Comprehensive Phenotype/Genotype Analyses of the Norepinephrine Transporter Gene (SLC6A2) in ADHD: Relation to Maternal Smoking during Pregnancy Geeta A. Thakur 1,4. , Sarojini M. Sengupta 4 * . , Natalie Grizenko 2,4" , Zia Choudhry 3,4 , Ridha Joober 1,2,3,4" 1 Integrated Program in Neuroscience, McGill University, Montreal, Quebec, Canada, 2 Department of Psychiatry, McGill University, Montreal, Quebec, Canada, 3 Department of Human Genetics, McGill University, Montreal, Quebec, Canada, 4 Douglas Mental Health University Institute, Montreal, Quebec, Canada Abstract Objective: Despite strong pharmacological evidence implicating the norepinephrine transporter in ADHD, genetic studies have yielded largely insignificant results. We tested the association between 30 tag SNPs within the SLC6A2 gene and ADHD, with stratification based on maternal smoking during pregnancy, an environmental factor strongly associated with ADHD. Methods: Children (6–12 years old) diagnosed with ADHD according to DSM-IV criteria were comprehensively evaluated with regard to several behavioral and cognitive dimensions of ADHD as well as response to a fixed dose of methylphenidate (MPH) using a double-blind placebo controlled crossover trial. Family-based association tests (FBAT), including categorical and quantitative trait analyses, were conducted in 377 nuclear families. Results: A highly significant association was observed with rs36021 (and linked SNPs) in the group where mothers smoked during pregnancy. Association was noted with categorical DSM-IV ADHD diagnosis (Z = 3.74, P = 0.0002), behavioral assessments by parents (CBCL, P = 0.00008), as well as restless-impulsive subscale scores on Conners’-teachers (P = 0.006) and parents (P = 0.006). In this subgroup, significant association was also observed with cognitive deficits, more specifically sustained attention, spatial working memory, planning, and response inhibition. The risk allele was associated with significant improvement of behavior as measured by research staff (Z = 3.28, P = 0.001), parents (Z = 2.62, P = 0.009), as well as evaluation in the simulated academic environment (Z = 3.58, P = 0.0003). Conclusions: By using maternal smoking during pregnancy to index a putatively more homogeneous group of ADHD, highly significant associations were observed between tag SNPs within SLC6A2 and ADHD diagnosis, behavioral and cognitive measures relevant to ADHD and response to MPH. This comprehensive phenotype/genotype analysis may help to further understand this complex disorder and improve its treatment. Clinical trial registration information – Clinical and Pharmacogenetic Study of Attention Deficit with Hyperactivity Disorder (ADHD); www.clinicaltrials.gov; NCT00483106. Citation: Thakur GA, Sengupta SM, Grizenko N, Choudhry Z, Joober R (2012) Comprehensive Phenotype/Genotype Analyses of the Norepinephrine Transporter Gene (SLC6A2) in ADHD: Relation to Maternal Smoking during Pregnancy. PLoS ONE 7(11): e49616. doi:10.1371/journal.pone.0049616 Editor: Takeo Yoshikawa, Rikagaku Kenkyu ¯ sho Brain Science Institute, Japan Received August 7, 2012; Accepted October 11, 2012; Published November 20, 2012 Copyright: ß 2012 Thakur et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by grants from the Fonds de la recherche en sante ´ du Que ´bec and the Canadian Institutes of Health Research to RJ. GAT holds a Frederick Banting and Charles Best Canada Graduate Scholarship doctoral award from CIHR. SS is a recipient of the 2008 NARSAD Young Investigator and 2009 Dr.Mortimer D. Sackler Developmental Psychology Investigator Awards. 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. * E-mail: sarojini.sengupta@douglas.mcgill.ca . These authors contributed equally to this work. " These authors also contributed equally to this work. Introduction Attention-deficit/hyperactivity disorder (ADHD) is a highly prevalent psychiatric disorder, with rates ranging from 5.9–7.1% in children and adolescents [1]. It is heterogeneous in its clinical expression, with core symptoms of poor sustained attention, impulsivity, and hyperactivity. It is often associated with cognitive deficits, particularly in executive function and sustained attention. ADHD has an important genetic component, with a mean heritability estimate of 76%, [2] and it has been suggested that multiple genes are involved, each having a small effect. [3]. Psychostimulants, mostly methylphenidate (MPH) [4] are the first-line of treatment for ADHD. These medications are known to block the dopamine (DA) and norepinephrine (NE) transporters, resulting in increased synaptic concentration of both neurotrans- mitters. [5,6,7] Short-term trials have concluded that MPH is efficacious in reducing ADHD symptoms in approximately 70% of affected children [4] and adults. [8] NE-specific pharmacological agents (including clonidine, guanfacine, desipramine, and atomox- etine) are effective in treating ADHD, thereby implicating this catecholamine as a major player in the pathophysiology of the disorder. [9] These studies reinforced the early evidence from PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e49616