The genetic overlap between Intellectual Disability and Attention-Deficit/Hyperactivity Disorder Anne van Pens Master thesis, written in October and November 2014 in Nijmegen, the Netherlands Supervisors: Marieke Klein, Alejandro Arias-Vasquez, Barbara Franke Affiliations: 1: Radboudumc, Nijmegen, The Netherlands 2; Donders Institute, Nijmegen, The Netherlands Corresponding author: Anne van Pens, [email protected] Abstract Objective: Attention-Deficit/Hyperactivity Disorder(ADHD) and Intellectual Disability(ID) co-occur more often than expected by chance, suggesting some genetic overlap. In four subprojects we investigated whether genes, affected by rare genetic variations in patients with ID, contribute to multifactorial ADHD. Methods: (1) Single nucleotide polymorphisms (SNPs) in 392 autosomal ID-related genes and (2) a subset involved in neurite outgrowth were tested for association with multifactorial ADHD risk, both on gene-set and gene-wide level, using data from the meta-analysis of the ADHD working group of the Psychiatric Genomic Consortium (PGC; 5, 621 cases and 13, 589 controls). (3) Twelve genes selected on frequent occurrence in copy number variants (CNVs) in patients with ADHD and ID and/orcongenital anomalies {PRODH, RBFOX1, PTPRD, CNTNAP2, NRXN1, XYLT1, PRIM2, FAM110C, SKI, NRG3, GRIN2A, NRG3and ERBB4}and(4) two genes selected because of suggested involvement in ID and ADHD in the literature {CHRNA7and NRXN1}were tested for gene-wideassociations with multifactorial ADHD risk using the ADHD PGC meta-analysis data and with symptom counts using data from the International Multicenter ADHD Genetics project (IMAGE; 930 cases). Single-SNP and gene-wideassociation analyses for CHRNA7and NRXN1were performed with regional brain volumes in 1302 healthy participants of the Brain ImagingGenetics (BIG) cohort. Single-SNPassociation analyses were also performed forvoxel-wide structural connectivity measurements. Results: SNPs in all autosomal ID-related genes, but not in the subset of neurite outgrowth genes, were significantly linked to ADHD as a group. The MEF2Cgeneshowed gene-wideassociation with ADHD risk. Other gene-wideand SNP-specificanalyses did not yield significantassociations. Conclusion: SNPs in 392 genes, and specifically the MEF2Cgene, affected by rare genetic variations in patients with ID, contribute to multifactorial ADHD risk as a group. This contribution to ADHD risk does not seem to be driven by neurite outgrowth genes. Keywords: Genetic Overlap, Attention-Deficit/Hyperactivity Disorder, Intellectual disability, Gene-set analysis, MEF2C, Brain Imaging Introduction Neurodevelopmental continuum Neurodevelopmental disorders are often accompanied by developmental or psychiatric comorbidities. For example, 68-87% ofAttention-Deficit/Hyperactivity Disorder (ADHD) patients have at least one co-morbid disorder (Ghanizadeh, 2009; Jensen & Steinhausen, 2014; Kadesjo & Gillberg, 2001; Kraut et al., 2013; Larson, Russ, Kahn, & Halfon, 2011). Because neurodevelopmental disorders share a common genetic etiology (Pettersson, Anckarsater, Gillberg, & Lichtenstein, 2013), it has been hypothesized that the same 'risk genes' often contribute to different neurodevelopmental disorders (Moreno-De-Lucaet al., 2013). Therefore, all neurodevelopmental disorders have been hypothesized to lie on a 'continuum', with a largely shared causality (Moreno- De-Lucaet al., 2013; Owen, 2012). The model of developmental brain dysfunction by Moreno-de- Luca et al. (2013) predicts that each particular genetic cause can manifest as a spectrum of impairments of varying severity in the cognitive, neurobehavioral and neuromotor domain. What specific impairments reach the threshold for clinical diagnosis, however, depends on the genetic background of the individual and on environmental factors. This explains why patients with neurodevelopmental disorders often fulfill also part of the criteria for other neurodevelopmental disorders. For example, many studies support the idea that 30-50% of all children with autism spectrum disorder (ASD) also fulfill the criteria for ADHD (de Bruin, Ferdinand, Meester, de Nijs, & Verheij, 2007; Gadow, DeVincent, & Pomeroy, 2006; Leyferet al., 2006; Schwenck& Freitag, 2014; Simonoffet al., 2008; Sinzig, Morsch, & Lehmkuhl, 2008; van Steenset, Bogels, & de Bruin, 2013). Martin et al. (2014) showed that there is substantial genetic overlap between the biological processes ofASD and ADHD, as for example large rare copy number variations (CNVs) contributing to the disorders disrupt the same biological processes. This supports the neurodevelopmental continuum hypothesis. Overlap between ADHD and Intellectual Disability Lesswell-studied than the overlap between ADHDand ASD, is the overlap between ADHDand intellectual disability (ID). The prevalence of ADHD in patients with ID seems to be twice as high as in the general population (Franke et al., 2012; Maulik, 2010). The real prevalence ofADHD in ID- patients may be much higher, though, because ADHD can be difficult to diagnose in children with ID. This is because ID-patients may not understand the questions of a psychiatric interview meant for people with a normal intelligence quotient (IQ) (Turygin, Matson, & Adams, 2014). In addition, one must take into account the patient's developmental age, rather than the biological age to assess if the observed hyperactive, impulsive and/or inattentive behavior is aberrant (Buitelaar, Kan, & Asherson, 2011). Apart from that, parents of a child diagnosed with ID, may easily assign ADHD-like symptoms of their child to ID, and never report them to a medical professional. In addition, the low IQ in ID-patients may cause inability to understand schoolwork, games, etcetera, and may therefore lead to decreased motivation to pay attention to these things. On the other hand, inattention problems in ADHD may lead to lower scores on IQtests (Styck & Watkins, 2014). All in all, research is needed to investigate whetherthe larger-than-expected shared prevalence is due to genetic factors, non-genetic factors or a combination of both. Attention-Deficit/Hyperactivity Disorder (ADHD) ADHD is a psychiatric disorder characterized by inattention and/or hyperactivity and impulsivity (American Psychiatric Association, 2013). The prevalence ofADHD is 5-6% in children and 2. 5-5% in adults (Franke et at., 2012). Although the disorderstarts in early childhood, it often persists into adulthood (Demian, 2011; Franke et al., 2012). According to the Diagnostic and Statistical Manual of Mental DisordersV (DSM-V), a first diagnosis of ADHDcan also be made in adulthood, provided that the symptoms have been visible since age 12 when assessed in retrospect (American Psychiatric Association, 2013). In both children and adults, ADHD is a heterogeneous disorder, since it is diagnosed by having six out of nine symptoms in at least one or two domains of the disorder (American Psychiatric Association, 2013). In addition, the majority of patients with ADHD has co-morbid disorders (Ghanizadeh, 2009; Jensen & Steinhausen, 2014; Kadesjo & Gillberg, 2001; Kraut et al., 2013; Larson et al., 2011), such as conduct/oppositional defiant disorders, language development disorders, motor development disorders, ASD and/or ID (Jensen & Steinhausen, 2014). When ADHD remains untreated, it can also lead to drop-out of school (Barbaresi, Katusic, Colligan, Weaver, & Jacobsen, 2007), mood disorders (Chen et al., 2014; Cubero-Millan et al., 2014) and drug abuse (Levy et al., 2014). Fortunately, early diagnosis and adequate treatment of ADHD can improve the quality of life of patients significantly. Insight in the etiology of ADHDand its co-morbidities can improve diagnosisand treatment of patients with ADHD. Since the heritability of ADHD is high, with an estimated 70 - 80% of the phenotypic variance explained by genetic factors (Franke et al., 2012), identification of the genes involved in ADHD can help us elucidate the pathogenesis ofADHD. IdentifyingADHD-genes appears challengingthough, since most cases ofADHD have a multifactorial etiology. This means that they are caused by the combination of multiple variants in many genes and by environmental factors, which all have a small effect on the risk for ADHD. Most of these gene variants are 'common', which means that they have a prevalence of at least 1% in the general population, as is the case for single nucleotide polymorphisms (SNPs), but also for certain CNVs. Interestingly, halfofthetop-ranked ADHD candidate genes, as found in five genome-wide association studies (GWASs) that were studied by Poelmans et al. (2011), contribute to a single biological process, i. e. neurite outgrowth. In addition to multifactorial ADHD, for an unknown percentage of ADHD cases the underlying genetic background is likely to be of a mono- or oligogenic nature. In these forms of the disorder, one or a few severe gene defects are sufficientto cause ADHD in an individual patient (Franke et al., 2012). However, less severe defects in the same genes may contribute to multifactorial forms ofADHD. The genetic heterogeneity of ADHD, i. e. the different combinations of risk genes in different patients with ADHD, may explain why symptoms and co-morbidities of patients with ADHD vary so widely ('phenotypic heterogeneity'). Genes may be associated with part of the symptoms or brain phenotypes of ADHD, rather than with all of them. It is assumed that abnormality of a certain behavioral, functional brain or structural brain domain lies in between the risk gene