Noncoding NOTCH1 mutations activate aberrant splicing in patients with CLL. (A) Scheme of NOTCH1 3′UTR splicing-specific PCR assays. The different NOTCH1 transcripts from unspliced process (746 base pairs [bp]) and alternative splicing (∼200 bp) can be distinguished. (B) PBMCs from healthy adults revealed alternatively spliced NOTCH1 141 variant (AG/AAAT) ∼200 bp in addition to unspliced NOTCH1 wt ∼750 bp. Patients 1 and 2 with NOTCH1 152 A>G and NOTCH1 145 A>G mutations had stronger spliced signals of ∼200 bp with barely seen unspliced signals of ∼750 bp, verified by the sequencing data with NOTCH1 152 (AG/AATC) and NOTCH1 145 (AG/AAAG) specific boundary sequence. Interestingly, patient 3 and patient 4 with NOTCH1 143∗ A>C were uncovered with a spliced NOTCH1 141 variant harboring specific boundary sequence (AG/AAAT) verified by sequencing data of patient 3 in addition to relatively weaker unspliced transcripts of ∼750 bp. (C) Quantification of splicing events in patients with NOTCH1 152-mutated CLL. Scheme of quantitative PCR primer pairs amplifying unspliced and spliced NOTCH1 transcripts (left). Relative expression of unspliced and spliced NOTCH1 mRNA in patients with CLL without (wt) or with NOTCH1 152 mutation (right). Expression is normalized to 3 housekeeping genes and log2 transformed. n > 7; median; 2-way analysis of variance (ANOVA) followed by Šídák multiple comparison test; P values are shown. GRCh37.p13, Genome Reference Consortium Human Build 37 patch release 13.