Optical Genome Mapping in Routine Human Genetic Diagnostics—Its Advantages and Limitations

In recent years, optical genome mapping (OGM) has developed into a highly promising method of detecting large-scale structural variants in human genomes. It is capable of detecting structural variants considered difficult to detect by other current methods. Hence, it promises to be feasible as a first-line diagnostic tool, permitting insight into a new realm of previously unknown variants. However, due to its novelty, little experience with OGM is available to infer best practices for its application or to clarify which features cannot be detected. In this study, we used the Saphyr system (Bionano Genomics, San Diego, CA, USA), to explore its capabilities in human genetic diagnostics. To this end, we tested 14 DNA samples to confirm a total of 14 different structural or numerical chromosomal variants originally detected by other means, namely, deletions, duplications, inversions, trisomies, and a translocation. Overall, 12 variants could be confirmed; one deletion and one inversion could not. The prerequisites for detection of similar variants were explored by reviewing the OGM data of 54 samples analyzed in our laboratory. Limitations, some owing to the novelty of the method and some inherent to it, were described. Finally, we tested the successful application of OGM in routine diagnostics and described some of the challenges that merit consideration when utilizing OGM as a diagnostic tool

Exclusion from spheroid formation identifies loss of essential cell-cell adhesion molecules in colon cancer cells

Many cell lines derived from solid cancers can form spheroids, which recapitulate tumor cell clusters and are more representative of the in vivo situation than 2D cultures. During spheroid formation, a small proportion of a variety of different colon cancer cell lines did not integrate into the sphere and lost cell-cell adhesion properties. An enrichment protocol was developed to augment the proportion of these cells to 100% purity. The basis for the separation of spheroids from non-spheroid forming (NSF) cells is simple gravity-sedimentation. This protocol gives rise to sub-populations of colon cancer cells with stable loss of cell-cell adhesion. SW620 cells lacked E-cadherin, DLD-1 cells lost -catenin and HCT116 cells lacked P-cadherin in the NSF state. Knockdown of these molecules in the corresponding spheroid-forming cells demonstrated that loss of the respective proteins were indeed responsible for the NSF phenotypes. Loss of the spheroid forming phenotype was associated with increased migration and invasion properties in all cell lines tested. Hence, we identified critical molecules involved in spheroid formation in different cancer cell lines. We present here a simple, powerful and broadly applicable method to generate new sublines of tumor cell lines to study loss of cell-cell adhesion in cancer progression.(VLID)463741

Genome amplification and cellular senescence are hallmarks of human placenta development

Genome amplification and cellular senescence are commonly associated with pathological processes. While physiological roles for polyploidization and senescence have been described in mouse development, controversy exists over their significance in humans. Here, we describe tetraploidization and senescence as phenomena of normal human placenta development. During pregnancy, placental extravillous trophoblasts (EVTs) invade the pregnant endometrium, termed decidua, to establish an adapted microenvironment required for the developing embryo. This process is critically dependent on continuous cell proliferation and differentiation, which is thought to follow the classical model of cell cycle arrest prior to terminal differentiation. Strikingly, flow cytometry and DNAseq revealed that EVT formation is accompanied with a genome-wide polyploidization, independent of mitotic cycles. DNA replication in these cells was analysed by a fluorescent cell-cycle indicator reporter system, cell cycle marker expression and EdU incorporation. Upon invasion into the decidua, EVTs widely lose their replicative potential and enter a senescent state characterized by high senescence-associated (SA) beta-galactosidase activity, induction of a SA secretory phenotype as well as typical metabolic alterations. Furthermore, we show that the shift from endocycle-dependent genome amplification to growth arrest is disturbed in androgenic complete hydatidiform moles (CHM), a hyperplastic pregnancy disorder associated with increased risk of developing choriocarinoma. Senescence is decreased in CHM-EVTs, accompanied by exacerbated endoreduplication and hyperploidy. We propose induction of cellular senescence as a ploidy-limiting mechanism during normal human placentation and unravel a link between excessive polyploidization and reduced senescence in CHM.Funding Agencies|Austrian Science Fund [P-25187, P-28417, P-31470]; Herzfeldersche Familienstiftung [00685]; Jubilaumsfonds Austrian National Bank [16517]; European Fund for Regional Development (EFRE) [IWB2020]; Federal State of Upper Austria; Stanford Child Health Research Institute</p