The Evolution of Techniques Used for the Detection of the F8 IVS 22 Inversion Mutation.

Almost half of patients with severe hemophilia A have a genomic rearrangement of the F8 gene, resulting in the separation of exons 1–22 from exons 23–26. This disruption is due to a recombination hotspot involving a 9.5kb region within intron 22 (int22h-1) and one of at least two extragenic copies (int22h-2 and inth22-3) located telomeric to the F8 gene. The 3 copies of int22h are estimated to be 99.9% homologous, with consistent differences between them confined to only 3 nucleotides. The more distal Int22h-3 lies in opposite orientation to the intragenic copy, and recombination between these two results in an inversion mutation, and a total lack of F8 protein. It was recently shown that int22h-2 is oriented in the same direction as int22h-1, and recombination events between these will result in either deletion or duplication rather than inversion.

Three techniques have been published for the detection of the IVS22 recombination mutations: Southern blot, Long-PCR and Inverse PCR. We present data on a significant number of patients tested by more than one technique, and highlight difficulties in the detection/positive identification of mutations by each. Southern blotting (the only available method for many years) requires a large amount of DNA, not readily available from immuno-compromised patients, relies on radioisotope activity, and takes several days to obtain a result. All forms of recombination can be distinguished, but polymorphic variants can be misinterpreted as rare inversions resulting in patients being incorrectly assigned as inversion positive. In 1998, a long range PCR method was published for the analysis of IVS22 inversions. This technique, more rapid and less hazardous than Southern blotting, has proved to be problematic in many laboratories. The primers degrade rapidly, do not consistently permit multiplexing, and amplification is highly dependant on freshly extracted, high purity DNA. In 2005, a technique based on genomic digestion, followed by self ligation and PCR was published. This inverse PCR methodology has proved to be robust and can be used for fresh or archived samples, generating reproducible results within 36 hours. Proximal and distal recombinations are indistinguishable, and non-causative polymorphisms are not detected. During evaluation of the emerging technologies this laboratory has parallel tested a significant number of samples. 36 patients, including 6 carrier females, gave concordant results with Southern blot and inverse PCR. 12 patients with polymorphic, non-haemophilia associated, banding patterns gave a normal result by inverse PCR. 3/12 had been incorrectly assigned as mutation positive by southern blot; with 1 discovered only by the subsequent detection of non-linkage in the family. 25 patients gave identical results by long PCR and inverse PCR, including 4 carrier females. 2 patients were wrongly defined as positive by long PCR due to poor quality DNA. A result was unobtainable for 23 patients by southern blot, and for 6 patients by long PCR, due to insufficient or poor quality DNA. Of these, 8/23 and 6/6 were subsequently resolved by inverse PCR. Of the remaining 15/23 failures, the causative mutation has since been identified elsewhere in the F8 gene in 8 patients, while 7 require repeat bleeding.

In conclusion, the inverse PCR technique provides a fast, reliable, reproducible and safe method for the detection of IVS 22 recombination mutations, and is recommended as the method of choice for diagnostic laboratories.