Performance of inverse shifting-PCR (IS-PCR) to investigate F8 intron 22 inversions and potential rearrangements involving int22h recombination

ABELLEYRO MM, RADIC CP, ROSSETTI LC, ZUCCOLI JR, LARRIPA IB, DE BRASI CD

Buenos Aires, Argentina

Congreso; XXIX Congreso Internacional de la federación mundial de hemofilia.; 2010

Federación mundial de hemofilia

Intron 22 inversions (Inv22) cause almost half of severe haemophilia A (HA) cases. Inv22 originates by recombination between a sequence within the F8 intron 22, int22h-1, and an extragenic-distal inversely-oriented copy of it. Recombination between int22h-1 with a third copy, extragenic-proximal and equally-oriented, predicts deletions or duplications. These duplications and deletions have not been found in well-characterised clinical cases so far. In 2008 we devised an approach, IS-PCR, which can differentiate all these rearrangements using two separate tests (i.e., diagnostic and complementary). The aim of this study is to test the performance of IS-PCR in clinical samples and to explore the presence of rare int22h-mediated rearrangements. Methods: We study 69 males with severe HA and 38 females, representing 145 Xchromosomes. IS-PCR involved three steps: BclI-restriction, self-ligation of restriction fragments, and two tests of multiplex-PCR analysis. Results: IS-PCR diagnostic tests yield 32 cases with Inv22 type I (-I), five Inv22 type II (- II), 10 Inv22-I carriers, three Inv22-II carriers, 42 cases with the Inv22 uninformative signal with this test (32 severe HA patients and 10 female relatives) and 15 non-carrier