohort (Table 1). Each of the CCK system SNPsand haplotypes were analysed against multiple different

Fig. 1. Schematic of CCK, CCKAR and CCKBR (NM_000729.4, NM_000730.2 and NM_176875.2 respectively) with the location of tag SNPs from custom designedblocks. The forward strand is shown and horizontal arrows indicate gene direction. CCK has two common splice variants as shown by boxes above (variant 1)and below (variant 2) the line.

905J. Wilson et al. / Journal of Affective Disorders 136 (2012) 902–908

phenotypes that have previously been found to be highly co-morbid with panic. These phenotypes included: bipolar dis-order I or II (BPD); PD, PA, any phobia or obsessive compul-sive disorder (OCD) (Anxiety); any panic co-morbid withany phobia (Panic+Phobia); any panic co-morbid with sub-stance use or abuse disorders (Panic+Substance); any panicco-morbid with any bipolar disorder (Panic+BP); and sub-groups of panic (PD, PD or PA, PA, PA or PS).

CCK variantsmost commonly demonstrated the strongest as-sociation with Anxiety phenotypes (Table 3). Using the nominalsignificance threshold (pb0.05), two CCK variants showed thestrongest association with Anxiety, one with panic co-morbidwith any phobia (Panic+Phobia), and one with BPD. ForCCKAR, the strongest significant associations were seen forpanic with co-morbid bipolar disorder (Panic+BP) (three vari-ants with pb0.05), BP no Panic (two variants with pb0.05) andBPD (one variant with pb0.017) (Table 3). For CCKBR, the stron-gest significant associations were seen with Any Panic or sub-groups of panic for seven variants, four with pb0.017. Anxietyalso showed an association with two CCKBR variants (pb0.05),and BPD with one (pb0.017).

4. Discussion

This study thoroughly investigated the three CCK systemgenes by genotyping multiple SNPs within each gene and,

unlike previous studies, also investigated whether other phe-notypes often found to be co-morbid with panic might alsobe associated with these genes. Our data supported an associa-tion of panic with CCKBR, and provided some evidence for anassociation with CCKAR. However, despite analysing multipleSNPs and haplotypes, there was no evidence that variationwithin CCK is associated with panic. Further analysis of thedata also indicated that CCKBR is associated with panic, whileCCKAR appeared to be specifically associated with the panic–bipolar co-morbid phenotype and CCK with various anxietydisorders. Our study cohortwas selected on the basis of bipolardisorder and approximately two thirds of thosewith panic alsohad co-morbid bipolar disorder. Consequently, any significantfindings for panic may have been false-positives due to associ-ations with bipolar disorder. To eliminate this possibility weperformed a control analysis, coding everyone with any bipo-lar disorder without panic as affected. The only statisticallysignificant associations we found were for CCKAR, making itunlikely that other resultswere due to the presence of co-morbidbipolar disorder.

Our findings provide the greatest support for the involve-ment of CCKBR variants in the pathogenesis of panic. Overhalf of the variants tested reached the threshold for nominalsignificance with Any Panic and most of these were moststrongly associatedwith panic. Although several previous stud-ies have found no association with CCKBR variants and panic

Table 2Associations between CCK system variants and Any Panic or BP no panic.

Variant Any Panica BP no panicb

Familiesc Trans.d p-value Familiesc Trans.d p-value

CCK-SNPsrs8192472 28 −0.2 0.961 29 −5.7 0.122rs11129949 25 −1.9 0.508 25 −4.1 0.156rs4602358 33 1.2 0.752 34 −1.6 0.638rs10865918 38 −5.2 0.246 34 −3.2 0.399rs747455 30 0.1 0.974 31 −3.3 0.294

HaplotypesGGG 37 −6.5 0.131 37 −3.1 0.408ATG 31 1.3 0.704 33 −3.5 0.264ATA 27 0.1 0.975 23 3.1 0.267GTG 21 4.4 0.097 20 3.4 0.280

CCKAR-SNPsrs7665027 13 2.3 0.144 15 −4.8 0.044rs2000978 21 5.3 0.043 26 3.5 0.231rs1800855 21 2.2 0.492 20 −2.8 0.337rs6448456 24 4.9 0.070 25 1.3 0.646

HaplotypesATT 35 −7.5 0.045 34 2.8 0.428ACT 22 5.4 0.035 25 2.9 0.292ATA 14 −0.3 0.881 13 −0.2 0.913GTA 12 1.7 0.348 16 −5.0 0.050

CCKBR-SNPsrs906895 33 1.6 0.697 38 3.7 0.462rs2929180 30 7.6 0.038 31 0.7 0.800rs2941025 32 8.3 0.021 29 0.0 0.987rs2929183 34 8.9 0.027 31 0.5 0.886rs2941023 34 8.9 0.027 30 −0.5 0.862rs2947025 31 6.7 0.077 27 0.1 0.966rs7943852 27 −4.3 0.219 36 1.8 0.720rs2880898 25 −6.1 0.032 26 1.2 0.800rs1112716 24 −5.6 0.046 26 1.2 0.800rs2947029 37 5.3 0.203 37 1.4 0.793rs2947028 36 11.0 0.009⁎ 32 0.7 0.844rs12364575 32 8.8 0.030 32 4.4 0.246rs1042048 36 −1.9 0.665 42 1.0 0.791

HaplotypesAGCAAGTT 38 −3.8 0.359 33 −2.2 0.697GGCAAGCT 25 −8.4 0.007⁎ 22 2.8 0.517GTTGGCTC 26 5.7 0.088 20 −0.9 0.734

Italicised SNPs were used to form haplotypes. Bold indicates pb0.05(nominal significance), asterisk indicates pb0.017 (adjusted significance).

a Includes individuals with any panic spectrum disorders (PD, PA and PS)as affected.

b A control analysis in which individuals with any bipolar disorder and noco-morbid panic spectrum disorder are coded as affected.

c Number of informative families for the variant/phenotype combination.d Number of over or under transmissions of the rare allele (see Supple-

mentary data) or haplotype. Values are not whole numbers as complete pa-rental genotypes are sometimes unknown.

Table 3Strongest significant associations between CCK system variants and variousphenotypes.

Variant Phenotypea Familiesb Trans.c p-value

CCK-SNPsrs8192472 BPD 41 −10.6 0.019rs10865918 Anxiety 45 −11.2 0.040

Haplotypesd

GGG Anxiety 41 −11.9 0.024GTG Panic+Phobia 14 4.8 0.023

CCKAR-SNPsrs7665027 BP no Panic 15 −4.8 0.044rs2000978 BPD 37 8.8 0.013rs6448456 Panic+BP 17 4.8 0.048

Haplotypesd

ATT Panic+BP 27 −7.2 0.011ACT Panic+BP 15 5.1 0.023GTA BP no Panic 16 −5.0 0.050

CCKBR-SNPsrs2929180 Anxiety 34 8.4 0.026rs2941025 Anxiety 33 8.4 0.020rs2929183 Any Panic 34 8.9 0.027rs2941023 Any Panic 34 8.9 0.027rs7943852 PA or PS 16 −5.6 0.022rs2880898 PA or PS 15 −6.0 0.004rs1112716 PA or PS 14 −5.5 0.006rs2947028 Any Panic 36 11.0 0.009rs12364575 BPD 50 11.8 0.011

Haplotypesd

GGCAAGCT PA or PS 15 −6.3 0.006

Only variants with pb0.05 (nominal significance) are shown, those withpb0.017 (adjusted significance) are in bold.

a Phenotypes analysed: any panic spectrum disorder (Any Panic); anybipolar disorder without co-morbid panic (BP no Panic); bipolar disorder I orII (BPD); PD, PA, any phobia or OCD (Anxiety); any panic co-morbid with anyphobia (Panic+Phobia); any panic co-morbid with substance use or abusedisorders (Panic+Substance); any panic co-morbid with any bipolardisorder (Panic+BP); and subgroups of panic.

b Number of informative families for the variant/phenotype combination.c Number of transmissions over or under the expected value for the rare

allele (see Supplementary data) or haplotype. Values are not whole numbersas complete parental genotypes are sometimes unknown.

d Haplotypes were formed from: CCK — rs4602358, rs10865918 andrs747455; CCKAR — rs7665027, rs2000978 and rs1800855; CCKBR —

rs906895, rs292180, rs2941025, rs2929183, rs2941023, rs2947025,rs2880898 and rs2947028.

906 J. Wilson et al. / Journal of Affective Disorders 136 (2012) 902–908

(Hamilton et al., 2001; Hattori et al., 2001b; Kato et al., 1996;Koefoed et al., 2010; Yamada et al., 2001), others have identi-fied significant associations (Gratacos et al., 2009; Hosing etal., 2004; Kennedy et al., 1999; Maron et al., 2005). Additionalsupport for our CCKBR results come from the finding of mis-sense mutations in the gene in a small number of PD cases(Koefoed et al., 2010) and an association between PD and a var-iant of a microRNA that represses CCKBR translation (Muinos-Gimeno et al., 2011). Furthermore, CCKB receptor is thoughtto be the target for CCK induced panic attacks as CCKB receptor

selective agonists will induce panic attacks, and selective an-tagonists block both the psychological and physiological symp-toms (Lines et al., 1995). Hence, CCKBR has both biologicalplausibility and genetic evidence for involvement in panic.

Studies of CCKAR variants and panic have typically shownno significant association (Gratacos et al., 2009; Ise et al.,2003; Kennedy et al., 1999; Koefoed et al., 2010), with haplo-types tending to be the exception to this rule (Koefoed et al.,2010; Miyasaka et al., 2004). This is similar to our findings ofnominal significance for three CCKAR variants, including twohaplotypes. The strongest associations we found for CCKARwere for the co-morbid panic–bipolar phenotype. These sec-ondary analyses are likely to have produced false-positive re-sults and individual variants should be viewed with caution.However, we found multiple variants in CCKAR that suggestedan association with bipolar disorder (with or without co-

907J. Wilson et al. / Journal of Affective Disorders 136 (2012) 902–908

morbid panic); over half of all variants tested showed thestrongest association with a BP phenotype and two of thesehad p-values below the adjusted threshold.

Although there has been little investigation of CCKAR, theco-morbid bipolar–panic phenotype has been studied previ-ously. Family studies of bipolar disorder have found highlevels of PD in bipolar probands and their relatives, resultingin the suggestion that the co-morbidity is a distinct subtypewith specific genetic risk alleles (MacKinnon et al., 1997,2002). Molecular research has also found some evidence forunique genetics in this co-morbid condition (Rotondo et al.,2002). Furthermore, a study following up on a region associ-atedwith BPD in GWAS, found CCKAR overlappedwith a haplo-type that was significantly associated with BPD (Christoforouet al., 2007) and the PD–major depressive disorder co-morbidphenotype has been found to be associated with a variant inCCKAR (Maron et al., 2005). Hence, further investigation ofCCKAR and mood disorders is warranted.

One of the most frequently studied CCK system variants isrs1799923 in CCK. We did not genotype this SNP directly, how-ever, we did use haplotypes to infer the subjects' genotype atrs1799923 and confirmed the accuracy of this by sequencing asubset of our sample; no significant association was seen withAny Panic (Supplementary data). Five previous studies alsofound no significant association (Hamilton et al., 2001; Hattoriet al., 2001a,b; Hosing et al., 2004; Kennedy et al., 1999;Maron et al., 2005), two found that the rare allele was over-represented in patients (Maron et al., 2005; Wang et al., 1998),and one found that the rare allele was under-represented inpatients (Koefoed et al., 2010). Most other CCK variants studiedhave also failed to produce significant results, with the excep-tions including: a SNP (not genotyped in our research) thatwas associated with PD at pb0.05 (Maron et al., 2005); anda repeat length polymorphism associated with PD (Hattoriet al., 2001a).

The conflicting data for associations between CCK and PDare typical for this area of research, as variants that showconsis-tent, reproducible results are unusual (Maron et al., 2010). Thislack of reproducibility, however, does not necessarily indicate alack of involvement for the genes concerned; there are severalpossible reasons for variation between studies. The specificpopulations studied vary frompaper to paper. If the variants in-vestigated are not causal, but are in linkage disequilibriumwithsuch variants, unpredictable associations between variants fordifferent ethnic groups may have affected the results. Similarly,gene–gene or gene–environment interactions may result in as-sociations with only one study population; for example, if thevariant is only causative under environmental conditions com-mon to just one of the study populations. The study populationsalso vary on phenotype(s) analysed: some studies have lookedat pure PD, others PD and PA, and there has been variation onthe inclusion or exclusion of co-morbid conditions. The resultsof our secondary analyses suggest that these differences mayhave a considerable effect on the study outcome. Finally, thestudies on CCK system genetics to date have suffered fromsmall sample sizes and, as such,many of these papers, includingour own, may have missed variants of small effect.

This research is a family based association study of CCK sys-tem genetics and panic. Most previous studies have used acase–control design, which requires careful selection of the con-trol cohort to avoid false-positive results due to stratification.

Since TDT does not require a control cohort, false-positive resultsfrom stratification are less likely to occur (Laird et al., 2000). Ad-ditionally, the analysis method we used only analyses transmis-sion to affected individuals (unaffected and unknown aretreated identically); hence, family members that developed adisorder after the survey would not have biassed the results.Our sample size, however, is small by current standards andour results need to be replicated in larger cohorts. This is themajor limitation of our study and has prevented the investiga-tion of pure phenotypes. Most previous studies of CCK systemgenes have only looked at one to a few polymorphisms pergene. Our study used data from the International HapMap Pro-ject to select multiple SNPs within each gene and, hence, thor-oughly characterise common variation within each gene.Therefore, we may have found associations that other studiesmissed. Conversely, the high number of polymorphisms investi-gated increases the likelihoodof finding false-positive results. Al-though we provided a corrected p-value to for the three genestested, we have not corrected for the total number of variants,as they are not independent of each other. Consequently, we be-lieve the focus should be on the overall pattern and number ofsignificant associations in each gene, not individual variants.Given this, our lack of significant associations with CCK SNPs orhaplotypes makes it unlikely that common variants at thisgene have a clinically significant effect on the risk of developingpanic; however, we cannot draw conclusions about specificpanic phenotypes, such as pure PD, as our cohort was not largeenough to investigate these.

Our findings add to the current evidence suggesting thatthe CCK system, particularly CCKB receptor, is involved inthe pathogenesis of panic. We also found evidence that CCKmay be involved in various co-morbid anxiety phenotypesand that CCKA receptor may be involved in the developmentof panic co-morbidwith bipolar disorder. Both of these findingsrequire further investigation.

Supplementary materials related to this article can befound online at doi:10.1016/j.jad.2011.09.011.

Role of funding sourceThe research was conducted during tenure of a Māori Health PhD Scholar-

ship of the Health Research Council of New Zealand and was supported by anHS and JC Anderson Trust Grant. Funders had no further role in: study design;the collection, analysis and interpretation of data; thewriting of the report; andthe decision to submit the paper for publication.

Conflict of interestThe authors of this study have no conflicts of interest.

AcknowledgementsThe authorswish to thank formermembers of the laboratory for their tech-

nical contribution to the genotyping of CCK and Kate Sears for her work to con-firm the relationships within the SIBS cohort.

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