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Reproducible Genetic Risk Loci for Anxiety: Results From
∼200,000 Participants in the Million Veteran Program
Daniel F. Levey, Ph.D., Joel Gelernter, M.D., Renato Polimanti, Ph.D., Hang Zhou, Ph.D., Zhongshan Cheng, Ph.D.,
Mihaela Aslan, Ph.D., Rachel Quaden, M.A., John Concato, M.D., M.P.H., Krishnan Radhakrishnan, M.D., Ph.D.,
Julien Bryois, Ph.D., Patrick F. Sullivan, M.D., the Million Veteran Program, Murray B. Stein, M.D., M.P.H.
Objective: Anxiety disorders are common and often dis-
abling. The goal of this study was to examine the genetic
architecture of anxiety disorders and anxiety symptoms,
which are also frequently comorbid with other mental dis-
orders, such as major depressive disorder.
Methods: Using one of the world’s largest biobanks including
genetic, environmental, and medical information, the Million
Veteran Program, the authors performed a genome-wide
association study (GWAS) of a continuous trait for anxiety
(based on score on the Generalized Anxiety Disorder 2-item
scale [GAD-2], N=199,611) as the primary analysis and self-
report of physician diagnosis of anxiety disorder (N=224,330)
as a secondary analysis.
Results: The authors identified five genome-wide significant
signals for European Americans and one for African Ameri-
cans on GAD-2 score. The strongest were on chromosome
3 (rs4603973) near SATB1, a global regulator of gene ex-
pression, and on chromosome 6 (rs6557168) near ESR1,
which encodes an estrogen receptor. The locus identified
on chromosome 7 (rs56226325, MAF=0.17) near MAD1L1
was previously identified in GWASs of bipolar disorder and
schizophrenia. The authors replicated these findings in the
summary statistics of two major published GWASs for anxiety,
and also found evidence of significant genetic correlation
between the GAD-2 score results and previous GWASs for
anxiety (r
g
=0.75), depression (r
g
=0.81), and neuroticism
(r
g
=0.75).
Conclusions: This is the largest GWAS of anxiety traits to date.
The authors identified novel genome-wide significant as-
sociations near genes involved with global regulation of gene
expression (SATB1) and the estrogen receptor alpha (ESR1).
Additionally, the authors identified a locus (MAD1L1) that may
have implications for genetic vulnerability across several
psychiatric disorders. This work provides new insights into
genetic risk mechanisms underpinning anxiety and related
psychiatric disorders.
AJP in Advance (doi: 10.1176/appi.ajp.2019.19030256)
Anxiety disorders are common, affecting 1 in 10 Americans
each year, and are a leading cause of disability worldwide (1).
An analysis of health expenditures in the United States found
that anxiety and depressive disorders together accounted for
about $90 billion in personal health spending in the United
States in 2013 (2). Given their prevalence, associated im-
pairment, and economic costs, anxiety disorders are a major
public health concern (3).
Anxiety is “a future-oriented mood state associated with
preparation for possible, upcoming negative events”(4) and is
usually a normal and adaptive behavioral response to ev-
eryday life. In anxiety disorders, anxiety is excessive or out of
proportion to the actual or anticipated event and is accom-
panied by clinically significant distress or disability (5).
Numerous risk factors for anxiety disorders have been
studied, including experiential and genetic factors (6). For
example, neurotic personality traits are predictive of the
onset of anxiety disorders (7). Twin studies demonstrate
a heritable component to anxiety disorders (6), but there
have been few published genome-wide association studies
(GWASs) to date investigating anxiety or anxiety-related
traits. The Anxiety Neuro Genetics Study (ANGST) (8)
meta-analysis was the first large GWAS to identify significant
genetic associations, finding one genome-wide significant
locus each for a categorical case-control design for any
anxiety disorder diagnosis and a quantitative factor score for
anxiety in a cohort of over 18,000 subjects. Another recent
large GWAS, from the Lundbeck Foundation Initiative for
Integrative Psychiatric Research (iPSYCH), identified a
significant genetic association with anxiety and stress-related
disorders in a cohort of 31,880 individuals in the national
Danish registers (9). Also of note is a study based on the UK
Biobank cohort, the second largest GWAS of anxiety to date
(10), which examined anxiety using case-control and clinical
ajp in Advance ajp.psychiatryonline.org 1
ARTICLES
cutoffs based on a score $10 on the Generalized Anxiety
Disorder 7-item scale (GAD-7). While progress is being made,
understanding of the genetics of anxiety disorders has lagged
behind other related disorders, such as major depression (11).
Only a third of individuals with anxiety disorders receive
treatment (12). For those who do enter treatment, psycho-
logical approaches such as cognitive-behavioral therapy
(CBT) have been shown to be effective (13), as have certain
pharmacotherapies (14). A recent systematic review of CBT
treatment response rates for anxiety disorders showed av-
erage rates of 49.5% at end of treatment and 53.6% at follow-
up (15). A better understanding of genetic risk factors and
determinants now informs other aspects of medicine, such as
oncology and cardiology, through identification of causal
mutations (16) and variants, and this approach will have
important implications for psychiatry (17). These precision-
medicine approaches are challenging in complex traits such
as anxiety, which are associated with many (perhaps hun-
dreds of thousands of ) variants of individually small effect
(18). The use of polygenic risk scores will require a suitably
large sample size to provide sufficient confidence in these
small individual effects that cumulatively account for so
much of the heritability (19). Underlying polygenic risk
factors from sufficiently large cohorts may inform an ap-
proach to identifying individuals with a predisposition to
anxiety disorders and to improving outcomes.
The Million Veteran Program (MVP), one of the world’s
largest biobanks including genetic, environmental, and
medical information, is based on data from U.S. military
veterans (20–22). Using this large genetic data set and the
Generalized Anxiety Disorder 2-item scale (GAD-2) (23) as
well as self-reported physician diagnosis of an anxiety dis-
order, we discovered novel genome-wide significant variants
associated with anxiety in European Americans and African
Americans. We examined replication and genetic overlap of
these results with previous studies of anxiety and traits with
which anxiety disorders are commonly comorbid—major
depression, PTSD, and neuroticism. We also examined ex-
pression quantitative trait loci (eQTLs) to identify possible
gene expression implications of these genetic variants, with
eQTL evidence for altered expression in the basal ganglia and
cerebellum. These findings, in the largestcohort of individuals
analyzed by GWAS for anxiety and anxiety disorders (199,611
subjects for the quantitative trait, 224,330 for binary self-
report diagnosis) to date, indicate shared genetic risk with
some other mental disorders but also point to loci that may be
especially important for anxiety and anxiety-related traits.
METHODS
Participants
The MVP cohort has been described previously (20). Results
were analyzed in two separate tranches based on when geno-
typing results were available. Ancestry was assigned using
10 principal components and the 1000 Genomes Project phase
3 EUR and AFR data as reference within each MVP tranche.
Genotyping, Imputation, and Quality Control
Genotyping, imputation, and quality control within MVP has
been previously described. Briefly, samples were genotyped
using a 723,305-SNP Affymetrix Axiom Biobank array, cus-
tomized for MVP (20). Imputation was performed with
minimac3 using data from the 1000 Genomes Project. For
postimputation quality control, SNPs with an imputation
INFO score ,0.3 or a minor allele frequency (MAF) ,0.001
were removed from analysis. For the first tranche of data,
22,183 SNPs were selected through linkage disequilibrium
(LD) pruning using PLINK 2.0 (24), and then Eigensoft (25)
was used to conduct principal component analysis on 343,286
MVP samples and 2,504 1000 Genomes samples. The ref-
erence population groups (EUR, EAS, AFR, AMR, or SAS) in
the 1000 Genomes samples were used to define European
American (N=241,541) and African American (N=61,796)
groups used in this analysis. Similar methods were used in the
second tranche of data, which contained 108,416 new MVP
samples and the same 2,504 1000 Genomes samples. In
tranche 2, 80,694 participants were defined as European
American and 20,584 as African American.
Phenotypic Assessment
We used the GAD-2 (23) for our primary analysis. The GAD-2
consists of two questions (see Table S1 in the online sup-
plement) in a self-report survey, each scored on a scale of 0–3.
Participants are asked to respond according to their symp-
toms during the past 2 weeks. Values for the two responses
are summed, resulting in a range of scores between 0 and 6,
which we treated as a continuous trait (Table 1). Mean GAD-2
scores in European American men (N=163,470) and women
(N=11,693) were 1.08 (SD=1.64) and 1.64 (SD=1.87), re-
spectively, and mean scores in African American men
(N=21,153) and women (N=3,295) were 1.57 (SD=10.21) and
1.94 (SD=10.64), respectively. The mean ages of the European
American and African American participants were 66.58
years (SD=11.62) and 60.6 years (SD=10.78), respectively.
Another anxiety phenotype—self-reported physician di-
agnosis of anxiety disorder—was analyzed based on data
collected from the MVP baseline survey. Participants were
asked, “Please tell us if you have been diagnosed with the
following conditions: anxiety reaction/panic disorder.”An-
swers were recorded as yes/no binary responses, and missing
responses were excluded from analysis. A total of 224,330
participants (34,189 case subjects who responded yes, 190,141
control subjects who responded no) had available phenotype
and genotype information and had assignments of either
European ancestry (28,525 cases, 163,731 controls) or African
ancestry (5,664 cases, 26,410 controls) (see Table S2 in the
online supplement).
Statistical Analysis
GWAS analysis was carried out by linear regression for each
ancestry group and tranche using PLINK 2.0 on geno-
type dosage data, covarying for age, sex, and the first 10 prin-
cipal components against the phenotype of GAD-2 score.
2ajp.psychiatryonline.org ajp in Advance
REPRODUCIBLE GENETIC RISK LOCI FOR ANXIETY
Ancestry-specificandtrans-ancestry meta-analysis were per-
formed using inverse variance weighting in the METAL software
package (European American, N=175,163; African American,
N=24,448; combined trans-ancestry, N=199,611). Logistic re-
gression was used for self-reported physician diagnosis of an
anxiety disorder, and the results obtained were combined using
the same meta-analytic approach. To identify independent
GWAS signals, we clumped results using an r
2
of 0.10 and
window size of 1,000 kb. Post-GWAS analyses were conducted
for what turned out to be the most genetically informative
phenotype based on z-scored heritability: GAD-2 score.
Linkage Disequilibrium Score Regression
and SNP-Based Heritability
We used linkage disequilibrium score regression through
LD Hub (26) to estimate SNP-based heritability and to
assess genetic correlation of GAD-2 anxiety with all traits
available in LD Hub. The traits from the ANGST GWAS of
anxiety case-control and factor scores (8) and iPSYCH
anxiety and stress-related disorders (9)—neither of which
were available in LD Hub—were calculated separately
with LD score regression software (LDSC) using sum-
mary statistics downloaded from the Psychiatric Geno-
mics Consortium (PGC) web site (https://www.med.
unc.edu/pgc/results-and-downloads/) or from the authors,
respectively.
Conditional Analysis for Major Depression
Considering the extensive comorbidity between major de-
pression and anxiety disorders (6), we ran a conditional
analysis with the multi-trait-based conditional and joint
analysis method (mtCOJO) (27) using the GCTA software
package. This method uses GWAS summary statistics from
one trait to perform conditional analysis on GWAS summary
statistics from another trait. We conditioned the MVP GAD-2
summary statistics as the primary analysis with the PGC
major depressive disorder (11) summary statistics for de-
pression. We quantified changes in variance explained by
using LD score regression to calculate heritability in the
depression-conditioned GAD-2 analysis and compared with
the original GAD-2 analysis.
Gene-Based Tests
Summary statistics from the
GWAS were loaded into the
FUMA (Functional Map-
ping and Annotation) GWAS
platform to test for gene-level
associations using Multi-
Marker Analysis of Geno-
Mic Annotation (MAGMA)
(28). Input SNPs were
mapped to 18,469 protein
coding genes. The genome-
wide significance thresh-
oldforthegene-basedtest
was defined in accordance
with Bonferroni multiple testing correction (p=0.05/
18,469=2.71310
26
).
Fine Mapping
Fine mapping was conducted using PAINTOR, version 3 (29).
A brain functional annotation set (30) was used to prioritize
causal SNPs. The z-scored GAD-2 GWAS summary statistics
served as the base analysis data set, with the aforementioned
brain data set serving as the functional annotation. We
enumerated all possible combinations and searched for a
single causal SNP within each locus.
RESULTS
Primary Analysis
GWAS of GAD-2 scores was conducted separately in two
tranches of each ancestry in the MVP sample, defined by the
time when data became available, and meta-analyzed to-
gether within ancestral group. One genomic locus was
genome-wide significant in the African American meta-
analysis (Figure 1A), and five loci were genome-wide sig-
nificant in the European American meta-analysis (Figure 1B).
The genome-wide significant result from the African
American analysis (rs575403075, MAF=0.06, p=2.82310
28
)
was near the TRPV6 (Transient Receptor Potential Cation
Channel Subfamily V Member 6) locus. The top signal in the
European American meta-analysis consisted of 64 genome-
wide significant SNPs in high LD at the SATB1-AS1 (Special
AT-Rich Sequence Binding 1 Antisense RNA 1) locus on
chromosome 3. The strongest finding (rs4603973, MAF=0.29,
p=6.18310
211
) was intronic at SATB1-AS1.Thesecond
strongest independent signal was on chromosome
6 (rs6557168, MAF=0.37, p=1.33310
29
) intronic at ESR1
(Estrogen Receptor 1) with 10 other genome-wide significant
SNPs in high LD. A third genome-wide significant association
for European Americans was found on chromosome
1 (rs12023347, MAF=0.48, p=8.88310
29
) near the long
noncoding RNA LINC01360 and LRRIQ3 (Leucine-Rich
Repeats and IQ motif containing 3). The fourth genome-
wide significant association found in European Americans
was on chromosome 7 (rs56226325, MAF=0.17, p=2.01310
28
)
TABLE 1. Subjects and phenotype distribution in a study of genetic risk loci for anxiety
a
European Americans African Americans
Age Age
GAD-2 Score N Mean SE Female (%) N Mean SE Female (%)
0 100,141 69.10 0.033 4.79 11,212 62.93 0.1 10.64
1 21,569 65.31 0.082 8.20 2,897 60.33 0.2 15.15
2 25,061 63.95 0.075 8.86 3,947 58.97 0.166 16.06
3 8,450 62.40 0.129 9.44 1,708 58.86 0.243 14.87
4 8,046 61.49 0.134 10.08 1,730 57.83 0.253 16.99
5 4,447 60.61 0.176 9.83 1,011 57.30 0.333 15.23
6 7,449 59.16 0.143 11.63 1,943 56.40 0.235 16.83
Total 175,163 66.58 0.028 6.68 24,448 60.59 0.069 13.48
a
GAD-2=Generalized Anxiety Disorder 2-item scale.
ajp in Advance ajp.psychiatryonline.org 3
LEVEY ET AL.
in an intron of MAD1L1 (Mitotic Arrest Deficient 1 Like 1). The
fifth association for European Americans was on chromosome
20 in and around the TCEA2 (Transcription Elongation Factor
A2), RGS19 (Regulator of G Protein Signaling 19), and OPRL1
(Opioid Related Nociceptin Receptor 1) genes (rs6090040,
MAF=0.48, p=3.28310
28
).
We conducted additional analyses using case-control
status for self-reported physician diagnosis of an anxiety
disorder. For the European American subjects, there were
two genome-wide significant signals for the GWAS of self-
reported physician diagnosis of an anxiety disorder, in a gene-
rich region nearest AURKB on chromosome 17 (rs35546597,
MAF=0.42, p=1.88310
28
) and on chromosome 7 in an IQCE
intron (rs10534613, MAF=0.41, p=4.92310
28
) close to the
MAD1L1 locus identified for GAD-2. There were no genome-
wide significant findings for this phenotype in African
Americans.
Replication
For replication, wetested our top five SNPsfrom the analysis of
GAD-2 scores in European Americans in three independent
GWASs with anxiety-related phenotypes. We considered a
replication to be significant if the p value was ,0.05. We in-
vestigated our lead genome-wide significant SNPs in GWASs
for the ANGST anxiety case-control (8), iPSYCH anxiety and
stress-related disorders (9), and UK Biobank, 23andMe, and
Genetics of Personality Consortium (GPC) neuroticism (31)
phenotypes (Table 2). The first two phenotypes are very
similar to our GAD-2 measure; the third is best considered a
related phenotype (r
g
=0.7174, p=1.95310
253
).
In the ANGST anxiety
study (8), which was the
smallest replication cohort,
all five of our top in-
dependent SNPs had the
same direction of effect, with
two being nominally signifi-
cant (p,0.05). In the
iPSYCH study of anxiety and
stress-related disorders (9),
four of five independent
SNPs had the same direction
of effect, with three being
nominally significant (p,0.05).
We also replicated the lead
SNP from iPSYCH near
PDE4B in our own study
(iPSYCH lead SNP: rs7528604,
p=5.39310
28
; present study
GAD-2: same SNP, p=0.015).
Only one of our findings, near
OPRL1, failed to replicate in at
least one independent study.
Our lead SNP on chro-
mosome 3 near the SATB1
locus, rs4603973, was not
available for lookup in the neuroticism GWAS (31), which we
used as a proxy replication of a related trait, so we used the
strongest LD-proxy SNP available (rs4390955 R
2
=0.91,
p=7.78E-11). In this study, four of our five independent SNPs
we looked up had the same direction of effect, three were
nominally significant (p,0.05), and one near MAD1L1 was
nearly genome-wide significant (UK Biobank neuroticism:
rs56226325, p=6.59310
28
; present study GAD-2: same SNP,
p=2.01310
28
).
Lastly, a preprint reported results for anxiety from the
UK Biobank using case-controlandtheGAD-7,scoredasa
dichotomous trait (10). We found significant replication
fortwooftheirfourfindings, with suggestive evidence
for a third (see Table S9 in the online supplement).
Genome-Wide Gene-Based Association Study for GAD-2
In the genome-wide gene-based association study, the top
gene identified was OPRL1 (p=1.15310
29
), which was also
significant in the SNP-wise analysis, as noted above. Thirty-
one genes were identified as genome-wide significant fol-
lowing Bonferroni correction for multiple comparisons. A
more permissive Benjamini-Hochberg correction performed
by step-up procedure, with genes ranked by p value and
corrected for 18,469 individual tests with a still relatively
restrictive 0.05 false discovery rate, identified 189 genes (see
Table S3 in the online supplement) in total for investigation
of biological relevance through the Ingenuity pathway en-
richment tool (32), Ingenuity Pathway Analysis (Ingenuity
Systems, Redwood City, Calif.; www.ingenuity.com) (see
Table S4 in the online supplement). Among the top enriched
FIGURE 1. Circle Manhattan plot for anxiety phenotypes in African Americans and European
Americans
a
a
The outer circle displays results of the Generalized Anxiety Disorder 2-item scale genome-wide association
study (GWAS), and the inner circle contains results for the case-control (self-report of physician diagnosis of
anxiety disorder) GWAS. Numbers outside the circle represent chromosomes. Red dots indicate genome-wide
significant findings (p,5 x10
28
) and yellow dots indicate suggestive findings (p,5310
26
). The scale on the
y-axis represents 2log
10
(p value). Vertical dashed gray lines are drawn through genome-wide significant
findings to indicate overlap between analyses. The genes nearest to the lead SNP are labeled adjacent to the
result. In most cases a genome-wide significant (red) locus from one phenotype overlaps with at least a
suggestive (yellow) locus in the other.
4ajp.psychiatryonline.org ajp in Advance
REPRODUCIBLE GENETIC RISK LOCI FOR ANXIETY
diseases or functional annotations were carcinoma (p=1.76310
27
)
and fear conditioning (p=3.62310
24
).
Expression Quantitative Trait Loci (eQTLs)
To identify causal implications for genetic variants, eQTLs
were assessed for the top genome-wide significant GAD-2
signals using GTEx version 7 brain tissue expression data.
Top genome-wide significant signals on chromosomes 7 and
20 had significant eQTLs (false discovery rate #0.05) for four
different genes: FTSJ2,RGS19,C20orf201, and OPRL1 (see
Table S7 in the online supplement). The top signals are
centered in the basal ganglia and cerebellum.
SNP-Based Heritability
SNP-based heritability using LDSC for the GAD-2 quantitative
trait was estimated to be 5.58% (SE=0.004, z-score=13.95). SNP-
based inflation was mild considering the sample size and
polygenic trait studied (l=1.19); the intercept (1.026) and at-
tenuation ratio (0.1177) estimated by LDSC showed negligible
evidence for inflation due to population stratification. SNP-
based heritability for the self-reported physician diagnosis of
an anxiety disorder binary trait was 8.79% (SE=0.0085, z-
score=10.34) on the liability scale assuming prevalence of 20%.
This value is similar to that reported for anxiety by Otowa et al.
(h
2
=0.095, SE=0.037, z-score=2.57) (8), depression by the PGC
(h
2
=0.087, SE=0.004, z-score=21.75) (11) and Howard et al.
(h
2
=0.089, SE=0.003, z-score=29.67) (33), and neuroticism by
Nagel et al. (h
2
=0.100, SE=0.003, z-score=33.33) (31), but some-
what lower than that reported by Meier et al. for anxiety and
stress-related disorders (h
2
=0.28, SE=0.027, z-score=10.37) (9).
Linkage Disequilibrium Score Regression Analysis
The traits most significantly genetically correlated with GAD-
2 score were depressive symptoms (r
g
=0.81, p=1.95310
253
)
and neuroticism (r
g
=0.72, p=6.53310
253
). We also investigated
genetic correlation within the MVP cohort for GAD-2 score and
self-reported physician diagnosis of an anxiety disorder. These
were high (r
g
=0.87, p=2.39310
2119
), and higher than the phe-
notypic correlation between these traits (r=0.64, p,2.2310
216
)
(Figure 2; see also Table S5 in the online supplement).
Polygenic Risk Score (PRS) Analysis
Summary statistics from the MVP GAD-2 analysis were used
as the base data for calculating polygenic risk scores (PRSs)
(using PRSice, version 1.25 [34]). Genetic overlap between
anxiety and PTSD or major depressive disorder was tested
using the “summary statistics to summary statistics”pro-
cedure, using the gtx R package incorporated into PRSice, in
the PGC major depressive disorder (11) and PTSD (35)
GWASs, respectively, and overlap with case-control anxiety
disorder was tested in the largest previously available studies
(8, 9). Significant overlap was identified: the MVP GAD-2 PRS
can explain up to 0.24% of the variance in major depressive
disorder in the PGC GWAS (p=2.05310
294
), 0.23% of the
variance in PTSD in the PGC GWAS (p=4.23310
212
), and
0.48% of the variance in both the ANGST (p=3.66310
220
)
and iPSYCH anxiety studies (p=6.68310
236
) (Table 3).
Multi-Trait-Based Conditional and Joint Analysis
Multi-trait-based conditional and joint analysis was used to
condition the GAD-2 MVP summary statistics for anxiety on
the PGC summary statistics for major depressive disorder
(11). There were no new signals and the significance levels of
the lead findings were reduced, but the results on chromo-
somes 3 (SATB1) and 6 (ESR1) remained genome-wide sig-
nificant. Degree of lost variance explained in the anxiety
GWAS when conditioned on major depressive disorder was
tested using LD score regression. Genetic correlation analysis
was performed between the original European American
GAD-2 GWAS summary statistics and the major depressive
disorder conditioned summary statistics, which served as an
internal control to show that the trait measured was still the
same (r
g
=1.0). Heritability dropped significantly (p=0.021)
from 0.0558 (SE=0.0041) in the original GWAS to 0.0429
(SE=0.0038) in the conditioned GWAS.
Fine Mapping
Fine mapping in PAINTOR, version 3, was used to predict
causal SNPs using functional brain annotations (see Figure S6
in the online supplement). In one case (chromosome 6,
rs6557168) the causal SNP identified was the same as the
TABLE 2. Replication in independent cohorts in a study of genetic risk loci for anxiety
a
GAD-2 MVP
European American
Meta-Analysis
b
iPSYCH Anxiety and
Stress-Related
Disorders (9)
UK Biobank,
23andMe, and GPC
Neuroticism (31) ANGST Anxiety (8)
RSID CHR Gene p Risk Allele p Risk Allele p Risk Allele p Risk Allele
rs4603973 3 SATB1-AS1 6.18E–11 G 0.181 G na na 0.0299 G
rs4390955 3 SATB1-AS1 7.78E–11 A 0.851 C 5.20E–04 A 0.2935 A
rs6557168 6 ESR1 1.33E–09 C 0.0128 C 0.367 C 0.170 C
rs12023347 1 LINC01360 /LRRIQ3 8.88E–09 T 6.61E–04 T 6.85E–04 T 0.00296 T
rs56226325 7 MAD1L1 2.01E–08 C 6.41E–04 C 6.59E–08 C 0.354 C
rs6090040 20 TCEA2 3.28E–08 C 0.461 A 0.444 A 0.867 C
a
Italic font indicates same direction of effect, and boldface indicates same direction of effect and nominal significance (p=0.05). The lead SNP on chromosome
3 near the SATB1 locus, rs4603973, was not available for lookup in the UK Biobank neuroticism genome-wide association study, so we used the strongest LD proxy
available (rs4390955, R
2
=0.91, p=7.78E–11). ANGST=Anxiety Neuro Genetics Study; CHR=chromosome; GAD-2=Generalized Anxiety Disorder 2-item scale;
GPC=Genetics of Personality Consortium; iPSYCH=Initiative for Integrative Psychiatric Resear ch; MVP=Million Veteran Program; RSID=reference SNP cluster ID.
b
Results from the present study.
ajp in Advance ajp.psychiatryonline.org 5
LEVEY ET AL.
GWAS lead SNP. On chromosome 20, there were several
genes in the region of our lead SNP, and several genes had
associated eQTLs. The fine mapping analysis prioritized a
likely causal SNP (rs8126001) within the 5:UTR of OPRL1.
DISCUSSION
We present the largest GWAS to date for anxiety traits,
employing a quantitative phenotype, the GAD-2 score, in
nearly 200,000 MVP sub-
jects, as well as self-reported
physician diagnosis of anxi-
ety/panic case-control phe-
notypes in .220,000 MVP
subjects. We identified novel
genetic variants in and
around several genes, some
of which have previously
known functional relation-
ships with anxiety. These
genes play roles in the
hypothalamic-pituitary-adrenal
(HPA) axis, neuronal develop-
ment, and global regulation of
gene expression.
There is high comorbidity
between anxiety, PTSD, and
depression. We used a PRS
derived from the MVP GAD-
2 analysis to identify genetic
overlap with the independent
PGC PTSD and major de-
pressive disorder GWASs
(Table3;seealsoFigureS7in
theonlinesupplement).We
found significant genetic
overlap between these traits,
providing biological evidence
that this known clinical
comorbidity is due at least in
part to shared genetic etiology.
Additionally, we performed
multi-trait-based conditional
and joint analysis, using a prior
GWAS of depression to con-
dition the results of the present
GAD-2 GWAS. In this analysis,
we show not only that the peak
signals for anxiety are reduced
in magnitude (see Figure S3 in
the online supplement) but
also that the overall heritabil-
ityforanxietysymptomsis
diminished, from 5.58% to
4.29%, when conditioned on
genetic liability to depression.
Via linkage disequilibrium score regression, we identified
substantial genetic correlations between anxiety and numerous
other traits (Figure 2). Particularly noteworthy were positive
correlations with depression and neuroticism as well as a
negative correlation with subjective well-being (Figure 2).
These findings replicate similar correlations found using a
case-control approach (9).
The genome-wide significant result in African Americans
is an insertion variant that is rare outside of African ancestry
FIGURE 2. Genetic correlation between Million Veteran Program GAD-2 score in European Americans
and other traits and disorders from LD score regression in LD Hub
a
–0.6 –0.4 –0.2 0 0.2 0.4 0.6 0.8 1
Intelligence 28530673
Age of first birth 27798627
Subjective well-being 27089181
Childhood IQ 23358156
Smoking cessation 20418890
College completion 23722424
Educational attainment 25201988
Years of schooling 2016 27225129
Years of schooling 2013 23722424
Parents’ age at death 27015805
Father’s age at death 27015805
Mother’s age at death 27015805
Parkinson’s disease 19915575
HDL cholesterol 20686565
Age at menopause 26414677
Urate 23263486
Overweight 23563607
Triglycerides 20686565
Coronary artery disease 26343387
Obesity class 2 23563607
Body mass index 20935630
Rheumatoid arthritis 24390342
Obesity class 1 23563607
Waist circumference 25673412
Body fat 26833246
Waist-to-hip ratio 25673412
Fasting insulin main eect 22581228
Obesity 23563607
PGC cross-disorder analysis 23453885
Schizophrenia 25056061
Number of children ever born 27798627
Smoking initiation 20418890
Lung cancer 27488534
Squamous cell lung cancer 27488534
Lung cancer (all) 24880342
ADHD 27663945
Amyotrophic lateral sclerosis 27455348
Lung adenocarcinoma 27488534
Lung cancer (squamous cell) 24880342
Insomnia 27992416
Insomnia 28604731
Major depressive disorder 22472876
Major depressive disorder 29700475
Neuroticism 27089181
Anxiety 26754954
Neuroticism 24828478
Depressive symptoms 27089181
rg
PubMed
Measure, Trait, or Disorder Identifier
a
All plotted traits survive 0.05 false discovery rate. Full results are presented in Table S5 in the online supplement.
GAD-2=Generalized Anxiety Disorder 2-item scale; LD=linkage disequilibrium; PGC=Psychiatric Genomics
Consortium.
6ajp.psychiatryonline.org ajp in Advance
REPRODUCIBLE GENETIC RISK LOCI FOR ANXIETY
and occurs in a genomic region proposed to be under recent
selection in Europeans (36). The lead SNP is at TRPV6, which
encodes a Ca
2+
-selective membrane cation channel associ-
ated with epithelial calcium transport and homeostasis in
kidney and intestine. The lead SNP rs575403075 has an MAF
range of between 0% and 1% in non-African populations and
would fall below MAF quality control thresholds used for
common variants in most non-African populations. In indi-
viduals of African ancestry, this variant is much more com-
mon, with an MAF in our study of 5.8%. This highlights the
importance of studying genetic risks in diverse populations—
otherwise these signals may be missed entirely.
The top genome-wide significant findings for European
Americans in the GAD-2 analysis were in and around SATB1
and the antisense gene SATB1-AS1.SATB1 is a global regu-
lator that influences expression of multiple genes involved
in neuronal development (37). One gene modulated in
expression is Corticotropin Releasing Hormone (CRH),
encoding the protein product of the same name that plays an
essential role in the HPA axis, which has frequently been
shown to modulate stress and fear/anxiety response (38). The
CRHR1 (Corticotropin Releasing Hormone Receptor 1) gene
was genome-wide significant in the gene-based association
analysis (p=3.60310
27
). CRHR1 has been a proposed target
for treatment for anxiety and stress-related disorders, with
evidence for anxiolytic-like effects of CRHR1 antagonists in
animal models although not yet in humans. Based on our
findings, we speculate that individuals with differing genetic
risk that does or does not involve this pathway may differ in
their responses to CRHR1-targeted and other glucocorticoid-
targeted therapeutic agents; this may be a reasonable path-
way to address via personalized medicine, and it presents a
testable hypothesis.
The estrogen receptor ESR1 (also known as estrogen re-
ceptor alpha) has been a focus in animal models of anxiety-
like behaviors, and these have provided mechanistic validity
for the role of ESR1. Studies of estradiol administration to
ovariectomized rats and ESR1 null mice have shown con-
sistent evidence that ESR1 is involved in anxiety-like behavior
(39). Our finding of an association between ESR1 and anxiety
may have implications for our understanding of sex differ-
ences in anxiety disorders and trauma and stressor-related
disorders such as PTSD, which are more common in females
(40). Although this female predominance is partially
explained by sex-specific
exposure to certain kinds of
traumatic events (e.g., do-
mestic violence, sexual as-
sault), there may also be
differential biological con-
text provided in part by the
role of the estrogen receptor.
Our study in a predominantly
male sample identifies ESR1
as genome-wide significant.
Estrogen is important in both
sexes, and a recent review has highlighted the important role
for estrogens in men (41). Studies with larger numbers of
women will be needed to more fully investigate sex differ-
ences in genetic risk for anxiety-related traits.
Previous genetic epidemiology studies have shown that
common genetic factors can underlie anxiety and depressive
traits (42). The lead SNP from the GAD-2 GWAS near the
LINC01360 and LRRIQ3 (rs2180945) loci is nominally sig-
nificant and has the same direction of effect in the 2018 PGC
major depressive disorder analysis (p=1.434310
26
) (11). This
variant may be linked to a common risk factor for both
disorders.
One genome-wide significant signal for GAD-2 was in a
gene-rich region on chromosome 20 near TCEA2,C20orf201,
RGS19, and OPRL1, with fine-mapping analysis prioritizing a
causal region in the latter gene. OPRLI (which encodes the
amygdala nociceptin/orphanin FQ receptor) is involved in
learning and memory and anxiety and fear-related behaviors
(43, 44) and has been hypothesized to play a role in anxiety
and stressor-related disorders such as PTSD (44). In-
terestingly, fear conditioning was also significantly enriched
in the pathway analysis. Taken together, these observations
suggest that OPRL1 and related systems should be further
explored as targets for anxiety and stressor-related thera-
peutics. eQTL data suggest that variants in this region reg-
ulate expression of RGS19 and OPRL1 in the cerebellum and in
the basal ganglia (see Table S7 in the online supplement). The
basal ganglia have long been implicated in obsessive-
compulsive disorder and anxiety disorders. A recent re-
view discussed cerebellum-linked neurocircuitry to anxiety
and fear behaviors in rodents and in humans (45). The cer-
ebellum is thought to play an important role in anticipation/
prediction processes. Given that anxiety has been defined as
“a future-oriented mood state associated with preparation for
possible, upcoming negative events”(4), these results may
provide further evidence for a role for the cerebellum in fear
and anxiety.
MAD1L1 (GAD-2 lead SNP rs56226325, MAF=0.17,
p=2.01310
28
; self-reported physician diagnosis of an anxiety
disorder lead SNP rs10534613, MAF=0.41, p=4.92310
28
)is
replicated in the iPSYCH anxiety GWAS data (9) (Table2;
p=6.85310
24
) and has been associated previously with bi-
polar disorder (46). One of the lead SNPs in the iPSYCH study
is also nominally associated with anxiety in the present study
TABLE 3. Polygenic risk scores generated from the Million Veteran Program genome-wide association
study (GWAS) of GAD-2 anxiety trait and predicting into other relevant GWAS summary statistics
a
Trait (Reference Number) GWAS p Threshold SNPs Tested (N) r
2
p
PGC depression (1) 0.145 89,152 0.002445 2.05E–94
PGC PTSD (35) 0.075 58,854 0.002330 4.23E–12
ANGST anxiety (8) 0.415 83,033 0.004884 3.66E–20
iPSYCH anxiety (9) 0.130 79,974 0.004853 6.68E–36
a
ANGST5Anxiety Neuro Genetics Study; GAD-2=Generalized Anxiety Disorder 2-item scale; iPSYCH=Initiative for
Integrative Psychiatric Research; PGC=Psychiatric Genomics Consortium; PTSD=posttraumatic stress disorder. The
best-fitting polygenic risk score (PRS) is shown for each trait. The first data column contains the threshold used for the
best-fitting PRS. The second column indicates the number of SNPs tested in the best-fitting PRS. The r
2
is the variance
explained by the GAD-2 SNPs at the p value threshold used to create the given PRS.
ajp in Advance ajp.psychiatryonline.org 7
LEVEY ET AL.
(rs11764590, p=3.363
27
); this SNP is in LD with our lead
SNP, rs56226325 (r
2
=0.69). A recent large GWAS of bipolar
disorder identified genome-wide significant SNPs in the
MAD1L1 locus, although their lead signal is not significant in
our study of anxiety (rs4236274, p=0.27) (47). This locus has
also been identified among 108 genome-wide significant loci
by the PGC schizophrenia study (rs58120505, p value=
6.43310
214
) (48), and our lead SNP is nominally significant in
that study (rs56226325, p=1.12310
23
). This SNP is also
nominally significant (6.71310
24
)inthe2018PGCde-
pression GWAS (11). Taken together, these observations
suggest that this locus may be a common risk factor for several
psychiatric disorders.
MAD1L1*rs56226325 is also an eQTL for expression of
FTSJ2 (seeTableS7intheonlinesupplement),andthis
variant is associated with decreased expression in the brain.
MAD1L1 is a mitochondrial RNA methyltransferase that is
important for the proper assembly of the mitochondrial
ribosome and cellular respiration (49). The protein product
of FTSJ2 is Mitochondrial rRNA Methyltransferase 2
(MRM2), which was implicated in a case study of a 7-year-
old Italian boy with a damaging mutation that reduced the
catalytic activity of MRM2, leading to an encephalopathy,
lactic acidosis, and stroke-like (MELAS) syndrome (50).
Larger-effect mutations at this locus can have devastating
effects on the brain; smaller-effect variations may be less
deleterious but still cumulatively influence development,
which may predispose to neurological and psychiatric
disorders.
The Brainstorm Consortium has investigated shared
heritability between psychiatric and neurological disorders
(51). Consistent with their findings, we find very strong ge-
netic correlation between anxiety (GAD-2) and psychiatric
traits such as depression (r
g
=0.81, p=2.48310
253
) and neu-
roticism (r
g
=0.72, p=7.09310
253
) but relatively weaker ge-
netic overlap with neurological disorders. Significant positive
genetic association is detected for amyotrophic lateral scle-
rosis (r
g
=0.39, p=3.00310
24
), and negative genetic associa-
tion with Parkinson’s disease (r
g
=20.19, p=4.70310
23
), but
no genetic overlap is seen for Alzheimer’s disease (r
g
=0.00,
p=1.00). Further work will be needed to better discern the
implications of these findings for understanding shared and
disparate disease mechanisms among these neuropsychiatric
conditions (11, 35, 51).
Limitations of this work include the fact that phenotypes
were based on self-reported survey data. The GAD-2 asks
questions that temporally reference the “past 2 weeks.”Al-
though the GAD-2 has demonstrated high sensitivity and
specificity for anxiety disorders (52), it falls short of the
desired trait (lifetime) anxiety measure. That our work re-
produces (and is reproduced by) other independent groups
who did use lifetime anxiety measures (8, 9) further supports
the utility of the GAD-2 in capturing a genetically meaningful
anxiety trait. Similarly, the question about diagnosis for
anxiety or panic that yielded our binary self-reported phy-
sician diagnosis of anxiety or panic disorder phenotype relies
on self-report. A further limitation is that MVP has pre-
dominantly male participants (92.5%). While women are
included in this analysis, clinically important interactions
between sex, phenotype, and genotype could not be
addressed. This cohort is growing, and future recruitment
will provide additional power to revisit sex-stratified analyses
of this sample. Males presumably have a higher genetic lia-
bility threshold for anxiety, as evidenced by lower rates of
anxiety disorders. Accordingly, affected males could reflect
higher genetic risk than females (because they must pass a
higher threshold to be affected), which would result in
greater power to detect risk loci in a mostly male compared
with a mostly female sample.
In summary, we have identified novel variants for anxiety
by performing GWASs in the large MVP cohort. We repli-
cated results in our GWASs for top findings from recent
anxiety and other relevant anxiety-related GWASs. We also
identified significant genetic overlap with major depressive
disorder, PTSD, and neuroticism using polygenic risk scores
and LD score regression. This work provides additional ge-
netic evidence for the overlap between disorders that are
frequently comorbid with anxiety and presents new mo-
lecular targets for investigation with a longer view toward the
development of new treatments.
AUTHOR AND ARTICLE INFORMATION
Division of Human Genetics, Department of Psychiatry, Yale University
School of Medicine, New Haven, Conn., and Department of Psychiatry,
Veterans Affairs Connecticut Healthcare Center, West Haven, Conn.
(Levey, Gelernter, Polimanti, Zhou, Cheng); VA Clinical Epidemiology
Research Center, VA Connecticut Healthcare System, West Haven, Conn.
(Aslan, Concato, Radhakrishnan); Department of Medicine, Yale University
School of Medicine, New Haven, Conn. (Aslan, Concato); Massachusetts
Veterans Epidemiology Research and Information Center, VA Boston
Healthcare System, Boston (Quaden); College of Medicine, University of
Kentucky, Lexington (Radhakrishnan); Department of Medical Epidemi-
ology and Biostatistics, Karolinska Institutet, Stockholm (Bryois, Sullivan);
Departments of Genetics and Psychiatry, University of North Carolina,
Chapel Hill (Sullivan); Psychiatry Service, VA San Diego Healthcare System,
San Diego, and Departments of Psychiatry and of Family Medicine and
Public Health, University of California San Diego, La Jolla (Stein).
Send correspondence to Dr. Gelernter (joel.gelernter@yale.edu) and
Dr. Stein (mstein@health.ucsd.edu).
Drs. Levey, Gelernter, and Stein contributed equally to this work.
Supported by funding from the Veterans Affairs Office of Research and
Development Million Veteran Program grant MVP011 and VA Cooperative
Studies Program CSP575B.
The authors thank the veterans who participate in the Million Veteran
Program.
Dr. Sullivan has served on an advisory committee and received grant
support from Lundbeck and served on a scientific advisory board for Pfizer.
Dr. Gelernter is named as co-inventor on PCT patent application 15/
878,640 (genotype-guided dosing of opioid agonists), filed January 24,
2018. Dr. Stein has served as a consultant for Aptinyx, Bionomics, Janssen,
Jazz Pharmaceuticals, Neurocrine, Pfizer, and Oxeia Biopharmaceuticals.
The other authors report no financial relationships with commercial
interests.
Received March 8, 2019; revisions received August 9 and October 14,
2019; accepted October 15, 2019.
8ajp.psychiatryonline.org ajp in Advance
REPRODUCIBLE GENETIC RISK LOCI FOR ANXIETY
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