Developing a new generation of tests for genotyping hemophilia-causative rearrangements involving int22h and int1h hotspots in the factor VIII gene

Authors

  • L. C. ROSSETTI,

    Search for more papers by this author
    • L. C. Rossetti and C. P. Radic contributed equally to this work and are both to be considered as first authors.

  • C. P. RADIC,

    Search for more papers by this author
    • L. C. Rossetti and C. P. Radic contributed equally to this work and are both to be considered as first authors.

  • I. B. LARRIPA,

    1. Departamento de Genética, Instituto de Investigaciones Hematológicas Mariano R. Castex, Academia Nacional de Medicina de Buenos Aires, Buenos Aires, Argentina
    Search for more papers by this author
  • C. D. DE BRASI

    1. Departamento de Genética, Instituto de Investigaciones Hematológicas Mariano R. Castex, Academia Nacional de Medicina de Buenos Aires, Buenos Aires, Argentina
    Search for more papers by this author

Liliana C. Rossetti, Sección Genética Molecular de la Hemofilia, Departamento de Genética, Instituto de Investigaciones Hematológicas Mariano R. Castex, Academia Nacional de Medicina, Pacheco de Melo 3081, 1425 – Buenos Aires, Argentina.
Tel.: +54 11 480 58803; fax: +54 11 4803 9475.
E-mail: rossetti@hematologia.anm.edu.ar

Abstract

Summary.  Background: Inversions of F8-intron 22 (Inv22) and F8-intron 1 (Inv1) are responsible for 45–50% of severe hemophilia A cases. Objective: In order to improve the molecular diagnosis of Inv22 and Inv1, and to enable rapid discrimination of Inv22-type 1 and Inv22-type 2 patterns, int22h-mediated deletions (Del22) and duplications (Dup22), we developed a genotyping system based on a novel inverse shifting-polymerase chain reaction (IS-PCR) approach. Methods: IS-PCR involved BclI restriction, followed by self-ligation to create ‘BclI circles’, and finally PCR analysis. Three PCR analysis tests were developed: (i) Inv22-diagnostic for a pattern-sensitive detection of deleterious mutations (Inv22 and Del22) from non-deleterious variants (Dup22 and normal); (ii) Inv1-diagnostic; and (iii) Inv22-complementary for discrimination between Inv22 and Del22, and between Dup22 and normal. For rapid molecular analysis of F8, the Inv22 and Inv1 diagnostic tests can be performed simultaneously. The optional Inv22-complementary test need only be used for specific purposes. Results and Conclusions: Diagnostic tests were validated using previously studied samples. IS-PCR evaluated carrier mosaicisms and performed robustly over wide ranges of DNA qualities and procedural conditions. IS-PCR improved the molecular diagnosis of hemophilia A. This genotyping strategy may potentially be adapted to virtually all known rearrangements in the human genome.

Ancillary