Page 202 - 2019_11 Resto del Mondo-web

B. Brenig et al.
and this was the first disorder in dogs characterized on the DNA level, data on hemophilia B cases in dogs remain rather scarce compared to data from humans.5-8 For instance, in the Cairn Terrier colony of the Francis Owen Blood Research Laboratory (University of North Carolina, Chapel Hill, NC, USA) a G>A transition (NC_006621.3:g.109,532,018G>A) in exon 8 causing an amino acid exchange (NP_001003323.1:p.Gly418Glu) that resulted in a complete lack of circulating FIX was detected in affected dogs.9 Due to a complete deletion of F9 in a Labrador Retriever, FIX inhibitors were produced after transfusion of canine blood products.10 In a study of Pit Bull Terrier mixed breed dogs and Airedale Terrier dogs a large deletion of the entire 5’ region of F9 extending to exon 6 was found in the former and a 5 kb insertion dis- rupting exon 8 was described in the latter.11 As in the Labrador Retriever with hemophilia B, FIX inhibitors were produced in both breeds. A mild form of hemophilia B in German Wirehaired Pointers was caused by a 1.5 kb Line- 1 insertion in intron 5 of F9 at position NC_006621.3:g.109,521,130.12 Until today, hemophilia B has been described in four mixed-breed dogs and nine dog breeds, i.e. German Shepherd, Lhasa Apso, Labrador Retriever, Rhodesian Ridgeback, Airedale Terrier, Cairn Terrier, Maltese, Mongrel and German Wirehaired Pointer.9-17
In the canine cases analyzed so far on the DNA level, mutations have been observed only in exons and introns of F9, whereas alterations of the F9 promoter have not yet been reported. In humans promoter variants have been detected and result in the so-called hemophilia B Leyden characterized by low levels of FIX until puberty, whereas after puberty FIX concentrations rise to almost normal levels.18-20 Since its first description, the genetic background of human hemophilia B Leyden was elucidat- ed by various studies identifying variants in different transcription factor binding sites in the F9 promoter including the androgen-responsive element (ARE), hepa- tocyte nuclear factor 4α (HNF4α), one cut homeobox (ONECUT1/2) and CCAAT/enhancing-binding protein α (C/EBPα) binding sites.21,22 HNF4α is a liver-enriched member of the nuclear receptor superfamily of ligand- dependent transcription factors and has been associated with several disorders, including diabetes, atherosclero- sis, hepatitis, cancer, and hemophilia.23 Promoter analyses have identified at least 140 genes with HNF4α binding sites. A recent, more detailed analysis using protein bind- ing microarrays identified an additional 1,400 potential binding sites.24,25 Hence, HNF4α plays an important role in the regulation of numerous genes especially in the main- tenance of many liver-specific functions. Liver-specific HNF4α-null mice have been used to study the involve- ment of hepatic HNF4α in blood coagulation. In the murine model it was shown that expression of factors V, XI, XII, and XIIIB depends directly on hepatic HNF4α and FIX expression was decreased with significantly pro-
26
Brandenburg.31,32 Unlike the classical hemophilia B Leyden, FIX levels in patients with these variants cannot be restored by testosterone-driven AR activity and remain low after puberty with no clinical recovery.21,32
Methods
Animals and genomic DNA isolation
Canine blood and/or hair samples were collected by local vet- erinarians. The collection of samples was approved by the Lower Saxony State Office for Consumer Protection and Food Safety (33.19-42502-05-15A506) according to §8a Abs. 1 Nr. 2 of the German Animal Protection Law (TierSchG). Blood collected into EDTA and/or hair samples were provided by different Hovawart and dog breeders with written consent from the dogs’ owners. DNA was extracted from 30-50 hair roots using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the man- ufacturer‘s instructions.33 A salting out procedure was used to obtain DNA from the EDTA blood samples.34 Additional DNA samples deposited with the Institute of Veterinary Medicine were used as controls. All samples were pseudonymized using internal identities.
Next-generation sequencing and genotyping
DNA from animals #4 and #6 was used for next-generation sequencing on an Illumina HiSeq2500. The quality of the fastq- files was analyzed using FastQC 0.11.7.35 Total reads of 1,029,601,630 (#4; sequencing depth 51x) and 1,000,503,256 (#6; sequencing depth 50x) were obtained and mapped to the refer- ence canine F9 gene (NC_006621.3, region 109,501,341 to 109,533,798; CanFam3.1) using DNASTAR Lasergene Genomics Suite SeqMan NGen 15.2.0 (130).36-40
Targeted genotyping of the promoter deletion was done by polymerase chain reaction (PCR) amplification with primers cfa_F9_Ex1_F (5’-CCACTGAGGGAGATGGACAC-3’) and cfa_F9_Ex1_R (5’-CCCACATGCTGACGACTAGA-3’) resulting in a fragment of 328 bp (wildtype) or 327 bp (deletion) spanning the variant position. The resulting PCR products were either directly sequenced on an ABI 3730 Genetic Analyzer (Thermo Fisher Scientific, Basel, Switzerland) or genotypes were deter- mined by restriction fragment length polymorphism analysis after cleavage with RsaI. The wildtype allele generated two fragments of 52 bp and 276 bp while the allele with the deletion remained uncut.
Electrophoretic mobility shift assay
For the electrophoretic mobility shift assay, biotin-labeled, dou- ble-stranded wildtype (cfa_F9n_wt_Biotin: 5’-CAGAAGTAAAT- ACAGCTCAACTTGTACTTTGGAACAACTGGTCAACC-3’) and mutated (cfa_F9n_mut_Biotin: 5’-CCAGAAGTAAAT- ACAGCTCAACTTGTATTTGGAACAACTGGTCAACC-3’) oligonucleotides were synthesized (Integrated DNA Technologies IDT, Leuven, Belgium) harboring the overlapping HNF4α and AR binding sites (underlined). The position of the deleted C- nucleotide is indicated in bold and italics. Recombinant human HNF4α and human AR overexpression lysate were purchased from Origene Technologies Inc. (Rockville, MD, USA).
DNA was detected using the Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Scientific, USA) with minor mod- ifications, i.e. membranes were incubated for 1 min in the sub- strate working solution.
Luciferase assay
pGL3 Luciferase Reporter Vectors (pGL3-Basic, pGL3-Control)
longed activated partial thromboplastin time (aPTT). Ten of the so far identified 28 5’-UTR variants (35.7%) are located within the overlapping binding sites of the andro- gen receptor (AR) and HNF4α in the human F9 promot- er.4,21 Four variants at positions -21, -20 and -19 only affect HNF4α binding and all of them have been shown to cause hemophilia B Leyden.19,27-30 The remaining six vari- ants at positions -26, -24 and -23, located in the overlap- ping region, cause the so-called hemophilia B
2308
haematologica | 2019; 104(11)