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Christmas disease in a Hovawart family
AB
Figure 2. Analysis of hepatocyte nuclear factor 4α and androgen receptor binding of wildtype and mutated F9 promoter regions using an electrophoretic mobility shift assay. (A, B) Human hepatocyte nuclear factor 4α (HNF4α) and (B) androgen receptor (AR) were used to bind biotin-labeled wildtype and mutated F9 promoter fragments (F9-wt, F9-mut). Specific shifted bands (solid arrowheads) are detected in lane 2 (A) for HNF4α and lanes 5 and 6 (B) for AR. To test specificity, binding reactions were also performed using bovine serum albumin [BSA; lanes 3 and 4 (A), lanes 1 and 2 (B)]. In lanes 5 and 6 (A) and lanes 3 and 4 (B) no protein was added. Binding reactions were separated on 12% Tris-Glycine gels. X-ray films were cropped using GIMP 2.8.22. The 70 kDa protein marker band (PageRuler Prestained Protein Ladder, Fermentas) is indicated with an asterisk (lane M). The open arrowhead indicates unbound, free DNA.
factor VIII (F8) and IX (F9) gene, respectively.47 Since the 1960s cases of canine hemophilia B have been reported and in 1989 the first description of the molecular cause in a Cairn Terrier population at the Francis Owen Blood Research Laboratory was published.5,8,9,44
Since then only six further types of variants, all of them affecting the coding region of the F9 gene, i.e. deletions, insertions, missense variants, have been described as causative for canine hemophilia B.10-12,15,16 The identifica- tion of a causative promoter variant in the Hovawart dogs described here is therefore unique in two respects: it is the first regulatory variant described in dogs and secondly this variant resembles a specific subtype of hemophilia B, known as hemophilia B Leyden, in humans.21 Hitherto, 21 distinct variants in the human F9 promoter have been determined in families affected by hemophilia B Leyden.48 These variants cluster in the so-called Leyden-specific region (LSR) and interfere with the binding of different transcription factors, e.g. AR, HNF4α, ONECUT, and C/EBPα.49 The deletion identified in the Hovawart dogs was located 73 bp upstream of the start codon of the canine F9 gene corresponding to position -23 in the third human promoter cluster harboring the overlapping bind- ing sites of AR and HNF4α.48 Similar to analyses in humans, it was possible to show by electrophoretic mobility shift assay that the deletion in the canine pro- moter abolished HNF4α binding because it affects the highly conserved core sequence of the HNF4α binding motif.24 On the other hand, binding of AR was not affect- ed. This might be due to the fact that AR DNA-binding
Figure 3. Dual-luciferase reporter analysis of F9 promoter activities in Hep G2 cells. Box and whisker plot showing the change of relative response ratios (RRR) between the wildtype (F9-wt) and mutant promoter (F9-mut) gene constructs. The lines in the boxes represent the medians. Whiskers indicate minimum and maximum RRR values. Values were normalized as described in the Online Supplementary Methods. P-values are indicated.
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