title: Deep intronic variations may cause mild hemophilia A.
abstract: BACKGROUND
  In about 10% of patients with mild hemophilia A, no candidate gene mutations are apparent after complete gene sequencing.
  
  
  OBJECTIVE
  To analyze factor VIII gene (F8) mRNA for mutations in five families with mild hemophilia A with no apparent genomic mutation and a reduced response to desmopressin.
  
  
  RESULTS
  In four cases, mRNA studies revealed the presence of an abnormal mRNA transcript in addition to normal F8 mRNA. Sequencing of the abnormal transcripts revealed complex abnormalities, which allowed the identification of three different intronic variations (c.2113+1152delA, c.5587-93C>T and c.5999-277G>A) at the DNA level, absent from 387 normal alleles. By in silico analysis, c.2113+1152delA and c.5587-93C>T were strongly predicted to result in the generation of new splice sites with the introduction of premature termination codons, while c.5999-277G>A was predicted to generate a new protein with 30 additional amino acids. However, these predictions were not homogeneous across the different mutations and programs used. The detrimental effect of two mutations was also confirmed by in vitro expression studies. These changes were also identified in related female carriers and in other mild HA patients not included in the original study. No mRNA abnormality was identified in the remaining patient.
  
  
  CONCLUSIONS
  Although rare, deep intronic variations may be responsible for mild hemophilia A where no other F8 mutations have been identified and may be associated with a reduced biologic response to desmopressin. F8 mRNA analysis is a useful tool for the identification of deep intronic variation not detectable by standard DNA sequencing.
authors.affiliation: Department of Cell Therapy and Hematology, Hemophilia and Thrombosis Center, San Bortolo Hospital, Vicenza, Italy. castaman@hemato.ven.it
authors.forename: G
authors.initials: G
authors.lastname: Castaman
authors.forename: S H
authors.initials: SH
authors.lastname: Giacomelli
authors.forename: M E
authors.initials: ME
authors.lastname: Mancuso
authors.forename: G
authors.initials: G
authors.lastname: D'Andrea
authors.forename: R
authors.initials: R
authors.lastname: Santacroce
authors.forename: S
authors.initials: S
authors.lastname: Sanna
authors.forename: E
authors.initials: E
authors.lastname: Santagostino
authors.forename: P M
authors.initials: PM
authors.lastname: Mannucci
authors.forename: A
authors.initials: A
authors.lastname: Goodeve
authors.forename: F
authors.initials: F
authors.lastname: Rodeghiero
mesh_terms: D000328:Adult; D001483:Base Sequence; D001777:Blood Coagulation / Q000187:drug effects / Q000235:genetics*; D016022:Case-Control Studies; D004252:DNA Mutational Analysis; D003894:Deamino Arginine Vasopressin / Q000627:therapeutic use; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism; D005260:Female; D020022:Genetic Predisposition to Disease; D057809:HEK293 Cells; D006467:Hemophilia A / Q000097:blood / Q000175:diagnosis / Q000188:drug therapy / Q000235:genetics*; D006490:Hemostatics / Q000627:therapeutic use; D040941:Heredity; D006801:Humans; D007438:Introns; D007558:Italy; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D010375:Pedigree; D010641:Phenotype; D022821:RNA Splice Sites; D012333:RNA, Messenger / Q000032:analysis; D017423:Sequence Analysis, RNA; D012720:Severity of Illness Index; D014162:Transfection; D055815:Young Adult
chemical_list: D006490:Hemostatics; D022821:RNA Splice Sites; D012333:RNA, Messenger; D005169:Factor VIII; D003894:Deamino Arginine Vasopressin