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      Need for high-resolution Genetic Analysis in iPSC: Results and Lessons from the ForIPS Consortium

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          Abstract

          Genetic integrity of induced pluripotent stem cells (iPSCs) is essential for their validity as disease models and for potential therapeutic use. We describe the comprehensive analysis in the ForIPS consortium: an iPSC collection from donors with neurological diseases and healthy controls. Characterization included pluripotency confirmation, fingerprinting, conventional and molecular karyotyping in all lines. In the majority, somatic copy number variants (CNVs) were identified. A subset with available matched donor DNA was selected for comparative exome sequencing. We identified single nucleotide variants (SNVs) at different allelic frequencies in each clone with high variability in mutational load. Low frequencies of variants in parental fibroblasts highlight the importance of germline samples. Somatic variant number was independent from reprogramming, cell type and passage. Comparison with disease genes and prediction scores suggest biological relevance for some variants. We show that high-throughput sequencing has value beyond SNV detection and the requirement to individually evaluate each clone.

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          Most cited references29

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          Common genetic variation drives molecular heterogeneity in human iPSCs

          Induced pluripotent stem cell (iPSC) technology has enormous potential to provide improved cellular models of human disease. However, variable genetic and phenotypic characterisation of many existing iPSC lines limits their potential use for research and therapy. Here, we describe the systematic generation, genotyping and phenotyping of 711 iPSC lines derived from 301 healthy individuals by the Human Induced Pluripotent Stem Cells Initiative (HipSci: http://www.hipsci.org). Our study outlines the major sources of genetic and phenotypic variation in iPSCs and establishes their suitability as models of complex human traits and cancer. Through genome-wide profiling we find that 5-46% of the variation in different iPSC phenotypes, including differentiation capacity and cellular morphology, arises from differences between individuals. Additionally, we assess the phenotypic consequences of rare, genomic copy number mutations that are repeatedly observed in iPSC reprogramming and present a comprehensive map of common regulatory variants affecting the transcriptome of human pluripotent cells.
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            Detectable clonal mosaicism from birth to old age and its relationship to cancer

            Clonal mosaicism for large chromosomal anomalies (duplications, deletions and uniparental disomy) was detected using SNP microarray data from over 50,000 subjects recruited for genome-wide association studies. This detection method requires a relatively high frequency of cells (>5–10%) with the same abnormal karyotype (presumably of clonal origin) in the presence of normal cells. The frequency of detectable clonal mosaicism in peripheral blood is low (<0.5%) from birth until 50 years of age, after which it rises rapidly to 2–3% in the elderly. Many of the mosaic anomalies are characteristic of those found in hematological cancers and identify common deleted regions that pinpoint the locations of genes previously associated with hematological cancers. Although only 3% of subjects with detectable clonal mosaicism had any record of hematological cancer prior to DNA sampling, those without a prior diagnosis have an estimated 10-fold higher risk of a subsequent hematological cancer (95% confidence interval = 6–18).
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              Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations

              Human pluripotent stem cells (hPSCs) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with acquisition of large copy number variants (CNVs) that provide mutant cells with a growth advantage in culture1–3. However, the nature, extent, and functional impact of other acquired genome sequence mutations in cultured hPSCs is not known. Here, we sequenced the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hESC) lines, including 26 lines prepared for potential clinical use4. We then applied computational strategies for identifying mutations present in a subset of cells5. Though such mosaic mutations were generally rare, we identified five unrelated hESC lines that carried six mutations in the TP53 gene that encodes the tumor suppressor P53. Notably, the TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We used droplet digital PCR to demonstrate that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that P53 mutation confers selective advantage. When we then mined published RNA sequencing data from 117 hPSC lines, we observed another nine TP53 mutations, all resulting in coding changes in the DNA binding domain of P53. Strikingly, in three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from loss of heterozygosity at the TP53 locus. As the acquisition and favored expansion of cancer-associated mutations in hPSCs may go unnoticed during most applications, we suggest that careful genetic characterization of hPSCs and their differentiated derivatives should be carried out prior to clinical use.
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                Author and article information

                Contributors
                andre.reis@ukerlangen.de
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                21 November 2018
                21 November 2018
                2018
                : 8
                : 17201
                Affiliations
                [1 ]Institute of Human Genetics, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Schwabachanlage 10, 91054 Erlangen, Germany
                [2 ]Department of Molecular Neurology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Schwabachanlage 6, Erlangen, Germany
                [3 ]Department of Stem Cell Biology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Glückstrasse 6, Erlangen, Germany
                [4 ]ISNI 0000 0001 1958 8658, GRID grid.8379.5, Stem Cell Biology and Regenerative Medicine Group, Institute of Anatomy and Cell Biology, , Julius-Maximilians-University of Würzburg, ; Würzburg, Germany
                [5 ]ISNI 0000 0004 1937 0650, GRID grid.7400.3, Institute of Medical Genetics, , University of Zurich, ; Schlieren, Zurich Switzerland
                Author information
                http://orcid.org/0000-0002-3679-1081
                http://orcid.org/0000-0002-4147-8866
                http://orcid.org/0000-0001-6099-7066
                http://orcid.org/0000-0002-1497-0564
                http://orcid.org/0000-0002-2172-7386
                http://orcid.org/0000-0002-6301-6363
                Article
                35506
                10.1038/s41598-018-35506-0
                6249203
                30464253
                00975964-dbd8-49a2-9996-7c19804370ea
                © The Author(s) 2018

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 8 August 2018
                : 1 November 2018
                Funding
                Funded by: FundRef https://doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft (German Research Foundation);
                Award ID: GRK2162
                Award ID: GRK2162
                Award ID: GRK2162
                Award Recipient :
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