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Mechanisms for Complex Chromosomal Insertions

View Article: PubMed Central - PubMed

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.

No MeSH data available.


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.
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pgen.1006446.g003: 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.

Mentions: Individual Cplex11 exhibited the most complicated rearrangement in this study. Array results demonstrated a duplication-normal-duplication-normal-deletion pattern at 13q33.2 to 13q34 and a duplication-normal-duplication-triplication-duplication pattern at Xq21.1 (Fig 3A); FISH analysis showed that both of the two duplicated regions on chr13 were inserted into chrX (S6 Fig, S1 Table). Breakpoint mapping further demonstrated that the rearrangement between chr13 and chrX could be potentially generated through 6 junctions (Fig 3B). With the exception of the hypothetical Junction 5, we were able to map the remaining 5 junctions to nucleotide resolution. Based on the information of the five junctions mapped and the CNVs observed, we proposed the existence of Junction 5 to most parsimoniously explain the observed overall rearrangement in this individual (Fig 3B). Upon careful examination of the junctions, we observed that sequences of Junction 2 contained an 8,192 bp insertion from Xq13.2, followed by a 5,167 bp insertion from 4q13.1, leading to the discovery of the involvement of a third chromosome, chromosome 4, in this individual’s CGR (Table 1, S7 Fig). The remaining mapped junctions showed 2 bp microhomology (Junction 6) or blunt ends (Junction 1, 3 and 4).


Mechanisms for Complex Chromosomal Insertions
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.
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pgen.1006446.g003: 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.
Mentions: Individual Cplex11 exhibited the most complicated rearrangement in this study. Array results demonstrated a duplication-normal-duplication-normal-deletion pattern at 13q33.2 to 13q34 and a duplication-normal-duplication-triplication-duplication pattern at Xq21.1 (Fig 3A); FISH analysis showed that both of the two duplicated regions on chr13 were inserted into chrX (S6 Fig, S1 Table). Breakpoint mapping further demonstrated that the rearrangement between chr13 and chrX could be potentially generated through 6 junctions (Fig 3B). With the exception of the hypothetical Junction 5, we were able to map the remaining 5 junctions to nucleotide resolution. Based on the information of the five junctions mapped and the CNVs observed, we proposed the existence of Junction 5 to most parsimoniously explain the observed overall rearrangement in this individual (Fig 3B). Upon careful examination of the junctions, we observed that sequences of Junction 2 contained an 8,192 bp insertion from Xq13.2, followed by a 5,167 bp insertion from 4q13.1, leading to the discovery of the involvement of a third chromosome, chromosome 4, in this individual’s CGR (Table 1, S7 Fig). The remaining mapped junctions showed 2 bp microhomology (Junction 6) or blunt ends (Junction 1, 3 and 4).

View Article: PubMed Central - PubMed

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.

No MeSH data available.