FROM SOUTHEN BLOT ANALYSIS TO INVERSE PCR: GENOTYPING THE HEMOPHILIA INVERSION HOTSPOT OVER THE LAST TEN YEARS IN ARGENTINA.

DE BRASI C, ROSSETTI L, RADIC P, LARRIPA I, CANDELA M, BIANCO R, DE TEZANOS PINTO M.

Vancouver, Canadá

Congreso; XXVII International Congress of the Word Federation of Hemophilia (WFH).; 2006

Word Federation of Hemophilia (WFH).

Hemophilia A (HA) is an X-chromosome inherited disorder associated with a deficient FVIII gene (F8). The intron 22 inversion (Inv22) is the most frequent mutation involved in 40-45% severe (se) HA cases worldwide. The objective is to present the Inv22 genotyping in Argentina, from the early days of Southern blot analysis to the development of inverse-PCR. Since 1996 to 2004 the Southern blot approach (BclI digests isotopically probed) allowed molecular diagnosis of 25 Inv22[+] families (19, 76% distal pattern and 6, 24% proximal) (including 27 probands, 17 mothers and 16 female relatives). Recently, a method based on inverse-PCR was developed by us and applied in severe HA. This strategy includes, (a) BclI-digestion, (b) ligation in large volumes to yield DNA-circles, and (c) PCR analysis with a set of 3 primers. Electrophoretic signals of 487bp and 587bp indicate the presence of wild-type and Inv22[+] circles, respectively. Inv22 inverse-PCR is rapid and permits the study of Inv22 carrier mosaicisms. This new approach allowed detection of five additional Inv22[+] families (5 probands, 3 mothers and 3 female relatives). All twenty mothers of Inv22[+] probands, including 14 cases with sporadic HA resulted carriers, thus confirming that the Inv22 originates from male meiosis. Our results confirm the value to investigate Inv22 mutations at first line in severe-HA, as it provides secure data for genetic counseling in ~43% of seHA families and specific information for the hematologist. Additionally, Inv22 inverse-PCR represents an ideal tool to be included in the routine of hemophilia molecular genetic laboratories.