Figure 4: Summary of RNA-seq findings.

(a) PIR for major (grey) and minor (orange) introns, for kindred 1 and kindred 2 subjects, showing specific minor intron retention in affected subjects compared with unaffected subjects. (b) Affected–unaffected average difference in percentage alternative splicing (dPSI) for different alternative splicing categories (ALTA, alternative 5′ splice site; ALTD, alternative 3′ splice site; EX, cassette exon; INT, major intron retention; INTmin: minor intron retention; MIC, micro-exon); significant deviation from 0 is observed only for major introns (small yet significant reduction in affected) and minor introns (significant increase in affected). (c) Log2 of the mean expression ratio between affected and unaffected, for minor intron genes and all other genes, displaying a slight shift of minor intron genes towards higher expression in affected subjects. (d) Scatterplot showing that increased expression in affected relative to unaffected subjects (y axis, log2 ratio of the mean expression in the two groups) is insufficient to compensate for the increased minor intron retention (x axis, log2 ratio of the mean correctly spliced fraction in the two groups), with 30% of the genes over-compensated and 70% of the genes under-compensated (separated by the red dashed line); the linear correlation between the expression ratio and correctly spliced ratio is negative but modest (grey dashed line).