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Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells

Abstract

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) has been suspected of causing de novo copy number variation1,2,3,4. To explore this issue, here we perform a whole-genome and transcriptome analysis of 20 human iPSC lines derived from the primary skin fibroblasts of seven individuals using next-generation sequencing. We find that, on average, an iPSC line manifests two copy number variants (CNVs) not apparent in the fibroblasts from which the iPSC was derived. Using PCR and digital droplet PCR, we show that at least 50% of those CNVs are present as low-frequency somatic genomic variants in parental fibroblasts (that is, the fibroblasts from which each corresponding human iPSC line is derived), and are manifested in iPSC lines owing to their clonal origin. Hence, reprogramming does not necessarily lead to de novo CNVs in iPSCs, because most of the line-manifested CNVs reflect somatic mosaicism in the human skin. Moreover, our findings demonstrate that clonal expansion, and iPSC lines in particular, can be used as a discovery tool to reliably detect low-frequency CNVs in the tissue of origin. Overall, we estimate that approximately 30% of the fibroblast cells have somatic CNVs in their genomes, suggesting widespread somatic mosaicism in the human body. Our study paves the way to understanding the fundamental question of the extent to which cells of the human body normally acquire structural alterations in their DNA post-zygotically.

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Figure 1: Characterization of candidate LM-CNVs with respect to passage number and total CNVs.
Figure 2: Validation and estimation of cell frequency of representative somatic CNVs in fibroblasts.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

The CNV array and sequencing data are available from Gene Expression Omnibus under accessions GSE41716 and GSE41563, and from https://ndarportal.nih.gov/ndarportal/NDARSTD289.

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Acknowledgements

We acknowledge support from the National Institutes of Health (NIH) and from the AL Williams Professorship fund and the Harris Professorship fund. We also acknowledge the Yale University Biomedical High Performance Computing Center and its support team (in particular, R. Bjornson and N. Carriero). We thank A. Klin for help with family recruitment. We thank M. V. Simonini for technical help, I.-H. Park for advice in the characterization of iPSC lines and the gift of the iPSC PGP1-1, and S. A. Duncan for the gift of the K3 iPSC line. We acknowledge the following grant support: NIMH MH089176 and MH087879, the Simons Foundation (SFARI 137055 F.V.) and the State of Connecticut, which funded the hiPSC generation and characterization; and NIH grant RR19895, which funded the instrumentation. We acknowledge the Yale Center for Clinical Investigation for clinical support in obtaining the biopsy specimens. We thank J. Overton for advice in carrying out DNA and RNA sequencing. Finally, we thank M. O’Huallachain and J. Li-Pook-Than for their advice on planning, carrying out and analysing the ddPCR experiments.

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Authors

Contributions

The authors contributed to this study at different levels, as described in the following. Study conception and design: F.M.V., A.A. and A.E.U. Family selection: E.L.G. Skin biopsy: A.S. Fibroblast culture: A.H. hiPSC generation and characterization: L.A.R.B., J.M. and L.T. Virus production: A.K. Microarrays data analysis: L.T. Neuronal differentiation: L.A.R.B., N.E.C. and J.M. Sequencing library preparation: L.A.R.B., J.M., L.T. and Y.Z. Processing and analysis of RNA-seq data: D.P. and A.A. Processing and analysis of DNAseq data: A.A. and M.W. qPCR validation: A.F.F. PCR validation: Y.Z. and A.A. aCGH hybridization and analysis: M.S.H. ddPCR experiments and analysis: M.S.H. and A.A. Human subjects: K.C. Coordination of analyses: F.M.V., S.W., A.E.U. and M.G. Display item preparation: A.A., F.M.V., L.T., D.P., J.M., N.E.C., Y.Z. and M.S.H. Manuscript writing: A.A., F.M.V. and A.E.U. The following authors contributed equally to the study: J.M., D.P., Y.Z., M.S.H. and L.T. All authors participated in discussion of results and manuscript editing.

Corresponding authors

Correspondence to Alexander Eckehart Urban or Mark Gerstein or Flora M. Vaccarino.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Text, Supplementary References, Supplementary Figures 1-57, Supplementary Tables 2, 5 and 6 and full legends for Supplementary Tables 1, 3 and 4 (see contents for details). (PDF 22730 kb)

Supplementary Table 1

This file contains the Gene expression microarray dataset - see Supplementary Information for full legend. (XLSX 2185 kb)

Supplementary Table 3

This file contains comprehensive information about all LM-CNV candidates - see Supplementary Information for full legend. (XLS 75 kb)

Supplementary Table 4

This file contains comprehensive information about LM-CNV candidates from aCGH - see Supplementary Information for full legend. (XLSX 12 kb)

Supplementary Data

This file shows the alignment of sequenced PCR bands to genomic regions with LM-CNVs. (TXT 197 kb)

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Abyzov, A., Mariani, J., Palejev, D. et al. Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells. Nature 492, 438–442 (2012). https://doi.org/10.1038/nature11629

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