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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 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.
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pgen.1006446.g004: 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.

Mentions: Previously, we reported a child (BAB1379) with PLP1 deletion that resulted from a maternal balanced insertion (BAB1381) of a segment on chrX containing the entire PLP1 gene translocated and inserted into the telomeric region of the q arm of chr19 (Fig 4A).[16] The PLP1 deletion breakpoint junction was mapped in the previous publication, showing an Alu-Alu mediated rearrangement (Junction 3 in S8 Fig, re-drawn in hg19). This junction was present in the mother (BAB1381) and her affected son with Pelizaeus-Merzbacher disease (BAB1379), but not in the unaffected son (BAB1380). To fine map other breakpoint junctions involving the insertion, we designed high-density aCGH targeting both the regions on chrX containing PLP1, and the potential insertion site at 19qter. Surprisingly, in the mother, we did not see complete copy-number neutral genomic intervals around the PLP1 region as expected for her balanced insertion, but instead observed small CNVs that map at the exact loci of both ends of the deletion position in her affected son (Fig 4B). More specifically, an ~10 kb deletion at the proximal boundary, and an ~22 kb duplication at the distal boundary of the deletion position in her son (Fig 4B). In addition, an ~182 kb duplication was detected at 19q13.4, the potential inserting site, in the mother (Fig 4B). Further breakpoint junction mapping in the mother revealed that the distal side of the duplication on chr19 joined the distal side of the small deletion on chrX (Junction 1 in Fig 4C), while the proximal side of the chr19 duplication joined the distal side of the small duplication on chrX (Junction 2 in Fig 4C). The two small CNVs detected on chrX in the mother were actually due to unbalanced insertion from chrX to chr19, together with a duplication at the inserting site at 19q13.4. Sequences of the junctions showed 3 bp microhomology (Junction 1) and 15 bp templated insertion from nearby sequences at Junction 2 (Table 1, S8 Fig).


Mechanisms for Complex Chromosomal Insertions
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.
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Related In: Results  -  Collection

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pgen.1006446.g004: 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.
Mentions: Previously, we reported a child (BAB1379) with PLP1 deletion that resulted from a maternal balanced insertion (BAB1381) of a segment on chrX containing the entire PLP1 gene translocated and inserted into the telomeric region of the q arm of chr19 (Fig 4A).[16] The PLP1 deletion breakpoint junction was mapped in the previous publication, showing an Alu-Alu mediated rearrangement (Junction 3 in S8 Fig, re-drawn in hg19). This junction was present in the mother (BAB1381) and her affected son with Pelizaeus-Merzbacher disease (BAB1379), but not in the unaffected son (BAB1380). To fine map other breakpoint junctions involving the insertion, we designed high-density aCGH targeting both the regions on chrX containing PLP1, and the potential insertion site at 19qter. Surprisingly, in the mother, we did not see complete copy-number neutral genomic intervals around the PLP1 region as expected for her balanced insertion, but instead observed small CNVs that map at the exact loci of both ends of the deletion position in her affected son (Fig 4B). More specifically, an ~10 kb deletion at the proximal boundary, and an ~22 kb duplication at the distal boundary of the deletion position in her son (Fig 4B). In addition, an ~182 kb duplication was detected at 19q13.4, the potential inserting site, in the mother (Fig 4B). Further breakpoint junction mapping in the mother revealed that the distal side of the duplication on chr19 joined the distal side of the small deletion on chrX (Junction 1 in Fig 4C), while the proximal side of the chr19 duplication joined the distal side of the small duplication on chrX (Junction 2 in Fig 4C). The two small CNVs detected on chrX in the mother were actually due to unbalanced insertion from chrX to chr19, together with a duplication at the inserting site at 19q13.4. Sequences of the junctions showed 3 bp microhomology (Junction 1) and 15 bp templated insertion from nearby sequences at Junction 2 (Table 1, S8 Fig).

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.