title: Characterisation of two novel large F8 deletions in patients with severe haemophilia A and factor VIII inhibitors.
abstract: Large deletions are found in approximately 5% of patients with severe haemophilia A, but only a few deletion breakpoints have been characterised precisely so far. In this study we characterised the deletion breakpoints of two patients with severe haemophilia A, large deletions and factor VIII (FVIII) inhibitors, and subsequently established deletion-specific assays for the identification of carriers. Patient 1 had a deletion of 37,410 bp comprising exon 1 and the F8 promoter region, and a 5 bp homology (GGGCC) is present at the chromosomal fusion site. In patient 2, a deletion of 22,230 bp including parts of intron 25, exon 26 and 3'-UTR was identified. No homologous repetitive elements were found at the breakpoints. However, both breakpoints were located within long terminal repeats of endogenous retroviruses and the DNA motif TTTAAA - known to be able to bend DNA molecules - was identified at the centromeric breakpoint. By deletion-specific PCR experiments we were able to identify a heterozygous state in mother 2 (carrier) while mother 1 presented only with wild-type alleles (non-carrier). Both deletions are most likely created by DNA double strand breaks and subsequent DNA repair by the non-homologous end joining DNA repair pathway (NHEJ). The exact identification of the deletion breakpoints provides a reliable diagnostic tool for carrier identification in affected families by means of a deletion-specific PCR.
authors.affiliation: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. Lukas.Roth@stud.uke.uni-hamburg.de
authors.forename: Lukas
authors.initials: L
authors.lastname: Roth
authors.forename: Rolf
authors.initials: R
authors.lastname: Marschalek
authors.forename: Johannes
authors.initials: J
authors.lastname: Oldenburg
authors.forename: Florian
authors.initials: F
authors.lastname: Oyen
authors.forename: Reinhard
authors.initials: R
authors.lastname: Schneppenheim
mesh_terms: D001323:Autoantibodies / Q000097:blood*; D001483:Base Sequence; D056905:Chromosome Breakpoints; D053903:DNA Breaks, Double-Stranded; D004252:DNA Mutational Analysis; D004260:DNA Repair; D005091:Exons; D005169:Factor VIII / Q000235:genetics* / Q000276:immunology*; D050939:Gene Fusion; D006580:Genetic Carrier Screening; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing / Q000379:methods; D006467:Hemophilia A / Q000097:blood / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D007438:Introns; D008297:Male; D008969:Molecular Sequence Data; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion; D012720:Severity of Illness Index
chemical_list: D001323:Autoantibodies; D005169:Factor VIII