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Clinical Chemistry 51: 1154-1158, 2005. First published April 28, 2005; 10.1373/clinchem.2004.046490
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(Clinical Chemistry. 2005;51:1154-1158.)
© 2005 American Association for Clinical Chemistry, Inc.

Hemostasis and Thrombosis

Genotyping the Hemophilia Inversion Hotspot by Use of Inverse PCR

Liliana Carmen Rossettia, Claudia Pamela Radic, Irene Beatriz Larripa and Carlos Daniel De Brasi

1 Department of Genetics, Instituto de Investigaciones Hematológicas Mariano R. Castex, Academia Nacional de Medicina de Buenos Aires, Buenos Aires, Argentina.

aAddress correspondence to this author at: Departamento de Genética, Instituto de Investigaciones Hematológicas Mariano R. Castex, Academia Nacional de Medicina, Pacheco de Melo 3081, 1425 Buenos Aires, Argentina. Fax 5411-4803-9475; e-mail rossetti{at}hematologia.anm.edu.ar.

Background: Factor VIII intron 22 inversions (Inv22) cause 40%–45% of severe cases of hemophilia A in all human populations. Currently, Inv22 can be analyzed either by Southern blotting or by rapid long-distance-PCR–based approaches. We describe an alternative method using inverse-PCR (I-PCR).

Methods: I-PCR involved 3 steps: (a) BclI restriction; (b) self-ligation of restriction fragments, providing BclI rings; and (c) standard multiplex-PCR analysis. PCR was achieved by use of a set of 3 primers that yielded a 487-bp amplicon for the nonrearranged intragenic allele and a 559-bp amplicon for the Inv22 allele. Specific primer sites were targeted by masking relevant regions for human repeats and low-complexity DNA. Inv22 I-PCR was applied to samples from 16 individuals (8 women and 8 men) representing 24 X chromosomes previously genotyped by Southern blotting. Additionally, we evaluated the sensitivity and the ability to assess eventual Inv22 carrier mosaicisms by experiments using artificial DNA mixtures (Inv22 + no-Inv22 male samples).

Results: Results for previously genotyped samples agreed with results of Southern blot analyses. As expected, cell composition of the artificial mosaic was linearly reflected by the relative intensities of Inv22 signals. I-PCR was estimated to detect Inv22-positive cells at concentrations as low as ~5%.

Conclusion: The proposed technique provides a rapid tool for Inv22 genotyping.




The following articles in journals at HighWire Press have cited this article:


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L. C. Rossetti, C. P. Radic, M. Candela, R. P. Bianco, M. de Tezanos Pinto, A. Goodeve, I. B. Larripa, and C. D. De Brasi
Sixteen novel hemophilia A causative mutations in the first Argentinian series of severe molecular defects
Haematologica, June 1, 2007; 92(6): 842 - 845.
[Abstract] [Full Text] [PDF]


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