Fig. 6: RNA-seq identifies an alternative 5′SS signature that differentiates severe from mild RNU4-2-related phenotypes.

a, PCA based on PSI values from 111 significant 5′SS events detected using rMATS, comparing 19 patients with RNU4-2 variants (6 mildly affected in teal and 13 severely affected in red) to 21 controls (purple). Triangles, n.64_65insT; circles, other variants. Yellow symbols correspond to three test samples—one variant of uncertain significance (VUS; n.45_46insT), one VUS that could be reclassified as LP and the recurrent variant n.64_65insT from a patient with a milder phenotype. b, Box plot showing raw spliceAI scores of the decreased 5′SS site and the increased 5′SS for the 50 events shared between mild (n = 6) and severe individuals (n = 13) and the 19 events only detected in severe individuals. SpliceAI scores for severe and shared 5′SS were not statistically different for decreased sites (P = 0.476) but were significant for increased sites (P = 0.014) using the two-sided Mann–Whitney U test. Box plot elements are defined as follows: centerline, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. c,d, Sashimi plots showing isoform shifts in MAP4K4 (c) and AKNA (d). Aggregated coverage and splicing-supporting reads from patients with RNU4-2 variants with mild (n.75C>G, n.76C>T and n.72_73del; n = 6) or severe (n.64_65insT, n.67A>G, n.68A>C and n.70T>C; n = 13) phenotypes and controls (n = 21) are shown. The MAP4K4 event is present in both patient groups, while the AKNA abnormality appears only in severe cases. e, Consensus nucleotide sequence of decreased and increased 5′SS for 50 shared events (left) and for the 19 severe-only events (right), in comparison to the consensus sequence of all 5′SS from MANE transcripts (top).