Figure 2: RNA aberrant expression detection and validation.
From: Genetic diagnosis of Mendelian disorders via RNA sequencing
(a) Aberrantly expressed genes (Hochberg corrected P value<0.05 and |Z-score|>3) for each patient fibroblasts. (b) Gene-wise RNA expression volcano plot of nominal P values (−log10 P value) against Z-scores of the patient #35791 compared against all other fibroblasts. Z-scores with absolute value >5 are plotted at ±5, respectively. (c) Same as (b) for patient #73804. (d) Sample-wise RNA expression is ranked for the genes TIMMDC1 (top) and MGST1 (bottom). Samples with aberrant expression for the corresponding gene are highlighted in red (#35791, #66744, and #73804). (e) Gene-wise comparison of RNA and protein fold changes of patient #35791 compared to the average across the fibroblast cell lines of all other patients. Subunits of the mitochondrial respiratory chain complex I are highlighted (red squares). Reliably detected proteins that were not detected in this sample are shown separately with their corresponding RNA fold changes (points below solid horizontal line). (f) Western blot of TIMMDC1, NDUFA13, NDUFB3 and NDUFB8 protein in three fibroblast cell lines without (#62346, #91324, NHDF) and three with a variant in TIMMDC1 (#35791, #66744 and #96687), and fibroblasts re-expressing TIMMDC1 (‘-T’) (#35791-T, #66744-T and #96687-T). UQCRC2 was used as loading control. CI, complex I subunit; CIII, complex III subunit; MW, molecular weight. (g) Blue native PAGE blot of the control fibroblasts re-expressing TIMMDC1 (NHDF-T), the control fibroblasts (NHDF), patient fibroblasts (#96687) and patient fibroblast re-expressing TIMMDC1 (#96687-T). Immunodecoration for complex I and complex III was performed using NDUFB8 and UQCRC2 antibodies, respectively. CI, complex I subunit; CIII, complex III subunit.
