A Theoretical Molecular Network for Dyslexia

Molecular Psychiatry (2011) 16, 365–382 & 2011 Macmillan Publishers Limited All rights reserved 1359-4184/11 www.nature.com/mp FEATURE REVIEW A theoretical molecular network for dyslexia: integrating available genetic findings G Poelmans1, JK Buitelaar1, DL Pauls2 and B Franke3,4 1Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 2Psychiatric and Neurodevelopmental Genetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; 3Department of Psychiatry, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands and 4Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

Developmental dyslexia is a common specific childhood learning disorder with a strong heritable component. Previous studies using different genetic approaches have identified several genetic loci and candidate genes for dyslexia. In this article, we have integrated the current knowledge on 14 dyslexia candidate genes suggested by cytogenetic findings, linkage and association studies. We found that 10 of the 14 dyslexia candidate genes (ROBO1, KIAA0319, KIAA0319L, S100B, DOCK4, FMR1, DIP2A, GTF2I, DYX1C1 and DCDC2) fit into a theoretical molecular network involved in neuronal migration and neurite outgrowth. Based on this, we also propose three novel dyslexia candidate genes (SLIT2, HMGB1 and VAPA) from known linkage regions, and we discuss the possible involvement of genes emerging from the two reported genome-wide association studies for reading impairment-related phenotypes in the identified network. Molecular Psychiatry (2011) 16, 365–382; doi:10.1038/mp.2010.105; published online 19 October 2010 Keywords: dyslexia; genetics; neurodevelopment; molecular network; bioinformatics

Introduction parietotemporal and ventral occipitotemporal systems. Functional brain imaging studies have Developmental dyslexia (or specific reading disabil- repeatedly demonstrated that dyslexic readers are ity) is defined as a specific and significant impair- characterized by a so-called ‘neural signature,’ in that ment in reading ability that cannot be explained by the anterior system of their left brain hemisphere is deficits in either intelligence, learning opportunity, slightly overactivated during reading tasks whereas, motivation or sensory acuity. Dyslexia is the most in contrast, the two posterior systems are under- common childhood learning disorder.1–3 activated.9,10 The prevalence of dyslexia ranges from 3.6% in Evidence from family and twin studies shows that primary school children in The Netherlands4 to as dyslexia is a highly heritable disorder, and up to 75% high as 10% in US school-aged children,5 which is of the phenotypic variance can be explained by likely due to the fact that many different psycho- genetic factors.2–4 Dyslexia behaves as a multifactorial metric tests and diagnostic criteria for dyslexia exist (or complex) disorder, in which combinations of (Box 1).2,3,6 Twice as many boys as girls are affected genetic and environmental factors contribute to with dyslexia.4,7,8 disease risk. This article will concentrate on the Results from functional magnetic resonance ima- genetic factors involved in dyslexia etiology. The ging studies and studies using other functional brain genetic model underlying most cases of dyslexia is imaging modalities (such as event-related potentials likely one in which several to multiple genetic and magnetoencephalography), indicate that three factors, all of small or moderate individual effect regions in the left brain hemisphere are important for size, contribute to disease risk.3,5 In the current study, fluent reading (for recent reviews, see Shaywitz we reviewed the available evidence for dyslexia loci and Shaywitz9 and Hoskyn10). These regions are an and genes, including linkage studies (candidate gene ‘anterior’ system, that is the inferior frontal gyrus based) association studies and reports of chromoso- (Broca’s area), and two ‘posterior’ systems, the dorsal mal aberrations cosegregating with dyslexia. We found that 10 of the 14 dyslexia candidate genes that Correspondence: Dr B Franke, Department of Human Genetics, were suggested by the literature had been shown to 855, Radboud University Nijmegen Medical Centre, P.O. Box directly or indirectly interact and could be integrated 9101, 6500 HB Nijmegen, The Netherlands. into a molecular signaling network contributing to E-mail: [email protected] Received 5 March 2010; revised 10 August 2010; accepted 22 dyslexia etiology, responsible for regulating neuronal September 2010; published online 19 October 2010 migration and neurite outgrowth. Our work builds on A theoretical molecular network for dyslexia G Poelmans et al 366 Box 1 Diagnosis of dyslexia been spent in trying to identify the dyslexia genes in a number of these regions by conducting candidate Many different psychometric tests and diagnostic criteria gene association studies, which will be discussed for dyslexia exist. For a recent review of the different 6 below and in Tables 2 and 3. For additional reviews of definitions and tests for dyslexia, we refer to Fletcher. Most the candidate gene association literature of dyslexia, of the linkage and association studies we refer to in this we also refer to Petryshen and Pauls,5 Scerri and article make use of five measures of the normal reading 8 40 process, that is single word reading (SWR), spelling, Schulte-Korne and Schumacher et al. phoneme awareness (PA), phonological decoding (PD) and Until now, no genome-wide association studies orthographic coding (OC).2,3 Specific tests have been (GWAS) for developmental dyslexia have been pub- developed for each of these five reading measures2 and are lished. However, Meaburn et al.41 reported a GWAS of used for the diagnosis of dyslexia. PA is the ability to reflect early reading (dis)ability in the general population on and manipulate the phonemes of words. PD could be using a quantitative trait loci approach. In addition, defined as the skill to convert written sublexical letter units the results of a GWAS for an electrophysiological (or graphemes) into their corresponding phonemes. The OC endophenotype of dyslexia were reported.42 reading component reflects the ability to recognize the specific letter patterns (or orthography) of whole words. OC DYX1 locus (15q21) is a particularly important skill in English, in which B frequently the same word sounds can be represented by Although located 7 Mb distal of the maximal 43,44 different letter patterns.2 It should be noted that linkage peak in the DYX1 locus, the DYX1C1 orthographic coding is less difficult in Dutch (or another gene was identified as the dyslexia candidate in the ‘open’ language like German or Italian) than in English, as 15q21 locus. In 2003, Taipale et al.43,45 reported the the Dutch language contains (much) less irregularities of mapping of DYX1C1 from the breakpoint of a grapheme to phoneme correspondences than exist in translocation (t(2;15)(q11;q21)) in four dyslexic mem- English. bers of an earlier described family. In the original Based on the five reading measures mentioned above, one article describing the mapping, nominally significant commonly used scheme for diagnosing dyslexia uses cutoffs genetic association was also reported between alleles of a standard score of below À2 s.d. in children and a standard score of below À1.5 s.d. in adults for at least two of of two single nucleotide polymorphisms (SNPs) in the the five measures. Apart from frequent impairments in SWR gene (rs3743205A and rs57809907T) and dyslexia. A and/or spelling, dyslexics usually show deficits in one or haplotype of these two alleles was associated with more of PA, PD and OC skills. Dyslexia should therefore be dyslexia as well.43 However, efforts to replicate these regarded as a continuum in which often, several measures original associations have produced mixed and some- of the normal reading process are affected to some extent. times conflicting results (Table 2). Four subsequent Moreover, the different reading measures seem to be association studies failed to replicate an association 2,3 (genetically) correlated with one another and with IQ. of any allele of rs3743205 and rs57809907 with dyslexia,46–49 whereas three other studies found a (nominally) significant association between dyslexia findings of other researchers in the field of dyslexia and/or specific reading measures and the opposite genetics,5,11–13 as well as existing knowledge about allele of rs374320550,51 and/or rs57809907.50–52 One genes involved in neuronal migration disorders study reported association for the two-SNP haplotype and the KEGG pathway for axon guidance (http:// that was reported in the original mapping study of www.genome.jp/dbget-bin/show_pathway?map043600). DYX1C1,53 and more recently, a haplotype of three Moreover, based on the signaling network, we SNPs including rs3743205 was found associated with propose three novel dyslexia candidate genes from dyslexia in female probands.54 The (nominally) linkage regions. significant genetic association between the DYX1C1 missense variant rs17819126 and three reading and spelling measures was also reported55 (Table 2). Linkage and association studies Because of the above-described association results, As shown in Table 1, a substantial number of genetic several researchers in the dyslexia genetics field now linkage studies for developmental dyslexia have been submit that either another dyslexia candidate gene in carried out. At least 11 loci have been repeatedly DYX1 exists that remains to be discovered or that linked to dyslexia and/or measures of the reading different causative variants in DYX1C1 might exist process known to be disturbed in dyslexia (Box 1), between different (language) populations.8,56 on chromosomes 1p35–1p36 (DYX8),14–18 2p11–2p16 Nevertheless, functional evidence points to (DYX3),2,17,19–24 2q22.3,25,26 3p12–3q13 (DYX5),2,18,27 DYX1C1 as a viable candidate for dyslexia etiology: 6p22 (DYX2),1,2,18,28–30 6q12–q15 (DYX4),31,32 DYX1C1 encodes DYX1C1 (alternative name: EKN1), 7q32,22,31 11q13.4,25,26 15q15–15q21 (DYX1),1,18,33–37 a cytoplasmic and nuclear protein containing three 18p11.2 (DYX6)2,31,38 and Xq26–Xq28 (DYX9).2,4,18,31 tetratricopeptide repeat domains, known protein– Additional (suggestive) linkage findings for chromo- protein interaction modules. DYX1C1 is expressed some regions 4p15.33–4p15.32,31 11p15.5 (DYX7),39 in several tissues, including the brain, where it 12q13.13–12q21.33,26 13q12.13–13q12.3,26 13q22.1,2 localizes to cortical neurons and white matter glial 17p13.3,31 18q22.2–18q22.32 and 21q21–21q222 still cells.43,57 In the promoter region of DYX1C1, a binding await replication (Table 1). Considerable effort has site for the ELK1 and TFII-I transcription factors is

Molecular Psychiatry A theoretical molecular network for dyslexia G Poelmans et al 367 Table 1 Linkage studies for developmental dyslexia and/or different measures of the reading process (Box 1)

Locus Markera Sample Phenotype Reported level Loci examined Reference origin of significanceb

1p35–1p36 D1S253 USA SWR Significant 1p36–1q23, 15 (DYX8) 6p21.3–6p23 D1S507, D1S199 USA PD Significant 1p36–1q23, 15 6p21.3–6p23 D1S507 Canada Dyslexia Replication 1p34–1p36 16 D1S507, D1S199 South Africac Dyslexia Replication Replication study 18 D1S552–D1S1622 Canada Dyslexia Significant 1p34–1p36 16 D1S199–D1S478 USA Dyslexia Suggestive Various loci 14 D1S199 The Netherlands Dyslexia Replication Replication study 17 MATN1 USA PA Significant 1p36–1q23 15 2p11–2p16 D2S2352–D2S378– Norway Dyslexia Significant Genome scan 19 (DYX3) D2S1337 D2S2352–D2S378– Canada PD Replication Replication study 20 D2S1337 D2S378 The Netherlands NWR Replication Replication study 17 D2S337–D2S286 USA Dyslexia Suggestive 2p12–2p16 21 D2S2368 USA PA Suggestive QTL genome scan 2 D2S286 Finland Dyslexia Replication Genome scan 22 rs917235 Finland Dyslexia Significant 2p12 24 rs917235 Germany Dyslexia Significant 2p12 24 D2S2114 USA PA, PD, OC Suggestive 2p12–2p16 21 rs730148 Finland Dyslexia Significant 2p12 24 rs730148 Germany Dyslexia Significant 2p12 24 D2S2216 Finland Dyslexia Significant Genome scan 22 D2S2216 Finland Dyslexia Replication 2p11–2p12 23 2q22.3 D2S1399 USA PDE Significant Genome scan 25 D2S1399 USA SWE Replication Genome scan 26 3p12–3q13 D3S1595–D3S3655 Finland Dyslexia Significant Genome scan 27 (DYX5) D3S1278 USA PD Significant QTL genome scan 2 D3S1278 South Africac Dyslexia Replication Replication study 18 4p15.33–4p15.32 D4S403 Australia IWR Suggestive Genome scan 31 D4S2633 Australia IWS Suggestive Genome scan 31 6p22 (DYX2) D6S461, D6S299 USA PA Significant 1p36–1q23, 1 6p21.3–6p23 D6S461 USA Dyslexia Replication 6p21.3–6p22 29 D6S299 UK PD Suggestive QTL genome scan 2 D6S299 South Africac Dyslexia Replication Replication study 18 JA04 USA Dyslexia Significant 6p21.3–6p22 29 D6S1554 USA Dyslexia Significant 6p22.3 30 6q12–6q15 D6S965 Canada PD Significant Chromosome 6 32 (DYX4) D6S462 Australia IWS Suggestive Genome scan 31 7q32 D7S530 Finland Dyslexia Significant Genome scan 22 D7S530 Australia NWS Replication Genome scan 31 11p15.5 HRAS–DRD4 Canada PD Significant 11p15.5 39 (DYX7) 11q13.4 D11S1314 USA PDE Suggestive Genome scan 25 D11S1314 USA SWE Replication Genome scan 26 12q13.13–12q21.33 D12S297 USA WID Suggestive Genome scan 26 D12S1064 USA SWE Suggestive Genome scan 26 13q12.13–13q12.3 D13S1304–ATA5A09 USA SWE Significant Genome scan 26 13q22.1 D13S156 USA OC Suggestive QTL genome scan 2 15q15–15q21 D15S994 UK Dyslexia Significant 15q 35 (DYX1) D15S143 USA SWR Significant 15pter–15qter 1 D15S143 Germany Spelling Replication Chromosome 15 34 D15S143 USA WID Replication Replication study 36 D15S143 Germany Dyslexia Replication Replication study 37 D15S143 South Africac Dyslexia Replication Replication study 18 17p13.3 D17S831 Australia IWS Suggestive Genome scan 31

Molecular Psychiatry A theoretical molecular network for dyslexia G Poelmans et al 368 Table 1 Continued

Locus Markera Sample Phenotype Reported level Loci examined Reference origin of significanceb

18p11.2 D18S464 UK SWR Significant QTL genome scan 2 (DYX6) D18S464 Australia RWR Replication Genome scan 31 D18S53 USA SWR Significant QTL genome scan 2 D18S53 USA Reading ability Replication Genome scand38 18q22.2–18q22.3 D18S61–D18S1161 USA SWR Significant QTL genome scan 2 D18S61–D18S1161 USA PD, OC Suggestive QTL genome scan 2 21q21–21q22 D21S1899–D21S266 USA OC Significant QTL genome scan 2 Xq26–Xq28 DXS1047 UK SWR Suggestive QTL genome scan 2 (DYX9) DXS1047 South Africac Dyslexia Replication Replication study 18 DXS1227–DXS8091 The Netherlands Dyslexia Significant Genome scan 4 DXS1227, DXS8091 Australia NWS Replication Genome scan 31 DXS1227 South Africac Dyslexia Replication Replication study 18

Abbreviations: IWR, irregular word reading; IWS, irregular word spelling; NWR, nonword reading; NWS, nonword spelling; OC, orthographic coding; PA, phoneme awareness; PD, phonological decoding; PDE, phonological decoding efficiency; QTL, quantitative trait loci; RWR, regular word reading; SWE, single word reading efficiency; SWR, single word reading; WID, word identification, which is a measure of single word reading accuracy. The genetic markers and single nucleotide polymorphisms (SNPs) that were found in more than one linkage study are indicated in bold. aAll markers are presented in the order they are localized on the chromosome (i.e. from centromeric to telomeric). bReplication indicates that at least suggestive evidence of linkage was found for the marker(s) that were identified in the original linkage study. cThis linkage study was conducted on a sample of Afrikaans-speaking/reading children with dyslexia of Dutch descent. dPerformance on the Wide-Range Achievement Test (WRAT), a test of reading ability, was linked to D18S53 in the Framingham sample.

present.43 This binding site contains rs3743205,44 one be important in brain development and neuronal of the SNPs associated with dyslexia in the original processes such as neuronal migration and synaptic DYX1C1 study.43 Experiments in rats using in utero plasticity.62 In addition, ERS1 and ERS2 are thought RNA interference against Dyx1c1 showed that the to be involved in cognitive processes such as learning gene product has a role in neuronal migration (that is and memory,62 which seem partially impaired in the migration of whole neurons to target regions dyslexia.63 during cerebral cortex development) in the developing neocortex. For this, the C-terminus of Dyx1c1 DYX2 locus (6p22) (which contains the three tetratricopeptide repeat A cluster of five genes at this locus (VMP, DCDC2, domains) is necessary and sufficient.57 The neuronal KIAA0319, TTRAP and THEM2) has been associated migration anomalies resulting from the in utero with dyslexia.30,64 Of these five genes, genetic variants RNA interference in rats appeared similar to those in DCDC2, KIAA0319 and TTRAP were reported to be observed in a relatively small set (n = 8) of post- associated with the disorder more than once3,52,65–77 mortem brains in humans with dyslexia (see Rosen (Table 3). et al.58 and references herein). Moreover, in rats, In 2005, DCDC2 was identified as a dyslexia these neuronal migration anomalies were shown to candidate gene in a fine mapping association study lead to distinct impairments in auditory processing of the DYX2 locus.65 The study reported a 2445-bp and spatial learning that have also been suggested deletion in intron 2 of DCDC2 that harbors a to have a role in dyslexia.58,59 Apart from its clear compound short tandem repeat polymorphism con- role in neuronal migration, several alternatively sisting of variable copy numbers of repeat units. spliced transcript variants of DYX1C1 appear to be Moreover, the combined allele of the deletion and biomarkers for colorectal cancer.60 In addition, minor alleles of the compound short tandem repeat through one of its tetratricopeptide repeat domains, marker (that is alleles with frequencies of < 5%) the DYX1C1 protein functions as a cochaperone yielded significant association with reading perfor- by interacting with HSP70 (heat shock protein 70) mance.65 However, a later association study failed to and HSP90 (heat shock protein 90) proteins in breast replicate this finding in a German dyslexia sample.66 cancer cells.61 Finally, in hippocampal neurons, Another study reported a haplotype consisting of DYX1C1 interacts with both the nuclear and cyto- specific alleles of two SNPs in intron 7 of DCDC2 plasmic estrogen receptors a (ESR1) and b (ESR2)62 (rs793862 and rs807701) to be associated with (see below). ESR1 and ESR2 have been recognized to dyslexia67 (Table 3). In 2007, the 2445-bp deletion in

Molecular Psychiatry A theoretical molecular network for dyslexia G Poelmans et al 369 Table 2 Reported associations of DYX1C1 at DYX1 with reading measures and dyslexia

Gene Study Sample Phenotype SNP and/or haplotype Reported level Reference population of significance origin

DYX1C1 Finland 109 cases and Dyslexia rs3743205 (À3A) P = 0.016 43 195 controls Dyslexia rs57809907 (1249T) P = 0.048 43 Dyslexia rs3743205 (À3A): P = 0.015 43 rs57809907(1249T) UK 264 families Dyslexia rs3743205 (À3G > A) Not significant 50 OC rs57809907 (1249G) P = 0.021 50 OC rs3743205 (À3G): P = 0.014 50 rs57809907 (1249G) Canada 148 families SWR rs3743205 (À3G) P = 0.027 51 WID rs3743205 (À3G) P = 0.032 51 Spelling rs3743205 (À3G) P = 0.027 51 PA rs3743205 (À3G) P = 0.023 51 PD rs3743205 (À3G) P = 0.023 51 Dyslexia rs57809907 (1249G > T) Not significant 51 Dyslexia rs3743205 (À3G): P = 0.026 51 rs57809907(1249G) UK 247 trios Dyslexia rs3743205;rs57809907; Not significant 46 rs3743205:rs57809907 Italy 158 families Dyslexia rs3743205;rs57809907; Not significant 47 rs3743205:rs57809907 USA 150 families Dyslexia rs3743205;rs57809907; Not significant 48 rs3743205:rs57809907 Italy 57 cases and Dyslexia rs3743205;rs57809907; Not significant 49 97 controls rs3743205:rs57809907 USA 191 trios Dyslexia rs3743205 (À3G > A) Not significant 52 Dyslexia rs57809907 (1249G) P = 0.040 52 Dyslexia rs3743205 (À3G > A): Not significant 52 rs57809907 (1249G > T) Italy 212 families Dyslexia rs3743205 (À3G > A) Not significant 53 Dyslexia rs57809907 (1249G > T) Not significant 53 Dyslexia rs3743205 (À3A): P = 0.047 53 rs57809907 (1249T) Germany 366 trios Dyslexia rs3743205 (À3G) P = 0.006 54 Australia 790 families IWR rs17819126 P = 0.020 55 NWR rs17819126 P = 0.0003 55 IWS rs17819126 P = 0.009 55

Abbreviations: IWR, irregular word reading; IWS, irregular word spelling; NWR, nonword reading; OC, orthographic coding; PA, phoneme awareness; PD, phonological decoding; SNP, single nucleotide polymorphism; SWR, single word reading; WID, word identification, which is a measure of single word reading accuracy. intron 2 of DCDC2 was reported to be nominally spelling measures in a general population sample70 associated with single word reading efficiency and (Table 3). spelling,52 and subsequently, healthy individuals DCDC2 is part of an 11-member doublecortin heterozygous for the 2445-bp deletion were reported (DCX)-repeat gene family, including the DCX gene, to exhibit significantly larger gray matter volumes in which is involved in neuronal migration and mutated reading/language-related brain regions.68 More in X-linked lissencephaly and double cortex syn- recently, the association findings for the intron 2 drome, two neuronal migration disorders.65,78,79 deletion as well as the two intron 7 SNPs were DCDC2 encodes a protein with two DCX domains. replicated in an independent case–control cohort.69 The known function of DCX domains is that they The same study also reported an association with associate with, bundle and stabilize neuronal micro- dyslexia for another SNP, in intron 6 of DCDC2 tubules, which are essential for neurite outgrowth and (rs807724), and for a risk haplotype consisting of the neuronal migration.12,13,69,78–80 The DCX domains are deletion in DCDC2 and specific alleles of rs807724, also ‘regulated’ through phosphorylation by MAP rs793862 and rs807701.69 Very recently, two other kinase proteins such as the extracellular signal- intronic SNPs in DCDC2 (rs1419228 and rs1091047) regulated kinase 1 (ERK1) and 2 (ERK2) proteins.80 were also reported to be associated with reading and DCDC2 is expressed in many organs and tissues,

Molecular Psychiatry A theoretical molecular network for dyslexia G Poelmans et al 370 Table 3 Reported associations of DCDC2, KIAA0319 and TTRAP at DYX2 with reading measures and dyslexia

Gene Study Sample Phenotype SNP and/or haplotype Reported level Reference population of significance origin

DCDC2 USA 153 families Reading 2445 bp del:compound P = 0.00002 65 STR minor alleles German 396 trios Dyslexia 2445 bp del:compound Not significant 66 STR minor alleles German 239 trios Dyslexia rs793862:rs807701 P = 0.001 67 USA 191 trios SWE 2445 bp del P = 0.046 52 Spelling 2445 bp del P = 0.035 52 German 72 cases and Dyslexia 2445 bp del P < 0.001 69 184 controls Dyslexia rs807724 P = 0.044 69 Dyslexia rs793862 P = 0.038 69 Dyslexia rs807701 P = 0.019 69 Dyslexia 2445 bp del:rs807724: P < 0.050 69 rs793862:rs807701 Australian 522 families RWR rs1419228 P = 0.002 70 IWR rs1419228 P = 0.004 70 Spelling rs1419228 P = 0.002 70 IWR rs1091047 P = 0.003 70 KIAA0319 UK 126 families SWR rs4504469:rs2038137: P = 0.0024 3 rs2143340 Spelling rs4504469:rs2038137: P = 0.025 3 rs2143340 OC rs4504469:rs2038137: P = 0.00007 3 rs2143340 US 124 families SWR rs4504469:rs2038137: P = 0.017 3 rs2143340 Spelling rs4504469:rs2038137: P = 0.037 3 rs2143340 PA rs4504469:rs2038137: P = 0.018 3 rs2143340 UK 248 cases and Dyslexia rs4504469 P = 0.002 71 273 controls Dyslexia rs2038137 P = 0.001 71 UK 126 families SWR rs4504469 P = 0.004 72 PA rs4504469 P = 0.010 72 PD rs4504469 P = 0.008 72 OC rs4504469 P = 0.001 72 SWR rs2038137 P = 0.0002 72 Spelling rs2038137 P = 0.006 72 PD rs2038137 P = 0.003 72 OC rs2038137 P = 0.001 72 UK 350 cases and Dyslexia rs4504469 P = 0.005 72 273 controls Dyslexia rs2038137 P = 0.005 72 Australia 440 families SWR rs4504469:rs2038137: P = 0.04 73 rs2143340 TTRAP UK B6000 childrena Reading rs2143340 P = 0.001 74 Spelling rs2143340 P = 0.008 74

Abbreviations: IWR, irregular word reading; OC, orthographic coding; PA, phoneme awareness; PD, phonological decoding; RWR, regular word reading; SNP, single nucleotide polymorphism; SWE, single word reading efficiency; SWR, single word reading. aTested for reading and spelling; sample ranges from 5254 to 7090 children depending on the phenotype measured.

including the brain. Within the human brain, DCDC2 migration anomalies that are similar to those seen in a is expressed, among others, in regions that are small set of post-mortem brains in humans with implicated in reading ability.65,78,79,81 Finally, as with dyslexia (see Rosen et al.58 and references herein65,81). DYX1C1 (see above), in utero RNA interference KIAA0319 was identified as a dyslexia candidate against the rat homolog of DCDC2 results in neuronal gene in two independent positional association

Molecular Psychiatry A theoretical molecular network for dyslexia G Poelmans et al 371 studies of the 6p22 DYX2 locus.3,71 Francks et al.3 An additional putative dyslexia gene on 6p22 is identified a three-SNP risk haplotype consisting TTRAP. As indicated above and in Table 3, rs2143340, of specific alleles of two SNPs (rs4504469 and an SNP in intron 2 of TTRAP, has yielded association rs2038137) in KIAA0319 and rs2143340 in intron 2 with variation in spelling73 and reading73,74 ability in of TTRAP that yielded association with several the general population. Moreover, this SNP is a part of reading measures in UK and US samples of families a three-SNP risk haplotype associated with dyslexia with dyslexic individuals (Table 3). Association with in studies of the KIAA0319 gene.3,75 However, as the rs4504469, rs203813771,72 and the three-SNP risk association findings for rs2143340 now seem to be haplotype73 was replicated in subsequent studies, due to its linkage disequilibrium with rs9461045 whereas the rs2143340 SNP was also shown to be affecting the expression of KIAA031976 (see above), associated with normal variation in reading and a role of TTRAP in dyslexia etiology has become spelling ability in the general population74 (Table 3). unlikely. In addition, the risk haplotype containing rs2143340 The two other genes in the cluster of five genes was associated with > 40% lower expression of at the DYX2 locus, that is VMP and THEM2, have KIAA0319 in lymphoblastoid cell lines from unaf- not been repeatedly associated with developmental fected individuals and in neuroblastoma cell lines.75 dyslexia. Recently, an SNP in the promoter region of KIAA0319, rs9461045, that is in complete linkage disequilibrium DYX3 locus (2p11–p16) with rs2143340 (and hence the risk haplotype for In 2002, Francks et al.21 tested the SEMA4F and OTX1 dyslexia) was shown to create a protein-binding site genes in the DYX3 locus for association with dyslexia. for the silencing transcription factor OCT-1.76 SEMA4F belongs to a protein family with a role in KIAA0319 expression from the risk haplotype axonal guidance, and the OTX1 protein is a transcrip- increased upon small interfering (si-)RNA-mediated tion factor involved in forebrain morphogenesis and knockdown of the OCT-1 gene in a neuronal cell line, cortex differentiation.21 However, no association with which implies that rs9461045 is the functional dyslexia was found.21 variant causing reduced expression of KIAA0319.76 In 2007, the C2ORF3 and MRPL19 genes were Interestingly, nominally significant genetic interac- identified by positional candidate association map- tion effects for the DCDC2 and KIAA0319 genes were ping of the DYX3 locus,24 though these data still reported and replicated, which suggests that both await replication. Both the C2ORF3 and MRPL19 genes independently contribute to dyslexia risk.72,77 genes are highly expressed in the adult and fetal KIAA0319 encodes a protein that consists of an human brain and although the function of both genes extracellular part with a MANSC (motif at N-terminus is unknown, it is interesting to note that C2ORF3 with seven cysteines) domain and several overlap- expression was found to correlate across brain regions ping fibronectin type III (FN III) and polycystic with that of DYX1C1, DCDC2 and ROBO1, whereas kidney disease (PKD) domains, a transmembrane MRPL19 expression was most highly correlated with domain and a cytoplasmic part (http://www.ensembl. the expression of KIAA0319.24 org/homo_sapiens). Expression of KIAA0319 is specific to the brain, with highest levels in the cerebral DYX5 locus (3p12–q13) cortex, hippocampus CA3 and dentate gyrus, puta- The ROBO1 gene was identified as the dyslexia men, amygdala and cerebellum of the adult human candidate gene in this locus through its position at brain.75,76,82 PKD domains have been implicated in the 3p12 translocation breakpoint of an individual cell–cell adhesion processes, which led to the with dyslexia carrying a translocation between chro- hypothesis that the KIAA0319 PKD domains have a mosomes 3 and 8 (t(3;8)(p12;q11)).85 Further exam- role in the adhesion between neurons and glial fibers ination of ROBO1 SNPs and haplotypes in a four- during neuronal migration.11,13,82 generation family determined that a haplotype cose- In utero RNA interference against the rat homolog gregated with dyslexia in 19 of 21 genotyped dyslexic of KIAA0319 indeed results in neuronal migration family members.85 To date, there has been no anomalies.75 Recently, embryonic knockdown of independent replication of this finding. Kiaa0319 in rats was found to result in significant The function of ROBO1—a gene that is highly arrest of neuronal migration and subsequently causes expressed in the brain—in brain development and specific types of neuronal migration anomalies in the neuronal migration is well established. ROBO1 postnatal rat brain whereas, in contrast, embryonic encodes a member of the ROBO family of axonal overexpression of Kiaa0319 does not cause gross guidance receptors. ROBO1 and other ROBO neuronal migration anomalies.83 Lastly, apart from a family proteins consist of an extracellular part main variant (variant A) of KIAA0319, two alterna- with five immunoglobulin (Ig) and three FN III tively spliced products of the gene exist (variants B domains, a transmembrane domain and a cytoplasmic and C), which encode KIAA0319 isoforms that lack a part (http://www.ensembl.org/homo_sapiens). The transmembrane domain. Isoform B was found to be ROBO (or Roundabout homolog) proteins are recep- secreted, suggesting that KIAA0319 could be involved tors for so-called SLIT proteins. not only in cell–cell interactions, but also in extra- These interact with the netrin proteins and their cellular signaling.82,84 receptor, DCC (deleted in colorectal carcinoma), to

Molecular Psychiatry A theoretical molecular network for dyslexia G Poelmans et al 372 regulate the direction and rate of axon/neurite out- Chromosomal aberrations growth85,86 (see below). Several chromosomal aberrations have been reported DYX8 locus (1p35–p36) to cosegregate with dyslexia. As reported above, the So far, only one candidate gene in this locus was DYX1C1 and ROBO1 genes in the DYX1 and DYX5 investigated. An association study of the KIAA0319L loci, respectively, were identified through their posi- gene using five tagging SNPs in a Canadian sample of tion in the breakpoints of translocations in dyslexic 291 nuclear families ascertained through a proband individuals. with reading difficulties found nominally significant Our own group recently reported PCNT, DIP2A, S100B evidence for association with dyslexia for one SNP and PRMT2 to be (partially) deleted in a small deletion (rs7523017; P = 0.042) and a haplotype (P = 0.032) in of chromosome band 21q22.3 that cosegregated with KIAA0319L in a subset of the sample (n = 156 dyslexia in a father and his three affected sons.56 Two families).87 This finding still awaits replication. of these genes seem valid candidate genes for dyslexia. KIAA0319L is expressed in several brain regions DIP2A encodes the ‘disco-interacting protein 2 (http://biogps.gnf.org/#goto = genereport&id = 79932) homolog A’ (DIP2A).95 (In our original report,56 based and although its function is still unknown, the gene on incorrect gene annotation information, we identi- has a high-protein sequence identity to KIAA0319 fied this gene as encoding the ‘DLX-interacting (http://www.ensembl.org/homo_sapiens), and may protein 2’, which is an alternatively spliced human therefore have similar functions. variant of glutamate receptor interacting protein 1.101) The Drosophila homolog of DIP2A is involved in DYX9 locus (Xq26–Xq28) neuronal connectivity in the visual system.102 In This chromosome region contains 15 known protein- humans, DIP2A is a nuclear protein that is ubiqui- coding genes (http://www.ensembl.org/homo_sapiens), tously expressed. In a complex with the transcription and due to their very high homology to the SLIT factors DMAP1,95,103 DNMT1 and HDAC2,103 it nega- proteins88 that bind ROBO1 (see above), SLITRK2 and tively regulates neurite outgrowth104 and synaptic SLITRK4 in the region have been hypothesized to be plasticity.105 Synaptic plasticity is crucial for cogni- dyslexia candidate genes.85 However, no mutations in tive processes such as learning and memory,105 which the coding regions of these genes nor those of CXORF1 seem partially impaired in dyslexia (see above).63 or FMR1, two other brain-expressed genes in the region, The S100B gene is mainly and highly expressed in were identified in a family showing linkage to the the brain (especially in the hippocampus).106 S100B DXS1227–DXS8091 region.4 That being said, the encodes a calcium-binding peptide that is mainly 50 UTR trinucleotide repeat in FMR1, which—when produced by astrocytes and that exerts paracrine and expanded—leads to Fragile X syndrome, the most autocrine effects on neurons and glial cells. S100B is common inherited form of intellectual disability, was known to bind to the receptor for advanced glycation not investigated in this family. Fragile X syndrome end products (RAGE) protein in (hippocampal) is often accompanied by deficient reading skills.89 neurons, which activates a signaling pathway result- Moreover, 8 of 15 investigated females with fragile X ing in neurite outgrowth.107 The protein has been syndrome and a normal intelligence were reported to reported to be involved in modulating synaptic have dyslexia,90 and males carrying a so-called FMR1 plasticity,108 although the mechanism by which this ‘premutation’—who are normally intelligent as well— is brought about is currently unknown.106 The con- display specific verbal working memory deficits91 often centration of the S100B peptide is increased in blood found to be present in dyslexics.63 and cerebrospinal fluid in various clinical brain FMR1 activates/phosphorylates the MAP kinase conditions, including psychiatric disorders such as proteins ERK1 and ERK2 in hippocampal neurons.92 schizophrenia and Tourette syndrome.106 In addition, FMR1 acts as an RNA-binding protein In a very recent study, a microdeletion of the involved in the localization and regulated translation DOCK4 gene was found to cosegregate—though of specific neuronal mRNAs encoding key molecules imperfectly—with dyslexia in individuals from two for neurodevelopmental processes such as synaptic independent families in a study on rare structural plasticity and neurite outgrowth,93–95 including the DNA variants predisposing to autism and/or reading mRNA of RAC1,96 a protein that has an important role impairment/dyslexia.109 in our putative dyslexia network (see below). Very DOCK4 encodes a peripheral membrane protein95 recently, direct genetic interaction was also reported that regulates dendritic growth and branching of between the Drosophila homologs of the FMR1 and hippocampal neurons110 and (neuronal) cell migra- FLNA genes.97 The human FLNA gene encodes filamin tion111 through the activation of RAC1. A, a protein that directly binds and modulates the actin A deletion of chromosome 7q11.23 results in filaments in the neuronal cytoskeleton.95,98 Mutations in Williams–Beuren syndrome (WBS), a human neuro- FLNA cause periventricular nodular heterotopia type 1, developmental syndrome that has multisystemic a neuronal migration disorder that—apart from neuro- manifestations including mild-to-moderate mental logical symptoms such as epilepsy—has been asso- retardation and cognitive deficits. WBS has also been ciated with dyslexia in the context of a normal associated with reading difficulties and ‘full’ dys- intelligence.99,100 lexia.112–114 The deletion of GTF2I as part of the WBS

Molecular Psychiatry A theoretical molecular network for dyslexia G Poelmans et al 373 deletion is responsible for the rather typical neuro- FMR1, PCNT, DIP2A, S100B, PRMT2, DOCK4 and cognitive profile associated with WBS and, more GTF2I. An analysis with the GOstat bioinformatics specifically, for the visuospatial construction deficits tool (http://gostat.wehi.edu.au/)120 showed that after that are very often seen in individuals with correction for multiple testing, 6 of the 10 most WBS.115,116 Visuospatial construction deficits are also significantly enriched gene ontology terms in these 14 found in people with dyslexia.117 Another indication candidate genes are related to nervous system devel- that GTF2I is involved in the etiology of language- opment in general and/or neurite outgrowth in related disorders is the fact that in a recent study of particular, that is generation of neurons, neurogen- people with a 7q11.23 microduplication syndrome esis, axonogenesis, neuron projection morphogenesis, involving GTF2I, all 13 subjects investigated in the neuron projection development and neuron develop- study also had some degree of specific language ment (Table 4). Whereas three genes from the set impairment,118 a disorder that is comorbid with carried gene ontology terms implicating them in dyslexia.8 In these individuals, the expression of only neurite outgrowth (that is DCDC2, ROBO1 and two genes in the duplicated region was upregulated, S100B), it was clear from the above review that more that is GTF2I and CYLN2.118 Interestingly, mice with genes could be linked to this process and the related haploinsufficiency for Cyln2, the other gene that was neurodevelopmental process of neuronal migration. upregulated in the duplicated region, have features of Therefore, we systematically searched PubMed WBS, including mild growth deficiency and deficits (http://www.ncbi.nlm.nih.gov/sites/entrez) and the in motor coordination.119 GTF2I is highly expressed Uniprot Protein Knowledgebase (http://www.uniprot. in the brain, where it has a role in normal brain org/uniprot)95 for the (proposed) function of the development. It encodes the transcription factor proteins that are encoded by the 14 dyslexia candi- TFII-I.44 As indicated above, TFII-I binds to the date genes that were described above. promoter of the DYX1C1 gene at a binding site As shown and described in (the legends of) Figures containing rs3743205,44 the SNP associated with 1 and 2, 10 of the 14 proposed dyslexia candidate dyslexia.43,50,51,53,54 TFII-I acts as an enhancer or genes (that is ROBO1, KIAA0319, KIAA0319L, S100B, suppressor of the transcription of DYX1C1 depending DOCK4, FMR1, DIP2A, GTF2I, DYX1C1 and DCDC2) on the context of other transcription factors.44 fit into a neurodevelopmental and theoretical molecular network. All the corresponding proteins could From single candidate genes to an integrated be directly or indirectly linked to processes regulating molecular network and modulating cytoskeletal microtubules and actin filaments that are involved in neuronal migration As discussed earlier, 14 genes from the literature had and/or the directed outgrowth of neurites/axons. The (at least some) evidence for involvement in the proposed network therefore fits and extends the etiology of dyslexia, that is DYX1C1, DCDC2, currently dominating hypothesis of dyslexia as a KIAA0319, C2ORF3, MRPL19, ROBO1, KIAA0319L, neuronal migration disorder.5,11–13,57–59,65,75,81–83

Table 4 Top 10 gene ontology (GO) terms significantly enriched in the 14 dyslexia candidate genes from the literature using the GOstat bioinformatics tool (http://gostat.wehi.edu.au/)120

GO term Genes TAG Significancea Adjusted significanceb

Cell projection morphogenesis PCNT, ROBO1, S100B 204 5.84EÀ05 4.05EÀ03 and organization Generation of neurons DCDC2, ROBO1, S100B 242 9.69EÀ05 5.04EÀ03 Neurogenesis DCDC2, ROBO1, S100B 262 1.22EÀ04 5.10EÀ03 Identical protein binding PRMT2, ROBO1, S100B 364 3.22EÀ04 1.12EÀ02 Nucleus C2ORF3, DIP2A, DYX1C1, FMR1, 6058 5.56EÀ04 1.37EÀ02 GTF2I, MRPL19, PRMT2, S100B Axonogenesis ROBO1, S100B 114 8.50EÀ04 1.37EÀ02 Neuron projection morphogenesis ROBO1, S100B 120 9.41EÀ04 1.37EÀ02 Intracellular organelle C2ORF3, DIP2A, DOCK4, DYX1C1, FMR1, 10763 1.05EÀ03 1.37EÀ02 GTF2I, MRPL19, PCNT, PRMT2, S100B Neuron projection development ROBO1, S100B 135 1.19EÀ03 1.37EÀ02 Neuron development ROBO1, S100B 154 1.54EÀ03 1.53EÀ02

For each GO term, the total number out of all currently annotated human genes (n = 33 972) that could be linked to this term (TAG) is indicated. The eight genes encoding proteins that could be directly placed in the theoretical molecular network for dyslexia are indicated in bold. aSingle test P-values. bMultiple test-corrected P-values using the Benjamini–Hochberg correction; only the 10 most significantly enriched GO terms with more than one gene and reaching a corrected statistical significance of P < 0.05 are shown.

Molecular Psychiatry A theoretical molecular network for dyslexia G Poelmans et al 374 In addition to the 14 candidate genes described In the DYX6 locus on 18p11.2, marker D18S464— above, the delineation of the molecular network which showed genetic linkage with (single word) enabled us to propose three additional dyslexia reading in two dyslexia linkage studies2,31 (Table 1)— candidate genes from linkage regions in which no is located immediately downstream from the VAPA candidates had been described thus far. gene, in a linkage disequilibrium block that contains

Molecular Psychiatry A theoretical molecular network for dyslexia G Poelmans et al 375 part of the VAPA gene (http://www.broad.mit.edu/ SLIT gene, that is SLIT3.129 In addition, HMGB1 mpg/haploview/). The VAPA gene is highly expressed enhances the ESR1-induced expression of genes in the brain. containing estrogen response elements130 (see The VAPA protein (other names: VAP-33 or VAMP- below). associated protein A) is an integral membrane protein In Figures 1 and 2, we have used a color code found in the (neuronal) cell membrane, membranes indicating the level of ‘evidence’ that implicates the of cellular organelles (such as the Golgi apparatus) above-described/proposed dyslexia candidates in the and associated with microtubules.121,122 VAPA was etiology of dyslexia. Proteins with relatively robust reported to have an important role in stimulating evidence for involvement in dyslexia are indicated in neurite outgrowth.123 In addition, it is involved in orange. These proteins are encoded by genes that synaptic plasticity124 and necessary for vesicular were disrupted by translocation breakpoints in dys- neurotransmission at the synapse.121,122,125 Like lexic individuals (DYX1C1 and ROBO1) and/or found DYX1C1,61 VAPA binds and forms a complex with associated with dyslexia, requiring repeated associa- HSP90, a protein with many functions including a tion findings for those genes not implicated by role in intracellular vesicle transport.122 Recently, translocations (DYX1C1, DCDC2, KIAA0319 and genetic association between several SNPs in VAPA ROBO1). Indicated in yellow are proteins with less and bipolar disorder has been reported.125 robust evidence of their involvement in dyslexia. Suggestive genetic linkage of ‘irregular word spel- These proteins are encoded by genes that were ling’ near marker D4S2633 on chromosome 4p15.32 repeatedly found to be deleted in people with was reported in the dyslexia linkage study by Bates dyslexia (PCNT, DIP2A, S100B, PRMT2, GTF2I and et al.31 (Table 1). The gene nearest to this marker is DOCK4), genes that showed (nominal) association SLIT2, which maps B1.3 Mb proximally. SLIT2 with dyslexia in only one study (C2ORF3, MRPL19 encodes a ligand of the ROBO family of axonal and KIAA0319L) and FMR1 that was mutated in guidance receptors of which the dyslexia candidate people with dyslexia. The proteins indicated in ROBO1 is a member.86,126 purple are encoded by the three novel dyslexia In the study by Igo et al.,26 linkage with ‘single word candidate genes we propose based on their location reading efficiency’ was reported for marker ATA5A09 in reported dyslexia linkage regions (VAPA, SLIT2 on 13q12.3 (Table 1). ATA5A09 is located within and HMGB1). the HMGB1 gene (http://www.ensembl.org/homo_ Signaling through the network can be initiated at sapiens). Like S100B, the encoded protein (HMGB1 the neuronal cell membrane by the interacting SLIT or Amphoterin) binds to the RAGE receptor and is and netrin axonal guidance pathways, which regulate involved in neurite outgrowth.107,127 the direction and rate of axon/neurite outgrowth HMGB1 can also directly activate the ETS1 (Figures 1a and 2). The molecular guidance ‘cues’ transcription factor,128 which upregulates another SLIT (including the dyslexia candidate SLIT2) and

Figure 1 Schematic representation of the dyslexia molecular signaling network for neurite outgrowth and neuronal migration. The color code we have used in Figures 1 and 2 to indicate the level of evidence that implicates the dyslexia candidate genes/ proteins in the etiology of dyslexia is described in more detail in the article text. In short, the orange proteins are encoded by genes for which there is relatively robust evidence. The yellow proteins represent genes for which we found less robust evidence, and the purple proteins are encoded by genes we proposed as dyslexia candidates based on their location in dyslexia linkage regions. SLIT and Netrin axonal guidance pathways at the neuronal cell membrane regulate the CDC42-extracellular signal-regulated kinase (ERK)1/2 cascade (a). This process is shown and described in more detail in (the legend of) Figure 2. Binding of S100B and HMGB1 to receptor for advanced glycation end product (RAGE) results in CDC42135 and ERK1/2132 activation (b). The latter is also induced by FMR1.92 DOCK4,110 S100B and HMGB1 binding to RAGE136,137 activate RAC1 (b), which in turn activates the transcriptional activity of NF-kB136 (c). FMR1 also activates ERK1 and ERK292 (d, e) and it is involved in the translation of the mRNA of RAC196 (d). Activated ERK1 and ERK2 (also) activate/phosphorylate the NF-kB,138 ETS1,139 ELK1140 and TFII-I141 transcription factors (c). ERK1 and ERK2 also activate/regulate DCDC280 (e). Activated NF-kB upregulates the transcription and expression of Netrin-1 (NTN1)142 and HSP90.143 It also upregulates the transcription of ELK1,144 a transcriptional activator of DYX1C1.43 ETS1 regulates the transcription of SLIT3129 and is directly activated by HMGB1128 (c). Activated (nuclear) TFII-I141 upregulates or downregulates the transcription of DYX1C1, depending on the context of other transcription factors.43,44 DIP2A binds DMAP1,102 which itself is a part of the DMAP1–DNMT1–HDAC2 repressive transcription complex.103 HDAC2 inhibits ELK195 and ESR1, the nuclear estrogen receptor a145 (c). When bound by their endogenous ligand 17b-estradiol, the nuclear ESR1 and ESR2 (estrogen receptor b) proteins—which form heterodimers— upregulate the expression of reporter genes containing estrogen response elements (ERE) by directly binding to the DNA sequences containing these ERE95 (c). This process is enhanced—for ESR1—by HMGB1130 and suppressed—for both ESR1 and ESR2—by DYX1C1.62 DYX1C1 also binds directly to nuclear ESR1 and ESR2, which promotes their proteosomal degradation62 (c). ESR1 and ESR2 are also found in the cytoplasm, where they are involved in activating ERK1/262,146 (d). The ESR–DYX1C1 complexes that promote the proteosomal degradation of ESR1 and ESR2 are also found in the cytoplasm,62 and we hypothesize that a complex of VAPA,122 HSP90 and DYX1C161 would be involved in degrading cytoplasmic ESR1 and ESR2 (d). Lastly, activated DCDC2 as well as FLNA directly bind and modulate neuronal microtubules and actin filaments65,69,78–81,95,98 (e). HSP90, heat shock protein 90; NF-kB, nuclear factor kappa-B; VAPA, VAMP-associated protein A.

Molecular Psychiatry A theoretical molecular network for dyslexia G Poelmans et al 376

Figure 2 Schematic representation of how the proteins from the Netrin and SLIT molecular guidance pathways interact to regulate the direction and rate of axon/neurite outgrowth. The color code we have used in Figures 1 and 2 to indicate the level of evidence that implicates the dyslexia candidate genes/proteins in the etiology of dyslexia is described in detail in the article text. In short, the orange proteins are encoded by genes for which there is relatively robust evidence. The yellow proteins represent genes for which we found less robust evidence, and the purple proteins are encoded by genes we proposed as dyslexia candidates based on their location in dyslexia linkage regions. In a simplified model of the interaction between the SLIT and Netrin ligands with their receptors, Netrin-1 binding to the DCC (deleted in colorectal carcinoma) receptor leads to the activation of CDC42,131 which in turn phosphorylates and activates the cytoplasmic Extracellular signal-regulated kinase (ERK) 1 and 2 proteins 132 (2). The activation of ERK1 and ERK2 leads to further downstream signaling through different second messenger systems resulting in changes in the cytoskeletal organization of microtubules and actin filaments. This, in turn, results in neurite attraction towards the netrin guidance cues and stimulation of neurite outgrowth.86 However, when both ROBO1 and DCC receptors are present in the neuronal plasma membrane (1), the binding of Netrin-1 to DCC still leads to the activation of CDC42 and subsequently of ERK1/2, but SLIT(2) binding to one of the immunoglobulin (Ig) domains of the ROBO1 receptor86 results in the cytoplasmic domains of ROBO1 and DCC interacting86 and in binding of an srGAP protein to the cytoplasmic domain of ROBO1. This in turn reduces/inhibits the activity of CDC42.133,134 The end result of the interaction between ROBO1 and DCC is still stimulation of neurite outgrowth, but the neurite attraction towards the Netrin guidance cues is silenced.86 When only a ROBO1 receptor is present in the neuronal plasma membrane (3), binding of SLIT(2) to one of the Ig domains of the ROBO1 receptor86 eventually leads—through the inhibition of CDC42—to stimulation of neurite outgrowth but repulsion away from the SLIT guidance cues.86,126,133,134

Netrin (mainly the Netrin-1 protein) bind to ROBO1 netrin-like molecules. However, these interactions are and DCC, respectively.86,126 As shown and described hypothetical and have not yet been confirmed by in more detail in (the legend of) Figure 2, the rate and experimental data, and therefore are not shown in direction of outgrowth is critically dependent on the Figures 1 and 2. Moreover, as suggested by others, exact receptor composition in the plasma membrane. KIAA0319,11,13,82 and possibly KIAA0319L, might As they have fibronectin type III domains very similar alternatively/additionally function in the direct adhe- to those of the DCC receptor protein (http://www. sion between neurons and glial fibers during neuronal ensembl.org/homo_sapiens), the KIAA0319 and migration through their polycystic kidney disease KIAA0319L proteins may function as receptors for domains.

Molecular Psychiatry A theoretical molecular network for dyslexia G Poelmans et al 377 As indicated in Figure 2, the activation of the activating ERK1/262,146 (Figure 1d). The ESR–DYX1C1 ROBO1 and DCC receptors leads to signaling via complexes that promote the proteosomal degradation CDC42131 and cytoplasmic ERK1 and ERK2 pro- of ESR1 and ESR2 are also found in the cytoplasm of teins132 resulting in changes in the cytoskeletal hippocampal neurites (Figure 1d), which suggests the organization of microtubules and actin filaments involvement of DYX1C1 in negatively regulating and (eventually) in neurite outgrowth.86,126,133,134 estrogen signaling cascades in these neurites.62 More- Two other dyslexia candidates, S100B and HMGB1, over, as both DYX1C161 and VAPA122 bind to HSP90, can also result in CDC42135 and ERK1/2132 activation this makes one speculate that a complex of VAPA, and neurite outgrowth by binding to RAGE106,127 HSP90 and DYX1C1 would be involved in degrading (Figure 1b). In addition, S100B or HMGB1 binding and negatively regulating cytoplasmic ESR1 and to RAGE also activates RAC1136,137 (Figure 1b). This ESR2 (Figure 1d). latter pathway is also activated by DOCK4, a periph- After being activated by any of the above-described eral membrane protein95 that regulates dendritic cascades, the ERK1/2 proteins also regulate the growth and branching of hippocampal neurons110 activity of the DCDC2 protein by phosphorylating its (Figure 1b). In the cytoplasm of hippocampal neu- DCX domains80 (Figure 1e). DCDC2 and FLNA—a rons, FMR1 also activates ERK1 and ERK292 (Figures protein that is encoded by a gene interacting with 1d and e). Moreover, FMR1 is involved in the FMR197 (see above)—directly bind and modulate translation of the mRNA of RAC196 (Figure 1d). neuronal microtubules and actin filaments that are Activated ERK1 and ERK2 activate/phosphorylate involved in neurite outgrowth and neuronal migra- the NF-kB (nuclear factor kappa-B),138 ETS1,139 tion11–13,65,69,78–81,95,98–100 (Figure 1e). ELK1140 and TFII-I141 transcription factors (Figure As already stated above, two GWAS related to 1c). NF-kB is also activated downstream of RAC1.136 reading disability have been reported to date. The first All four transcription factors seem to have a role in was a GWAS using a quantitative trait loci approach the dyslexia neurodevelopmental network. Activated comparing the two extremes of the reading ability NF-kB upregulates the transcription of the Netrin-1142 spectrum of the normal population in 1502 children and HSP90143 genes. It also upregulates the transcrip- from the UK Twins Early Development Study TEDS.41 tion of the ELK1 gene,144 a transcriptional activator of The authors used a pooled design and SNP arrays of DYX1C1.43 ELK1 is a brain-expressed transcription only 100.000 SNPs, which greatly reduced the power factor, and its activation has been associated with of the study. Nevertheless, they nominally replicated neurite outgrowth and hippocampus-dependent some of their findings in 4258 additional samples learning in rats.43 The ETS1 transcription factor from TEDS, resulting in 10 SNPs of interest for regulates the transcription and expression of the reading ability in early childhood.41 Six of these SLIT3 gene.129 Activated (nuclear) TFII-I141 upregu- SNPs are located in introns of protein-coding genes lates or downregulates the transcription and expres- (http://www.ensembl.org/homo_sapiens), three of sion of the DYX1C1 gene, depending on the context of which could be directly linked to our molecular other transcription factors.43,44 network for dyslexia: CDC42BPA (rs1320490; The nuclear protein DIP2A binds DMAP1,102 which P = 0.030), TIAM1 (rs2409411; P = 0.004) and DPF3 is a part of the DMAP1–DNMT1–HDAC2 repressive (rs2192595; P = 0.003).41 CDC42BPA is a downstream transcription complex103 (Figure 1c). HDAC2— effector of CDC42 involved in modulating the neuro- through which the DMAP1–DNMT1–HDAC2 com- nal cytoskeleton95 and hence neurite outgrowth.12 plex negatively regulates neurite outgrowth104—is TIAM1 is a cytoplasmic protein that is involved in involved in repressing the transcriptional activity of neurite outgrowth by directly activating RAC1.147 ELK1,95 which results in a reduced expression of DPF3 is a nuclear protein that belongs to the DYX1C1. HDAC2 also directly inhibits ESR1, the neuron-specific chromatin remodeling complex (also nuclear estrogen receptor a.145 called the nBAF complex),95 which has an important The nuclear ESR1 and ESR2 proteins—when role in neural development and dendritic outgrowth activated—upregulate the expression of genes con- of neurons.148 taining estrogen response elements by directly bind- Recently, the results of a GWAS for an electro- ing to the DNA sequences containing these elements95 physiological endophenotype of dyslexia (that is the (Figure 1c). This process—that is important in brain so-called ‘mismatch negativity component’ or MMN) development and neuronal processes such as neuro- in a discovery sample of 200 and a replication sample nal migration and synaptic plasticity62 (see above)—is of 186 dyslexic children were reported.42 Genome- enhanced—for ESR1—by HMGB1130 and sup- wide significance was reached for two SNPs in high pressed—for both ESR1 and ESR2—by DYX1C1.62 linkage disequilibrium in CLSTN2 (rs1365152 and DYX1C1 also binds directly to nuclear ESR1 and rs2114167) in the discovery sample, though this ESR2, which promotes their proteosomal degradation finding was not replicated. As the replication sample and hence negatively regulates the function of these was less strongly affected with dyslexia than the proteins.62 discovery sample, the authors suggest that CLSTN2 In addition to their role in the cell nucleus, ESR1 should not yet be discarded as a dyslexia candidate.42 and ESR2 are also found in the cytoplasm of The gene encodes calsyntenin 2, a brain-specific hippocampal neurites, where they are involved in protein localizing to postsynaptic specializations of

Molecular Psychiatry A theoretical molecular network for dyslexia G Poelmans et al 378 asymmetric synapses in the adult mouse brain149 and genes and functional relationships between genes shows association with verbal working memory,150 a that need to be directly tested in future studies. cognitive process that is repeatedly found to be Therefore, we think that a number of additional impaired in dyslexic subjects.42,63 genetic association and mutation studies as well as Although its function is currently unknown, in functional studies—for example neuroimaging stu- analogy with its homologue calsyntenin 1,151 CLSTN2 dies and studies in animal models—will be needed to might have a role in axonal transport, and may sustain some of the claims made here. In this respect, therefore fit well into our proposed neuronal migra- we also hope that in the next few years, results of tion and neurite outgrowth network. additional, large genome-wide studies of dyslexia and In addition to the finding in CLSTN2, genome-wide reading-related phenotypes will become available in significant association was reported in the combined order to provide more clues for further unraveling the discovery and replication sample between the late genetic etiology of dyslexia. component of the MMN and rs4234898 on 4q32.1, an SNP in a gene desert, that ‘trans-regulates’ the Conflict of interest expression of SLC2A3 (other name: GLUT3)on 12p13.31. SLC2A3 encodes the predominant facil- The authors declare no conflict of interest. itative glucose transporter in the brain. Therefore, the authors suggest that the ‘trans-regulation’ effect of Acknowledgments rs4234898 (and a haplotype of this SNP with rs11100040) on SLC2A3 might lead to decreased We gratefully acknowledge the families who have glucose uptake in dyslexic children that could in made all these studies possible. We are also indebted turn explain their attenuated MMN results.42 The to the many investigators whose work drives the expression of the SLC2A3 gene is known to be dyslexia genetics field forward. This work was sup- upregulated by NF-kB152 and through estradiol/ESR1 ported by a research grant from the ‘Hersenstichting activation.153 Moreover (glutamatargic) excitation of Nederland’ (Brain Foundation of The Netherlands). the neuronal cell membrane causes a translocation of SLC2A3 to the cell membrane leading to increased References glucose uptake,154 which implies that SLC2A3 is a dyslexia candidate that may have an indirect role in 1 Grigorenko EL, Wood FB, Meyer MS, Hart LA, Speed WC, Shuster neuronal migration and/or neurite outgrowth as an A et al. Susceptibility loci for distinct components of develop- energy supplier. mental dyslexia on chromosomes 6 and 15. Am J Hum Genet 1997; 60: 27–39. 2 Fisher SE, Francks C, Marlow AJ, MacPhie IL, Newbury DF, Looking forward Cardon LR et al. Independent genome-wide scans identify a chromosome 18 quantitative-trait locus influencing dyslexia. 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