Analysis of F8 intron 22 inversions (Inv22), int22h related rearrangements, and F8 intron 1 inversions (Inv1) by inverse shifting-PCR (IS-PCR)

ROSSETTI LILIANA; RADIC P; MARTÍN M ABELLEYRO; DE BRASI C

World Federation for Hemophilia Genetics Laboratory Manual.

World Federation of Hemophilia

Lugar: Montreal; Año: 2022;

IS-PCR based detection of the F8-intron 22 (Inv22) and intron 1 inversion (Inv1) (1) removes the requirement for Southern blot analysis or long range PCR for the former and double-PCR for the latter. As an approach derived from the classical inverse PCR (2), IS-PCR includes three steps: (1st) genomic DNA restriction, (2nd) fragment ends ligation and (3rd) PCR analysis. However, results can be obtained within 48 hours. Modifications from previous rapid and no pattern-sensitive methods for Inv22 genotyping, IS-PCR diagnostic test provides a rapid detection of Inv22-type 1, Inv22-type 2 and non-Inv22 alleles. An optional Inv22 complementary test (useful for academic research so far) permits further discrimination of other int22h-mediated rearrangements whose real existence and phenotipical consequences are still under debate (i.e., deletions, Del22, and duplications, Dup22) (3). For rapid genotyping of both severe haemophilia A-causative inversions, Inv1 diagnostic test can be performed streamlined from the same substrate, or even side-by-side at the same time. IS-PCR can evaluate carrier mosaicisms and performs robustly over wide ranges of DNA-qualities and procedure conditions.