Genotyping the hemophilia inversion hotspot by use of inverse PCR

Clin Chem. 2005 Jul;51(7):1154-8. doi: 10.1373/clinchem.2004.046490. Epub 2005 Apr 28.


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 approximately 5%.

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

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Southern
  • Chromosome Inversion*
  • Chromosomes, Human
  • Factor VIII / genetics
  • Female
  • Genotype
  • Hemophilia A / genetics*
  • Heterozygote
  • Humans
  • Introns
  • Male
  • Polymerase Chain Reaction / methods


  • Factor VIII