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, 8 (8), e72182
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Breakpoint Features of Genomic Rearrangements in Neuroblastoma With Unbalanced Translocations and Chromothripsis

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Breakpoint Features of Genomic Rearrangements in Neuroblastoma With Unbalanced Translocations and Chromothripsis

Valentina Boeva et al. PLoS One.

Abstract

Neuroblastoma is a pediatric cancer of the peripheral nervous system in which structural chromosome aberrations are emblematic of aggressive tumors. In this study, we performed an in-depth analysis of somatic rearrangements in two neuroblastoma cell lines and two primary tumors using paired-end sequencing of mate-pair libraries and RNA-seq. The cell lines presented with typical genetic alterations of neuroblastoma and the two tumors belong to the group of neuroblastoma exhibiting a profile of chromothripsis. Inter and intra-chromosomal rearrangements were identified in the four samples, allowing in particular characterization of unbalanced translocations at high resolution. Using complementary experiments, we further characterized 51 rearrangements at the base pair resolution that revealed 59 DNA junctions. In a subset of cases, complex rearrangements were observed with templated insertion of fragments of nearby sequences. Although we did not identify known particular motifs in the local environment of the breakpoints, we documented frequent microhomologies at the junctions in both chromothripsis and non-chromothripsis associated breakpoints. RNA-seq experiments confirmed expression of several predicted chimeric genes and genes with disrupted exon structure including ALK, NBAS, FHIT, PTPRD and ODZ4. Our study therefore indicates that both non-homologous end joining-mediated repair and replicative processes may account for genomic rearrangements in neuroblastoma. RNA-seq analysis allows the identification of the subset of abnormal transcripts expressed from genomic rearrangements that may be involved in neuroblastoma oncogenesis.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Genome-wide profile of predicted SVs in CLB-Ga and CLB-Re cell lines.
All inter-chromosomal rearrangements and intra-chromosomal SVs encompassing regions longer than 50 kb identified in mate-pair sequencing are visualized using Circos . Chromosomes appear as ideograms. The outer ring shows a representation of copy number as determined by sequencing data (grey: normal copy number; blue: deletion, orange: gain). The inner circle shows the two endpoints of each rearrangement identified (black: inverted duplication, blue: deletion, green: inversion, orange: large duplication, red: unbalanced inter-chromosomal rearrangements). Most of the identified unbalanced rearrangements correspond to unbalanced translocations previously identified by 24-color karyotyping. Line’s thickness is related to the numbers of pairs identified in each link. A, CLB-Ga cell line, B, CLB-Re cell line, C, zoom on the cluster of links between chromosomes 2p (0–43.95 Mb) and 3p (58.58–78.99 Mb) in CLB-Re. This dense clustering of SVs was unexpected considering previous characterization of the rearrangements present in this cell line by array-CGH and 24-color karyotyping.
Figure 2
Figure 2. Molecular characterization of unbalanced translocations in the CLB-Ga cell line.
A, Characterization at the base-pair level of an acquired unbalanced translocation der(4)t(4;17). The translocation was initially demonstrated by 24-color karyotyping (left panel) and loss of 4q as well as gain of 17q was consistently observed by read coverage analysis with the FREEC algorithm (middle panels). NGS data identified abnormally mapped reads corresponding to this inter-chromosomal rearrangement. Further PCR analysis confirmed that this rearrangement was somatic as detected in CLB-Ga cell line as well as in a bone marrow sample contaminated with tumor cells but not in the corresponding lymphoblastoid LL-Ga cell line (right panel). B, Characterization at the base-pair level of an acquired unbalanced translocation der(5)t(5;11). The translocation was initially evidenced by 24-color karyotyping (left panel) and loss of 5q as well as gain of 11p was consistently observed by FREEC (middle panels). NGS data identified reads corresponding to an unbalanced translocation between 5q and 11p; however, orientation of the stuck fragments was not compatible with the observed derivative chromosome. NGS data also revealed an intra-chromosomal inverted duplication at 5q close to the rearrangement between 5q and 11p. The FREEC profile indeed revealed a small region of gain preceding the 5q deletion. Further PCR analysis confirmed both rearrangements and indicated that they were somatic as detected in the CLB-Ga cell line as well as in the bone marrow sample contaminated with tumor cells but not in the LL-Ga cell line (right panels). Sequencing of the PCR products indicates that the first breakpoint falls into the first intron of the CDX1 gene, which is consequently truncated. An inverted duplication is observed between positions 149 499 753 and 149 551 306 comprising the first exon of the CDX1 gene as well as exons 1 to 17 of the PDGFRB gene. The breakpoint at position 41 211 793 on chromosome 11 corresponds to the first intron of the LRRC4C gene. Thus, this junction may result in a chimeric transcript including PDGFRB and LRRC4C.
Figure 3
Figure 3. Chromothripsis in two primary NB tumors.
FREEC analysis showing the shattering of: A, chromosome 1 in NB1141; D, chromosome 6 in NB1142. All inter-chromosomal rearrangements and intra-chromosomal SVs encompassing regions longer than 50 kb identified in mate-pair sequencing are visualized using the Circos tool. B, NB1141 tumor; E, NB1142 tumor; C, zoom on chromosome 1 and distal part of chromosome 2p arm (0–35 Mb) in NB1141; F, zoom on chromosomes 6 and 19 in NB1142. Line’s thickness is related to the numbers of pairs identified in each link.
Figure 4
Figure 4. Examples of rearrangements with templated insertion of nearby sequences.
A, Rearrangement SV1 between chromosomes 1 and 7 in CLB-Re; B, intra-chromosomal rearrangement SV3 on chromosome 1 in NB1141. In each panel, the top graph shows the schematic representation of normal chromosomes. Below is the schematic diagram of the SV. The nucleotide sequence of the rearrangement region (black box) is indicated below. Alignments in boxes illustrate the structure of each breakpoint within the rearrangement. Each partner in the rearrangement is shown by a different color. The parts of chromosomes that were lost during rearrangement are shown in grey. Regions that were duplicated (A) or inverted (B) are shown by arrows of a darker color in the diagram; corresponding sequences are underlined.
Figure 5
Figure 5. Microhomology at breakpoint junctions is found more frequently than expected by chance in chromothripsis and non-chromothripsis associated breakpoints.
The histogram shows theoretical (grey) and observed frequencies of microhomology on validated breakpoint junctions for chromothripsis (orange) and non-chromothripsis cases (yellow).

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Grant support

The U830 Inserm laboratory is supported by grants from the Institut National du Cancer, the Ligue Nationale contre le Cancer (Equipe labellisée), the Institut National du Cancer, the ICGEX program, the Association Hubert Gouin, Les Bagouz à Manon, les amis de Claire and Enfants et Santé. GS is supported by the Annenberg Foundation. The U900 Inserm laboratory is supported by the Ligue Nationale Contre le Cancer (Equipe labellisée). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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