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.
OBJECTIVES: 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.