Elsevier

Biological Psychiatry

Volume 80, Issue 12, 15 December 2016, Pages 943-954
Biological Psychiatry

Archival Report
An Ultraconserved Brain-Specific Enhancer Within ADGRL3 (LPHN3) Underpins Attention-Deficit/Hyperactivity Disorder Susceptibility

https://doi.org/10.1016/j.biopsych.2016.06.026Get rights and content

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.

Section snippets

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 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

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, 31, 32). For that reason, an analysis with species more closely related to humans than fish is required to

Discussion

Few molecular studies have attempted to explain the molecular effects of ADHD-associated genetic variants. Studies on dopamine transporter (DAT1) (37, 38, 39, 40), tryptophan hydroxylase 2 (TPH2) (41, 42), and T-cadherin (CDH13) (43) have examined the functional properties of rare missense mutations of moderate and large effects, but these findings fail to explain the higher incidence of ADHD and larger phenotypic variance observed in populations. Instead, the common disease/common variant

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.

References (90)

  • B.S. Molina et al.

    Adolescent substance use in the multimodal treatment study of attention-deficit/hyperactivity disorder (ADHD) (MTA) as a function of childhood ADHD, random assignment to childhood treatments, and subsequent medication

    J Am Acad Child Adolesc Psychiatry

    (2013)
  • M. Nogueira et al.

    Early-age clinical and developmental features associated to substance use disorders in attention-deficit/hyperactivity disorder in adults

    Compr Psychiatry

    (2014)
  • D.A. Pineda et al.

    The role of neuropsychologic tests in the diagnosis of attention deficit hyperactivity disorder

    Pediatr Neurol

    (2007)
  • B.A. Barres

    The mystery and magic of glia: A perspective on their roles in health and disease

    Neuron

    (2008)
  • B. Andersen et al.

    Brn-5 is a divergent POU domain factor highly expressed in layer IV of the neocortex

    J Biol Chem

    (1993)
  • J.P. Burbach et al.

    Molecular programming of stem cells into mesodiencephalic dopaminergic neurons

    Trends Neurosci

    (2006)
  • M.J. Morgan et al.

    YY1 regulates the neural crest-associated slug gene in xenopus laevis

    J Biol Chem

    (2004)
  • H.J. Kwon et al.

    Yin Yang 1, a vertebrate polycomb group gene, regulates antero-posterior neural patterning

    Biochem Biophys Res Commun

    (2003)
  • J.D. Gaudreau et al.

    Psychotogenic drugs and delirium pathogenesis: The central role of the thalamus

    Med Hypotheses

    (2005)
  • S. Xia et al.

    Thalamic shape and connectivity abnormalities in children with attention-deficit/hyperactivity disorder

    Psychiatry Res

    (2012)
  • X. Cao et al.

    Abnormal resting-state functional connectivity patterns of the putamen in medication-naive children with attention deficit hyperactivity disorder

    Brain Res

    (2009)
  • L.J. Seidman et al.

    Structural brain imaging of attention-deficit/hyperactivity disorder

    Biol Psychiatry

    (2005)
  • F.X. Castellanos et al.

    Characterizing cognition in ADHD: Beyond executive dysfunction

    Trends Cogn Sci

    (2006)
  • S.V. Faraone et al.

    Attention-deficit/hyperactivity disorder

    Nat Rev Dis Primers

    (2015)
  • S.N. Visser et al.

    Trends in the parent-report of health care provider-diagnosed and medicated attention-deficit/hyperactivity disorder: United States, 2003-2011

    J Am Acad Child Adolesc Psychiatry

    (2014)
  • S.N. Visser et al.

    Increasing prevalence of parent-reported attention-deficit/hyperactivity disorder among children—United States, 2003 and 2007

    MMWR Morb Mortal Wkly Rep

    (2010)
  • Diagnostic and Statistical Manual of Mental Disorders

    (1994)
  • J. Biederman et al.

    Attention deficit hyperactivity disorder: A worldwide concern

    J Nerv Ment Dis

    (2004)
  • G.C. Akutagava-Martins et al.

    Genetics of attention-deficit/hyperactivity disorder: Current findings and future directions

    Expert Rev Neurother

    (2013)
  • M. Arcos-Burgos et al.

    A common genetic network underlies substance use disorders and disruptive or externalizing disorders

    Hum Genet

    (2012)
  • M. Ribases et al.

    Contribution of LPHN3 to the genetic susceptibility to ADHD in adulthood: A replication study

    Genes Brain Behav

    (2011)
  • M. Arcos-Burgos et al.

    A common variant of the latrophilin 3 gene, LPHN3, confers susceptibility to ADHD and predicts effectiveness of stimulant medication

    Mol Psychiatry

    (2010)
  • M.T. Acosta et al.

    A two-locus genetic interaction between LPHN3 and 11q predicts ADHD severity and long-term outcome

    Transl Psychiatry

    (2011)
  • M. Jain et al.

    A cooperative interaction between LPHN3 and 11q doubles the risk for ADHD

    Mol Psychiatry

    (2012)
  • Z. Choudhry et al.

    LPHN3 and attention-deficit/hyperactivity disorder: Interaction with maternal stress during pregnancy

    J Child Psychol Psychiatry

    (2012)
  • A. Labbe et al.

    Refining psychiatric phenotypes for response to treatment: Contribution of LPHN3 in ADHD

    Am J Med Genet B Neuropsychiatr Genet

    (2012)
  • E.M. Bruxel et al.

    LPHN3 and attention-deficit/hyperactivity disorder: A susceptibility and pharmacogenetic study

    Genes Brain Behav

    (2015)
  • J. Song et al.

    Association of SNAP-25, SLC6A2, and LPHN3 with OROS methylphenidate treatment response in attention-deficit/hyperactivity disorder

    Clin Neuropharmacol

    (2014)
  • S. Domene et al.

    Screening of human Lphn3 for variants with a potential impact on ADHD susceptibility

    Am J Med Genet B Neuropsychiatr Genet

    (2011)
  • M.L. Bravo et al.

    Polymorphisms and phyletic relationships of the Paisa community from Antioquia (Colombia)

    Gene Geogr

    (1996)
  • D.A. Pineda et al.

    Potential cognitive endophenotypes in multigenerational families: Segregating ADHD from a genetic isolate

    Atten Defic Hyperact Disord

    (2011)
  • I. Ovcharenko et al.

    ECR Browser: A tool for visualizing and accessing data from comparisons of multiple vertebrate genomes

    Nucleic Acids Res

    (2004)
  • J. Ernst et al.

    Mapping and analysis of chromatin state dynamics in nine human cell types

    Nature

    (2011)
  • J. Ernst et al.

    Discovery and characterization of chromatin states for systematic annotation of the human genome

    Nat Biotechnol

    (2010)
  • J. Wang et al.

    Factorbook.Org: A wiki-based database for transcription factor-binding data generated by the encode consortium

    Nucleic Acids Res

    (2013)
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    1

    HL is currently affiliated with the Institute for Developmental Biology, Cologne University, Cologne, Germany.

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