Performance of inverse shifting-PCR (IS-PCR) to investigate F8 intron 22

M. ABELLEYRO; P. RADIC; L. ROSSETTI; J. ZUCCOLI; I. LARRIPA; C. DE BRASI

Buenos Aires, Argentina

Congreso; XXIX International Congress of the World Federation of Hemophilia; 2010

World Federation of Hemophilia

Intron 22 inversions (Inv22) cause almost half of severe Haemophilia A (HA). 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. We study 69 males with severe HA and 38 females, representing 145 X-chromosomes. IS-PCR involved three steps: BclI-restriction, self-ligation of restriction fragments and two tests of multiplex-PCR analysis.          IS-PCR diagnostic test 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 females. Complementary test in these groups indicated neither X-chromosomes bearing duplications, nor deletions. In two related cases IS-PCR diagnostic test failed to present any signal and the complementary test permitted the molecular diagnosis of Inv22-I. In conclusion, the IS-PCR system performs accurately and robustly to investigate both frequent and potential int22h-rearrangements and therefore is an ideal method to be applied at first line in severe HA cases.