Attention-deficit/hyperactivity disorder (ADHD) is the most common neurodevelopmental behavioral disorder, affecting about 10% of children and adolescents worldwide. It frequently persists into adulthood and can have serious life-long health consequences. Affected individuals are at increased risk for poor educational achievement, low income, underemployment, legal difficulties, and impaired social relationships. The annual societal burden of ADHD was conservatively estimated to reach $42.5 billion in the U.S. In addition, ADHD increases the risk of substance use disorder (SUD) and disruptive (externalizing) disorders such as oppositional defiant disorder (ODD), conduct disorder (CD). SUD is characterized by compulsive drug seeking behavior and drug use in the face of severe adverse consequences. The World Health Organization estimates that there are worldwide at least two billion alcohol users, one billion tobacco users and almost 185 million illicit drug users. Genetic factors are strongly implicated in ADHD. During the last few years, our research on the genetics of ADHD made seminal advances to understand: 1) the innate susceptibility to ADHD and associated comorbidities, 2) the interaction of genetic, demographic and environmental factors underpinning the risk of developing ADHD, 3) how much these factors shape the response of ADHD patients to pharmacological interventions (pharmacogenetics of ADHD), 4) the overrepresentation of functional and ontological gene-based networks implicated in determining synapse structure, and 5) the use of advanced genetic-epidemiological models with potential for use in clinical practice (translational genomics). Over the last 15 years, our research has made significant contributions to the genetics of ADHD. We collected families clustering ADHD and associated comorbidities from disparate regions around the world. Using genome-wide data, we found evidence of linkage of ADHD to chromosomes 4q13.2, 5q33.3, 8q11.23, 11q22, and 17p11.93 Linkage and association analyses revealed co-segregation between ADHD and disruptive behaviours linking genetics to the natural history of the disease.83 The Mendelian co-segregation of ADHD with these genomic regions and their high genotypic penetrance identify them as major loci predisposing to ADHD. Using positional cloning approaches we identified latrophilin 3 (LPHN3) variants in 4q13.2 that predispose to ADHD. We and others have found a significant homogeneous genetic effect of LPHN3 variants predisposing to ADHD in children, adolescents, and adults using cohorts of thousands of individuals from Colombia, Germany, Norway, Spain, South Korea, Brazil, two U.S. populations, and two from Canada; some of them used in pharmacogenetic studies. Altogether, these results show that: i) LPHN3 variants underpin the predisposition to ADHD in all these cohorts; ii) LPHN3 common variants are associated with ADHD, correlate with brain metabolism, predict ADHD severity and comorbidity with disruptive symptoms (CD and ODD), and substance use disorder (SUD), and long-term outcome; iii) these LPHN3 common variants predict the response to treatment with stimulant medication, introducing a new theme for translational applications; and iv) recently we linked these LPHN3 variants to ADHD endophenotypes. To the best of our knowledge, this is the most highly positively replicated genetic study of association to ADHD and to the response to stimulant treatment. As a measure of the epidemiological impact, the population attributable risk of LPHN3 susceptibility variants was estimated at 8.99 (95% confidence interval = 3.90-14.12), which means that the incidence of ADHD would be reduced by 9% if we were able to control the effect of this gene. LPHN3 encodes a member of the latrophilin subfamily of adhesion G-protein coupled receptors and is highly expressed in brain regions implicated in the dopaminergic systems. LPHN3 endogenous ligand has been identified as FLRT3, a postsynaptic membrane protein involved in axon guidance and neuronal cell migration during embryonic development. More importantly, the LPHN3-FLRT3 synaptic pair regulates excitatory transmission in vitro and in vivo. Animal models also support LPHN3 implication in ADHD pathophysiology. In addition, we discovered a genetic interaction between LPHN3 and a haplotype in chromosome 11q that doubles the risks of developing ADHD and worsens ADHD severity, and predicts the response to stimulant medication and differences in brain neurochemistry. This haplotype encompasses NCAM1 gene, which has a fundamental role in neural development. This gene is adjacent to DRD2, which has been previously associated with ADHD, and to TTC12 and ANKK1, two genes associated with disruptive symptoms and SUD. Moreover, we have proposed a common genetic network underlying ADHD and disruptive symptoms. Our model suggests that behavioral disorders involving ADHD, disruptive disorders and SUD may share similar signs and symptoms, pathophysiological and psychopathological mechanisms, and genetic factors. During this year year, we published a high impact manuscript in Biological Psychiatry on the characterization of a non-coding LPHN3 risk haplotype associated with ADHD. In the past we interrogated the entire LPHN3 coding region looking for coding variations that might affect the LPHN3 protein function. However, we could not identify any LPHN3 non-synonymous or canonical splice site mutations associated with ADHD, suggesting that intronic non-coding variants instead are the likely pathological contributors. Using a combination of evolutionary sequence conservation and regulatory annotation data we identified candidate sequences within LPHN3 with potential regulatory function. Several variants revealed significant association with ADHD, disruptive disorders, substance use disorder and/or neuropsychological endophenotypes, with a three-marker haplotype within a highly evolutionary conserved sequence (ECR47) showing the highest level of association across the board. This result suggests that ECR47 may participate in a common neurobiological pathway to ADHD. Functional testing in vitro identified ECR47 as a transcriptional enhancer. ECR47 showed activity in cultured neurons and astrocytes and its function was disrupted by the ADHD risk haplotype C/T/A defined by variants rs17226398/ rs56038622/rs2271338 in complete linkage disequilibrium. We also found that ECR47 functions as a brain enhancer in zebrafish sharing specific aspects of endogenous lphn3.1 expression. Functional analysis of ECR47 risk variant allele substitutions demonstrated disruption of YY1 repressor binding site. Overall, our work continues to support a functional role for LPHN3 in the pathophysiology of ADHD.
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