Identification of Pathway-Biased and Deleterious Melatonin Receptor Mutants in Autism Spectrum Disorders and in the General Population

et al. (2010) Identification of Pathway-Biased and Deleterious Melatonin Receptor Mutants in Autism Spectrum Disorders and in the General Population. PLoS ONE 5(7): e11495. doi:10.1371/journal.pone.0011495 Identification of Pathway-Biased and Deleterious Melatonin Receptor Mutants in Autism Spectrum Disorders and in the General Population Pauline Chaste 0 Nathalie Clement 0 Oriane Mercati 0 Jean-Luc Guillaume 0 Richard Delorme 0 Hany Goubran Botros 0 Ce cile Pagan 0 Samuel Pe rivier 0 Isabelle Scheid 0 Gudrun Nygren 0 Henrik 0 Anckarsa ter 0 Maria Rastam 0 Ola Sta hlberg 0 Carina Gillberg 0 Emilie Serrano 0 Nathalie Lemie` re 0 Jean Marie Launay 0 Marie Christine Mouren-Simeoni 0 Marion Leboyer 0 Christopher Gillberg 0 Ralf Jockers 0 Thomas Bourgeron 0 Rafael Linden, Universidade Federal do Rio de Janeiro (UFRJ), Brazil 0 1 Human Genetics and Cognitive Functions, Institut Pasteur, Paris, France , 2 CNRS URA 2182 ''Genes, synapses et cognition'' , Institut Pasteur, Paris, France, 3 Institut Cochin, Universite Paris Descartes, CNRS (UMR 8104), Paris, France, 4 INSERM U1016, Paris, France, 5 Service de Psychopathologie de l9Enfant et de l9Adolescent, H opital Robert Debre , Assistance Publique-Hopitaux de Paris, Paris, France, 6 Department of Child and Adolescent Psychiatry, G o teborg University, G o teborg, Sweden, 7 Institute of Clinical Sciences, Lund University , Malmo , Sweden , 8 Department of Clinical Sciences in Lund, Lund University, Lund, Sweden, 9 Service de Biochimie, IFR 139, H opital Lariboisie`re, Assistance Publique-Ho pitaux de Paris EA 3621, Paris, France, 10 INSERM U955, Institut Mondor de Recherche Biome dicale, Universite Paris XII , Cre teil, France, 11 Foundation Fondamental, Cre teil , France , 12 Saint George's Hospital Medical School , London , United Kingdom , 13 University Denis Diderot Paris 7 , Paris , France Melatonin is a powerful antioxidant and a synchronizer of many physiological processes. Alteration of the melatonin pathway has been reported in circadian disorders, diabetes and autism spectrum disorders (ASD). However, very little is known about the genetic variability of melatonin receptors in humans. Here, we sequenced the melatonin receptor MTNR1A and MTNR1B, genes coding for MT1 and MT2 receptors, respectively, in a large panel of 941 individuals including 295 patients with ASD, 362 controls and 284 individuals from different ethnic backgrounds. We also sequenced GPR50, coding for the orphan melatoninrelated receptor GPR50 in patients and controls. We identified six non-synonymous mutations for MTNR1A and ten for MTNR1B. The majority of these variations altered receptor function. Particularly interesting mutants are MT1-I49N, which is devoid of any melatonin binding and cell surface expression, and MT1-G166E and MT1-I212T, which showed severely impaired cell surface expression. Of note, several mutants possessed pathway-selective signaling properties, some preferentially inhibiting the adenylyl cyclase pathway, others preferentially activating the MAPK pathway. The prevalence of these deleterious mutations in cases and controls indicates that they do not represent major risk factor for ASD (MTNR1A case 3.6% vs controls 4.4%; MTNR1B case 4.7% vs 3% controls). Concerning GPR50, we detected a significant association between ASD and two variations, D502-505 and T532A, in affected males, but it did not hold up after Bonferonni correction for multiple testing. Our results represent the first functional ascertainment of melatonin receptors in humans and constitute a basis for future structure-function studies and for interpreting genetic data on the melatonin pathway in patients. - Funding: This work was supported by the Institut Pasteur, University Denis Diderot Paris 7, University Paris Descartes, INSERM, CNRS, Fondation pour la Recherche Medicale (Equipe FRM, RJ), Foundation Orange, Fondation de France, Foundation FondaMentale, and by the Swedish Science Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Melatonin is synthesized in the pineal gland during the night and is involved in various physiologic functions, including sleep induction, circadian rhythm regulation, and immune response [1]. Melatonin synthesis requires serotonin, which is first acetylated by aryl alkylamine N-acetyltransferase (AA-NAT) and then converted to melatonin by acetyl serotonin methyl transferase (ASMT also known as hydroxyindole O-methyltransferase or HIOMT) [1]. Melatonin signaling is mainly mediated by the guanine nucleotide binding (G) protein-coupled receptors (GPCRs) MTNR1A (MT1) and MTNR1B (MT2) that are expressed in the suprachiasmatic nuclei (SCN) but are also present in other hypothalamic nuclei, retina, immune cells, and other peripheral organs. Established downstream cellular effects of melatonin receptor activation are inhibition of the adenylyl cyclase pathway and activation of the MAPK pathway [2]. The melatonin related receptor GPR50 is an orphan GPCR with no affinity for melatonin, but as a dimer with MT1, it inhibits melatonin signaling [3]. Abnormal melatonin signaling has been reported as a risk factor for medical conditions as diverse as diabetes mellitus, circadian rhythm and psychiatric disorders [4,5,6,7,8,9]. The role of melatonin in the susceptibility to these disorders remains unclear, but an alteration of melatonin as a powerful antioxidant molecule and/or as a Zeitgeber (time giver) could alter and/or desynchronize many physiological processes related to a broad range of disorders. Sleep disorders are common in the general population, and their incidence is higher in many psychiatric disorders [9]. Abnormal sleep patterns have often been reported in patients with autism [10,11,12] or Asperger syndrome [13,14,15], a condition characterized by the presence of markedly autistic behavior in people of normal general intelligence. Several studies have indicated that melatonin treatment improves sleep in autism [16,17,18,19], and in patients with Asperger syndrome [20]. The main effect on sleep seems to be a reduction of sleep onset latency. We therefore decided to study the genetic variability of the melatonin receptor MTNR1A and MTNR1B genes and the GPR50 gene in autism spectrum disorder (ASD) patients, in parallel to a normal European control population as well as in individuals of the human genome pluriethnic diversity panel (HGDP), by direct sequencing of the coding regions. Several non-synonymous mutants were identified for MT1 and MT2 and their functional properties determined in different in vitro assays. Identification of MTNR1A non-synonymous variants Six non-synonymous mutations were identified for MTNR1A. The MT1-I49N variant was detected in a patient with ASD, but not in our control sample (Tabl (...truncated)