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 variation. 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.

Figure 1. Characterization of candidate line manifested CNVs (LM-CNVs) with respect to passage number and total CNVs

a, The number of LM-CNVs does not show significant changes with respect to passage, irrespective of the sensitivity of our detection criterion. Throughout this paper, conservative criteria (blue symbols) were used unless noted. b, Percentage of LM-CNVs of all CNVs detected in hiPSCs by comparison with the reference human genome; square symbols represent data obtained at increased (20X) coverage. LM-CNVs represent a small fraction of all CNVs in a person. c, Counts of LM-CNVs in hiPSC using fibroblasts from different individuals as a baseline. Genomes of hiPSC are different in roughly 40 CNVs (gray bars) when compared to fibroblasts from unrelated persons, that is, individuals from the other family. In contrast, genomes of hiPSC differ by less than 10 CNVs as compared to their fibroblasts of origin (blue bars). LM-CNVs in hiPSC as compared to fibroblasts represent a small increment to the already existing genetic diversity in human population.

Figure 2. Validation and estimation of cell frequency of representative somatic CNVs in fibroblasts

a, Two of the three hiPSC lines obtained from fibroblast sample S1123-01 had the same duplication on chromosome X not detected in parental fibroblasts. b, PCR amplification across CNV breakpoints revealed that the duplication was present in parental fibroblasts at lower frequency (FBR=fibroblasts; CTRL=negative control) c, Scatter plot showing signal intensities associated with the PCR amplification across the breakpoints of the LM-CNV (Y axis, green). Signal for parallel amplification of a control region is shown on the X axis (blue). Each dot represents a single PCR event. There are significantly fewer dots for PCR in CNV regions rather than for PCR in control region. d, The frequency of cells harboring the LM-CNV in fibroblasts is calculated assuming that frequency of such cells in hiPSCs is 100%, after normalizing event numbers for LM-CNVs by the control region. Counts of ddPCR events for the LM-CNV (green bars) and the control region (blue bars) allows estimating cell frequency in fibroblast of 12.6%. e, Duplication on chromosome 7 that was undetectable in parental fibroblasts by RD but detected as a faint band by PCR. f, This event had an estimated cell frequency in fibroblasts of 14.6% by ddPCR. g, Deletion on chromosome 8 that was undetectable in parental fibroblasts both by RD and PCR. h, This event had an estimated cell frequency in fibroblasts of 0.8% by ddPCR.