Websites: www.humangenetics.nl; www.ncmls.eu

1. Genome-wide scan of copy number variation in Attention deficit hyperactivity disorder (ADHD) Barbara Franke1,2, Jayne Hehir-Kwa1, Alejandro AriasVasquez1,2, Joris Veltman1, Bart Bloemen1, J. Lasky-Su3, P. Asherson4, M. Gill5, J. Sergeant6, R. Ebstein7, A. Rothenberger8, HC. Steinhausen9, T. Banaschewski10, R. Oades11, E. Sonuga-Barke12, A. Miranda13, H. Royers14, J. Buitelaar2, S.V. Faraone15, for the IMAGE consortium Departments of 1Human Genetics and 2Psychiatry, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 3Boston, MA, USA; 4London, UK; 5Dublin, Ireland; 6Amsterdam, NL; 7Jerusalem, Israel; 8Göttingen, Germany; 9Zurich, Switzerland; 10Mannheim and 11Essen, Germany; 12Southampton, UK; 13Valencia, Spain; 14Ghent, Belgium; 15SUNY Upstate Medical University, Syracuse, NY, USA correspondence to: b.franke@antrg.umcn.nl Abstract Recent data suggest that copy number variants (CNVs) can contribute to complex disease susceptibility. The relative impact of CNVs compared to single nucleotide polymorphisms (SNPs) on one of the processes underlying disease vulnerability, variable gene expression, has been estimated at 18% (1). The involvement of CNVs in ADHD etiology has not been investigated. Within the International Multi-site ADHD Genetics (IMAGE) study, sponsored by the Genetic Association Information Network (GAIN), a whole genome association study investigating over 500.000 SNPs has been carried out on 949 European Caucasian parent-child trios with offspring meeting the DSM-IV combined-type criteria for ADHD. Families were collected in the Netherlands, Ireland, UK, Germany, Belgium, Switzerland, Spain and Israel. Using the intensity data from the SNP analysis carried out at Perlegen Sciences, copy number information is extracted for each individual. Pilot data are presented. In the next analysis steps, we plan to identify known and new CNVs in the patients and their parents. By comparing parents with offspring we will investigate which CNVs are inherited, which are de novo. For inherited CNVs a TDT-based association study will be carried out. For those that occur de novo in the patients, we will investigate the gene content to find out if the CNV carries genes that can explain the presence of ADHD in the particular patient. In conclusion: The Perlegen SNP-microarrays used in the GAIN projects contain copy number information. The CNV analysis can potentially identify new candidate genes and new risk alleles for ADHD. Introduction Preliminary Results and Future Plans Copy number variants (CNVs) comprise a newly identified type of structural variation within the human genome. CNVs include insertions, deletions and duplications and encompass relatively large genomic segments of 1kb to several Mb in size. Detection of CNVs can be achieved by karyotyping, (array) CGH and SNP microarrays (reviewed in (2)), depending on their size (3). CNVs are very common in the human genome (around 12% of the genome) and their frequencies can reach high percentages in the population. Depending on their location, they can include whole genes and influence their regulation due to dosage effects (1). For many of the rare CNVs there is a clear link with human disease (4-6); they often occur de novo in monogenic disorders. This link with disease is less clear for the polymorphisms within the CNV category (those with frequencies above 1% in the population). However, a number of studies have already found evidence that common CNVs can be risk factors for disease, e.g. in autoimmune conditions inlcuding glomerulonephritis, Crohn’s disease, systemic lupus erythematosus, polyangiitis and Wegener’s granulomatosis (7-10) and in HIV susceptibility/progression (11). In the current study we set out to study the role of CNVs in the etiology of ADHD. This highly heritable disorder is the most common neuropsychiatric disorder in children (seen in 3-5% of school-aged children). It also has a prevalence of at least 1% in the adult population. ADHD is characterized by early-onset, age-inappropriate and persistent hyperactive, inattentive and impulsive behavior. Raw Log2 intensity ratios of the sample versus a reference pool were calculated using the QC unfiltered allele 1 and 2 intensity data. For this the intensity of the two different alleles is combined to give an average intensity for each SNP. This is compared to the average intensity in a reference (pool), calculated also across both alleles. These Log2 intensity ratios are plotted for each SNP according to their chromosomal position (Figure 1). The plots indicate the presence of both loss and gain CNVs. The signal to noise in these plots will be further improved to allow the detection of smaller CNVs, and a statistical algorithm (Hidden Markov Model) will be incorporated to automatically detect all CNVs present. Chromosome 14 Experimental design Within the IMAGE project, 1400 European Caucasian families with at least one child aged 5-19 affected with combined subtype ADHD have been recruited from 8 countries (Netherlands, UK, Ireland, Germany, Israel, Spain, Switzerland, Belgium). Diagnosis of ADHD was based on the PACS (Parental Account of Childhood Symptom) combined with the teacher-rated Conners’ ADHD subscale (CTRS-R:L) and situational pervasiveness (see dbGAP website for more information: http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000016). As part of the Genetic Association Information Network (GAIN (12)), a total of 949 child-parent triads (2835 samples) were genotyped on a newly developed SNP-array by Perlegen Sciences. SNP selection for the Perlegen array was guided by a linkage disequilibrium (LD) analysis of the integrated Phase I and Phase II HapMap data, using a minimum pairwise r2 threshold of 0.8, for SNPs with a minor allele frequency (MAF) of 0.05 or higher. The set comprises almost 600,000 SNPs, yielding nearly complete coverage of the European Caucasian (CEU) map. In addition to the LD-based SNP selection, 20,000 non-synonymous SNPs culled from the HapMap, Celera’s exon resequencing data, and Perlegen data were included. For Caucasian samples, the average r2 between a SNP in the HapMap and a working assay is 0.94. The percentage of SNPs in the HapMap with r2 > 0.8 with a Perlegen SNP is 93%. Figure 1: Example of the detection of known CNVs on chromosomes 14 and 22 in the same ADHD patient from the GAIN project (phs000016). The arrowheads indicate a copy number gain on chromosome 14 and a copy number loss on chromosome 22. The table above shows that these CNVs have been reported before in healthy individuals, as registered in the ‘Database of Genomic Variants’ (http://projects.tcag.ca/variation) (13). Chromosome 22 • The following analyses will be carried out after extraction of the copy number information in all patient-parent trios: • Common, inherited CNVs (≥ 5% allelic frequency): • Association study with ADHD using TDT and qTDT. • Are the CNVs tagged by SNPs (LD / r2 pattern)? • Are the findings from the SNP WGAS and the CNV analysis comparable? • Rare, inherited CNVs: • A sum score of CNVs will be created and transmission to children will be tested • De novo CNVs: • What genes present in the CNV? • Are the genes possibly related to ADHD? • Are the CNVs present in regions found in linkage/association studies in ADHD? References (1) Stranger et al., Science. 2007 Feb 9;315(5813):848-53. (2) Feuk, Carson, Scherer. Nat Rev Genet. 2006 Feb;7(2):85-97. (3) Hehir-Kwa et al., DNA Res. 2007 Feb 28;14(1):1-11. (4) Vissers et al., Nat Genet. 2004 Sep;36(9):955-7. (5) de Vries et al., Am J Hum Genet. 2005 Oct;77(4):606-16. (6) Koolen et al., Nat Genet. 2006 Sep;38(9):999-1001. (7) Aitman et al., Nature. 2006 Feb 16;439(7078):851-5. (8) Fellermann et al., Am J Hum Genet. 2006 Sep;79(3):439-48. (9) Fanciulli et al., Nat Genet. 2007 Jun;39(6):721-3. (10) Yang et al., Am J Hum Genet. 2007 Jun;80(6):1037-54. (11) Gonzalez et al., Science. 2005 Mar 4;307(5714):1434-40. (12) The GAIN Collaborative Research Group et al., Nat Genet. 2007 Sep;39(9):1045-1051. (13) Redon et al., Nature. 2006 Nov 23;444(7118):444-54. Websites: www.humangenetics.nl; www.ncmls.eu