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

Abstract

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.

A unifying model that explains the origins of human inverted copy number variants

“…These duplications are illustrated using NAHR at repetitive elements, but similar structures could be generated at non-repetitive sequences through repair of DNA breaks by NHEJ, MMEJ or MMBIR. This particular outcome has recently been reported at the Factor 8 locus where NAHR at long, highly homologous inverted repeats generates one of the junctions and the second junction occurs at regions of little to no homology [35]. It produces the pattern of a deletion and inverted duplication separated by a stretch of copy-neutral DNA in between (1-0-1-2-1; Figure 4A; Figure 5; Table 1, examples 24-25, 39, 44-45).…”

“…A) The original sequence near the Xq telomere; F8 is the Factor 8 gene, highlighted in blue. Diagram is adapted from [35]. Exons of three adjacent genes (green arrows) and three different low-copy repeats (LCRs, black and gray arrows) are highlighted.…”

“…Exons of three adjacent genes (green arrows) and three different low-copy repeats (LCRs, black and gray arrows) are highlighted. B) Stylized aCGH data for the male patient with Factor 8 deficiency (reported in [35] 6: Mechanism that generates an inverted triplication of the SUL1 region in yeast (ODIRA)-modified from Figure 1 in Martin et al, 2023 [13]. A) The structure of the SUL1 locus in yeast includes an origin of replication (ARS228) and adjacent short (~5 nt) inverted repeats separated by 40-80 nt of sequence.…”

A unifying model that explains the origins of human inverted copy number variants

“…These duplications are illustrated using NAHR at repetitive elements, but similar structures could be generated at non-repetitive sequences through repair of DNA breaks by NHEJ, MMEJ, or MMBIR. This particular outcome has recently been reported at the Factor 8 locus where NAHR at long, highly homologous inverted repeats generates one of the junctions and the second junction occurs at regions of little to no homology [ 37 ]. It produces the pattern of a deletion and inverted duplication separated by a stretch of copy-neutral DNA in between (1-0-1-2-1; Figs 4A and 5 and Table 1 , examples 24, 25, 39, 44, 45).…”

Complete F9 Gene Deletion, Duplication, and Triplication Rearrangements: Implications for Factor IX Expression and Clinical Phenotypes

“…After a distance of reverse replication, the second template switch occurs through MMBIR, restoring the original direction of replication for subsequent DNA synthesis. 35 36 In other words, one rearrangement breakpoint is fixed within inverted LCRs, whereas the second breakpoints are relatively random. Therefore, the impact of LCR-F9A/LCR-F9B-induced DUP–TRP/INV–DUP rearrangements on the F9 copy number depends on the length of backward-directed replication and the position of the second template switch.…”