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Open Access Research article

Evidence of perturbations of cell cycle and DNA repair pathways as a consequence of human and murine NF1-haploinsufficiency

Alexander Pemov1, Caroline Park2, Karlyne M Reilly3 and Douglas R Stewart1*

Author Affiliations

1 Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, 20892, USA

2 Albert Einstein College of Medicine, Bronx, New York, 10461, USA

3 National Cancer Institute at Frederick, Fort Detrick Building 50, Frederick, Maryland, 21702, USA

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BMC Genomics 2010, 11:194  doi:10.1186/1471-2164-11-194

Published: 22 March 2010

Additional files

Additional file 1:

Intersection analysis of Coriell-18, ECACC-6 and Nf1 -Mouse-12 expression datasets. The most differentially expressed genes (top 5 per cent) from the human and mouse sets were used for intersection analysis. The results of a pair-wise and three-way intersection analysis are shown. See also Figure 3.

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Additional file 2:

Comparison of fold difference values of the most differentially expressed genes on Illumina and NHGRI microarrays. Samples from Coriell-18 set were analyzed in parallel on Illumina and NHGRI microarrays. Intersection analysis of the most differentially expressed transcripts from two platforms was performed, and the expression fold difference (Affecteds vs. Unaffecteds) was calculated for each transcript found in the overlap between the two platforms. See also Figure 4.

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Additional file 3:

Quantitative PCR validation of microarray data for select human and mouse genes. Twelve human genes were subject to qPCR validation in the human Coriell-18 and ECACC-6 sets. Ten mouse genes were subject to qPCR validation in the murine Nf1-Mouse-12 set. Both microarray and qPCR expression values in NF1-affecteds and Nf1+/- mice were normalized to expression values in NF1-unaffecteds or wild-type mice, respectively. Expression values in NF1-unaffecteds and wild-type mice were arbitrary set at 1.0. Red bars denote mean expression in NF1-affecteds on microarrays, blue bars denote mean expression in NF1-unaffecteds on microarrays; orange bars denote mean expression in NF1-affecteds by qPCR; and green bars denote mean expression in NF1-unaffecteds by qPCR. Gene names are shown below each set of bars. Sample set names are shown on top of each plot. Error bars are equal to one standard deviation. Asterisks above bars denote genes validated by qPCR (nominal P value < 0.05).

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Additional file 4:

Leading edge analysis identifies transcripts that are most frequently present in the significant gene sets. Leading edge analysis of significant GSEA gene sets was performed, and the transcripts were ranked by the total number of times they are present in statistically significant gene sets. See also Figure 5.

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Additional file 5:

Ontological annotation of transcripts identified by leading edge analysis. Up- and down-regulated transcripts identified by leading edge analysis were ontologically annotated, and similar or related categories were combined. The resulting categories were ranked by the number of transcripts included in each category.

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Additional file 6:

Ontological analysis of differentially expressed genes with Onto-Express. Functional profiles based on Gene Ontology (GO) biological processes terms were created for up- and down-regulated transcripts in two human and a mouse sample sets. For each set of transcripts, statistically significant ontological categories were determined by the hypergeometric test, followed by a multiple testing correction procedure (FDR). Only categories with FDR below the threshold (0.05) are shown. Note that there are no significant categories in the lists of down-regulated transcripts in ECACC-6 and Nf1-Mouse-12 sets.

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Additional file 7:

Pathway level analysis of differentially expressed genes with Pathway-Express. Differentially expressed genes in two human and a mouse sample sets were compared to known cellular pathways in the KEGG database, followed by impact factor computation and FDR correction. Statistically significant pathways (FDR < 0.05) and their impact factors (IF) are shown for each sample set.

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Additional file 8:

Ontological analysis of differentially expressed genes with MetaCore tools. Ontological profiling similar to that described in Additional file 7 was performed using proprietary MetaCore software and GO database. P values and FDR (not shown in the tables) were calculated for each category. Only categories with FDR below threshold (0.05) are shown. Note that there are no significant categories in the set of Nf1-Mouse-12 down-regulated transcripts.

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Additional file 9:

Pathway level analysis of differentially expressed genes with MetaCore tools. Up- and down-regulated genes in two human and a mouse sample sets were analyzed with GeneGo pathway maps database. P values and FDR (not shown in the tables) were calculated for each pathway. Only pathways with FDR below threshold (0.05) are shown. Note that there are no significant pathways in the sets of down-regulated transcripts.

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Additional file 10:

Dynamic change of neurofibromin level in lymphoblastoid cell lines in response to serum deprivation. We determined the effects of serum deprivation on neurofibromin level as a way to establish the physiologic relevance of lymphoblastoid cell lines (LCLs) in the study of NF1-haploinsufficiency. We measured levels of neurofibromin in two LCLs (one each from NF1-affected, and -unaffected individuals) that were serum-deprived (0.1% serum) for 16 hours, and then released for variable amounts of time in complete (10% serum; supports cell proliferation), or incomplete (1% serum; does not support cell proliferation) medium. Western blot analysis and quantitation of relative abundance of neurofibromin in an NF1-unaffected individual (A) and an NF1-affected individual (B). NF1 abundance data is shown to the right from respective western blot and is plotted as percentage relative to NF1 abundance in exponentially growing cells. "E" - exponentially growing LCLs; "S" - serum starved LCLs; 5', 30', 7 h - cells released into media containing either 1% or 10% FBS for 5 min, 30 min or 7 hours, respectively. Our experiment showed that the amount of neurofibromin increased approximately two-fold in serum-starved cells as compared to that in exponentially growing LCLs from both affected and unaffected individuals. When the cells were released into complete medium (10% FBS), the neurofibromin level quickly returned to pre-starvation levels in both NF1-affected and -unaffected LCLs. In contrast, the neurofibromin level continued to increase during prolonged incubation of the cells in incomplete medium (1% FBS). We conclude that in LCLs neurofibromin level is sensitive to environmental conditions.

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