Deep intronic ‘mutations’ cause hemophilia A: application of next generation sequencing in patients without detectable mutation in F 8 c DNA

Summary Background In a small group of typical hemophilia A ( HA ) patients no mutations in the F 8 coding sequence ( cDNA ) could be found. In the current study, we performed a systematic screening of genetic and non‐genetic parameters associated with reduced FVIII : C levels in a group of mostly mild HA (only one moderate) patients with no detectable mutations in F 8 c DNA . Methods We determined FVIII and VWF activity and antigen levels and performed VWF ‐ FVIII binding ( VWF : FVIIIB ) and VWF ‐collagen binding assays ( VWF : CB ) as well as VWF multimer analysis. VWF was completely sequenced to exclude mutations. The F 8 locus, including the introns, was sequenced using overlapping long‐range PCR s ( LR ‐ PCR s) combined with a next generation sequencing ( NGS ) approach. Moreover, the F 8 m RNA was analyzed quantitatively and qualitatively by real‐time PCR ( qRT ) and overlapping reverse transcription ( RT ) PCR s, respectively. Results All VWF tests were normal. The LR ‐ PCR s demonstrated the integrity of the F 8 locus. Eight unique polymorphisms were found in the patients, with two being recurrent. Furthermore, RT ‐ PCR s analysis confirmed that two of the unique variants create detectable new cryptic splice sites in the patients that result in the introduction of intronic DNA sequences into the m RNA and create premature stop codons. Conclusion By systematically excluding all possible causes of HA , we could with great certainty conclude that deep intronic mutations in F 8 , although rare, cause abnormal m RNA splicing, leading to mild HA .