Large-scale Chromosomal Aberrations Research Articles

Chromosome 8p engineering reveals increased metastatic potential targetable by patient-specific synthetic lethality in liver cancer.

Large-scale chromosomal aberrations are prevalent in human cancer, but their function remains poorly understood. We established chromosome-engineered hepatocellular carcinoma cell lines using CRISPR-Cas9 genome editing. A 33-mega-base pair region on chromosome 8p (chr8p) was heterozygously deleted, mimicking a frequently observed chromosomal deletion. Using this isogenic model system, we delineated the functional consequences of chr8p loss and its impact on metastatic behavior and patient survival. We found that metastasis-associated genes on chr8p act in concert to induce an aggressive and invasive phenotype characteristic for chr8p-deleted tumors. Genome-wide CRISPR-Cas9 viability screening in isogenic chr8p-deleted cells served as a powerful tool to find previously unidentified synthetic lethal targets and vulnerabilities accompanying patient-specific chromosomal alterations. Using this target identification strategy, we showed that chr8p deletion sensitizes tumor cells to targeting of the reactive oxygen sanitizing enzyme Nudix hydrolase 17. Thus, chromosomal engineering allowed for the identification of novel synthetic lethalities specific to chr8p loss of heterozygosity.

Refined detection and phasing of structural aberrations in pediatric acute lymphoblastic leukemia by linked-read whole-genome sequencing

Structural chromosomal rearrangements that can lead to in-frame gene-fusions are a leading source of information for diagnosis, risk stratification, and prognosis in pediatric acute lymphoblastic leukemia (ALL). Traditional methods such as karyotyping and FISH struggle to accurately identify and phase such large-scale chromosomal aberrations in ALL genomes. We therefore evaluated linked-read WGS for detecting chromosomal rearrangements in primary samples of from 12 patients diagnosed with ALL. We assessed the effect of input DNA quality on phased haplotype block size and the detectability of copy number aberrations and structural variants in the ALL genomes. We found that biobanked DNA isolated by standard column-based extraction methods was sufficient to detect chromosomal rearrangements even at low 10x sequencing coverage. Linked-read WGS enabled precise, allele-specific, digital karyotyping at a base-pair resolution for a wide range of structural variants including complex rearrangements and aneuploidy assessment. With use of haplotype information from the linked-reads, we also identified previously unknown structural variants, such as a compound heterozygous deletion of ERG in a patient with the DUX4-IGH fusion gene. We conclude that linked-read WGS allows detection of important pathogenic variants in ALL genomes at a resolution beyond that of traditional karyotyping and FISH.

Clustered DNA double-strand break formation and the repair pathway following heavy-ion irradiation.

Photons, such as X- or γ-rays, induce DNA damage (distributed throughout the nucleus) as a result of low-density energy deposition. In contrast, particle irradiation with high linear energy transfer (LET) deposits high-density energy along the particle track. High-LET heavy-ion irradiation generates a greater number and more complex critical chromosomal aberrations, such as dicentrics and translocations, compared with X-ray or γ irradiation. In addition, the formation of >1000 bp deletions, which is rarely observed after X-ray irradiation, has been identified following high-LET heavy-ion irradiation. Previously, these chromosomal aberrations have been thought to be the result of misrepair of complex DNA lesions, defined as DNA damage through DNA double-strand breaks (DSBs) and single-strand breaks as well as base damage within 1–2 helical turns (<3–4 nm). However, because the scale of complex DNA lesions is less than a few nanometers, the large-scale chromosomal aberrations at a micrometer level cannot be simply explained by complex DNA lesions. Recently, we have demonstrated the existence of clustered DSBs along the particle track through the use of super-resolution microscopy. Furthermore, we have visualized high-level and frequent formation of DSBs at the chromosomal boundary following high-LET heavy-ion irradiation. In this review, we summarize the latest findings regarding the hallmarks of DNA damage structure and the repair pathway following heavy-ion irradiation. Furthermore, we discuss the mechanism through which high-LET heavy-ion irradiation may induce dicentrics, translocations and large deletions.

The genomic landscape of tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is a rare genetic disease causing multisystem growth of benign tumours and other hamartomatous lesions, which leads to diverse and debilitating clinical symptoms. Patients are born with TSC1 or TSC2 mutations, and somatic inactivation of wild-type alleles drives MTOR activation; however, second hits to TSC1/TSC2 are not always observed. Here, we present the genomic landscape of TSC hamartomas. We determine that TSC lesions contain a low somatic mutational burden relative to carcinomas, a subset feature large-scale chromosomal aberrations, and highly conserved molecular signatures for each type exist. Analysis of the molecular signatures coupled with computational approaches reveals unique aspects of cellular heterogeneity and cell origin. Using immune data sets, we identify significant neuroinflammation in TSC-associated brain tumours. Taken together, this molecular catalogue of TSC serves as a resource into the origin of these hamartomas and provides a framework that unifies genomic and transcriptomic dimensions for complex tumours.

A novel system for correcting large-scale chromosomal aberrations: ring chromosome correction via reprogramming into induced pluripotent stem cell (iPSC).

Approximately 1 in 500 newborns are born with chromosomal abnormalities that include trisomies, translocations, large deletions, and duplications. There is currently no therapeutic approach for correcting such chromosomal aberrations in vivo or in vitro. When we attempted to produce induced pluripotent stem cell (iPSC) models from patient-derived fibroblasts that contained ring chromosomes, we found that the ring chromosomes were eliminated and replaced by duplicated normal copies of chromosomes through a mechanism of uniparental isodisomy (Bershteyn et al. 2014, Nature 507:99). The discovery of this previously unforeseen system for aberrant chromosome correction during reprogramming enables us for the first time to model and understand this process of cell-autonomous correction of ring chromosomes during human patient somatic cell reprograming to iPSCs. This knowledge could lead to a potential therapeutic strategy to correct common large-scale chromosomal aberrations, termed "chromosome therapy".

A genome-wide association analysis of chromosomal aberrations and Hirschsprung disease

Hirschsprung disease (HSCR) is a neurocristopathy characterized by the absence of intramural ganglion cells along variable lengths of the gastrointestinal tract. Although the RET proto-oncogene is considered to be the main risk factor for HSCR, only about 30% of the HSCR cases can be explained by variations in previously known genes including RET. Recently, copy number variation (CNV) and loss of heterozygosity (LOH) have emerged as new ways to understand human genomic variation. The goal of this present study is to identify new HSCR genetic factors related to CNV in Korean patients. In the genome-wide genotyping, using Illumina's HumanOmni1-Quad BeadChip (1,140,419 markers), of 123 HSCR patients and 432 unaffected subjects (total n=555), a total of 8,188 CNVs (1 kb∼1 mb) were identified by CNVpartition. As a result, 16 CNV regions and 13 LOH regions were identified as associated with HSCR (minimum P=0.0005). Two top CNV regions (deletions at chr6:32675155-32680480 and chr22:20733495-21607293) were successfully validated by additional real-time quantitative polymerase chain reaction analysis. In addition, 2 CNV regions (6p21.32 and 22q11.21) and 2 LOH regions (3p22.2 and 14q23.3) were discovered to be unique to the HSCR patients group. Regarding the large-scale chromosomal aberrations (>1 mb), 11 large aberrations in the HSCR patients group were identified, which suggests that they may be a risk factor for HSCR. Although further replication in a larger cohort is needed, our findings may contribute to the understanding of the etiology of HSCR.

Uniparental disomy of the entire X chromosome in Turner syndrome patient-specific induced pluripotent stem cells.

The human induced pluripotent stem cell (iPSC) technique promises to provide an unlimited, reliable source of genetically matched pluripotent cells for personalized therapy and disease modeling. Recently, it is observed that cells with ring chromosomes 13 or 17 autonomously correct the defects via compensatory uniparental disomy during cellular reprogramming to iPSCs. This breakthrough finding suggests a potential therapeutic approach to repair large-scale chromosomal aberrations. However, due to the scarceness of ring chromosome samples, the reproducibility of this approach in different individuals is not carefully evaluated yet. Moreover, the underlying mechanism and the applicability to other types of chromosomal aberrations remain unknown. Here we generated iPSCs from four 45,X chorionic villous fibroblast lines and found that only one reprogrammed line acquired 46,XX karyotype via uniparental disomy of the entire X chromosome. The karyotype correction was reproducible in the same cell line by either retroviral or episomal reprogramming. The karyotype-corrected iPSCs were subject to X chromosome inactivation and obtained better colony morphology and higher proliferation rate than other uncorrected ones. Further transcriptomic comparison among the fibroblast lines identified a distinct expression pattern of cell cycle regulators in the uncorrectable ones. These findings demonstrate that the iPSC technique holds the potential to correct X monosomy, but the correction rate is very low, probably due to differential regulation of cell cycle genes between individuals. Our data strongly suggest that more systematic investigations are needed before defining the iPSC technique as a novel means of chromosome therapy.

Pediatric KIT–Wild-Type and Platelet-Derived Growth Factor Receptor α–Wild-Type Gastrointestinal Stromal Tumors Share KIT Activation but not Mechanisms of Genetic Progression with Adult Gastrointestinal Stromal Tumors

Fewer than 15% of gastrointestinal stromal tumors (GIST) in pediatric patients harbor KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutations in contrast to a mutation rate of 80% in adult GISTs. However, some therapeutic inhibitors of KIT have efficacy in pediatric GIST, suggesting that KIT may, nevertheless, play an important role in oncogenesis. In adult GIST, characteristic cytogenetic changes occur during progression to malignancy. A better understanding of mechanisms of genetic progression and KIT and PDGFRA transforming roles in pediatric GIST might facilitate treatment advances. KIT and PDGFRA mutation analysis was done in 27 pediatric GISTs. The activation status of KIT, PDGFRA, and downstream signaling intermediates was defined, and chromosomal aberrations were determined by single nucleotide polymorphism assays. Mutations in KIT or PDGFRA were identified in 11% of pediatric GISTs. KIT and the signaling intermediates AKT and mitogen-activated protein kinase were activated in pediatric GISTs. In particular, most pediatric KIT-wild-type GISTs displayed levels of KIT activation similar to levels in adult KIT-mutant GISTs. Pediatric KIT-wild-type GISTs lacked the typical cytogenetic deletions seen in adult KIT-mutant GISTs. Notably, most pediatric KIT-wild-type GISTs progress to malignancy without acquiring large-scale chromosomal aberrations, which is a phenomenon not reported previously in malignant solid tumors. KIT activation levels in pediatric KIT-wild-type GISTs are comparable with those in KIT-mutant GISTs. Therapies that inhibit KIT activation, or crucial KIT signaling intermediates, should be explored in pediatric KIT-wild-type GIST.