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  • Several possibilities could explain the aberrant expression

    2018-11-09

    Several possibilities could explain the aberrant expression of ATM in Q3SC. Exon skipping has been observed in several genes (Dutertre et al., 2010; Nevo et al., 2012) and is believed to be influenced by ATM function (Tresini et al., 2015). By amplifying cDNA with PCR primers targeting sites outside of exons which contain mutations in Q3 (sequences found in Table S1), only full-length product was identified, providing no evidence for mRNA skipping those exons (Figure 1G). We also counted the frequencies of sequenced, cloned PCR products corresponding to each allele and found only the c.7792 C > T allele mixed with wild-type sequence in Q3SC: there was no evidence of the c.217_218 delGA allele in the mRNA, as was found in CAR3 (Figure 1H). This would be consistent with gene reversion, because a simple deletion could not generate a wild-type allele. The mutation sites were directly examined by sequencing of crude PCR-amplified segments of genome. As shown in Figure 2A, Q3SA exhibits the mutations identified in the diagnostic genotyping originally provided for subject JHU_Q3 (Figure 1A). Each mutated location was clearly heterozygous in Q3SA, as evidenced by the mixed trace from the unpurified PCR product (highlighted in yellow). CAR3 exhibited the c.217_218 pkc pathway delGA mutation, which is consistent with a parental relationship to JHU_Q3. Q3SC, on the other hand, is missing the c.217_218 delGA mutation, because only wild-type sequence is visible in the PCR products. The c.7792 C > T mutation is present in both Q3SA and Q3SC. The same pattern of mutations appears in sequenced, PCR-amplified segments of cDNA (not shown), suggesting that both pkc pathway are transcribed. These results predict that Q3SC carries one allele capable of encoding a full-length ATM protein, while Q3SA does not. Because the ATM western blot (Figure 1D) appears to show different quantities of ATM protein at different passage numbers of Q3SC, we wondered if early passage cells might have a different genotype from later passage cells. Early passage (P4) DNA prepared from Q3SC exhibited c.217_218 delGA in one allele, matching Q3SA and the diagnostic genotype for the JHU_Q3 subject, but later passage cells (P10) lacked this mutation (Figure 2B). This suggests that the Q3SC iPSC culture may have acquired a spontaneous gene reversion during early passaging. Alternatively, this culture could have suffered from cross-contamination. The latter seems unlikely, because the ATM mutation genotype for Q3SC does not match the maternal heterozygous carrier that we had thawed and reprogrammed (CAR3) but instead matches the imputed genotype and gender of cells from the father (CAR4), from whom we stored blood samples that have never been thawed or cultured in our laboratory. To rule out the possibility of cross-contamination, we evaluated large numbers of genomic variations using SNP arrays. Clustering with the Euclidean distance between called SNPs, DNA from both Q3SA and Q3SC are quite similar to DNA prepared from the amplified JHU_Q3 T cells originally used for reprogramming (Q3SA: 699,368 SNPs match JHU_Q3 out of 701,230 called SNPs [99.73%]; Q3SC: 99.85%; Figure 2C). Less closely related is the DNA from the parent CAR3 (71.95%), the unrelated Q1SA iPSC line (55.65%), or its source JHU_Q1 T cells (55.64%). This confirms that Q3SA and Q3SC are iPSC lines made from the same source individual (JHU_Q3), who is related to JHU_CAR3, but not to the source T cells or iPSC prepared from JHU_Q1. Furthermore, on the basis of SNPs, Q3SA and Q3SC are confirmed as male and both Q1SA and CAR3 are confirmed as female, consistent with designated cell sources. SNPs overlapping those recommended for use in cell line identification (Yu et al., 2015) are shown in Table S2. The ATM-expressing Q3SC, then, is not due to cross-contamination of cultures. We then searched for evidence of sequence variation in Q3SC. The karyotype of Q3SC was normal (Figure 2D; 46,XY in all 20 cells counted). SNP data were evaluated for evidence of loss of heterozygosity (LOH). Plots of the B allele frequency over the entire genome (not shown) or over the region of chr11 surrounding the ATM gene (Figure S2A) show no clear evidence for LOH overall but a lack of any heterozygous SNPs within the ATM gene, precluding the ability to assess LOH within the gene itself. However, the presence of SNP heterozygosity flanking ATM in all cell lines argues against a single crossing-over event between alleles, as this would have produced LOH distal from the event. By highlighting the 1,034 SNPs that distinguish Q3SA or Q3SC from source T cells (Figure S2B, colored dots) it is apparent that there is no concentrated genomic region of LOH between these two sublines, but that sequence variation has accumulated throughout the genome. Similar plots of the signal intensity of SNPs (log R ratio, not shown) show little evidence for copy-number variation (CNV) near the ATM gene. Not all known SNPs are included on the SNP array, so a 3.5 kb region surrounding the c.217_218 delGA site was re-sequenced. Only one SNP, rs2066734, was found to be heterozygous in Q3SA (red dot, Figure 2E). This SNP was homozygous for the major allele in Q3SC (green) and homozygous for the minor allele in CAR3 (blue). By estimating the rate of random SNP LOH using the SNP array data, a Fisher’s exact test indicates that the probability of finding a specific LOH for rs2066734 within ATM but unrelated to the loss of c.217_218 delGA is p = 0.00014. This indicates that the most likely source of reversion was a short gene correction event, including at least ∼1.5 kb upstream of c.217_218 delGA to include rs2066734. This result also confirms that the maternal allele, containing the c.217_218 delGA deletion as well as the minor rs2066734 allele, is lost in Q3SC and is replaced by a copy of the paternal allele sequence, explaining the absence of mutation at this position. Because mutation at c.7792 is heterozygous, reversion of c.217_218 delGA to wild-type sequence would be sufficient to produce a genotype of ATM+/−.