Spontaneous ATM Gene Reversion in A-T iPSC to Produce an Isogenic Cell Line

Abstract

A spontaneously reverted iPSC line was identified from an A-T subject with heterozygous ATM truncation mutations. The reverted iPSC line expressed ATM protein and was capable of radiation-induced phosphorylation of CHK2 and H2A.X. Genome-wide SNP analysis confirmed a match to source T cells and also to a distinct, non-reverted iPSC line from the same subject. Rearranged T cell receptor sequences predict that the iPSC culture originated as several independently reprogrammed cells that resolved into a single major clone, suggesting that gene correction likely occurred early in the reprogramming process. Gene expression analysis comparing ATM(-/-) iPSC lines to unrelated ATM(+/-) cells identifies a large number of differences, but comparing only the isogenic pair of A-T iPSC lines reveals that the primary pathway affected by loss of ATM is a diminished expression of p53-related mRNAs. Gene reversion in culture, although likely a rare event, provided a novel, reverted cell line for studying ATM function.

Figure 1

A-T iPSC Characterization (A) Subject and iPSC line information. Six subjects were collected from the Johns Hopkins University Medical Center A-T Clinic with the characteristics shown. The names of iPSC lines derived from each subject are listed separately from source subject codes. ^∗Subject samples used for iPSC reprogramming. The predicted patterns of ATM translation from each allele are shown for JHU_Q1 and JHU_Q3. (B) Pluripotency markers in iPSC cultures. Example immunocytochemical images for each iPSC line showing nuclear OCT4 (green) and cell surface TRA-1-60 (red) staining. The scale bar represents 10 μm. (C) Gene expression analysis (RNA-seq) results were clustered to show expression patterns consistent with pluripotency. Samples of unrelated iPSC, hESC-derived NSC at 0 or 5 days of neuronal differentiation (NSC0 and NSC5), and iPSC-derived midbrain-like DAN were included for comparison. NSC0, NSC5, DAN, and iPSC, n = 3; Q1SA, Q3SA, and Q3SC, n = 2. (D) Western blot to detect ATM protein expression in Q3-specific iPSC sublines. See also Figures S1A and S1B. Results were replicated in three western blots. (E) Western blot to detect XR-induced pCHK2. Cultures were pre-treated with 10 μM KU or DMSO vehicle 1 hr prior to irradiation. For both NR and XR blots, the white space separates two regions from the same blot. Results were replicated in two western blots. (F) Immunocytochemical detection of γH2A.X nuclear puncta induced by XR. Controls (NR and XR with/without KU inhibition) are shown in Figure S1C. The scale bar represents 10 μm. (G) Assays to detect exon skipping in ATM mRNA. PCR primers were designed for exons upstream or downstream of mutations in JHU_Q3. The predicted product for skipping Exon 4 would be 187 bp and intact Exon 4 would be 333 bp. Only the latter size product was detected. Skipping Exon 53 would produce a 95 bp product, which was not observed. (H) Allele frequencies in ATM mRNA. PCR products including the mutation sites were prepared from cDNA, cloned into plasmids to isolate individual molecules, and sequenced. The p value indicates the probability of observing these frequencies compared with an expected 50:50 ratio, according to chi-square analysis. The observed numbers of sequences were CAR3 c.216-217 delAG = 3, wild-type = 4; Q3SC c.216-217 delAG = 0, and c.7792 C > T = 8.

Figure 2

Genetic Analysis of A-T iPSC (A) Sequencing traces of unpurified genomic PCR products. Homozygous sequences appear as a single pattern, and heterozygous sequences show a mixture of the two alleles distal from the point of difference (highlighted in yellow). The presence of the c.217_218 delGA deletion is visible as multiple overlapping traces in Q3SA and CAR3. The c.7792 C > T substitution is visible in Q3SA and Q3SC. Other samples were wild-type (similar to unrelated SC1) at these locations. (B) Loss of the c.217_218 delGA variation over passaging of Q3SC. DNA samples from passage 4 (P4) or 10 (P10) identified the presence of this variation at P4 but not at P10. (C) Hierarchical SNP clustering demonstrates genetic relationships among iPSC from genome-wide SNP arrays. Clustering used Euclidean distance by substituting 1 for AA, 0 for AB, and −1 for BB. Also indicated are the imputed genders based on SNPs. (D) Karyotype of Q3SC. A representative chromosomal spread is shown for Q3SC, which was found to be 46 XY in 20 of 20 cells. (E) Map of SNPs detected by re-sequencing genomic DNA near c.217_218 delGA. The positions of the deletion and the one SNP exhibiting LOH are identified by arrows.

Figure 3

Genetic Variation in A-T iPSC (A) FISH analysis identifies similar proportions of cells with normal, “SD” (unequal number of puncta for ATM compared with chr11 centromere), and “AP” (aneuploid) in CAR3, Q1SA, Q3SA, Q3SC, or unrelated control (SC1). Example images are shown for comparison (green, ATM; red, chr11 centromere). The scale bar represents 10 μm. Cells classified as “AP” are likely apoptotic. Total cells counted: SC1, n = 111; CAR3, n = 131; Q3SA, n = 123; Q3SC, n = 132; and Q1SA, n = 100. (B) XR-induced pATM in iPSC following mutagenesis. Cultures were treated with 1 Gy XR once to induce mutagenesis and then again 1 week later to activate ATM autophosphorylation. The fraction of XR-induced pATM-positive nuclei is plotted for each iPSC line, with XR or without (NR) stimulation (n = 4 wells). This assay was repeated with cells from a different passage number with essentially identical results (not shown). (C) Examples of immunocytochemically detected pATM and γH2A.X puncta are identified with arrows. The scale bar represents 10 μm. (D) Clustering of PCR-amplified TCRβ junction sequences from various passages of Q3SC. The vertical dashed line cleaves the dendrogram into three clusters, labeled A, B, and C. Sequence alignments matching this dendrogram are shown in Figure S3A. As a comparison, clustering samples from several iPSC lines show distinct patterns generally specific for each line (Figure S3B). (E) Interpretation of cluster sequence frequencies calculated from results in (D).

Figure 4

Gene Expression Analysis in A-T iPSC (A) Volcano plot comparing changes in transcript (as log₂ fold change of fragments per kilobase of transcript per million mapped reads) and significance (−log₁₀ of the FDR) for Q3SA versus Q3SC. Sample names are color-coded by genotype: red for ATM^−/− and blue for ATM^+/−. The significance threshold of 5% FDR is shown as a horizontal dashed line (n = 2 per group). Smaller gray dots indicate transcripts with no significant difference. Colors of dots indicate whether transcripts overlap Q1SA versus Q3SC or not: see key and the colors of overlapping regions in the Venn diagram (C) for color interpretation. (B) Volcano plot comparing Q1SA with Q3SC, with dot coloring to show overlap with Q1SA versus Q3SC. (C) Venn diagram indicating the numbers of significantly different transcripts in each contrast and the numbers of overlaps among the contrasts. (D) Pathway analysis of transcripts that are decreased in abundance in Q3SA compared with Q3SC (top) or increased (bottom). The bar indicates the p value calculated by Fisher’s exact test, and the color intensity depicts the ratio of pathway genes included in this differentially expressed set.