The handedness-associated PCSK6 locus spans an intronic promoter regulating novel transcripts

Human Molecular Genetics, 2016, Vol. 25, No. 9 1771–1779 doi: 10.1093/hmg/ddw047 Advance Access Publication Date: 21 February 2016 Original Article ORIGINAL ARTICLE The handedness-associated PCSK6 locus spans an intronic promoter regulating novel transcripts 1 School of Medicine, University of St Andrews, St Andrews KY169TF, UK, 2Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK, 3School of Life and Health Sciences, Aston University, Birmingham, UK and 4Department of Physiology, Anatomy & Genetics, Parks Rd., Oxford OX1 3PT, UK *To whom correspondence should be addressed at: School of Medicine, University of St Andrews, St Andrews KY16 9TF, UK. Tel: +44 1334 463542; Fax: +44 1334 463482; Email: Abstract We recently reported the association of the PCSK6 gene with handedness through a quantitative genome-wide association study (GWAS; P < 0.5 × 10−8) for a relative hand skill measure in individuals with dyslexia. PCSK6 activates Nodal, a morphogen involved in regulating left–right body axis determination. Therefore, the GWAS data suggest that the biology underlying the patterning of structural asymmetries may also contribute to behavioural laterality, e.g. handedness. The association is further supported by an independent study reporting a variable number tandem repeat (VNTR) within the same PCSK6 locus to be associated with degree of handedness in a general population cohort. Here, we have conducted a functional analysis of the PCSK6 locus combining further genetic analysis, in silico predictions and molecular assays. We have shown that the previous GWAS signal was not tagging a VNTR effect, suggesting that the two markers have independent effects. We demonstrated experimentally that one of the top GWAS-associated markers, rs11855145, directly alters the binding site for a nuclear factor. Furthermore, we have shown that the predicted regulatory region adjacent to rs11855415 acts as a bidirectional promoter controlling the expression of novel RNA transcripts. These include both an antisense long non-coding RNA (lncRNA) and a short PCSK6 isoform predicted to be coding. This is the first molecular characterization of a handedness-associated locus that supports the role of common variants in non-coding sequences in influencing complex phenotypes through gene expression regulation. Introduction Handedness is one of the most researched human behavioural traits, yet one of the least understood. The vast majority of people worldwide prefer using their right hand for writing and performing most tasks. This observation implies a disadvantage for left-handedness and a large body of research has investigated whether being left-handed can increase susceptibility to particular traits or disorders (1). In this context, language-related disorders have been a particular focus of attention due to handedness showing a correlation, albeit weak, with language dominance lateralization (2). However, no robust evidence supports the association of handedness with disease risk. † Present address: Department of Psychiatry, University of California, San Diego, La Jolla, California 92093, USA. Received: September 29, 2015. Revised: February 8, 2016. Accepted: February 15, 2016 © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1771 Robert Shore1, Laura Covill2, Kerry A. Pettigrew1, William M. Brandler2,†, Rebeca Diaz1, Yiwang Xu1, Javier A. Tello1, Joel B. Talcott3, Dianne F. Newbury2, John Stein4, Anthony P. Monaco2 and Silvia Paracchini1, * 1772 | Human Molecular Genetics, 2016, Vol. 25, No. 9 reached statistical significance, other genes and pathways involved in the determination of left–right structural asymmetries showed association with handedness both in the cohort selected for dyslexia and in the epidemiological cohort. For example, the top associated gene in the general population cohort was located in proximity of GPC3 (P = 2.08×10−6) (6), a gene implicated in cardiac laterality defects (9). These data suggest that the same biological pathways controlling structural laterality may be partly implicated in contributing to behavioural laterality. The single-nucleotide polymorphisms (SNPs) at the PCSK6 locus that showed the highest association (6) lie close to an intronic region predicted to have a regulatory function (10) (Fig. 1 and Supplementary Material, Fig. S1). Both short-sense mRNA and antisense long non-coding RNA (lncRNA) molecules are predicted to be regulated by this region (Fig. 1B). The same locus has been found to be associated with degree of handedness, assessed by the Edinburgh questionnaire (11), in an independent sample representative of a general German population (12). This study did not find association with the rs11855415 marker [selected for replication of the first GWAS results (5)] but reported association with a 33 bp variable number tandem repeat (VNTR) rs10523972 (Fig. 1D). VNTRs have been shown to modulate gene Figure 1. PCSK6 locus associated with relative hand skills. (A) PCSK6 is located on chromosome 15q26.3 as indicated by the red box. (B) PCSK6 structure and expanded view of the region where top associations with relative hand skill cluster (5,6). Both sense (black) and antisense (green) transcripts are predicted to originate within this region, as shown by the human spliced EST and GENCODE tracks from the UCSC Genome Browser (http://genome-euro.ucsc.edu), respectively. A 1.8 kb region (orange box) was cloned into luciferase reporter vectors in both directions (black and green arrows). (C) This region is predicted to contain regulatory elements as shown by UCSC tracks for Chip-SEQ ENCODE (10). From top, the tracks show H3K27Ac and H3K4Me3 histone marks in different cell lines where the highest peak corresponds to the K562 cell line. RNA polymerase II binding for the K562 cell is shown in the third track. (D) The genetic associations cluster within a 12.7 kb LD region, visualized as D′ values, in HapMap CEPH data. The black bar indicates the position of the rs10523972 VNTR. Rs7182874, the strongest associated marker in the most recent GWAS (6), is in high LD (black diamond) with rs11855415, which was the top associated marker in the discovery sample (5) and shows functional allelic effects in the present study. LD visualization of the surrounding region is shown in Supplementary Material, Figure S1. Handedness presents a weak but very consistent heritability across different studies, estimated to be ∼25% (3). The assessment of hand preference is relatively trivial and has been collected for tens of thousands of individuals for which genome-wide genotype data are available. However, no gene or variant has yet been identified to be as (...truncated)