title: Analysis of mRNA in hemophilia A patients with undetectable mutations reveals normal splicing in the factor VIII gene.
abstract: BACKGROUND
  haemophilia A (HA) is characterized by partial or total deficiency of factor VIII (FVIII) protein activity. It is caused by a broad spectrum of mutations in the FVIII gene. Despite tremendous improvements in mutation screening methods, in about 2% of HA patients no DNA change could be found, even after sequencing the whole coding part of the FVIII gene including the flanking splice sites, as well as the promotor and the 3' UTR regions.
  
  
  OBJECTIVE
  In the present study we performed a detailed RNA analysis of three groups of patients. The first included control patients with known splicing defects, the second included two patients with already identified nucleotide changes close to splicing sites, that could potentially alter the normal splicing process, and a third group of 11 unrelated patients whose genomic DNA have already been screened for mutations by DHPLC and direct sequencing with no mutation being identified.
  
  
  RESULTS
  Both candidate splice site mutations were shown to result in either skipping or alternative splicing of at least one exon, therefore these DNA changes must be considered as causal for the patients' HA phenotype. In contrast, no abnormalities on the RNA level were observed in any of 11 unrelated patients without mutations in the FVIII gene.
  
  
  CONCLUSIONS
  These findings exclude mutations that could be located deep in the introns and affecting either normal splicing or lead to mechanisms causing some unknown rearrangements of the FVIII gene. In fact, our results point to the presence of still unknown factor(s) causing HA, which might be either allelic or in the close proximity of the FVIII gene or non-allelic associated with other genetic loci that are involved in the processing of the FVIII protein.
authors.affiliation: Institute of Experimental Haematology and Transfusion Medicine, Bonn, Germany.
authors.forename: O
authors.initials: O
authors.lastname: El-Maarri
authors.forename: U
authors.initials: U
authors.lastname: Herbiniaux
authors.forename: J
authors.initials: J
authors.lastname: Graw
authors.forename: J
authors.initials: J
authors.lastname: Schröder
authors.forename: A
authors.initials: A
authors.lastname: Terzic
authors.forename: M
authors.initials: M
authors.lastname: Watzka
authors.forename: H H
authors.initials: HH
authors.lastname: Brackmann
authors.forename: W
authors.initials: W
authors.lastname: Schramm
authors.forename: P
authors.initials: P
authors.lastname: Hanfland
authors.forename: R
authors.initials: R
authors.lastname: Schwaab
authors.forename: C R
authors.initials: CR
authors.lastname: Müller
authors.forename: J
authors.initials: J
authors.lastname: Oldenburg
mesh_terms: D004252:DNA Mutational Analysis; D005091:Exons; D005169:Factor VIII / Q000235:genetics*; D006467:Hemophilia A / Q000209:etiology / Q000235:genetics*; D006801:Humans; D009154:Mutation; D022821:RNA Splice Sites; D012326:RNA Splicing; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D022821:RNA Splice Sites; D012333:RNA, Messenger; D005169:Factor VIII