Christmas disease in a Hovawart family resembling human hemophilia B Leyden is caused by a single nucleotide deletion in a highly conserved transcription factor binding site of the F9 gene promoter

Abstract

Hemophilia B is a classical monogenic, X-chromosomal, recessively transmitted bleeding disorder caused by genetic variants within the coagulation factor IX gene (F9). Although hemophilia B has been described in dogs, it has not yet been reported in the Hovawart breed. Here we describe the identification of a Hovawart family transmitting typical signs of an X-linked bleeding disorder. Five males were reported to suffer from recurrent hemorrhagic episodes. A blood sample from one of these males with only 2% of the normal concentration of plasma factor IX together with samples from seven relatives were provided. Next-generation sequencing of the mother and grandmother revealed a single nucleotide deletion in the F9 promoter. Genotyping of the deletion in 1,298 dog specimens including 720 Hovawarts revealed that the mutant allele was only present in the aforementioned Hovawart family. The deletion is located 73 bp upstream of the F9 start codon in the conserved overlapping DNA binding sites of hepatocyte nuclear factor 4α (HNF-4α) and androgen receptor (AR). The deletion only abolished binding of HNF-4α, while AR binding was unaffected as demonstrated by electrophoretic mobility shift assay using human HNF-4α and AR with double-stranded DNA probes encompassing the mutant promoter region. Luciferase reporter assays using wildtype and mutated promoter fragment constructs transfected into Hep G2 cells showed a significant reduction in expression from the mutant promoter. The data provide evidence that the deletion in the Hovawart family caused a rare type of hemophilia B resembling human hemophilia B Leyden.

Figure 1.

Pedigree section of the hemophilia B Leyden Hovawart family and DNA sequence comparison of the mutant hepatocyte nuclear factor 4α/androgen receptor binding site in the promoter of canine F9 in the hemophilic male (#3) and relatives (#4 grandmother, #5 sister, #6 mother, #7 cousin). Pedigree symbols are according to the standardized human pedigree nomenclature. Individuals are pseudonymized using internal identities. DNA samples were available from individuals indicated with an arrow. DNA sequences of heterozygous bitches #4 and #6 (female carriers) show overlapping peaks with similar heights 5′ of the deletion position. For males #48, #51, #53 and #60, signs of hemophilia (Online Supplementary Table S1) were reported and the dogs had to be euthanized after recurrent hemorrhages. X_m: maternal X chromosome; X_p: paternal X chromosome; HNF4α: hepatocyte nuclear factor 4α binding site (consensus sequence: 5′-TGNACTTTG-3′);^, AR: 3′-part of the androgen receptor binding site (consensus sequence: 5′-AGNACANNNTGTNCT-3′).^,

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.

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.