Functional characterization of the duplicated region. (A) In silico identification of regulatory elements in the duplicated DNA region. The duplicated region identified by WGS of 3 patients from both families is shown at the top. The genomic coordinates of the putative enhancer regions A, B, C, D, E, and F (supplemental Tables 5 and 6) are indicated. The F8 proximal promoter, exon 1, and intron 1, color coded as in Figure 3, are shown for additional reference. The locations of H3K27Ac peaks, DNase I hypersensitivity sites, and binding sites for specific TFs (from ENCODE), as well as repeating and regulatory elements (extracted from ORegAnno) and IME motifs (identified with FIMO), are illustrated in the genome browser tracks below the F8 gene bar. (B) Regions A to F were cloned (1 by 1) in a reporter vector upstream of a minimal promoter (ie, a TATA-box promoter element already present in the empty vector) driving Firefly luciferase expression and transfected into HUVECs and HepG2 cells together with a vector expressing Renilla luciferase. (C-D) After 12 hours, cells were lysed, and luciferase activities were determined in HUVECs (C) and HepG2 cells (D). Data are normalized for empty vector (containing only the minimal promoter) and Renilla fluorescence. The bars represent means and standard deviations of 6 (HUVECs) or 4 (HepG2 cells) replicates. *P < .05, **P < .001, ***P < .0001. LINE, long interspersed nuclear element; LTR, long terminal repeat; SINE, short interspersed nuclear element.