Archival Report Biological

Psychiatry

An Ultraconserved Brain-Specific Enhancer

Within ADGRL3 (LPHN3) Underpins Attention-
Deficit/Hyperactivity Disorder Susceptibility
Ariel F. Martinez, Yu Abe, Sungkook Hong, Kevin Molyneux, David Yarnell, Heiko Löhr,
Wolfgang Driever, Maria T. Acosta, Mauricio Arcos-Burgos, and Maximilian Muenke

ABSTRACT
BACKGROUND: Genetic factors predispose individuals to attention-deficit/hyperactivity disorder (ADHD). Previous
studies have reported linkage and association to ADHD of gene variants within ADGRL3. In this study, we
functionally analyzed noncoding variants in this gene as likely pathological contributors.
METHODS: In silico, in vitro, and in vivo approaches were used to identify and characterize evolutionary conserved
elements within the ADGRL3 linkage region ( 207 Kb). Family-based genetic analyses of 838 individuals (372
affected and 466 unaffected patients) identified ADHD-associated single nucleotide polymorphisms harbored in
some of these conserved elements. Luciferase assays and zebrafish green fluorescent protein transgenesis tested
conserved elements for transcriptional enhancer activity. Electromobility shift assays were used to verify transcription
factor–binding disruption by ADHD risk alleles.
RESULTS: An ultraconserved element was discovered (evolutionary conserved region 47) that functions as a transcriptional
enhancer. A three-variant ADHD risk haplotype in evolutionary conserved region 47, formed by rs17226398, rs56038622, and
rs2271338, reduced enhancer activity by 40% in neuroblastoma and astrocytoma cells (pBonferroni , .0001). This enhancer
also drove green fluorescent protein expression in the zebrafish brain in a tissue-specific manner, sharing aspects of
endogenous ADGRL3 expression. The rs2271338 risk allele disrupts binding of YY1 transcription factor, an important factor
in the development and function of the central nervous system. Expression quantitative trait loci analysis of postmortem
human brain tissues revealed an association between rs2271338 and reduced ADGRL3 expression in the thalamus.
CONCLUSIONS: These results uncover the first functional evidence of common noncoding variants with potential
implications for the pathology of ADHD.
Keywords: ADGRL3, ADHD, Cis-acting regulatory element, Enhancer, Evolutionary conserved regions, Genetics,
Latrophilin, LPHN3, Zebrafish
http://dx.doi.org/10.1016/j.biopsych.2016.06.026

Attention-deficit/hyperactivity disorder (ADHD) is a complex
heritable trait that affects more children and adolescents than
any other psychiatric disorder. Approximately 5.3% of the
world’s population is estimated to be affected (1,2). ADHD
increases the risk of disruptive symptoms, substance use,
legal problems, and underemployment, which reduces the
quality of life of ADHD sufferers and their families (3–6). ADHD
heritability has been estimated at 76%, suggesting a strong
genetic component (7). Candidate gene and genome-wide
studies of single nucleotide polymorphism (SNP) and copy
number variants have identified a number of ADHD suscept-
ibility loci (8), but few molecular studies have attempted to
elucidate the functional effects of risk variants.
Common genetic variants within the adhesion G protein–
coupled receptor L3 gene (ADGRL3, also known as latrophilin
3 or LPHN3) in 4q13.2 are strongly linked and associated with
ADHD (9–19). The ADHD-linked region in ADGRL3 (hereafter
referred to as the minimal critical region [MCR]) spans exons 4
through 19 of the gene ( 207 Kb). Searching for variants that
might affect ADGRL3 protein function, Domene et al. (20)
sequenced the entire coding region of the gene in a small
cohort of subjects with ADHD, but no missense coding
changes or canonical splice site alterations were found to be
associated with the disorder. This suggests that intronic
noncoding variants are the likely pathological contributors.
In the present study, we interrogated the ADGRL3 MCR,
aiming to identify transcriptional enhancer elements with
potential functional implications for ADHD.
METHODS AND MATERIALS
Subjects
Individuals with and without ADHD were ascertained from
the metropolitan area of Medellin (Antioquia, Colombia). The
Paisa community is considered a genetic isolate of Caucasian

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Published by Elsevier Inc on behalf of Society of Biological Psychiatry. 1

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Biological
Psychiatry
descent with low admixture with Amerindian and Negroid
ethnicities (21). The cohort consisted of 14 multigenerational
families and 125 nuclear families for a total of 838 individuals
(372 affected and 466 unaffected individuals; 335 children and
adolescents [3–16 years of age] and 503 adults [$17 years of
age]). The multigenerational families had an average size of 28
members (range, 9–57 family members) and an average of
2.93 generations (range, 2–4 generations). Full details of the
clinical, demographic, and genetic ascertainment features and
the methodology of neuropsychological evaluation have been
published elsewhere (22–24). The study was reviewed and
approved by the Institutional Review Board of the National
Human Genome Research Institute (Protocol 00-HG-0058)
and the University of Antioquia Ethics Committee (Protocol
11-13-342).
Genetic Statistical Analyses
SNP genotyping methods are presented in Supplement 1.
Genotype data were imported into Golden Helix SVS software
(version 8.3.1; Golden Helix, Bozeman, MT) for family-based
association testing. Genotype and allelic frequencies were
estimated by maximum likelihood. Family-based association
testing analyses using ADHD status as a categorical variable
were applied to the entire set of markers that passed quality
control. We also performed family-based haplotype analyses
to compare with the results at the marker level. Comorbid
conditions (e.g., substance use and disruptive symptoms) and
neuropsychological endophenotypes (e.g., Wechsler Intelli-
gence Scale for Children [WISC] block design, WISC perform-
ance intelligence quotient [PIQ], WISC full-scale intelligence
quotient [FSIQ], “A”-cancellation and vigilance test correct
responses [ACVTCR] and omissions [ACVTO], and the Rey–
Osterrieth Complex Figure Test [ROCFT copy]) were used as
ADHD-interacting variables. We used endophenotype data
only for 255 children and adolescents (170 affected and 85
unaffected individuals) with an FSIQ $ 81 and regular school
grades adequate for their age in order to exclude participants
with potential learning disabilities.
Because the ADGRL3 genomic region under study is
known to be linked to ADHD (11), the null hypothesis of
linkage and no association was tested. Individual genotypes
inconsistent with Mendelian transmission were excluded from
the analyses. All markers were tested for deviations from the
Hardy-Weinberg equilibrium. Allelic tests of association were
applied using a dominant model of inheritance.
Identification of Evolutionary Conserved Elements
Within the ADGRL3 MCR and Prediction of
Regulatory Function
We used the Evolutionary Conserved Region (ECR) Browser
(25) to identify highly conserved elements within the
ADGRL3 MCR (accession no. NG_033950.2, GRCh37.p13/
hg19 assembly; coordinates, chr4:62,688,419-62,895,842).
We looked at DNA sequence conservation across several
vertebrate species, including human, mouse, chicken, and
zebrafish, using a sliding window of .150 base pairs long and
.70% identity. To predict transcriptional regulatory function,
we gathered annotation data from three regulation tracks of
the University of California, Santa Cruz (UCSC) table browser:
2 Biological Psychiatry ]]], 2016; ]:]]]–]]] www.sobp.org/journal
Brain Enhancer Associated With ADHD
chromatin state segmentation by a hidden Markov model
(Broad ChromHMM) (26,27), EP300 transcription factor (TF)
ChIP-seq (28), and uniform DNAse I hypersensitivity sites (29).
Additional information on these annotation tracks can be
found in Supplement 1.
Luciferase Assays
The generation of ECR-luciferase constructs, cell culture
conditions, and transient transfections are described in
Supplement 1. After 24 hours of transfection, culture super-
natants containing secreted luciferases were collected. Luci-
ferase assays were performed using the Pierce Luciferase
Flash Assay kits (Thermo Fisher Scientific, Waltham, MA).
Each ECR-luciferase construct was assayed in triplicate, and
each transfection experiment was repeated at least three
times (n 5 9). Luciferase activity data were analyzed using a
one-way analysis of variance with Bonferroni correction, as
implemented in Prism 6 software (GraphPad Software, La
Jolla, CA).
Electromobility Shift Assays
Electromobility shift assays were performed using standard
procedures. ECR47 DNA probes were incubated with Myc-
DDK-tagged recombinant TFs or whole-cell expression lysates
(Origene, Rockville, MD). For supershift reactions, an anti-DDK
(FLAG) monoclonal antibody (Origene) was added.
Zebrafish Bioassays
Zebrafish stocks and manipulations followed the animal care
and use protocols used in our Zebrafish Core facility (National
Institutes of Health/National Human Genome Research Insti-
tute). Zebrafish transgenesis and in situ hybridization were
performed following standard procedures (see Supplement 1
for details).
RESULTS
Identification of Potential Enhancer Elements Within
ADGRL3
Using the ECR Browser, we identified highly conserved
elements harbored in the ADGRL3 MCR. Although it is
currently well established that the intergenome comparisons
of distant species (e.g., humans and fish) are powerful in
identifying critical distant regulatory elements, only 5% of the
genes in the human genome contain a human/fugu noncoding
ECR in their genomic neighborhood (30–32). For that reason,
an analysis with species more closely related to humans than
fish is required to identify regulatory elements for many human
genes. We therefore used the human/chicken alignment to
select candidate sequences. The human/opossum alignment
(an evolutionarily closer species) was arbitrarily used to give an
identity to the conserved elements, thereby naming 51 regions
ECR1 through ECR51 (Supplemental Figure S1 in Supplement
1 and Supplemental Table S1 in Supplement 2).
Given that enhancer activity has been correlated with
certain properties of chromatin (33), we gathered p300 binding
site, histone mark, and DNase I hypersensitivity site annotation
data to predict ECR enhancer function (Supplemental Table S1
 Biological
Brain Enhancer Associated With ADHD Psychiatry

Table 1. Association Between Evolutionary Conserved Region Common Variants and Attention-Deficit/Hyperactivity Disorder, Disruptive Symptoms, Substance

ACVTCR, “A”-cancellation and vigilance test correct responses; ACVTO, “A”-cancellation and vigilance test omissions; ADHD, attention-deficit/hyperactivity disorder; CD, conduct
disorder; ECR, evolutionary conserved region; FDR, Benjamini–Hochberg false discovery rate; NS, not significant; ODD, oppositional defiant disorder; ROCFT, Rey–Osterrieth complex figure
.00261 Coding-syn
Coding-syn

.04088 Coding-syn
Function
in Supplement 2). From the list of elements conserved in

.00228 Intronic
.02775 Intronic
.02775 Intronic
.02775 Intronic

Intronic
.00928 Intronic
.02775 Intronic

Intronic
chicken, we excluded those ECRs not predicted to be func-
tional by any of the annotation tracks above and that did not
contain SNPs with a minor allele frequency . 1%, because

Nicotine p Value Alcohol p Value ACVTCR p Value ACVTO p Value ROCFT p Value
FDR
genetic studies predominantly suggest that ADHD risk is

NS

NS
NS
primarily explained by common variants (1,7,8). Following this
approach, the number of candidate ECRs was reduced to 10

.00017 .00015

.00186

.00006 .000004 .00006 .00035

.00098 .01295
.00098 .01295
.00098 .01295
.00098 .01295

.00013 .02180
Raw

NS

NS
NS
(i.e., ECR1, ECR2, ECR4, ECR9, ECR20, ECR22, ECR26,
ECR37, ECR46, and ECR47). Because coding regions can
overlap enhancer sequences (34,35), we did not exclude those

.00373
.00913
FDR

NS
NS
elements containing ADGRL3 exons (i.e., ECR20, ECR26,
ECR37, and ECR47).

.00003
.00046
.00046
.00046
.00046

.00199

.00002
.00548
Raw

NS
NS
Genetic Statistical Analyses of ECR Variants

.00016
.00098
.00098
.00098
.00098

.00376

.00013
.00920
FDR
Supplemental Table S2 in Supplement 1 shows a list of the

NS
NS
common variants harbored in ECR sequences that are pre-
dicted to be functional. We performed family-based associa-

.000004
.00003
.00046
.00046
.00046
.00046

.00201

.00002
.00552
Raw
tion tests in 838 individuals from the same Colombian

NS
NS
multigenerational and nuclear families that previously showed
linkage and association of ADHD to 4q13.2 (11,22). Genotype

.00199
.00199
.00199

.000001 .00001
.00199

.00255

.03866
.00204
FDR
proportions did not significantly depart from the Hardy-

NS

NS
NS
Weinberg equilibrium. SNPs in 6 of the 10 ECRs showed
significant association with ADHD, comorbid disorders, or
.00066
.00066
.00066

.03092
.00066

.00119

.02062

.04748
.00082
Raw

NS
endophenotypes after correction by false discovery rate
(FDR) (Table 1). ECR46, ECR47, and ECR4 showed the highest
association with ADHD (all five variants, pFDR 5 .00219),
.00203
.00203
.00203
.00203

.04241

.04241
.04241
.04241
FDR

NS

NS
NS
followed by ECR26 (both pFDR 5 .00342), ECR2 (variant
rs1868790, pFDR 5 .00988), and ECR37 (variant rs1397548,
pFDR 5 .03173). Interestingly, only variants in ECR46
.00054
.00054
.00054
.00054
.03740
.02262

.02062
.02191
.02223

.04169
Raw

NS
(rs11131352) and ECR47 (rs17226398, rs56038622, and
rs2271338) consistently showed association with disruptive
.00002 .00005 .00020 .00076
.00002 .00005 .00020 .00076
.00002 .00005 .00020 .00076
.00002 .00005 .00020 .00076

.02291 .03819 .01442 .04327

symptoms (i.e., oppositional defiant disorder [all pFDR 5
FDR
CD p Value

NS
NS

NS
NS
NS

NS
.00005] and conduct disorder [all pFDR 5 .00076]; substance
use of alcohol [all pFDR 5 .00199] and nicotine [all pFDR 5
Use, and Neuropsychological Endophenotypes in the Paisa Dataset

.03274
.00155 .00342 .000003 .00003 .02510
.00160 .00342 .000003 .00003 .02641

.01799 .03372 .02892

Raw

NS
NS

.00203]; and endophenotypes ACVTCR [all pFDR 5 .00098],

ACVTO [all pFDR 5 .00098], and ROCFT [all pFDR 5 .02775]).
.00272 .00582
FDR

Variants in ECR1, ECR9, and ECR22 did not show associa-

ODD p Value

NS

NS

tions with ADHD, comorbid conditions, or endophenotypes

and therefore were not considered for functional analysis.
.03921
Raw

NS

None of the variants showed association with WISC block

design, WISC PIQ, or WISC FSIQ. Haplotype analyses for
ADHD affection status are presented in Supplemental Table
ADHD p Value

.00073 .00219
.00073 .00219
.00073 .00219
.00073 .00219
.00063 .00219

.00527 .00988

.01904 .03173
FDR

NS

NS

S3 in Supplement 1.
Given the identical p values for some single-variant
Raw

associations and the physical proximity of variants, we

NS

NS

calculated the linkage disequilibrium (LD) between SNPs. We

used phased genotype data from the 1000 Genomes Project
Allele (Frequency)

for the Northern Europeans from Utah population. For our

G (0.278)

G (0.064)

G (0.059)
C (0.132)

A (0.132)

A (0.427)

A (0.308)
T (0.132)

T (0.132)

T (0.313)
T (0.281)

dataset, we performed an LD pairwise analysis using Golden

Helix SVS software. Variants rs2305339 and rs734644 in
ECR26 were in complete LD (r2 5 .99, D′ 5 1.00, haplotypes
AC/GT [protective/risk]) as were variants rs11131352
test; syn, synonymous.

(i.e., ECR46), rs17226398, rs56038622, and rs2271338

rs11131352
rs17226398
rs56038622

rs10021694

rs73823249
rs2271338

rs2305339
rs1868790

rs1397548
rs1397547
rs734644

(i.e., ECR47) (r2 5 1.00, D′ 5 1.00, haplotypes AGAG/TCTA

Variant

[protective/risk]) (Supplemental Table S4 in Supplement 1).

For initial evaluation in this study, risk alleles in complete
LD were tested in luciferase assays as a haplotype rather
ECR ID
ECR46
ECR47

ECR26

ECR37
ECR4

ECR2

than individually and were compared to the protective

haplotype.

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Biological
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ECR46 and ECR47 appear as two independent conserved
elements in chickens, but they are part of a single “core” ECR
($350 base pairs long, $77% identity) (31) in species that are
evolutionarily closer to humans, such as opossum, mouse,
and chimpanzee. For that reason, these two sequences were
evaluated in luciferase assays independently and together.
ECR47 Functions as a Brain-Specific Enhancer
We tested the ADHD-associated ECRs for enhancer activity using
a dual secreted luciferase reporter assay in four different cell lines
(Figure 1). We compared luciferase activity of ECRs containing the
protective versus the risk allele/haplotype. As described above,
ECR26 and ECR47 contain markers in complete LD; we therefore
compared haplotypes rather than pairwise combinations of alleles.
ECR37 and ECR47 were the only elements to stimulate luciferase
expression. ECR37 showed weak enhancer activity in all cell lines;
however, its ADHD-associated variant rs1397548 did not affect
enhancer function. ECR47 also showed weak enhancer activity,
but only in B35 neuroblastoma and U87 astrocytoma cells,
suggesting tissue-specific activity. Unlike ECR37, the ECR47 risk

Figure 1. Secreted luciferase assays testing attention-deficit/hyperactivity disorder–associated evolutionary conserved region (ECR) sequences for
enhancer activity. Four different cell lines were transfected with the ECR-luciferase constructs for 24 hours. Luciferase activity was normalized against a
constitutive Gaussia luciferase plasmid and expressed as relative luciferase activity (Cypridina/Gaussia ratio). Results are presented as the stimulation of
luciferase activity above the basal, enhancer-less vector containing only the minimal promoter. Letters in parentheses indicate the alleles or haplotypes being
tested. ECR37 and ECR47 showed weak enhancer activity compared to the strong control enhancers Simian vacuolating virus 40 (SV40) and human Bicore1.
ECR47 risk haplotype CTA decreased enhancer activity in B35 neuroblastoma and U87 astrocytoma cells (pBonferroni , .0001). Statistical differences were
defined using one-way analysis of variance with correction for multiple comparisons. CHO-K1, Chinese hamster ovary K1 cells; P19, mouse embryonic
carcinoma cells.

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 Biological
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haplotype (CTA) reduced luciferase activity by approximately 40% like transcription factor 1 (GRHL1); PAX2; PAX3; YY1 tran-
in both cells lines (pBonferroni , .0001). ECR2, ECR4, ECR26, and scription factor; hypoxia responsive elements; Tax-1/cyclic
ECR46 showed no stimulation of luciferase activity in any of the AMP-responsive element-binding protein 1 (CREB) complex;
cell lines. The activity of core element ECR46/47 was similar to LIM homeobox 3; POU domain, class 3, transcription factor 2
that of ECR47 alone (Supplemental Figure S2 in Supplement 1), (POU3F2); POU domain, class 4, transcription factor 3
indicating that regulatory activity resides on the ECR47 moiety. (POU4F3); POU domain, class 6, transcription factor 2
Subsequently, we evaluated the ability of the human ECR47 (POU6F2); pituitary-specific positive transcription factor 1
sequence to drive green fluorescent protein (GFP) reporter (PIT1); and cone–rod homeobox protein (CRX) (Table 2).
expression in stable transgenic zebrafish lines. A clear GFP signal
was detected in the zebrafish brain that was restricted to the YY1 Binding to ECR47 Is Disrupted by the rs2271338
ventral forebrain, the midbrain, and the hindbrain of embryos 28 to ADHD Risk Allele
30 hours postfertilization (Figure 2A). In contrast with the weak
The binding sites of three important neurodevelopmental TFs
enhancer activity detected in luciferase assays, a strong GFP
were predicted to be disrupted by ECR47 risk allele substitu-
signal was observed in zebrafish. This may be explained by the
tions. PAX2 and YY1 were predicted to be disrupted by
fact that multicopy integration events can occur during trans-
rs2271338 G.A and GRHL1 was predicted to be disrupted
genesis or that ECR47 may function as a developmental
by rs17226398 G.C (Table 2). While GRHL1 and PAX2 did not
enhancer, therefore behaving differently during embryogenesis
bind to their predicted sites (Figure 3B, C), YY1 produced a
compared to differentiated cells in culture. Interestingly, the
clear gel shift when added to a 27–base pair fragment contain-
ECR47-driven GFP expression pattern shared specific aspects
ing the rs2271338 protective allele. YY1 binding was abrogated
of endogenous adgrl3.1 expression in forebrain, midbrain, and
by addition of excess unlabeled protective sequence, but not
hindbrain, but not in telencephalon and retina, as evaluated by
by the sequence containing the risk allele (Figure 3A).
in situ hybridization (Figure 2B, C). Because the zebrafish
We next examined whether YY1 affected endogenous
enhancer detection vector system is not robust enough to allow
ADGRL3 expression. Real-time polymerase chain reaction analy-
for quantitative measurement of enhancer activity in vivo, we did
ses revealed a strong expression of endogenous ADGRL3
not investigate the effect of the ECR47 risk haplotype (CTA) on
messenger RNA (mRNA) in SH-SY5Y neuroblastoma but not in
enhancer function in the zebrafish. A number of variables make it
U87 astrocytoma cells (Supplemental Figure S4A in Supplement 1),
challenging to compare ECR47 risk and protective haplotypes
suggesting that expression of this gene may be specific to the
quantitatively using the zebrafish enhancer detection system,
neuronal lineage in the CNS. After YY1 small interfering RNA
including 1) different copy number integration during zebrafish
transfection, we could not detect any significant changes in
transgenesis; 2) the structural complexity of transgene integration
ADGRL3 mRNA expression in these cell lines (Supplemental
loci in the genome (heterochromatin vs. euchromatin); 3) DNA
Figure S4B in Supplement 1). This result might suggest that
methylation effects; and 4) zebrafish tissue autofluorescence and
the ECR47–YY1 pair is functional only during development or
nonspecific, background GFP expression.
that its spatiotemporal regulation of ADGRL3 expression is
It is important to highlight that ECR47 is also conserved in
complex and cannot be modeled properly outside the native
zebrafish (Supplemental Figure S3 in Supplement 1), which
regulatory circuitry of the brain.
makes it an ultraconserved element that is likely to have an
important biological function.
rs2271338 Is Associated With Decreased ADGRL3
mRNA Expression in the Thalamus
TFs Preferentially Associated With Brain Function
Are Overrepresented in ECR47 Unlike other behavioral disorders, such as schizophrenia and
major depressive disorder, brain tissue from ADHD patients is
Analysis of the TF–binding profiles of ECR47 and 144 in vivo
not readily available. However, SNPs associated with complex
tested brain enhancers from the VISTA Enhancer Browser (36)
diseases are likely to function as expression quantitative trait
(Supplemental Table S5 in Supplement 3) revealed a signifi-
loci, and the tissues of unaffected individuals can be used for
cant overrepresentation of developmental TF families prefer-
gene expression association analyses. Expression quantitative
entially associated with brain tissue, such as distal-less
trait loci analysis of brain tissue from 137 neuropathologically
homeodomain (V$DLXF), NK6 homeobox (V$NKX6), paired
confirmed controls (16–102 years of age) revealed a significant
box (PAX) homeodomain (V$PAXH), and Brn-5 POU (V$BRN5)
association between the rs2271338 AA risk genotype and
TFs, as suggested by the high Z scores (Supplemental Table
reduced ADGRL3 expression in the thalamus (p , .01)
S6 in Supplement 1). Homeobox TFs (V$HBOX), Brn POU
(Supplemental Figure S5 and Supplemental Table S7 in
domain (V$BRNF), cocaine- and amphetamine-regulated tran-
Supplement 1). rs2271338 was either absent or not associated
script 1 (V$CART), and Lim homeodomain (V$LHXF) factors
with ADGRL3 expression in the National Institutes of Health
were also overrepresented, but while they may participate in
Common Fund’s Genotype-Tissue Expression (GTEx), Colum-
neural development and function, they are not preferentially
bia University’s SNPExpress, and the National Heart, Lung,
associated with the central nervous system (CNS).
and Blood Institute’s GRASP databases (Supplement 1).
The potential effects of ECR47 SNP allele substitutions
on TF binding were examined using SNPInspector software
(Genomatix, Munich, Germany). All three ADHD risk alleles DISCUSSION
(CTA) were predicted to produce loss or gain of binding sites Few molecular studies have attempted to explain the molec-
for important neurodevelopmental TFs, such as grainyhead- ular effects of ADHD-associated genetic variants. Studies on

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Biological
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Figure 2. In vivo enhancer testing and correlation with adgrl3.1 expression in the zebrafish. Evolutionary conserved region 47–driven green fluorescent
protein (GFP) expression was monitored in the brain of transgenic embryos at 28 to 30 hours postfertilization (hpf). (A) Stable transgenic F2 embryo showing
GFP expression restricted to the central nervous system (i.e., the forebrain, midbrain, and hindbrain). (B, C) Expression of adgrl3.1 was detected by in situ
hybridization of embryos at (B) 36 hpf and (C) 48 hpf (top row, left midsagittal and right retina in focus). Evolutionary conserved region 47–driven GFP
expression represents several specific aspects of endogenous adgrl3.1, consistent with the location of neuronal tissue in the developing brain. Endogenous
adgrl3.1 messenger RNA expression is detected in the telencephalon, the ventral forebrain, the midbrain, the hindbrain, and the retina throughout the
analyzed developmental stages. The anterior is to the left in all images, and the dorsal side is up in all lateral views [scale bar 5 100 mm; all images in part (C)
are in the same scale]. FB, forebrain; HB, hindbrain; MB, midbrain; MHB, midbrain–hindbrain boundary; r, rostral; v, ventral.

dopamine transporter (DAT1) (37–40), tryptophan hydroxylase moderate and large effects, but these findings fail to explain
2 (TPH2) (41,42), and T-cadherin (CDH13) (43) have examined the higher incidence of ADHD and larger phenotypic variance
the functional properties of rare missense mutations of observed in populations. Instead, the common disease/

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Table 2. Transcription Factor–Binding Sites Predicted to be Affected by the Attention-Deficit/Hyperactivity Disorder–

Associated Haplotype in Evolutionary Conserved Region 47
Major Minor Allele Predicted TF Family/ Optimized Core Matrix
SNP rsID Allele Allele Change Effect Matrix TF Name Thresholda Strand Similarityb Similarityc
rs17226398 G C G.C Gained V$PAX3/ Pax-3 paired domain protein 0.85 1 1 0.893
PAX3.02
Gained V$HIFF/ Hypoxia-responsive element 0.97 – 1 0.978
HRE.02
Lost V$GRHL/ Grainyhead-like 1 0.86 1 1 0.864
GRHL1.01
Lost V$CP2F/ Transcription factor CP2-like 1 0.87 1 0.815 0.894
TCFCP2L1.01
rs56038622 A T A.T Gained V$CREB/ Tax/CREB complex 0.71 1 0.75 0.739
TAXCREB.02
rs2271338 G A G.A Gained V$LHXF/ LIM-homeodomain 3 0.82 1 1 0.84
LHX3.02
Gained V$BRNF/ Brn-3, POU-IV protein class 0.78 – 0.75 0.815
BRN3.01
Gained V$BRN5/ Retina-derived POU-domain 0.76 1 0.81 0.779
POU6F2.01 factor 1, dimeric
Gained V$BRNF/ POU class 3 homeobox 2 0.82 1 1 0.827
BRN2.04 (POU3F2)
Gained V$OCT1/ Octamer-binding protein 1 0.85 1 0.767 0.854
OCT1.03 (POU2F1)
Gained V$PIT1/ Pituitary transcription factor 1 0.81 1 1 0.821
PIT1.02 (POU1F1)
Gained V$HNF6/ One CUT-homeodomain 0.82 1 1 0.876
OC2.01 protein
Lost V$PAX2/ Zebrafish PAX2 paired domain 0.78 1 0.789 0.784
PAX2.01 protein
Lost V$YY1F/ Yin and Yang 1 repressor 0.94 – 1 0.979
YY1.02
Lost V$BCDF/ Cone–rod homeobox protein 0.94 1 1 0.946
CRX.01
Searches were performed using the SNPInspector function within the Genomatix Software Suite (Genomatix, Munich, Germany). The analyses
are based on the MatInspector and Genomatix libraries of matrix descriptions for transcription factor–binding sites (MatBase).
SNP, single nucleotide polymorphism; TF, transcription factor.
a
At the matrix similarity and optimized threshold, a minimum number of matches is found in nonregulatory test sequences (i.e., with this matrix
similarity, the number of false positive matches is minimized).
b
Core similarity refers to the degree of matching to the “core sequence” of a matrix (i.e., the highest conserved positions of the matrix, usually 4
bases). Maximum core similarity (1.0) is reached when the highest conserved bases of a matrix match exactly in the sequence.
c
Matrix similarity refers to the degree of matching to each sequence position in the matrix. A perfect match to the highest conserved nucleotide
at each position gets a score of 1.00. A “good” match usually has a similarity of . 0.80. Mismatches in highly conserved positions of the matrix
decrease the matrix similarity more than mismatches in less conserved regions.

common variant hypothesis is better supported by a sub-
stantial number of genetic epidemiological studies, with
common variants accounting for approximately 40% of ADHD
heritability (7,8).
ADGRL3 is a strong ADHD candidate gene. ADGRL3
common variants predispose individuals to ADHD, modulate
brain metabolism, and predict ADHD severity and comorbidity
with disruptive symptoms and substance use disorder
(11,13,15,16). When combined with other risk factors, ADGRL3
risk variants improve the prediction of ADHD severity, dysfunc-
tional comorbidity, long-term outcome, and response to treat-
ment with stimulant medication (10–16). Recent work from our
group also showed the genetic linkage and association of
ADGRL3 variants to neuropsychological endophenotypes, pro-
viding a more powerful framework for ADHD clinical classifica-
tion and for the identification of causative genetic variation (44).
ADGRL3 encodes a member of the latrophilin subfamily of
adhesion G protein–coupled receptors that is highly expressed
in brain regions implicated in dopaminergic systems (11,45).
ADGRL3 endogenous ligand has been identified as fibronectin
leucine rich transmembrane protein 3, a postsynaptic mem-
brane protein involved in axon guidance and neuronal cell
migration during embryonic development (46). More impor-
tantly, the ADGRL3–fibronectin leucine rich transmembrane
protein 3 synaptic pair regulates excitatory transmission both
in vitro and in vivo (47). Animal models also support ADGRL3
implication in ADHD pathophysiology (48,49).
Using a combination of evolutionary sequence conservation
and regulatory annotation data, we identified candidate
sequences within the ADGRL3 MCR with potential regulatory
function. Several variants revealed significant association with
ADHD, comorbid disorders, or neuropsychological endophe-
notypes, with a four-marker haplotype in the ECR46/ECR47
core element showing the highest level of association across
the board. This result may suggest that ECR46/47 participates
in a common neurobiological pathway to ADHD. Given the

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Biological
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Figure 3. YY1 transcription factor binding to evolutionary conserved region 47 (ECR47) enhancer is disrupted by the rs2271338 risk allele. (A) A biotin-
labeled DNA fragment containing a rs2271338 attention-deficit/hyperactivity disorder protective allele was incubated with a human embryonic kidney 293
(HEK293) whole cell lysate (WCL) expressing DDK (FLAG)-tagged human YY1 (lanes 2–5 from left to right). A mobility shift in lane 2 indicates protein binding to
the probe, which was abrogated by incubation with molar excess of the unlabeled fragment (lane 3), but not of an unlabeled fragment containing the
rs2271338 risk allele (lane 4). Higher molecular weight shift with the addition of anti-DDK antibody identifies YY1 as the binding factor (lane 5). Lanes 6 to 10
correspond to a known YY1 binding sequence used as positive control (87). Lanes 7 to 10 show protein binding to the control probe, with the anti-DDK
antibody producing a similar supershift, thereby confirming YY1 identity (lane 8). Molar excess of the unlabeled ECR47-protective fragment was capable of
reducing protein binding (lane 9), but the risk fragment was not (lane 10). No binding was detected for grainyhead-like transcription factor 1 (GRHL1) (B) or
paired box 2 (PAX2) (C) transcription factors. (B) Lanes 1 to 5, biotin-labeled fragment containing the rs17226398 protective allele; lanes 6 to 10, GRHL1-
positive control sequence (88). Interestingly, no shift was observed in the positive control except when the antibody was added (lane 8). Apparently, the
addition of the antibody stabilizes the GRHL1–DNA interaction, which otherwise is labile under the experimental conditions used. (C) Left panel, lanes 1 to 5,
same DNA fragment used for YY1 in (A); lanes 6 to 10, PAX2-positive control sequence (89). Neither the protective nor the risk ECR47 fragments affected
PAX2 binding to the positive control probe (lanes 9 and 10). The right panel shows the results for a longer ECR47 probe (Protective 2) extending to the 3ʹ end
of the sequence. PAX2 still did not bind to DNA. Position weight matrices were taken from MotifMap for human hg19 (e.g., YY1 and PAX2) and Drosophila
dm3 (GRHL1) assemblies (90).

complexity of the ADHD phenotype, we must expect complex The genetic association of ADGRL3 with disruptive behav-
genetic interactions within the ADGRL3 locus and with other iors and substance use supports previous observations.
genomic regions, which may explain the different levels of Families with ADHD cluster oppositional defiant disorder,
association observed across ECRs (Table 1). conduct disorder, and substance use disorder (23); children

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Brain Enhancer Associated With ADHD
diagnosed with ADHD monitored during the transition into
adolescence have higher rates of alcohol, tobacco, and
psychoactive drug use than unaffected children (50,51); the
lifetime risk for substance use is approximately 50% in
patients with childhood ADHD that persists into adulthood
(52,53).
Linkage and association of ADGRL3 with neuropsycholog-
ical endophenotypes was recently reported by Mastronardi
et al. (44). In agreement with their findings, we show a
significant association of ECR variants with lower ACVTCR
scores and higher ACVTO scores. Impaired response inhib-
ition and poor sustained attention, as measured by these two
tests, are fundamental components of the executive dysfunc-
tion present in patients with ADHD (54–57). In the same vein,
the inattentive and hyperactive/impulsive motor phenotype
associated with patients with ADHD is expected to affect the
ability to perform the ROCFT copy test, and therefore the
significant association with ECR risk variants. However, the
complexity of the multiple cognitive domains assessed by
ROCFT (i.e., visuospatial constructional ability, visual memory,
and several components of executive function) can confound
the interpretation of scores, which might explain the lower
level of significance (58). We did not detect any association
with the WISC measures (i.e., block design, PIQ, or FSIQ),
which might indicate that ADGRL3 variation does not contrib-
ute to the cognitive deficits evaluated by these particular tests.
Functional testing in vitro identified ECR47 as a transcrip-
tional enhancer. ECR47 was active in cultured neurons and
astrocytes in a tissue-specific manner, and its function was
disrupted by the ADHD risk haplotype defined by the variants
rs17226398, rs56038622, and rs2271338 (CTA) in complete
LD. The nonrandom association between these risk alleles
may suggest evolutionary selective pressure to conserve an
important biological function. Although neurons and astro-
cytes have distinct transcriptome profiles, they both share a
common neuroepithelial origin, and over the past two decades
it has become clear that astrocytes participate in a wide
variety of complex functions in the CNS, including crucial roles
in synaptic transmission and information processing in neural
circuits (59–61). Evidence also suggests that glial cells may
play a role in the pathogenesis of neuropsychiatric disorders,
including ADHD (62,63). However, additional studies are
required to determine whether ECR47 function in astrocytes
is functionally relevant for the pathology of ADHD, because
ADGRL3 expression in astrocytoma cells was low compared
to human neuroblastoma cells (Supplemental Figure S4 in
Supplement 1).
Strikingly, we also found that ECR47 functions as a brain
enhancer in vivo. ECR47 was able to drive GFP expression in
the zebrafish brain, which is consistent with various aspects of
endogenous adgrl3.1 expression. These results are in concert
with a previous report by Lange et al. (49) showing wide
adgrl3.1 expression in the zebrafish brain at 24, 48, and 72
hours postfertilization, which suggests that ECR47 activation
may share brain-specific factors with the adgrl3.1 transcrip-
tional machinery during zebrafish development.
Enhancer elements have signatures that define tissue
specificity (64,65). Analysis of ECR47 and a large set of
in vivo–tested brain enhancers revealed a significant over-
lapping of binding sites for homeobox TF families that are
Biological
Psychiatry
preferentially associated with neurodevelopmental processes.
Distal-less (i.e., DLX-1, DLX-2, and DLX-5), BRN5/POU6F1,
PAX (i.e., PAX2, PAX3, PAX5, PAX6, PAX7, and PAX8), and
NKX6 (NKX6.2) homeodomain factors are known to play
prominent roles in the development and function of the CNS
(66–70). This finding supports our results showing tissue-
specific activity of ECR47.
The risk allele substitution of rs2271338 disrupts YY1
binding to ECR47. YY1 is a controversial and versatile tran-
scriptional regulator (71); it can either activate or repress gene
expression depending on the cofactors it recruits (72). The
potential function of YY1 in the developing nervous system
was first suggested by the phenotypic analysis of Yy11/– mice.
Null Yy1 mice show early embryonic lethality, but a subset of
Yy11/– mice (20%) display growth retardation and neural
tube defects. The brains of Yy11/– mouse embryos show
exencephaly, asymmetric structure, and the presence of
pseudoventricles (73). Morpholino-knockdown studies in Xen-
opus reveal similar neurulation defects (74). While a significant
reduction of XYY1 protein levels results in early embryonic
lethality, a partial depletion results in anteroposterior pattern-
ing defects and reduction of head structures (74,75). At the
molecular level, Xyy1 ablation in Xenopus embryos reveals
downregulation of TFs involved in neural patterning (i.e.,
homeobox genes, engrailed2, otx2, and krox20) and neural
crest cell specification and migration (i.e., slug and snai1) (76).
Studies using other systems have also shown dysregulation of
neurotransmitter signaling and metabolism genes, such as
dynamin-1 and dopamine β-hydroxylase in neurons, and Glast
glutamate/aspartate transporter in astrocytes (76).
Analysis of the effect of rs2271338 risk allele on ADGRL3
expression in cultured human cells and postmortem brain
tissue showed apparently contradictory results. While YY1
downregulation did not affect ADGRL3 expression in neuro-
blastoma cells, the rs2271338 risk allele was associated with
reduced expression in the adult thalamus. The thalamus was
one of the earliest brain areas considered in the pathophysi-
ology of ADHD (77) because of its key role in filtering
information and stimulus processing (78). Morphological
abnormalities of the thalamus (79) and thalamic volume
reduction (80) have been shown previously in children with
ADHD. Various methods, including functional connectivity
analysis, have uncovered thalamic abnormalities in patients
with ADHD (81,82). Morphological abnormalities have also
been found across a number of cortical regions in children and
adolescents with ADHD (83), supporting the view that cortico-
striato-thalamo-cortical loops (84) play a key role in the
pathogenesis of ADHD.
Although additional studies are required to establish the
precise role of ECR47, the results presented here suggest an
important neurological function. While brain expression data
indicate an ADGRL3 expression maximum across fetal and
infant stages (Supplemental Figure S6 in Supplement 1),
relatively high expression levels are maintained throughout
life, suggesting that this gene is necessary for proper brain
function from conception to demise. The lack of effect of YY1
knockdown on endogenous ADGRL3 expression in differ-
entiated cells suggests that ECR47 may only be active during
developmental stages; however, the association between
rs2271338 and reduced ADGRL3 expression levels in the
Biological Psychiatry ]]], 2016; ]:]]]–]]] www.sobp.org/journal 9
Biological
Psychiatry
adult thalamus hampers our understanding of ADGRL3 spa-
tiotemporal regulation by ECR47–YY1. Elucidation of this
genetic interaction will help to decipher the molecular mech-
anisms underlying ADHD pathogenesis.
The experimental methodology used in this research could
be a paradigm for the evaluation of noncoding risk variants
associated with complex traits.
Limitations
We tested risk variants only for their effects on transcriptional
enhancer activity. While other transcriptional regulatory func-
tions (e.g., those mediated by silencer and insulator elements)
were not investigated, functional testing of these types of
sequence have proven difficult to design, and there are
currently no robust in vivo assays in widespread use (85,86).
We must also consider the possible effect of risk variants on
ADGRL3 mRNA splicing—especially synonymous changes
and noncoding variants within 50 base pairs of exon–intron
junctions—and on the expression and function of overlapping
noncoding RNAs.
ACKNOWLEDGMENTS AND DISCLOSURES
This work was supported by intramural resources from the National Human
Genome Research Institute of the U.S. National Institutes of Health.
We thank Paul Kruszka, M.D., for his detailed revision of the manuscript
and helpful comments.
This study used the computational capabilities of a demo license to
Genomatix Software (Munich, Germany).
The authors report no biomedical financial interests or potential conflicts
of interest.
ARTICLE INFORMATION
From the Medical Genetics Branch (AFM, YA, SH, KM, DY, MTA, MA-B,
MM), National Human Genome Research Institute, National Institutes of
Health, Bethesda, Maryland; Institute of Biology I (HL, WD), University of
Freiburg, Freiburg, Germany; and the Genome Biology Department (MA-B),
John Curtin School of Medical Research, The Australian National University,
Canberra, Australia.
HL is currently affiliated with the Institute for Developmental Biology,
Cologne University, Cologne, Germany.
Address correspondence to Maximilian Muenke, M.D., Medical Genetics
Branch, National Human Genome Research Institute, National Institutes
of Health, 35 Convent Drive, Room 1B-203, Bethesda, MD 20892-3717;
E-mail: mamuenke@mail.nih.gov.
Received Dec 11, 2015; revised Jun 28, 2016; accepted Jun 30, 2016.
Supplementary material cited in this article is available online at
http://dx.doi.org/10.1016/j.biopsych.2016.06.026.
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