title: Preimplantation genetic diagnosis for hemophilia A using indirect linkage analysis and direct genotyping approaches.
abstract: BACKGROUND
  Preimplantation genetic diagnosis (PGD) is an appealing option for couples at risk of having a child with hemophilia A (HA). Although many clinics offer PGD for HA by gender selection, an approach that detects the presence of the underlying F8 mutation has several advantages.
  
  
  OBJECTIVE
  To develop and validate analysis protocols combining indirect and direct methods for identifying F8 mutations in single cells, and to apply these protocols clinically for PGD.
  
  
  METHODS
  A panel of microsatellite markers in linkage disequilibrium with F8 were validated for single-cell multiplex polymerase chain reaction. For point mutations, a primer extension genotyping assay was included in the multiplex. Amplification efficiency was evaluated using buccal cells and blastomeres. Four clinical PGD analyses were performed, for two families.
  
  
  RESULTS
  Across all validation experiments and the clinical PGD cases, approximately 80% of cells were successfully genotyped. Following one of the PGD cycles, healthy twins were born to a woman who carries the F8 intron 22 inversion. The PGD analysis for the other family was complicated by possible germline mosaicism associated with a de novo F8 mutation, and no pregnancy was achieved.
  
  
  CONCLUSIONS
  PGD for the F8 intron 22 inversion using microsatellite linkage analysis was validated by the birth of healthy twins to one of the couples. The other family's situation highlighted the complexities associated with de novo mutations, and possible germline mosaicism. As many cases of HA result from de novo mutations, these factors must be considered when assessing the reproductive options for such families.
authors.affiliation: Canterbury Health Laboratories, PO Box 151, Christchurch, New Zealand. andrew.laurie@cdhb.govt.nz
authors.forename: A D
authors.initials: AD
authors.lastname: Laurie
authors.forename: A M
authors.initials: AM
authors.lastname: Hill
authors.forename: J R
authors.initials: JR
authors.lastname: Harraway
authors.forename: A P
authors.initials: AP
authors.lastname: Fellowes
authors.forename: G T
authors.initials: GT
authors.lastname: Phillipson
authors.forename: P S
authors.initials: PS
authors.lastname: Benny
authors.forename: M P
authors.initials: MP
authors.lastname: Smith
authors.forename: P M
authors.initials: PM
authors.lastname: George
mesh_terms: D004624:Embryo Transfer; D005169:Factor VIII / Q000235:genetics*; D005260:Female; D005307:Fertilization in Vitro; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing; D005838:Genotype; D006467:Hemophilia A / Q000175:diagnosis* / Q000235:genetics*; D006801:Humans; D007438:Introns; D015810:Linkage Disequilibrium; D050498:Live Birth; D008297:Male; D018895:Microsatellite Repeats; D009030:Mosaicism; D010375:Pedigree; D010641:Phenotype; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D011247:Pregnancy; D019836:Preimplantation Diagnosis / Q000379:methods*; D015203:Reproducibility of Results; D014427:Twins
chemical_list: D005169:Factor VIII