Fig. 2: Individuals with RNU4-2 variants have systematic changes in 5′ splice-site use.
From: De novo variants in the RNU4-2 snRNA cause a frequent neurodevelopmental syndrome
a, Boxplots of the number of abnormal splicing events (detected by FRASER2, ref. 40) at unannotated 5′ splice sites. The individuals with RNU4-2 variants (n = 5 individuals) have significantly more outlier events than both controls with non-NDD phenotypes (n = 378 individuals) and controls matched on genetic ancestry, sex and age at consent (n = 10 individuals, two per case; Wilcoxon P = 4.0 × 10−5 (W test statistic, 1,766) and P = 5.7 × 10−3 (W test statistic, 45.5), respectively). Centre line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; maxima and minima represented as points. FDR, false discovery rate. b, The distribution of the number of abnormal splicing events at unannotated 5′ splice sites shared between two or more out of five randomly selected control individuals over 10,000 permutations (grey histogram). The number of shared events in individuals with RNU4-2 variants is indicated as a dotted teal vertical line (n = 11). c, DNA sequence motifs around 5′ splice sites with increased and decreased use in individuals with RNU4-2 variants. Each plot shows the proportion of sites with each base at each position. 5′ splice sites with increased use (top) have an increase in T at the +3 position (eight out of 19 versus zero out of 36; Fisher’s P = 6.2 × 10−5; OR = Inf; 95%CI: 5.92-Inf) and an increase in C at the +4 (four out of 19 versus zero out of 36; Fisher’s P = 0.011; OR = Inf; 95%CI: 1.37-Inf) and +5 (six out of 19 versus 1/36; Fisher’s P = 0.0051; OR = 15.3; 95%CI: 2.09-Inf)) positions compared to decreased 5′ splice sites (bottom). The consensus sequence at 5′ splice sites in matched annotation from NCBI and EMBL-EBI (MANE) transcripts41 is shown in Supplementary Fig. 4. d, The structure of the U6 snRNA paired with the 5′ splice site after 5′ splice-site transfer. The three bases of the U6 ACAGAGA that directly pair with the 5′ splice site are shown in pink. The paired positions of the 5′ splice site (5′SS) are shown in green (A + 3 and A + 4) and yellow (G + 5). Statistical tests in a and c are one-sided with unadjusted P values.
