An Accurate, Simple, and Inexpensive Assay to Diagnose F8 Gene Inversion Mutations in Hemophilia a Patients and Carriers

The most frequent mutations resulting in hemophilia A are an intron 22 or intron 1 F8 gene inversion, which together result in ~50% of severe hemophilia A cases. We report simple and accurate RNA- and DNA-based assays to detect these mutations in patients and carrier mothers. They do not require specialized equipment or expensive reagents, and therefore they may provide useful and economic protocols that could be standardized for central laboratory testing.

RNA is purified from a whole-blood RNA-stabilized sample or from peripheral blood mononuclear cells, and a two-step nested PCR (RT-PCR followed by PCR) reaction is carried out to amplify complementary DNA (cDNA) fragments with F8 sequences spanning the exon 22-23 splice site (intron-22 inversion test) or the exon 1-2 splice site (intron-1 inversion test). These sequences will be amplified from F8 mRNA without an intron-22 or intron-1 inversion mutation, respectively. Additional RT-PCR reactions are carried out to amplify a unique intron 22 inverted-gene mRNA sequence extending from F8 exon 19 to the first in-frame stop codon within intron 22, or a chimeric intron 1 inverted-gene transcript containing sequences from F8 exon 1 and the adjacent VBP1 gene. These latter two DNA products will be amplified only from F8 mRNA isolated from patients with an intron-22 or an intron-1 inversion mutation, respectively. Amplification of a FVIII C2 domain sequence serves as an internal control for the RT-PCR reactions, as this product will be generated from samples with and without an inversion mutation. Primers were designed to amplify PCR products having different lengths (between 200 and 600 bp) that could be clearly distinguished following electrophoresis on agarose gels. Samples from carrier mothers amplified the normal control as well as the inversion-mutation-specific bands, as expected.

A nested PCR protocol reliably produced interpretable results from low template mRNA concentrations, while a one-step multiplexed RT-PCR version of the assay produced the expected bands, with fewer experimental steps, from higher template mRNA concentrations.

The standard DNA-based inverse-shifting PCR (IS-PCR) reaction may be used for primary diagnosis and/or to classify intron 22 inversions as type 1 or type 2 (reflecting the specific homologous recombination site). We utilized a commercially available Bcl I enzyme that efficiently cleaves DNA in 5 minutes, a substantial improvement over previous IS-PCR protocols that required overnight DNA digests.

These rapid, accurate protocols can economically detect or rule F8 inversion mutations via same-day testing of patient samples, providing timely information that is useful to families and in clinical evaluations.