The Dyslexic
Gene
KIAA0319
April 2005

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Journal List > Am J Hum Genet > v.76(4); Apr 2005

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Cope, N. 
Harold, D. 
Hill, G. 
Williams, J. 

Am J Hum Genet. 2005 April; 76(4): 581­591.
Published online 2005 February 16.

Copyright © 2005 by The American Society of Human Genetics. All rights reserved.

Strong Evidence That KIAA0319 on Chromosome 6p Is a Susceptibility Gene for Developmental Dyslexia

Natalie  Cope,1 Denise  Harold,1 Gary  Hill,1 Valentina  Moskvina,2 Jim  Stevenson,3 Peter  Holmans,2 Michael J.  Owen,1 Michael C.  O'Donovan,1 and Julie  Williams1
1Department of Psychological Medicine and 2Biostatistics and Bioinformatics Unit, Wales College of Medicine, Cardiff University, Cardiff; and 3School of Psychology, University of Southampton, Southampton, United Kingdom

Address for correspondence and reprints: Dr. Michael O'Donovan, Department of Psychological Medicine, Henry Wellcome Building, Cardiff University, Academic Avenue, Heath Park, Cardiff CF14 4XN, United Kingdom. E-mail: odonovanmc@cardiff.ac.uk

Received December 7, 2004; Accepted January 21, 2005.
This article has been cited by other articles in PMC.


Abstract

Linkage between developmental dyslexia (DD) and chromosome 6p has been replicated in a number of independent samples. Recent attempts to identify the gene responsible for the linkage have produced inconsistent evidence for association of DD with a number of genes in a 575-kb region of chromosome 6p22.2, including VMP, DCDC2, KIAA0319, TTRAP, and THEM2. We aimed to identify the specific gene or genes involved by performing a systematic, high-density (Å`2­3-kb intervals) linkage disequilibrium screen of these genes in an independent sample, incorporating family-based and case-control designs in which dyslexia was defined as an extreme representation of reading disability. Using DNA pooling, we first observed evidence for association with 17 single-nucleotide polymorphisms (SNPs), 13 of which were located in the KIAA0319 gene (P<.01­.003). After redundant SNPs were excluded, 10 SNPs were individually genotyped in 223 subjects with DD and 273 controls. Those SNPs that were significant at P.05 were next genotyped in a semi-independent sample of 143 trios of probands with DD and their parents, to control for possible population stratification. Six SNPs showed significant evidence of association in both samples (P.04­.002), including a SNP (rs4504469) in exon 4 of the KIAA0319 gene that changes an amino acid (P=.002; odds ratio 1.5). Logistic regression analysis showed that two SNPs (rs4504469 and rs6935076) in the KIAA0319 gene best explained DD status. The haplotype composed of these two markers was significantly associated with DD (global P=.00001 in the case-control sample; P=.02 in trios). This finding was largely driven by underrepresentation of the most common haplotype in cases (P=.00003 in the case-control sample; P=.006 in trios; 1­degree-of-freedom tests). Our data strongly implicate KIAA0319 as a susceptibility gene for dyslexia. The gene product is expressed in brain, but its specific function is currently unknown.

Introduction

Developmental dyslexia (DD [MIM 600202]), or reading disability, is a relatively common, complex cognitive disorder that affects 5%­10% of school-aged children (Shaywitz et al. 1992). The disorder is characterized by an impairment of reading performance despite adequate motivational, educational, and intellectual opportunities and in the absence of sensory or neurological disability. Although the pathophysiology of DD is unknown, there is strong evidence that genes make a substantial contribution to individual variation in risk of DD, with twin studies reporting heritability estimates of up to 0.71 (Fisher 1905; Hinshelwood 1907; DeFries et al. 1987, 1991; Stevenson et al. 1987; Pennington et al. 1991; Schulte-Körne et al. 1996). The mode of transmission is unknown, but DD is almost certainly a complex genetic disorder in which multiple genes play a role (Hohnen and Stevenson 1999).

Genetic linkage and association studies have implicated a number of chromosomal regions that may harbor susceptibility genes for DD. Regions showing replicated evidence for a role in DD include chromosome 1p (Rabin et al. 1993; Grigorenko et al. 2001; Tzenova et al. 2004), 2p (Fagerheim et al. 1999; Francks et al. 2002; Petryshen et al. 2002; Kaminen et al. 2003; Chapman et al. 2004), 6p (Cardon et al. 1994, 1995; Grigorenko et al. 1997, 2000, 2003; Fisher et al. 1999; Gayán et al. 1999; Kaplan et al. 2002; Turic et al. 2003; Chapman et al. 2004), 15q (Grigorenko et al. 1997; Schulte-Körne et al. 1998; Morris et al. 2000), and 18p (Fisher et al. 2002; Marlow et al. 2003; Chapman et al. 2004).

We have sought to identify a gene(s) in the most consistently supported region on chromosome 6p that shows association with DD. The broadest evidence for linkage stretches from marker D6S109 (Grigorenko et al. 1997) to marker D6S291 (Fisher et al. 1999), a distance of Å`16 Mb, with numerous studies implicating regions between these markers (D6S105­TNFB [Cardon et al. 1994, 1995], D6S109­D6S306 [Grigorenko et al. 1997] D6S276­D6S105 [Gayán et al. 1999] D6S464­D6S273 [Grigorenko et al. 2000], D6S109­JA01, D6S299­D6S1621, and D6S105­D6S265 [Grigorenko et al. 2003]). A region between D6S461 and D6S105 shows greatest overlap between studies, spanning Å`4.2 Mb.

Recently, Deffenbacher and colleagues (2004) examined 10 candidate genes (VMP, DCDC2, MRS2L, GPLD1, ALDH5A1, KIAA0319, TTRAP, THEM2, C6orf62, and GMNN) in the Å`680 kb between markers D6S276 and D6S1554, a region that yielded maximal evidence for linkage in their sample. Thirty-one SNPs were selected at a marker density of Å`1 marker per 20 kb; 13 of the SNPs provided some evidence for association with at least one of the phenotypes analyzed (discrepancy score, phoneme awareness, phoneme deletion, word reading, and orthographic coding). These SNPs were located in five genes: two in VMP, eight in DCDC2, and one each in KIAA0319, TTRAP, and THEM2. Francks and colleagues (2004) examined eight genes (ALDH5A1, KIAA0319, TTRAP, THEM2, C6orf32, SCGN, BTN3A1, and BTN2A1) on chromosome 6p as candidates for DD on the basis of known brain expression. Fifty-seven SNPs were analyzed in 89 U.K. families (sample 1). Association was detected in a 77-kb region spanning TTRAP and the first four exons of KIAA0319. In a second U.K. sample of 175 families (sample 2), 20 of the SNPs were analyzed; weaker evidence of association was found. It is noteworthy that the effect was most pronounced in a sample of families with probands representing the lower end of the reading-ability spectrum. Twenty-one SNPs were also analyzed in a U.S. sample of 159 families (sample 3). Again, association was observed in selected families representing the lower end of the reading-ability spectrum. A three-marker haplotype was significantly associated in both the U.S. sample and the combined U.K. sample, with different alleles forming the most significant haplotype in each sample.

Our study focused on the 575-kb region of chromosome 6p22.2, which was selected to include all genes implicated in recent candidate-gene association studies (Deffenbacher et al. 2004; Francks et al. 2004) and is situated within a region that shows the most consistent evidence of linkage (Deffenbacher et al. 2004; Francks et al. 2004). Our analysis employs a high-density (137 SNPs in seven genes, at 2­3-kb intervals) screen for linkage disequilibrium (LD) and uses both case-control and family-based designs to take account of possible population stratification.

The targeted genes comprise vesicular membrane protein p24 (VMP), doublecortin domain­containing 2 (DCDC2), kidney-associated antigen 1 (KAAG1), magnesium homeostasis factor (MRS2L), KIAA0319, TRAF and TNF receptor associated protein (TTRAP), thioesterase superfamily member 2 (THEM2), and chromosome 6 ORF 62 (C6orf62).

VMP is a neuron-specific vesicular membrane protein that is thought to play a role in vesicular organelle transport and neurotransmission (Cheng et al. 2002). DCDC2 is a ubiquitously expressed gene with a doublecortin-homology domain. Doublecortin itself has been implicated as a cause of X-linked lissencephaly (Gleeson et al. 1998) and is involved in neuronal migration in the CNS, including in the cortex (Gleeson et al. 1999).

KAAG1 is encoded on the strand that is opposite to-and overlaps with-DCDC2. It is a kidney antigen­associated gene, found in numerous tumors and normal testis and kidney (Van Den Eynde et al. 1999). Although it has no known CNS function, it was included in our analysis since it is encoded on the strand that is opposite to the DCDC2 gene and since it was covered by the SNP grid encompassing that gene. MRS2L is a ubiquitously expressed gene that is thought to encode a magnesium-transporter protein (Zsurka et al. 2001).

KIAA0319 is a protein of unknown function that is highly expressed in brain (Londin et al. 2003). The four polycystic kidney disease (PKD) domains found in KIAA0319 show homology to the extracellular domains of the PKD protein PKD1, which are involved in cell-adhesive functions (Streets et al. 2003).

TTRAP encodes a tumor necrosis factor receptor­associated protein. It has been shown to inhibit nuclear factor-É»B (NF-É»B) activation and subsequent downstream activation of transcription (Pype et al. 2000). NF-É»B transcription has been shown to play a role in long-term potentiation and synaptic plasticity associated with learning and memory. In mice, inhibition of NF-É»B has been shown to result in neurodegenerative-like phenotypes (Fridmacher et al. 2003). TTRAP can also interact with the cytoplasmic TNF receptor­associated factors (TRAFs) and with cytoplasmic domains of some members of the TNF-receptor superfamily (Pype et al. 2000).

THEM2 encodes an uncharacterized hypothalamus protein and is part of the thioesterase superfamily. The thioesterase superfamily catalyzes the hydrolysis of long-chain fatty acyl-CoA thioesters. It has been suggested that abnormal fatty-acid metabolism plays a role in DD (Richardson and Ross 2000; Richardson et al. 2000; Taylor and Richardson 2000).

C6orf62 is a gene with unknown function that is expressed ubiquitously, including in brain. Although not part of the present study, aldehyde dehydrogenase 5 family, member A1 (ALDH5A1 [succinate-semialdehyde dehydrogenase]), and glycosylphosphatidylinositol-specific phospholipase D1 (GPLD1) on 6p22.2-p22.3, were previously examined using a direct gene-analysis approach that is based on de novo polymorphism discovery and analysis of all detected variants. Extensive analysis of both genes failed to provide evidence for association with DD (authors' unpublished data).

This study thus provides a systematic, high-density LD screen spanning putative functional candidates in a region that shows the most consistent evidence of linkage to DD. Our strategy employed both case-control and family-based designs in which dyslexia was defined, to capture the extreme end of the reading ability/disability continuum.

Material and Methods

Ethical approval was obtained from local ethics committees in the United Kingdom; appropriate and informed written consent was obtained from subjects' parents. All participants were of white U.K. origin. Children with DD-and, if available, their parents and siblings-were ascertained in the United Kingdom through contacts with local education authorities and schools specializing in the education of children with reading difficulties. The inclusion criteria for probands were an IQ of 85 and a reading age 2.5 years behind that expected from chronological age. No age-IQ discrepancy measures were employed. Four subtests from the WISC-III UK were used to provide a prorated, full-scale IQ score: Vocabulary, Similarities, Block Design, and Picture Completion (Wechsler 1992). The accuracy score from the Neale (1989) analysis of reading ability was used to determine reading age, except when probands were aged >13 years, in which case, we used the accuracy score of British Ability Scale (BAS) single-word reading (Elliot 1983).

Initially, controls were adult white U.K. blood donors. Subsequently, control children matched for age and sex were ascertained from the same schools as the children with DD. Children classed as controls were required to have an IQ of 85 and a reading delay (RD) of no more than 6 mo. Control children were assessed using the accuracy score of the Neale (1989) analysis of reading ability and/or BAS single-word reading (Elliot 1983) to calculate reading age; the Vocabulary, Similarities, Block Design, and Picture Completion subsets of WISC-III UK enabled calculation of prorated IQ.

As a first pass, SNPs were analyzed in DNA pools. Because of sample availability, early analyses (n=56 SNPs) used case pools containing 140 unrelated probands with DD (mean age [± SD] 13.22±2.3 years; mean RD [± SD] -5.07±1.76; mean IQ [± SD] 100.13±11.03; 116 males, 24 females) and 550 adult blood-donor controls (mean age 41.39±12.5 years; 391 male, 159 female) (fig. 1, Start Point A). Later, pooled analyses were based on an extension of the original sample of subjects with DD (n=240 unrelated probands with DD; mean age 13.17±2.18 years; mean IQ 98.88±18.38; mean RD -4.93±1.87; 204 males, 36 females) and pools containing 312 age-matched and screened controls (mean age 11.98±2.39 years; mean IQ 103.35±11.97; mean RD +1.14±1.45; 178 males, 134 females) (fig. 1, Start Point B).

DNA was extracted from venous blood or 25 ml saline mouthwashes by use of standard procedures (Morris et al. 2000). SNPs were selected from Ensembl or SNPper (CHIP Bioinformatics Tools) for each of the eight positional/functional candidate genes, at intervals of Å`2­3 kb between each SNP. SNPs were also chosen for analysis if they showed association in the study by either Deffenbacher et al. (2004) or Francks et al. (2004), excluding SNPs in LD (r20.80) with those we had already typed on the basis of HapMap data. A total of 137 SNPs were analyzed across the region. With the exception of DCDC2, SNP grids extended from 3 kb upstream to the predicted start of transcription across all exons and introns. Introns 2, 7, and 8 of DCDC2 would have required 60­90 SNPs. Therefore, for pragmatic reasons, we restricted our analysis at these introns to 3 kb of flanking sequence on either side of each of the exons.

Genotyping of DNA pools was undertaken using the SNaPshot (Applied Biosystems) primer-extension method described by Norton and colleagues (2002). Pools were carefully constructed by a serial dilution method, each stage accompanied by quantitation of DNA concentration by use of the PicoGreen method, as we have described in detail elsewhere (Norton et al. 2004). Markers were selected for individual genotyping if pooled analysis revealed evidence for association with DD at an estimated level of P.05 in the pooled samples of 240 cases and 312 controls. However, to allow for the smaller sample of cases (n=140) in the early analysis, those markers showing only a trend for association (P.1) were regenotyped in the larger case pools and were selected for individual genotyping if the trend was confirmed at P.05 (see fig. 1). The pooling data presented in our tables reflect the analysis that is based on the larger of the samples in which it was conducted. SNPs selected for individual genotyping were examined, and those subjects for whom we had sufficient DNA (223 DD cases and 273 controls) were included in the pooling experiments. Those markers for which individual genotyping in this case-control sample confirmed the pooling data (P.05) were then genotyped in a semi-independent sample of 143 parent-proband trios to ensure that the results were not attributable to population stratification. Mean age of probands in the trio sample was 13.17±2.08 years; mean IQ was 104.01±11.88; mean RD was -5.06±1.76. All but 25 of the probands in the parent-proband trios were also included in the case-control sample. When genotypes were available for these 25 individuals, they were included in the final analysis of case-control association.

Individual genotyping was undertaken using a proprietary Amplifluor (Serologicals) genotyping method. Amplifluor reactions were performed in 5-É l reactions containing 50 ng DNA, in accordance with manufacturer instructions. Primers were designed using Amplifluor AssayArchitect and were obtained from Sigma-Genosys. PCR reactions were performed under standard conditions, with an initial denaturation stage of 96°C for 4 min, then 19 cycles at 96°C for 10 s, at 58°C or 60°C for 5 s, and at 72°C for 10 s; followed by 22 or 27 cycles at 96°C for 10 s, 20 s at 55°C, and 40 s at 72°C; and a final extension step at 72°C for 3 min. Genotypes were read on an LJL Biosystems Analyst. When we were unable to optimize Amplifluor, RFLP analysis of PCR products was undertaken. PCR reactions were performed under standard conditions in 12-É l reaction volumes with 32 ng of genomic DNA. Digests were undertaken in 17-É l reactions by use of the appropriate restriction enzyme (New England Biolabs), in accordance with manufacturer instructions. Products were visualized on 3% agarose gels stained with ethidium bromide.

All genotypes were tested for Hardy-Weinberg equilibrium with a É'2 goodness-of-fit test (see the Simple Interactive Statistical Analysis Web site). Analysis of LD between markers (r2 and DÅå) was performed using Haploview. Standard contingency tables were used for single-marker case-control analysis. Trios were analyzed using UNPHASED (Dudbridge 2003) (see the Rosalind Franklin Centre for Genomics Research Web site). Haplotypes were analyzed using EHPlus (Zhao et al. 2000) and UNPHASED (Dudbridge 2003). Logistic and conditional logistic regression analyses were performed on case-control and trio data, respectively.

Results

Of the 137 SNPs analyzed in pools, 17 yielded evidence for association at P.05, and 13 of these were located within KIAA0319 (see table 1 for results of the pooled genotyping). Of these SNPs, 15 were then typed for 42 subjects with DD and 48 controls, to identify redundant markers on the basis of marker-marker LD (the two remaining SNPs, rs1555090 and rs926529, were known to be in LD with other SNPs analyzed on the basis of HapMap data). In an attempt to replicate the most significant haplotype of Francks and colleagues (2004), we also genotyped rs2143340, although this SNP did not show significant evidence for association in DNA pools. Perfect LD (r2=1) was noted for several of the markers in KIAA0319 (see table 2). Genotyped markers showing LD, as assessed by r2, of at least 0.8 were dropped. On this basis, a minimal set of 10 markers was chosen for individual genotyping in the case-control sample (see table 3).

All genotypes were in Hardy-Weinberg equilibrium for cases and controls and probands and parents, except for SNP rs2038137) (located near the exon 1/intron 1 boundary of KIAA0319), which showed slight distortion in the controls (P=.03). Our most significant results (P.01) were found in KIAA0319, MRS2L, and THEM2 in the case-control sample (see table 3). To ensure that these data did not arise from population stratification, the seven markers that were significant in the case-control sample at P.05 were then individually genotyped in a sample of 143 parent-proband trios (table 4). This also provides for a degree of independence, since the control (nontransmitted) alleles are independent of the controls in the case-control study. After this step, six SNPs remained significant-three in the KIAA0319 gene (family-based, P=.04­.002; case-control, P=.007­.001), one in MRS2L (family-based, P=.04; case-control, P=.003), and two in or flanking the THEM2 gene (family-based, P=.03­.01; case-control, P=.03­.008). Two of these SNPs (rs4504469 and rs2179515) were reported in the study by Francks et al. (2004) to be associated with a number of componential measures of DD in the combined U.K. sample. Both are located in the KIAA0319 gene (r2=0.55).

To determine which SNPs accounted for the association, stepwise logistic regression analyses were performed on the case-control and trio data on the basis of all SNPs for which we had individual genotyping data (see tables 3 and 4). For the case-control sample, the SNPs-rs2793422 (MRS2L); rs4504469, rs6911855, rs6939068, rs2179515, rs6935076, and rs2038137 (KIAA0319); rs2143340 (TTRAP); rs3777664 (THEM2); and Intergenic rs1053598-were initially submitted into the logistic regression model (P=.029; 10 df). The stepwise procedure reduced the number of SNPs to three-rs2793422 (MRS2L) and rs4504469 and rs6935076 (KIAA0319)-that showed a highly significant fit (P=.00002; 3 df). For the proband-parent trios, the probands were considered as cases, and nontransmitted alleles were employed to create pseudocontrols (Cordell and Clayton 2002). These data were submitted into conditional logistic regression analyses (P=.347; 7 df). The best model was again identified by use of a stepwise procedure (P=.02; 2 df) that removed every SNP except SNPs rs4504469 and rs6935076. The addition of rs2793422 did not significantly improve the model (P=.10 [log-likelihood ratio test]). rs4504469 is a nonsynonymous SNP in exon 4 (AlaŮThr), and rs6935076 is located in intron 1 of the KIAA0319 gene (see fig. 2).

On the basis of the results of the regression analysis, we analyzed the two-marker haplotype that consisted of the KIAA0319 SNPs rs4504469 and rs6935076 in the case-control and trio samples (see tables 5 and 6). Significant evidence for association was obtained on the basis of the global test (P=.0001 in the case-control sample; P=.02 in trios). In each sample, the 1-2 haplotype was associated with DD, but more striking is the significant underrepresentation of haplotype 2-1 in the cases based on the case-control (P=.00003; odds ratio [OR] 0.53; 95% CI 0.40­0.70) and family-based (P=.006; OR 0.57; 95% CI 0.39­0.84) analyses. (Fig. 2 summarizes our results.)

We also analyzed the three-marker haplotype that consisted of rs4504469, rs2038137, and rs2143340, which was reported as significantly associated with DD by Francks and colleagues (2004). This haplotype did not yield global evidence for association in our sample (see table 7). Two individual haplotypes did, however, show evidence for association with DD. The 1-1-1 haplotype was more frequent in subjects with DD than in control individuals (P=.03). The 2-2-1 haplotype, which was significantly associated with the READ phenotype in the combined U.K. sample of the study by Francks et al. (2004), also displayed evidence of association with DD in our case-control sample (P=.01) and showed the same direction of effect (in their study, the 2-2-1 haplotype was associated with better performance; in our sample, this haplotype was more frequent in control individuals). In our sample, this haplotype is, in fact, perfectly defined by the first two SNPs (since there was no observation of the 2-2-2 haplotype). We therefore excluded rs2143340 and looked at the two-marker haplotype (2-2) that consisted of the other two SNPs in our family-based sample, but, although it was undertransmitted to the probands, this was not significant (P=.10). It should be noted that, since rs4504469 shows more significance individually than does the 2-2-1 haplotype, no extra information was obtained from this haplotype in our sample. Moreover, the 1-1-2 haplotype that was reported to show association with componential measures of DD in both the U.S. sample and the combined U.K. sample in the study by Francks et al. (2004) was not significantly associated with DD in our sample (P=.21).

Discussion

Previous linkage and association studies of DD and chromosome 6p have implicated a region between markers D6S461 and D6S105. More recently, following other positional candidate-gene studies, VMP, DCDC2, KIAA0319, TTRAP, and THEM2 have been suggested as possible susceptibility genes within this region (Deffenbacher et al. 2004; Francks et al. 2004). Our study tested for association with each of these genes (VMP, DCDC2, KAAG1, MRS2L, KIAA0319, TTRAP, THEM2, and C6orf62) by use of a high-density SNP map and an independent sample. Initially, we genotyped DNA pools from subjects and controls and followed up those findings with individual genotyping in a case-control sample and a nested family-based association sample. In both samples, we observed evidence for association with three SNPs in KIAA0319 (rs4504469, P=.002; rs2179515, P=.007; and rs6935076, P=.006), with one SNP in MRS2L (rs2793422, P=.003) and in THEM2 (rs3777664, P=.008), and with an intergenic SNP (rs1053598, P=.02). Two of these SNPs, rs4504469 and rs2179515 (both located in KIAA0319), have been reported elsewhere to display significant association with a number of componential measures of DD in a U.K. sample (Francks et al. 2004). Our results support existing data (Deffenbacher et al. 2004; Francks et al. 2004) that implicate genes in this region in DD, and our results extend the previous findings by demonstrating that the source of the signal is likely to be variation in KIAA0319. The study by Francks and colleagues (2004) implicated a region containing KIAA0319, TTRAP, and THEM2, whereas that of Deffenbacher and colleagues (2004) implicated KIAA0319, DCDC2, VMP, TTRAP, and THEM2. Combining their data with our own produces a pattern of evidence that implicates KIAA0319 as a susceptibility gene for DD.

This is compatible with the logistic regression and conditional logistic regression analyses in our case-control and proband-parent trio samples, respectively. Case-control data analyses with the use of a stepwise procedure revealed three SNPs that account for the association observed: rs2793422 in MRS2L and rs4504469 and rs6935076 in KIAA0319 (P=.00002). Analysis of the proband-parent trios identified two SNPs that account for the association observed: rs4504469 and rs6935076 (P=.03). The results, therefore, are consistent with crude inspection of the genes showing overlap between the studies and provide strong evidence that KIAA0319 SNPs rs4504469 and rs6935076 are responsible for the association with DD observed in this study. A haplotype comprising these two SNPs is highly significantly associated with DD in both the case-control sample (P=.00003) and the trio sample (P=.006). This effect is largely driven by haplotype 2-1 as a "protective" haplotype.

Although the logistic regression analyses imply that, of the markers tested, rs4504469 and rs6935076 can account for the association signal, this does not imply that they are the direct susceptibility alleles, per se. However, interestingly, rs4504469 (one of the two SNPs that makes up our most significant haplotype) is a nonsynonymous SNP (AlaŮThr), which suggests the possibility that this might, in part, contribute directly to the association. However, in our own sample, the threonine at this locus that is present on the protective rs4504469/rs6935076 2-1 haplotype is also present on the 2-2 haplotype (tables 5 and 6), which is more common in cases, albeit not significantly more so. This suggests that, if the nonsynonymous change at rs4504469 can influence risk of DD directly, then its effects can be modified by a second susceptibility allele in the gene. Given that the SNP showed the same pattern of allelic association in the U.K. sample of Francks and colleagues (2004) but not their U.S. sample, perhaps a more likely explanation is that this SNP does not directly influence susceptibility to DD. Thus, although our study provides strong evidence that variation in the KIAA0319 gene is associated with increased risk of developing dyslexia, the true susceptibility alleles remain to be identified.

Acknowledgments

We thank all the parents and children who took part in this study, and we are grateful to the Health Foundation (reference number 2263/1921), the Cardiff University Ph.D. Fund, and the U.K. Medical Research Council Cooperative Group Grant (APP1485) for funding this research. We also thank the Welsh Assembly Government for supporting the Biostatistics and Bioinformatics Unit, in which V.M. is employed.

Electronic-Database Information

The URLs for data presented herein are as follows:

Amplifluor AssayArchitect https://apps.serologicals.com/AAA/.
CHIP Bioinformatics Tools, http://snpper.chip.org/ (for SNPper).
Ensembl Genome Browser, http://www.ensembl.org/.
Haploview, http://www.broad.mit.edu/mpg/haploview/index.php.
International HapMap Project, http://www.hapmap.org/ (for LD data).
Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for DD).
Rosalind Franklin Centre for Genomics Research, http://www.hgmp.mrc.ac.uk/ (for the UNPHASED application).
Sigma-Genosys, http://orders.sigma-genosys.eu.com.
Simple Interactive Statistical Analysis, http://home.clara.net/sisa/ (for É'2 tests of

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Figures and Tables

Figure 1
Flow diagram of the samples used at each stage of the analysis
Figure 2
Location of candidate genes on chromosome 6p. The location of SNPs found to be significant (P.05) in our case-control sample are shown relative to nearby markers. The direction of transcription is shown for each gene. LD blocks across the region (more ...)
Table 1
All Pooled Genotyping Data[Note]
Table 2
LD between SNPs Showing Significance (P<.05) in DNA Pools[Note]
Table 3
Genotype and Allele Counts for Selected SNPs in the Case-Control Sample[Note]
Table 4
Transmission of Alleles at Selected SNPs in the Parent-Proband Trios[Note]
Table 5
Analysis of Haplotypes in KIAA0319 Comprising SNPs rs4504469 and rs6935076 in 248 Subjects with DD and 273 Controls
Table 6
Analysis of Haplotypes in KIAA0319 Comprising SNPs rs4504469 and rs6935076 in a Sample of 143 Families with DD
Table 7
Haplotype Analysis Spanning KIAA0319 and TTRAP[Note]
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