GSEA of CD235⁻ cells. (A-I) Comparison of the transcriptome of normal CD41⁻/CD44⁺/CD235⁻ cells to those from GATA1- or RP-mutated DBA patient CD41⁻/CD44⁺/CD235⁻ cells, as well as differences in direct comparison of RP and GATA1 specimens, by GSEA. Genes are ranked by signal-to-noise ratio according to differential expression, with the position of genes comprising each set (heme, GATA1, snoRNA) illustrated by the vertical black bars below the plot in each comparison. Overrepresentation of genes from a set at the top (left) or bottom (right) of the ranked list indicates over- and underexpression and is illustrated by a peak (positive) or valley (negative) in the running enrichment score (green) shifted to the left or right, respectively. The gray line represents no change. The normalized enrichment score and false discovery rate q-value are shown for each comparison illustrated. Evaluation of heme synthesis genes (from the MSigDB hallmark set) demonstrated (A) modest downregulation in GATA1-mutated samples when compared with normal controls that was not statistically significant but showed significant overexpression in RP-mediated DBA in comparison with normal controls (D) and GATA1-mutated patients (G). Patients with GATA1 mutations did not show underexpression of any tested GATA1 gene set (B; GATA1 TRANSFAC set from Ref. ²⁶  illustrated), whereas patients with RP mutations showed marked and statistically significant GATA1 target gene overexpression in comparison with both normal controls and patients harboring GATA1 mutations (E, H; GATA1 GSE628 set from Ref. ²⁶  illustrated). Similarly, evaluation of small nucleolar RNA transcription (gene set derived from all snoRNA annotated on the array) showed highly significant downregulation in patients with GATA1 mutations in comparison with normal controls (A) and DBA patients with RP mutations (I), but no significant changes in expression of RP-mediated DBA patients when compared with normal controls (F). NES, normalized enrichment score.