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Christmas disease in a Hovawart family
were used for the luciferase assay (Promega, Mannheim, Germany). The wildtype F9 promoter fragment (971 bp wildtype) was generated by PCR using primers cfa9_HindIII_F_neu (5’- CGTAGACTTAGCACTGTTCAAAGCTTCACACA- CACAGTTCTTAAAT-3’) and cfa9_HindIII_R_neu (5’-ATGGC- TAGCAACCGTCTAAGAAGCTTAATTGTGCAAGGAG- CAAGG-3’). The mutated F9 promoter fragment (970 bp) was generated by PCR using primers cfa9_HindIII_F (5’-ATCGT- CAAGCTTCACACACACAGTTCTTAAAT-3’) and cfa9_HindIII_R (5’-CGTACGAAGCTTAATTGTGCAAGGAG- CAAGG-3’). For cloning into the HindIII restriction site of pGL3, primers were designed with an unspecific random 5’-tag (italics) followed by a HindIII restriction site (underlined) (Online Supplementary Figure S1). DNA from female carrier #6 served as a template for amplification. Low expression levels of C/EBP in Hep G2 cells were complemented by co-transfection of a C/EBPα expression vector.22 Data are presented as relative response ratios.41 A Mann-Whitney U test was used to determine statistical signifi- cance. Values were considered statistically significant when P<0.05 (weakly significant), P<0.01 (medium-strength signifi- cance) and P<0.001 (strongly significant).
Othermethods
Further details of the study methods are given in the Online Supplementary Appendix.
Results
Hemophilias are rare diseases in dogs and hence it was rather coincidental that a case in a Hovawart (#3, Figure 1) was reported to us. With the reconstruction of the pedi- gree using the online dog breed database and pedigree data of individual dogs provided by the owners it was possible to trace the disease back to the female carrier #39 (Figure 1, Online Supplementary Figure S2).42 In the studied family the hemophilia was transmitted to animals #19, #4 and #6. Bitch #19 had one litter with three hemophilic males (#48, #51, #53). Bitches #4 and #6 had litters with one affected male each, #60 and #3, respectively. Although DNA samples from animals #48, #51, #53 and #60 were not available, blood parameters and medical reports about recurrent hemorrhagic episodes were provided (Online
Figure 1. Pedigree section of the hemo- philia 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 hemo- philic male (#3) and relatives (#4 grandmother, #5 sister, #6 mother, #7 cousin). Pedigree symbols are accord- ing to the standardized human pedigree nomenclature.61 Individuals are pseudo- nymized using internal identities. DNA samples were available from individuals
indicated with
sequences of heterozygous bitches #4 and #6 (female carriers) show overlap- ping 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 eutha- nized after recurrent hemorrhages. Xm: maternal X chromosome; Xp: paternal X chromosome; HNF4α: hepatocyte nuclear factor 4α binding site (consen- sus sequence: 5’-TGNACTTTG-3’);21,48 AR: 3’-part of the androgen receptor binding site (consensus sequence: 5’- AGNACANNNTGTNCT-3’).21,48
an arrow.
DNA
haematologica | 2019; 104(11)
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