Development of a Single‐Tube Reverse Transcription Real‐Time PCR Assay for Diagnosis of F8 Intron 22 Inversion in Patients and Carriers of Haemophilia A

ABSTRACT Introduction Intron 22 inversion mutation of F8 (inv22) is the most frequent cause of Haemophilia A (HA) and is present in approximately 45% of severe HA cases. This mutation disrupts F8 gene continuity, leading to a truncated protein. Traditional methods for detecting inv22, including inverse‐shifting PCR and long‐range PCR, are accurate but labour‐intensive. F8 inv22 truncated mRNA transcript contains a short (51 base pairs) abnormal exon 23. Thus, reverse‐transcription PCR has been proposed for the diagnosis of inv22 in patients with HA. Aim The aim of this study was to design and validate a multiplex reverse‐transcription real‐time PCR (RT‐qPCR) assay capable of detecting and differentiating between normal and inv22 F8 transcripts in a single reaction. Methods We designed an RT‐qPCR assay that employs specific primers and TaqMan probes to detect the exon 22 to 23 junction present in the normal F8 transcript, and the junction between exon 22 and the abnormal sequence only present in F8 transcripts harbouring inv22. We tested our assay in 14 HA patients (six with inv22 and eight with other mutations), four HA female carriers (two with inv22 and two with other mutations), and six negative controls. Results F8 expression in peripheral blood RNA was sufficient to be detected by RT‐qPCR. The assay showed perfect concordance within the cohort to identify inv22 in both patients and carriers. Conclusion RT‐qPCR is an accurate method for diagnosing inv22 in patients and HA carriers. Moreover, it is simpler and faster than previous methods.