A Common Variant Associated with Dyslexia Reduces
Expression of the KIAA0319 Gene
Megan Y. Dennis1,2, Silvia Paracchini1, Thomas S. Scerri1, Ludmila Prokunina-Olsson3, Julian C. Knight1,
Richard Wade-Martins4, Penny Coggill5, Stephan Beck6, Eric D. Green2*, Anthony P. Monaco1*
1Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom, 2Genome Technology Branch, National Human Genome Research Institute,
National Institutes of Health, Bethesda, Maryland, United States of America, 3Laboratory of Translational Genomics, Division of Cancer Epidemiology and Genetics,
National Cancer Institute, National Institutes of Health, Gaithersburg, Maryland, United States of America, 4Department of Physiology, Anatomy, and Genetics, University
of Oxford, Oxford, United Kingdom, 5Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge, United Kingdom, 6UCL Cancer Institute, University College
London, London, United Kingdom
Numerous genetic association studies have implicated the KIAA0319 gene on human chromosome 6p22 in dyslexia
susceptibility. The causative variant(s) remains unknown but may modulate gene expression, given that (1) a dyslexia-
associated haplotype has been implicated in the reduced expression of KIAA0319, and (2) the strongest association has been
found for the region spanning exon 1 of KIAA0319. Here, we test the hypothesis that variant(s) responsible for reduced
KIAA0319 expression resides on the risk haplotype close to the gene’s transcription start site. We identified seven single-
nucleotide polymorphisms on the risk haplotype immediately upstream of KIAA0319 and determined that three of these are
strongly associated with multiple reading-related traits. Using luciferase-expressing constructs containing the KIAA0319
upstream region, we characterized the minimal promoter and additional putative transcriptional regulator regions. This
revealed that the minor allele of rs9461045, which shows the strongest association with dyslexia in our sample (max p-
value=0.0001), confers reduced luciferase expression in both neuronal and non-neuronal cell lines. Additionally, we found
that the presence of this rs9461045 dyslexia-associated allele creates a nuclear protein-binding site, likely for the
transcriptional silencer OCT-1. Knocking down OCT-1 expression in the neuronal cell line SHSY5Y using an siRNA restores
KIAA0319 expression from the risk haplotype to nearly that seen from the non-risk haplotype. Our study thus pinpoints a
common variant as altering the function of a dyslexia candidate gene and provides an illustrative example of the strategic
approach needed to dissect the molecular basis of complex genetic traits.
Citation: Dennis MY, Paracchini S, Scerri TS, Prokunina-Olsson L, Knight JC, et al. (2009) A Common Variant Associated with Dyslexia Reduces Expression of the
KIAA0319 Gene. PLoS Genet 5(3): e1000436. doi:10.1371/journal.pgen.1000436
Editor: Lisa Stubbs, University of Illinois at Urbana-Champaign, United States of America
Received December 5, 2008; Accepted February 24, 2009; Published March 27, 2009
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This work was supported by the Wellcome Trust (MYD, SP, TSS, JCK, RWM, PC, SB, and APM), the Intramural Research Programs of the National Human
Genome Research Institute (MYD and EDG) and National Cancer Institute (MPO), and the NIH/Ox-Cam Graduate Partnership Program (MYD). The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (EDG); firstname.lastname@example.org (APM)
Dyslexia, or reading disability (RD), is a condition that affects an
individual’s ability to read and spell in the absence of any obvious
sensory or neurological impairment and despite adequate
intelligence and educational opportunity . RD is one of the
most common learning disabilities in school-aged children, with a
prevalence ranging from 5% to 17.5% [2,3]. Although the specific
causes of the disorder have yet to be elucidated, it is generally
accepted that RD has a strong genetic component [4,5]. Family
studies have estimated a high heritability of RD, reporting an
incidence of about 40% in siblings of affected individuals [6,7];
twin studies have shown a concordance rate of 68% in
monozygotic twins versus 38% in dizygotic twins .
Numerous candidate genes have emerged from genetic
association studies and the characterization of chromosomal
translocations in individuals with RD, including DYX1C1 on
15q21 [9–11], ROBO1 on 3p12 , DCDC2 [13,14] and
KIAA0319 [15–19] on 6p22, and MRPL19 and C2ORF3 on 2p12
. Several of these genes have been implicated in brain
development . In particular, RNAi-knockdown studies suggest
that DYX1C1 [22–24], DCDC2 [13,25], and KIAA0319  play a
role in neuronal migration during the development of the rat
cortex. Interestingly, altered neuronal migration has been
implicated in RD based on the only post-mortem anatomical
study conducted to date ; specifically, the brains of dyslexic
individuals were found to have subtle structural anomalies
consistent with defective neuronal migration.
We previously detected an RD-associated ‘risk haplotype’
through an association analysis of candidate genes residing at
the chromosome 6p22 locus, which is one of the most consistently
identified candidate regions by linkage studies . Single-
nucleotide polymorphisms (SNPs) were selected within brain-
expressed genes and used for subsequent genetic analyses of RD. A
77-kb region of high inter-marker linkage disequilibrium (LD) that
includes the first four exons of KIAA0319, all of TTRAP, and the
region immediately upstream of THEM2 (Figure 1A) showed
significant associations with RD. Three SNPs captured most of the
genetic variation and described the most common haplotype in the
77-kb region. One of these haplotypes, which was effectively
PLoS Genetics | www.plosgenetics.org1 March 2009 | Volume 5 | Issue 3 | e1000436
tagged by the rs2143340 marker, was found to be significantly
associated with RD. The association between this risk haplotype
and reading-related traits was detected in two independent family-
based sample sets of U.K. and U.S. origin . Association with
the same region was reported in a completely independent study
. Most recently, we replicated the association between the risk
haplotype and reading-related phenotypes in an unselected sample
of more than 6,000 children from the Avon Longitudinal Study of
Parents and Children (ALSPAC) . Using a quantitative allele-
specific gene expression assay, we showed that there is reduced
expression of KIAA0319 (but not TTRAP and THEM2) from the
risk haplotype in both lymphoblastoid and neuronal cell lines .
These data are consistent with the findings of a comprehensive
association study, which tested an identical set of SNPs within the
chromosome 6p22 locus in two independent U.K. sample sets
. The strongest association with RD was found with SNPs near
the first exon of KIAA0319 in both sample sets. Taken together,
these data suggest that the risk haplotype might harbor a
regulatory variant that alters KIAA0319 transcription.
Here, we report additional genetic and functional character-
ization of the risk haplotype, specifically focusing on variants
within the putative regulatory element(s) immediately upstream of
KIAA0319. Our results implicate one variant as the likely cause of
reduced KIAA0319 expression from the risk haplotype. More
broadly, these findings are relevant for further understanding the
role of KIAA0319 in RD and brain development as well as for
establishing the role of non-coding mutations in complex genetic
Thirteen human bacterial artificial chromosomes (BACs)
spanning the 77-kb RD-associated region were obtained either
from Children’s Hospital Oakland Resource Institute or the
California Institute of Technology. The BACs were genotyped for
the three risk haplotype-tagging SNPs (rs4504469, rs2038137, and
rs2143340 ) using the Sequenom platform, according to the
manufacturer’s instructions. BACs RP11-195J19 [containing the
risk haplotype(‘risk BAC’);
CR925830] and RP11-948M1 [containing a non-risk haplotype
(‘non-risk BAC’); GenBank accession number CR942205] were
chosen for their similar genomic coverage. Both BACs were
sequenced at the Wellcome Trust Sanger Institute. Variants were
detected by pair-wise comparisons using AlignX in the Vector
NTI Advance 9 program (Invitrogen).
The collection of families used for quantitative trait association
has been extensively described . Briefly, all probands and
siblings from our complete Oxford set of 264 unrelated nuclear
families were identified from the dyslexia clinic at the Royal
Berkshire Hospital (Reading, U.K.) and were administered a
battery of psychometric tests. The following reading-related
measures were used for statistical analyses: orthographic coding
using irregular words (OC-irreg), phonological decoding ability
(PD), orthographic coding assessed by forced word choice test
(OC-choice), single-word reading ability (READ), spelling ability
(SPELL), phonemic awareness (PA), and measures of IQ [verbal
(SIM) and nonverbal (MAT)]. The scores were adjusted for age
and IQ, and then standardized against a normative control data
set as described [28,29].
SNP genotyping was performed using either the MassARRAY
hME or iPLEX system (Sequenom), according to the manufac-
turer’s instructions (all primer sequences are available upon
request). Marker-trait association was evaluated using the ‘‘total’’
association model with the QTDT package . Variants were
initially tested for association with the reading-related traits in a
sample set consisting of 89 U.K. families previously described by
Francks et al. , referred to as ‘sample 1.’ The LD among SNPs
in this sample was determined using Haploview version 4.0
(http://www.broad.mit.edu/mpg/haploview) . SNPs showing
significant associations were tested in the entire sample of 264
families, referred to as ‘entire U.K. set,’ as well as in a
phenotypically severe sample subset consisting of 126 families
described previously [15,17] and referred to as ‘severe U.K.
subset.’ Briefly, the severe U.K. subset was chosen based on scores
.0.5 SD below a composite mean score of the PD and OC-irreg
traits, the two measures that contribute to the greatest degree to
the chromosome 6p22 linkage peak .
Multi-Species Sequence Comparisons
Genomic sequences orthologous to the interval between TTRAP
and KIAA0319 were obtained from publicly available databases
(http://genome.ucsc.edu for chimpanzee, orangutan, macaque,
marmoset, dog, mouse, and rat; http://www.ncbi.nlm.nih.gov/
blast/Blast.cgi for horse, pig, and elephant). A multi-sequence
alignment of these sequences was generated with MultiPipMaker
(http://pipmaker.bx.psu.edu/pipmaker) using the sequence of the
non-risk BAC as the human reference .
Haplotype-Specific KIAA0319 Promoter Region
The genomic segment immediately upstream of KIAA0319
[24,028 bp to +77 bp relative to the transcription start site (TSS)]
from the non-risk BAC was cloned into the luciferase-expressing
pGL3-Basic vector (Promega) using BAC recombineering .
Specifically, the pGL3-Basic vector was linearized with the
restriction enzyme KpnI (New England Biolabs) and gel-purified
(Qiagen). PCR amplification (Bioxact Long, Bioline) was per-
formed using the linearized pGL3-Basic vector as the template and
appropriate recombineering primers (see Text S1 for sequences).
Electrocompetent cells containing the non-risk BAC were
generated as described . Column-purified (Qiagen) PCR
product (2 mg), consisting of linearized pGL3-Basic vector flanked
Dyslexia, or reading disability, is a common disorder
caused by both genetic and environmental factors.
Genetic studies have implicated a number of genes as
candidates for playing a role in dyslexia. We functionally
characterized one such gene (KIAA0319) to identify
variant(s) that might affect gene expression and contribute
to the disorder. We discovered a variant residing outside of
the protein-coding region of KIAA0319 that reduces
expression of the gene. This variant creates a binding site
for the transcription factor OCT-1. Previous studies have
shown that OCT-1 binding to a specific DNA sequence
upstream of a gene can reduce the expression of that
gene. In this case, reduced KIAA0319 expression could lead
to improper development of regions of the brain involved
in reading ability. This is the first study to identify a
functional variant implicated in dyslexia. More broadly, our
study illustrates the steps that can be utilized for
identifying mutations causing other complex genetic
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by homologous sequence to the non-risk BAC, was electroporated
into 25 ml of temperature-induced SW102 E. coli  containing
the non-risk BAC, and the cells were plated onto LB agar
containing 100 mg/ml ampicillin and incubated at 32uC for
Constructs harboring various deletions were engineered by
removing the segment between the restriction sites for EcoRV
(24,026 bp from the TSS) and the following: PmlI (22,802 bp),
BstXI (22,185 bp), NsiI (21,728 bp), PvuII (2940 bp), StuI
(2544 bp), Bpu10I (2216 bp), Bsu36I (297 bp), and BssHII
Figure 1. KIAA0319 promoter region SNPs residing on the RD-associated risk haplotype. (A) Graphical representation of the location of the
seven SNPs (corresponding to SNPs 1–7) on the RD-associated risk haplotype within the genomic region 4,028 bp upstream and 77 bp downstream
of the KIAA0319 TSS. The asterisk within TTRAP depicts the location of rs2143340, the risk haplotype-tagging SNP. (B) Associations of each SNP variant
with the six indicated quantitative reading-related measures, as assessed by genotyping sample 1 (see Methods for details). (C) Representation of LD
across the genomic region harboring these variants (calculated from the same genotyping data as in B), evaluated by Haploview version 4.0. The
indicated numbers represent absolute D prime (D’) between two loci. An empty red box represents complete LD, while an empty blue box indicates
low LD. (D) MultiPip (percent identity plot) alignment of genomic sequence from the indicated vertebrate species across the region containing SNPs
1–7 compared to the human sequence derived from the non-risk BAC. Red indicates .75% identity between that species’ sequence and the human
sequence over 100 nucleotides; green indicates .50% identity between that species’ sequence and the human sequence over 100 nucleotides; grey
corresponds to sequence missing in that species; white corresponds to no sequence from that species aligning with the human sequence for the
indicated interval. At the bottom, the nucleotide-level alignments are provided for the immediate regions encompassing SNPs 2, 4, and 5 (with the
position of the SNP highlighted in each case; note that the depicted human sequence reflects the non-risk haplotype). A dot indicates that the
corresponding base in that species matches the base in the human reference sequence.
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(224 bp). Site-directed mutagenesis of the full-length construct
was performed using the QuikChange XL Site-Directed Muta-
genesis kit (Stratagene) according to the manufacturer’s protocol
(primer sequences used for the mutagenesis are provided in Text
S1). All mutated constructs were sequenced to ensure the absence
of unwanted additional mutations.
SHSY5Y, SK-N-MC, and HEK293T cell lines were grown
according to ECACC guidelines at 37uC with 5% CO2. All three
cell lines were grown in 96-well plates at a concentration of
2.46104cells/well for SHSY5Y, 3.56104cells/well for SK-N-
MC, and 1.56104cells/well for HEK293T. After 24 hours, the
cells were co-transfected with 0.05 pmol of the pGL3-derived
construct (e.g., containing the non-risk haplotype, deletions, or
mutations; note that a promoter-less pGL3-Basic construct was
used as a negative control) and 2 ng of pRL-CMV with 20 ml
Lipofectamine 2000 (Invitrogen). At 3–4 hours post-transfection,
the medium was replaced. At 48 hours post-transfection when the
cells had reached approximately 90% confluency, cells were lysed,
and the luminescence was assayed using the Dual Luciferase
Reporter Assay (Promega). The luminescence of 20 ml of lysis
product was measured using a microplate luminometer (Lumi-
noskan Ascent, Thermo Fisher Scientific).
The transfection efficiencies were normalized to the level of
pRL-CMV renilla luciferase activity, and the results reflected as
‘relative luciferase activity’ (RLA). The RLA for each transfection
were scaled so that the pGL3-Basic construct (in the case of
constructs harboring deletions) or the full-length non-risk haplo-
type-containing construct (in the case of mutagenized constructs)
yielded a 1.0 RLA. All transfections were performed in
quadruplicate and repeated at least three times (twelve biological
replicates in total). An unpaired two-sided t-test was used to
compare the RLAs between the non-risk haplotype and mutagen-
Electrophoretic Mobility Shift Assays (EMSA)
To create double-stranded EMSA probes carrying risk and non-
risk alleles of the RD–associated SNPs, complementary oligonu-
cleotides (see Text S1 for sequences) were annealed, end-labeled
with [c-32P]ATP (PerkinElmer) using 10 units of T4 polynucle-
otide kinase (Promega), and column-purified (GE Healthcare).
Equal amounts of nuclear extract from the SHSY5Y cell line,
prepared using a nuclear extraction kit (Cayman Chemical), were
pre-incubated with or without an unlabeled double-stranded
‘competitor’ DNA in the presence of DNA-binding buffer
(Promega) for 10 minutes at room temperature, and then
incubated with the relevant32P-labeled probe (17.5 fmol/sample)
for 20 minutes at room temperature. For the ‘supershift EMSA’
, 2 mg of appropriate EMSA-grade concentrated antibody
[OCT-1 (octamer-1), sc-232x and CRX (cone-rod homeobox), sc-
30150x; Santa Cruz Biotechnology] was then added, and the
sample was incubated at 4uC overnight. DNA-protein complexes
were electrophoretically separated on a 6% polyacrylamide 0.56
TBE DNA retardation gel (Invitrogen) at room temperature, dried
at 80uC for 1 hour, and visualized using a Fujifilm FLA-5000
RNA Silencing Studies
SHSY5Y cells, chosen because of their heterozygosity for the
RD-associated risk haplotype, were reverse-transfected with
corresponding siRNA cocktails or with Lipofectamine only
(‘mock-transfected’). Briefly, SHSY5Y cells (46105cells/well)
were plated in 24-well plates just before transfection and mixed
with pre-incubated siRNA cocktails. For the cocktails, siRNAs for
OCT-1 (sc-36119, Santa Cruz Biotechnology), a positive control
[GAPDH (glyceraldehyde 3-phosphate dehydrogenase), AM4605,
Ambion], or a scrambled negative control (AM4636, Ambion)
were diluted with Opti-MEM media and pre-incubated with
Lipofectamine 2000 (Invitrogen). Two concentrations of siRNA
were used for all experiments: 1.5 and 3.0 mM. The results were
consistent with both concentrations, but there was less variation
and the results were more statistically significant with the 1.5 mM
concentration. After incubation at 37uC with 5% CO2 for
24 hours, the medium was replaced, and the cells were incubated
for an additional 24 hours. All siRNA transfections were
performed in 6 biological replicates for each concentration of
siRNA. Subsequently, total RNA from the cells was prepared with
Trizol reagent (Invitrogen) and the RNeasy miniprep kit (Qiagen).
cDNA was synthesized from 1 mg of total RNA using the
Superscript III First Strand Reverse Transcriptase Kit and
random hexamers (Invitrogen).
Effects on gene expression by OCT-1 and GAPDH siRNAs,
compared to scrambled siRNA, were evaluated by quantitative
real-time PCR (qRT-PCR) with TaqMan expression assays
(4333764F for GAPDH and HS00231250_m1 for OCT-1,
Applied Biosystems). Expression was measured in siRNA-trans-
fected and mock-transfected samples, and normalized to the level
of expression of endogenous B2M (ß2-microglobulin), which is not
affected by siRNA transfection (assay HS00187842_m1, Applied
Biosystems). For each sample, expression was measured in 4
technical replicates, and average values were used for analysis.
Allele-specific expression in cDNA samples from different
transfections was measured in quadruplicate by use of allele-
discriminating TaqMan genotyping assays for SNPs rs807541 and
rs4504469 (C___3073667_1_ and C___390135_10, respectively;
Applied Biosystems). Both SNPs are located within coding
sequence of KIAA0319, and therefore both alleles could be
detected in cDNA. The alleles of these SNPs represent the risk
and non-risk haplotypes: the risk haplotype allele of rs4504469 was
established previously , while the risk haplotype allele of
rs807541 was established by sequencing cloned cDNA derived
from SHSY5Y cells. For each assay, a standard curve consisting of
10 dilutions of two HapMap DNA samples homozygous for either
the risk or non-risk haplotype allele was generated (rs4504469:
NA10847, NA12761; rs807541: NA10847, NA18858). The
standard curve was used to validate the assay quality and to
generate a regression equation necessary for determining the
relative allelic ratio in the experimental samples. The relative ratio
of the two alleles (A and B) was measured as the ratio between VIC
and FAM fluorofores, which were attached to the two different
corresponding allele-specific probes in each case. Specifically, the
Ct(cycle at threshold) values were averaged between technical
replicates, and the differences between the two alleles were
FAM)=dCt. The ratios of known dilutions of the HapMap
DNA samples were plotted relative to dCt, and a linear regression
model fitted to the data. The allelic ratios for the experimental
samples were calculated using dCtin the regression equation. An
unpaired two-sided t-test was used to compare the means between
groups of samples.
Identification and Analysis of Sequence Variants on the
Pair-wise sequence comparison of the risk and non-risk BAC
sequences revealed eight variants within the 4-kb region between
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TTRAP and KIAA0319: one simple repeat and seven SNPs
[designated SNP 1 through SNP 7 (Figure 1A)]. These seven
KIAA0319 promoter region SNPs were genotyped in sample 1 (see
Methods) and tested for association with various reading-related
traits. In addition to the previously reported associations with
rs3212236 (SNP 4)  and rs9467247 (SNP 5) , we found
that rs9461045 (SNP 2) is significantly associated with many
reading-related traits (Figure 1B and Table S1). Specifically, the
minor alleles of these SNPs are most significantly associated with
OC-irreg (P=0.0002, SNP 2; P=0.0002, SNP 4; P=0.0001, SNP
5). An evaluation of LD across the region (Figure 1C) showed that
all SNPs but rs28501680 (SNP 1) are in strong LD with rs2143340,
the previously implicated risk haplotype-tagging SNP (residing
within the TTRAP gene) . SNPs 2 and 5 are in perfect LD with
each other, sharing the same minor allele frequency of 0.19 (i.e.,
the minor alleles of both SNPs always occur together), with the
slight differences in association P-values likely reflecting different
genotyping success rates (Table S2). We followed up these findings
by genotyping SNPs 2, 4, and 5 in both the entire U.K. set (Table 1
and Table S3) and the severe U.K. subset (Table 1 and Table S4).
SNPs 2 and 5 show the strongest association detected so far with
these samples. Both SNPs are most significantly associated with
OC-irreg (P=0.0046, SNP 2; P=0.0025, SNP 5) in the entire
U.K. set. Additional significant associations were found with the
severe U.K. subset for OC-irreg (P=0.0006, SNP 2; P=0.0003,
SNP 5), OC-choice (P=0.0003, SNP 2; P=0.0001, SNP 5), and
READ (P=0.0003, SNP 2; P=0.0002, SNP 5).
We also performed comparative analyses of the genomic region
4 kb upstream of the KIAA0319 TSS using sequences from 11
vertebrate species (Figure 1D). Multi-species sequence compari-
sons can reveal genomic segments under evolutionary constraint
due to their functional importance [36–39], such as serving a role
in transcriptional regulation. Overall, there is little conservation of
this upstream region across species, evident by a paucity of multi-
species conserved sequences identified on the UCSC Genome
Browser (http://genome.ucsc.edu)  ‘Most Conserved’ track
(Figure S1), which compares orthologous sequences from 12
different species. The most pronounced conservation across
species extends from the TSS to approximately 1 kb upstream of
KIAA0319. This region includes SNPs 5, 6, and 7, and likely
encompasses the promoter and perhaps other upstream elements
important in regulating KIAA0319 expression. Examination of this
region using the UCSC Genome Browser reveals a predicted CpG
island, a DNase I hypersensitive site, a FirstEF-predicted
promoter, and evidence for sequence conservation in certain
species (Figure S1). SNP 5 is the only variant within this conserved
region showing association with RD; analysis of SNP 5 reveals that
the nucleotide on the non-risk haplotype (G allele) is conserved
across primates only (Figure 1D). In the case of the other two
associated variants (SNPs 2 and 4), the SNP 2 nucleotide on the
non-risk haplotype (G allele) is conserved across all species
examined except marmoset and horse, while the SNP 4 nucleotide
on the non-risk haplotype (A allele) is conserved across all species
examined except pig. Note that the sequences encompassing SNPs
2 and 4 could not be aligned with orthologous mouse or rat
Characterization of the Putative KIAA0319 Promoter
We generated a series of luciferase-expressing constructs
containing progressively smaller segments of the genomic region
immediately upstream of KIAA0319 (derived from the non-risk
BAC), and tested each construct in two neuronal cell lines,
SHSY5Y and SK-N-MC (Figure 2A). Neuronal cell lines were
chosen based on the strong expression of KIAA0319 in the
developing human brain . These studies indicated the
presence of promoter activity between 224 and 297 bp of the
KIAA0319 TSS. TRANSFAC  analysis of this interval revealed
predicted binding sites for the transcription factors RFX1
(regulatory factor X, 1) and ETF (epidermal growth factor
receptor transcription factor); we also identified the same RFX1-
binding site using the UCSC Human Genome Browser (Figure
S1). Site-directed mutagenesis of the RFX1- or ETF-binding site
significantly reduced luciferase expression (Figure 2B), although
neither mutated site was associated with a complete loss of
promoter activity. Interestingly, ETF is known to drive transcrip-
tion from promoters that are GC-rich and lack a TATA box ;
this is the case for the putative promoter of KIAA0319, which
includes an in silico-predicted CpG island (Figure S1). None of the
seven SNPs we identified between TTRAP and KIAA0319 reside in
this putative promoter region.
Additionally, transcriptional silencing activity appeared to be
associated with the region from 297 to 2216 bp of the KIAA0319
TSS, an interval in which TRANSFAC predicted a Pax-6 (paired
box gene 6) binding site. While SNP 7 falls within this region, it
does not interrupt this predicted binding site or show strong
association with any reading-related traits.
Influence of Risk Variants on Gene Expression and
Nuclear Protein Binding
We next investigated the effect of the three variants highly
associated with reading-related traits (SNPs 2, 4, and 5) on
luciferase expression using mutagenized versions of the above-
described non-risk haplotype construct (Figure 3A). In these
studies, we directly compared the non-risk versus risk allele for
each SNP, measuring luciferase expression in SHSY5Y and SK-
N-MC cells as well as in HEK293T, a human embryonic kidney
Table 1. Genetic associations for markers genotyped in selected U.K. sample sets.
P-value for Traita
Entire U.K. set (630 Siblings, 264 Families) Severe U.K. subset (313 Siblings, 126 Families)
OC-irregPD OC-choiceREADSPELL OC-irregPD OC-choiceREADSPELL
rs9461045 (SNP 2) 0.00460.00970.01040.0006 0.0489 0.00030.00030.0018
rs3212236 (SNP 4)0.01750.018 0.02090.00130.0006 0.00080.0024
rs9467247 (SNP 5)0.0025 0.00440.0084 0.00030.0362 0.0001 0.0002 0.0020
aUncorrected for multiple testing with only significant P-values,0.05 shown. All associations with low reading scores are with the minor alleles of the SNPs. None of the
SNPs were associated with the PA trait in either sample set.
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cell line; this allowed examination of promoter activity in neuronal
as well as non-neuronal cell lines. Introduction of the SNP 2 risk
variant significantly reduced luciferase expression (by 35–57%) in
all three cell lines. The SNP 4 risk variant increased luciferase
expression in SHSY5Y cells, but not in SK-N-MC or HEK293T
cells; the SNP 5 risk variant had a negligible effect on luciferase
expression in these cell lines. These findings suggest that SNP 2
may contribute to the reduced KIAA0319 expression seen from the
EMSAs were performed to investigate the potential role of SNP
2 in modulating transcription factor binding. A probe correspond-
ing to the risk (but not the non-risk) allele of SNP 2 binds nuclear
protein(s) in an EMSA (Figure 3B). No allele-specific nuclear
protein binding was detected by EMSA for either SNP 4 or 5 (data
not shown). In silico analysis of the sequence encompassing SNP 2
using TRANSFAC revealed that the risk variant creates a putative
binding site for CRX and OCT-1. Accordingly, we performed an
EMSA in the presence of unlabeled competitors containing known
binding sites for human CRX and OCT-1, respectively. Both
competitors ablated binding of the nuclear protein(s) to the probe
containing the SNP 2 risk variant (Figure 3C). We also performed
a supershift EMSA (see Methods) with anti-CRX or anti-OCT-1
polyclonal antibody, and found that the presence of the anti-OCT-
1 (but not anti-CRX) antibody decreased the observed binding
(Figure 3D). These data provide in vitro evidence of a functional
mechanism by which the SNP 2 risk allele contributes to the
reduced KIAA0319 expression through creation of a binding site
for the transcription factor OCT-1.
Figure 2. Luciferase-based expression analysis of the putative KIAA0319 promoter region. (A) Luciferase-expressing constructs containing
different portions of the KIAA0319 promoter region from the non-risk haplotype were generated. Restriction sites relevant to the creation of the
depicted ‘‘deletion series’’ of constructs are shown, as are the locations of SNPs 1–7 (see Figure 1A). Each construct was transfected into SHSY5Y and
SK-N-MC neuronal cell lines and subsequent luciferase expression measured; all assays were performed in quadruplicate and repeated at least three
times. RLA for each construct was scaled such that pGL3-Basic activity equaled 1.0. Error bars represent the standard error of the mean. The green box
in each construct represents the proximal end of KIAA0319, with the arrow indicating the TSS. (B) Additional studies were performed with constructs
containing disrupted RFX1- or ETF-binding sites (represented by a red triangle and red square, respectively).
A Functional Variant of a Dyslexia Candidate Gene
PLoS Genetics | www.plosgenetics.org6 March 2009 | Volume 5 | Issue 3 | e1000436
Allele-Specific Effect of an OCT-1 Knock-Down on
Using an siRNA, we knocked-down OCT-1 expression in
SHSY5Y cells, which Paracchini et al.  previously showed
express KIAA0319,andareheterozygousforthe riskhaplotype.This
siRNA reduced OCT-1 expression by about half (versus transfec-
tion with a scrambled siRNA, P=0.0002). We then measured the
effect of OCT-1 knock-down on KIAA0319 expression using allele-
specific qRT-PCR assays for two heterozygous coding SNPs
residing within KIAA0319 (rs807541 and rs4504469). These SNPs
showed mean allelic ratios significantly lower than 1.0 (Figure 4)
after transfection of a scrambled siRNA (negative control); in
particular, the results for both SNPs indicate that KIAA0319
expression from the risk haplotype is lower than from the non-risk
haplotype, with risk:non-risk allelic ratios of Xrs807541=0.4860.13
and Xrs4504469=0.5760.08 (in agreement with values previously
reported by Paracchini et al. ). Following OCT-1 knock-down,
(Xrs807541=0.8160.06 and Xrs4504469=0.8560.04), consistent with
an increase in KIAA0319 expression from the risk haplotype.
In this study, we sought to identify a variant(s) on the RD-
associated risk haplotype  that decreases expression of
KIAA0319. Our experimental data consistently indicate that the
minor allele of rs9461045 (SNP 2) is likely to be functionally
relevant for the development of RD. Specifically, we have shown
that the risk allele of rs9461045: (1) is one of the markers most
significantly associated with RD in our set of families; (2) decreases
gene expression in luciferase-based assays; and (3) creates a
binding site for a nuclear protein(s), likely to include the
transcriptional silencer OCT-1. Moreover, the role of OCT-1
was further supported by the increase in KIAA0319 expression
from the risk haplotype upon siRNA-mediated knock-down of
The chromosome 6p22 risk haplotype is a well-established
genetic risk factor for reading problems in populations of
European descent, showing association in at least two sets of
families with RD  and a large unselected set of additional
individuals . The data we present here help to provide an
explanation for previous contradictory reports that failed to
replicate an RD-association with the risk haplotype. Specifically,
Luciano et al.  detected an opposite trend of association,
showing that the same haplotype was associated with good (as
opposed to poor) reading skills in an unselected Australian sample
set. A different LD structure of the region in the population
examined in this latter study can explain these apparently
divergent findings, as previously suggested . HapMap samples
were analyzed for both markers, rs2143340 (the risk haplotype-
Figure 3. Effect of risk versus non-risk variants on luciferase expression and nuclear protein binding. (A) Luciferase expression from
constructs containing the risk versus non-risk variants of SNPs 2, 4, and 5 was measured in SHSY5Y, SK-N-MC, and HEK293T cells. White and red circles
represent the non-risk and risk variants, respectively (see Figure 2 for additional features of the depicted constructs). All assays were performed in
quadruplicate and repeated at least three times. Error bars represent the standard error of the mean. The vertical dashed line represents the RLA
measured for the construct containing the non-risk haplotype (set at 1.0 RLA); note that this reflects a different scale than the RLA depicted in
Figure 2. For all three cell lines, only the construct containing the SNP 2 risk variant (denoted with a red arrow) yielded a significant RLA difference
compared to the construct containing the non-risk haplotype, as analyzed using an unpaired two-sided t-test (P=8.32610210, SHSY5Y;
P=3.9261027, SK-N-MC; P=4.0261028, HEK293T). (B) EMSA testing the binding of SHSY5Y nuclear protein(s) to probes containing the SNP 2 risk
versus non-risk variant. The presence of a competitor is denoted above each lane: -, no competitor; R, risk competitor; N, non-risk competitor; AP2,
competitor containing an AP2-binding site (negative control); and *, 10-fold and **, 100-fold excess of competitor, respectively. (C) EMSA testing the
binding of SHSY5Y nuclear protein(s) to probes containing the SNP 2 risk variant in the presence of competitors containing binding sites for CRX,
OCT-1, and AP2 (negative control). (D) Supershift EMSA testing the binding of SHSY5Y nuclear protein(s) to probes containing the SNP 2 risk variant in
the presence of anti-CRX or -OCT-1 antibody or general rabbit antiserum.
A Functional Variant of a Dyslexia Candidate Gene
PLoS Genetics | www.plosgenetics.org7 March 2009 | Volume 5 | Issue 3 | e1000436
tagging SNP) and rs9461045 (SNP 2), as shown in Figure S2; the
detected LD differs among populations. LD is strong in the CEPH
population (European descent), implying that haplotypes contain-
ing the minor allele of rs9461045 will also harbor the minor allele
of rs2143340; LD between these two markers is not seen in three
other HapMap populations. As such, the two markers will be
present in all the possible haplotypes within these other
populations, which makes it possible that, by chance, the minor
allele of rs9461045 will appear more frequently in combination
with the major allele of rs2143340. This scenario can explain why
we see conflicting association results between studies using
different populations, as is often the case in replication analyses
of disease/trait associations . This could certainly be the case
for the Australian sample set, which is at least partially admixed.
Thus, our study provides an empirical explanation for apparently
contradictory complex trait-related genetic associations.
The precise function of KIAA0319 has yet to be elucidated, but
it appears to play a role in neuronal migration during brain
development, similar to other RD candidate genes  and as
evidenced by its specific pattern of expression in the developing
human and mouse neocortex . Additionally, KIAA0319 is
strongly expressed in human adult brain, specifically in the
superior parietal cortex, primary visual cortex, and occipital cortex
, areas thought to be important in reading . Our studies
identified two regions that may contribute to this expression
specificity (Figure 2A). First, the KIAA0319 promoter has a
potential binding site for RFX1, a protein shown to regulate
differentiation of ciliated sensory neurons in C. elegans  and
Drosophila . Second, the region implicated as a likely silencer
element contains a predicted binding site for Pax-6, a transcription
factor known to play a major role in regulating cortex
development . It is notable that the Pax-6 and KIAA0319
genes have similar expression patterns in the developing mouse
and human brains , consistent with their potential transcrip-
tional regulatory interactions.
The rs9461045 risk variant creates potential binding sites for
CRX and OCT-1 transcription factors, although we could only
find evidence for OCT-1 binding to the risk haplotype (Figure 3D).
Both CRX and OCT-1 contain DNA-binding homeobox domains
with similar recognition sites [49,50]; it is thus possible that OCT-
1 was able to bind both CRX and OCT-1 competitors, which
would explain the observed ablation of risk probe-binding in the
EMSA with either competitor (Figure 3C). OCT-1, also known as
POU2f1 (POU domain, class 2, transcription factor 1), is a
ubiquitously expressed member of the POU domain factor family
. This protein is involved in many biological processes, and has
been shown to play a role in the formation of radial glia, the cells
that provide a scaffold structure for neuronal migration .
OCT-1 can act as a transcriptional silencer by binding to an 8-bp
AT-rich target (‘octamer’) near a promoter . Notably,
rs9461045 falls in a 120-bp AT-rich genomic region that has
relatively higher sequence identity with the orthologous regions in
the horse, pig, and elephant genomes compared to the
surrounding region (Figure 1D). Further, it has been shown that
such AT-rich regions are important for unzipping DNA during
transcription  and are susceptible to binding by nuclear matrix
attachment proteins, such as OCT-1 . While the specific
region encompassing rs9461045 is not highly conserved across
mammals, recent findings suggest that upwards of 50% of
authentic transcription factor-binding sites are not heavily
conserved, at least not based on the methods used to date for
identifying multi-species sequence conservation . Since the
rs9461045 risk variant appears to create a human-specific
transcription factor-binding site that reduces gene expression, this
site may not be under evolutionary constraint.
The studies reported here provide for the first time strong
evidence implicating a specific variant to be functionally relevant
for RD. Our findings provide new insights for understanding the
role of KIAA0319 in RD and brain development as well as for
establishing the role of non-coding mutations in complex genetic
diseases. A growing body of evidence suggests that variants
residing in transcriptional regulatory elements (as opposed to
coding regions) underlie many such disorders [56,57]. Therefore,
the experimental strategies described here more broadly illustrate
a general approach that can be used for investigating the
molecular basis of genetically complex diseases. Our findings also
provide the first example, to our knowledge, of using siRNA to
define the functional basis of allele-specific effects of genetic
variants, and highlight the different approaches needed to
implicate functional variants in complex genetic diseases.
(http://genome.ucsc.edu) using data from the Human May 2004
Assembly (chr6:24,753,365–24,758,893). Depicted is the region
between TTRAP and KIAA0319 showing (from top to bottom) the
interval covered by the luciferase deletion series (see Figure 2A),
the seven KIAA0319 promoter region SNPs (see Figure 1A),
TTRAP and KIAA0319 genes, and various ‘Regulation and
Comparative Genomics’ tracks.
Figure S1. UCSC Human Genome Browser snapshot
Figure 4. Effect of OCT-1 knock-down on KIAA0319 expression
in SHSY5Y neuronal cells. KIAA0319 expression from the risk versus
non-risk haplotype measured in SHSY5Y neuronal cells transfected with
a scrambled versus OCT-1–specific siRNA. Allele-specific KIAA0319
expression of all samples was quantified by measurements of the
allelic ratios of two heterozygous coding SNPs in KIAA0319 (rs807541
and rs4504469). The results are presented as the global mean6the
standard error of the mean of the measurements in the six biological
replicates (*P=0.004; **P=0.0003). The horizontal dashed line at 1.0
represents equal KIAA0319 expression from the risk and non-risk
A Functional Variant of a Dyslexia Candidate Gene
PLoS Genetics | www.plosgenetics.org8 March 2009 | Volume 5 | Issue 3 | e1000436
Found at: doi:10.1371/journal.pgen.1000436.s001 (0.14 MB PPT)
at the KIAA0319 locus. D’ values are indicated, as represented
through Haploview version 3.32. The black diamonds indicate LD
values between rs2143340 (the risk haplotype-tagging SNP) and
rs9461045 (SNP 2). Strong LD (red squares) between rs2143340
and SNP 2 is detected only in the population of European origin.
Adapted from Paracchini et al. .
Found at: doi:10.1371/journal.pgen.1000436.s002 (0.40 MB PPT)
Figure S2. LD structure of four HapMap populations
1 (89 families).
Found at: doi:10.1371/journal.pgen.1000436.s003 (0.06 MB PDF)
Table S1. Marker-trait association P-values in sample
Found at: doi:10.1371/journal.pgen.1000436.s004 (0.04 MB PDF)
Table S2. Genotype statistics for KIAA0319 promoter
in the entire U.K. set (264 families) - adapted from Harold et al.
Found at: doi:10.1371/journal.pgen.1000436.s005 (0.07 MB PDF)
Table S3.Marker-trait associationP-values
severe U.K. subset (126 families) - adapted from Harold et al. .
Found at: doi:10.1371/journal.pgen.1000436.s006 (0.07 MB PDF)
Table S4. Marker-trait association P-values in the
Found at: doi:10.1371/journal.pgen.1000436.s007 (0.07 MB PDF)
Text S1. Supplementary methods.
We thank Dr. Neal Copeland for providing the SW102 E. coli strain used
for BAC recombineering, Drs. Je ´ro ˆme Nicod and Anthony Antonellis for
providing advice and DNA constructs used for the luciferase assays, Shih-
Queen Lee-Lin for technical assistance, Monica Berger and Tony Gill for
editorial assistance, and Drs. Clotilde Le ´vecque, Antonio Velayos-Baeza,
David Bodine, and Laura Elnitski for helpful discussions and advice.
Conceived and designed the experiments: MYD SP TSS LPO JCK RWM
EDG APM. Performed the experiments: MYD SP TSS LPO. Analyzed the
data: MYD SP TSS LPO JCK RWM EDG APM. Contributed reagents/
materials/analysis tools: MYD LPO RWM EDG APM. Wrote the paper:
MYD SP LPO EDG APM. Made significant contributions to editing the
paper: TSS JCK RWM. Sequenced the BACs: PC SB.
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