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. 2009 Mar;5(3):e1000436.
doi: 10.1371/journal.pgen.1000436. Epub 2009 Mar 27.

A common variant associated with dyslexia reduces expression of the KIAA0319 gene

Megan Y Dennis  1 , Silvia Paracchini, Thomas S Scerri, Ludmila Prokunina-Olsson, Julian C Knight, Richard Wade-Martins, Penny Coggill, Stephan Beck, Eric D Green, Anthony P Monaco
Affiliations

A common variant associated with dyslexia reduces expression of the KIAA0319 gene

Megan Y Dennis et al. PLoS Genet. 2009 Mar.

Abstract

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
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.
Figure 2
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).
Figure 3
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 = 8.32×10−10, SHSY5Y; P = 3.92×10−7, SK-N-MC; P = 4.02×10−8, 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.
Figure 4
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 mean±the 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 haplotypes.
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