Because Q SA and Q SC represent an
Because Q3SA and Q3SC represent an isogenic pair of iPSC lines from the same individual but with contrasting ATM genotypes (ATM−/− versus ATM+/−, respectively), we examined gene expression differences using RNA sequencing (RNA-seq), evaluating matches at the transcript level. Relatively few mRNAs were significantly different in the isogenic Q3 pair (206, false discovery rate [FDR] ≤ 5%), as contrasted with a much larger number of differences between Q1SA and Q3SC (1,349), even though both contrasts compared ATM−/− with ATM+/− (Figures 4A–4C). This indicates that observed gene expression differences due to variations in genetic backgrounds generally overwhelm any analysis of single-gene, variant-specific differences. Out of the 206 transcripts different in the isogenic pair of cell lines (Table S3), 113 (54.8%) also differed between Q1SA and Q3SC (Figure 4A). On the other hand, a large proportion (601 of 1,349 [44.6%]) of transcripts differing between Q1SA and Q3SC were in common with the Q1SA versus Q3SA comparison (Figure 4B), even though the latter compared two different ATM−/− cell lines. Nayler et al. (2012) identified 7,920 genes as significantly different in A-T iPSC compared with wild-type EPI-001 using the same selection criteria. This list translated to 7,402 unique RefSeq transcript identifiers, and only 50 of these overlapped with the 206 Q3SA versus Q3SC transcripts found here. Three hundred sixty-four of Nayler’s selected transcripts overlapped with the broader Q1SA versus Q3SC list, again indicating that comparisons between isogenic pairs are distinct from those between cell lines with different genetic backgrounds.
Looking to functional characterization of the transcripts selected as diminished in Q3SA compared with Q3SC, the top enriched pathway was p53 signaling (p = 1.63 × 10−4; Figure 4D). This is reinforced by upstream analysis, in which TP53 mRNA was reduced ∼24-fold in the absence of ATM, and 18 of 19 genes predicted to be transcriptionally regulated by p53 were affected (p = 3.46 × 10−5). Other pathways enriched by the reduced transcripts included IGF-1 signaling, acute phase response signaling, and the Wnt/Ca+2 pathway (Figure 4D). Few pathways were enriched in the list of transcripts increased in Q3SA compared with Q3SC, and these had no clear biological interpretation (Figure 4D). Under normal conditions in the absence of ATM stimuli such as double-stranded breaks, the primary cellular effect is a reduction in p53 signaling.
Discussion Spontaneous genetic variation has been described in cultured cells almost from the beginning of genetic analysis in cell culture. In one example, published in 1968, Atkins and Gartler proved that the mutation rate in culture is the same whether selection is applied or not (Atkins and Gartler, 1968). However, there has been relatively little report of spontaneous reversion in iPSC created from genetic variant cells, except for cases in which naturally occurring reversion of cultured primary cells have been reprogrammed into iPSC (Tolar et al., 2014; Maclean et al., 2012). Careful studies demonstrate that much of the variation between iPSC lines may be attributed to genetic variation in source skin fibroblasts (Abyzov et al., 2012) and that skin cells accumulate mutations in situ (Martincorena et al., 2015). Our results show that cultured iPSC have the capacity to undergo genetic rearrangement, in this case a reversion from ATM−/− to ATM+/−. All available evidence, including at least the 1.4 kb spanning the c.217_218 delGA position to the location of rs2066734, supports the conclusion that gene conversion of ATM occurred, replacing the maternal allele with a copy of the paternal allele to create an allele encoding full-length ATM protein capable of phosphorylating pCHK2 and γH2A.X upon XR. This likely occurred between initial reprogramming of the iPSC line (P0) and P10, as DNA prepared from P4 had not reverted, or at least not detectably. One puzzling aspect was that the ATM protein levels detected by western blotting appeared to change over passaging (Figure 1D). It is not clear if this is meaningful in the context of our study, as ATM protein remained detectable after the reversion event, even if levels were reduced. Alternatively, ATM gene reversion could have occurred in circulating T cells prior to reprogramming. Even if this were the case, Figure S2B demonstrates that independent SNP variants accumulate after reprogramming, proving that iPSC cultures accumulate genetic variants during culture.