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B. Brenig et al.
sites display an exceptional amount of sequence variation.50 Although the C-deletion is located in the con- sensus TGTNCT-motif of class I AR-binding sites several alternative motives, e.g. TGTTTC in the stomatin-like protein 3 gene or TGTATC in the prostate-specific antigen gene enhancer III region, have been reported.50-52 Therefore, it can be assumed that the affected males would have recovered from hemophilia during puberty. To what extent a sex- or possibly also age-dependent ame- lioration of the hemophilia B Leyden, as described in humans and also a mouse model, might be present in the affected Hovawarts remains to be determined.49,53 In this respect the Hovawart family could also be of interest as a model in the comparative analysis of age-dependent nor- malization of F9 expression in symptomatic carriers of hemophilia B Leyden in humans.
To analyze whether the deletion not only affected HNF4α binding but also resulted in a downregulation of F9 expression, we performed luciferase assays. Although relative response ratios do not directly reflect F9 mRNA or FIX levels in vivo, silencing of the promoter activity due to the deletion was obvious from these experiments. Similar findings have been made using HNF4α-null mice.26 In the murine model it was shown that expression of factors V, XI, XII, and XIIIB directly depends on hepatic HNF4α. Northern blot analysis also demonstrated that F9 expres- sion was decreased with a significantly prolonged aPTT in the HNF4α-null mice.26 The finding that F9 expression was not completely dependent on HNF4α is in agreement with the observation that control of F9 transcription in mice and humans is complex and depends on a plethora of factors.26 For instance, in earlier studies it was shown, using DNaseI footprinting, that there are further binding
sites of HNF4α and other factors, e.g. ARP1, COUP/Ear3, in the human F9 promoter influencing F9 expression.54 However, the classical HNF4α binding site at position -26 to -19 only binds HNF4α. When analyzing the canine F9 promoter using transcription factor binding site prediction algorithms, further potential binding motifs for HNF4α and other transcription factors were predicted (data not shown).55-57 As ectopic expression of F9 in vivo can be excluded or at least ignored according to recent RNA- sequencing analyses,58 a remaining reduced hepatic activi- ty of the mutated promoter is in agreement with the clin- ical findings of residual FIX activity in the affected males (Online Supplementary Table S1) and the results of the elec- trophoretic mobility shift assay showing binding of AR in androgen-dependent promoter activation.
In summary, we have identified and elucidated the causative genetic variant for hemophilia B Leyden in Hovawarts. This is the first report on a single nucleotide deletion within the binding sites of HNF4α and AR in the F9 promoter causing hemophilia B Leyden in dogs. As the deletion only abolishes the binding of HNF4α, it can be assumed that male dogs will most likely recover during puberty, as reported in humans.30,59,60 However, to prevent any risk of a further propagation of the disorder, genotyp- ing of females is recommended in further breeding.
Acknowledgments
The authors are grateful to S. Pach for expert technical assis- tance and L. Binder for support. The owners of Hovawarts who have provided blood samples are thanked for their generous help. A. Leutz and E. Kowenz-Leutz are thanked for providing the C/EBPα expression vector. S. Shan and F. Xu are supported by a fellowship from the Chinese Scholarship Council (CSC).
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