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

Abstract

BACKGROUND Genetic factors predispose 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 non-coding 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 on 838 individuals (372 affected and 466 unaffected) identified ADHD-associated SNPs harbored in some of these conserved elements. Luciferase assays and zebrafish GFP 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 (ECR47) that functions as a transcriptional enhancer. A three-variant ADHD risk haplotype in ECR47, formed by rs17226398, rs56038622 and rs2271338, reduced enhancer activity by 40% in neuroblastoma and astrocytoma cells (PBonferroni<0.0001). This enhancer also drove GFP expression in the zebrafish brain in a tissue-specific manner, sharing aspects of endogenous ADGRL3 expression. The rs2271338 risk allele disrupts binding of YY1, an important factor in the development and function of the central nervous system. Expression quantitative trait loci analysis of post-mortem 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 non-coding variants with potential implications for the pathology of ADHD.

Conserved non-coding elements: developmental gene regulation meets genome organization

“…A single nucleotide variant associated with IRX1, IRX2 and IRX4 , located within a CNE, was also recently found to be involved in the pathogenesis of kyphoscoliosis ( 97 ). Beyond malformations, variations within CNEs can even be linked to complex behavioral or neurological disorders, evident from research linking cases of attention-deficit/hyperactivity disorder ( 98 ), autism ( 99 ) and restless leg syndrome ( 100 ) to single point mutations within CNEs.…”

Pharmacological analysis of zebrafish lphn3.1 morphant larvae suggests that saturated dopaminergic signaling could underlie the ADHD-like locomotor hyperactivity

“…The ADHD-risk gene Latrophilin 3 (LPHN3 ; recently renamed Adhesion G protein-coupled receptor L3 ( ADGRL3 )) was identified by linkage analysis followed by fine mapping of human patients ( Arcos-Burgos et al, 2010 ; Domené et al, 2011 ; Martinez et al, 2016 ). A polymorphism in LPHN3 has been found to predict the efficacy of ADHD treatment drugs ( Arcos-Burgos et al, 2010 ; Labbe et al, 2012 ) and the LPHN3 risk haplotype alters cognitive control in human patients ( Fallgatter et al, 2013 ).…”

Identification and evolution of latrophilin receptor gene involved in Tribolium castaneum devolopment and female fecundity

“…In Homo sapiens , lph‐3 is involved in alcohol/nicotine dependence based on genome‐wide linkage analyses (Jain et al, ). In addition, family‐based genetic analyses of mass individuals also have identified attention‐deficit/hyper activity disorder (ADHD)‐associated single nucleotide polymorphisms harbored in lph ‐ 3 gene (Martinez et al, ). Simultaneously, down‐regulation of lph‐3 caused ADHD in zebrafish accompanied a reduction and misplacement of dopamine‐positive neurons in the ventral diencephalon, which was rescued by the ADHD treatment drugs methylphenidate (psychostimulants; Lange et al, ).…”