Identifying Large F8 Gene Deletions

Large F8 gene deletions were identified by consistent failure of PCR amplification of a single exon or adjacent F8 exons, as indicated by missing or altered bands upon electrophoresis of PCR products. At least three separate attempts to amplify missing fragments from the subject’s genomic DNA were performed using the same primers and PCR conditions, alongside successful amplification and sequencing of the exons flanking the suggested deletion. For detection of deletion breakpoints, we carried out LD-PCR using primers for amplification of the exons framing the deletion and GoTaq^® Long PCR Master Mix following the manufacturer’s protocol. We were able to identify deletion breakpoints for two patients. To accomplish this, we designed primers flanking the copies of the Alu element that could be involved in the deletion formation. For patient A375, we used primers F8-delF (5′-GTTTGTTTACATTTGTCCCAACT-3′, c.787+2045_2067, intron 6) and F8-delR (5′-TGCAACTCAAAGGACTAAACA-3′, c.1903 +1569_1589, intron 12). For patient A469, we used primers F8-5D [32 (link)] and Del6R (5′-CAGTTGACTCTTGAACAATACA-3′, c.787+2976_2995, intron 6).
Large duplications that could not be identified using routine PCR-based analysis methods were tested with multiplex ligation-dependent probe amplification (MLPA). MLPA was carried out using the F8 SALSA MLPA kit P178 (MRC Holland, Amsterdam, The Netherlands) according to the manufacturer’s instructions. Exon dosage was calculated using Coffalyser.Net software (MRC Holland).