Article

Pleiotropic Locus for Emotion Recognition and AmygdalaVolume Identified Using Univariate and Bivariate Linkage

Emma EM Knowles PhD

D Reese McKay PhD

Jack W Kent Jr PhD

Emma Sprooten PhD

Melanie A Carless PhD

Joanne E Curran PhD

Marcio AA de Almeida PhD

Thomas D Dyer PhD

Harald HH Goumlring PhD

Rene L Olvera MD

Ravi Duggirala PhD

Peter T Fox MD

Laura Almasy PhD

John Blangero PhD

David C Glahn PhD

Objective The role of the amygdala inemotion recognition iswell established andamygdala volume and emotion recognitionperformance have each been shown sepa-rately to be highly heritable traits but thepotential role of common genetic influenceson both traits has not been explored Theauthors investigated the pleiotropic influ-ences of amygdala volume and emotionrecognition performance

Method In a sample of randomly selectedextended pedigrees (N=858) the authorsused a combination of univariate and bi-variate linkage to investigate pleiotropybetween amygdala volume and emotionrecognition performance and followed upwith association analysis

Results The authors found a pleiotropicregion for amygdala volume and emotionrecognition performance on chromosome

4q26 (LOD score=440) Association analysisconducted in the region underlying thebivariate linkage peak revealed a variantmeeting the corrected significance level(Bonferroni-corrected p=50131025) withinan intron of PDE5A (rs2622497 p=4431025)as being jointly influential on both traitsPDE5A has been implicated previously inrecognition-memory deficits and is ex-pressed in subcortical structures that arethought to underlie memory ability in-cluding the amygdala

Conclusions This study extends our un-derstanding of the shared etiology betweenthe amygdala and emotion recognition byshowing that the overlap between amyg-dala volume and emotion recognition per-formance is due at least in part to commongenetic influencesMoreover this study iden-tifies a pleiotropic locus for the two traitsand an associated variant which localizesthe genetic signal evenmore precisely Theseresultswhen taken in the context of previousresearch highlight the potential utility ofPDE5 inhibitors for ameliorating emotionrecognition deficits in individuals sufferingfrom mental or neurodegenerative illness

Am J Psychiatry Knowles et al AiA1ndash9

The ability to successfully process and label the emo-tions of others is crucial to human social interaction (1)Impaired emotion recognition is a hallmark of a numberof psychiatric disorders including schizophrenia bipolardisorder and major depressive disorder (2 3) Emotionrecognition deficits also occur in neurodegenerative ill-nesses like Parkinsonrsquos disease and Alzheimerrsquos disease (45) Furthermore there is considerable evidence for indi-vidual differences in emotion recognition in healthy pop-ulations (6) as well as evidence that a substantial portionof that variation appears to be under genetic influence (7)While multiple neural systems putatively subserve emo-tion recognition the amygdala appears to have a preferen-tial role in affect processing in both healthy and mentallyill individuals (1 8ndash10) As the structure of the amygdala isinfluenced by genetic factors (11 12) it is possible that thesame genes that influence emotion recognition also in-fluence amygdala volume and vice versa However it re-mains unclear whether the association between amygdalavolume and emotion recognition is due to common geneticinfluences and if so which genes in particular are involved

Identifying genes with pleiotropic influence on both traitsmight reveal those molecular mechanisms that alter brainarchitecture or function which in turn affect emotion recog-nition performance In an effort to further our understandingof themolecular underpinnings of emotion recognition ouraim in this study was to isolate genes that jointly influenceemotion recognition and amygdala volume in randomlyselected extended pedigreesNumerous neural systems are implicated in emotion

recognition and those systems reside in particular in thefrontal and temporal lobes which together make up theneural pathways responsible for the interpretation of visualemotional stimuli (9 10) After a visual stimulus is processedvia the lateral geniculate nucleus and the primary visualcortex (V1) the amygdala becomes the focus of furtherprocessing in the brain receiving input from both corticaland subcortical streams (13) The amygdala was first im-plicated in emotional capacity by Brown and Shafer (14)who noted that monkeys with bilateral temporal lobelesions were rendered tame and docile Later this typeof lesion was linked to emotion processing and more

AJP in Advance ajppsychiatryonlineorg 1

specifically to fearful responses (15 16) Subsequent workin primates and rodents narrowed the region of interest foremotion processing down to the amygdala (17 18) The roleof the amygdala in emotion recognition and in particularthe recognition of negative emotions was confirmed inhumans by lesion studies (19 20) as well as functionalimaging studies (10 21 22) While there is evidence thatvariation in the serotonin transporter gene influences theamygdalarsquos response to emotive faces (23) the completegenetic architecture of amygdala function and structureis largely unknown (24)

There have been a handful of candidate gene studieswith a focus on emotion recognition ability (25 26) as wellas amygdala volume in healthy and depressed individuals(27ndash31) A genome-wide linkage study isolated a significantquantitative trait locus (QTL) for emotion recognitionon chromosome 1p36 in a sample selected for schizo-phrenia (32) However there have been no genome-widesearches related to amygdala volume nor have there beenattempts to disentangle the pleiotropic effects on the traitsusing multivariate analyses which are statistically morepowerful than univariate ones if traits are genetically cor-related (33ndash35)

We report here on bivariate linkage and associationanalysis in a sample ofMexican American individuals fromextended pedigrees Using bivariate linkage analysis weidentified a region of chromosome 4 as being truly pleio-tropic for bilateral amygdala volume and emotion recogni-tion performance Using association analysis of commonvariants within that linkage region we identified a genePDE5A as being influential on both traits

Method

Participants

The sample comprised 858 Mexican American individualsfrom extended pedigrees (115 families average family size 753people range 1ndash89 people) from the San Antonio Family StudyThe sample was 63 female and had a mean age of 4478 years(SD=1519 range=18ndash97) Individuals in this cohort have activelyparticipated in research for more than 18 years and were randomlyselected from the community with the constraints that they had tobe of Mexican American ancestry be part of a large family and livewithin the San Antonio region (see reference 36 for recruitmentdetails)

All participants provided written informed consent on formsapproved by the institutional review board at the University ofTexas Health Science Center at San Antonio

Neuropsychological Assessment

As part of the ldquoGenetics of Brain Structure and Functionrdquoprotocol each participant completed a 90-minute neuropsycho-logical test battery consisting of standard and computerizedmeasures (37 38) including the Penn Emotion Recognition Task(39) This computer-based emotion recognition task consists of40 color photographs of facial expression of emotions includinghappy sad angry fearful and neutral (Figure 1) The stimuli arebalanced for gender and ethnicity across emotions During thetask participants are required to identify which of the five emotions

best describes each face stimulus The emotion recognitionphenotype in the present study is a summed score across allstimuli

MRI Acquisition and Processing

All images were acquired on a research-dedicated Siemens 3-TTim Trio scanner and a high-resolution phase array head coilhoused in the Research Imaging Institute University of TexasHealth Science Center at San Antonio Seven high-resolutionT1-weighted three-dimensional turbo-flash sequences with anadiabatic inversion contrast pulse were acquired in each subjectusing the following parameters TE=304 ms TR=2100 ms TI=785 msflip angle=13deg 800 mm isotropic resolution (40)

The software package FreeSurfer (41 42 httpsurfernmrmghharvardedu) as implemented in our group (12) was usedto extract amygdala volume for subsequent genetic analysesThese methods have been described elsewhere briefly Fischland colleagues (43 44) developed a procedure for automaticallyand accurately labeling each voxel in the brain as part of one of40 subcortical structures (thalamus hippocampus amygdalaand so on) This procedure is based on modeling the segmentationas a nonstationary anisotropic Markov random field in whichthe probability of a neuroanatomic label is modulated by thatof its neighbors Probabilities were computed separately at eachposition in an atlas resulting in a maximum a posteriori estima-tion of each voxelrsquos label in each image Amygdala volume wasaveraged across hemispheres yielding an average volume phenotypefor each subject

Data Analysis

Genotyping Subjects were genotyped for approximately 1million single-nucleotide polymorphisms (SNPs) using Illu-mina HumanHap550v3 HumanExon510Sv1 Human1Mv1 andHuman1M-Duov3 BeadChips according to the Illumina Infiniumprotocol (Illumina San Diego) SNP loci were checked for Mendelianconsistency using SimWalk2 (45) SNPs or samples exhibiting highcalling rate failures or requiring excessive blanking (ie if 95of the genotypes are retained) were eliminated from analysesMissing genotypes were imputed according to Mendelian lawsbased on available pedigree data using MERLIN (46) Maximumlikelihood techniques accounting for pedigree structure wereused to estimate allelic frequencies (47) For linkage analysesmultipoint identity-by-descent matrices were calculated basedon 28387 SNPs selected from the 1 million-SNP genome-wideassociation study panel as follows Using genotypes for 345founders SNPs on each chromosome were selected to be at least1 kb apart with a minor allele frequency $5 and linkage dis-equilibrium (LD) within a 100-kb sliding window not exceeding anabsolute value of 015 for rho The resulting selection averagedseven to eight SNPs per centimorgan For each centimorganlocation in the genome multipoint identity-by-descent prob-ability matrices were calculated using a stochastic Markovchain Monte Carlo procedure implemented in the software pack-age LOKI (48)

Confirmatory factor analysis Given that the aim of this studywas to identify genes with pleiotropic effects on both amygdalavolume and emotion recognition performance it was importantto be able to conduct multivariate analyses beyond bivariatelinkage To this end a simple one-factor confirmatory factormodel was built using two items amygdala volume and emotionrecognition performance in which to ensure that each traitcontributed to the factor score equally factor loadings wereconstrained to be equal which is also a requirement of themodel being identified This model was built using Mplus (49)in which family structure was taken into account using theldquoclusterrdquo command

2 ajppsychiatryonlineorg AJP in Advance

PLEIOTROPIC LOCUS FOR EMOTION RECOGNITION AND AMYGDALA VOLUME

Quantitative genetic analyses All genetic analyses were per-formed in SOLAR (33) SOLAR implements a maximum likelihoodvariance decomposition to determine the contribution of genesand environmental influence to a trait by modeling the covari-ance among family members as a function of expected allele shar-ing given the pedigree In the simplest such decomposition theadditive genetic contribution to a trait is represented by theheritability index (h2) First univariate variance decompositionanalysis was applied to both left and right amygdala volume andemotion recognition performance Both traits were normalizedusing an inverse Gaussian transformation Age age squared sexand their interactions were included as covariates Second bi-variate analysis was applied to the two variables with the pheno-typic covariance between the traits decomposed into its geneticand environmental constituents to determine the extent to whichthey are influenced by shared genetic effects (eg genetic cor-relation rhog)

Linkage and association analyses Quantitative trait linkageanalysis was performed to localize specific chromosomal loca-tions influencing amygdala volume and emotion recognitionability (33) Initially this was done under a univariate model foreach trait Model parameters were estimated using maximumlikelihood The hypothesis of significant linkage was assessed bycomparing the likelihood of a classical additive polygenic modelwith that of a model allowing for both a polygenic componentand a variance component due to linkage at a specific chromo-somal location (as evidenced by the location-specific identity-by-descent probability matrix) The LOD score given by the log10 ofthe ratio of the likelihoods of the linkage and the polygenic nullmodels served as the test statistic for linkage Genome-widethresholds for linkage evidence were computed for this exactpedigree structure and density of markers using a methodderived from Feingold et al (50) a LOD score of 169 is requiredfor suggestive significance (likely to happen by chance less thanonce in a genome-wide scan) and a LOD score of 29 is required

for genome-wide significance Regions showing potential pleiot-

ropy were subjected to bivariate linkage analysis for comparison

to the univariate results the resulting LOD score was converted

to a one degree of freedom equivalent based on the p value for

the two degrees of freedom test (linkage to both traits versus

linkage to neither) (51) To ensure that the bivariate LOD scores

were truly driven by both and not one of the traits we tested

the null hypothesis of the absence of pleiotropy (that is co-

occurrence of linkage is by chance) versus the alternative of

complete pleiotropy by comparing the likelihoods of the relevant

nested models To this end we maximized two models one in

which the genetic correlation between linkage peaks was allowed

to vary freely and a null in which this correlation was constrained

to be zero the likelihoods of these two models were then com-

pared with twice the difference between these two log-likelihoods

being distributed as a chi-square with one degree of freedom This

method has been established as a powerful approach to detecting

pleiotropic effects (52)Genomic regions meeting bivariate genome-wide significance

for linkage were investigated in greater detail using association

analysis of the emotion and amygdala confirmatory factor score

and the genetic variants encapsulated by the linkage peak

Statistical significance levels were established according to the

effective number of tested variants given the LD structure in the

region to this end the pairwise genotypic correlations are cal-

culated in an effort to establish the effective number of in-

dependent tests carried out during association analysis This

method proposed by Moskvina and Schmidt (53) is considered

to be conservative and entails computing the eigenvalues of the

genotypic correlation matrix A corrected p value is obtained

from a Bonferroni correction based on the nominal alpha (005)

and the total number of independent tests

FIGURE 1 Examples of Facial Emotion Stimuli From the Penn Emotion Recognition Taska

a The emotions depicted from left to right (where the upper image is a mild expression of emotion and the lower is intense) are happy sadangry fearful and neutral

AJP in Advance ajppsychiatryonlineorg 3

KNOWLES MCKAY KENT ET AL

Results

Heritability and Linkage Analysis

Both amygdala volume (h2=072 SE=007 p=49531025mean=306672 SD=43447) and emotion recognition (h2=032SE=006 p=512310210 happymean=778 SD=147 sadmean=615 SD=140 fear mean=661 SD=136 angermean=556 SD=221 neutral mean=558 SD=437) werehighly heritable and a bivariate model indicated signif-icant genetic overlap between the two traits (rhog=025SE=013 p=0048) For amygdala volume and emotionrecognition age and sex were significant covariates (seeTable S1 in the data supplement that accompanies theonline edition of this article) and hence the effect of ageand sex was covaried in all subsequent analyses Thefactor score derived for emotion recognition and amyg-dala volume was also highly heritable (h2=045 SE=006p=568310222) and the factor loadings for both traits weredeemed to be significant at the p0001 level goodness-of-fit statistics were not available because of saturation ofthe model

For amygdala volume one genome-wide significant locuswasobservedonchromosome4at 120 cM(LODscore=4065)A LOD score of 2175 was observed for the emotion rec-ognition task on chromosome 2 at 71 cM meeting criteriafor suggestive significance while the chromosome 4 locusshowed some evidence for linkage to emotion recognition(LOD score=1217) (Figure 2) Bivariate linkage revealed agenome-wide significant QTL for both amygdala and emo-tion recognition on chromosome 4 at 122 cM (1 df-equivalentLOD score=4399) which suggests that this region of chro-mosome4mediates both amygdala volumeandperformanceon the emotion recognition task The test for pleiotropyversus coincident linkage confirmed the presence ofpleiotropy for the two traits at this locus (x2=2012 df=1p=3631026) The factor score derived from the one-factor model of emotion recognition and amygdalavolume showed genome-wide significant linkage at pre-cisely the same region on chromosome 4 at 122 cM (LODscore=3336)

The possible confounding role of intracranial volumeat this locus was investigated using trivariate linkage Uni-variate linkage revealed a QTL of suggestive significancefor intracranial volume on chromosome 16 at 37 cM (LODscore=265) with little evidence for genetic influence on thistrait on chromosome 4 at 122 cM (LOD score=047) More-over in a trivariate linkage model of the emotion rec-ognition task amygdala volume and intracranial volume onchromosome 4 at 122 cM showed genome-wide signifi-cance (1 df-equivalent LOD score=3546) and even withinthis trivariate model the pleiotropy test supported com-plete pleiotropy between amygdala and emotion recog-nition (x2=774 p=2731023) Furthermore the possibilitythat the right or left amygdala might be driving the resultwas addressed by running univariate and bivariate linkage

in the same region Left amygdala volume (h2=07019SE=00788 p=203310221) had a univariate LOD score of3315 on chromosome 4 at 120 cM and right amygdalavolume (h2=06958 SE=00754 p=143310223) had a uni-variate LOD score of 3103 in the same location Moreoverbivariate linkage analysis with left amygdala volume andemotion recognition (rhog=024) revealed a bivariate LODscore of 3282 on chromosome 4 at 122 cM while bivariatelinkage analysis with right amygdala volume and emotionrecognition (rhog=027) revealed a bivariate LOD score of36812 on chromosome 4 at 122 cM These results supportthe idea that the shared genetic influence on amygdalavolume and emotion recognition is not lateralized to eitherthe left or right amygdala

Association Analysis

Association analysis was conducted for all genetic var-iants under the 1 ndash LOD score confidence interval of thebivariate linkage peak (defined as 120ndash124 cM) and a factorscore derived from amygdala volume and emotion recog-nition In total there were 2053 SNPs in this region but aftertaking LD into account (53) there were 1023 effective SNPsnecessitating a Bonferroni-corrected alpha of 50131025One variant met the adjusted-significance level rs2622497(p=44031025) located within an intron of the gene PDE5A(phosphodiesterase 5A cGMP specific) Several other var-iants met a suggestive level of significance that also fellwithin PDE5A in addition to a number of variants down-stream of the gene all of which were in varying degrees ofLD with the top-ranked variant (see Table 1 and Figure 3)Univariate association analysis for each individual traitfor rs2622497 did not reach significance either for amyg-dala volume (p=1031023) or for emotion recognition(p=4131023)When the SNP rs2622497 was included as a covariate in

the linkage analysis of the factor score derived from emo-tion recognition and amygdala volume the LOD scoreobserved without the covariate (3336) was reduced (2506)and no longer significant This linkage conditional onassociation test gives additional support for the associationbetween rs2622497 emotion recognition performance andamygdala volumeGiven the significant effect of age and sex on amygdala

volume the interactive effects of genotype sex and agewere included as covariates in the association analysis Forour top SNPs therewas a significant interactionwith sex (sexby rs2622497 b=09823664 p=00032331 sex by rs2715021b=09887387 p=00032051 sex by rs9884801 b=09857738p=00038682 see Figures S1ndashS3 in the online data supple-ment) but not with age In each case the sex-by-SNP in-teractions indicated that the effect was marginally morepronounced in men than in women although the directionof the effect was the same in both groups Consequently aninteraction term for sex by genotype was included in allassociation analyses

4 ajppsychiatryonlineorg AJP in Advance

PLEIOTROPIC LOCUS FOR EMOTION RECOGNITION AND AMYGDALA VOLUME

Discussion

While numerous studies have highlighted an associationbetween the amygdala and emotion recognition (10 19ndash22)thepresent study extends thisfindingbyproviding evidencefor a pleiotropic locus on chromosome 4 using bivariatelinkage Furthermore by examining variants within thequantitative locus we identified a variant (rs2622497) with-in an intron of PDE5A that appears to jointly influenceamygdala volume and emotion recognition performanceTo our knowledge this is the first study to formally testthe common genetic influences on amygdala volume andemotion recognition ability using a bivariate model It iswell established that the implementation of a bivariate asopposed to a univariate model is beneficial as the jointanalysis of multiple traits confers greater power and pre-cision in the mapping of a QTL (33 54 55)The cyclic nucleotide phosphodiesterases (PDEs) are

a family of enzymes with twomain subtypes cAMP- andcGMP-specific nucleotides The PDE5A gene codes forPDE5 a cGMP-specific PDE (56) cGMP a second mes-senger is crucial to signal transduction between cells aswell as synapse communication and synaptic plasticity(57) Hence PDEs are important for effective cell-to-cell

communication in the central nervous system (56) In-deed there is widespread expression of PDE5A throughoutthe body and it is expressed in various regions of the brainincluding in the amygdala (58) the pyramidal cells of thehippocampus Purkinje cells in the cerebellum and someareas of the cortex (59 60) Thus PDE5 has emerged asa potential drug target for treating cognitive deficits (56 61)Through the use of mouse models it has emerged thatadministration of sildenafil a PDE5 inhibitor improvesmemory including recognition memory as well as spatialand fear-conditioningmemory in aged rats (62 63) Sildenafilalsoamelioratescognitivedeficits associatedwithHuntingtonrsquoschorea and Alzheimerrsquos disease by increasing cGMP levelsin the hippocampus (64ndash66) Thus the results of the pres-ent study are in line with previous research As in previousstudies the results presented here show an association be-tween recognition memorymdashof which emotion recognitioncould be considered a sub-type (67)mdashand PDE5Recognition is predicated on knowledge retention which

is enhanced by contextual association In the case of emo-tion recognition this might include associations formed byprevious experiences in which a particular facial configu-ration has come to be associated with a particular emotionSuch examples might include life events that precede or

FIGURE 2 Chromosome 4 Multipoint Plot for Univariate and Bivariate Linkage Analysesa

45

40

35

30

25

20

15

10

05

00

LOD

Sco

re

Location (cM)

0 50 100 200150

Emotion recognition performance

Amygdala volume

Emotion recognition performance and amygdala volume

a Univariate analysis reveals a genome-wide significant quantitative trait locus for amygdala volume and near suggestive significance foremotion recognition performance and bivariate analysis reveals a genome-wide significant linkage signal for both traits

AJP in Advance ajppsychiatryonlineorg 5

KNOWLES MCKAY KENT ET AL

co-occur with the expression of a particular emotion withwhat a person saidwhile experiencing that emotion orwhatwas said about them during that time with how one felt onseeing the expression and so on (67) If this is the case thenemotion recognition should require access to long-termmemory evenwhen the emotional stimulus is portrayed bya stranger so it seems plausible that emotion recognition issupported by a distributed neural network that includesthose brain regions typically implicated in memory per-formance in addition to the amygdala (10 68) In this con-text PDE5A a gene expressed particularly in the cerebellumand hippocampus is especially interesting Indeed thecerebellum and the hippocampus and for that matter theamygdala have been implicated in long-term memoryactivation in humans (69) It is of note that in the presentsample there was a significant genetic correlation between

amygdala and both cerebellum (rhog=030 p=2531023)and hippocampus (rhog=066 p=215310214) A tentativehypothesis generated from the results of this study is thatemotion recognition might be improved with the admin-istration of a PDE5 inhibitor which would be in line withsome of the research cited above (62 63) and would havesubstantial implications for those individuals for whomemotion recognition proves difficult such as schizophreniapatients (2)Rare variation is a likely source of family-based linkage

signals associated with complex traits (70 71) Thereforeit is unsurprising that those SNPs showing the strongestassociation with amygdala volume and emotion recogni-tion in the present study are relatively rare (Table 1) Theuse of extended pedigrees such as those used this studyimproves the chances of detecting association with rare

FIGURE 3 Quantitative Trait Locus (QTL)-Specific Association Analysis for the Genome-Wide Significant QTL RegionAssociated With Amygdala Volume and Emotion Recognition Performance on Chromosome 4a

MYOZ2

USP53

C4or

f3

FABP2

PDE5

A

050

100r2

000

40

50

30

20

10

00

ndashlo

g10(p

)

Chromosome 4 (Mb)

1202 1204 1206 1208

a Intergenic regions are pale gray and genes are represented by dark gray bars with the gene name shown at the top of the plot The top-ranked variant in this region is represented by a diamond and the degree of linkage disequilibrium (r2) with this variant is represented by thecolor scale

TABLE 1 Estimates for the Top Five Single-Nucleotide Polymorphisms (SNPs) From the Quantitative Trait Locus-SpecificAssociation Analysis for the Bivariate Emotion Recognition and Amygdala Volume Factor Score

SNP x2 p b Variance Explained Minor Allele Frequency pa

rs2622497 1667 0000044 ndash007 002 0008 084rs2715021 1642 0000051 ndash007 002 0008 073rs9884801 1576 0000072 ndash007 002 0006 088rs2389894 1410 0000173 ndash005 001 0014 013rs2714982 1222 0000472 ndash005 001 0015 062a Hardy-Weinberg equilibrium p value

6 ajppsychiatryonlineorg AJP in Advance

PLEIOTROPIC LOCUS FOR EMOTION RECOGNITION AND AMYGDALA VOLUME

variants as pedigree-based studies represent an implicitenrichment strategy for identifying rare variants Mende-lian transmissions from parents to offspring maximize thechances that multiple copies of rare variants exist in thepedigree Thus pedigree-based studies have optimal powerto detect effects of rare variants so it is unlikely that theassociations we observed here are false positives partic-ularly given that the variants in question do not show asignificant departure from the Hardy-Weinberg equilib-rium (Table 1) Our top-ranked variant (rs2622497) appearsto be similarly rare across populations (72 see also Table S2in the online data supplement)The results of this study could be called into question if

the genetic effects detected were in fact univariate in nature(eg driven by only one of the phenotypes perhaps inparticular by amygdala volume) However the linkagesignal was subjected to a pleiotropy test and was shownto be truly pleiotropic Furthermore the factor score de-rived from the factormodel can be said to be driven by bothtraits equally as the factor loadings which are used todetermine each individualrsquos factor score were constrainedto be equal It is true however that no formal test can beapplied to the association analysis to further underscore thebivariate underpinnings of the signalSome evidence from previous research is suggestive of a

modulatory effect of age on amygdala volume and emo-tion recognition (73ndash75) as well as interactions betweensex and genotype on amygdala volume (76) In the presentstudy the effects of age and sex were controlled for inall analyses including in the association analysis wherean additional interaction covariate (sex by SNP) was in-cluded A significant sex-by-SNP interaction was evi-dent for amygdala volume for our top three SNPs (seeFigures S1ndashS3 in the online data supplement) such thatthe effect of genotype was slightly more pronounced inmen than in women but the direction of effect of wasthe samePatients with schizophrenia exhibit substantial and ro-

bust impairments in emotion recognition ability (39 77) Itis interesting then that the gene implicated in this studyPDE5 codes for a member of the phosphodiesterase en-zyme family as phosphodiesterase genes and in particularcAMP-specific PDE4 have been implicated in schizophreniarisk (78 79) Moreover administration of rolipram (a PDE4inhibitor) reduces phencyclidine-induced cognitive impair-ments in humans (phencyclidine is an established phar-macologicalmodel of schizophrenia symptomatology) (80)Phosphodiesterases have also been shown to have po-tential utility in the treatment of Alzheimerrsquos disease andin particular the associated cognitive impairment (81 82)Furthermore the administration of a PDE5 inhibitor wasshown in a recent placebo-controlled study (83) to reducesymptoms of depression and cognitive impairment The es-tablished role of phosphodiesterases in psychopathologyand cognitive impairment in psychiatric illness taken to-gether with the results of the present study highlight the

potential utility of PDE5 inhibitors in the treatment of emo-tion recognition impairments in schizophrenia depressionand Alzheimerrsquos diseaseIn summary the linkage and association findings pre-

sented here highlight a pleiotropic gene PDE5A for amyg-dala volume and emotion recognition ability To ourknowledge this is the first study to identify a commongenetic locus that influences these two traits Although thisstudy was conducted in healthy individuals when taken inthe context of previous research which has shown thepotential utility of PDE5 inhibitors as cognitive enhancersit suggests that PDE5A may be an important target forameliorating emotion recognition deficits in patients suf-fering from mental or neurodegenerative illness

Received March 10 2014 revision received June 13 2014accepted July 18 2014 (doi 101176appiajp201414030311) Fromthe Department of Psychiatry Yale University School of MedicineNew Haven Conn Olin Neuropsychiatric Research Center Instituteof Living Hartford Hospital Hartford Conn the Department of Ge-netics Texas Biomedical Research Institute San Antonio the De-partment of Psychiatry and the Research Imaging Institute Universityof Texas Health Science Center at San Antonio and South TexasVeterans Health System San Antonio Address correspondence toDr Knowles (emmaknowlesyaleedu)The authors report no financial relationships with commercial

interestsSupported by NIMH grants MH078143 (principal investigator

Dr Glahn) MH078111 (principal investigator Dr Blangero) andMH083824 (principal investigator Dr Glahn) SOLAR is supportedby NIMH grant MH059490 (to Dr Blangero)

References

1 LeDoux JE Emotion circuits in the brain Annu Rev Neurosci2000 23155ndash184

2 Mikhailova ES Vladimirova TV Iznak AF Tsusulkovskaya EJSushko NV Abnormal recognition of facial expression of emo-tions in depressed patients with major depression disorder andschizotypal personality disorder Biol Psychiatry 1996 40697ndash705

3 Kohler CG Bilker W Hagendoorn M Gur RE Gur RC Emotionrecognition deficit in schizophrenia association with symptom-atology and cognition Biol Psychiatry 2000 48127ndash136

4 Gray HM Tickle-Degnen L A meta-analysis of performance onemotion recognition tasks in Parkinsonrsquos disease Neuropsychol-ogy 2010 24176ndash191

5 Hargrave R Maddock RJ Stone V Impaired recognition of facialexpressions of emotion in Alzheimerrsquos disease J Neuropsychi-atry Clin Neurosci 2002 1464ndash71

6 Petrides KV Jackson CJ Furnham A Levine SZ Exploring issuesof personality measurement and structure through the de-velopment of a short form of the Eysenck Personality Profiler JPers Assess 2003 81271ndash280

7 Anokhin AP Golosheykin S Heath AC Heritability of individualdifferences in cortical processing of facial affect Behav Genet2010 40178ndash185

8 Phillips ML Drevets WC Rauch SL Lane R Neurobiology ofemotion perception II implications for major psychiatric dis-orders Biol Psychiatry 2003 54515ndash528

9 Adolphs R Damasio H Tranel D Damasio AR Cortical systemsfor the recognition of emotion in facial expressions J Neurosci1996 167678ndash7687

AJP in Advance ajppsychiatryonlineorg 7

KNOWLES MCKAY KENT ET AL

10 Phan KL Wager T Taylor SF Liberzon I Functional neuroanat-omy of emotion a meta-analysis of emotion activation studiesin PET and fMRI Neuroimage 2002 16331ndash348

11 Bohlken MM Brouwer RM Mandl RC van Haren NE Brans RGvan Baal GC de Geus EJ Boomsma DI Kahn RS Hulshoff Pol HEGenes contributing to subcortical volumes and intellectual abil-ity implicate the thalamus Hum Brain Mapp 2014 352632ndash2642

12 McKay DR Knowles EE Winkler AA Sprooten E Kochunov POlvera RL Curran JE Kent JW Jr Carless MA Goumlring HH Dyer TDDuggirala R Almasy L Fox PT Blangero J Glahn DC Influenceof age sex and genetic factors on the human brain Brain Im-aging Behav 2014 8143ndash152

13 Pessoa L Adolphs R Emotion processing and the amygdalafrom a ldquolow roadrdquo to ldquomany roadsrdquo of evaluating biological sig-nificance Nat Rev Neurosci 2010 11773ndash783

14 Brown S Schafer EA An investigation into the functions of theoccipital and temporal lobes of the monkeyrsquos brain Philos TransR Soc Lond B Biol Sci 1888 179303ndash327

15 Kluver H Bucy PC ldquoPsychic blindnessrdquo and other symptomsfollowing bilateral temporal lobectomy in rhesus monkeys AmJ Physiol 1937 119352ndash353

16 Kluumlver H Bucy PC Preliminary analysis of functions of thetemporal lobes in monkeys 1939 J Neuropsychiatry Clin Neu-rosci 1997 9606ndash620

17 Weiskrantz L Behavioral changes associated with ablation ofthe amygdaloid complex in monkeys J Comp Physiol Psychol1956 49381ndash391

18 LeDoux JE Emotion and the amygdala in The AmygdalaNeurobiological Aspects of Emotion Memory and Mental Dys-function Edited by Aggleton JP New York Wiley-Liss 1992 pp339ndash351

19 Adolphs R Tranel D Damasio H Damasio A Impaired recog-nition of emotion in facial expressions following bilateral dam-age to the human amygdala Nature 1994 372669ndash672

20 Calder AJ Young AW Rowland D Perrett D Hodges JR EtcoffNL Facial emotion recognition after bilateral amygdala dam-age differentially severe impairment of fear Cogn Neuropsy-chol 1996 13699ndash745

21 Morris JS Frith CD Perrett DI Rowland D Young AW Calder AJDolan RJ A differential neural response in the human amygdalato fearful and happy facial expressions Nature 1996 383812ndash815

22 Morris JS Friston KJ Buchel C Frith CD Young AW Calder AJDolan RJ A neuromodulatory role for the human amygdala inprocessing emotional facial expressions Brain 1998 121(Pt 1)47ndash57

23 Heinz A Braus DF Smolka MN Wrase J Puls I Hermann DKlein S Gruumlsser SM Flor H Schumann G Mann K Buumlchel CAmygdala-prefrontal coupling depends on a genetic variationof the serotonin transporter Nat Neurosci 2005 820ndash21

24 Zirlinger M Kreiman G Anderson DJ Amygdala-enriched genesidentified by microarray technology are restricted to specificamygdaloid subnuclei Proc Natl Acad Sci USA 2001 985270ndash5275

25 Lin MT Huang KH Huang CL Huang YJ Tsai GE Lane HY METand AKT genetic influence on facial emotion perception PLoSONE 2012 7e36143

26 Baune BT Dannlowski U Domschke K Janssen DG Jordan MAOhrmann P Bauer J Biros E Arolt V Kugel H Baxter AG SuslowT The interleukin 1 beta (IL1B) gene is associated with failure toachieve remission and impaired emotion processing in majordepression Biol Psychiatry 2010 67543ndash549

27 Furman DJ Chen MC Gotlib IH Variant in oxytocin receptorgene is associated with amygdala volume Psychoneuroendo-crinology 2011 36891ndash897

28 Stjepanovic D Lorenzetti V Yuumlcel M Hawi Z Bellgrove MAHuman amygdala volume is predicted by common DNA varia-tion in the stathmin and serotonin transporter genes TranslPsychiatr 2013 3e283

29 Cerasa A Quattrone A Gioia MC Magariello A Muglia M Assogna FBernardini S Caltagirone C Bossugrave P Spalletta G MAO A VNTRpolymorphism and amygdala volume in healthy subjects Psy-chiatry Res 2011 19187ndash91

30 Montag C Weber B Fliessbach K Elger C Reuter M The BDNFVal66Met polymorphism impacts parahippocampal and amyg-dala volume in healthy humans incremental support for agenetic risk factor for depression Psychol Med 2009 391831ndash1839

31 Scherk H Gruber O Menzel P Schneider-Axmann T Kemmer CUsher J Reith W Meyer J Falkai P 5-HTTLPR genotype influ-ences amygdala volume Eur Arch Psychiatry Clin Neurosci 2009259212ndash217

32 Greenwood TA Swerdlow NR Gur RE Cadenhead KS CalkinsME Dobie DJ Freedman R Green MF Gur RC Lazzeroni LCNuechterlein KH Olincy A Radant AD Ray A Schork NJ SeidmanLJ Siever LJ Silverman JM Stone WS Sugar CA Tsuang DWTsuang MT Turetsky BI Light GA Braff DL Genome-wide link-age analyses of 12 endophenotypes for schizophrenia from theConsortium on the Genetics of Schizophrenia Am J Psychiatry2013 170521ndash532

33 Almasy L Blangero J Multipoint quantitative-trait linkageanalysis in general pedigrees Am J Hum Genet 1998 621198ndash1211

34 Bearden CE Freimer NB Endophenotypes for psychiatric dis-orders ready for primetime Trends Genet 2006 22306ndash313

35 Glahn DC Curran JE Winkler AM Carless MA Kent JW JrCharlesworth JC Johnson MP Goumlring HH Cole SA Dyer TDMoses EK Olvera RL Kochunov P Duggirala R Fox PT AlmasyL Blangero J High dimensional endophenotype ranking inthe search for major depression risk genes Biol Psychiatry2012 716ndash14

36 Olvera RL Bearden CE Velligan DI Almasy L Carless MA CurranJE Williamson DE Duggirala R Blangero J Glahn DC Commongenetic influences on depression alcohol and substance usedisorders in Mexican-American families Am J Med Genet BNeuropsychiatr Genet 2011 156B561ndash568

37 Glahn DC Almasy L Blangero J Burk GM Estrada J Peralta JMMeyenberg N Castro MP Barrett J Nicolini H Raventoacutes HEscamilla MA Adjudicating neurocognitive endophenotypes forschizophrenia Am J Med Genet B Neuropsychiatr Genet 2007144B242ndash249

38 Glahn DC Almasy L Barguil M Hare E Peralta JM Kent JW JrDassori A Contreras J Pacheco A Lanzagorta N Nicolini HRaventoacutes H Escamilla MA Neurocognitive endophenotypes forbipolar disorder identified in multiplex multigenerationalfamilies Arch Gen Psychiatry 2010 67168ndash177

39 Kohler CG Turner TH Bilker WB Brensinger CM Siegel SJ KanesSJ Gur RE Gur RC Facial emotion recognition in schizophreniaintensity effects and error pattern Am J Psychiatry 2003 1601768ndash1774

40 Kochunov P Lancaster JL Glahn DC Purdy D Laird AR Gao F FoxP Retrospective motion correction protocol for high-resolutionanatomical MRI Hum Brain Mapp 2006 27957ndash962

41 Dale AM Fischl B Sereno MI Cortical surface-based analysis Isegmentation and surface reconstruction Neuroimage 1999 9179ndash194

42 Fischl B Sereno MI Dale AM Cortical surface-based analysis IIinflation flattening and a surface-based coordinate systemNeuroimage 1999 9195ndash207

43 Fischl B Salat DH Busa E Albert M Dieterich M Haselgrove Cvan der Kouwe A Killiany R Kennedy D Klaveness S MontilloA Makris N Rosen B Dale AM Whole brain segmentation

8 ajppsychiatryonlineorg AJP in Advance

PLEIOTROPIC LOCUS FOR EMOTION RECOGNITION AND AMYGDALA VOLUME

automated labeling of neuroanatomical structures in the hu-man brain Neuron 2002 33341ndash355

44 Fischl B Salat DH van der Kouwe AJ Makris N Seacutegonne FQuinn BT Dale AM Sequence-independent segmentation of mag-netic resonance images Neuroimage 2004 23(suppl 1)S69ndashS84

45 Sobel E Lange K Descent graphs in pedigree analysis ap-plications to haplotyping location scores and marker-sharingstatistics Am J Hum Genet 1996 581323ndash1337

46 Abecasis GR Cherny SS Cookson WO Cardon LR Merlin rapidanalysis of dense genetic maps using sparse gene flow trees NatGenet 2002 3097ndash101

47 Boehnke M Allele frequency estimation from data on relativesAm J Hum Genet 1991 4822ndash25

48 Heath SC Markov chain Monte Carlo segregation and linkageanalysis for oligogenic models Am J Hum Genet 1997 61748ndash760

49 Mutheacuten LK Mutheacuten BO Mplus Userrsquos Guide 6th ed LosAngeles Mutheacuten amp Mutheacuten 2011

50 Feingold E Brown PO Siegmund D Gaussian models for ge-netic linkage analysis using complete high-resolution maps ofidentity by descent Am J Hum Genet 1993 53234ndash251

51 Almasy L Dyer TD Blangero J Bivariate quantitative trait link-age analysis pleiotropy versus co-incident linkages Genet Epi-demiol 1997 14953ndash958

52 Williams JT Begleiter H Porjesz B Edenberg HJ Foroud T ReichT Goate A Van Eerdewegh P Almasy L Blangero J Joint mul-tipoint linkage analysis of multivariate qualitative and quanti-tative traits II alcoholism and event-related potentials Am JHum Genet 1999 651148ndash1160

53 Moskvina V Schmidt KM On multiple-testing correction ingenome-wide association studies Genet Epidemiol 2008 32567ndash573

54 Jiang C Zeng ZB Multiple trait analysis of genetic mapping forquantitative trait loci Genetics 1995 1401111ndash1127

55 Knott SA Haley CS Multitrait least squares for quantitative traitloci detection Genetics 2000 156899ndash911

56 Menniti FS Faraci WS Schmidt CJ Phosphodiesterases in theCNS targets for drug development Nat Rev Drug Discov 20065660ndash670

57 Wallace TL Ballard TM Pouzet B Riedel WJ Wettstein JG Drugtargets for cognitive enhancement in neuropsychiatric disor-ders Pharmacol Biochem Behav 2011 99130ndash145

58 Allen Institute for Brain Science Allen Human Brain Atlas 2012httphumanbrain-maporgmicroarraysearchshowexact_match=falseampsearch_term=PDE5ampsearch_type=gene

59 van Staveren WC Glick J Markerink-van Ittersum M Shimizu MBeavo JA Steinbusch HW de Vente J Cloning and localization ofthe cGMP-specific phosphodiesterase type 9 in the rat brain JNeurocytol 2002 31729ndash741

60 Bender AT Beavo JA Specific localized expression of cGMP PDEsin Purkinje neurons and macrophages Neurochem Int 200445853ndash857

61 Blokland A Schreiber R Prickaerts J Improving memory a rolefor phosphodiesterases Curr Pharm Des 2006 122511ndash2523

62 Baratti CM Boccia MM Effects of sildenafil on long-term reten-tion of an inhibitory avoidance response in mice Behav Phar-macol 1999 10731ndash737

63 Prickaerts J Sik A van Staveren WC Koopmans G SteinbuschHW van der Staay FJ de Vente J Blokland A Phosphodiesterasetype 5 inhibition improves early memory consolidation of ob-ject information Neurochem Int 2004 45915ndash928

64 Puzzo D Staniszewski A Deng SX Privitera L Leznik E Liu SZhang H Feng Y Palmeri A Landry DW Arancio O Phospho-diesterase 5 inhibition improves synaptic function memoryand amyloid-beta load in an Alzheimerrsquos disease mouse modelJ Neurosci 2009 298075ndash8086

65 Cuadrado-Tejedor M Hervias I Ricobaraza A Puerta E Peacuterez-Roldaacuten JM Garciacutea-Barroso C Franco R Aguirre N Garciacutea-Osta ASildenafil restores cognitive function without affecting b-amyloid

burden in a mouse model of Alzheimerrsquos disease Br J Pharmacol2011 1642029ndash2041

66 Saavedra A Giralt A Arumiacute H Alberch J Peacuterez-Navarro E Regu-lation of hippocampal cGMP levels as a candidate to treat cog-nitive deficits in Huntingtonrsquos disease PLoS ONE 2013 8e73664

67 Adolphs R Recognizing emotion from facial expressions psy-chological and neurological mechanisms Behav Cogn NeurosciRev 2002 121ndash62

68 Haxby JV Hoffman EA Gobbini MI The distributed humanneural system for face perception Trends Cogn Sci 2000 4223ndash233

69 Svoboda E McKinnon MC Levine B The functional neuro-anatomy of autobiographical memory a meta-analysis Neu-ropsychologia 2006 442189ndash2208

70 Bowden DW An SS Palmer ND Brown WM Norris JM HaffnerSM Hawkins GA Guo X Rotter JI Chen YD Wagenknecht LELangefeld CD Molecular basis of a linkage peak exome se-quencing and family-based analysis identify a rare genetic vari-ant in the ADIPOQ gene in the IRAS Family Study HumMol Genet2010 194112ndash4120

71 Martin LJ Benson DW Congenital heart disease in Genomicand Personalized Medicine 2nd ed vols 1ndash2 Edited by GinsburgGS Willard HF London Academic Press 2012 pp 624ndash634

72 International HapMap Consortium The International HapMapProject Nature 2003 426789ndash796

73 Bucks RS Garner M Tarrant L Bradley BP Mogg K Interpretationof emotionally ambiguous faces in older adults J Gerontol BPsychol Sci Soc Sci 2008 63337ndash343

74 Wright CI The human amygdala in normal aging and Alzheimerrsquosdisease in The Human Amygdala Edited by Whalen PJ PhelpsEA New York Guilford Press 2009 pp 382ndash402

75 Suzuki A Akiyama H Cognitive aging explains age-related dif-ferences in face-based recognition of basic emotions except foranger and disgust Neuropsychol Dev Cogn B Aging Neuro-psychol Cogn 2013 20253ndash270

76 Cerasa A Quattrone A Piras F Mangone G Magariello A FagioliS Girardi P Muglia M Caltagirone C Spalletta G 5-HTTLPR anx-iety and gender interaction moderates right amygdala volume inhealthy subjects Soc Cogn Affect Neurosci (Epub ahead of printSept 29 2013)

77 Kohler CG Walker JB Martin EA Healey KM Moberg PJ Facialemotion perception in schizophrenia a meta-analytic reviewSchizophr Bull 2010 361009ndash1019

78 Millar JK Pickard BS Mackie S James R Christie S Buchanan SRMalloy MP Chubb JE Huston E Baillie GS Thomson PA Hill EVBrandon NJ Rain JC Camargo LM Whiting PJ Houslay MDBlackwood DH Muir WJ Porteous DJ DISC1 and PDE4B areinteracting genetic factors in schizophrenia that regulate cAMPsignaling Science 2005 3101187ndash1191

79 Pickard BS Thomson PA Christoforou A Evans KL Morris SWPorteous DJ Blackwood DH Muir WJ The PDE4B gene conferssex-specific protection against schizophrenia Psychiatr Genet2007 17129ndash133

80 Jentsch JD Roth RH The neuropsychopharmacology of phen-cyclidine from NMDA receptor hypofunction to the dopaminehypothesis of schizophrenia Neuropsychopharmacology 199920201ndash225

81 Siuciak JA The role of phosphodiesterases in schizophreniatherapeutic implications CNS Drugs 2008 22983ndash993

82 Garciacutea-Osta A Cuadrado-Tejedor M Garciacutea-Barroso C OyarzaacutebalJ Franco R Phosphodiesterases as therapeutic targets for Alz-heimerrsquos disease ACS Chem Neurosci 2012 3832ndash844

83 Shim YS Pae CU Cho KJ Kim SW Kim JC Koh JS Effects ofdaily low-dose treatment with phosphodiesterase type 5inhibitor on cognition depression somatization and erec-tile function in patients with erectile dysfunction a double-blind placebo-controlled study Int J Impot Res 2014 2676ndash80

AJP in Advance ajppsychiatryonlineorg 9

KNOWLES MCKAY KENT ET AL