FoSTeS, MMBIR and NAHR at the human proximal Xp region and the mechanisms of human Xq isochromosome formation

Abstract

The recently described DNA replication-based mechanisms of fork stalling and template switching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR) were previously shown to catalyze complex exonic, genic and genomic rearrangements. By analyzing a large number of isochromosomes of the long arm of chromosome X (i(Xq)), using whole-genome tiling path array comparative genomic hybridization (aCGH), ultra-high resolution targeted aCGH and sequencing, we provide evidence that the FoSTeS and MMBIR mechanisms can generate large-scale gross chromosomal rearrangements leading to the deletion and duplication of entire chromosome arms, thus suggesting an important role for DNA replication-based mechanisms in both the development of genomic disorders and cancer. Furthermore, we elucidate the mechanisms of dicentric i(Xq) (idic(Xq)) formation and show that most idic(Xq) chromosomes result from non-allelic homologous recombination between palindromic low copy repeats and highly homologous palindromic LINE elements. We also show that non-recurrent-breakpoint idic(Xq) chromosomes have microhomology-associated breakpoint junctions and are likely catalyzed by microhomology-mediated replication-dependent recombination mechanisms such as FoSTeS and MMBIR. Finally, we stress the role of the proximal Xp region as a chromosomal rearrangement hotspot.

Figure 1

Microarray-determined breakpoints of idic(Xq) in relation to regional genomic architecture. (A) Expanded view of the 7 Mb proximal Xp region in which all the idic(Xq) breakpoints were localized. (B) Breakpoint annotation in relation to the underlying genomic architecture. The arms of the LCR palindromes i(Xq)-P1 to i(Xq)-P6 are denoted as paired green triangles (P1–P6). The LINE, L1 palindrome i(Xq)-LINEP is shown as a red triangle pair (LINEP). Breakpoint sequences in regions of no extended homology are denoted as blue rectangles. (C) High-resolution custom oligo aCGH breakpoints of idic(Xq). Red bars denote proximal Xp sequences that are duplicated on the idic(Xq). Lighter red bars denote breakpoint intervals. Color gradients in four cases indicate breakpoint uncertainty (see text). (D) WGTPA breakpoints of idic(Xq). Blue bars denote proximal Xp sequences that are duplicated on the idic(Xq). Lighter blue bars denote breakpoint intervals. (E) Segmental duplications in proximal Xp. The large, complex LCR cluster which includes i(Xq)-P1 to i(Xq)-P5 can be seen between ~52 and 53 Mb.

Figure 2

The fine-scale structure of palindrome-associated idic(Xq) breakpoints. (A) idic(Xq) breakpoint characterized by WGTPA CGH. Highlighted breakpoint clones exhibit intermediate ratios in relation to duplicated and deleted clones. (B) Expanded view of highlighted breakpoint region showing non-repeat-filtered aCGH data from the high-resolution oligo array (GK28-HRO) and repeat-filtered aCGH data from the ultra-high-resolution oligo array (GK28-UHRO_RF) in relation to i(Xq)-P1. Light-red highlighted probes represent sequences immediately distal to the left palindrome arm and are deleted. Light-blue highlighted probes represent sequences immediately proximal to the right palindrome arm and are duplicated. Light-yellow highlighted probes represent sequences corresponding to the palindrome arms and unique-sequence spacer and are present in one copy on the isochromosome. For clarity, chromosome X is not shown to scale.

Figure 3

Microhomology-associated breakpoints. (A) Replication of the entire Xq arm and a small part of proximal Xp, represented as a solid horizontal line above the chromosome X illustration, followed by a template switch (dotted line) more distally and replication in the reverse direction to the end of the chromosome can catalyze the formation of idic(Xq) that have non-recurrent breakpoints. Proximal and distal junctions (P.J. and D.J.) represent the sites of replication disruption and resumption, respectively. The template switch region (T.S.R.) is intact on the idic(Xq) (present in one copy). Microhomologies are illustrated as vertical red lines. Alternatively, the proximal-distal template switch could occur in the reverse order. (B) Three sequenced breakpoint junctions. Plus strand proximal reference sequence is shown at the top. Minus strand distal reference sequence is shown at the bottom. Breakpoint sequence is shown in the middle. Microhomology bases (green letters) are boxed. All sequences are shown 5′–3′.