HPAEC

Differential Gene Expression in Pulmonary Artery Endothelial Cells Exposed to Sickle Cell Plasma

Elizabeth S. Klings1, Surinder Safaya2, Adeboye H. Adewoye2, Adam Odhiambo1, Garrett Frampton3, Marc Lenburg3, Norman Gerry3, Paola Sebastiani4, Martin H. Steinberg2, Harrison W. Farber1


1–The Pulmonary Center,
2–Division of Hematology/Oncology, Department of Medicine,
3–Department of Genetics/Genomics, Boston University School of Medicine,
4–Department of Biostatistics, Boston University School of Public Health, Boston, MA 02118.

 Supplementary material

  • Original manuscript: Under revision. Contact the corresponding author Elizabeth S. Klings.
  • Data: The original data with a description of the experimental protocol are available from Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE1849)
  • Brief description of the analysis: The quality of gene expression data was assessed by evaluating the reproducibility of repeated samples. Genes expression were annotated by the percentage of absent calls and analyzed with BADGE (Bayesian anaysis of differential gene expression) to identify genes with evidence of differential expression (posterior probability that the fold exceeds 1 greater than 0.999 or smaller than 0.001). The robustness of these genes was further assessed by extrinsic leave-one-out cross validation (ECV), and genes that were retained at least 70% of time in ECV (predictive-score) were selected. These genes were further annotated by the % of absent calls. With this analysis we finally selected as reliable only those genes that had a predictive score larger than 70% and more than 40% present calls across each pair-wise comparison. 
     
  • Results of individual comparisons: 

    • Gene expression of HPAEC incubated with plasma from SCD patients at steady state versus HPAEC incubated with plasma from normal volunteers.
      • Table A.1: Initial selection of 642 genes.
      • Table A.2: Refined list of genes, with annotation. Highlighted in yellow are the genes that are not very reliable because the percentage of absent calls is above 50%. The excel file contains the annotation from Unchip and NetAffx.
      • Figure 1: Heatmap of the 56 probes (50 genes with 4 that appear in duplicates and one that appears in triplicates) for which there is very strong evidence of differential expression. The first 9 columns are the gene expression of HPAEC incubated with plasma from SCD subjects. Red cells denote expression values that are above the average and green cells denote expression values that are below the average, so the first 22 rows (20 genes) are genes that are upregulated in SCD and the last 34 rows (30 genes) are genes that are downregulated in SCD.  
    • Gene expression of HPAEC incubated with plasma from sickle ACS patients versus HPAEC incubated with plasma from SCD patients at steady state.
      • Table A.4: Refined list of genes, with annotation.
      • Figure 2: Heatmap of the 62 probes (58 genes with 4 that appear in duplicates) for which there is very strong evidence of differential expression. The first 9 columns are the gene expression of HPAEC incubated with plasma from ACS subjects. Red cells denote expression values that are above the avarege and green cell denote expression values that are below the average, so the first 37 rows (35 genes) are genes that are upregulated in ACS and the last 25 rows (23 genes) are genes that are downregulated in ACS.  
    • Gene expression of HPAEC incubated with plasma from sickle ACS patients versus HPAEC incubated with plasma from normal volunteers.
      • Table A.5: Initial selection of 249 genes.
      • Table A.6: Refined list of genes, with annotation.
      • Figure 3: Heatmap of the 65 probes (53 genes with 4 that appear in duplicates and 4 that appears in triplicates) for which there is very strong evidence of differential expression. The first 9 columns are the gene expression of HPAEC incubated with plasma from ACS subjects. Red cells denote expression values that are above the avarege and green cell denote expression values that are below the average, so the first 24 rows (21 genes) are genes that are upregulated in ACS and the last 41 rows (32 genes) are genes that are downregulated in ACS. 
         
  • Overall Summary:
    • Table A.7: 24 genes change between HPAEC incubated with plasma from SCD patients at steady state versus HPAEC incubated with plasma from normal volunteers (column SCD2norm), between HPAEC incubated with plasma from sickle ACS patients versus HPAEC incubated with plasma from SCD patients at steady state (column ACS2SCD), and between HPAEC incubated with plasma from sickle ACS patients versus versus HPAEC incubated with plasma from normal volunteers (column ACS2norm).
  • Gender specific genes:
    •  Because the majority of normal and SCD patients were females, we tested whether some of the selected genes were “gender-specific”. To this end, we created a fictitious data set comprising the expression of genes in HPAEC incubated with plasma from normal, SCD and sickle ACS patients in 9 females and 9 males, and then searched for genes that were differentially expressed between males and females
    • Badge Report
    • None of the “gender-specific” genes appears in any of the three comparisons above.  
  • Table A.8 (Matched Samples): We performed an additional analysis of data derived when plasma samples were obtained from the same individuals (n=2) at steady-state and during ACS. This analysis demonstrated that 914 genes changed expression by at least 1.5 fold. Twenty five of the high intensity genes identified in our prior groups were differentially expressed in these samples as well, confirming the reliability of our findings.

updated 8.28.09