Fig. 1: Mutations from B-ALL patients cause CD19 mis-splicing. | Nature Communications

Fig. 1: Mutations from B-ALL patients cause CD19 mis-splicing.

From: High-throughput mutagenesis identifies mutations and RNA-binding proteins controlling CD19 splicing and CART-19 therapy resistance

Fig. 1

a Patient #4 shows increased CD19 intron 2 retention after CART-19 therapy relapse. Re-analysed RNA-seq data from Orlando et al.5. Selected isoforms (GENCODE) are shown. b Intron 2 retention increases in B-ALL patients after CART-19 therapy relapse. Intron 2 retention frequency (as % of all isoforms) is shown for nine patients with matched RNA-seq data at screening and after relapse. P value = 0.009, one-sided paired Wilcoxon signed-rank test. Grey lines connect matched samples. Boxes represent quartiles, centre lines denote 50th percentile, and whiskers extend to most extreme values within 1.5× interquartile range (IQR). c, d Intron2-retention is the predominant isoform in B-ALL patients and pre B cells. Stacked bar chart shows relative usage (percent selected index, PSI; left y axis) of junctions originating from exon 3 (Supplementary Fig. 1a) in 220 patients from the TARGET B-ALL programme (c) and normal B cells44, 75 (n = 21) (d). Black dots in d indicate total CD19 mRNA expression, in transcripts per million (TPM; right y axis). Cell lines NALM-6 and K562 are shown for comparison. e The CD19 minigene spans exons 1–3 and the intervening introns from the CD19 gene. f, g The minigene generates the same isoforms as the endogenous CD19 gene in NALM-6 cells. Gel-like representation (f) and quantification (g) of semiquantitative RT-PCR showing isoforms intron2-retention (blue), inclusion (grey) and skipping (turquoise) for the WT minigene in NALM-6 cells. Isoforms of CD19 gene in NALM-6 cells are shown for comparison. Asterisk indicates a previously reported RT-PCR artefact66 (see Methods). Error bars indicate standard deviation of mean (s.d.m.), n = 3 replicates. P value > 0.1 for all isoforms, one-way ANOVA. h, i Patient mutations cause splicing changes in the CD19 minigene. Top: Location of the tested mutations. Patient IDs as reported in Orlando et al.5. 14.1 and 14.2 correspond to distinct mutations from patient #14. Gel-like representation (h) and quantification (i) of semiquantitative RT-PCR as in f, g. Additional isoform alt-exon2 (purple) includes a truncated version of exon 2. Error bars indicate s.d.m., n = 3 replicates. *P value < 0.05, **P value < 0.01, ***P value < 0.001, two-sided Student’s t test. Source data including P values are provided as a Source Data file.

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