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. Although hemophilia B has been described in dogs, 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 were reported to suffer from recurrent hemorrhagic episodes. A blood sample of one of these males with only 2% of the normal concentration of plasma factor IX together with samples of 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 and androgen receptor. The deletion only abolished binding of hepatocyte nuclear factor 4α, while androgen receptor binding was unaffected as demonstrated by electrophoretic mobility shift assay using human hepatocyte nuclear factor 4α and androgen receptor 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 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.