Modeling Global Genomic Instability in Chronic Myeloid Leukemia (CML) Using Patient-Derived Induced Pluripotent Stem Cells (iPSCs).

METHODS: We used a patient-specific induced pluripotent stem cell (iPSC) line treated with the mutagenic agent N-ethyl-N-nitrosourea (ENU). Genomic instability was validated using γ-H2AX and micronuclei assays and CGH array for genomic events. RESULTS: An increased number of progenitors (x5-Fold), which proliferated in liquid cultures with a blast cell morphology, was observed in the mutagenized condition as compared to the unmutagenized one. CGH array performed for both conditions in two different time points reveals several cancer genes in the ENU-treated condition, some known to be altered in leukemia (BLM, IKZF1, NCOA2, ALK, EP300, ERG, MKL1, PHF6 and TET1). Transcriptome GEO-dataset GSE4170 allowed us to associate 125 of 249 of the aberrations that we detected in CML-iPSC with the CML progression genes already described during progression from chronic and AP to BC. Among these candidates, eleven of them have been described in CML and related to tyrosine kinase inhibitor resistance and genomic instability. CONCLUSIONS: These results demonstrated that we have generated, for the first time to our knowledge, an in vitro genetic instability model, reproducing genomic events described in patients with BC.

A Central Role of Telomere Dysfunction in the Formation of a Unique Translocation within the Sub-Telomere Region Resulting in Duplication and Partial Trisomy.

Telomeres play a major role in maintaining genome stability and integrity. Putative involvement of telomere dysfunction in the formation of various types of chromosomal aberrations is an area of active research. Here, we report a case of a six-month-old boy with a chromosomal gain encompassing the 11q22.3q25 region identified by SNP array analysis. The size of the duplication is 26.7 Mb and contains 170 genes (OMIM). The duplication results in partial trisomy of the region in question with clinical consequences, including bilateral renal dysplasia, delayed development, and a heart defect. Moreover, the karyotype determined by R-banding and chromosome painting as well as by hybridization with specific sub-telomere probes revealed the presence of an unbalanced t(9;11)(p24;q22.3) translocation with a unique breakpoint involving the sub-telomere region of the short arm of chromosome 9. The karyotypes of the parents were normal. Telomere integrity in circulating lymphocytes from the child and from his parents was assessed using an automated high-throughput method based on fluorescence in situ hybridization (FISH) with telomere- and centromere-specific PNA probes followed by M-FISH multicolor karyotyping. Very short telomeres, as well as an increased frequency of telomere loss and formation of telomere doublets, were detected in the child's cells. Interestingly, similar telomere profiles were found in the circulating lymphocytes of the father. Moreover, an assessment of clonal telomere aberrations identified chromosomes 9 and 11 with particularly high frequencies of such aberrations. These findings strongly suggest that telomere dysfunction plays a central role in the formation of this specific unbalanced chromosome rearrangement via chromosome end-to-end fusion and breakage-fusion-bridge cycles.

Telomere and Centromere Staining Followed by M-FISH Improves Diagnosis of Chromosomal Instability and Its Clinical Utility.

Dicentric chromosomes are a relevant marker of chromosomal instability. Their appearance is associated with telomere dysfunction, leading to cancer progression and a poor clinical outcome. Here, we present Telomere and Centromere staining followed by M-FISH (TC+M-FISH) for improved detection of telomere dysfunction and the identification of dicentric chromosomes in cancer patients and various genetic syndromes. Significant telomere length shortening and significantly higher frequencies of telomere loss and deletion were found in the peripheral lymphocytes of patients with cancer and genetic syndromes relative to similar age-matched healthy donors. We assessed our technique against conventional cytogenetics for the detection of dicentric chromosomes by subjecting metaphase preparations to both approaches. We identified dicentric chromosomes in 28/50 cancer patients and 21/44 genetic syndrome patients using our approach, but only 7/50 and 12/44, respectively, using standard cytogenetics. We ascribe this discrepancy to the identification of the unique configuration of dicentric chromosomes. We observed significantly higher frequencies of telomere loss and deletion in patients with dicentric chromosomes (p < 10-4). TC+M-FISH analysis is superior to classical cytogenetics for the detection of chromosomal instability. Our approach is a relatively simple but useful tool for documenting telomere dysfunction and chromosomal instability with the potential to become a standard additional diagnostic tool in medical genetics and the clinic.