Mechanisms for Complex Chromosomal Insertions

Abstract

Chromosomal insertions are genomic rearrangements with a chromosome segment inserted into a non-homologous chromosome or a non-adjacent locus on the same chromosome or the other homologue, constituting ~2% of nonrecurrent copy-number gains. Little is known about the molecular mechanisms of their formation. We identified 16 individuals with complex insertions among 56,000 individuals tested at Baylor Genetics using clinical array comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH). Custom high-density aCGH was performed on 10 individuals with available DNA, and breakpoint junctions were fine-mapped at nucleotide resolution by long-range PCR and DNA sequencing in 6 individuals to glean insights into potential mechanisms of formation. We observed microhomologies and templated insertions at the breakpoint junctions, resembling the breakpoint junction signatures found in complex genomic rearrangements generated by replication-based mechanism(s) with iterative template switches. In addition, we analyzed 5 families with apparently balanced insertion in one parent detected by FISH analysis and found that 3 parents had additional small copy-number variants (CNVs) at one or both sides of the inserting fragments as well as at the inserted sites. We propose that replicative repair can result in interchromosomal complex insertions generated through chromothripsis-like chromoanasynthesis involving two or three chromosomes, and cause a significant fraction of apparently balanced insertions harboring small flanking CNVs.

Fig 1. Proposed mechanisms in individuals with basic complex insertions.

(A) Upper panel: CMA and high-density aCGH results of Cplex4. Lower panel: breakpoint junction sequences in Cplex4. Microhomology between distal and proximal sequences are highlighted in red. HD array, high-density aCGH; (+), sequences in the positive strand in the hg19 reference genome. (B) Chromosome idiograms of individual Cplex4 demonstrating the duplicated fragment (segment highlighted in red) on chr14 was inserted and translocated to chr13 with where the deletion of chr13 (segment highlighted in green) occurred. (C) Chromosome idiograms of individual Cplex9 demonstrating the duplicated fragment (right facing arrow) on chr9 was inserted and translocated to chr13 with where the deletion of chr13 (segment highlighted in green) occurred. (D) Chromosome idiograms of individual Cplex12 demonstrating the duplicated fragment (right facing arrow) on chr6 was inserted and translocated to chr5 with where the deletion of chr5 (segment highlighted in green) occurred. Note that in Cplex9 and Cplex12, the inserted fragments were both inverted after the insertion in comparison to the reference genome (left facing arrows). Jct1, Junction 1; Jct2, Junction 2. Polymorphism is defined as the observation that similar CNVs have been documented in multiple healthy, clinically unaffected individuals according to the Database of Genomic Variants (DGV).

Fig 2. Proposed mechanisms in individuals Cplex5 and Cplex6 with chromothripsis-like, chromoanasynthesis insertions.

(A) CMA and high-density aCGH results of Cplex5. (B) Chromosome idiograms of individual Cplex5 demonstrating a duplication (segment highlighted in magenta) and two deletions (segments highlighted in green and blue, respectively) on chr6, plus a duplication (segment “b” highlighted in red) on chrX. Breakpoint junction mapping indicated that the duplicated fragment of Xq28 (red segment “b”) was inserted to 6q24.2, replacing the deleted region of 6p24.2 (green segment) through Junction 1 and 2, while the duplicated fragment of 6p21 (magenta segment “a”) was inserted to 6q25.1, again replacing the other deleted region of 6q25.1q25.3 (blue segment) through Junction 3 and 4. The overall result in Cplex5 was a rearranged chr6 with inserted fragment from chrX. (C) Chromosome idiograms of individual Cplex6 demonstrating a duplication (segment “a” highlighted in red) at 5pter and a triplication (segment “c” highlighted in blue) embedded in a duplication (segment “b+c+d” highlighted in orange) at Xqter. Junction 1 and 2 led to the insertion and joining of the duplicated region on chr5 (red segment “a”) to the triplication (blue segment “c”) on chrX, both in an inverted orientation in comparison to the reference genome. Note that Junction 3 was a hypothetical breakpoint junction to most parsimoniously explain the putative mechanism for this rearrangement. The overall result in individual Cplex6 was a rearranged chrX with inserted fragment from chr5. Jct1, Junction1; Jct2, Junction 2; Jct3, Junction 3. Dashed purple lines represent potential template switching paths during the generation of the CGRs. ‘??’ indicates hypothetical junction.

Fig 3. Proposed mechanisms in individual Cplex11 with chromothripsis-like, chromoanasynthesis insertions.

(A) CMA and high-density aCGH results of Cplex11. (B) Chromosome idiograms of individual Cplex11 demonstrating two duplications (segments “a” and “b” highlighted in red and cyan) and a deletion (segment highlighted in green) on chr13, plus a duplication (segment “c” highlighted in magenta) and a triplication embedded in the other duplication (segment “e” highlighted in blue embedded in segment “d+e+f” in orange) on chrX. Junction 1 joined the distal side of the chr13 deletion (green) to the proximal side of the first duplication on chr13 (red “a”). Junction 2 joined the distal side of the first duplication on chr13 (red “a”) to the proximal side of the first duplication on chrX (magenta “c”). Junction 3 joined the distal side of the first duplication on chrX (magenta “c”) to the proximal side of the triplication on chrX (blue “e”). Junction 4 joined the distal side of the triplication on chrX (blue “e”) to the distal side of the second duplication on chr13 (cyan “d”). Junction 5 (note this junction is hypothetical) joined the proximal side of the second duplication on chr13 (cyan “d”) to the proximal side of the second duplication on chrX (orange “d+e+f”). Lastly, Junction 6 joined the distal side of the second duplication on chrX (orange “d+e+f”) to the proximal side of the deletion on chr13 (green). The overall result in individual Cplex11 was a rearranged chr13 with multiple inserted fragments from chrX. Jct1 to Jct6, Junction 1 to Junction 6. Dashed purple lines represent potential template switching paths during the generation of the CGRs. ‘??’ indicates hypothetical junction.

Fig 4. Proposed mechanisms in the PLP1 deletion/insertion family with apparently balanced insertion in the mother.

(A) Pedigree of the family. (B) High-density aCGH results of BAB1379 and BAB1381. (C) Chromosome idiograms of the mother (BAB1381) demonstrating a duplication (segment “a” highlighted in red) on chr19 and a small deletion (segments highlighted in green) plus a small duplication (segment “b” highlighted in magenta) on chrX. The insertion event from chrX to chr19 were generated through Junction 1 and 2: Junction 1 joined the distal side of the duplication on chr19 (red “a”) to the distal side of the small deletion on chrX (green), while Junction 2 joined the distal side of the small duplication on chrX (magenta “b”) to the proximal side of the duplication on chr19 (red “a”). The deletion event on chrX was generated through Junction 3 joining the proximal side of the small deletion on chrX (green) to the proximal side of the small duplication on chrX (magenta “b”). Her affected son (BAB1379) inherited the chrX with the deletion and an intact chr19, while her unaffected son (BAB1380) inherited an intact chrX and a chr19 with the insertion. Note that colored fragments are not in proportion to the actual CNVs’ sizes; i.e. not to scale. Jct1, Junction1; Jct2, Junction 2; Jct3, Junction 3. Dashed purple lines represent potential template switching paths during the generation of the CGRs. Polymorphism is defined as the observation that similar CNVs have been documented in multiple healthy, clinically unaffected individuals according DGV.

Fig 5. High-density aCGH results and breakpoint junction in Family 3 with apparently balanced insertion in the mother.

Upper panel: High-density aCGH showed the deletion at 7p15.2p14.3 in P3, and a small deletion in Mat3 at the distal boundary of the large deletion in her child P3. Lower panel: deletion breakpoint junction sequences shared in P3 and Mat3. An 815 bp insertion from chr9:5874574–5875388 was observed at this junction. (+), sequences in the positive strand in the hg19 reference genome. Jct1, Junction1.