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 28 dog breeds and four mixed-breed dogs hitherto, it has not yet been reported in the Hovawart. Here we describe the identification of a Hovawart family transmitting typical signs of an X-linked bleeding disorder. Five males had been reported to suffer from recurrent hemorrhagic episodes, four of them had to be euthanized finally and one died due to severe blood loss. A blood sample of one of these males with only 2% of the normal concentration of plasma factor IX (FIX) together with samples of seven relatives including the mother and grandmother were provided for further analysis. Next generation sequencing of DNA of the mother and grandmother revealed a single nucleotide deletion in the F9 promoter (NC_006621.3:g.109,501,492delC; CanFam3.1). Genotyping of the deletion in 1,298 dog specimens (81 different breeds) 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 highly conserved overlapping DNA binding sites of hepatocyte nuclear factor 4 (HNF4) and androgen receptor (AR). The deletion only abolishes binding of HNF4 as demonstrated by electrophoretic mobility shift assay (EMSA) using purified recombinant human HNF4 and a transient overexpression lysate of human AR with double-stranded DNA probes encompassing the mutant promoter region. Luciferase reporter assays using wild type and mutated promoter fragment constructs transfected into Hep G2 cells showed a 65.3% reduction in expression from the mutant promoter. The data presented here provide evidence that the deletion identified in the Hovawart family caused a rare type of hemophilia B resembling human hemophilia B Leyden.\n\nAuthor summaryHemophilia B is the rarer form of classical hemophilias resulting from the absence or residual activity of blood clotting factor IX. Due to its X-linked recessive inheritance normally only males are affected. In a disease subtype, termed hemophilia B Leyden, factor IX activities increase during puberty resulting in spontaneous improvement of bleeding symptoms or even clinical recovery. This surprising development-related alteration is caused by nucleotide variants in important developmental and hormone-responsive regulatory regions of the factor IX gene promoter interfering with transcription factor binding. Although hemophilia B has been reported in several dog breeds, subtypes resembling human hemophilia B Leyden were unknown hitherto. In addition, the single nucleotide deletion reported here in Hovawarts in the overlapping binding sites of transcription factor HNF4 and androgen receptor only affecting HNF4 binding, was unexpected. Although it is advisable to genotype females in the future to prevent a further spread of this subtype of the disorder, our findings also open up the possibility not to euthanize affected males inevitably but to treat until puberty if necessary.