Abstract
Background: A paradigm shift in our understanding of women and girls with hemophilia A is evolving. van Galen et al published an ISTH SSC-recommended nomenclature based on bleeding phenotype and baseline FVIII activity levels to reflect current clinical wisdom- women/girls with mild, moderate, or severe hemophilia (FVIII activity >0.05 and <0.40 IU/ml, 0.01-0.05 IU/ml, and <0.01 IU/ml, respectively) and symptomatic and asymptomatic carriers (FVIII activity ≥0.40 IU/ml with and without a bleeding phenotype, respectively). Therefore, it is imperative to identify the F8 variants these women and girls harbor.
Methods: Genetic testing for F8 variants was obtained as a component of clinical practice. Each patient or legal guardian provided written permission for genetic testing. Genomic DNA was isolated via standard technique. Inverse-shifting polymerase chain reaction was used to screen for intron 1 and 22 inversions if there was a history of severe hemophilia A or no family history. If negative, Sanger sequencing of the coding region, intron/exon boundaries, and areas 5' upstream and downstream of the coding region was performed. If Sanger sequencing was negative, multiplex ligation-dependent probe amplification assay was performed to detect large structural variants. If a candidate causative variant was found confirmation on a second aliquot of genomic DNA was performed.
Results: 235 females underwent F8 variant testing. All but 17 provided a family history of hemophilia A. For 168 individuals a familial variant and for 65 baseline FVIII activity (3-180%) was provided. A candidate causative variant (CCV) was detected in 146 individuals. 89 females without a CCV detected had a median FVIII activity of 60% (30-180%; n=16) or were normal (familial variant was not found) with a median baseline FVIII activity of 94% (20-159%; n=6), 76 had predicted non-null variants with a median baseline FVIII activity of 35% (5.3- 171%; n=20) and 69 had predicted null variants with a median baseline FVIII activity of 38.4% (15-157%; n=19). One woman harboring a splice variant that could not be classified as non-null or null had a FVIII activity of 3%. In the individuals with FVIII < 40% and no family history of hemophilia A, a CCV was found in 3 of 8, suggesting an alternative diagnosis for the low FVIII in most. One individual was identified with a deletion only after it was subsequently identified in her son, and she was retested with variant-specific primers. Of the 76 females with predicted non-null variants 63 harbored missense variants, 10 synonymous variants, 2 large duplications, and 1 a variant in the 5' untranslated region of F8. Of the 69 females with predicted null variants 35 harbored intron 22 inversions, 16 frameshift deletions or insertions, 13 nonsense variants, and 5 large deletions (Table 1).
Conclusion: As our clinical knowledge of women and girls affected by hemophilia expands so must our knowledge of the genetic variants that they harbor. Baseline FVIII activity levels are not predictive of variant type. Knowledge of the F8 variant in the patient and her family is important in reproductive decision making and obstetrical management to reduce the risk of consequential delivery methods, as well as understanding bleeding symptoms, inhibitor risks, and potentially treatment choices for her and her family. Knowledge of the variant in affected males improves variant detection or exclusion in females.
Disclosures
Sandoval:OctapharmaUSA: Current Employment. Johnsen:Takeda: Consultancy, Honoraria; CSL Behring: Consultancy, Honoraria; Octapharma: Consultancy, Honoraria, Research Funding. Konkle:Pfizer: Honoraria, Research Funding; Uniqure: Honoraria, Research Funding; Sigilon: Honoraria, Research Funding; Baxalta: Research Funding; Sanofi: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; CSL Behring: Honoraria; BioMarin: Honoraria; Spark: Honoraria.
