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. 2024 Apr 4;111(4):714–728. doi: 10.1016/j.ajhg.2024.03.004

Figure 2.

Figure 2

Rescue of the metabolic phenotype after ABE-mediated edition of the ASLD c.1153C>T variant

(A) Urea cycle diagram.

(B) mRNA levels of ASL. Representative mRNA samples from different individuals, differentiation batches, stages (day 0, 7, 13, and 18), and with different genotypes (control = HEL24.3, edited, and not edited) were analyzed by qPCR. The mRNA levels are expressed in fold change and normalized to the HepG2 commercial hepatocarcinoma cell line (illustrated with a yellow line at the fold change 1 on the y axis). Each point represents an independent hiPSC line, which we consider a biological replicate. Data are represented as the mean ± SEM. Statistical significance based on Tukey test; p > 0.05 (ns, not significant), p < 0.05 (∗), p < 0.01 (∗∗), p < 0.001 (∗∗∗), p < 0.0001 (∗∗∗∗).

(C and D) The sum of the absolute abundance of all the metabolites detected by LC-MS in the cell lysate (C) and the media (D). Each shape represents independent differentiation batches (circle, square, diamond, triangle). We employed day-18 hiPSC-derived hepatocyte-like cells from two different individuals. We analyzed two independently edited hiPSC lines per proband (four biological replicates), two not edited independent hiPSC lines per proband (four biological replicates), and HEL24.3 as a control (two biological replicates). We processed five technical replicates of each sample in the LC-MS. The sum of the absolute abundance of all the metabolites in each sample was employed as a normalization to calculate the relative abundance of individual metabolites. Data are represented as the mean ± SEM. Statistical significance based on Tukey test; p > 0.05 (ns, not significant), p < 0.05 (∗), p < 0.01 (∗∗), p < 0.001 (∗∗∗), p < 0.0001 (∗∗∗∗).

(E–H) Relative abundance of intracellular and media ASA (E and F) and citrulline (G and H) detected by LC-MS in the same samples described in the previous graph. Each shape represents independent differentiation batches (circle, square, diamond, triangle). As expected, in some of the control and edited samples, the ASA levels were below the detection limit of the mass spectrometer. We did not consider these values for the graph bar or the statistical analysis, but we illustrated these cases with a shape containing a black center. Relative abundance is the absolute abundance value normalized to the sum of all metabolites. Data are represented as the mean ± SEM. Statistical significance based on Tukey test; p > 0.05 (ns, not significant), p < 0.05 (∗), p < 0.01 (∗∗), p < 0.001 (∗∗∗), p < 0.0001 (∗∗∗∗).