Noncarrier mothers of hemophilia A patients with Intron 22 inversions often have other rearrangements.

BACKGROUND

Inversions involving intron 22 (Inv22) of the F8 gene are detected in approximately 45% of all severe hemophilia A (HA) patients. Digital droplet polymerase chain reaction using milepost assays and multiplex ligation-dependent probe amplification (MLPA) allows robust diagnosis of inversions, copy number variations, and more complex rearrangements in F8.

OBJECTIVES

A total of 350 HA patients and 280 of their mothers from the Hemophilia Inhibitor Genetics Study cohort were analyzed to identify additional F8 mutations.

METHODS

Digital droplet polymerase chain reaction and MLPA were used to investigate archival samples.

RESULTS

Of 350 patients analyzed, 13 were found to harbor previously unidentified mutations: 3 with inversions (Inv22 type 1) and 10 with large deletions. In addition, 7 patients had complex rearrangements with duplications, in addition to Inv22 type 1. Of 138 mothers of patients with inversions, 7 were noncarriers. Five of these were found to have the same duplications as those detected in their 7 sons, indicating that most, if not all, noncarrier mothers have other rearrangements in the F8 region. MLPA showed that these duplications had breakpoints in intron 22, with either duplication of the first part of the gene (exons 1-22) or the last part of the gene (exons 23-26).

CONCLUSION

Mutation detection in F8 can be improved by dedicated analysis for inversions and duplications/deletions using milepost assays. Noncarrier mothers of HA patients with Inv22 often have other rearrangements.