Genetic Variants Associated with Anxiety and Stress-Related Disorders a Genome-Wide Association Study and Mouse-Model Study

Research

JAMA Psychiatry | Original Investigation Genetic Variants Associated With Anxiety and Stress-Related Disorders A Genome-Wide Association Study and Mouse-Model Study

Sandra M. Meier, PhD; Kalevi Trontti, PhD; Kirstin L. Purves, MSc; Thomas Damm Als, PhD; Jakob Grove, PhD; Mikaela Laine, MSc; Marianne Giørtz Pedersen, MSc; Jonas Bybjerg-Grauholm, PhD; Marie Bækved-Hansen, PhD; Ewa Sokolowska, PhD; Preben B. Mortensen, DrMedSc; David M. Hougaard, DrMedSc; Thomas Werge, PhD; Merete Nordentoft, PhD; Gerome Breen, PhD; Anders D. Børglum, MD; Thalia C. Eley, PhD; Iiris Hovatta, PhD; Manuel Mattheisen, MD; Ole Mors, PhD

Editorial page 889

IMPORTANCE Anxiety and stress-related disorders are among the most common mental Supplemental content disorders. Although family and twin studies indicate that both genetic and environmental factors play an important role underlying their etiology, the genetic underpinnings of anxiety and stress-related disorders are poorly understood.

OBJECTIVES To estimate the single-nucleotide polymorphism–based heritability of anxiety and stress-related disorders; to identify novel genetic risk variants, genes, or biological pathways; to test for pleiotropic associations with other psychiatric traits; and to evaluate the association of psychiatric comorbidities with genetic findings.

DESIGN, SETTING, PARTICIPANTS This genome-wide association study included individuals with various anxiety and stress-related diagnoses and controls derived from the population-based Lundbeck Foundation Initiative for Integrative Psychiatric Research (iPSYCH) study. Lifetime diagnoses of anxiety and stress-related disorders were obtained through the national Danish registers. Genes of interest were further evaluated in mice exposed to chronic social defeat. The study was conducted between June 2016 and November 2018.

MAIN OUTCOMES AND MEASURES Diagnoses of a relatively broad diagnostic spectrum of anxiety and stress-related disorders.

RESULTS The study sample included 12 655 individuals with various anxiety and stress-related diagnoses and 19 225 controls. Overall, 17 740 study participants (55.6%) were women. A total of 7308 participants (22.9%) were born between 1981-1985, 8840 (27.7%) between 1986-1990, 8157 (25.6%) between 1991-1995, 5918 (18.6%) between 1996-2000, and 1657 (5.2%) between 2001-2005. Standard association analysis revealed variants in PDE4B to be associated with anxiety and stress-related disorder (rs7528604; P =5.39×10−11; odds ratio = 0.89; 95% CI, 0.86-0.92). A framework of sensitivity analyses adjusting for mental comorbidity supported this result showing consistent association of PDE4B variants with anxiety and stress-related disorder across analytical scenarios. In mouse models, alterations in Pde4b expression were observed in those mice displaying anxiety-like behavior after exposure to chronic stress in the prefrontal cortex (P = .002; t = −3.33) and the hippocampus (P = .001; t = −3.72). We also found a single-nucleotide polymorphism heritability of 28% (standard error = 0.027) and that the genetic signature of anxiety and stress-related overlapped with psychiatric traits, educational outcomes, obesity-related phenotypes, smoking, and reproductive success.

CONCLUSIONS AND RELEVANCE This study highlights anxiety and stress-related disorders as Author Affiliations: Author complex heritable phenotypes with intriguing genetic correlations not only with psychiatric affiliations are listed at the end of this article. traits, but also with educational outcomes and multiple obesity-related phenotypes. Corresponding Author: Sandra M. Furthermore, we highlight the candidate gene PDE4B as a robust risk locus pointing to Meier, PhD, Department of the potential of PDE4B inhibitors in treatment of these disorders. Psychiatry, Dalhousie University, 5850/5980 University Ave, PO Box JAMA Psychiatry. 2019;76(9):924-932. doi:10.1001/jamapsychiatry.2019.1119 9700, Halifax, NS B3K 6R8, Canada Published online May 22, 2019. ([email protected]).

924 (Reprinted) jamapsychiatry.com

Downloaded From: https://jamanetwork.com/ on 09/29/2021 Genetic Variants Associated With Anxiety and Stress-Related Disorders Original Investigation Research

nxiety disorders are characterized by excessive and inappropriate fear and anxiety triggered by stimuli per- Key Points ceived as threatening. They are among the most com- A Question Which genetic variants are associated with anxiety and mon mental disorders with a lifetime prevalence of more than stress-related disorders and do they correlate with other traits? 20%.1 Given the prevalence and the immense social and eco- Findings In this study of genome-wide association data, PDE4B nomic burden of these disorders,2 it is of strong interest to variants were associated with anxiety and stress-related disorders, identify their risk factors. and their genetic signature overlapped with other psychiatric Family and twin studies indicate that both genetic and en- traits, educational outcomes, obesity-related phenotypes, vironmental factors are of relevance in the etiology of anxiety smoking, and reproductive success. disorders, with levels of familial aggregation and heritability at Meaning Large samples are needed to validly identify genetic 30% to 50%.3 Although stress-related disorders share many variants associated with anxiety and stress-related disorders. symptom characteristics with anxiety disorders and both con- ditions are highly comorbid, they have recently been moved to a separate diagnostic category. Interestingly, susceptibility fac- Biobank.23 Following protocols for DNA extraction and am- tors common to different anxiety and stress-related disorders plification (described elsewhere24), all samples were geno- seem to account for a larger proportion in heritability than fac- typed using Illumina’s PsychChip (Illumina). Through the na- tors predisposing to individual disorders.4 This indicates the tional research registers,25,26 we identified individuals with potential of combining these phenotypes to identify their shared an anxiety and stress-related diagnosis assigned by a psychia- genetic underpinnings. Genome-wide association studies trist during routine clinical care according to the Interna- (GWAS) have proven to be an effective tool for the identifica- tional Statistical Classification of Diseases and Related Health tion of common genetic variants increasing the susceptibility Problems, Tenth Revision (F40.0-F41.9; F43.0-F43.9). Indi- to complex disorders. Recently,GWAS of panic disorder,5-8 post- viduals with comorbid autism were excluded. Although pa- traumatic stress disorder,9-15 generalized anxiety disorders,16 tients with autism experience anxiety, their anxiety is often phobias,17,18 and a composite indicator of anxiety disorders19,20 reflecting their core autistic symptomatology and lacks the have been published. However, besides the study by Purves social component central to many anxiety and stress-related et al,20 most of these efforts were limited in sample size result- diagnoses.27 Exclusion criteria for control individuals were ing in low overall power to detect significant associations. International Statistical Classification of Diseases and Related In this study, we conducted a GWAS aggregating indivi- Health Problems, Tenth Revision diagnoses of anxiety, stress- duals in the Lundbeck Foundation Initiative for Integrative related disorders, and mood disorders. Characteristics of the Psychiatric Research (iPSYCH) study with varying diagnoses of sample are displayed in Table 1. The study was conducted anxiety and stress-related disorders to identify their common between June 2016 and November 2018. This study was ap- genetic factors, extending previous successful attempts.19,20 As proved by the Danish Data Protection Agency. By Danish law, most individuals with these disorders experience another co- registry-based studies do not require informed consent. morbid mental disorder, especially depression,21 we explored the association of mental comorbidity with the genetics of anxi- Quality Control, GWAS, and Gene-Based Analysis ety and stress-related disorders. Genes identified through this Quality control, imputation, and primary association analy- effort were further followed up in a mouse model of chronic so- ses in iPSYCH have been described elsewhere.28 We used the cial defeat. Our effort represents the first genetic study of this bioinformatics pipeline Ricopili29 developed by the Psychiat- magnitude to explicitly target comorbidity of anxiety and stress- ric Genomics Consortium.30 To avoid potential study effects related disorders, to our knowledge. of the 23 genotyping batches within the iPSYCH cohort, each batch was processed separately. Standard procedures for stringent quality control included filters for call rate, Hardy- Methods Weinberg equilibrium, and heterozygosity rates. Each batch was phased and imputed using the 1000 Genomes Project Participants phase 3 imputation reference panel31 using SHAPEIT32 and The GWAS sample under analysis included 12 655 individuals IMPUTE2,33 respectively. Cryptic relatedness and population with anxiety and stress-related diagnoses as well as 19 225 structure were assessed on high-quality single-nucleotide poly- controls from Denmark. All study participants were enrolled morphisms (SNPs) with low linkage disequilibrium (LD). in iPSYCH, a study designed to unravel risk factors of severe Genome-wide association studies for the 23 batches in mental disorders, including individuals with schizophrenia, iPSYCH were performed using logistic regression models with autism, attention-deficit/hyperactivity disorder, anorexia ner- the imputed marker dosages including the first 4 principal com- vosa, and affective disorders (referred to in this article as ponents to control for remaining population stratification.34 design phenotypes) as wells as population-based controls. More Subsequently, the results were meta-analyzed using an inverse information on iPSYCH can be found elsewhere.22 Of these de- variance–weighted fixed-effects model, implemented in METAL.35 sign individuals, 4584 individuals were diagnosed as having Additionally,the analyses were stratified for anxiety disorders and an anxiety disorder and 9831 as having a stress-related disor- stress-related disorders and different subtypes. Most individu- der, of which 1760 received both diagnoses. DNA samples for als were diagnosed as having an additional mental disorder iPSYCH were taken from the Danish Neonatal Screening owing to their comorbidity with 1 of the design phenotypes in

jamapsychiatry.com (Reprinted) JAMA Psychiatry September 2019 Volume 76, Number 9 925

Downloaded From: https://jamanetwork.com/ on 09/29/2021 Research Original Investigation Genetic Variants Associated With Anxiety and Stress-Related Disorders

SNP Heritability and Genetic Correlation With Other Traits Table 1. Sample Characteristics Linkage disequilibrium score regression was used to dissect No. (%) the relative contribution of polygenic effects and confound- Individuals Controls Total Characteristic (n = 12 655) (n = 19 225) (N = 31 880) ers (eg, cryptic relatedness, sample overlap, and population Sex stratification) to deviation from the null in the genome-wide Male 4130 (32.6) 10 010 (52.1) 14 140 (44.4) distribution of GWAS χ2 statistics.38,39 Prevalence was speci- Female 8525 (67.4) 9215 (47.9) 17 740 (55.6) fied at 20%.40,41 Using LD score regression, SNP heritability Birth year was also partitioned by functional category and tissue 1981-1985 3886 (30.7) 3422 (17.8) 7308 (22.9) association.38,39 Partitioning was performed for 53 overlap- 1986-1990 4510 (35.6) 4330 (22.5) 8840 (27.7) ping functional categories as well as 220 cell-type–specific 1991-1995 3239 (25.6) 4918 (25.6) 8157 (25.6) annotations grouped into 10 cell-type categories.42 Genetic cor- 1996-2000 876 (6.9) 5042 (26.2) 5918 (18.6) relations were tested for 228 phenotypes with publicly avail- 2001-2005 144 (1.1) 1513 (7.9) 1657 (5.2) able GWAS summary statistics and 596 traits from the UK a Anxiety diagnosis Biobank study43 using LD Hub.44 In addition, polygenic risk Agoraphobia 524 (4.1) NA 524 (1.6) scores45 were constructed in our sample to explore polygenic Generalized 758 (6.0) NA 758 (2.4) anxiety disorder heterogeneity across diagnostic subtypes and comorbidities Panic disorder 910 (7.2) NA 910 (2.9) (eMethods in the Supplement). Social phobia 1323 (10.5) NA 1323 (4.1) Specific phobia 214 (1.7) NA 214 (0.7) Mouse Model of Chronic Psychosocial Stress Mixed 580 (4.6) NA 580 (1.8) Given that stress is known to increase the risk of anxiety and anxiety disorder stress-related disorders, we aimed to establish the association Other/unspecific 1875 (14.8) NA 1875 (5.9) anxiety disorder of chronic psychosocial stress with the brain gene expression >1 Disorder 1600 (12.6) NA 1600 (5.0) levels of significant genes. We used the chronic social defeat Stress-related disorder stress (CSDS) model46 including 2 inbred strains: C57/BL6NCrl diagnosisa Acute stress reaction 1230 (9.7) NA 1230 (3.9) (B6, innately nonanxious strain) and DBA/2NCrl (D2, innately Posttraumatic stress 615 (4.9) NA 615 (1.9) anxious strain). Twenty-four hours after the last CSDS session, disorder we tested all mice for social aversion comparing their explor- Adjustment disorder 6809 (53.8) NA 6809 (21.4) ative behavior in the area around a cylinder with and without Other stress disorder 3573 (28.2) NA 3573 (11.2) a Clr-CD1 mouse. One week after the end of CSDS, we dis- >1 Disorder 2396 (18.9) NA 2396 (7.5) sected the medial prefrontal cortex (mPFC) and ventral hippo- Comorbidity campus (vHPC) for RNA sequencing. All animal procedures were Attention-deficit/ 2594 (20.5) 222 (1.2) 2816 (8.8) hyperactivity disorder approved by the Regional State Administration Agency for Anorexia nervosa 686 (5.4) 38 (0.2) 724 (2.3) Southern Finland (license numbers ESAVI-3801-041003-2011 Bipolar disordera 679 (5.4) NA 679 (2.1) and ESAVI/2766/04.10.07/2014) and carried out according to di- Depressiona 8759 (69.2) NA 8759 (27.5) rective 2010/63/EU of the European Parliament and of the Schizophrenia 1095 (8.7) 28 (0.1) 1123 (3.5) Council and the Finnish Act on the Protection of Animals Used Abbreviation: NA, not applicable. for Science or Educational Purposes (497/2013). a These data are only reported for the individuals and total because these specific categories have been excluded from the controls. Gene Expression Profiling Total RNA was extracted using TriReagent. For RNA sequenc- iPSYCH. We therefore aimed to adjust for mental comorbidity in ing, we carried out ribosomal RNA depletion using Ribo-Zero a framework of sensitivity analyses (eMethods in the Supplement). (Illumina) followed by fragmentation with an S2 ultrasonicator There is currently no other study available with the same pheno- (Covaris Inc). Messenger RNA sequencing libraries were pre- type definition, to our knowledge; however, we sought for rep- pared with Nextera (Illumina; vHPC samples) or ScriptSeq ver- lication of our index SNP and correlated genome-wide significant sion 2 (Epicenter; mPFC samples) kits. Whole transcriptome variants (0.2 < r2 <0.5,rs7528604 and rs7539350)inrelated level multiple testing correction was done with the Benjamini- phenotypes, such as anxiety20 and neuroticism.36 Finally, as Hochberg method,47 after which the expression levels of sig- obsessive-compulsive disorder has in previous classifications nificant genes were extracted from the data set (eMethods in systems been categorized under anxiety disorders, analyses were the Supplement). A 2-sided P value less than .05 was consid- supplemented by also including individuals with obsessive- ered statistically significant. compulsive disorder. Gene-based associations were calculated with MAGMA37 using the summary statistics from the main GWAS analyses. Results Association was tested using the SNP-wise mean model, in which the sum of –log (SNP P value) for SNPs located within the GWAS and Gene-Based Analysis transcribed region was used as test statistic. MAGMA37 con- At a single locus on chromosome 1, 68 genetic variants trols for gene size, number of SNPs in a gene, and LD between exceeded the threshold for genome-wide significance in this markers estimated from 1000 Genomes Project phase 3 samples. GWAS for anxiety and stress-related disorders (ie, without

926 JAMA Psychiatry September 2019 Volume 76, Number 9 (Reprinted) jamapsychiatry.com

Downloaded From: https://jamanetwork.com/ on 09/29/2021 Genetic Variants Associated With Anxiety and Stress-Related Disorders Original Investigation Research

Figure 1. Manhattan Plot of the Results From the Genome-Wide Association Study of Anxiety and Stress-Related Disorders

) 8 P ( 10

Log 7

12345678910111213141516171819202122 Chromosome

This plot displays 12 665 individuals and 19 225 controls. Single-nucleotide polymorphisms in green are in linkage disequilibrium with the index single-nucleotide polymorphisms (diamonds) and have a P value less than .001. Index variants located with a distance less than 400 kilobase are considered as 1 locus.

Table 2. Results for Index Variants in the Top 10 Loci Associated With Anxiety and Stress-Related Disordersa

Chromo- Base Allele Allele some Position SNP Genes 1 2 Frequency OR (95% CI) P Value Phenotypes 1 66407352 rs7528604 PDE4B A G 0.385 0.890 (0.860-0.921) 5.39 × 10−11 SCZ48,49 11 81047274 rs1458103 NA A C 0.741 0.898 (0.864-0.933) 6.19 × 10−8 NA 9 2511193 rs113209956 NA T C 0.085 0.828 (0.773-0.887) 6.36 × 10−8 NA 7 3676002 rs6462203 SDK1 A C 0.265 0.901 (0.867-0.936) 1.09 × 10−7 Cannabis50 20 41070559 rs6030245 PTPRT T C 0.795 1.120 (1.072-1.170) 5.06 × 10−7 NA 15 41024303 rs11855560 KNL1, RAD51-AS1, T C 0.469 1.089 (1.053-1.126) 6.96 × 10−7 NA RAD51, RMDN3, GCHFR, DNAJC17, C15orf62, ZFYVE19 5 7748796 rs2451828 ADCY2 T C 0.019 1.340 (1.193-1.505) 7.37 × 10−7 BP51 8 87643741 rs16916239 CNGB3 A G 0.783 0.903 (0.867-0.941) 8.96 × 10−7 NA 2 233649290 rs79928194 GIGYF2, KCNJ13 T C 0.905 0.862 (0.812-0.915) 1.26 × 10−6 SCZ30,48,49 5 83470986 rs342422 EDIL3 A G 0.530 0.920 (0.880-0.952) 1.28 × 10−6 SCZ49 Abbreviations: BP, base pair; NA, not applicable; OR, odds ratio; location (chromosome and base position), SNP, alleles 1 and 2, allele SCZ, schizophrenia; SNP, single-nucleotide polymorphism. frequency, OR of the effect with respect to allele 1, and association P value of a Index variants are linkage disequilibrium independent (r2 < 0.1) and are the index variant along with genes are within 100 kilobases of the locus. merged into 1 locus when located with a distance less than 400 kilobases. The

correction for the study design). The locus overlaps with 1 gene ure 6 in the Supplement). In addition, results from analyses (PDE4B) with the lead SNP rs7528604 (P =5.39×10−11;odds mimicking the comorbidity pattern of population-based ratio [OR] = 0.89; 95% CI, 0.86-0.92) (Figure 1, Table 230,48-51; samples (rs7528604; P =1.20×10−11; OR = 0.88; 95% CI, 0.85- eFigures 1 and 2 in the Supplement). We found no evidence 0.91) and including psychiatric phenotypes as covariates for significant heterogeneity between genotyping batches for (rs7528604; P =2.32×10−8; OR = 0.90; 95% CI, 0.87-0.93) were this marker (eFigure 3 in the Supplement). Stratified analyses in line with our initial GWAS results (eFigures 7 and 8 in the focusing on anxiety and stress-related disorder phenotype defi- Supplement). Even in the stringent sensitivity analyses, asso- nitions separately supported the identified locus (eFigures 4 ciations within the PDE4B gene were among the top signals and 5 in the Supplement), permutation tests indicated that the although with a different lead SNP (rs17128482; P =1.43×10−5; weaker signals can be attributed to a loss in sample size. Per- OR = 1.12; 95% CI, 1.07-1.17; eFigure 9 in the Supplement). Re- mutation tests also showed the difference in significance lev- sampling analyses by removing individuals with comorbid de- els across diagnostic subgroups could be attributed to their pression from our data did not show a significant association sample size (for exclusion of adjustment disorders see eFig- of depression with outcomes of our result at the PDE4B locus

jamapsychiatry.com (Reprinted) JAMA Psychiatry September 2019 Volume 76, Number 9 927

Downloaded From: https://jamanetwork.com/ on 09/29/2021 Research Original Investigation Genetic Variants Associated With Anxiety and Stress-Related Disorders

Figure 2. Significant Genetic Correlations Between Anxiety and Stress-Related Disorders and Other Heritable Traits

Trait Depressive Symptoms Major Depressive Disorder Subjective Well-being Neuroticism (SSGAC) Neuroticism (GPC) Schizophrenia PGC Cross-Disorder Analysis Insomnia Insomnia Excessive Daytime Sleepiness Years of Schooling (SSGAC v2) Years of Schooling (Proxy) Years of Schooling (SSGAC v1) College Completion Childhood IQ Intelligence Ever vs Never Smoked Cigarettes Smoked per Day Lung Cancer Lung Cancer (all) Obesity Class 1 Obesity Class 2 Waist-to-Hip Ratio Body Mass Index Bonferroni correction P <2.19×10−4. Overweight In total, 228 traits were tested, Body Fat including psychiatric traits, Age at Menarche educational outcomes, Age of First Birth obesity-related phenotypes, smoking, and reproductive success Number of Children Ever Born (eTable 4 in the Supplement). Error Mother’s Age at Death bars indicate 95% confidence limits. Coronary Artery Disease GPC indicates Genetics of Personality Consortium; PGC, Psychiatric –0.8 –0.6 –0.4 –0.2 0 0.2 0.4 0.6 0.8 1.0 Genomics Consortium; SSGAC, Social Genetic Correlation Science Genetic Association Consortium.

(P = .46). To assess whether our PDE4B signal might have been estimate is comparable with those reported in previous stud- based on comorbid schizophrenia, we excluded individuals ies for posttraumatic stress disorder52 and anxiety disorders.20 with schizophrenia and still observed genome-wide signifi- Partitioning heritability based on functional annotations re- cance and the same OR for that locus (P =3.58×10−10; vealed significant enrichment in the heritability for SNPs OR = 0.89; 95% CI, 0.86-0.91; eFigure 10 in the Supplement). located in conserved regions (enrichment = 2.01; SE = 0.32; Analyses including individuals with obsessive-compulsive dis- P < .001), supporting the general biological importance of con- order are displayed in eFigure 11 in the Supplement. A sum- served regions and their potential association with suscepti- mary of all analyses can be found in eTable 1 in the Supple- bility of anxiety and stress-related disorders (eFigure 12 in the ment. Genome-wide analyses using MAGMA37 identified Supplement). Cell-type–specific analyses revealed a signifi- 7 genome-wide significant genes, whereas no pathways were cant enrichment in the heritability by SNPs located in central significant after correction for multiple testing (eTables 2 and nervous system specific enhancers and promoters (enrich- 3intheSupplement). ment = 2.93; SE = 0.50; P =4.32×10−4; eFigures 13 and 14 in the Supplement). SNP Heritability and Genetic Correlation With Other Traits Using LDhub,44 31 phenotypes displayed significant ge- Linkage disequilibrium–score regression38,39 was used to cal- netic correlation with anxiety and stress-related disorders af- culate SNP heritability of our naive anxiety and stress-related ter Bonferroni correction (P =2.19×10−4), including psychi- disorders GWAS and the genetic correlation with other phe- atric traits, educational outcomes, obesity-related phenotypes, notypes. Assuming a population prevalence of 20% for anxi- smoking, and reproductive success (Figure 2). An overview of ety and stress-related disorders, we estimate the liability- all genetic correlations can be found in eTables 4 and 5 in the scale SNP heritability at 0.28 (standard error [SE] = 0.027). This Supplement. Linkage disequilibrium–score regression also

928 JAMA Psychiatry September 2019 Volume 76, Number 9 (Reprinted) jamapsychiatry.com

Downloaded From: https://jamanetwork.com/ on 09/29/2021 Genetic Variants Associated With Anxiety and Stress-Related Disorders Original Investigation Research

Figure 3. Differential Pde4b Gene Expression Levels in Mice Exposed to Chronic Psychosocial Stress

A B6 mPFC B B6 vHPC P =.002 P =.001 200 200 P =.006 P =.003

150 150

CPM 100 CPM 100 Pde4b Pde4b

50 50

0 0 Control Resilient Susceptible Control Resilient Susceptible

C D2 mPFC D D2 vHPC

200 200

150 150 Each sample is represented by a dot superimposed on the box plot.

CPM CPM Number of mice per group: B6 strain 100 100 controls = 6 (mPFC and vHPC),

Pde4b Pde4b resilient = 6 (mPFC) and 8 (vHPC), susceptible = 6 (mPFC) and 3 (vHPC); 50 50 D2 strain controls = 6 (mPFC and vHPC), resilient = 0, susceptible = 8 (mPFC) and 5 (vHPC). CPM indicates 0 0 log counts per million; mPFC, medial Control Susceptible Control Susceptible prefrontal cortex; vHPC, ventral hippocampus.

revealed a strong genetic correlation (r for genetic correla- date20 (rs7528604; P = .18; OR = 0.97; 95% CI, 0.93-1.02 and tion = 0.55; P =6.79×10−17) with the largest anxiety GWAS20 rs7539350; P = .02; OR = 1.05; 95% CI, 1.01-1.09) and to date. We performed the same LD score regression–based neuroticism36 (rs7528604; P =9.01×10−3; OR = 0.99; 95% CI, analyses also for the results from our framework of sensitiv- 0.99-0.99 and rs7539350; P =2.59×10−4; OR = 1.01; 95% CI, ity analyses. The association of the analyses mimicking the co- 1.01-1.01), which is often used as a proxy for anxiety and stress- morbidity pattern of population-based samples on SNP heri- related disorders. tability and the genetic correlations with other traits was marginal (eTables 6 and 7 in the Supplement). Owing to the Lower Pde4b Expression Levels in Mice overrepresentation of psychiatric phenotypes in the control We determined the gene expression levels of Pde4b in mice sample, the design including psychiatric phenotypes as co- exposed to CSDS using RNA sequencing (Figure 3). Stress- variates and the propensity score matched design resulted in susceptible mice from the B6 strain had lower expression lev- lower SNP heritability and genetic correlations (eTables 8 to els of Pde4b in the mPFC compared with both control (P < .01; 11 in the Supplement). An overview with regard to SNP heri- t = −3.33; adjusted P = .06) and stress-resilient mice (P = .01; tability reflecting the increased prevalence of anxiety and t = −2.97; adjusted P = .10), as well as in the vHPC compared stress-related disorders in psychiatric phenotypes can be found with control (P < .01; t = −3.72; adjusted P < .01) and stress- in eTable 12 in the Supplement. Using polygenic risk scores resilient mice (P = .003; t = −3.29; adjusted P = .01). D2 mice trained on different phenotypes, we only observed limited are highly susceptible to CSDS and therefore, we were only able heterogeneity across diagnostic and comorbidity groups (eFig- to compare stress-susceptible mice with controls. There were ures 15 and 16 in the Supplement). no differences in Pde4b expression levels between these groups in either brain region. Interestingly, the innately highly anx- Study Results in Context ious D2 control mice had lower Pde4b expression levels com- We sought for replication of our index SNP and correlated pared with the nonanxious B6 control mice in the vHPC genome-wide significant variants (rs7528604; P =5.39×10−11; (P =4.6×10−5; t = −4.81; adjusted P = .00016) but not in the OR = 0.89; 95% CI, 0.86-0.92 and rs7539350; P =1.46×10−8; mPFC (P = .13; t = −1.57; adjusted P = .26), suggesting that OR = 1.10; 95% CI, 1.06-1.14) in related phenotypes.20,36 PDE4B especially in the vHPC Pde4b expression levels may also was associated with anxiety disorders in the largest GWAS to contribute to innate anxiety levels.

jamapsychiatry.com (Reprinted) JAMA Psychiatry September 2019 Volume 76, Number 9 929

Downloaded From: https://jamanetwork.com/ on 09/29/2021 Research Original Investigation Genetic Variants Associated With Anxiety and Stress-Related Disorders

negative associations between these disorders and educa- Discussion tional attainment have been reported,58 and our results suggest that genetic factors may partially account for these To our knowledge, we conducted the largest GWAS on reported associations. anxiety and stress-related disorders to date and extend previous findings about shared genetic associations with other Strengths and Limitations phenotypes. Specifically, we aggregated 12 655 individuals A major strength of this study is the aim to identify genetic and 19 225 control individuals with the aim of identifying variants that play a central but nonspecific role in the common variants underlying the etiology of these susceptibility of anxiety and stress-related disorders. This disorders. We tried to capture the clinical complexity of contrasts with the approach taken in most psychiatric anxiety and stress-related phenotypes in a sample enriched genetic studies, which generally focus on specific clinical for individuals with comorbid mental illnesses and identi- diagnoses. However, it has long been recognized that clini- fied genetic variants that were associated with disease cal diagnoses poorly reflect etiological mechanisms, as both susceptibility. genetic and environmental factors have been found to have The most consistent association signal across our differ- nonspecific effects across a wide range of diagnoses.59 ent analyses was observed for genetic variants located within Given how critical fear and anxiety are for human survival, the PDE4B gene, which regulates intracellular cyclic adeno- it is very likely that conserved genes common to a range of sine monophosphate signaling and is strongly expressed in the anxiety and stress-related disorder regulate these basic human brain. PDE4B has been proposed as a candidate gene biological processes. for anxiety, in particular, panic disorder53; however, replica- Making use of registry-based diagnoses as a proxy for tion has so far been lacking. Pharmacologic profiles of selec- mental disorders in a research study that was primarily tive PDE4B inhibitors have demonstrated clear antidepres- ascertained for closely related traits constitute the major sant and anxiolytic benefits.54 Furthermore, mice deficient in limitation, but it is also a strength of our study enabling the Pde4b exhibit behavioral changes in a range of tests sensitive characterization of anxiety and stress-related disorders sus- to anxiolytic drugs.55 We found the expression of Pde4b to be ceptibility in the context of mental comorbidity. Similar to pre- altered in B6 mice susceptible to chronic psychosocial stress vious studies, the generalizability to milder forms of anxiety compared with controls and stress-resilient mice. Lower ex- and stress-related disorders or truly population-based samples pression levels were observed in brain regions (mPFC and is difficult to assess. vHPC), which are known to regulate emotional and social be- Through different sensitivity analyses, we aimed to ad- havior in mice and humans. In innately anxious (D2 inbred) dress some of the limitations (to the extent possible with the mice, Pde4b expression did not differ from controls after data at hand) and gained new insights that probably would not chronic psychosocial stress exposure indicating a genetic have been possible otherwise. Importantly, despite this lack background effect. in generalizability, these limitations are unlikely to lead to false In line with other GWAS,19,52 our SNP heritability esti- positive associations in a narrow sense. However, they do ask mate of 28% for anxiety and stress-related disorders indi- for a reflection on the tested hypothesis in the sensitivity cates a substantial role for common genetic variation, account- analyses under consideration. However, we cannot exclude the ing for a sizable portion of twin-based heritability.3 Conserved possibility that our results rather reflect the genetic underpin- regions and regions containing enhancers and promoters of nings of stress-related disorders than anxiety or specific diag- expression in the central nervous system tissues were found nostic subgroups (ie, adjustment disorders) given their differ- to be enriched for associations with anxiety disorders, con- ences in sample sizes, although our permutation analyses did sistent with findings for schizophrenia, bipolar disorder, and not suggest so. As a final point, we would like to stress that our depression.42 More work is needed to unravel the nature of the replication efforts were limited by the fact that our study is genetic correlations described in this article and how differ- including a wider range of both anxiety and stress-related ent designs in our analytical framework affected these find- disorders than previous efforts.19,20,52 ings. Nevertheless, the range of genetic correlations with psychiatric traits, educational outcomes, obesity-related phenotypes, and smoking helps to broaden our conceptualiza- Conclusions tion of anxiety and stress-related disorders. First, the strong positive genetic correlations of these disorders with depres- In summary, our results highlight anxiety and stress-related sion and neuroticism in our naive GWAS reinforce clinical and disorders to be a complex heritable phenotype. We highlight epidemiologic observations. Anxiety and stress-related dis- the candidate gene PDE4B as a robust risk locus (through stud- orders are commonly comorbid with depression, often pre- ies in mice and humans), pointing to the potential of PDE4B cede depression, and even affect the course of the depression.56 inhibitors in treatment of these disorders. Future studies are Second, the positive genetic correlations with schizophrenia needed to confirm these findings via independent replica- and the cross–psychiatric disorder phenotype firmly anchor tion and to detect additional loci, not only identifying poten- anxiety and stress-related disorders with other psychiatric traits tial pleiotropic effects across the full spectrum of anxiety and and reflect the substantial evidence for partially shared ge- stress-related disorders, but also loci associated specifically netic susceptibility across many psychiatric disorders.57 Third, with individual disorders.

930 JAMA Psychiatry September 2019 Volume 76, Number 9 (Reprinted) jamapsychiatry.com

Downloaded From: https://jamanetwork.com/ on 09/29/2021 Genetic Variants Associated With Anxiety and Stress-Related Disorders Original Investigation Research

ARTICLE INFORMATION Statistical analysis: Meier, Trontti, Purves, Als, mouse experiments. None of these individuals Accepted for Publication: March 20, 2019. Grove, Bybjerg-Grauholm, Bækved-Hansen, Breen, received compensation for their work. Mattheisen. Published Online: May 22, 2019. Obtained funding: Mortensen, Werge, Nordentoft, REFERENCES doi:10.1001/jamapsychiatry.2019.1119 Breen, Børglum, Eley, Hovatta, Mors. 1. Kessler RC, Avenevoli S, Costello EJ, et al. Author Affiliations: Psychosis Research Unit, Administrative, technical, or material support: Prevalence, persistence, and sociodemographic Aarhus University Hospital, Risskov, Denmark (Meier, Bybjerg-Grauholm, Bækved-Hansen, Mortensen, correlates of DSM-IV disorders in the National Mors); The Lundbeck Foundation Initiative for Hougaard, Werge, Nordentoft, Breen, Børglum, Comorbidity Survey Replication Adolescent Integrative Psychiatric Research, iPSYCH, Mattheisen, Mors. Supplement. Arch Gen Psychiatry. 2012;69(4):372- Copenhagen, Denmark (Meier, Als, Grove, Supervision: Sokolowska, Mortensen, Werge, 380. doi:10.1001/archgenpsychiatry.2011.160 Pedersen, Bybjerg-Grauholm, Bækved-Hansen, Nordentoft, Breen, Børglum, Eley, Hovatta, 2. WittchenHU,JacobiF,RehmJ,etal.Thesizeandbur- Mortensen, Hougaard, Werge, Nordentoft, Mattheisen, Mors. denofmentaldisordersandotherdisordersofthebrain Børglum, Mattheisen, Mors); Department of Conflict of Interest Disclosures: Dr Mortensen in Europe 2010. Eur Neuropsychopharmacol. 2011;21(9): Psychiatry, Psychosomatics and Psychotherapy, reports grants from the Lundbeck Foundation during 655-679. doi:10.1016/j.euroneuro.2011.07.018 Center of Mental Health, University Hospital the conduct of the study and outside the submitted 3. Hettema JM, Neale MC, Kendler KS. A review Würzburg, Würzburg, Germany (Meier, work. Dr Hougaard reports grants from the and meta-analysis of the genetic epidemiology of Mattheisen); now with the Department of Lundbeck Foundation during the conduct of the anxiety disorders. Am J Psychiatry. 2001;158(10): Psychiatry, Dalhousie University, Halifax, Nova study and personal fees from the Statens Serum 1568-1578. doi:10.1176/appi.ajp.158.10.1568 Scotia, Canada (Meier); Research Program of Institut outside the submitted work. Dr Nordentoft Molecular and Integrative Biosciences, Faculty of 4. Tambs K, Czajkowsky N, Røysamb E, et al. reports grants from the Lundbeck Foundation during Biological and Environmental Sciences, Department Structure of genetic and environmental risk factors the conduct of the study. Dr Breen reports grants for dimensional representations of DSM-IV anxiety of Psychology and Logopedics, Medicum, and from the UK National Institute for Health Research disorders. Br J Psychiatry. 2009;195(4):301-307. SleepWell Research Program, University of Helsinki, during the conduct of the study. Dr Børglum reports doi:10.1192/bjp.bp.108.059485 Helsinki, Finland (Trontti, Laine, Sokolowska, grants from the Lundbeck Foundation during the Hovatta); Social, Genetic and Developmental 5. Erhardt A, Czibere L, Roeske D, et al. TMEM132D, conduct of the study and outside the submitted Psychiatry Centre, Institute of Psychiatry, a new candidate for anxiety phenotypes: evidence work. Dr Eley reports grants from the UK Medical Psychology & Neuroscience, King’s College London, from human and mouse studies. Mol Psychiatry. Research Council, UK National Institute for Health 2011;16(6):647-663. doi:10.1038/mp.2010.41 London, United Kingdom (Purves, Breen, Eley); Research, and Fondation Peters during the conduct Department of Biomedicine, Aarhus University, 6. Otowa T, Yoshida E, Sugaya N, et al. of the study. Dr Hovatta reports grants from Aarhus, Denmark (Als, Grove, Børglum, Genome-wide association study of panic disorder in European Research Council, Sigrid Juselius Mattheisen); Centre for integrative Sequencing the Japanese population. J Hum Genet. 2009;54 Foundation, and University of Helsinki during the (iSEQ), Aarhus University, Aarhus, Denmark (Als, (2):122-126. doi:10.1038/jhg.2008.17 conduct of the study. Dr Mattheisen reports grants Grove, Mortensen, Børglum); National Centre for 7. Otowa T, Tanii H, Sugaya N, et al. Replication of a from the Lundbeck Foundation during the conduct Register-Based Research, Aarhus University, genome-wide association study of panic disorder in of the study. Dr Mors reports grants from the Aarhus, Denmark (Pedersen, Mortensen); Danish a Japanese population. J Hum Genet. 2010;55(2): Lundbeck Foundation during the conduct of the Centre for Neonatal Screening, Department for 91-96. doi:10.1038/jhg.2009.127 study. No other disclosures were reported. Congenital Disorders, Statens Serum Institut, 8. OtowaT,KawamuraY,NishidaN,etal.Meta-analysis Copenhagen, Denmark (Bybjerg-Grauholm, Funding/Support: This article was funded by the of genome-wide association studies for panic disorder Bækved-Hansen, Hougaard); Institute of Biological Lundbeck Foundation (grants R102-A9118 and in the Japanese population. Transl Psychiatry. 2012;2: Psychiatry, Mental Health Centre Sct Hans, R155-2014-1724); the Novo Nordisk Foundation, e186. doi:10.1038/tp.2012.89 Copenhagen University Hospital, Roskilde, who provided support for the Danish National 9. Logue MW, Baldwin C, Guffanti G, et al. Denmark (Werge); Department of Clinical Medicine, Biobank resource; the European Research Council A genome-wide association study of post-traumatic University of Copenhagen, Copenhagen, Denmark Starting Grant GenAnx; and the Sigrid Jusélius stress disorder identifies the retinoid-related (Werge); Mental Health Centre Copenhagen, Foundation. The research in this study was partly orphan receptor alpha (RORA) gene as a significant Faculty of Health Sciences, University of funded by the National Institute for Health risk locus. Mol Psychiatry. 2013;18(8):937-942. Copenhagen, Copenhagen, Denmark (Nordentoft); Research Biomedical Research Centre at South doi:10.1038/mp.2012.113 National Institute for Health Research Biomedical London and Maudsley NHS Foundation Trust and 10. Xie P, Kranzler HR, Yang C, Zhao H, Farrer LA, Research Centre for Mental Health, South London King’s College London. Ms Purves’s work was Gelernter J. Genome-wide association study and Maudsley National Health Service Trust, funded by the Alexander von Humboldt identifies new susceptibility loci for posttraumatic London, United Kingdom (Breen); Department of Foundation. Dr Eley is partly funded by a program stress disorder. Biol Psychiatry. 2013;74(9):656-663. Clinical Neuroscience, Centre for Psychiatric grant from the UK Medical Research Council doi:10.1016/j.biopsych.2013.04.013 (MR/M021475/1). Research, Karolinska Institutet, Stockholm, 11. Guffanti G, Galea S, Yan L, et al. Genome-wide Sweden (Mattheisen). Role of the Funder/Sponsor: The funders had no association study implicates a novel RNA gene, the Author Contributions: Drs Meier and Mattheisen role in the design and conduct of the study; lincRNA AC068718.1, as a risk factor for had full access to all of the data in the study and take collection, management, analysis, and post-traumatic stress disorder in women. responsibility for the integrity of the data and the interpretation of the data; preparation, review, or Psychoneuroendocrinology. 2013;38(12):3029-3038. accuracy of the data analysis. Drs Hovatta, approval of the manuscript; and decision to submit doi:10.1016/j.psyneuen.2013.08.014 Mattheisen, and Mors contributed equally. the manuscript for publication. 12. Stein MB, Chen CY, Ursano RJ, et al; Army Study Concept and design: Meier, Hougaard, Werge, Disclaimer: The views expressed are those of the to Assess Risk and Resilience in Servicemembers Nordentoft, Breen, Børglum, Mattheisen, Mors. authors and not necessarily those of the National (STARRS) Collaborators. Genome-wide association studies of posttraumatic stress disorder in 2 cohorts Acquisition, analysis, or interpretation of data: Health Service, the National Institute for Health of US Army soldiers. JAMA Psychiatry. 2016;73(7): Meier, Trontti, Purves, Als, Grove, Laine, Pedersen, Research, or the Department of Health. Bybjerg-Grauholm, Bækved-Hansen, Sokolowska, 695-704. doi:10.1001/jamapsychiatry.2016.0350 Additional Contributions: We thank Naomi Wray, Mortensen, Hougaard, Werge, Breen, Børglum, 13. Ashley-Koch AE, Garrett ME, Gibson J, et al; PhD (University of Queensland, Brisbane), and Jack Eley, Hovatta, Mattheisen, Mors. Veterans Affairs Mid-Atlantic Mental Illness Research, Hettema, MD, PhD (Virginia Commonwealth Drafting of the manuscript: Meier, Trontti, Laine, Education,andClinicalCenterWorkgroup.Genome-wide University), for helpful comments and suggestions Breen, Hovatta, Mattheisen. association study of posttraumatic stress disorder in a during the preparation of the manuscript. We also Critical revision of the manuscript for important cohort of Iraq-Afghanistan era veterans. J Affect Disord. thank Ingrid Balcells, PhD; Natalia Kulesskaya, PhD; intellectual content: Purves, Als, Grove, Pedersen, 2015;184:225-234. doi:10.1016/j.jad.2015.03.049 Zuzanna Misiewicz, PhD; Suvi Saarnio, MSc; Jenni Bybjerg-Grauholm, Bækved-Hansen, Sokolowska, 14. Nievergelt CM, Maihofer AX, Mustapic M, et al. Lahtinen, MSc; and Sanna Kängsep, PhD, from the Mortensen, Hougaard, Werge, Nordentoft, Breen, Genomic predictors of combat stress vulnerability Hovatta Lab for technical help and discussions on Børglum, Eley, Mors. and resilience in U.S. Marines: a genome-wide

jamapsychiatry.com (Reprinted) JAMA Psychiatry September 2019 Volume 76, Number 9 931

Downloaded From: https://jamanetwork.com/ on 09/29/2021 Research Original Investigation Genetic Variants Associated With Anxiety and Stress-Related Disorders

association study across multiple ancestries 30. Schizophrenia Working Group of the Psychiatric 45. Purcell SM, Wray NR, Stone JL, et al; implicates PRTFDC1 as a potential PTSD gene. Genomics Consortium. Biological insights from 108 International Schizophrenia Consortium. Common Psychoneuroendocrinology. 2015;51:459-471. doi:10. schizophrenia-associated genetic loci. Nature. 2014;511 polygenic variation contributes to risk of 1016/j.psyneuen.2014.10.017 (7510):421-427. doi:10.1038/nature13595 schizophrenia and bipolar disorder. Nature. 2009; 15. Kilaru V, Iyer SV, Almli LM, et al. Genome-wide 31. Abecasis GR, Altshuler D, Auton A, et al; 1000 460(7256):748-752. gene-based analysis suggests an association GenomesProjectConsortium.Amapofhumangenome 46. Golden SA, Covington HE III, Berton O, Russo between Neuroligin 1 (NLGN1) and post-traumatic variation from population-scale sequencing. Nature. SJ. A standardized protocol for repeated social stress disorder. Transl Psychiatry. 2016;6:e820. doi: 2010;467(7319):1061-1073. doi:10.1038/nature09534 defeat stress in mice. Nat Protoc. 2011;6(8):1183-1191. 10.1038/tp.2016.69 32. Delaneau O, Marchini J, Zagury JF. A linear doi:10.1038/nprot.2011.361 16. Dunn EC, Sofer T, Gallo LC, et al. Genome-wide complexity phasing method for thousands of 47. Hochberg Y, Benjamini Y. More powerful association study of generalized anxiety symptoms genomes. Nat Methods. 2011;9(2):179-181. doi:10. procedures for multiple significance testing. Stat Med. in the Hispanic Community Health Study/Study of 1038/nmeth.1785 1990;9(7):811-818. doi:10.1002/sim.4780090710 Latinos. Am J Med Genet B Neuropsychiatr Genet. . Howie BN, Donnelly P, Marchini J. A flexible and . Pardiñas AF, Holmans P, Pocklington AJ, et al; 2017;174(2):132-143. doi:10.1002/ajmg.b.32448 33 48 accurate genotype imputation method for the next GERAD1 Consortium; CRESTAR Consortium. 17. Walter S, Glymour MM, Koenen K, et al. generation of genome-wide association studies. Common schizophrenia alleles are enriched in Performance of polygenic scores for predicting PLoS Genet. 2009;5(6):e1000529. doi:10.1371/ mutation-intolerant genes and in regions under phobic anxiety. PLoS One. 2013;8(11):e80326. doi: journal.pgen.1000529 strong background selection. Nat Genet. 2018;50 10.1371/journal.pone.0080326 34. Price AL, Patterson NJ, Plenge RM, Weinblatt (3):381-389. doi:10.1038/s41588-018-0059-2 18. Stein MB, Chen CY, Jain S, et al; Army STARRS ME, Shadick NA, Reich D. Principal components 49. Li Z, Chen J, Yu H, et al. Genome-wide Collaborators. Genetic risk variants for social analysis corrects for stratification in genome-wide association analysis identifies 30 new susceptibility anxiety. Am J Med Genet B Neuropsychiatr Genet. association studies. Nat Genet. 2006;38(8):904- loci for schizophrenia. Nat Genet. 2017;49(11): 2017;174(2):120-131. doi:10.1002/ajmg.b.32520 909. doi:10.1038/ng1847 1576-1583. doi:10.1038/ng.3973 19. Otowa T, Hek K, Lee M, et al. Meta-analysis of . Willer CJ, Li Y, Abecasis GR. METAL: fast and . PasmanJA,VerweijKJH,GerringZ,etal;23andMe genome-wide association studies of anxiety 35 50 efficient meta-analysis of genomewide association ResearchTeam;SubstanceUseDisordersWorkingGroup disorders. Mol Psychiatry. 2016;21(10):1391-1399. scans. Bioinformatics. 2010;26(17):2190-2191. doi: of the Psychiatric Genomics Consortium; International doi:10.1038/mp.2015.197 10.1093/bioinformatics/btq340 Cannabis Consortium. GWAS of lifetime cannabis use 20. PurvesKL,ColemanJRI,MeierSM,etal.Amajorrole 36. Nagel M, Watanabe K, Stringer S, Posthuma D, reveals new risk loci, genetic overlap with psychiatric for common genetic variation in anxiety disorders. Bio traits, and a causal influence of schizophrenia. Nat Rxiv. https://www.biorxiv.org/content/10.1101/ van der Sluis S. Item-level analyses reveal genetic heterogeneity in neuroticism. Nat Commun. 2018;9 Neurosci. 2018;21(9):1161-1170. doi:10.1038/s41593- 203844v2. Published April 11, 2019. Accessed April 15, 018-0206-1 2019. (1):905. doi:10.1038/s41467-018-03242-8 37. de Leeuw CA, Mooij JM, Heskes T, Posthuma D. 51. Mühleisen TW, Leber M, Schulze TG, et al. 21. Kessler RC, Sampson NA, Berglund P, et al. Genome-wide association study reveals two new Anxious and non-anxious major depressive disorder MAGMA: generalized gene-set analysis of GWAS data. PLoS Comput Biol. 2015;11(4):e1004219. doi: risk loci for bipolar disorder. Nat Commun. 2014;5: in the World Health Organization World Mental 3339. doi:10.1038/ncomms4339 Health Surveys. Epidemiol Psychiatr Sci. 2015;24(3): 10.1371/journal.pcbi.1004219 210-226. doi:10.1017/S2045796015000189 38. Bulik-Sullivan BK, Loh PR, Finucane HK, et al; 52. is Duncan LE, Ratanatharathorn A, Aiello AE, Schizophrenia Working Group of the Psychiatric et al. Largest GWAS of PTSD (N=20 070) yields 22. Pedersen CB, Bybjerg-Grauholm J, Pedersen MG, genetic overlap with schizophrenia and sex et al. The iPSYCH2012 case-cohort sample: new Genomics Consortium. LD Score regression distinguishes confounding from polygenicity in differences in heritability. Mol Psychiatry. 2018;23 directions for unravelling genetic and environmental (3):666-673. doi:10.1038/mp.2017.77 architectures of severe mental disorders. Mol genome-wide association studies. Nat Genet. 2015; Psychiatry. 2018;23(1):6-14. 47(3):291-295. doi:10.1038/ng.3211 53. Otowa T, Kawamura Y, Sugaya N, et al. 39. Bulik-SullivanB,FinucaneHK,AnttilaV,etal;Repro- Association study of PDE4B with panic disorder in 23. Nørgaard-Pedersen B, Hougaard DM. Storage the Japanese population. Prog Neuropsychophar- policies and use of the Danish Newborn Screening GenConsortium;PsychiatricGenomicsConsortium;Ge- neticConsortiumforAnorexiaNervosaoftheWellcome macol Biol Psychiatry. 2011;35(2):545-549. doi:10. Biobank. J Inherit Metab Dis. 2007;30(4):530-536. 1016/j.pnpbp.2010.12.013 doi:10.1007/s10545-007-0631-x TrustCaseControlConsortium3.Anatlasofgeneticcor- relations across human diseases and traits. Nat Genet. 54. ZhangC,XuY,ZhangHT,GurneyME,O’DonnellJM. 24. Hollegaard MV, Grove J, Thorsen P, Nørgaard- 2015;47(11):1236-1241. doi:10.1038/ng.3406 Comparisonofthepharmacologicalprofilesofselective PedersenB,HougaardDM.High-throughputgenotyping 40. Pedersen CB, Mors O, Bertelsen A, et al. PDE4B and PDE4D inhibitors in the central nervous on archived dried blood spot samples. Genet Test Mol system. Sci Rep. 2017;7:40115. doi:10.1038/srep40115 Biomarkers. 2009;13(2):173-179. doi:10.1089/gtmb. A comprehensive nationwide study of the incidence 2008.0073 rate and lifetime risk for treated mental disorders. 55. ZhangHT,HuangY,MasoodA,etal.Anxiogenic-like JAMA Psychiatry. 2014;71(5):573-581. doi:10.1001/ behavioralphenotypeofmicedeficientinphosphodies- 25. Lynge E, Sandegaard JL, Rebolj M. The Danish jamapsychiatry.2014.16 terase 4B (PDE4B). Neuropsychopharmacology. 2008; national patient register. Scand J Public Health. 2011;39 33(7):1611-1623. doi:10.1038/sj.npp.1301537 (7)(suppl):30-33. doi:10.1177/1403494811401482 41. KesslerRC,BerglundP,DemlerO,JinR,Merikangas KR, Walters EE. Lifetime prevalence and age-of-onset 56. Meier SM, Petersen L, Mattheisen M, Mors O, 26. Mors O, Perto GP, Mortensen PB. The Danish distributions of DSM-IV disorders in the National Mortensen PB, Laursen TM. Secondary depression psychiatric central research register. Scand J Public Comorbidity Survey Replication. Arch Gen Psychiatry. in severe anxiety disorders: a population-based Health. 2011;39(7)(suppl):54-57. doi:10.1177/ 2005;62(6):593-602. doi:10.1001/archpsyc.62.6.593 cohort study in Denmark. Lancet Psychiatry. 2015;2 1403494810395825 42. Finucane HK, Bulik-Sullivan B, Gusev A, et al; (6):515-523. doi:10.1016/S2215-0366(15)00092-9 27. van Steensel FJ, Bögels SM, Perrin S. Anxiety ReproGenConsortium;SchizophreniaWorkingGroupof 57. Cross-Disorder Group of the Psychiatric Genomics disorders in children and adolescents with autistic thePsychiatricGenomicsConsortium;RACIConsortium. Consortium.Identificationofrisklociwithsharedeffects spectrum disorders: a meta-analysis. Clin Child Fam Partitioning heritability by functional annotation using on five major psychiatric disorders: a genome-wide Psychol Rev. 2011;14(3):302-317. doi:10.1007/ genome-wide association summary statistics. Nat analysis. Lancet. 2013;381(9875):1371-1379. doi:10.1016/ s10567-011-0097-0 Genet. 2015;47(11):1228-1235. doi:10.1038/ng.3404 S0140-6736(12)62129-1 28. Grove J, Ripke S, Als TD, et al; Autism Spectrum 43. UK Biobank: Neale lab. http://www.nealelab.is/ 58. de Lijster JM, Dieleman GC, Utens EMWJ, et al. So- Disorder Working Group of the Psychiatric Genomics uk-biobank. Accessed April 15, 2019. cial and academic functioning in adolescents with anxi- Consortium; BUPGEN; Major Depressive Disorder 44. Zheng J, Erzurumluoglu AM, Elsworth BL, et al; ety disorders: a systematic review. J Affect Disord. 2018; WorkingGroupofthePsychiatricGenomicsConsortium; 230:108-117. doi:10.1016/j.jad.2018.01.008 23andMe Research Team. Identification of common Early Genetics and Lifecourse Epidemiology genetic risk variants for autism spectrum disorder. Nat (EAGLE) Eczema Consortium. LD Hub: a centralized 59. Hettema JM, Prescott CA, Myers JM, Neale MC, Genet. 2019;51(3):431-444. doi:10.1038/s41588-019- database and web interface to perform LD score Kendler KS. The structure of genetic and 0344-8 regression that maximizes the potential of environmental risk factors for anxiety disorders in summary level GWAS data for SNP heritability and men and women. Arch Gen Psychiatry. 2005;62(2): 29. Ripkelab/ricopili. Main ricopili repo for public genetic correlation analysis. Bioinformatics. 2017;33 182-189. doi:10.1001/archpsyc.62.2.182 releases. https://github.com/Ripkelab/ricopili. (2):272-279. doi:10.1093/bioinformatics/btw613 Accessed April 15, 2019.

932 JAMA Psychiatry September 2019 Volume 76, Number 9 (Reprinted) jamapsychiatry.com

Downloaded From: https://jamanetwork.com/ on 09/29/2021