FIGURE 3. Functional Follow-Up of rs6781009 in the SCN5A Locus.

(A) In vivo activity of 4 exemplar human cardiac enhancers in embryonic transgenic mice stained for LacZ enhancer reporter activity (dark blue) are shown. (For additional examples of previously described enhancers near lead SNPs, see Online Figure 13.) (B) Position of the regulatory element containing rs6781009 on the SCN5A-SCN10A locus. GWAS signals are plotted on a −log(p) scale in dark blue. The regulatory element is bound by TBX3, TBX5, and P300 (lower black traces) in mice, and the contact profile of the SCN5A promoter obtained by 4C-seq human cardiac ventricular tissue revealed an interaction between this regulatory element and the SCN5A promoter (upper black trace and contact profile). Normalized contact intensities (gray dots) and their running median trends (black line) are depicted for the SCN5A promoter viewpoint. Medians are computed for 4-kb windows, and the gray band displays the 20% to 80% percentiles for these windows. Below the profile, statistical enrichment across differently scaled window sizes (from 2 kb [top row] to 50 kb [bottom rom]) is depicted of the observed number of sequenced ligation products over the expected total coverage of captured products, with the latter being estimated on the basis of a probabilistic background model. Local changes in color codes indicate regions that are statistically enriched for captured sequences. The lowest box shows the linkage disequilibrium pattern for the HapMap CEU population. (C) Luciferase assay performed in H10 cells showing a high constitutive activity for the enhancer core element (0.6 kb) containing the major allele for rs6781009, which is reduced for the minor allele in both a large enhancer construct (1.5 kb), as well as in the core enhancer element (0.6 kb). *p < 0.01. (D) Dorsal views of hearts containing the human regulatory element with the major versus minor allele for rs6781009 in a LacZ reporter vector, showing specific expression of the enhancer in the interventricular septum (ivs) for the major allele, which is absent for the minor allele. *p < 0.05. la = left atrium; lv = left ventricle; ra = right atrium; rv = right ventricle; other abbreviations as in Figure 1.