Molecular Psychiatry (2006) 11, 1085–1091 & 2006 Nature Publishing Group All rights reserved 1359-4184/06 $30.00 www.nature.com/mp ORIGINAL ARTICLE Further evidence that the KIAA0319 gene confers susceptibility to developmental dyslexia D Harold1,6, S Paracchini2,6, T Scerri2, M Dennis2,3, N Cope1, G Hill1, V Moskvina4, J Walter5, AJ Richardson5, MJ Owen1, JF Stein5, ED Green3, MC O’Donovan1, J Williams1,4 and AP Monaco2 1Department of Psychological Medicine, Cardiff University, Heath Park, Cardiff, UK; 2Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, UK; 3Genome Technology Branch, National Human Genome Research Institute, Bethesda, MD, USA; 4Biostatistics and Bioinformatics Unit, Cardiff University, Heath Park, Cardiff, UK and 5Department of Physiology, University of Oxford, Parks Road, Oxford, UK The DYX2 locus on chromosome 6p22.2 is the most replicated region of linkage to developmental dyslexia (DD). Two candidate genes within this region have recently been implicated in the disorder: KIAA0319 and DCDC2. Variants within DCDC2 have shown association with DD in a US and a German sample. However, when we genotyped these specific variants in two large, independent UK samples, we obtained only weak, inconsistent evidence for their involvement in DD. Having previously found evidence that variation in the KIAA0319 gene confers susceptibility to DD, we sought to refine this genetic association by genotyping 36 additional SNPs in the gene. Nine SNPs, predominantly clustered around the first exon, showed the most significant association with DD in one or both UK samples, including rs3212236 in the 50 flanking region (P = 0.00003) and rs761100 in intron 1 (P = 0.0004). We have thus refined the region of association with developmental dyslexia to putative regulatory sequences around the first exon of the KIAA0319 gene, supporting the presence of functional mutations that could affect gene expression. Our data also suggests a possible interaction between KIAA0319 and DCDC2, which requires further testing. Molecular Psychiatry (2006) 11, 1085–1091. doi:10.1038/sj.mp.4001904; published online 10 October 2006 Keywords: reading disability; susceptibility locus; genetic association; KIAA0319; DCDC2; epistasis Introduction ibility locus. In independent samples from the UK and the US, we previously found evidence that Developmental dyslexia (DD [MIM 600202]), or read- variation in the KIAA0319 gene confers risk of ing disability, is a relatively common, complex dyslexia.17–19 A 77 kb region was found to be asso- cognitive disorder that affects 5–10% of school-aged ciated with dyslexia in two independent samples of children.1 The disorder is characterized by an nuclear families from the UK (hereafter referred to as impairment of reading performance despite adequate the Oxford sample) and the US.17 The region motivational, educational and intellectual opportu- extended from the first four exons of KIAA0319 to nities. Although the pathophysiology of DD is the first exon of THEM2, covering the entire TTRAP unknown, there is strong evidence that genes make gene. A specific three-SNP haplotype across this a substantial contribution to individual variation in region was significantly associated with a range of risk of DD, with twin studies reporting heritability reading-related measures in both the Oxford UK and estimates of up to 0.71.1–8 Linkage studies have US samples, particularly when the samples were identified several genomic regions that may harbor selected for severity of phenotype. In a comple- susceptibility quantitative trait loci (QTL) for DD, and mentary study, Cope et al.18 employed a case/control the most consistently replicated of these is DYX2, sample in which a severe definition of dyslexia was which lies on chromosome 6p22.2.1,9–16 used to ascertain probands (hereafter referred to as the A number of association studies have now been Cardiff sample). By performing a systematic, high- performed attempting to identify the DYX2 suscept- density linkage disequilibrium screen of candidate genes within the DYX2 region, we found that Correspondence: Professor J Williams, Department of Psycholo- variation in the KIAA0319 gene was most strongly gical Medicine, Henry Wellcome Building, Cardiff University, implicated in dyslexia susceptibility. Subsequent Heath Park, Cardiff CF14 4XN, UK. functional analysis showed that the risk haplotype E-mail: [email protected] identified by Francks et al.17 is associated with a 6These authors contributed equally to this paper. Received 13 July 2006; revised 31 August 2006; accepted 5 reduction of expression of KIAA0319, but does not September 2006; published online 10 October 2006 affect either THEM2 or TTRAP.19 KIAA0319, DCDC2 and DD D Harold et al 1086 Two recent studies have indicated that another gene et al.17 As previous evidence had indicated that the within the DYX2 QTL, DCDC2, may also play a role in DYX2 locus influences the most severely affected developmental dyslexia. Meng et al.20 identified two with reading disability,17,21,25 we used the same sub- SNPs within DCDC2 (rs807724 and rs1087266) asso- sample of sibships as in Francks et al.,17 that is ciated with a weighted composite measure of reading scoring below a mean measure calculated on phono- and spelling subtests (discriminant score) in a sample logical decoding ability and orthographic coding, of US families. They also described an intronic which were the two measures contributing most to deletion containing a compound simple tandem the linkage signal. This selection yielded 126 families, repeat (STR). The deletion, in combination with 10 including 313 siblings. For the Cardiff sample, rare STR alleles, showed significant association with children with DD were ascertained in Wales and different reading traits. Using a categorical definition England through contacts with local education autho- of dyslexia based on spelling impairment, Schuma- rities and schools specializing in the education of cher et al.21 also found association with variation children with reading difficulties. The inclusion within the DCDC2 gene in two independent trio criteria for probands were an IQ of X85 and a reading samples of German descent, most significantly with a age X2.5 years behind that expected from chrono two-marker haplotype in intron 7 comprised of the logical age. The accuracy score from the Neale SNPs rs793862 and rs807701. This association ap- analysis of reading ability26 was used to determine peared strongest in the most severely affected patients reading age, except when probands were aged > 13 of both samples. years, in which case the accuracy score of British Given the evidence implicating both the KIAA0319 Ability Scale (BAS) single-word reading was used.27 and DCDC2 genes in developmental dyslexia, we Children classed as controls were required to have an aimed to study this genomic region in more detail, in IQ of X85 and a reading delay of no more than 6 two large, independent UK samples. We have analyzed months. A total of 350 cases and 273 controls were a new selection of SNPs covering KIAA0319 and included. As probands were ascertained on the basis DCDC2, including the DCDC2 markers that showed of a severe definition of dyslexia, no further pheno- significant association with DD in previous studies.20,21 typic selection was performed for genetic analysis. The READ measure in the Oxford sample (which is based on BAS single-word reading) is most similar to Materials and methods the Cardiff definition of dyslexia. Comparison of Subjects standardized BAS single-word reading scores in the Individuals with DD were ascertained as previously severe Oxford sibships and all Cardiff cases indicated described.17,18 Briefly, the Oxford sample included a similar level of reading disability (data not shown). 264 unrelated nuclear families identified from the All participants in both samples were collected dyslexia clinic at the Royal Berkshire Hospital in within the UK and were of white European descent. Reading, UK. The majority of families were recruited Ethical approval for the Oxford and Cardiff sample on the basis of having at least one proband whose was obtained from the NHS Research Ethics Commit- single-word reading ability was > 2 s.d. below that tee and the Multi-Centre Research Ethics Committee predicted by tests of verbal or nonverbal reasoning22,23 for Wales, respectively. Appropriate and informed and where at least one sibling displayed evidence of written consent was obtained from all participants. DD. The remaining families were recruited through probands that were required to have single-word Genotyping reading ability X1 s.d. below that predicted for their The Cardiff group identified polymorphisms within age, with a minimum IQ of 90. All probands and KIAA0319 through dbSNP or by denaturing high- siblings were administrated a battery of psychometric performance liquid chromatography as previously tests and we age-adjusted and standardized their described,28 while the Oxford group selected new scores against a normative control data set as markers that were located within multi-species con- previously described.22,23 These included the follow- served sequences (MCSs).29 SNPs with a minor ing measures used in the present study for quantita- allele frequency < 5% were included if they were of tive analysis: orthographic coding using irregular putative functional significance (e.g. lying within words (OC-irreg), phonological decoding ability MCSs), and as such could be directly associated with (PD), orthographic coding assessed by a forced word DD. SNP genotyping was performed using either the choice test (OC-choice), single-word reading ability MassARRAY system (Sequenom) or the Amplifluor (READ), spelling ability (SPELL), phonemic aware- SNPs Genotyping System (Serologicals), in accor- ness (PA), and tests of verbal (SIM) and nonverbal dance with the manufacturers’ instructions. For (MAT) reasoning. Multivariate linkage analysis had Sequenom genotyping, PCR primers and primer- previously shown that the DYX2 QTL influenced extension probes were designed with the Spectro- variability shared by all the DD measures but did not DESIGNER software.