Detailed molecular cytogenetic characterisation of the myeloid cell line U937 reveals the fate of homologous chromosomes and shows that centromere capture is a feature of genome instability

Background The U937 cell line is widely employed as a research tool. It has a complex karyotype. A PICALM-MLLT10 fusion gene formed by the recurrent t(10;11) translocation is present, and the myeloid common deleted region at 20q12 has been lost from its near-triploid karyotype. We carried out a detailed investigation of U937 genome reorganisation including the chromosome 20 rearrangements and other complex rearrangements. Results SNP array, G-banding and Multicolour FISH identified chromosome segments resulting from unbalanced and balanced rearrangements. The organisation of the abnormal chromosomes containing these segments was then reconstructed with the strategic use of targeted metaphase FISH. This provided more accurate karyotype information for the evolving karyotype. Rearrangements involving the homologues of a chromosome pair could be differentiated in most instances. Centromere capture was demonstrated in an abnormal chromosome containing parts of chromosomes 16 and 20 which were stabilised by joining to a short section of chromosome containing an 11 centromere. This adds to the growing number of examples of centromere capture, which to date have a high incidence in complex karyotypes where the centromeres of the rearranged chromosomes are identified. There were two normal copies of one chromosome 20 homologue, and complex rearrangement of the other homologue including loss of the 20q12 common deleted region. This confirmed the previously reported loss of heterozygosity of this region in U937, and defined the rearrangements giving rise to this loss. Conclusions Centromere capture, stabilising chromosomes pieced together from multiple segments, may be a common feature of complex karyotypes. However, it has only recently been recognised, as this requires deliberate identification of the centromeres of abnormal chromosomes. The approach presented here is invaluable for studying complex reorganised genomes such as those produced by chromothripsis, and provides a more complete picture than can be obtained by microarray, karyotyping or FISH studies alone. One major advantage of SNP arrays for this process is that the two homologues can usually be distinguished when there is more than one rearrangement of a chromosome pair. Tracking the fate of each homologue and of highly repetitive DNA regions such as centromeres helps build a picture of genome evolution. Centromere- and telomere-containing elements are important to deducing chromosome structure. This study confirms and highlights ongoing evolution in cultured cell lines.

Detailed molecular cytogenetic characterisation of the myeloid cell line U937 reveals the fate of homologous chromosomes and shows that centromere capture is a feature of genome instability

BackgroundThe U937 cell line is widely employed as a research tool. It has a complex karyotype. A PICALM-MLLT10 fusion gene formed by the recurrent t(10;11) translocation is present, and the myeloid common deleted region at 20q12 has been lost from its near-triploid karyotype. We carried out a detailed investigation of U937 genome reorganisation including the chromosome 20 rearrangements and other complex rearrangements. ResultsSNP array, G-banding and Multicolour FISH identified chromosome segments resulting from unbalanced and balanced rearrangements. The organisation of the abnormal chromosomes containing these segments was then reconstructed with the strategic use of targeted metaphase FISH. This provided more accurate karyotype information for the evolving karyotype. Rearrangements involving the homologues of a chromosome pair could be differentiated in most instances.Centromere capture was demonstrated in an abnormal chromosome containing parts of chromosomes 16 and 20 which were stabilised by joining to a short section of chromosome containing an 11 centromere. This adds to the growing number of examples of centromere capture, which to date have a high incidence in complex karyotypes where the centromeres of the rearranged chromosomes are identified.There were two normal copies of one chromosome 20 homologue, and complex rearrangement of the other homologue including loss of the 20q12 common deleted region. This confirmed the previously reported loss of heterozygosity of this region in U937, and defined the rearrangements giving rise to this loss.Conclusions Centromere capture, stabilising chromosomes pieced together from multiple segments, may be a common feature of complex karyotypes. However, it has only recently been recognised, as this requires deliberate identification of the centromeres of abnormal chromosomes. The approach presented here is invaluable for studying complex reorganised genomes such as those produced by chromothripsis, and provides a more complete picture than can be obtained by microarray, karyotyping or FISH studies alone. One major advantage of SNP arrays for this process is that the two homologues can usually be distinguished when there is more than one rearrangement of a chromosome pair. Tracking the fate of each homologue and of highly repetitive DNA regions such as centromeres helps build a picture of genome evolution. Centromere- and telomere-containing elements are important to deducing chromosome structure. This study confirms and highlights ongoing evolution in cultured cell lines.

Praenatalisan diagnosztizált Pallister–Killian-szindróma esete

Pallister–Killian syndrome (PKS) is a rare, sporadic genetic disorder that is caused by the mosaic presence of a supernumerary marker chromosome, isochromosome 12p. The syndrome is a polydysmorphic condition characterized by mental retardation, craniofacial dysmorphism, hypotonia, seizures, epilepsy and certain organic malformations (diaphragmatic hernia, congenital heart disease). Prenatal diagnosis is challenging due to the mosaic tissue-specific distribution of the chromosomal disorder and highly variable phenotype. Prenatal diagnosis is often accidental, however, appropriate laboratory techniques based on the second trimester ultrasound anomalies provide accurate prenatal diagnosis. We report a case of a 36-year-old primipara with second trimester ultrasound markers (polyhydramnion, ventriculomegaly, rhizomelic micromelia, abnormal facial profile). The patient underwent amniocentesis, the conventional karyotyping revealed a supernumerary chromosome in nearly 50 percent of amniocytes. FISH and targeted multicolour FISH probes verified mosaic tetrasomy of the short arm of chromosome 12 of the fetus. Fetopathological examinations and analysis of fetal tissues and blood confirmed the prenatal diagnosis. To our knowledge, this is the first reported case of prenatally diagnosed Pallister–Killian syndrome in Hungary. Orv Hetil. 2018; 159(21): 847–852.

A NOTCH1-Independant Pathway of c-Myc Oncogenesis in TAL1+ Human T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of developing thymocytes afflicting both children and adults. Although the outcome has significantly improved over the past decades, further advances in targeted therapy will require an accurate stratification of T-ALL subtypes based on the precise understanding of the mechanisms involved. TAL1 is among the most frequently deregulated oncogenes in T-ALL. Studies on TAL1 transgenic animals have recently shown that leukemic growth and survival is largely dependent on the NOTCH1-controlled c-Myc pathway. We recently identified a particular aggressive paediatric T-ALL with t(1;14)(p32;q11)-mediated TAL1 overexpression and, in agreement with a central role of c-Myc in human T-ALL, displaying high levels of c-Myc transcripts. However, the absence of NOTCH1/FBW7 mutations in this case suggested an alternative c-Myc deregulation process. Multicolour FISH analysis indeed revealed a cryptic t(7;8)(q34;q24) within the same t(1;14)+ T-ALL clone, and breakpoint cloning confirmed the juxtaposition of c-Myc to the TCRβ locus. To further investigate the likely scenario of neoplastic development in this patient, we undertook a detailed geno/phenotyping of the tumor sample. In good agreement with the proposed oncogenic role of TAL1 in blocking thymocytes at late stages of maturation, immunophenotypic, transcriptional and genotype profiles clearly converged towards a maturation arrest of the malignant clone at the late cortical CD8 SP stage. By contrast, analysis of translocation breakpoints indicated that both events resulted from V(D)J recombination mistakes, and congruently occurred during the early stages of TCRδ and TCRβ rearrangements (DN2/DN3). Thus, the activation of both c-Myc and TAL1 was clearly decoupled from maturation arrest. As thymocyte progression from DN3 to SP includes extensive proliferation at the ISP/DP transition before rearrangement of the TCRα chain, the tumor clone was expected to show polyclonal TCRα rearrangements. Instead, LR-LMPCR and TCRα multiplex PCR revealed the presence of a monoclonal TCRα rearrangement. Thus, out of hundreds of thymocytes carrying t(1;14) and t(7;8), only one progressed towards malignant transformation after the onset of TCRα rearrangements, suggesting the necessity of yet a “3rd hit”. Most importantly, this indicates that in absence of NOTCH1, TAL1 and c-Myc cooperation was not sufficient to trigger aggressive proliferation. One likely candidate for this 3rd hit is LMO2, which we also found significantly over-expressed in the tumour clone. As previously underlined in animal models, the synergy between LMO2, TAL1 and c-MYC oncogenes likely accounts for the aggressive tumor phenotype in this patient, and illustrates the complex cooperative oncogenic pathways in human T-ALL pathogenesis. Intriguingly, the lack of major mediastin involvement in a clone with such a fulgurating growth raises the alternative possibility that proliferation was actually fired in the periphery through cognate triggering of the expressed TCRαβ, and thus that proliferation is uncoupled from differentiation block.