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 OCT-1.
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-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 containing 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 (). 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
genes have similar expression patterns in the developing mouse and human brains 
, consistent with their potential transcriptional 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 (). Both CRX and OCT-1 contain DNA-binding homeobox domains with similar recognition sites 
; 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 (). OCT-1, also known as POU2f1 (POU
domain, class 2
, transcription f
), 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 (). 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 
. 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.