Aberrant firing of replication origins potentially explains intragenic nonrecurrent rearrangements within genes, including the human DMD gene

Abstract

Non-allelic homologous recombination (NAHR), non-homologous end joining (NHEJ), and microhomology-mediated replication-dependent recombination (MMRDR) have all been put forward as mechanisms to explain DNA rearrangements associated with genomic disorders. However, many nonrecurrent rearrangements in humans remain unexplained. To further investigate the mutation mechanisms of these copy number variations (CNVs), we performed breakpoint mapping analysis for 62 clinical cases with intragenic deletions in the human DMD gene (50 cases) and other known disease-causing genes (one PCCB, one IVD, one DBT, three PAH, one STK11, one HEXB, three DBT, one HRPT1, and one EMD cases). While repetitive elements were found in only four individual cases, three involving DMD and one HEXB gene, microhomologies (2-10 bp) were observed at breakpoint junctions in 56% and insertions ranging from 1 to 48 bp were seen in 16 of the total 62 cases. Among these insertions, we observed evidence for tandem repetitions of short segments (5-20 bp) of reference sequence proximal to the breakpoints in six individual DMD cases (six repeats in one, four repeats in three, two repeats in one, and one repeat in one case), strongly indicating attempts by the replication machinery to surpass the stalled replication fork. We provide evidence of a novel template slippage event during replication rescue. With a deeper insight into the complex process of replication and its rescue during origin failure, brought forward by recent studies, we propose a hypothesis based on aberrant firing of replication origins to explain intragenic nonrecurrent rearrangements within genes, including the DMD gene.

Figure 1.

(Left) Comparative genomic hybridization data for the DMD gene locus (chrX:31,137,345–33,229,673) for DMD patients using a custom-designed 385K high-density array from NimbleGen. (Right) The zoomed-in view of the corresponding array on the left, with the deleted exons highlighted in red.

Figure 2.

Comparative genomic hybridization data for the IVD gene locus (chr15:40,697,686–40,713,512) in patient 4551 using a custom-designed 385K high-density array from NimbleGen. The zoomed-in view of the corresponding array highlights the breakpoints for a patient with a deletion mutation encompassing exons 10–12 with breakpoints in intron 9 and the 3′ UTR.

Figure 3.

The presence of microhomologies at breakpoint junctions. The graphs represent the extent of microhomologies at the deletion breakpoint junction sequences in DMD (A) and other genes (B). The x-axis represents the number of bases homologous in both junction sequences of a deletion. The y-axis represents the number of samples that have been observed to have the indicated microhomology.

Figure 4.

Template slippages at deletion breakpoints in DMD cases 5024, 9250, 1148, 5904, 5078, and 6194. In all the cases shown in the figure, short segments of the inserted sequence align with the template (reference genome) sequence proximal to the breakpoint as shown (each such segment of the inserted sequence is highlighted in a different color for each case). These indicate repeated slippage of the replication machinery along the template and re-replication. Each lightning symbol shown in the figure represents a slippage event of the replication machinery. Template sequence proximal to the breakpoints on both 5′ and 3′ ends of the deletion (gray bars); deletion (red dotted lines); breakpoints (vertical black bars). Below this, for each case, the gray arrow bars (with sequence enclosed) refer to the normal replication, while template slippages and re-replications leading to insertion are shown by colored arrows. Each cycle of re-replication is represented in a different color. The introns in which the deletion breakpoints lie are shown above each breakpoint. Each re-replicated sequence is also shown enclosed in corresponding colored boxes on the template DNA.

Figure 5.

Illustration of the proposed deletion mechanism as explained by failure of replication origins in different patient cases. As shown for each of the six patient cases (5024, 6194, 1148, 5904, 9250, and 5708), failure/delay in origin firing may result in incomplete replication, causing DNA deletion. For example, in patient 5904, failure of origins in introns 28 and 43 may have led to deletions of exons 20–44. The horizontal bar at the top represents the DMD gene with the five mapped replication origins (green circles) and the six termination regions (red rhomboids), along with adjacent numbered exons (vertical black bars). The numbers in bold italics represent the patient sample. For each patient sample illustrated above, we show the template with origins and terminal regions adjacent to the deletion (gray arrowheads showing the direction of replication forks). (Brown horizontal bars) Deletion breakpoints (or replication cease points); (blue bars) patient DNA (or newly replicated DNA); (blue arrowheads) the replication fork direction at breakpoint. The directions of the replication forks are predicted based on the presence of re-replicated sequences at either breakpoint junction. For example, in sample 5024, the template slippage was found at the breakpoint in intron 7, whereas in sample 6194, the template slippage and sequence repetition was found to be at the breakpoint in intron 47. (Red horizontal braces) Corresponding deletion, with the deleted exons mentioned. The crossed-out origins represent replication origins that probably failed to fire during S phase.