case_images: PMC4503302_01
case_images: Expression heatmaps of RNA analyzed from whole blood.. All genes are displayed that reach significance after correction for multiple testing FDR and have an FPKM above 1. Interesting dysregulated pathways and subpathways are: Regulation of apoptosis (p = 3.0 x 10-8), DNA replication, with affected sub-pathways regulation of DNA replication (p = 1.4 x 10-5), M/G1 transition (p = 5.2 x 10-5) and the synthesis of DNA (p = 2.1 x 10-4)). DNA repair is also dysregulated, especially the fanconi anemia pathway (Translocation of ub-FANCD2 and ub-FANCI to chromatin; p = 4.0 x 10-4 and regulation of the Fanconi anemia pathway 4.4 x 10-3). Also membrane trafficking is affected, especially subpathway Endosomal Sorting Complex Required For Transport (ESCRT) (p = 1.8 x10-5). The citric acid (TCA) cycle and respiratory electron transport pathway is significantly dysregulated (p = 9.3x10-6) and cellular responses to stress as well (p = 8.6 x 10-3). For signal transduction, especially signaling by Wnt is affected (p = 3.2 x 10-4).
case_images: pone.0131797.g001.jpg
case_images: PMC4503302_02_pone.0131797.g001.jpg
case_images: pone.0131797.g001
case_images: Sanger sequencing in the patient and parents. Studying mRNA extracted from the patient's whole blood revealed that both mutant and wild type CAV1 were transcribed (Fig B in S1 File), and that the RNA from the mutant allele was neither degraded by nonsense mediated RNA decay; nor was the second allele silenced by a regulatory mutation. Most significantly dysregulated pathways are: Translational regulation, RNA metabolism and protein metabolism and most interesting pathways are shown in Fig 1.
case_images: PMC4503302_02
case_images: Expression heatmaps of RNA analyzed from whole blood.. All genes are displayed that reach significance after correction for multiple testing FDR and have an FPKM above 1. Interesting dysregulated pathways and subpathways are: Regulation of apoptosis (p = 3.0 x 10-8), DNA replication, with affected sub-pathways regulation of DNA replication (p = 1.4 x 10-5), M/G1 transition (p = 5.2 x 10-5) and the synthesis of DNA (p = 2.1 x 10-4)). DNA repair is also dysregulated, especially the fanconi anemia pathway (Translocation of ub-FANCD2 and ub-FANCI to chromatin; p = 4.0 x 10-4 and regulation of the Fanconi anemia pathway 4.4 x 10-3). Also membrane trafficking is affected, especially subpathway Endosomal Sorting Complex Required For Transport (ESCRT) (p = 1.8 x10-5). The citric acid (TCA) cycle and respiratory electron transport pathway is significantly dysregulated (p = 9.3x10-6) and cellular responses to stress as well (p = 8.6 x 10-3). For signal transduction, especially signaling by Wnt is affected (p = 3.2 x 10-4).
case_images: pone.0131797.g001.jpg
case_images: PMC4503302_02_pone.0131797.g001.jpg
case_images: pone.0131797.g001
case_images: Sanger sequencing in the patient and parents. Studying mRNA extracted from the patient's whole blood revealed that both mutant and wild type CAV1 were transcribed (Fig B in S1 File), and that the RNA from the mutant allele was neither degraded by nonsense mediated RNA decay; nor was the second allele silenced by a regulatory mutation. Most significantly dysregulated pathways are: Translational regulation, RNA metabolism and protein metabolism and most interesting pathways are shown in Fig 1.
case_images: A. Expression bar plots of RNA analyzed from whole blood involved in other progeroid syndromes. B. AGPAT2, lipodystrophy. C. Known Cav1 pathways and D. Direct interaction partners. * Reach significance after correction for multiple testing (FDR). LPIN1 is not significantly different between case and controls. 95% FPKM cufflinks confidence intervals for estimates were obtained using a Bayesian inference method based on importance sampling from the posterior distribution.
case_images: pone.0131797.g002.jpg
case_images: PMC4503302_02_pone.0131797.g002.jpg
case_images: pone.0131797.g002
case_images: In addition, we looked at genes involved in other progeroid and lipodystrophy syndromes (Fig 2). Berardinelli-Seip Congenital Lipodystrophy (AGPAT2) is very significantly underexpressed (padj = 0.002; Fig 2). BSCL2, PTRF and LPIN1 lipodystrophy genes are not differentially expressed. Of these 102, 30 were significantly different between our case and the controls (29.4%) (Table B in S1 File), and 72 (73.3%) are down regulated (Fig 2). MAPK3 (p44) is underexpressed, and HCK and FGR are also significantly underexpressed (SRC-family tyrosine kinases) (Fig 2). In this pathway, TRADD, TRAF2, TNFRSF1A and TNFRSF1B are also significantly underexpressed (Fig 2).
case_images: A. western blot of lysates from case and control fibroblasts. Two antibodies were used, one that recognized an internal epitope in CAV1 (LS-B192), and one that recognized a C-terminal epitope (ORB47981; would only recognize WT CAV1). Beta-actin was used as a loading control. Calculation of intensity of the CAV1 isoform bands compared to the loading control, illustrated that WT expression is decreased in the case compared to the control. B: Immunocytochemistry of case and control fibroblast cells with anti-PRTF and anti-CAV1 antibodies. In the control, co-localization of CAV1 and PTRF (Caveolae marker) is very clear. In the proband's cells, this co-localization is less consistant. Green bar = 28um.
case_images: pone.0131797.g003.jpg
case_images: PMC4503302_02_pone.0131797.g003.jpg
case_images: pone.0131797.g003
case_images: All blots showed a significant decrease in expression of CAV1 compared to the control that was similar between an antibody that detected WT CAV1 only and an antibody detecting both WT + MT CAV1, suggesting MT CAV1 is not expressed or expressed at a very low level (Fig 3). In addition, fibroblasts were grown on glass coverslips, fixed and stained for localization of CAV1 and PTRF (Caveolae marker) (Fig 3). In the control, co-localization of CAV1 and the caveolae marker was perfect, however, the co-localization was not entirely fully concordant in the case (Fig 3).