Tandem and inverted duplications in haemophilia A: Breakpoint characterisation provides insight into possible rearrangement mechanisms.

Introduction: Approximately half of patients with severe haemophilia A are caused by structural variants in the F8 gene. Unlike inversions or deletions directly impairing the integrity of F8, some duplications do not completely disrupt the open reading frame or even retain an intact F8 copy. Currently, only a few duplication breakpoints were precisely characterized, and the corresponding rearrangement mechanisms and clinical outcomes remain to be further investigated.

Aim: Establishing an effective strategy for breakpoint characterization of duplications and revealing their rearrangement mechanisms.

Methods: AccuCopy is used for the detection of duplications, long-distance PCR for the characterization of tandem duplications, genome walking technique and whole genome sequencing for the characterization of inverted duplications.

Results: Four F8 duplication rearrangements were successfully characterized at the nucleotide level: one tandem duplication (exons 7-11) and three inverted duplications (exons 7-22, exons 2-26, and exons 15-22). Two shared features of inverted duplication were found after carefully analysing our results and breakpoint information in the literature: 1, an inverted fragment was inserted into the original chromosome via two junctions; 2, one junction is mediated by a pair of inverted repetitive elements, while the other consists of two breakpoints with microhomology.

Conclusion: Similar breakpoint features motivated us to propose a DNA replication-based model to explain the formation of duplication rearrangements. Based on our model, we further divide the inverted duplications into three basic types: type I with a DEL-NOR/INV-DUP pattern, type II with a DUP-NOR/INV-DUP pattern and type III with a DUP-TRP/INV-DUP pattern.